Whenever DNA is replicated, DNA polymerase, the enzyme that links together the nucleic acids, is unable to add nucleotides to the 3’ end of the chromosome. It requires several nucleotides as overlap in order to “hang on” to the DNA molecule. In order to prevent the chromosome from being shortened significantly with every replication, the cell produces an enzyme called telomerase. Telomerase adds a short sequence of nucleotides to the 3’ end of the DNA molecule. This provides a handle for the polymerase so that it can complete replication of the chromosome. Telomerase gets its name from telomeres, the extra stretches of nucleotides at the end of the chromosome that help protect the chromosome from being shortened during replication. Telomeres can be thought of as analogous to the little plastic ends of shoelaces, which help prevent the lace from unraveling after use.

Telomerase is generally very active during early development, in order to allow for the necessary replication of cells to help the organism grow. Eventually, the telomerase becomes less active, or is even turned off completely. This is believed to help prevent the cell from replicating too many times or too frequently. The absence of functional telomerase is associated with aging. As the cells replicate more and more, the chromosomes have a greater chance of being damaged due to shortening of the telomeres. This damage to the DNA can cause many symptoms of aging. Some scientists have proposed that by reactivating telomerase in cells, the aging process can be prevented. This could be particularly important in adult stem cells, which lose their ability to differentiate effectively as telomeres shorten. However, reactivating telomerase may also have many drawbacks. It could permit uncontrolled replication of cells that have developed mutations, leading to cancer.

Telomerase has been shown to be over-expressed in many different types of cancers. This may be due to the fast replication of cancer cells. Because cancer cells replicate indiscriminately, they need telomerase to help prevent the chromosomes from being shortened and damaged with each replication. Researchers have suggested that by stopping the action of telomerase in tumor cells, they could potentially stop the replication of the cancer cells. Indeed, early studies demonstrated that many cancer cells had telomeres that were dramatically shorter than those seen in normal cells. When researchers treated cells with viral proteins to induce constant replication, many of the cells died after the telomeres had been depleted. However, some cells eventually were able to produce telomerase, and survived, albeit with much shorter telomeres than normal. This indicated to the researchers that telomerase was not activated immediately in rapidly replicating cells. Once the telomerase is activated, however, it can prolong survival of the cells. The highly active telomerase in cancer cells can maintain the shortened telomeres enough to allow for rapid proliferation of the cancer.

Recently, researchers discovered that regulation of telomerase appears to be different than regulation of many other genes. This regulation is related to alternative splicing of the gene. In eukaryotes, alternative splicing allows one gene to encode multiple proteins. The parts of the gene that are spliced together to make the mature mRNA are generally determined by codes in the DNA that are near or within the gene. However, researchers noted that the regulatory DNA sequences for splicing the telomerase gene were more distant from the protein coding portion of the gene. This indicates that alternative splicing of telomerase is regulated in a manner different than most other genes.

Many of the alternative splice variants of the telomerase gene produce an inactive protein. However, the researchers also found some splice variants that functioned, albeit at a much lower efficacy than normal telomerase. By targeting the manner in which telomerase splicing is regulated, the researchers propose that they could effectively turn of telomerase genes in cancer cells. This would result in telomeres eventually disappearing in the cancer cells, and the cancer cells being unable to continue replicating at their advanced rate. Eventually, the cancer cells would die off, due to the damage to their chromosomes. The researchers are excited about this new strategy to control expression of telomerase. By decreasing telomerase activity, they propose to treat cancer. By increasing telomerase expression, they could potentially be able to treat conditions associated with aging.


References:

http://www.sciencedaily.com/releases/201...160713.htm

www.scientificamerican.com/article.cfm?id=telomeres-telomerase-and

http://en.wikipedia.org/wiki/Telomerase

Currently, the Centers for Disease Control and Prevention (CDC) recommend that all Americans above the age of six months receive an annual flu vaccine. This is important, because the predominant strain of influenza virus in the population can change from year to year. Influenza virus has a genome composed of RNA. Because RNA polymerase is highly error prone, the virus is prone to mutations. These mutations can change the proteins expressed on the surface of the virus. This process is called genetic drift. Therefore, even if the predominant strain remains the same for more than one year, the virus can still accumulate enough mutations that would make it difficult for the adaptive immune system to recognize. Scientists have been interested in developing a universal influenza vaccine, which could develop broadly neutralizing antibodies capable of recognizing proteins on many strains of the virus. This would require finding commonalities between different strains, so that antibodies would be able to recognize multiple strains. Development of a universal vaccine against multiple strains of influenza would be important for public health. Fewer vaccinations would need to be given to protect the population, resulting in improved compliance. Compliance among individuals able to receive the vaccine is important in providing herd immunity, which can help protect individuals who are not able to receive the vaccine. In addition, a universal influenza vaccine might also protect against newly emerging strains of influenza, thus reducing the likelihood of a worldwide pandemic.

The two major proteins on the surface of the influenza virus that are recognized by the immune system are hemagglutinin (H) and neuraminidase (N). The specific strain of influenza virus is indicated by the H and N molecules expressed on the surface of the virus; for example, H1N1 influenza has hemagglutinin type 1 and neuraminidase type 1 on its surface. Each year, scientists determine which strains of influenza will predominate, and use those strains to develop a vaccine. The virus is grown in hen eggs, and the proteins are isolated from the virus for use in the vaccine. As mentioned above, even if the primary circulating strain is H1N1 multiple years in a row, annual vaccination is recommended due to the virus’s ability to mutate. Many people are hesitant to be vaccinated annually, which may result in increased transmission of the virus, causing more damage during flu season.

Recently, Inovio Pharmaceuticals announced positive results from a trial of a universal H1N1 influenza virus vaccine. Unlike traditional seasonal influenza vaccines, the universal influenza vaccine was not designed to target a specific strain of influenza. Rather, it has been designed to recognize a broad range of H1N1 influenza strains. Scientists have been unsure whether this approach would result in an effective vaccination, due to the high mutability of influenza virus, and the difficulty in generating broadly neutralizing antibodies that would recognize multiple strains of virus.

The vaccine was able to induce antibody responses at a level comparable to that of the current seasonal vaccine against the current predominant influenza strain. Sixty percent of the volunteers who received the highest dose of the universal influenza vaccine were able to seroconvert, meaning they had developed protective levels of antibody against the current circulating influenza strain. This was similar to the percent of volunteers who received the seasonal vaccine. Importantly, the adverse effects noted during the study only including minor irritation at the injection site, demonstrating the safety of the universal influenza vaccine as well.

In addition to developing protective levels of antibody against the current influenza strain, many of the volunteers that received the universal influenza vaccine developed antibody responses against a variety of other H1N1 influenza viruses. This included the strain that caused the 1918 influenza pandemic, as well as many of the H1N1 strains that have been included in seasonal vaccines since 1986. The volunteers that received the universal influenza vaccine developed much more potent antibody responses against all these strains, and were more likely to seroconvert, than volunteers who received the current seasonal influenza vaccine. This helps demonstrate the broad cross reactivity of the immune response induced by the Inovio universal H1N1 influenza vaccine. Inovio is obviously very excited about the broad cross reactivity, and states that the information gained from this trial will also be useful in developing other universal vaccines and determining proper dosing.


References:

http://finance.yahoo.com/news/inovio-pha...00189.html

The appetite-regulation hormone called leptin was discovered in 1994, and ever since then scientists have been trying to understand the mechanisms that control and direct its action. It was known that leptin was produced by fat cells, that it reduced appetite and interacted with insulin, but the precise molecular schematics of its function, details that might enable the creation of a new treatment for obesity, remained unsolved. The discovery and other recent research into leptin and obesity-related genes can be found here. Reports about the research into linking neural activity and brain physiology with obesity can be found here.

University of Texas Medical Branch at Galveston scientists have uncovered a significant part of one of the leptin-related mechanisms, identifying a protein that can interfere with the brain's response to leptin. They've also created an experimental compound that blocks the protein's action, which can be a potential precursor to an anti-obesity drug.

The experiments were done with mice that were fed a high-fat diet; scientists from UTMB and the University of California, San Diego collaborated to explore the role of the newly identified protein, known as Epac1, in interfering and blocking leptin's activity in the brain. They found that mice who were genetically engineered as to be unable to produce Epac1 had significantly lower body weights, lower body fat percentages, lower blood-plasma leptin levels and an increased glucose tolerance compared to normal mice.

Then the researchers applied a specially developed "Epac inhibitor" onto brain-slice cultures taken from normal laboratory mice, and they found that elevated levels of proteins were associated with greater leptin sensitivity. Similar results were shown in the genetically engineered mice that lacked the Epac1 gene. Normal mice treated with the inhibitor had marked lower levels of leptin in their blood plasma, which is an indication that Epac1 also affected their leptin levels.

"We found that we can increase leptin sensitivity by creating mice that lack the genes for Epac1 or through a pharmacological intervention with our Epac inhibitor. The knockout mice gave us a way to tease out the function of the protein, and the inhibitor served as a pharmacological probe that allowed us to manipulate these molecules in the cells." - said UTMB professor Xiaodong Cheng, lead author of a paper on the study that recently appeared on the cover of Molecular and Cellular Biology.

The research team had long suspected there to be a connection between Epac1 and leptin because Epac1 is activated by cyclic AMP, a signaling molecule which is severely linked to metabolism and leptin production and secretion, as shown in previous studies. Cyclic AMP has already been tied to a multitude of other cell signaling processes, many of which are targeted by a multitude of current drugs. Cheng and his team believe that exploring how cyclic AMP and Epac1 and its alternative form Epac2 interact and compile might provide great potential for research into the leptin metabolism, and eventually provide with a long-sought after achievement – A potential drug to treat obesity.

"We refer to these Epac inhibitors as pharmacological probes, and while they are still far away from drugs, pharmaceutical intervention is always our eventual goal. We were the first to develop Epac inhibitors, and now we're working very actively with Dr. Jia Zhou, a UTMB medicinal chemist, to modify them and improve their properties. In addition, we are collaborating with colleagues at the NIH National Center for Advancing Translational Sciences in searching for more potent and selective pharmacological probes for Epac proteins." – Said Dr. Cheng


This was a large collaboration paper with many authors. Other authors of the Molecular and Cellular Biology paper include research associates Jingbo Yan, Jingna Wei and Sonja Stutz, research scientists Fang C. Mei and Igor Patrikeev, graduate assistant Yaohua Hu, visiting physician Dapeng Hao, professors Massoud Motamedi and Kathryn A. Cunningham, associate professor Kelly T. Dineley and assistant professor Jonathan D. Hommel, all from UTMB. Authors from the University of California, San Diego include postdoctoral fellows Hongqiang Cheng and Dieu Hung Lao, and professor Ju Chen.

Resouces:
J. Yan, F. C. Mei, H. Cheng, D. H. Lao, Y. Hu, J. Wei, I. Patrikeev, D. Hao, S. J. Stutz, K. T. Dineley, M. Motamedi, J. D. Hommel, K. A. Cunningham, J. Chen, X. Cheng.Enhanced Leptin Sensitivity, Reduced Adiposity, and Improved Glucose Homeostasis in Mice Lacking Exchange Protein Directly Activated by Cyclic AMP Isoform 1. Molecular and Cellular Biology, 2012

A virus is a small infectious agent that enters a host cell and uses the cellular machinery to replicate its genome. In order for the host to fight the viral infection, it uses components from the adaptive immune system. Cells involved in the adaptive immune response include CD4+ T cells, CD8+ T cells, and B cells. CD4+ T cells are termed helper T cells, and work by activating and recruiting other immune cells to fight the infection. CD8+ T cells can recognize specific antigens being presented on the surface of the infected cell, and directly kill the cell. B cells produce special proteins called antibodies. The antibody recognizes specific antigens, and targets the antigen for killing by other immune cells.

Antibodies produced by B cells have been an important tool in biological research. Antibodies against specific markers on cells can be used to help determine the number and percentage of specific cell types in a population, and to determine what functions theses cells have. The antibodies used in these applications are labeled with a type of marker, or can be detected with a secondary antibody that is conjugated to a marker. The marker is used for detection of the antibody. The antibodies are used in a variety of assays, to detect proteins, cell surface markers, and other organic molecules. This approach is very valuable in many types of research, and can even be used to separate different cell types from large, mixed populations. Antibodies are currently the best tool scientists have for isolating cells, viruses, and other large molecules for study. In addition, antibodies are used in imaging studies to label cells and parts of the cell. The antibodies are generally conjugated to a fluorescent molecule, which can be detected as different colors by the microscope.

While antibodies are an effective tool for these studies, they have some drawbacks. First, antibodies can be difficult and time consuming to produce. Antibodies for scientific research must be developed by inoculating a producer animal with an antigen in order to activate the B cells. This inoculation also generally requires the use of an adjuvant, to increase the immune response. Once the animal has developed a strong immune response to the antigen, B cells must be isolated from the blood. B cells producing antibody that recognizes the specific antigen are isolated from other B cells, and individually seeded into plates. The B cells are generally hybridized with a tumor cell, so that the B cell can live indefinitely and continue to produce large quantities of antibody. This is called a hybridoma. Each hybridoma is tested to determine which has the optimum antibody for the application. Once the hybridoma has been selected, it can be grown, and antibody can finally be isolated.

The antibody must be purified, and for many research projects, it must be conjugated to another molecule, such as a fluorophore or enzyme, to assist with detection. Once the antibody has been produced, it may be unstable, and will generally require specific storage conditions. In addition, antibodies are fairly large proteins. Some isotypes of antibodies are arranged as five antibody molecules. The large size of the antibody may make it inappropriate for use in certain studies. Smaller materials would be better for studies involving small molecules, and for many imaging applications. If synthetic material could be developed that directly incorporate fluorescent properties, this could provide a big step forward to imaging small molecules on and within the cell.

Synthetic materials could be easier to produce, cheaper, and more stable than antibodies. The problem has been developing synthetic materials that can recognize molecules as specifically and efficiently as antibody does. Recently, researchers in Switzerland were able to produce synthetic nanoparticles capable of specifically recognizing a family of viruses. The viruses were bound to silica nanoparticles. Then, a layer of carbon and silicone containing compounds, called organosilanes, was grown around the viruses. When the viruses were removed, the oragnosilanes still maintained the shape of the viruses. They were also able to recognize the chemical properties of the virus. The organosilanes could specifically bind to the template virus, but not to other similarly structured viruses. This technology could be adapted to recognize not only virus particles, but cells and other organic molecules as well.



References:

http://www.sciencenews.org/view/generic/...ze_viruses

In a process dubbed “Cellular Alchemy” researchers at Case Western Reserve School of Medicine have succeeded at a technique that converts skin cells to the type of brain cells compromised or destroyed in patients with multiple sclerosis, cerebral palsy and other so-called myelin disorders.

The breakthrough enables on demand, immediate production of myelinating cells, oligodendrocytes, which produce a vital layer of insulation that protects neurons from detirioration and enables the transfer of brain impulses to the rest of the body. In patients with multiple sclerosis, cerebral palsy, and rare genetic disorders called leukodystrophies, myelinating cells are destroyed and cannot be replaced by the body.

Diseases that result in destruction of the oligodendroglial cells include demyelinating diseases such as multiple sclerosis and leukodystrophies. Trauma such as spinal cord injury can also cause demyelination to incur. Cerebral palsy, or periventricular leukomalacia, is caused by the deterioration of the developing oligodendrocytes in the brain areas in and around the cerebral ventricles. In cerebral palsy, spinal cord injury, stroke and possibly multiple sclerosis, oligodendrocytes are thought to be damaged by excessive release of the neurotransmitter glutamate. Oligodendrocyte dysfunction may also be connected to the pathophysiology of schizophrenia and bipolar disorder.

The new technique involves directly converting fibroblasts - an abundant structural cell present in the skin and most organs - into oligodendrocytes. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the architecture of connective tissues and the area around the cells, and it plays a critical role in wound healing. Fibroblasts are the most abundant cells of connective tissue in animals.

"Its 'cellular alchemy,'" explained Paul Tesar, PhD, assistant professor of genetics and genome sciences at Case Western Reserve School of Medicine and senior author of the study.
"We are taking a readily accessible and abundant cell and completely switching its identity to become a highly valuable cell for therapy."

In a process called "cellular reprogramming," the scientists manipulated the levels of three naturally occurring proteins to induce fibroblast cells extracted from skin tissue to become usable precursors to oligodendrocytes called oligodendrocyte progenitor cells.

The team, led by Case Western Reserve researchers and co-first authors Fadi Najm and Angela Lager, rapidly produced billions of these induced oligodendtrocyte precursors. In an even more important step, they showed that induced oligodendrocyte precursors could regenerate new myelin coatings around nerves after being transplanted to mice—a result that gives that hope the technique might be usable to treat human myelin disorders.

When oligodendrocytes become dysfunctional they lose the ability to insulate neurons with myelin layers, thus exposing them to damage and severely crippling the neurons ability to conduct signals. A cure for diseases caused this way would have to regenerate the myelin layer by introducing new, functional oligodendrocytes.

Until now, oligodendrocyte precursor cells and oligodendrocytes could only be obtained from fetal tissue or pluripotent stem cells, which is rare and hard to come by. These techniques have been useful, but with severe limitations.

"The myelin repair field has been hampered by an inability to rapidly generate safe and effective sources of functional oligodendrocytes. The new technique may overcome all of these issues by providing a rapid and streamlined way to directly generate functional myelin producing cells." - explained co-author of the study and myelin expert Robert Miller, PhD, professor of neurosciences at the Case Western Reserve School of Medicine.

This particular study utilized mouse subjects. The next step would have to be proof of concept in human tissue, and testing its safety and feasibility in human patients with myelin disorders. If successful, this approach would provide great hope for patients suffering from one of the disorders caused by myelin instability.

"The progression of stem cell biology is providing opportunities for clinical translation that a decade ago would not have been possible. It is a real breakthrough." - said Stanton Gerson, MD, professor of Medicine-Hematology/Oncology at the School of Medicine and director of the National Center for Regenerative Medicine and the UH Case Medical Center Seidman Cancer Center.

Resoruces:
Transcription factor–mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells; published April 12th in Nature Biotechnology

The recent outbreak of avian influenza in China is causing concern to public health officials. The H7N9 avian influenza virus has so far infected nearly 90 people, and has been fatal in 17 of these cases.

Influenza is a very concerning virus for public health scientists. Because it can be transmitted easily between people, cause severe disease, and leave the host prone to other respiratory complications, influenza could cause a deadly worldwide pandemic. One of the worst pandemics in recorded history was caused by influenza virus in 1918. The Spanish Flu caused an estimated 20 to 40 million deaths worldwide. It caused severe disease in young adults, which is not common with most flu viruses. The most susceptible populations are normally the very young, and the very old. In addition, the virus left many susceptible to secondary infections, such as pneumonia. The virus was so severe, the average life expectancy in the US dropped by 10 years. Part of the reason why the pandemic may have spread so quickly was due to international travel. Many soldiers, who had been living in close quarters during World War I, suddenly returned to their homes all over the world, bringing the virus with them.

Influenza is particularly prone to causing such devastating pandemics due to the nature of its genome. The genetic information is stored in 8 separate RNA molecules. Because RNA polymerase has a lower fidelity that DNA polymerase, the genome of influenza is very susceptible to mutation. This is called genetic drift. When enough mutations accumulate, it can make the proteins produced by the virus difficult for the host immune system to recognize, and prevent the virus from being attacked. In addition, some influenza viruses can infect multiple species. If a virus that can infect both humans and birds is in a bird, it can exchange some of the RNA genome with another, avian-origin influenza virus. This results in drastically new viruses, which can be unrecognizable by the human immune system. This process is called genetic shift. Genetic drift and genetic shift are some reasons why public health officials were particularly worried about the 2009 H1N1 influenza virus. It had origins from both human and swine influenza viruses, which indicated that the virus may not be easily recognized by the human immune system. In addition, many of those who had severe disease from the virus were young adults, which is similar to the 1918 Spanish Influenza outbreak.

Not every influenza virus is able to cause a pandemic. Most viruses that have circulated through the human population recently are not candidates for a severe pandemic, as many people would have adaptive immune responses to the virus. Rather, viruses from a different animal source, such as pigs or birds, are much more likely to cause a pandemic. These would not be rapidly recognized by the immune system, and would be able to cause more damage to many people before being controlled by adaptive immune responses.

The recent outbreak of avian influenza in China is causing concern to public health officials. The H7N9 avian influenza virus has so far infected nearly 90 people, and has been fatal in 17 of these cases. What is concerning to investigators is how the virus is being transmitted. Estimates state that as many as forty percent of the patients have not had direct contact with poultry, which indicates that the virus might be able to spread from human to human. However, due to the processing of poultry in markets in China, it is possible that these patients indeed contracted the virus directly from birds. Chinese poultry markets are often crowded, with many droppings accumulating, and the processing of fowl in preparation for the consumer can result in droplets being dispersed. Even if a patient had not directly been in contact with the poultry, they could have inhaled these droplets in the market and become infected.

The H7N9 virus is concerning to public health scientists because of its avian origins. The virus would be novel in the human population, meaning that no one would have memory immune responses against the virus. Without any prior protection in the population, the virus would be free to spread to virtually any human on earth. With rapid travel available between almost every location on earth, a pandemic could quickly spread throughout the world, causing rapid devastation. Public health officials are constantly watching and monitoring any novel influenza virus that is reported, in hopes of preventing a global pandemic.


References:

http://news.yahoo.com/experts-unclear-ch...27149.html

http://www.cnn.com/2013/04/18/world/asia...index.html

http://www.nytimes.com/2013/04/19/world/....html?_r=0

http://virus.stanford.edu/uda/

miRNA’s can be utilized for a number of purposes, and recent research in this field has been prolific and detailed. Along with miRNA’s great potential as biomarkers for different pathological states and as useful diagnostic tools, miRNA’s can also be used as potent therapeutics. Benjamin tenOever, Ph.D., professor of medicine at Mount Sinai School of Medicine, has been working on figuring out how miRNAs can be utilized in engineered RNA-based vectors for therapeutic purposes.

The majority of the discovered and known miRNA genes is intergenic or oriented antisense to adjacent genes and is therefore thought to be transcribed as independent units. However, it has been discovered that in some cases a microRNA gene is transcribed together with its host gene which provides a means for coupled regulation of miRNA and a protein-coding gene. The main function of miRNAs appears so far to be gene regulation. For this purpose, a miRNA is usually complementary to a part of one or more messenger RNAs.

Dr. tenOever’s lab has successfully developed a new method for exploiting endogenous miRNAs to regulate tissue tropism of viral vectors.

“Because viruses lack a mechanism for antagonizing miRNA function, we can exploit miRNA expression to control viruses. We have shown that if you incorporate targets for a cell-specific miRNA into an RNA virus genome, you can create a virus that looks identical at a protein level but would be selectively blocked from infecting these particular cells.” – says Dr. tenOever

On the other side, Dr. tenOever’s research has focused on engineering viral vectors that can produce functional miRNAs, which can be used as a therapeutic platform, of the vector type, to deliver miRNAs or any other small RNAs to practically any tissue in the body. Dr. tenOever states that, while it has already been known that RNA viruses do not produce miRNAs, it was not clear whether viruses simply lack the capacity to do so, or whether this specific activity was perhaps detrimental, or in some other way harmful, to the viral life cycle.
The researchers decided to approach this question by incorporating primary miRNAs (pri-miRNA) into various available RNA viral vectors that are known to localize to the nucleus or the cytoplasm in different tissues. In all their tests, mature miRNAs were properly processed and loaded into the RISC complex. In the case of cytoplasmic viruses, an RNase named Drosha was exported out of the nucleus into the cytoplasm to process the artificial pri-miRNA.
After the experiments the team concluded that it appears most RNA viruses are in fact capable of producing functional miRNAs, but avoid to do so in natural cases, which is not yet clear as to why, presumably to prevent some degree of self-attenuation.

“What we can capitalize on now is to convert an miRNA that behaves like a tailor-made, sequence-specific siRNA. We can then adapt any RNA virus, regardless of tropism, and use it to generate siRNAs to silence a desired host gene,” Dr. tenOever reported.

The artificial RNA viruses only produce miRNAs for 7–10 days afer being implanted, but nonetheless, they have show a great capacity for usefulness, and many transient cytoplasmic RNA viruses have become very useful in Dr. tenOever’s lab for a great variety of applications, some of them as reprogramming of fibroblast cells into iPSCs by introducing a few miRNAs.
“The advantage of our system is that we are not going into the nucleus with our vectors and we’re not integrating into the genome; we’re only there for 7–10 days and then our vectors disappear, but by then we have reprogrammed our cells to become pluripotent stem cells.”

This new approach using the recently discovered miRNA’s has great potential, in research as well as medicine and the clinical approach. By using miRNA in combination with recombinant RNA viruses as transition vectors, future research might offer a way of ‘reprogramming’ the body’s cells for a variety of purposes, ranging from therapeutics, diagnosis and prevention of various diseases and pathological states.


Resources:
Tuddenham L, Jung JS, Chane-Woon-Ming B, Dölken L, Pfeffer S (February 2012)."Small RNA deep sequencing identifies microRNAs and other small noncoding RNAs from human herpesvirus“
Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R (2008). "MicroRNA-373 induces expression of genes with complementary promoter sequences"
Krol J, Sobczak K, Wilczynska U, Drath M, Jasinska A, Kaczynska D, Krzyzosiak WJ (2004). "Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design".

New progress in the field of evaluating miRNAs in Circulation


Adittional to SomaGenics’ method for miRNA plasma measurement, other scientists are also working on new methods to measure the miRNA circulating in the plasma, especially those associated with certain pathological states. Dominik M. Duelli, Ph.D., assistant professor at Rosalind Franklin University of Medicine and Science, has been experimenting with circulating miRNAs, and he and his lab have evaluated the most accurate and reproducible methods for detecting and quantifying miRNA from plasma using qRT-PCR.

Dr. Duelli and his lab got the idea for testing these various parameters of miRNA profiling from inconsistencies in detecting certain miRNAs in plasma.

“When we first started our work, the original idea for a biomarker measured in blood plasma was that it should reflect what’s inside the malignancy, so an miRNA that’s high inside the cell should also be high outside the cell,” Dr. Duelli explained. “We profiled miRNAs in an array of cell-line cultures and saw that 60–70% of the miRNAs had the same profile outside and inside cells. But some miRNAs appeared to be exclusively released 90% or more out of the cell. Another category was miRNAs that were retained and were not released at all.”
Dr. Duelli tried to evaluate whether this was normal, a physiological phenomenon, or were the miRNAs simply not detectable based on their methodology and using that specific technology?
They began their work by measuring miR-16, which is highly abundant in plasma and miR-223 with a low abundance in plasma, in fresh plasma from 16 subjects to determine the optimal parameters needed for miRNA profiling. They noticed that anticoagulants like, for instance, citrate or KOx/NaF are a much better option than heparin for miRNA quantitation in plasma. Heparin has a habit of inhibiting RT and polymerases, and detecting the miRNA’s had been possible only if they diluted the heparin or treated raw plasma with heparinase.

Heparin, also known as unfractionated heparin, is a highly sulfated glycosaminoglycan, and widely used as an injectable anticoagulant. It also has the highest negative charge density of any known biological molecule. It is often used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines, and in recent times is used to prevent coagulation in plasma and blood samples used for diagnostics and lab-work.


Dr. Duelli and his lab tried different methods for RNA isolation and noticed that some reagents can selectively precipitate certain miRNAs, which can cause some miRNAs not to be detected at all in a sample. Instead, by using a silica membrane or beads for RNA extraction they can prevent polymerase inhibitors from co-purifying with miRNA’s. This methof provided better purity and yield.

In addition, Dr. Duelli says that using a Taq polymerase, Hemo KlenTaq™, from New England Biolabs, can solve this problem because some of the more significant inhibitors cannot bind to this truncated polymerase. Simply by including Hemo KlenTaq in the reaction Dr. Duelli’s lab observed a 30-fold increase in miR-16 and miR-223 expression.
Dr. Duelli’s lab also used fluorescent SmartFlare™ RNA Detection Probes, EMD Millipore, which utilize a non enzymatic approach for fast and direct miRNA quantification in live samples. They were also able to detect and quantify miR-16 expression in plasma within minutes to one hour after venipuncture, he says, which greatly contributes to the effectiveness of the process.

Dr. Duelli’s lab was focused, among other things, on making this technology applicable in the clinical setting, allowing for a many fold increase in diagnostic methods, and bringing medicine one step closer not only to personal, individualized medicine, but also helping doctors “detect and prevent” disease, instead of just curing and managing the symptoms.

“You can measure miRNA levels right after chemotherapy and correlate it with tumor progression; if the tumor is shrinking, you can measure if these miRNAs go away. It comes at a cost to sensitivity because there is no amplification, but it can be used in plasma or other samples.”

Malaria is one of the top leading causes of death due to infectious disease worldwide. Every year, there are approximately 100 to 300 million cases of malaria, resulting in approximately one million deaths. Most of the cases of malaria occur in Sub-Saharan Africa, as well as in parts of South East Asia, and Central and South America. A host is infected with malaria through the bite of a mosquito. The malaria parasite first grows in the liver, before it develops into a stage that can infect red blood cells. Once the malaria infects red blood cells, symptoms such as fever, malaise, and chills develop. Fatal malaria is often caused by one specific type of malaria, P. falciparum, which can infect both mature and young red blood cells. In addition, P. falciparum can cause the red blood cells to stick together, causing blockages in blood vessels due to the clumps of cells. If this occurs in the brain, it is called cerebral malaria, and can often be fatal. Quick, effective treatment of P. falciparum malaria is essential to help prevent death, as well as to prevent transmission of the parasite and the development of drug resistance.

Malaria effects impoverished areas disproportionately, which contributes to maintaining the cycle of poverty. When people are sick with malaria, they are unable to work, and must spend their money on treatments. However, accessing diagnostics and treatment may be difficult for many individuals in impoverished areas. Additionally, if multiple doses of medicine are required, the patient may not be able to obtain all the doses. This leads to drug-resistance, and compounds the detrimental effects of malaria in the area. Traditional, affordable medications such as chloroquine are not as useful as they once were due to the emergence of drug-resistant malaria.

An anti-malarial treatment that is gaining favor is artemisinin. Artemisinin is a drug that has been used in China for thousands of years to treat malaria. It is derived from the leaves of the sweet wormwood plant. Artemisinin is very effective, and because it has not been used extensively, there is currently a very low incidence of resistance, particularly in P. falciparum. However, artemisinin can sometimes be difficult to obtain. Because it comes from a plant source, production of the drug is dependent on proper growth conditions for the plants. The plants take months to grow, depending on environmental conditions. In addition, a limited number of manufacturers of artemisinin means that supply can vary considerably. Because the availability of artemisinin varies, the price can vary as well, making it difficult for people in impoverished areas to afford. In order for an anti-malarial drug to be usable worldwide, it must be easy to obtain and easy to afford for the populations most at-risk.

Fortunately, scientists have been able to produce precursors to artemisinin in yeast. The researchers had previously developed yeast that can produce amorphadiene, which is a precursor to artemisinic acid. As described in a recent publication in Nature, the researchers have been able to increase the yield of amorphadiene from the yeast, and have developed protocols to convert artemisinic acid to artemisinin. This could provide a considerable increase in the amount of artemisinin available to patients, compared to using only the standard approach to manufacturing the drug. The increased availability will also translate to reduced cost, and could help break the cycle of poverty, malaria, and drug resistance.

Indeed, being able to produce large quantities of anti-malarial drugs and distributing them is an important goal of many organizations dedicated to helping treat malaria. One model of malaria drug distribution is termed the Coca-cola model. This model poses the question: “How does Coca-cola get its product into the most remote areas of the world?” The answer is by selling the product at very low cost to retailers, so that the product is affordable in the area. Public health experts have suggested a similar strategy to get effective anti-malarial drugs to these areas. Manufacturers would sell the drugs to retailers for pennies, instead of dollars, with costs to the manufacturer offset by donors. The retailers could then sell the effective anti-malarial drugs for a cost similar to or below that of generic, knock-off anti-malarials. The cheap, rapid production of artemisinin using yeast could make this goal more realistic and help millions of people.



References:

http://www.sciencenews.org/view/generic/...kers_yeast

http://www.cdc.gov/Malaria/

http://www.sciencemag.org/content/322/5905/1174.long

Almost 120,000 Americans are currently waiting for live-saving organ donations. However, there are far too few donors available, and finding appropriate matches between donor and recipient can be difficult. Being able to grow organs in vitro could potentially save tens of thousands of lives. Kidneys are one organ that many Americans need. While dialysis can sustain life for kidney patients, it is only a temporary solution. A kidney transplant is the only available cure for kidney disease. Some groups of scientists have been able to engineer devices to help assist kidney function, but none of these devices can be implanted directly as a donor kidney would be. Recently, however, scientists at Massachusetts General Hospital were able to successfully transplant kidneys grown in lab dishes into rats.

The team removed kidneys from deceased rats, and stripped away the tissues using detergent. This left a bare structure that the scientists termed scaffolding, which could use as a base to make a new kidney. The scaffolding was then seeded with human umbilical cord cells to help regenerate blood vessels, and kidney cells removed from newborn rats to create functional kidney cells.

The kidneys were able to perform the necessary function of filtering the blood and producing urine in the rats. However, they did not function as well as a normal kidney, indicating that further work needs to be performed to help optimize the bioengineered kidneys. The researchers suggest that the implanted kidneys were too immature to function, and might need additional time to properly develop. In addition, because the blood vessels were developed from human umbilical vein cells, they may not have been compatible with the rat cells. Also, as the researchers continue their work, they can determine how to properly build and place kidney cells to develop even more functional bioengineered kidneys. Even if they aren’t able to make kidneys that are one-hundred percent functional, the researchers still believe these bioengineered kidneys will be very helpful. Because dialysis is not begun in patients until the kidneys have dropped to fifteen percent efficiency, the kidneys do not need to have full efficiency in order to be useful. Being able to help a patient avoid dialysis while waiting for a donor kidney would be a big success.

One advantage that the bioengineered kidneys have is that they can be transplanted into the patient, acting as a permanent cure similar to a donor kidney. Another advantage is that the kidneys can be custom made for each patient, thereby seriously reducing the potential of organ rejection due to improper matching. The researchers propose that the scaffolding used to build the bioengineered kidneys could come from deceased donors. The stripping and rebuilding process would allow them to build a new kidney that would be tolerated by the recipient. Another option would be to use the patient’s own damaged kidney as a scaffolding. Again, the kidney would be stripped down and rebuilt to restore proper function.

While the concept of bioengineered organs for transplant is exciting, there are many obstacles involved in moving the technology from rodents to humans. As mentioned above, the kidneys were not fully functional. More research is required to determine how to develop all of the proper cell types, in the proper positions, in the bioengineered kidneys. While finding scaffolding for the kidneys would be fairly straightforward, either from a deceased donor or directly from the patient, obtaining the materials to rebuild the kidneys might be more difficult. Stem cells from human umbilical tissue can be obtained through umbilical blood which is sometimes donated after the birth of a child, but these donations may not be sufficient to meet the needs of all the patients in need of kidney transplants. In addition, the original bioengineered kidneys were rebuilt using kidney cells from newborn rats. It would certainly be unethical to take cells from newborn humans for this purpose. The most likely source of cells to rebuild kidneys would likely be from stem cells grown in culture. This would require additional research to determine how to differentiate the stem cells into the proper kidney cells.

While the use of bioengineered kidneys in humans is likely a long way off, the research performed still provides great insights into how cells in the kidney are developed and organized. This information alone will allow future research that could help provide more options to kidney patients.



References:

http://www.nature.com/news/lab-grown-kid...ts-1.12791

http://donatelife.net/understanding-dona...tatistics/

Amyotrophic lateral sclerosis (ALS), common as Lou Gehrig’s disease, is a neurological disease that affects the motor neurons. In ALS, both the upper motor neurons (UMNs) and the lower motor neurons degenerate (LMNs). The disease is characterized by the loss of motor neurons. Motor neurons are subsequently replaced by supporting cells of the nervous system.

Definitive cause of ALS is unknown. Current research focuses on many different possibilities, with some pertaining to enzyme deficiencies, infections, environmental factors, and a whole slew of other possibilities.

Upper motor neurons signs are problems you would foresee with the loss of the normal inhibitory input the UMNs usually have on the LMNs. That would lead one to see a hyperactive state in the musculature, which is indeed the case. Specific findings related to UMN degeneration include hyperreflexia, increased tone, and weakness. As opposed to the UMN, the LMN provides an excitatory component to the muscle groups so that a loss of LMN health leads to a different set of signs and symptoms. LMN signs include fasciculations, atrophy, and weakness. A combination of both UMN and LMN signs often leads a neurologist to consider ALS as the diagnosis, but not before exhausting other possible diagnoses, such as multiple sclerosis, myasthenia gravis, Eaton-Lambert syndrome, and others. For that reason, ALS is termed a diagnosis of exclusion. The ultimate cause of death in ALS patients is the loss of muscle strength to properly breathe.

Treatment

The first drug ever approved for the treatment of ALS is still used today. It is Riluzole. Its exact mechanism of action in prolonging survival in ALS patients is unknown. Riluzole has been shown to decrease glutamate release, preventing any possible toxic effects to motor neurons that could have been caused by overexcitation, something that often involves glutamate. Trials with the drug have shown a median increase in survival time of three months. It should be stressed, however, that Riluzole is by no means a cure for ALS.

The potential for stem cell research lies in the ability to regenerate both UMNs and LMNs. What some researchers emphasize is the importance of understanding the underlying principles, such as the importance of timing and cell delivery, immune modulation, and the need for a multidisciplinary approach. With a better comprehension of these factors, the treatment of amyotrophic lateral sclerosis has a better chance for being successful in patient care.

Neural Stem Cells in ALS Treatment on Mice

Transplantation of neural stem cells or their more mature progeny is considered a potentially curative therapy for patients suffering from neurodegenerative disorders. Because adult neural stem cells, in contrast with fetal neural stem cells, have a more limited capacity to proliferate in vitro, fetal neural stem cells may be the most promising cells for cellular therapy of neurodegenerative disorders. Several studies in animals have transplanted adult neural stem cells. Because the cells receive signals from the brain microenvironment, further maturation to either glial support cells or neurons can be seen. In some cases these cells integrate and contribute to physiological neural circuits. In other cases the cells make glial support cells that can also have significant effects in some animal models of disease.

In a new study, researchers used mice as experimental models. The human neural stem cells were treated with growth factors, and directed to become motor neurons. The mice were treated first with a chemical to induce amyotrophic lateral sclerosis (ALS), and then they received a transplant of the new motor neurons that had been derived from human neuron stem cells taken from human induced pluripotent stem cells.

Pluripotent stem cells are adult cells such as skin cells that have been genetically reprogrammed to an embryonic stem cell-like state. After transplantation, the stem cells migrated to the spinal cord of the mice, matured and multiplied. That study found that human neural stem cell transplantation significantly extended the lifespan of the mice by 20 days and improved their neuromuscular function by 15 percent.

Conclusion

This study showed promise for testing stem cell transplantation in human clinical trials. In amyotrophic lateral sclerosis, motor neurons die, leading to paralysis. In preclinical animal work, neural stem cells made synaptic contact with the host motor neurons and expressed neurotrophic growth factors, which are protective of cells. By analogy to mice neural stem cells, these observations may allow the development of neural stem cells transplantation for a range of disorders. In the future, patients with ALS will be treated with injection of human fetal-derived neural stem cells into the lumbar region of the spinal cord, where they will exert a neuroprotective effect. However, since the preclinical date (safety, dosage, long-term survival, post-mortem biopsy) are insufficient and clinical evidence of improvement is weak, more preclinical studies are needed prior to the development of further clinical applications. Neural stem cells may be the best way to avoid the problems. They can self-renew, make more neural stem cells and differentiate into nerve cells, in this case into motor neurons. They can also rescue nerve cells that don't work properly and help preserve and regenerate neural tissue.

There are currently no clinical trials, but a few unpublished efforts have been disclosed using neural stem cells in humans. With all the excitement and possibilities stem cells have to offer as a therapy, it is important that scientists and clinicians are careful, plan severe studies and most importantly focus on laboratory experiments that will provide answers to the many challenges that still face this therapeutic approach. To be safe and have potential in the clinic, it is important that the appropriate studies are conducted to learn more about the properties and complexities of the various stem cells.

Biotech Company “Firefly BioWorks”, develops a new technology, FirePlex™, a method of miRNA detection that can been used for both academic and clinical research.

miRNAs have recently emerged as key fine-tune regulators of a large number of genes that control many cellular processes and signaling pathways. These molecules also have specific roles in a variety of diseases, such as cancer and inflammation, and have thus sparked interest for research into their potential as therapeutic and diagnostic biomarker tools.
“microRNA: Targets and Tools for Therapeutic Development” is an upcoming conference by CHI, upon which the latest discoveries regarding miRNA-mediated disease regulation will be address and new methodologies for conducting these studies in a laboratory or clinical setting will be introduced.

To be properly utilized, they must first be detected. Much of the latest research regarding miRNA has focused on improving detection methods. Daniel Pregibon, Ph.D., CTO of Firefly BioWorks, will talk about his firm’s latest technology, FirePlex™, a new method of miRNA detection that has been used by both academic and clinical researchers.

Dr. Pregibon explains that , using microarrays or deep sequencing to profile all miRNAs in a single sample, or using single assays to examine individual miRNA expression across many samples are the only two types of technology available for miRNA profiling studies. Unfortunately, these approaches are not suitable for performing biomarker validation studies necessary for developing diagnostic tests.
“What we’re seeing is that there was really nothing in that middle range, if you wanted to look at 25–50 miRNAs over hundreds or thousands of different samples,” he says. FirePlex was designed specifically to to help researchers with these problems and allow high-throughput validation of a certain subset of miRNAs they are interested in studying.
Because each scientist working on miRNA research may be interested in looking at a unique and different set of miRNAs, Dr. Pregibon reported that 99% of the panels they make are custom panels, which can be quickly prepared, one week at the most. FirePlex panels have been developed to run on benchtop flow cytometers, so additional or specialized equipment does not need to be purchased to run an assay.
The FirePlex technology is encoded on hydrogel particles, instead of a glass surface, as with most microarrays. Dr. Pregibon explains that using a hydrogel instead of a glass surface allows each molecule to have many more degrees of freedom, which is useful from a thermodynamics standpoint. This hydrogel base for capturing miRNAs allows for greater sensitivity and specificity. Polyethylene glycol (PEG) is used as a substrate to eliminate nonspecific binding of proteins, lipids, or other complexes.

The FirePlex assay is also based on post-hybridization labeling (the miRNAs are labeled after being hybridized throughout the hydrogel volume), so it can be used with crude samples as well. Any debris will be washed away after immobilizing the miRNAs. Dr. Pregibon states that minimal manipulation of the sample also reduces any bias or mistakes that are consequences of the RNA isolation method used, which can lead to variable RNA yield or selective enrichment. FirePlex has also been expanded to profile miRNAs in cell lysates, fresh tissue, FFPE, and serum/plasma.

Another company, SomaGenics, has developed a method, miR-IDirect, for the direct detection of miRNAs from a plasma sample without total RNA isolation required. Sumedha Jayasena, Ph.D., vp of technology and therapeutic development, says the miR-IDirect platform incorporates SomaGenics’ qPCR-based miR-ID technology. It functions by detecting circulating miRNA in plasma.
This method provides quantification of small RNAs by circularizing them as a first step, then, as the second step creating cDNA by rolling circle amplification of the miRNA circles, and finally amplifying the cDNA further by qPCR using 5´-overlapping PCR primers. miR-ID has been already applied for miRNA detection in total cellular RNA as well as detecting purified RNA from various biological fluids.
miR-ID assays have been developed for about 100 different miRNAs up until now, with even more in development. The miR-ID technology can distinguish miRNAs with terminal modifications, such as 2´-OMe groups at 3´-ends, according to Dr. Jayasena.

A new company in the biotech sector, CytoVas, has developed a new concept for vascular health analysis, using a relatively new filed approach, cytomics, to screen the blood for viomarkers relating to vascular disease.

The early-stage company, dealing with in vitro diagnosis, is working to improve its Vascular Health Profile (VHP) concept. This new approach is based on cytomics, which analyzes cell systems and networks. VHP is a blood test that can potentially give a complete view of a person’s cardiovascular health status. The firm says VHP also can also be potentially used by pharmaceutical companies to develop drugs in a safer way.

In contrast to genomics, which deals with genetic and DNA analysis, or proteomics, doing th same with proteins; cytomics, a relatively new approach that arose in the last few years, is based on the combination of the two. It offers comprehensive detailed results based on protein and gene interaction, coupled with intracellular and extracellular interactions, and environmental factors. It uses data acquired from biomarkers in combination with data mining and modulating environmental factors to create highly personalized and individual vascular health profiles.

VHP uses a panel of biomarkers that indicate damage and repair to the cardiovascular system. The biomarkers measured are related to microparticles and endothelial progenitor cells. Microparticles indicate cell death levels or damage to the blood vessels, whereas endothelial progenitor cells reflect blood vessel repair capacity and speed. At this point, 13 biomarkers are measured, and the VHP panel can be customized to specific applications and different optimizations. “That’s part of our strategy to appeal to different markets,” says Pascal Yvon, Pharm.D., CEO.

VHP is based on discoveries made by researchers at the University of Pennysylvania. It combines cytomics, single-cell analysis, and data mining. Three Penn researchers—Emile Mohler, M.D., Jonni Moore, Ph.D., and Wade Rogers, Ph.D.—and Penn’s Upstart Incubator co-founded CytoVas in 2010.

Cardiovascular disease, as many other diseases, has an extremely complex biology which is subject to both individual differences and environmental factors. Cytomics presents a method to look at both the genetic and environmental influences of a disease. Compared to genomics, which coveres genes, and proteomics, which deals with proteins, cytomics captures the molecular integration of genes and proteins, as well as environmental factors like smoking and diet. “The beauty of cytomics is that it shows single-cell phenotypes of individuals resulting from genes and environmental exposures,” says Dr. Yvon.
Cytomics takes a top-down approach to diagnosing at a disease. “We start with phenotypes and link them to what we observe about a disease,” says Dr. Yvon. This is, in basis, easier than genomics or proteomics, which are bottom-up approaches. A top-down approach looks at the single-cell level of an individual, while genomics and proteomics look at biomolecular events in cell populations. This way of looking at things allows for a much more personalized approach to vascular health.
“Cytomics offers an efficient alternative to systemically explore the biocomplexity of human organisms and is more closely related to explaining a disease state,” -Dr. Yvon.
The technology available at CytoVas analyzes millions of cells at a rate of 100,000 events/second, and the large-output and high-dimensional analysis is sensitive and specific, says Dr. Yvon. It relies on flow cytometry and cytometric fingerprinting, which interprets and gives context to the huge volume of data gathered. Cytometric fingerprinting identifies specific patterns or signatures that are essential for a clinical diagnosis in an unbiased and correct way.

Microparticles are derivative of vascular damage caused by smoking, high cholesterol levels, high blood pressure, diabetes, and other stressors. High levels of microparticles occur in clinical conditions where vascular dysfunction and inflammation happen, such as coronary artery disease.
On the other hand, endothelial progenitor cells are indicative of the bodies repair mechanism. They originate in the bone marrow. When endothelial damage occurs, cellular signals direct endothelial progenitor cells to the site of damage for repair. “In a healthy person, damage and repair mechanisms are in balance. But in an unhealthy person, damage overwhelms repair,” says Dr. Yvon.

CytoVas predicts a great market potential for their new diagnostic approach, and they indicate it might be used in pharmaceutical research to predict drug toxicity concerning the vascular system, and allow for quicker and more effective drug tests.

“You want to identify and include patients who will respond best to a new drug,” says Dr. Yvon. “Companies also can use VHP as a companion diagnostic to their cardiovascular drugs.”

It can also find application in medicine, as a powerful diagnostic tool, used by physicians to diagnose and monitor their patients.

“Physicians can use the information from the VHP to decide a course of treatment for a patient,” says Dr. Yvon



CytoVas
Location: 3160 Chestnut Street, Suite 200, Philadelphia, PA 19104
Phone: (609) 705-3854
Website: www.cytovas.com
Principal: Pascal Yvon, Pharm.D., CEO
Focus: CytoVas develops advanced cell- and particle-based in vitro diagnostic assays to evaluate the health of the cardiovascular system in each individual.

Electron microscopes are powerful tools for studying small cells, viruses, and molecules within cells. An electron microscope can magnify an image up to 10 million times, allowing scientists to view objects on a smaller scale than with a conventional light microscope. Because the size of the electron is much smaller than the wavelength of visible light, electron microscopes have much greater resolving power than light microscopes. There are two major types of electron microscopes used by scientists. Transmission electron microscopy (TEM) allows for viewing of the internal structures of the specimen being studied. Scanning electron microscopy (SEM) permits viewing of the surface of the specimen, and can produce a three-dimensional image.

One major drawback to electron microscopy is that the process of preparing the specimen, as well as the actual process of examining the specimen, results in death of the cell or organism. Electron microscopes utilize a vacuum in order to allow the electrons to penetrate the specimen. Living organisms cannot survive in this vacuum, so all electron micrographs show only dead cells. These cells may even be distorted somewhat during the process of examination, due to the excessive processing needed to prepare the specimen, as well as the extremely low pressure environment of the microscope.

Researchers have recently noted that some small invertebrates are able to withstand being in a vacuum for short periods of times. Beetle larvae, ticks, and tardigrades (also called water bears) have all been previously shown to be able to withstand extremely low pressure environments. When these animals are observed using a scanning electron microscope, the electrons seemed to coat the animals, forming a protective barrier. Normally, when an animal, such as a small insect, is placed in a vacuum, they will dehydrate due to the low pressure and die. While the scanning electron microscope was being used, fruit fly larvae were able to survive up to one hour in the low pressure conditions. After being imaged, the larvae developed normally into adult fruit flies.

Fruit fly larvae are naturally coated with biological molecules. The researchers believed that the electron beams from the microscope caused the biological molecules to polymerize, or join together. These polymers were able to form a protective shield around the larvae. Other organisms that have a similar coating, such as a type of honeybee, required plasma irradiation to form the protective polymers. However, after being irradiated, these organisms were also able to survive the vacuum environment of the electron microscope. In order to confirm that the polymerized coating was indeed protecting these organisms in the vacuum, the researchers applied a similar artificial coating to animals that don’t naturally have one. After plasma irradiation, these organisms were also protected from the vacuum.

When the protected organisms were studied with the electron microscope, the structures of the organisms appeared quite different from traditionally prepared specimens. This might be because the polymer coating helped preserve the natural structure of the specimen. The environment of the electron microscope, as well as sample preparation, may cause distortions in the structure of the specimen. However, it is possible that since the researchers were using relatively low magnification settings, they obtained results that differed from prior, conventional electron microscopy examinations.

The data obtained, however, is interesting for a number of different reasons. First, it is encouraging to scientists trying to discover extraterrestrial life. If some small organisms from earth can survive in a vacuum with the protective polymer coating, it is possible that organisms from another world might also be able to survive using a similar mechanism. This would make it possible for the organism to be transported through the vacuum of outer space. In addition, this information might have result in big advancements for electron microscopy. Scientists may eventually be able to adapt the polymer coats to other organisms, or even single cells and viruses. This would permit the study of live organisms, and may even permit scientists to visualize specific interactions. For example, scientists could directly view a virus invading a host cell, and be able to figure out the process of infection. In addition, because traditional electron microscopy may result in distortion of the specimen, the protective coating may help scientists develop more accurate images.



References:

http://www.nature.com/news/nano-suit-shi...id-1.12799

Premature infants are susceptible to a number of different short and long term health problems. This is because various systems in the infant’s body have not fully matured. The lungs and digestive tract can be particularly worrisome when an infant is born too early. These health concerns can be fatal if the newborn does not receive proper care. One condition, called necrotizing enterocolitis, is particularly common in premature infants.

Necrotizing enterocolitis is an affliction in which the cells of the intestinal wall are killed. This condition normally affects premature infants; however, the precise cause of the necrotizing enterocolitis is unknown. The condition can be fatal, with approximately 25% of infants who contract the disease dying. It is believed that the digestive tract of a premature infant, not being fully developed, may be more susceptible in premature infants to damage from bacteria or even food. It has long been known that premature infants that have been fed formula are at a higher risk for developing necrotizing enterocolitis than premature infants who have been fed breast milk, but the reasons for this are not fully known. To determine what causes necrotizing enterocolitis and how to prevent it in premature infants, scientists have researched the different microbiota in the guts of premature infants, and have looked at the effects of different food sources on cells obtained from premature infants. Scientists have already found that breast milk-fed babies have a different array of bacteria in their intestines compared to forumula-fed babies. The different bacteria present may be involved in the development or prevention of necrotizing enterocolitis.

Recently, researchers tested to see whether digested food could cause damage to intestinal cells. They utilized nine different infant formulas, designed specifically for either premature infants or full term infants, and human breast milk. The formulas and breast milk were digested with enzymes from the pancreas and intestinal fluid. The digested formulas and breast milk were then incubated with several cell types from the intestines, including intestinal epithelial cells, intestinal endothelial cells, and neutrophils, which are a type of white blood cell involved in the innate immune system. They then measured if the formula and milk digests killed any of the cells, and how long this killing took. All of the infant formulas tested resulted in rapid cytotoxity, while the breast milk did not cause such severe damage to cells. For example, the digested formula caused death in forty-seven to ninety-nine percent of the neutrophils, while breast milk only resulted in about six percent of the neutrophils.

Researchers had previously shown that adult intestinal cells could be killed by digested food as well. This killing was attributed to the formation of free fatty acids from digestion of food. The free fatty acids were also attributed with cell killing in the above study by digested infant formula. Even though free fatty acids can be formed by many types of digested food and kill intestinal cells from premature infants and adults, premature infants are the most likely group to contract necrotizing enterocolitis. Scientists attribute this to a layer of mucus in adult intestines that protects cells from the cytotoxic effects of free fatty acids. This layer may be absent or immature in premature infants, so their intestinal cells are not protected. The researchers proposed that human breast milk is specially designed so as not to create as many damaging free fatty acids, and is therefore well tolerated by the immature intestines present in premature infants.

This research indicates a very specific potential benefit of breast milk for premature infants. Many neonatal units are beginning to focus more on providing breastfeeding support to new mothers and babies. This could help prevent many serious conditions, such as necrotizing enterocolitis, particularly in premature or other sick infants. However, some premature infants may not be able to breastfeed due to physical limitations from being born too soon. Donated or expressed breast milk from the mother are good options to help the infant thrive and prevent necrotizing enterocolitis, though this may not always be feasible for every family. The development of formulas for premature infants that does result in the production of free fatty acids could therefore help prevent the development of necrotizing enterocolitis in premature infants that are unable to receive breast milk.


References:

http://ucsdnews.ucsd.edu/pressrelease/in...c_to_cells

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0002133/

Study shows that inhibiting interferons may help clear the body of persistent viral infection.

Persistent viral infections have long baffled virologists, who were unable to explain how some viruses manage to avoid the bodies critical immune response and remain ‘hidden’. A new study, published April 12th in Science Magazine, may shed some new light on this phenomenon.

Scientists at The Scripps Research Institute have made a new discovery that led to counterintuitive conclusions that gives potential to clear persistent infection that is the hallmark of such diseases as AIDS, hepatitis B and hepatitis C.

The study focused on the activity of the body's type 1 interferon proteins. Since its discovery over 50 years ago, IFN-I has been believed to be an especially powerful antiviral agent that marshals the immune system's response against the body's foreign invaders. But in the new study, scientists document that IFN-I initiates persistent infection and limits the generation of an effective antiviral immune response in mice.

"Our findings illuminate an unexpected role for IFN-I proteins in persistent infections, which has major implications for how we treat these infections," said Michael B. A. Oldstone, a professor in the Department of Immunology and Microbial Science at TSRI and senior investigator for the study.

For decades, virologists around the world have been trying to figure out how some viruses manage to persist in their hosts and avoid detection by the immune system.

A discovery made in recent years shows that some of these viruses are specialized at getting into cells of the immune system known as dendritic cells. These cells serve as key detectors in cases of infection and normally respond to viral infections by producing IFN-I proteins. They also produce cytokines and chemokines, both immune-enhancing proteins that drive forward immune responses, as well as immune-suppressing proteins including interleukin-10 (IL-10) and PD-1, which act as a warning system that balances the immune response to keep it within healthy limits, and prevent auto-immune responses.

It has been shown that persistent viruses can turn this immune-suppressing effect and use it for their own purposes. In several experimental models of persistent infections in humans, a rise in IL-10 and PD-L1 is rapidly followed by declines in the function and numbers of antiviral T-cells. Many of the surviving T cells are rendered ineffective, an occurrence called "T-cell exhaustion" or "hyporesponsiveness."

To figure out this immune-suppressing response, Oldstone and his team looked at the early events in a persistent viral infection. The team used a standard animal model that Oldstone himself developed almost 30 years ago: laboratory mice infected with lymphocytic choriomeningitis virus (LCMV) Clone (Cl) 13 strain.

One initial observation was surprising. "A day after infection, bloodstream levels of IFN-I were at least several times higher in the persistent infection, compared to a non-persistent LCMV infection," said Teijaro, one of the lead authors of the study.

The persistent LCMV Cl 13 strain also showed to be much better at infecting plasmacytoid dendritic cells which are considered the main source of IFN-I proteins during viral infections. This specific strain is Differs from others only in three amino-acids, of which just two are important; one in the glycoprotein for binding and entry into dendritic cells and the other in the viral polymerase that enhances viral replication.
Up until now, production of IFN-Is by plasmacytoid dendritic cells has been considered a normal and beneficial part of the immune reaction to a viral infection. "We usually think of IFN-I proteins as antiviral proteins, so that more IFN is better," said Ng, another of the lead researchers on this project. Once she and Teijaro used a monoclonal antibody to block IFN-I-alpha-beta receptor, activity just before or after infection with Cl 13, they noticed a sharp drop in the production of IL-10 and PD-L1, a reduction in cytokine and chemokine expression levels and maintenance of normal secondary lymphoid tissue structure.

Over a longer time period, as the scientists found, levels of immune-suppressing IL-10, as well as PD-L1, both inducers of T-cell exhaustion noticeably drop, and that was associated with restoration of antiviral immune response and virus clearance. Even though blocking the IFN-I-a-b receptor led to higher bloodstream levels of the viruses in the first days of the infection, it soon brought about a stronger, infection-clearing response.

"Even when we blocked IFN-I-a-b receptor after a persistent infection had been established and T-cell exhaustion had set in, we still saw a significantly earlier clearance of the virus," Ng said.

The team now plans to study IFN-I signaling pathways in greater detail. In particular, they hope to determine whether the IFN-I-a-b receptor blocking strategy can work against chronic viral infections in humans. They will also be seeking small molecules which can mimic the function of receptor blocking.

"Most of our findings in the LCMV model mirror what has been observed in human persistent infections, namely the up regulation of IL-10 and PD-L1, and the disruption of lymphoid architecture," said Oldstone.


"Persistent LCMV infection is controlled by blockade of type 1 interferon signaling” Kathleen C. F. Sheehan and Robert D. Schreiber of Washington School of Medicine at St. Louis; and Megan J. Welch, Andrew M. Lee, and Juan Carlos de la Torre of TSRI.
The study was supported by the National Institutes of Health grants AI009484, AI057160 and AI077719, as well as an American Heart Association Fellowship (11POST7430106).

The problems of donor-organ shortage are well known in modern medicine. In case of kidney failure alone, only 18 out of 100 people receive a donor organ, the rest remain of waiting lists until critical failure and ultimately, death. Those that do receive a donor kidney are forced to live on immunosuppressant medication for the rest of their lives, and face a multitude of complications, either from rejection, or other complications concerning their immunocompromised state.

Several projects have been on the way, some for more than a decade now, attempting to engineer kidneys eligible for transplants. This approach, would, theoretically, avoid any immune system complications, and solve the organ-shortage problem.

Researchers of the Massachusetts General Hospital have bioengineered rat kidneys that successfully produced urine both in a laboratory apparatus and after being transplanted into living animals, an achievement long sought after in organ engineering research. In their research report, published in Nature Medicine, the scientists describe building functional replacement kidneys on the structure of donor organs from which living cells had been stripped off, an approach previously used to create bioartificial hearts, lungs, and livers.

“What is unique about this approach is that the native organ's architecture is preserved, so that the resulting graft can be transplanted just like a donor kidney and connected to the recipient's vascular and urinary systems. If this technology can be scaled to human-sized grafts, patients suffering from renal failure who are currently waiting for donor kidneys or who are not transplant candidates could theoretically receive new organs derived from their own cells.” - explains Harald Ott, M.D., Ph.D., of the MGH Center for Regenerative Medicine, senior author of the paper.

Dr. Ott discovered, as a research fellow at the University of Minnesota, a new technique to bioengineer organs, and that technique was utilized in this project. It involves stripping the living cells from a donor organ with a detergent solution without damaging the fiber architecture of the organ, and then repopulating the collagen scaffold that remains with the appropriate cell type, in this case endothelial cells and nephrocytes. In this case, human endothelial cells were used to replace the lining of the vascular system and kidney cells were taken from newborn rats. The research team first decellularized rat kidneys to confirm that the organ's complex structures would not be destroyed or altered. They also showed the technique worked on a larger scale by stripping cells from pig and human kidneys.

To make sure that appropriate cells are in their appropriate positions, the researchers had to administer the endothelial lining of the blood vessels through the vascular system of the organ, and the nephrocytes through the ureter. By precisely adjusting the pressures of the solutions the researchers enabled the cells to be dispersed throughout the whole organs, which were then cultured and grown in a bioreactor for up to 12 days. The researchers first tested the repopulated organs in a device that passed blood through its vascular system and drained off any urine, which revealed evidence of limited filtering of blood, molecular activity, and urine production, proving the concept that this organs in fact, worked, though at limited capacity.
Bioengineered kidneys transplanted into living rats from which one kidney had been removed began producing urine as soon as the blood supply was restored, with no evidence of any complications such as bleeding or clot formation. The overall function and capacity of the regenerated organs was significantly reduced compared with that of normal, healthy kidneys, something the researchers believe may be attributed to the immaturity of the neonatal cells used to repopulate the scaffolding, it nonetheless proves a principle.
“Further refinement of the cell types used for seeding and additional maturation in culture may allow us to achieve a more functional organ. Based on this initial proof-of- principle, we hope that bioengineered kidneys will someday be able to fully replace kidney function just as donor kidneys do. In an ideal world, such grafts could be produced ‘on demand’ from a patient's own cells, helping us overcome both the organ shortage and the need for chronic immunosuppression. We're now investigating methods of deriving the necessary cell types from patient-derived cells and refining the cell-seeding and organ culture methods to handle human-sized organs.” Stated Dr. Ott.

Results published online, pre-print version; Nature Medicine

Twenty three years ago, in 1990, scientists began working together on one of the largest biological research projects ever proposed. The project proposed to sequence the 3 billion nucleotides in the human genome. It was met with great hope, from discovering the causes of many human diseases, to the eventual discovery of new treatments for these diseases. The project took 13 years to complete, at a cost of approximately three billion dollars.

When the project began, scientists estimated they would find about 100,000 distinct genes in the human genome. As the project progressed, they were stunned to find humans had only about 25,000 genes. How could an organism as complex as a human being have a number of genes similar to that of a worm? The answer was alternative splicing. Splicing is a process that occurs during transcription, or the production of mRNA, in eukaryotic cells. Introns, which are noncoding regions of nucleotides, are removed, while the exons, the coding regions, are fused, or spliced, together. This forms a mature mRNA that can direct protein production. Sometimes, though, different combinations of exons may be spliced together. These different combinations of exons allow one gene to code for the production of multiple proteins.

Another surprise to scientists was how difficult it was to determine the functions of the genes that were sequenced during the human genome project. Sequencing the genome merely told scientists which of the four DNA bases was in each position in the human genome. After the genome was sequenced, scientists had to begin the process of determining what function each gene has. Some genes had already been studied, and were easily identified after the genome was sequenced. Others were, and still are, unknown. Alternative splicing patterns, which can create multiple proteins from a single gene, further complicate the determination of gene function.

In addition to sequencing the coding regions of DNA, which tell the cell how to make proteins, scientists are now interested in determining the function of noncoding, or junk, regions of DNA. These repetitive nucleotide sequences are found in every person’s genome, and make up approximately 98% of the bases. For many years, scientists believed that these noncoding sequences had no function. They were believed to be made of remnants of retroviruses that had previously integrated into the genome and had since become inactive. Transposons, pieces of DNA that can move from one location to another, are also believed to be a component of the junk DNA. In recent years, scientists have noticed that noncoding DNA may actually play a role in the cell. Some of this junk DNA may actually function as transcription factors. These are sequences of DNA that help recruit the enzymes required for transcription of DNA into mRNA. Another idea is that the junk DNA provides variation in the population, which is important for helping the human population continue to evolve. These sequences might even help explain differences between humans and other closely related animals. This may be directly linked to the transcription factors found in noncoding DNA. The timing and amount of gene expression may lead to many of the differences between humans and other mammals.

As with many scientific endeavors, the human genome project generated far more questions than answers. However, the information gained must not be undersold. In addition to learning more about how genes function, we have developed exponentially superior technology over the past ten years. While the human genome project took 13 years and cost billions of dollars, a normal human genome can be sequenced in a few days and a cost of only a few thousand dollars. The technology has become faster, more widely available, and cheaper. Individual genome sequencing is already being used to help people determine their risk for various genetically –linked disorders. Rapid and affordable genome sequencing is also helpful for researchers who want to find differences between patients afflicted with a disorder, and volunteers without the disorder. Because of the research resulting from the sequencing of the human genome, scientists have links between genes and many human disorders, including cancer, some neurodegenerative disease, and more. By studying the defects in genes that cause these disorders, researchers might be more able to develop rational treatments. Indeed, many advancements have been made in the past 10 years, and many more are sure to come in the next 10.


References:

http://news.yahoo.com/human-genome-proje...31005.html

http://www.nytimes.com/2013/04/16/scienc....html?_r=0

http://www.sciencedaily.com/releases/200...180928.htm

REPORT FIELD TRIP on March 28th 2013:
LAWRENCE BERKELEY LABORATORY FIELD TRIP
Student reporter: Katherine Miller, Biotech Club, CCSF

Tuesday, April 2, 2013

Dear fellow club members,

I would like to represent all the students and staff instructors in the field trip group from the CCSF Biotech Club to thank Doctor Corie Ralston, Doctor Simon Morton, and Dr. Peter Zwart, who gave our group the opportunity to witness the fascinating giant synchrotron system, the Advanced Light Souce (ALS) at Lawrence Berkeley National Laboratory. The ALS generates forty beam-lines, each of which is used for a different application. I also want personally to thank Rebecca D’ Urso, the field trip coordinator who had thoroughly prepared the tour, leading to its success.

The group gathered at the conference room 2202 on the second floor of building 6 of Lawrence Berkeley National Laboratory. We had a thirty-minute brief introduction with short video illustration of one of the most fascinating applications of synchrotron and its beam-line system: crystallography.

Crystallography is an essential technique to image 3-D structure of proteins. To understand why it is essential to know protein 3-D structures, we need to understand what proteins are and why they are so important in biotechnology and how they relate to the pharmaceutical industry.

Proteins (their four main atoms are linked together with multiple linkage styles between carbon, hydrogen, oxygen, and nitrogen, in addition with numerous of other atoms such as sulfur, iron, zinc, copper, etc.) in biotechnology field, are the huge bio-molecule with myriad bio-functions depending on their myriad shapes. Their shapes, in 3-D structures, are too convoluted and small to photograph by the technique of regular X-ray imaging. That is why synchrotron, the high-energy magnetic field circular system, generates electron beam; then, the beam-line system is one of the end-points that take up this high energy beam to create high-fidelity-X-ray beams at the level of photon-beam that can increase the signal-to-noise level more than thousand times that of non-synchrotron X-ray sources. Once diffraction data is collected on a target protein, its structure into the level of their atoms and their linkages can be solved.

In the pharmaceutical industry, a protein found is often the target for patho-physiology cause of a disease. Researchers try to make other proteins to either activate or inactivate this protein depending on the practical reason of the treatment for that disease. For example, incretins are a group of gastrointestinal hormones to increase the body’s sensitivity to insulin (a pancreas protein) in the treatment of diabetes mellitus. Activation of incretins by creating a linkage or breaking a linkage in their structure would lead to the increased sensitivity of the body to insulin. Incretin is made into a drug for this purpose.

How to do crystallography on a protein? Dr. Ralston instructed the group to create crystals of lysozyme, a cell enzyme for cleaning up debris inside cell matrix. Once protein crystals are made, they will be frozen in liquid nitrogen and transferred to the beam-line for imaging its structure. The beam-line has robot arms that can accurately fish up the crystal hook pockets and positions it in front of the beam-line ending box right in front of the diffraction detector camera for snap-shot one by one, and then the diffraction images are transferred into the computer system where a scientist interprets the result and determines the protein structure. Multiple snap-shots of the crystals of the same protein are generated and the more accurate image is extrapolated by the computer system.

Doctor Ralston explained that Lawrence Lab takes contracts with many pharmaceutical companies for using the beam-line system for their target proteins in their new drug manufacture, and that is paid-service contract for at least a few years for one drug! Lawrence Berkeley National Laboratory, though independently operates from UC Berkeley, has some collaborative and supportive activities in academics and beam line researches with UC Berkeley to a certain extent.

The synchrotrons system is also essential for high-tech discovery in other fields; the beam line can also be used in tomography, in imaging computer chips, etc. There are four nationally-funded synchrotrons in the US, two in California, one in Chicago, one in Long Island. There are also many synchrotrons around the world; a few are built in China, Europe, etc. These are often national and government funded system; however, private smaller synchrotrons with less investment dollars can be built separately from a large-scale synchrotron which is a multi-million dollar investment. Stanford has built an even brighter X-ray source called LCLS, which stands for Linac Coherent Light source.

The tour ended half an hour later than planned because everybody was so interested in asking many questions.

Thank you again Dr. Ralston, Dr. Morton, and Dr. Zwart and all of their staffs have given us a great opportunity to learn about this fascinating technology.

VIDEO LINKS:

• Click here to watch Video 1
  
• Click Here to watch Video 2
  

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Biomimicing nano-particles may provide with a new effective food preservation technique

Engineers and researchers at Rensselaer Polytechnic Institute have been working to develop a new method to kill deadly pathogenic bacteria, including listeria, for a range of uses, dominantly the food industry. This industrial innovation presents an alternative to the use of antibiotics or chemical decontamination in food supply systems.

Using inspiration derived from Nature, the researchers successfully adhered a cell lytic enzyme to food-safe silica nanoparticles, thus creating a coating with the demonstrated ability to selectively kill listeria-dangerous foodborne bacteria that causes an estimated 500 deaths every year in the United States. The coating kills the bacteria upon contact, even at very high concentrations, within a short time span without affecting other bacteria or organisms. The lytic enzymes can also, using this method, be attached to starch nanoparticles that are commonly used in food packaging.

Listeria is a bacterial genus containing seven species. Named after the English pioneer of sterile surgery, Joseph Lister, Listeria species are Gram-positive bacilli and are typified by ''L. monocytogenes'', and they are the the causative agents of listeriosis. This infection occurs primarily in newborn infants, elderly patients, and patients who are immune-compromised.

This new method is very flexible, and by using different lytic enzymes, could be modified to create surfaces that selectively target other deadly bacteria such as anthrax, bacillus or cocii, said Jonathan Dordick, vice president for research at Rensselaer, who helped lead the study.

This research took place in the Rensselaer Center for Biotechnology and Interdisciplinary Studies and the Rensselaer Nanoscale Science and Engineering Center for the Directed Assembly of Nanostructures, and combined the efforts and skills of chemical engineers, biotechnologists and material scientists. Collaborating with Dordick were Ravi Kane, Professor of Chemical and Biological Engineering, and Linda Schadler, associate dean for academic affairs for the Rensselaer School of Engineering.

"In this study, we have identified a new strategy for selectively killing specific types of bacteria. Stable enzyme-based coatings or sprays could be used in food supply infrastructure-from picking equipment to packaging to preparation-to kill listeria before anyone has a chance to get sick from it," said Kane. "What's most exciting is that we can adapt this technology for all different kinds of harmful or deadly bacteria."

This recent study by the same team builds upon their success in 2010 of creating a coating for killing methicillin resistant Staphylococcus aureus (MRSA), the primal culprit responsible for antibiotic resistant infections. While this coating was intended for use on surgical equipment and hospital walls, the development of a listeria-killing coating had to be food-safe, which presented additional challenge.

The team found inspiration in nature; the lytic enzymes used by viruses. Phages are viruses that infect bacterial cells, and once they have inserted their DNA and reproduced, the new viroids must escape the cells to be able to spread the infection. At that point the original phage releases a cue that activates the inserted gene that synthesizes lytic enzymes, which break down the bacterial wall from within, releasing the new generation of viruses. The team used this principle to break the walls from outside.

The next task was to stabilize the listeria-killing lytic enzymes, called Ply500, so they attached them to U.S. Food and Drug Administration-approved silica nanoparticles to create an ultra-thin film. The researchers used maltose as a binding protein to attach Ply500 to conventional, edible starch nanoparticles commonly used in food packaging. Both formulations showed as effective. Within 24 hours all listeria at concentrations as high as 100,000 bacteria per milliliter were killed. This concentrations are significantly higher than normally found in food contamination situations.

Results of the study are detailed in the paper "Enzyme-based Listericidal Nanocomposites," published April 3. in the journal Scientific Reports from the Nature Publishing Group.