Missing Link in Parkinson's Disease Found: Discovery Also Has Implications for Heart Failure

Researchers at Washington University School of Medicine in St. Louis have described a missing link in understanding how damage to the body's cellular power plants leads to Parkinson's disease and, perhaps surprisingly, to some forms of heart failure.

These cellular power plants are called mitochondria. They manufacture the energy the cell requires to perform its many duties. And while heart and brain tissue may seem entirely different in form and function, one vital characteristic they share is a massive need for fuel.

Working in mouse and fruit fly hearts, the researchers found that a protein known as mitofusin 2 (Mfn2) is the long-sought missing link in the chain of events that control mitochondrial quality.

The findings are reported April 26 in the journal Science.

The new discovery in heart cells provides some explanation for the long known epidemiologic link between Parkinson's disease and heart failure.

"If you have Parkinson's disease, you have a more than two-fold increased risk of developing heart failure and a 50 percent higher risk of dying from heart failure," says senior author Gerald W. Dorn II, MD, the Philip and Sima K. Needleman Professor of Medicine. "This suggested they are somehow related, and now we have identified a fundamental mechanism that links the two."

Heart muscle cells and neurons in the brain have huge numbers of mitochondria that must be tightly monitored. If bad mitochondria are allowed to build up, not only do they stop making fuel, they begin consuming it and produce molecules that damage the cell. This damage eventually can lead to Parkinson's or heart failure, depending on the organ affected. Most of the time, quality-control systems in a healthy cell make sure damaged or dysfunctional mitochondria are identified and removed.

Over the past 15 years, scientists have described much of this quality-control system. Both the beginning and end of the chain of events are well understood. And since 2006, scientists have been working to identify the mysterious middle section of the chain -- the part that allows the internal environment of sick mitochondria to communicate to the rest of the cell that it needs to be destroyed.

"This was a big question," Dorn says. "Scientists would draw the middle part of the chain as a black box. How do these self-destruct signals inside the mitochondria communicate with proteins far away in the surrounding cell that orchestrate the actual destruction?"

"To my knowledge, no one has connected an Mfn2 mutation to Parkinson's disease," Dorn says. "And until recently, I don't think anybody would have looked. This isn't what Mfn2 is supposed to do."

Mitofusin 2 is known for its role in fusing mitochondria together, so they might exchange mitochondrial DNA in a primitive form of sexual reproduction.

"Mitofusins look like little Velcro loops," Dorn says. "They help fuse together the outer membranes of mitochondria. Mitofusins 1 and 2 do pretty much the same thing in terms of mitochondrial fusion. What we have done is describe an entirely new function for Mfn2."

The mitochondrial quality-control system begins with what Dorn calls a "dead man's switch."

"If the mitochondria are alive, they have to do work to keep the switch depressed to prevent their own self-destruction," Dorn says.

Specifically, mitochondria work to import a molecule called PINK. Then they work to destroy it. When mitochondria get sick, they can't destroy PINK and its levels begin to rise. Then comes the missing link that Dorn and his colleague Yun Chen, PhD, senior scientist, identified. Once PINK levels get high enough, they make a chemical change to Mfn2, which sits on the surface of mitochondria. This chemical change is called phosphorylation. Phosphorylated Mfn2 on the surface of the mitochondria can then bind with a molecule called Parkin that floats around in the surrounding cell.

Once Parkin binds to Mfn2 on sick mitochondria, Parkin labels the mitochondria for destruction. The labels then attract special compartments in the cell that "eat" and destroy the sick mitochondria. As long as all links in the quality-control system work properly, the cells' damaged power plants are removed, clearing the way for healthy ones.

"But if you have a mutation in PINK, you get Parkinson's disease," Dorn says. "And if you have a mutation in Parkin, you get Parkinson's disease. About 10 percent of Parkinson's disease is attributed to these or other mutations that have been identified."

According to Dorn, the discovery of Mfn2's relationship to PINK and Parkin opens the doors to a new genetic form of Parkinson's disease. And it may help improve diagnosis for both Parkinson's disease and heart failure.

"I think researchers will look closely at inherited Parkinson's cases that are not explained by known mutations," Dorn says. "They will look for loss of function mutations in Mfn2, and I think they are likely to find some."

Similarly, as a cardiologist, Dorn and his colleagues already have detected mutations in Mfn2 that appear to explain certain familial forms of heart failure, the gradual deterioration of heart muscle that impairs blood flow to the body. He speculates that looking for mutations in PINK and Parkin might be worthwhile in heart failure as well.

"In this case, the heart has informed us about Parkinson's disease, but we may have also described a Parkinson's disease analogy in the heart," he says. "This entire process of mitochondrial quality control is a relatively small field for heart specialists, but interest is growing."

This work was supported by the National Institutes of Health (NIH) grants R01 HL059888 and R21 HL107276.

Journal Reference:

1. Y. Chen, G. W. Dorn. PINK1-Phosphorylated Mitofusin 2 Is a Parkin Receptor for Culling Damaged Mitochondria. Science, 2013; 340 (6131): 471 DOI:10.1126/science.1231031

Courtesy: ScienceDaily

Hundreds of Potential Drug Targets to Starve Cancer Tumors Identified

A massive study analyzing gene expression data from 22 tumor types has identified multiple metabolic expression changes associated with cancer. The analysis, conducted by researchers at Columbia University Medical Center, also identified hundreds of potential drug targets that could cut off a tumor's fuel supply or interfere with its ability to synthesize essential building blocks. The study was published today in the online edition ofNature Biotechnology.

The results should ramp up research into drugs that interfere with cancer metabolism, a field that dominated cancer research in the early 20th century and has recently undergone a renaissance.

"The importance of this new study is its scope," said Dennis Vitkup, PhD, associate professor of biomedical informatics (in the Initiative in Systems Biology) at CUMC, the study's lead investigator. "So far, people have focused mainly on a few genes involved in major metabolic processes. Our study provides a comprehensive, global view of diverse metabolic alterations at the level of gene expression."

Cell metabolism is a dynamic network of reactions inside cells that process nutrients, such as glucose, to obtain energy and synthesize building blocks needed to produce new cellular components. To support uncontrolled proliferation, cancer needs to significantly reprogram and "supercharge" a cell's normal metabolic pathways.

The first researcher to notice cancer's special metabolism was German biochemist Otto Warburg, who in 1924 observed that cancer cells had a peculiar way of utilizing glucose to make energy for the cell. "Although a list of biochemical pathways in normal cells was comprehensively mapped during the last century," said Dr. Vitkup. "We still lack a complete understanding of their usage, regulation, and reprogramming in cancer."

"Right now we have something like a static road map. We know where the streets are, but we don't know how traffic flows through the streets and intersections," said Jie Hu, PhD, a postdoctoral researcher at Columbia and first author of the study. "What researchers need is something similar to Google Traffic, which shows the flow and dynamic changes in car traffic."

Drs. Hu and Vitkup's study is an important step toward achieving this dynamic view of cancer metabolism. Notably, the researchers found that the tumor-induced expression changes are significantly different across diverse tumors. Although some metabolic changes -- such as an increase in nucleotide biosynthesis and glycolysis -- appear to be more frequent across tumors, others, such as changes in oxidation phosphorylation, are heterogeneous.

"Our study clearly demonstrates that there are no single and universal changes in cancer metabolism," said Matthew Vander Heiden, MD, PhD, assistant professor at MIT, and a co-author of the paper. "That means that to understand transformation in cancer metabolism, researchers will need to consider how different tumor types adapt their metabolism to meet their specific needs."

The researchers also found that expression changes can mimic or cooperate with cancer mutations to drive tumor formation. A notable example is the enzyme isocitrate dehydrogenase. In several cancers, such as glioblastoma and acute myeloid leukemia, mutations in this enzyme are known to produce a specific metabolite -- 2-hydroxyglutarate -- that promotes tumor growth. The Columbia team found that isocitrate dehydrogenase expression significantly increases in tumors with the recurrent mutations. Such an overexpression may create an efficient enzymatic factory for overproduction of 2-hydroxyglutarate.

The analysis also led the researchers to an interesting finding in colon cancer. In several other cancers, mutations in two enzymes -- succinate dehydrogenase and fumarate hydratase -- can promote tumor formation as a result of efflux from mitochondria and accumulation of their substrates, fumarate and succinate. The researchers found that in colon cancer, accumulation of these metabolites may be caused by a significant decrease in the enzymes' expression. This was confirmed when metabolomics data from colon tumor patients showed significantly higher concentrations of fumarate in tumors than in normal tissue.

"These are just several examples of how cancer cells use various creative mechanisms to hijack the metabolism of native cells for their own purposes," said Dr. Vitkup.

For cancer researchers looking for new drug targets, Dr. Vitkup's team also found hundreds of differences between normal and cancer cells' use of isoenzymes. This opens up additional possibilities for turning off cancer's fuel and supply lines. Isoenzymes often catalyze the same reactions, but have different kinetic properties: Some act quickly and sustain rapid growth, while others are more sluggish. In kidney and liver cancers, for example, a quick-acting aldolase isoenzyme -- suitable for fast cell proliferation -- was found to be more prevalent than the more typical slow-moving version found in normal kidney and liver tissue. Although a few examples of differential isoenzyme expression in tumors were already known, the Columbia researchers identified hundreds of isoenzymes with cancer-specific expression patterns.

"Inhibiting specific isoenzymes in tumors may be a way to selectively hit cancer cells without affecting normal cells, which could get by with other isoenzymes," said Dr. Hu.

In fact, a recent study from Matthew Vander Heiden's laboratory demonstrated the potential of targeting a specific isoenzyme, pyruvate kinase M2, expression of which often increases in tumors. "The comprehensive expression analysis suggests that a similar approach could potentially be applied in multiple other cases," said Dr. Vander Heiden.

Targeting metabolism may be a way to strike cancer at its roots. "Cancer cells usually have multiple ways to turn on their growth program," said Dr. Vitkup. "You can knock out one, but the cells will usually find another pathway to turn on proliferation. Targeting metabolism may be more powerful, because if you starve a cell of energy or materials, it has nowhere to go."

The paper is titled, "Heterogeneity of tumor-induced gene expression changes in the human metabolic network." The other authors are Jason W. Locasale (Cornell University), Jason H. Bielas (Fred Hutchinson Cancer Research Center, Seattle, Wash.; and University of Washington, Seattle, Wash.), Jacintha O'Sullivan (St. Vincent's University Hospital, Dublin, Ireland), Kieran Sheahan St. Vincent's University Hospital, Dublin, Ireland), and Lewis C. Cantley (Harvard Medical School).

Dr. Vander Heiden is a consultant and advisory board member, and Dr. Cantley is a consultant and founder, of Agios Pharmaceuticals. The authors report no other financial or potential conflicts of interest.

This work was supported by National Institutes of Health grant GM079759 to Dr. Vitkup and National Centers for Biomedical Computing grant U54CA121852 to Columbia University. Dr. Locasale is supported by an NIH Pathway to Independence Award R00CA168997. Dr. Bielas is supported by an Ellison Medical Foundation New Scholar award AG-NS-0577-09, a National Institute of Environmental Health Sciences grant R01ES019319, and New Development Funds from the Fred Hutchinson Cancer Research Center. Dr. Vander Heiden acknowledges support from the Burroughs Wellcome Fund, the Damon Runyon Cancer Research Foundation, the Smith Family, and the National Cancer Institute.

Journal Reference:

1. Jie Hu, Jason W Locasale, Jason H Bielas, Jacintha O'Sullivan, Kieran Sheahan, Lewis C Cantley, Matthew G Vander Heiden, Dennis Vitkup. Heterogeneity of tumor-induced gene expression changes in the human metabolic network. Nature Biotechnology, 2013; DOI:10.1038/nbt.2530

Courtesy: ScienceDaily

Stem Cell Transplant Restores Memory, Learning in Mice

For the first time, human embryonic stem cells have been transformed into nerve cells that helped mice regain the ability to learn and remember.

A study at the University of Wisconsin-Madison is the first to show that human stem cells can successfully implant themselves in the brain and then heal neurological deficits, says senior author Su-Chun Zhang, a professor of neuroscience and neurology.

Once inside the mouse brain, the implanted stem cells formed two common, vital types of neurons, which communicate with the chemicals GABA or acetylcholine. "These two neuron types are involved in many kinds of human behavior, emotions, learning, memory, addiction and many other psychiatric issues," says Zhang.

The human embryonic stem cells were cultured in the lab, using chemicals that are known to promote development into nerve cells -- a field that Zhang has helped pioneer for 15 years. The mice were a special strain that do not reject transplants from other species.

After the transplant, the mice scored significantly better on common tests of learning and memory in mice. For example, they were more adept in the water maze test, which challenged them to remember the location of a hidden platform in a pool.

The study began with deliberate damage to a part of the brain that is involved in learning and memory.

Three measures were critical to success, says Zhang: location, timing and purity. "Developing brain cells get their signals from the tissue that they reside in, and the location in the brain we chose directed these cells to form both GABA and cholinergic neurons."

The initial destruction was in an area called the medial septum, which connects to the hippocampus by GABA and cholinergic neurons. "This circuitry is fundamental to our ability to learn and remember," says Zhang.

The transplanted cells, however, were placed in the hippocampus -- a vital memory center -- at the other end of those memory circuits. After the transferred cells were implanted, in response to chemical directions from the brain, they started to specialize and connect to the appropriate cells in the hippocampus.

The process is akin to removing a section of telephone cable, Zhang says. If you can find the correct route, you could wire the replacement from either end.

For the study, published in the current issue of Nature Biotechnology, Zhang and first author Yan Liu, a postdoctoral associate at the Waisman Center on campus, chemically directed the human embryonic stem cells to begin differentiation into neural cells, and then injected those intermediate cells. Ushering the cells through partial specialization prevented the formation of unwanted cell types in the mice.

Ensuring that nearly all of the transplanted cells became neural cells was critical, Zhang says. "That means you are able to predict what the progeny will be, and for any future use in therapy, you reduce the chance of injecting stem cells that could form tumors. In many other transplant experiments, injecting early progenitor cells resulted in masses of cells -- tumors. This didn't happen in our case because the transplanted cells are pure and committed to a particular fate so that they do not generate anything else. We need to be sure we do not inject the seeds of cancer."

Brain repair through cell replacement is a Holy Grail of stem cell transplant, and the two cell types are both critical to brain function, Zhang says. "Cholinergic neurons are involved in Alzheimer's and Down syndrome, but GABA neurons are involved in many additional disorders, including schizophrenia, epilepsy, depression and addiction."

Though tantalizing, stem-cell therapy is unlikely to be the immediate benefit. Zhang notes that "for many psychiatric disorders, you don't know which part of the brain has gone wrong." The new study, he says, is more likely to see immediate application in creating models for drug screening and discovery.

Journal Reference:

1. Yan Liu, Jason P Weick, Huisheng Liu, Robert Krencik, Xiaoqing Zhang, Lixiang Ma, Guo-min Zhou, Melvin Ayala, Su-Chun Zhang. Medial ganglionic eminence–like cells derived from human embryonic stem cells correct learning and memory deficits. Nature Biotechnology, 2013; DOI: 10.1038/nbt.2565

Courtesy: ScienceDaily

Special E. Coli Bacteria Produce Diesel On Demand

It sounds like science fiction but a team from the University of Exeter, with support from Shell, has developed a method to make bacteria produce diesel on demand. While the technology still faces many significant commercialisation challenges, the diesel, produced by special strains ofE. coli bacteria, is almost identical to conventional diesel fuel and so does not need to be blended with petroleum products as is often required by biodiesels derived from plant oils.

This also means that the diesel can be used with current supplies in existing infrastructure because engines, pipelines and tankers do not need to be modified. Biofuels with these characteristics are being termed 'drop-ins'.

Professor John Love from Biosciences at the University of Exeter said: "Producing a commercial biofuel that can be used without needing to modify vehicles has been the goal of this project from the outset. Replacing conventional diesel with a carbon neutral biofuel in commercial volumes would be a tremendous step towards meeting our target of an 80% reduction in greenhouse gas emissions by 2050. Global demand for energy is rising and a fuel that is independent of both global oil price fluctuations and political instability is an increasingly attractive prospect."

E. coli bacteria naturally turn sugars into fat to build their cell membranes. Synthetic fuel oil molecules can be created by harnessing this natural oil production process. Large scale manufacturing using E. coli as the catalyst is already commonplace in the pharmaceutical industry and, although the biodiesel is currently produced in tiny quantities in the laboratory, work will continue to see if this may be a viable commercial pathway to 'drop in' fuels.

Rob Lee from Shell Projects & Technology said: "We are proud of the work being done by Exeter in using advanced biotechnologies to create the specific hydrocarbon molecules that we know will continue to be in high demand in the future. While the technology still faces several hurdles to commercialisation, by exploring this new method of creating biofuel, along with other intelligent technologies, we hope they could help us to meet the challenges of limiting the rise in carbon dioxide emissions while responding to the growing global requirement for transport fuel."

Journal Reference:

1. Thomas P. Howard, Sabine Middelhaufe, Karen Moore, Christoph Edner, Dagmara M. Kolak, George N. Taylor, David A. Parker, Rob Lee, Nicholas Smirnoff, Stephen J. Aves, and John Love. Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. PNAS, April 22, 2013 DOI: 10.1073/pnas.1215966110

Courtesy: ScienceDaily

Vaccine Adjuvant Uses Host DNA to Boost Pathogen Recognition

Aluminum salts, or alum, have been injected into billions of people as an adjuvant to make vaccines more effective. No one knows, however, how they boost the immune response. In the March 19, 2013, issue of the Proceedings of the National Academy of Sciences, researchers at National Jewish Health continue unraveling the mystery of adjuvants with a report that host DNA coats the alum adjuvant and induces two crucial cells to interact twice as long during the initial stimulation of the adaptive immune system.

"Alum makes T cells take a longer look at the antigen, which produces a better immune response," said Philippa Marrack, PhD, senior author and professor of immunology at National Jewish Health. "Understanding how adjuvants work could help us make more effective vaccines. That is very important. Vaccines have saved millions of lives and been among the greatest advances in medical history."

Live vaccines, containing weakened forms of an infectious organism, generally work fine by themselves. But vaccines containing dead organisms (inactivated vaccines) or pieces of the infectious organisms or their toxins (acellular or recombinant vaccines) generally need adjuvants to boost their effectiveness.Aluminum salts, known as alum, are the only adjuvant approved for use in the United States for routine preventive vaccines.

Adjuvants were first discovered as the result of empirical experiments with tetanus early in the 20th century. They have been widely used in many vaccines since the 1940s, including the Diphtheria/Tetanus/Pertussis (DtaP), Hepatitis, Haemophilus influenzae (Hib), typhoid and some flu vaccines. No one fully understands why adjuvants boost the effectiveness of nonliving vaccines.

Recently a Belgian team showed that DNA is involved in the adjuvant effect. When they administered a vaccine with adjuvant and DNase, an enzyme that digests DNA, the vaccine was less effective. The National Jewish Health team built on those findings to reveal the role that DNA plays.

The National Jewish Health team had previously shown that the process starts with a series of events similar to those that initiate responses to bacterial infections. Neutrophils, and other early responders in the immune system, flood into a site of potential infection, attack the foreign agent, in this case the alum vaccine, then quickly die in massive numbers.

Upon death the neutrophils release large amounts of DNA, which uncoils from its chromatin spools and acts somewhat like a net to entangle the foreign agent. Other cells then engulf the DNA-alum-vaccine complex. These antigen-presenting cells display small fragments of the vaccine on their surfaces for T-cells to recognize. T-cells drive the adaptive immune response, the one that recognizes and attacks the specific infectious agent, as opposed to the more general innate immune response.

T-cells are also the basis for effective vaccines. Some T-cells, and the B-cells stimulated by the T-cells, transform into memory cells once the infection has been cleared. Those memory cells help mount a quicker and stronger immune response if they see that organism again.

The National Jewish Health team showed that the DNA coating the adjuvant doubles the time that the T-cell engages the vaccine fragment on the surface of the antigen-presenting cell. When they added DNase to digest DNA, the T-cell engaged the vaccine fragment half as long, and the vaccine was less effective. Several of the findings were made possible by an innovative use of multi-photon microscopy to film the interaction of T-cells and antigen-presenting cells.

"The DNA makes the antigen-presenting cell stickier," said Amy McKee, PhD, Instructor at the University of Colorado, and lead author of the paper. "We believe that extended engagement provides a stronger signal to the T-cell, which makes the immune response more robust."

The researchers are not sure exactly what makes the antigen-presenting cell 'stickier.' When that an antigen-presenting cell engulfs free-floating DNA, the researchers believe it recognizes that something is amiss (DNA should not normally be floating around outside an intact cell nucleus) and becomes more activated. It may respond with an additional co-receptor to engage the T-cell or release a molecule that stimulates the T-cell. The researchers are now working to understand that process.

Journal Reference:

1. A. S. McKee, M. A. Burchill, M. W. Munks, L. Jin, J. W. Kappler, R. S. Friedman, J. Jacobelli, P. Marrack. Host DNA released in response to aluminum adjuvant enhances MHC class II-mediated antigen presentation and prolongs CD4 T-cell interactions with dendritic cells. Proceedings of the National Academy of Sciences, 2013; 110 (12): E1122 DOI: 10.1073/pnas.1300392110

Courtesy: ScienceDaily

Pathogen's Scissor-Like Enzyme Provides New Clues to Treatment of Infectious Disease

UT Southwestern Medical Center researchers report that a pathogen annually blamed for an estimated 90 million cases of food-borne illness defeats a host's immune response by using a fat-snipping enzyme to cut off cellular communication.

"Our findings provide insight into severe bacterial infectious diseases, as well as some forms of cancer, in which the attachment of fat molecules to proteins is an essential feature of the disease process," said Dr. Neal Alto, assistant professor of microbiology and senior author of the study in today's print edition ofNature. The study's first author is Nikolay Burnaevskiy, a graduate student in microbiology.

The research group discovered a scissor-like enzyme that specifically cuts off functionally-essential fatty acids from proteins. "The one we studied in particular -- a 14-carbon saturated fatty acid called myristic acid- has received a lot of attention due to its crucial role in the transformation of normal cells to cancer cells and for promoting cancer cell growth," Dr. Alto said.

Because of the fat's importance in human disease, researchers have tried for years to identify effective methods to remove them from proteins. "To our amazement, bacteria have invented the precise tool for the job," Dr Alto said.

The bacteria used in this study,Shigella flexneri, are able to cross the intestinal wall and infect immune cells. Other intestinal bacteria, such as E. coli, are unable to do this. Once Shigella encounters immune system cells, including white blood cells such as macrophages, the bacteria use a needle-like complex to inject the cells with about 20 bacterial toxins.

The UTSW researchers conducted a series of experiments to characterize one of those toxins, called IpaJ, chosen in part because so little was known about the protein. They not only discovered IpaJ's fat-cutting ability, but also determined how the protein disables the immune system's communication infrastructure, which Dr. Alto compared to knocking out a bridge needed to deliver a package.

"Normally, a macrophage will engulf an invading bacteria and send out cytokines, proteins that act as cellular alert signals, which in turn recruit more immune cells to the site of infection," Dr. Alto said. "When the macrophages engulf Shigella, however, the bacteria use IpaJ to cut fatty acids from proteins, which need those fats attached in order to sound the alarm. Doing so buys more time for the bacteria to grow and survive.

"It's very interesting from a disease process point of view, but it's also important because we now have a potential drug target," said Dr. Alto. The next step, he said, will be to identify small molecule inhibitors that are specific to this fat-snipping protease and that might be developed into drugs.

The study in Nature received support from the National Institutes of Health, the Welch Foundation, and the Burroughs Wellcome Fund.

Journal Reference:

1. Nikolay Burnaevskiy, Thomas G. Fox, Daniel A. Plymire, James M. Ertelt, Bethany A. Weigele, Andrey S. Selyunin, Sing Sing Way, Steven M. Patrie, Neal M. Alto. Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ. Nature, 2013; 496 (7443): 106 DOI: 10.1038/nature12004

Courtesy: ScienceDaily

3-D Printer Can Build Synthetic Tissues

A custom-built programmable 3D printer can create materials with several of the properties of living tissues, Oxford University scientists have demonstrated.

The new type of material consists of thousands of connected water droplets, encapsulated within lipid films, which can perform some of the functions of the cells inside our bodies.

These printed 'droplet networks' could be the building blocks of a new kind of technology for delivering drugs to places where they are needed and potentially one day replacing or interfacing with damaged human tissues. Because droplet networks are entirely synthetic, have no genome and do not replicate, they avoid some of the problems associated with other approaches to creating artificial tissues -- such as those that use stem cells.

The team report their findings in this week's Science.

'We aren't trying to make materials that faithfully resemble tissues but rather structures that can carry out the functions of tissues,' said Professor Hagan Bayley of Oxford University's Department of Chemistry, who led the research. 'We've shown that it is possible to create networks of tens of thousands connected droplets. The droplets can be printed with protein pores to form pathways through the network that mimic nerves and are able to transmit electrical signals from one side of a network to the other.'

Each droplet is an aqueous compartment about 50 microns in diameter. Although this is around five times larger than living cells the researchers believe there is no reason why they could not be made smaller. The networks remain stable for weeks.

'Conventional 3D printers aren't up to the job of creating these droplet networks, so we custom built one in our Oxford lab to do it,' said Professor Bayley. 'At the moment we've created networks of up to 35,000 droplets but the size of network we can make is really only limited by time and money. For our experiments we used two different types of droplet, but there's no reason why you couldn't use 50 or more different kinds.'

The unique 3D printer was built by Gabriel Villar, a DPhil student in Professor Bayley's group and the lead author of the paper.

The droplet networks can be designed to fold themselves into different shapes after printing -- so, for example, a flat shape that resembles the petals of a flower is 'programmed' to fold itself into a hollow ball, which cannot be obtained by direct printing. The folding, which resembles muscle movement, is powered by osmolarity differences that generate water transfer between droplets.

Gabriel Villar of Oxford University's Department of Chemistry said: 'We have created a scalable way of producing a new type of soft material. The printed structures could in principle employ much of the biological machinery that enables the sophisticated behaviour of living cells and tissues.'

Journal Reference:

1. Gabriel Villar, Alexander D. Graham, Hagan Bayley. A Tissue-Like Printed Material. Science, 5 April 2013: Vol. 340 no. 6128 pp. 48-52 DOI: 10.1126/science.1229495.

Courtesy: ScienceDaily

Parkinson's Disease Protein Gums Up Garbage Disposal System in Cells

Clumps of α-synuclein protein in nerve cells are hallmarks of many degenerative brain diseases, most notably Parkinson's disease.

"No one has been able to determine if Lewy bodies and Lewy neurites, hallmark pathologies in Parkinson's disease can be degraded," says Virginia Lee, PhD, director of the Center for Neurodegenerative Disease Research, at the Perelman School of Medicine, University of Pennsylvania.

"With the new neuron model system of Parkinson's disease pathologies our lab has developed recently, we demonstrated that these aberrant clumps in cells resist degradation as well as impair the function of the macroautophagy system, one of the major garbage disposal systems within the cell."

Macroautophagy, literally self eating, is the degradation of unnecessary or dysfunctional cellular bits and pieces by a compartment in the cell called the lysosome.

Lee, also a professor of Pathology and Laboratory Medicine, and colleagues published their results in the early online edition of the Journal of Biological Chemistry this week.

Alpha-synuclein (α-syn ) diseases all have clumps of the protein and include Parkinson's disease (PD), and array of related disorders: PD with dementia , dementia with Lewy bodies, and multiple system atrophy. In most of these, α-syn forms insoluble aggregates of stringy fibrils that accumulate in the cell body and extensions of neurons.

These unwanted α-syn clumps are modified by abnormal attachments of many phosphate chemical groups as well as by the protein ubiquitin, a molecular tag for degradation. They are widely distributed in the central nervous system, where they are associated with neuron loss.

Using cell models in which intracellular α-syn clumps accumulate after taking up synthetic α-syn fibrils, the team showed that α-syn inclusions cannot be degraded, even though they are located near the lysosome and the proteasome, another type of garbage disposal in the cell.

The α-syn aggregates persist even after soluble α-syn levels within the cell are substantially reduced, suggesting that once formed, the α-syn inclusions are resistant to being cleared. What's more, they found that α-syn aggregates impair the overall autophagy degradative process by delaying the maturation of autophagy machines known as autophagosomes, which may contribute to the increased cell death seen in clump-filled nerve cells. Understanding the impact of α-syn aggregates on autophagy may help elucidate therapies for α-syn-related neurodegeneration.

Co-authors are Selcuk A. Tanik, Christine E. Schultheiss, Laura A. Volpicelli-Daley, and Kurt R. Brunden, all from Penn.

This research was funded by the National Institutes of Neurological Diseases (NS053488), the JPB Foundation, and the Jeff and Anne Keefer Fund.

Journal Reference:

1. S. A. Tanik, C. E. Schultheiss, L. A. Volpicelli-Daley, K. R. Brunden, V. M. Y. Lee. Lewy body-like  -synuclein aggregates resist degradation and impair macroautophagy. Journal of Biological Chemistry, 2013; DOI: 10.1074/jbc.M113.457408

Courtesy: ScienceDaily

Biological Transistor Enables Computing Within Living Cells

When Charles Babbage prototyped the first computing machine in the 19th century, he imagined using mechanical gears and latches to control information. ENIAC, the first modern computer developed in the 1940s, used vacuum tubes and electricity. Today, computers use transistors made from highly engineered semiconducting materials to carry out their logical operations.

And now a team of Stanford University bioengineers has taken computing beyond mechanics and electronics into the living realm of biology. In a paper to be published March 28 in Science, the team details a biological transistor made from genetic material -- DNA and RNA -- in place of gears or electrons. The team calls its biological transistor the "transcriptor."

"Transcriptors are the key component behind amplifying genetic logic -- akin to the transistor and electronics," said Jerome Bonnet, PhD, a postdoctoral scholar in bioengineering and the paper's lead author.

The creation of the transcriptor allows engineers to compute inside living cells to record, for instance, when cells have been exposed to certain external stimuli or environmental factors, or even to turn on and off cell reproduction as needed.

"Biological computers can be used to study and reprogram living systems, monitor environments and improve cellular therapeutics," said Drew Endy, PhD, assistant professor of bioengineering and the paper's senior author.

The biological computer

In electronics, a transistor controls the flow of electrons along a circuit. Similarly, in biologics, a transcriptor controls the flow of a specific protein, RNA polymerase, as it travels along a strand of DNA.

"We have repurposed a group of natural proteins, called integrases, to realize digital control over the flow of RNA polymerase along DNA, which in turn allowed us to engineer amplifying genetic logic," said Endy.

Using transcriptors, the team has created what are known in electrical engineering as logic gates that can derive true-false answers to virtually any biochemical question that might be posed within a cell.

They refer to their transcriptor-based logic gates as "Boolean Integrase Logic," or "BIL gates" for short.

Transcriptor-based gates alone do not constitute a computer, but they are the third and final component of a biological computer that could operate within individual living cells.

Despite their outward differences, all modern computers, from ENIAC to Apple, share three basic functions: storing, transmitting and performing logical operations on information.

Last year, Endy and his team made news in delivering the other two core components of a fully functional genetic computer. The first was a type of rewritable digital data storage within DNA. They also developed a mechanism for transmitting genetic information from cell to cell, a sort of biological Internet.

It all adds up to creating a computer inside a living cell.

Boole's gold

Digital logic is often referred to as "Boolean logic," after George Boole, the mathematician who proposed the system in 1854. Today, Boolean logic typically takes the form of 1s and 0s within a computer. Answer true, gate open; answer false, gate closed. Open. Closed. On. Off. 1. 0. It's that basic. But it turns out that with just these simple tools and ways of thinking you can accomplish quite a lot.

"AND" and "OR" are just two of the most basic Boolean logic gates. An "AND" gate, for instance, is "true" when both of its inputs are true -- when "a" and "b" are true. An "OR" gate, on the other hand, is true when either or both of its inputs are true.

In a biological setting, the possibilities for logic are as limitless as in electronics, Bonnet explained. "You could test whether a given cell had been exposed to any number of external stimuli -- the presence of glucose and caffeine, for instance. BIL gates would allow you to make that determination and to store that information so you could easily identify those which had been exposed and which had not," he said.

By the same token, you could tell the cell to start or stop reproducing if certain factors were present. And, by coupling BIL gates with the team's biological Internet, it is possible to communicate genetic information from cell to cell to orchestrate the behavior of a group of cells.

"The potential applications are limited only by the imagination of the researcher," said co-author Monica Ortiz, a PhD candidate in bioengineering who demonstrated autonomous cell-to-cell communication of DNA encoding various BIL gates.

Building a transcriptor

To create transcriptors and logic gates, the team used carefully calibrated combinations of enzymes -- the integrases mentioned earlier -- that control the flow of RNA polymerase along strands of DNA. If this were electronics, DNA is the wire and RNA polymerase is the electron.

"The choice of enzymes is important," Bonnet said. "We have been careful to select enzymes that function in bacteria, fungi, plants and animals, so that bio-computers can be engineered within a variety of organisms."

On the technical side, the transcriptor achieves a key similarity between the biological transistor and its semiconducting cousin: signal amplification.

With transcriptors, a very small change in the expression of an integrase can create a very large change in the expression of any two other genes.

To understand the importance of amplification, consider that the transistor was first conceived as a way to replace expensive, inefficient and unreliable vacuum tubes in the amplification of telephone signals for transcontinental phone calls. Electrical signals traveling along wires get weaker the farther they travel, but if you put an amplifier every so often along the way, you can relay the signal across a great distance. The same would hold in biological systems as signals get transmitted among a group of cells.

"It is a concept similar to transistor radios," said Pakpoom Subsoontorn, a PhD candidate in bioengineering and co-author of the study who developed theoretical models to predict the behavior of BIL gates. "Relatively weak radio waves traveling through the air can get amplified into sound."

Public-domain biotechnology

To bring the age of the biological computer to a much speedier reality, Endy and his team have contributed all of BIL gates to the public domain so that others can immediately harness and improve upon the tools.

"Most of biotechnology has not yet been imagined, let alone made true. By freely sharing important basic tools everyone can work better together," Bonnet said.

Journal Reference:

1. Jerome Bonnet, Peter Yin, Monica E. Ortiz, Pakpoom Subsoontorn, and Drew Endy. Amplifying Genetic Logic Gates. Science, 28 March 2013 DOI:10.1126/science.1232758

Courtesy: ScienceDaily

You Are What You Eat -- Even the Littlest Bites: Dietary Influences Tied to Changes in Gene Expression

Sometimes you just can't resist a tiny piece of chocolate cake. Even the most health-conscious eaters find themselves indulging in junk foods from time to time. New research by scientists at the University of Massachusetts Medical School (UMMS) raises the striking possibility that even small amounts of these occasional indulgences may produce significant changes in gene expression that could negatively impact physiology and health.

A pair of papers published in Cell by A.J. Marian Walhout, PhD, co-director of the Program in Systems Biology and professor of molecular medicine at UMMS, describe how metabolism and physiology are connected to diet. Using C. elegans, a transparent roundworm often used as a model organism in genetic studies, Dr. Walhout and colleagues observed how different diets produce differences in gene expression in the worm that can then be linked to crucial physiological changes.

"In short, we found that when C. elegans are fed diets of different types of bacteria, they respond by dramatically changing their gene expression program, leading to important changes in physiology," said Walhout. "Worms fed a natural diet of Comamonas bacteria have fewer offspring, live shorter and develop faster compared to worms fed the standard laboratory diet of E. coli bacteria."

Walhout and colleagues identified at least 87 changes in C. elegans gene expression between the two diets. Surprisingly, these changes were independent of the TOR and insulin signaling pathways, gene expression programs typically active in nutritional control. Instead, the changes occur, at least in part, in a regulator that controls molting, a gene program that determines development and growth in the worm. This connection provided one of the critical links between diet, gene expression and physiology detailed in "Diet-induced Development Acceleration Independent of TOR and Insulin in C. elegans." "Importantly, these same regulators that are influenced by diet in the worms control circadian rhythm in humans," said Lesley MacNeil, PhD, a postdoctoral student in the Walhout Lab and first author on the paper. "We already know that circadian rhythms are affected by diet. This points to the real possibility that we can now use C. elegans to study the complex connections between diet, gene expression and physiology and their relation to human disease."

Strikingly, Walhout and colleagues observed that even when fed a small amount of the Comamonas bacteria in a diet otherwise composed of E. coli bacteria, C. elegans exhibited dramatic changes in gene expression and physiology. These results provide the tantalizing possibility that different diets are not "healthy" or "unhealthy" but that specific quantities of certain foods may be optimal under different conditions and for promoting different physiological outcomes.

"It's just as true that a small amount of a 'healthy' food in an otherwise unhealthy diet could elicit a beneficial change in gene expression that could have profound physiological effects," said Walhout.

Additional research by the Walhout Lab further explored the possibility of using C. elegans as a model system to answer complex questions about disease and dietary treatment in humans. Detailed in the "Integration of Metabolic and Gene Regulatory Networks Modulates the C. elegans Dietary Response," Walhout and colleagues found that disrupting gene expression involved with C. elegans metabolism lead to metabolic imbalances that interfered with the animal's dietary response; a result that may have a direct correlation to the treatment of a class of human genetic diseases.

"To better understand the molecular mechanisms by which diet effects gene expression in the worm, we performed complimentary genetic screens looking for genes that gave an abnormal response to diet," said Emma Watson, a doctoral student in the Walhout Lab and co-first author on the secondCell study together with Dr. MacNeil. "What we discovered was a large network of metabolic and regulator genes that can integrate internal cellular nutritional needs and imbalances with external availability," said Watson. "This information is then communicated to information processing genes in the worm to illicit the appropriate response in the animal."

These findings suggest the existence of a genetic regulatory network that facilitates rapid responses to internal physiological and external environmental cues in order to maintain a metabolic balance in the worm. Interestingly, a similar phenomenon is involved in mutations that lead to inborn metabolic diseases in humans; classes of genetic diseases resulting from defects in genes that code for enzymes which help convert nutrients into usable materials in the cell. These diseases are usually treated by dietary interventions designed to avoid build-up of toxins and to supplement patients with metabolites that may be depleted.

According to Dr. Walhout, it may be possible to use this genetic regulatory network in C. elegans to compare how certain dietary regimens can be used to mitigate these metabolic diseases. It may also be used to screen for drugs or other small molecules that can produce the same results as dietary treatments.

Though Walhout and colleagues started out asking a fundamental dietary question in the worm, what they got was an answer directly related to disease and treatment in humans, thus establishing C. elegans as a model system for elucidating the mechanisms for dietary responses, inborn metabolic diseases and the connections between them.

"It's very hard to answer questions about the complex interaction between diet, gene expression and physiology in humans for many reasons," said Walhout. "Now, we can use a very tractable system -- namely C. elegans -- to ask precise questions about which components in diet can effect gene expression and physiological traits and ultimately disease, in humans."

Journal Reference:

1. Lesley T. MacNeil, Emma Watson, H. Efsun Arda, Lihua Julie Zhu, Albertha J.M. Walhout. Diet-Induced Developmental Acceleration Independent of TOR and Insulin in C. elegans. Cell, 2013; 153 (1): 240 DOI:10.1016/j.cell.2013.02.049

Courtesy: ScienceDaily