Modeling Plant Metabolism to Optimize Oil Production

Posted by Biomechanism 

Computational studies aim to increase use of plant oils as renewable resource.

(“Biomechanism.com“) — Scientists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory have developed a computational model for analyzing the metabolic processes in rapeseed plants — particularly those related to the production of oils in their seeds. Their goal is to find ways to optimize the production of plant oils that have widespread potential as renewable resources for fuel and industrial chemicals.

Developing embryos after being excised from a growing rapeseed plant. The embryos accumulate seed oils which represent the most energy-dense form of biologically stored sunlight, and have great potential as renewable resources for fuel and industrial chemicals.

The model, described in two “featured articles” in the August 1, 2011, issue ofthe Plant Journal (now available online*), may help to identify ways to maximize the conversion of carbon to biomass to improve the production of plant-derived biofuels.

“To make efficient use of all that plants have to offer in terms of alternative energy, replacing petrochemicals in industrial processes, and even nutrition, it’s essential that we understand their metabolic processes and the factors that influence their composition,” said Brookhaven biologist Jorg Schwender, who led the development of the model with postdoctoral research associate Jordan Hay.

In the case of plant oils, the scientists’ attention is focused on seeds, where oils are formed and accumulated during development. “This oil represents the most energy-dense form of biologically stored sunlight, and its production is controlled, in part, by the metabolic processes within developing seeds,” Schwender said.

One way to study these metabolic pathways is to track the uptake and allotment of a form of carbon known as carbon-13 as it is incorporated into plant oil precursors and the oils themselves. But this method has limits in the analysis of large-scale metabolic networks such as those involved in apportioning nutrients under variable physiological conditions.

“It’s like trying to assess traffic flow on roads in the United States by measuring traffic flow only on the major highways,” Schwender said.

To address these more complex situations, the Brookhaven team constructed a computational model of a large-scale metabolic network of developing rapeseed (Brassica napus) embryos, based on information mined from biochemical literature, databases, and prior experimental results that set limits on certain variables. The model includes 572 biochemical reactions that play a role in the seed’s central metabolism and/or seed oil production, and incorporates information on how those reactions are grouped together and interact.

The scientists first tested the validity of the model by comparing it to experimental results from carbon-tracing studies for a relatively simple reaction network — the big-picture view of the metabolic pathways analogous to the traffic on U.S. highways. At that big-picture level, results from the two methods were largely consistent, providing validation for both the computer model and the experimental technique, while identifying a few exceptions that merit further exploration.

The scientists then used the model to simulate more complicated metabolic processes under varying conditions — for example, changes in oil production or the formation of oil precursors in response to changes in available nutrients (such as different sources of carbon and nitrogen), light conditions, and other variables.

A network illustrating some of the reactions and chemical pathways involved in oil production in rapeseed plants. By modeling these interacting pathways, scientists may find ways to optimize plant oil production so the oils can be used as fuels or raw materials for industrial processes.

“This large-scale model is a much more realistic network, like a map that represents almost every street,” Schwender said, “with computational simulations to predict what’s going on.” Continuing the traffic analogy, he said, “We can now try to simulate the effect of ‘road blocks’ or where to add new roads to most effectively eliminate traffic congestion.”

The model also allows the researchers to assess the potential effects of genetic modifications (for example, inactivating particular genes that play a role in plant metabolism) in a simulated environment. These simulated “knock-out” experiments gave detailed insights into the potential function of alternative metabolic pathways — for example, those leading to the formation of precursors to plant oils, and those related to how plants respond to different sources of nitrogen.

“The model has helped us construct a fairly comprehensive overview of the many possible alternative routes involved in oil formation in rapeseed, and categorize particular reactions and pathways according to the efficiency by which the organism converts sugars into oils. So at this stage, we can enumerate, better than before, which genes and reactions are necessary for oil formation, and which make oil production most effective,” Schwender said.

The researchers emphasize that experimentation will still be essential to further elucidating the factors that can improve plant oil production. “Any kind of model is a largely simplified representation of processes that occur in a living plant,” Schwender said. “But it provides a way to rapidly assess the relative importance of multiple variables and further refine experimental studies. In fact, we see our model and experimental methods such as carbon tracing as complementary ways to improve our understanding of plants’ metabolic pathways.”

The scientists are already incorporating information from this study that will further refine the model to increase its predictive power, as well as ways to extend and adapt it for use in studying other plant systems.

This work was supported by the DOE Office of Science.

Are cancers newly evolved species?

Posted by Biomechanism 

(“CANCER RESEARCH”) — Cancer patients may view their tumors as parasites taking over their bodies, but this is more than a metaphor for Peter Duesberg, a molecular and cell biology professor at the University of California, Berkeley.

Cancerous tumors are parasitic organisms, he said. Each one is a new species that, like most parasites, depends on its host for food, but otherwise operates independently and often to the detriment of its host.

A karyograph is one way to display the number of copies of each chromosome in a clone of cells from an individual or a cancer. Here, the karyograph shows the chromosomes of 20 individual cells (represented by black lines) of a normal human male. Each cell has precisely two copies of 22 chromosomes and one copy of each sex chromosome, demonstrating that human cells have a fixed and stable karyotype. Photo: Robert Sanders/Univ. of Cali

In a paper published in the July 1 issue of the journalCell Cycle, Duesberg and UC Berkeley colleagues describe their theory that carcinogenesis – the generation of cancer – is just another form of speciation, the evolution of new species.

“Cancer is comparable to a bacterial level of complexity, but still autonomous, that is, it doesn’t depend on other cells for survival; it doesn’t follow orders like other cells in the body, and it can grow where, when and how it likes,” said Duesberg. “That’s what species are all about.”

This novel view of cancer could yield new insights into the growth and metastasis of cancer, Duesberg said, and perhaps  new approaches to therapy or new drug targets. In addition, because the disrupted chromosomes of newly evolved cancers are visible in a microscope, it may be possible to detect cancers earlier, much as today’s Pap smear relies on changes in the shapes of cervical cells as an indication of chromosomal problems that could lead to cervical cancer.

Carcinogenesis and evolution

The idea that cancer formation is akin to the evolution of a new species is not new, with various biologists hinting at it in the late 20^th century. Evolutionary biologist Julian S. Huxley wrote in 1956 that “Once the neoplastic process has crossed the threshold of autonomy, the resultant tumor can be logically regarded as a new biologic species ….”

Last year, Dr. Mark Vincent of the London Regional Cancer Program and University of Western Ontario argued in the journal Evolution that carcinogenesis and the clonal evolution of cancer cells are speciation events in the strict Darwinian sense.

The evolution of cancer “seems to be different from the evolution of a grasshopper, for instance, in part because the cancer genome is not a stable genome like that of other species. The challenging question is, what has it become?” Vincent said in an interview. “Duesberg’s argument from karyotype is different from my argument from the definition of a species, but it is consistent.”

Vincent noted that there are three known transmissible cancers, including devil facial tumor disease, a “parasitic cancer” that attacks and kills Tasmanian devils. It is transmitted from one animal to another by a whole cancer cell. A similar parasitic cancer, canine transmissible venereal tumor, is transmitted between dogs via a single cancer cell that has a genome dating from the time when dogs were first domesticated. A third transmissible cancer was found in hamsters.

“Cancer has become a successful parasite,” Vincent said.

Mutation theory vs. aneuploidy

Duesbeg’s arguments derive from his controversial proposal that the reigning theory of cancer – that tumors begin when a handful of mutated genes send a cell into uncontrolled growth – is wrong. He argues, instead, that carcinogenesis is initiated by a disruption of the chromosomes, which leads to duplicates, deletions, breaks and other chromosomal damage that alter the balance of tens of thousands of genes. The result is a cell with totally new traits – that is, a new phenotype.

“I think Duesberg is correct by criticizing mutation theory, which sustains a billion-dollar drug industry focused on blocking these mutations,” said Vincent, a medical oncologist. “Yet very, very few cancers have been cured by targeted drug therapy, and even if a drug helps a patient survive six or nine more months, cancer cells often find a way around it.”

Chromosomal disruption, called aneuploidy, is known to cause disease. Down syndrome, for example, is caused by a third copy of chromosome 21, one of the 23 pairs of human chromosomes. All cancer cells are aneuploid, Duesberg said, though proponents of the mutation theory of cancer argue that this is a consequence of cancer, not the cause.

Key to Duesberg’s theory is that some initial chromosomal mutation – perhaps impairing the machinery that duplicates or segregates chromosomes in preparation for cell division – screws up a cell’s chromosomes, breaking some or making extra copies of others. Normally this would be a death sentence for a cell, but in rare cases, he said, such disrupted chromosomes might be able to divide further, perpetuating and compounding the damage. Over decades, continued cell division would produce many unviable cells as well as a few still able to divide autonomously and seed cancer.

Duesberg asserts that cancers are new species because those viable enough to continue dividing develop relatively stable chromosome patterns, called karyotypes, distinct from the chromosome pattern of their human host. While all known organisms today have stable karyotypes, with all cells containing precisely two or four copies of each chromosome, cancers exhibit a more flexible and unpredictable karyotype, including not only intact chromosomes from the host, but also partial, truncated and mere stumps of chromosomes.

“If humans changed their karyotype – the number and arrangement of chromosomes – we would either die or be unable to mate, or in very rare cases become another species,” Duesberg said. But cancer cells just divide and make more of themselves. They don’t have to worry about reproduction, which is sensitive to chromosomal balance. In fact, as long as the genes for mitosis are still intact, a cancer cell can survive with many disrupted and unbalanced chromosomes, such as those found in an aneuploid cell, he said.

The karyotype does change as a cancer cell divides, because the chromosomes are disrupted and thus don’t copy perfectly. But the karyotype is “only flexible within a certain margin,” Duesberg said. “Within these margins it remains stable, despite its flexibility.”

Karyographs display karyotype variability

Duesberg and his colleagues developed karyographs as a way to display the aneuploid nature of a cell’s karyotype and its stability across numerous cell cultures. Using these karyographs, he and his colleagues analyzed several cancers, clearly demonstrating that the karyotype is amazingly similar in all cells of a specific cancer line, yet totally different from the karyotypes of other cancers and even the same type of cancer from a different patient.

In contrast to normal cells, cervical cancer cells (HeLa) have flexible chromosomes. The 23 normal chromosomes have between 0 and 4 copies, while the several dozen hybrid or “marker” chromosomes have between 0 and 2. The copy numbers differ in the 20 individual HeLa cells shown, but they are nearly clonal, varying around an average clonal number. Photo: Robert Sanders/Univ. of Cali

HeLa cells are a perfect example. Perhaps the most famous cancer cell line in history, HeLa cells were obtained in 1951 from a cervical cancer that eventually killed a young black woman named Henrietta Lacks. The 60-year-old cell line derived from her cancer has a relatively stable karyotype that keeps it alive through division after division.

“Once a cell has crossed that barrier of autonomy, it’s a new species,” Duesberg said. “HeLa cells have evolved in the laboratory and are now even more stable than they probably were when they first arose.”

The individualized karyotypes of cancers resemble the distinct karyotypes of different species,, Duesberg said. While biologists have not characterized the karyotypes of most species, no two species are known that have the same number and arrangement of chromosomes, including those of, for example, gorillas and humans, who share 99 percent of their genes.

Duesberg argues that his speciation theory explains cancer’s autonomy, immortality and flexible, but relatively stable, karyotype. It also explains the long latency period between initial aneuploidization and full blown cancer, because there is such a low probability of evolving an autonomous karyotype.

“You start with a chromosomal mutation, that is, aneuploidy perhaps from X-rays or cigarettes or radiation, that destabilizes and eventually changes your karyotype or renders it non-viable,” he said. “The rare viable aneuploidies of cancers are, in effect, the karyotypes of new species.”

Duesberg hopes that the carcinogenesis-equals-speciation theory will spur new approaches to diagnosing and treating cancer. Vincent, for example, suspects that cancers are operating right at the edge of survivability, maintaining genomic flexibility while retaining the ability to divide forever. Driving them to evolve even faster, he said, “might push them over the edge.”

Duesberg’s colleagues are postdoctoral fellow Daniele Mandrioli and research associate Amanda McCormack of UC Berkeley and graduate student Joshua M. Nicholson in the Department of Biological Sciences at Virginia Polytechnic Institute.

Duesberg’s research is funded by the Abraham J. and Phyllis Katz Foundation, philanthropists Dr. Christian Fiala, Rajeev and Christine Joshi, Robert Leppo and Peter Rozsa of the Taubert Memorial Foundation, other private sources and the Forschungsfonds der Fakultät für Klinische Medizin Mannheim der Universität Heidelberg.

Staining chromosomes with different dyes highlights the orderly nature of the normal human karyotype (left), that is, humans have precisely two copies of each chromosome with no leftovers. A bladder cancer cell (right) has extra copies of some chromosomes, a few missing normal chromsomes, and a lot of hybrid or marker chromosomes, which characterize cancer cells. Photo: Robert Sanders/Univ of California

UBC researchers create more powerful “lab-on-a-chip” for genetic analysis

Posted by Biomechanism 

(“Biomechanism.com“) — UBC researchers have invented a silicone chip that could make genetic analysis far more sensitive, rapid, and cost-effective by allowing individual cells to fall into place like balls in a pinball machine.

Microfluidic Chip

The UBC device – about the size of a nine-volt battery – allows scientists to simultaneously analyze 300 cells individually by routing fluid carrying cells through microscopic tubes and valves. Once isolated into their separate chambers, the cells’ RNA can be extracted and replicated for further analysis.

By enabling such “single-cell analysis,” the device could accelerate genetic research and hasten the use of far more detailed tests for diagnosing cancer.

Single-cell analysis is emerging as the gold standard of genetic research because tissue samples, even those taken from a single tumour, contain a mixture of normal cells and various types of cancer cells – the most important of which may be present in only very small numbers and impossible to distinguish.

So standard genetic tests, which require large numbers of cells, capture only an average “composite picture” of thousands or millions of different cells – obscuring their true nature and the interactions between them.

“It’s like trying to trying to understand what makes a strawberry different from a raspberry by studying a blended fruit smoothie,” says Carl Hansen, an assistant professor in the Dept. of Physics and Astronomy and the Centre for High-Throughput Biology, who led the team that developed the device.

The device, described and validated in this week’s issue of the Proceedings of the National Academy of Sciences, was developed by Hansen’s team, in collaboration with  researchers from BC Cancer Agency and the Centre for Translational and Applied Genomics.

The device’s ease of use and cost-effectiveness arise from its integration of almost the entire process of cell analysis – not just separating the cells, but mixing them with chemical reagents to highlight their genetic code and analyzing the results by measuring fluorescent light emitted from the reaction. Now all of that can be done on the chip.

“Single-cell genetic analysis is vital in a host of areas, including stem cell research and advanced cancer biology and diagnostics,” Hansen says. “But until now, it has been too costly to become widespread in research, and especially for use in health care. This technology, and other approaches like it, could radically change the way we do both basic and applied biomedical research, and would make single-cell analysis a more plausible option for treating patients – allowing clinicians to distinguish various cancers from one another and tailor their treatments accordingly.”

The research was funded by Genome BC, Genome Canada, Western Economic Diversification Canada, the Canadian Institutes of Health Research, the Terry Fox Foundation, and the Natural Sciences and Engineering Research Council.

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‘Fragile’ gene risks dementia

Monash University

The research could change the way health professionals identify and treat late-onset dementia. Image: annedehaas]/iStockphoto New research could change the way health professionals identify and treat late-onset dementia. Monash researcher Professor Kim Cornish and colleagues at the University of London examined impulsivity, attention and working memory skills of men aged 18 to 69 years, who were “carriers” of the FMR1 (Fragile X Mental Retardation 1) gene. Their findings will be published in the August edition of the prestigious journal Neurology. Fragile X Syndrome (FXS) is a leading cause of inherited intellectual disability worldwide and one of the few known single-gene causes of autism. It occurs when the FMR1 gene, found on the X chromosome, mutates. Approximately 70,000 Australian men and women will be carriers, and for many years it was assumed that carriers were unaffected by any of the challenges faced by those with FXS. The Monash research showed that, despite having none of the obvious symptoms of the syndrome earlier in life, carriers may be at high risk of developing severe dementia as they age. The FMR1 gene contains a DNA sequence which is prone to excessive repetition. In these cases, the gene is said to undergo “expansion”. Individuals whose FMR1 gene is affected by this excessive repetition are usually considered to be in one of two categories: small-medium expansion and large expansion. Those with small-medium expansions are known as carriers. They have the gene in a premutation (PM) state and do not have Fragile X Syndrome, but can pass it on to successive generations. Those with large expansions are considered to have the “full mutation” of the gene. These individuals will have Fragile X syndrome. The men were tested for their ability to phase out irrelevant information as well as actively store short-term information. These core brain functions decline with late-stage dementia. The research found that carriers of the gene who were at the upper end of the medium expansion range were more likely to have problems with inhibition and remembering materials, demonstrating cognitive dementia symptoms, whereas those who had expansions just within the medium range appeared risk-free. The findings will make it easier to accurately identify men who may go on to develop Fragile X-associated dementia and influence current approaches to diagnosing, preventing and treating the disorder. Professor Cornish, who conceptualised and designed the study, said that it provided the first clear evidence that being a male carrier with a larger expansion may infer some risk. "Until 10 years ago, it was assumed that carriers of FXS would remain free of symptoms as they grew older," Professor Cornish said. "It is now well-documented that approximately 30 to 40 per cent of PM males will develop FXS-related late-stage dementia." Recognising the need to identify risk factors in Australian carriers of the FXS gene, a new study funded by the Australian Research Council and led by Professor Cornish will for the first time chart the history of strengths and challenges facing carriers across their lifespan.

How tumours kill devils

University of Tasmania

“The unique ability of DFTD to hide from the devil’s immune system is intriguing and baffling." Image: altmarkfoto/iStockphoto The secret behind the spread of the Devil Facial Tumour Disease is due to more than just a lack of genetic diversity within the species, research published by scientists working with the Save the Tasmanian Devil Program has revealed. DFTD is a transmissible cancer that is spread from animal to animal through biting. Previously, it was thought that the transmitted tumour cells weren’t rejected because of the lack of genetic variation within the Tasmanian devil population. But Associate Professor Greg Woods, from the Menzies Research Institute Tasmania, said the latest research suggests that DFTD is more the fault of the tumour than the devil. “The unique ability of DFTD to hide from the devil’s immune system is intriguing and baffling,” said Assoc. Prof. Woods. “We know that a lack of genetic diversity is part of the reason, but we were keen to test the severity of this limited diversity - and the simple way to do this was through skin grafts.” To help with this procedure, the researchers called in the assistance of Royal Hobart Hospital plastic surgeon, Mr Frank Kimble. Skin samples from both healthy and diseased devils were grafted on to other devils. The theory was that if the grafts would take, then those devils must be very genetically similar. “It has been a great honour to be involved in this important research,” Mr Kimble said. “Undertaking these procedures on the devils was quite difficult and one of the hardest aspects was getting the devils to keep their dressings on, just like working with some of my paediatric patients! “This work certainly demonstrated to me the complexity of both the devils and the DFTD.” All of the five successful skin allografts performed by Mr Kimble were rejected within 14 days of surgery. Assoc. Prof. Woods said this result indicated there is enough genetic diversity within the species to produce a protective immune response. “That result brings us back to the tumour,” Assoc. Prof. Woods said. “What is special about the tumour cells that they can avoid rejection by the host devil? “A lack of genetic diversity is still part of the answer, but there must be something else. Something is missing from those tumour cells.” The challenge for the team now, said the manager of the Save the Tasmanian Devil Program, Mr Andrew Sharman, is to understand more about the tumour itself – particularly its ability to hide from the devil’s immune system. The paper, titled ‘Allorecognition in the Tasmanian devil (Sarcophilus harrisii), an Endangered Marsupial Species with Limited Genetic Diversity’, was authored by Dr Alexandre Kreiss (Menzies Research Institute Tasmania), Ms Yuan Yuan Cheng (University of Sydney), Mr Frank Kimble (Royal Hobart Hospital), Mr Barrie Wells (The University of Tasmania), Dr Shaun Donovan (Royal Hobart Hospital), Associate Professor Kathy Belov (University of Sydney) and Associate Professor Greg Woods (Menzies Research Institute Tasmania). It was published in PLoS ONE, an interactive open-access journal for the communication of all peer-reviewed scientific and medical research. The paper is available online at: http://dx.plos.org/10.1371/journal.pone.0022402.

How to curb unplanned pregnancies

University of Otago

Image: milanklusacek/iStockphoto Repeat abortions are significantly reduced if women use long-acting reversible contraceptive methods such as intrauterine devices (IUDs) after an abortion. The new research by the Women's Health Research Centre, University of Otago, Wellington, has just been published in the American Journal of Obstetrics and Gynecology. It involved a study that followed 510 women aged from 13-44 (mean 25) after they had an abortion in a public clinic in Wellington and chose either a free long-acting reversible contraceptive (LARC) method or a non-LARC method. LARC methods included a multiload or Mirena IUD or a Depoprovera (DMPA) injection. Women were then followed up at two years to determine whether they had another abortion. The study showed only 6.45 per cent of women who used LARC methods had a repeat abortion, whereas those who used some other method of contraception had a 14.5 per cent return rate. “These results tell us that if women who have an abortion then use a long-acting contraceptive, particularly an IUD, they are far less likely to have a repeat abortion within two years than women who use the pill, condoms or some other method,” says lead author Dr Sally Rose. Co-author and Women’s Health Research Centre director, Dr Beverley Lawton, says use of LARCs in New Zealand has historically been relatively low compared to other methods for a range of reasons including high upfront costs and lack of knowledge amongst providers and patients. Guidelines and best practice support doctors discussing use of LARC methods with all reproductive aged women. “These are almost 100 per cent effective at preventing pregnancy and are reversible if you don’t like them. It’s interesting that 80 per cent of women who chose IUDs were under the age of 25 in our study. Historically IUDs have been thought of as more suitable for older women,” Dr Lawton says. Dr Rose says the recent addition of contraceptive implants to government funded long-acting methods (DMPA and the multiload IUD) is a positive step for women wanting effective long-acting methods, but PHARMAC now needs to extend this funding to include the Mirena hormonal IUD. Removal of cost as a barrier to use of long-acting methods will be key in achieving more widespread use. "We’re very excited about the potential public health impact of these findings. By improving access to long-acting contraception, women will not only have better choices to control their fertility, but we now know they will also reduce their chance of a further unplanned pregnancy" Dr Rose says.