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DNA’s dynamic nature makes it well-suited to serve as the blueprint of life

A new study could explain why DNA and not RNA, its older chemical cousin, is the main repository of genetic information. The DNA double helix is a more forgiving molecule that can contort itself into different shapes to absorb chemical damage to the basic building blocks — A, G, C and T — of genetic code. In contrast, when RNA is in the form of a double helix it is so rigid and unyielding that rather than accommodating damaged bases, it falls apart completely.

The research, published August 1, 2016 in the journal Nature Structural and Molecular Biology, underscores the dynamic nature of the DNA double helix, which is central to maintaining the stability of the genome and warding off ailments like cancer and aging. The finding will likely rewrite textbook coverage of the difference between the two purveyors of genetic information, DNA and RNA.

“There is an amazing complexity built into these simple beautiful structures, whole new layers or dimensions that we have been blinded to because we didn’t have the tools to see them, until now,” said Hashim M. Al-Hashimi, Ph.D., senior author of the study and professor of biochemistry at Duke University School of Medicine.

DNA’s famous double helix is often depicted as a spiral staircase, with two long strands twisted around each other and steps composed of four chemical building blocks called bases. Each of these bases contain rings of carbon, along with various configurations of nitrogen, oxygen, and hydrogen. The arrangement of these atoms allow G to pair with C and A to pair with T, like interlocking gears in an elegant machine.

When Watson and Crick published their model of the DNA double helix in 1953, they predicted exactly how these pairs would fit together. Yet other researchers struggled to provide evidence of these so-called Watson-Crick base pairs. Then in 1959, a biochemist named Karst Hoogsteen took a picture of an A-T base pair that had a slightly skewed geometry, with one base rotated 180 degrees relative to the other. Since then, both Watson-Crick and Hoogsteen base pairs have been observed in still images of DNA.

Five years ago, Al-Hashimi and his team showed that base pairs constantly morph back and forth between Watson-Crick and the Hoogsteen configurations in the DNA double helix. Al-Hashimi says that Hoogsteen base pairs typically show up when DNA is bound up by a protein or damaged by chemical insults. The DNA goes back to its more straightforward pairing when it is released from the protein or has repaired the damage to its bases.

“DNA seems to use these Hoogsteen base pairs to add another dimension to its structure, morphing into different shapes to achieve added functionality inside the cell,” said Al-Hashimi.

Al-Hashimi and his team wanted to know if the same phenomenon might also be occurring when RNA, the middleman between DNA and proteins, formed a double helix. Because these shifts in base pairing involve the movement of molecules at an atomic level, they are difficult to detect by conventional methods. Therefore, Al-Hashimi’s graduate student Huiqing Zhou used a sophisticated imaging technique known as NMR relaxation dispersion to visualize these tiny changes. First, she designed two model double helices — one made of DNA and one made of RNA. Then, she used the NMR technique to track the flipping of individual G and A bases that make up the spiraling steps, pairing up according to Watson-Crick or Hoogsteen rules.

Prior studies indicated that at any given time, one percent of the bases in the DNA double helix were morphing into Hoogsteen base pairs. But when Zhou looked at the corresponding RNA double helix, she found absolutely no detectable movement; the base pairs were all frozen in place, stuck in the Watson-Crick configuration.

The researchers wondered if their model of RNA was an unusual exception or anomaly, so they designed a wide range of RNA molecules and tested them under a wide variety of conditions, but still none appeared to contort into the Hoogsteen configuration. They were concerned that the RNA might actually be forming Hoogsteen base pairs, but that they were happening so quickly that they weren’t able to catch them in the act. Zhou added a chemical known as a methyl group to a specific spot on the bases to block Watson-Crick base pairing, so the RNA would be trapped in the Hoogsteen configuration. She was surprised to find that rather than connecting through Hoogsteen base pairs, the two strands of RNA came apart near the damage site.

“In DNA this modification is a form of damage, and it can readily be absorbed by flipping the base and forming a Hoogsteen base pair. In contrast, the same modification severely disrupts the double helical structure of RNA,” said Zhou, who is lead author of the study.

The team believes that RNA doesn’t form Hoogsteen base pairs because its double helical structure (known as A-form) is more compressed than DNA’s (B-form) structure. As a result, RNA can’t flip one base without hitting another, or without moving around atoms, which would tear apart the helix.

“For something as fundamental as the double helix, it is amazing that we are discovering these basic properties so late in the game,” said Al-Hashimi. “We need to continue to zoom in to obtain a deeper understanding regarding these basic molecules of life.”

https://www.sciencedaily.com/  Science Daily

https://www.sciencedaily.com/releases/2016/08/160801113823.htm Original web page at Science Daily

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Artificial pancreas likely to be available by 2018

 

The artificial pancreas — a device which monitors blood glucose in patients with type 1 diabetes and then automatically adjusts levels of insulin entering the body — is likely to be available by 2018, conclude authors of a paper in Diabetologia (the journal of the European Association for the Study of Diabetes). Issues such as speed of action of the forms of insulin used, reliability, convenience and accuracy of glucose monitors plus cybersecurity to protect devices from hacking, are among the issues that are being addressed.

Currently available technology allows insulin pumps to deliver insulin to people with diabetes after taking a reading or readings from glucose meters, but these two components are separate. It is the joining together of both parts into a ‘closed loop’ that makes an artificial pancreas, explain authors Dr Roman Hovorka and Dr Hood Thabit of the University of Cambridge, UK. “In trials to date, users have been positive about how use of an artificial pancreas gives them ‘time off’ or a ‘holiday’ from their diabetes management, since the system is managing their blood sugar effectively without the need for constant monitoring by the user,” they say.

One part of the clinical need for the artificial pancreas is the variability of insulin requirements between and within individuals — on one day a person could use one third of their normal requirements, and on another 3 times what they normally would. This is dependent on the individual, their diet, their physical activity and other factors. The combination of all these factors together places a burden on people with type 1 diabetes to constantly monitor their glucose levels, to ensure they don’t end up with too much blood sugar (hyperglycaemic) or more commonly, too little (hypoglycaemic). Both of these complications can cause significant damage to blood vessels and nerve endings, making complications such as cardiovascular problems more likely.

There are alternatives to the artificial pancreas, with improvements in technology in both whole pancreas transplantation and also transplants of just the beta cells from the pancreas which produce insulin. However, recipients of these transplants require drugs to supress their immune systems just as in other organ transplants. In the case of whole pancreas transplantation, major surgery is required; and in beta cell islet transplantation, the body’s immune system can still attack the transplanted cells and kill off a large proportion of them (80% in some cases). The artificial pancreas of course avoids the need for major surgery and immunosuppressant drugs.

Researchers globally continue to work on a number of challenges faced by artificial pancreas technology. One such challenge is that even fast-acting insulin analogues do not reach their peak levels in the bloodstream until 0.5 to 2 hours after injection, with their effects lasting 3 to 5 hours. So this may not be fast enough for effective control in, for example, conditions of vigorous exercise. Use of the even faster acting ‘insulin aspart’ analogue may remove part of this problem, as could use of other forms of insulin such as inhaled insulin. Work also continues to improve the software in closed loop systems to make it as accurate as possible in blood sugar management.

A number of clinical studies have been completed using the artificial pancreas in its various forms, in various settings such as diabetes camps for children, and real life home testing. Many of these trials have shown as good or better glucose control than existing technologies (with success defined by time spent in a target range of ideal blood glucose concentrations and reduced risk of hypoglycaemia). A number of other studies are ongoing. The authors say: “Prolonged 6- to 24-month multinational closed-loop clinical trials and pivotal studies are underway or in preparation including adults and children. As closed loop devices may be vulnerable to cybersecurity threats such as interference with wireless protocols and unauthorised data retrieval, implementation of secure communications protocols is a must.”

The actual timeline to availability of the artificial pancreas, as with other medical devices, encompasses regulatory approvals with reassuring attitudes of regulatory agencies such as the US Food and Drug Administration (FDA), which is currently reviewing one proposed artificial pancreas with approval possibly as soon as 2017. And a recent review by the UK National Institute of Health Research (NIHR) reported that automated closed-loop systems may be expected to appear in the (European) market by the end of 2018. The authors say: “This timeline will largely be dependent upon regulatory approvals and ensuring that infrastructures and support are in place for healthcare professionals providing clinical care. Structured education will need to continue to augment efficacy and safety.”

The authors say: “Cost-effectiveness of closed-loop is to be determined to support access and reimbursement. In addition to conventional endpoints such as blood sugar control, quality of life is to be included to assess burden of disease management and hypoglycaemia. Future research may include finding out which sub-populations may benefit most from using an artificial pancreas. Research is underway to evaluate these closed-loop systems in the very young, in pregnant women with type 1 diabetes, and in hospital in-patients who are suffering episodes of hyperglycaemia.”

They conclude: “Significant milestones moving the artificial pancreas from laboratory to free-living unsupervised home settings have been achieved in the past decade. Through inter-disciplinary collaboration, teams worldwide have accelerated progress and real-world closed-loop applications have been demonstrated. Given the challenges of beta-cell transplantation, closed-loop technologies are, with continuing innovation potential, destined to provide a viable alternative for existing insulin pump therapy and multiple daily insulin injections.”

https://www.sciencedaily.com/  Science Daily

https://www.sciencedaily.com/releases/2016/06/160630214503.htm  Original web page at Science Daily

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Copper-induced misfolding of prion proteins

Iowa State University researchers have described with single-molecule precision how copper ions cause prion proteins to misfold and seed the misfolding and clumping of nearby prion proteins.

The researchers also found the copper-induced misfolding and clumping is associated with inflammation and damage to nerve cells in brain tissue from a mouse model.

Prions are abnormal, pathogenic agents that are transmissible and induce abnormal folding of a specific type of protein called prion proteins, according to the Centers for Disease Control and Prevention. Prion proteins are mostly found in the brain. The abnormal folding of prion proteins leads to brain damage and symptoms of neurodegenerative disease. A similar cycle of neuronal protein misfolding and clumping is observed in other neurodegenerative disorders, including Parkinson’s and Alzheimer’s diseases.

“Our study establishes a direct link, at the molecular level, between copper exposure and prion protein neurotoxicity,” the researchers wrote in a summary of the paper.

The findings were published today in the journal Science Advances. The corresponding author is Sanjeevi Sivasankar, an Iowa State University associate professor of physics and astronomy; the first author is Chi-Fu Yen, an Iowa State doctoral student in electrical and computer engineering. Co-authors are Anumantha Kanthasamy, an Iowa State Clarence Hartley Covault Distinguished Professor in Veterinary Medicine, chair of biomedical sciences and director of the Iowa Center for Advanced Neurotoxicology; and Dilshan Harischandra, an Iowa State doctoral student in biomedical sciences.

Grants from the National Institute of Environmental Health Sciences at the National Institutes of Health supported the project, including one from the Virtual Consortium for Transdisciplinary Environmental Research.

Although this study determined that copper-induced misfolding and clumping of prion proteins is associated with the degeneration of nerve tissues, Sivasankar cautioned that the study does not directly address the infectivity of prion diseases.

“There are different strains of misfolded prion proteins and not all of them are pathogenic,” Sivasankar said. “Although we do not show that the strains generated in our experiments are infectious, we do prove that copper ions trigger misfolding of prion proteins which causes toxicity in nerve cells.”

The Sivasankar and Kanthasamy research groups plan to perform additional studies to determine if the copper-induced misfolding causes disease.

Integrating approaches Sivasankar also noted that a unique aspect of this project was the integration of biophysical and neurotoxicological research approaches. He said the combination has the potential to transform studies of the molecular basis for neurodegenerative diseases.

A fluorescence-based technique that identified misfolded prion proteins with single-molecule sensitivity and determined the role of metal ions in misfolding. The researchers used this technique to show that misfolding begins when copper ions bind to the unstructured tail of the prion protein. A single-molecule atomic force microscopy assay that measured the efficiency of prion protein clumping. The researchers used this technique to show that misfolded prion proteins stick together nearly 900 times more efficiently than properly folded proteins.

The Kanthasamy and Sivasankar research groups worked together on a real-time, quaking-induced conversion assay to demonstrate that misfolded prion proteins serve as seeds that trigger the misfolding and clumping of nearby prion proteins. Kanthasamy’s research group also used its expertise in neurotoxicology to show the copper-induced, misfolded prion proteins damage nerve cells in slices of brain tissue from mice.

Taken together, the results identify the biophysical conditions and mechanisms for copper-induced prion protein misfolding, clumping and neurotoxicity, the researchers wrote.

“This was a very comprehensive study,” Sivasankar said. “We took it from single molecules all the way to tissues.”

And, although the study doesn’t address the infectious nature of prion diseases, Kanthasamy said it is still important: “This study has major implications to our understanding the role of metals in protein misfolding diseases including prion, Alzheimer’s and Parkinson’s diseases.”

https://www.sciencedaily.com/ Science Daily

https://www.sciencedaily.com/releases/2016/07/160701183556.htm Original web page at Science Daily

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* Research suggests a way to identify animals at risk of blood clots

Patients who are critically ill, be they dog, cat or human, have a tendency toward blood clotting disorders. When the formation of a clot takes too long, it puts them at risk of uncontrolled bleeding. But the other extreme is also dangerous; if blood clots too readily, it can lead to organ failure or even death if a clot goes to the lungs, brain or heart.

This latter condition, where blood clots to an excessive degree, is known as hypercoagulability. Despite its risks, veterinarians have few tools to identify patients experiencing it. With new findings from a retrospective study, a team at the University of Pennsylvania School of Veterinary Medicine has found that a common diagnostic tool often used to identify patients at risk of bleeding may also be used to identify those predisposed to clot excessively.

The study was conducted by three clinicians in Penn Vet’s Department of Clinical Studies: lead author Jennifer Song, an intern at the time of the study and now a resident at The Ohio State University; Kenneth J. Drobatz, a professor of critical care and director of emergency services; and senior author Deborah C. Silverstein, an associate professor of critical care. Their work was published in the Journal of Veterinary Emergency and Critical Care.

Two blood tests are commonly used to measure a dog’s ability to clot: prothrombin time, or PT, and activated partial thromboplastin time, or aPTT. These tests have an established normal reference range. Animals with results that are longer than normal are considered at risk of abnormal bleeding. However, when a clotting time was shorter than normal, clinicians have typically dismissed it.

“In the past,” Silverstein said, “we’ve always said, no, it’s probably that you pulled the sample incorrectly or the handling of the sample was inappropriate, even though logically you would think that a shorter time might indicate the animal is hypercoagulable.

“This study was attempting to say, can we actually use a shortened prothombin time or activated partial thromboplastin time to identify patients with hypercoagulability,” she added.

To do so, the Penn Vet team looked through the medical records of hundreds of dogs treated at Penn’s Ryan Veterinary Hospital between 2006 and 2011, searching for animals who had a diagnostic test called a TEG run. A TEG, or thromboelastogram, is considered the gold standard for evaluating clotting dynamics but is conducted on equipment that is expensive and not commonly found in primary veterinary practices. Of the 540 dogs the researchers considered, they found 23 that had a shortened PT or aPTT recorded in the same 24-hour period as the TEG test. Twenty-three other dogs with normal PT and aPTT served as a control group.

They then looked at the medical records for indications of a clinical finding of hypercoagulability, such as clots formed within the intravenous catheter or in the circulatory system, or of a suspected blood clot in the lungs, known as pulmonary thromboembolism.

Comparing these readings and clinical signs between the group of dogs with shortened PT or aPTT times and the control group, the researchers found statistically significant differences: more dogs with shortened PT and aPTT times had clinical signs of hypercoagulability and suspected pulmonary thromboembolism compared with the control group.

They also found a correlation between dogs with shortened PT and aPTT results and increased level of D-dimer, a protein fragment that is produced when a clot is being broken down.

“If your body is forming a lot of clots and is trying to break them down,” Silverstein said, “the D-dimer is a one of the metabolites from the clot that will appear in elevated levels in the blood. So seeing that animals that have shortened coagulation time also have elevated D-dimers was consistent with the suspicion that they might be hypercoagulable and therefore forming and breaking down excessive clots.”

Silverstein cautioned that the study was based on a relatively small group, but she finds the results compelling enough to push clinicians to consider further diagnostics and anti-coagulant treatment in certain high-risk cases.

“I think based on this retrospective study, we should pay more attention to shortened clotting times and look at them with a degree of diagnostic value,” she said. “In this patient population of critically ill dogs, it may help in identifying patients at risk of thrombosis.”

Silverstein would like to follow up on the work, looking at more animals and examining how different diseases may impact clotting risk. She’d also like to pursue a similar study in cats, a species susceptible to devastating arterial clots.

https://www.sciencedaily.com/  Science Daily

https://www.sciencedaily.com/releases/2016/05/160519144852.htm  Original web page at Science Daily

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Using magnetic forces to control neurons, study finds brain is vital in glucose metabolism

A new tool to control the activity of neurons in mice avoids the downfalls of current methods by using magnetic forces to remotely control the flow of ions into specifically targeted cells. Applying this method to a group of neurons in the hypothalamus, researchers found that the brain plays a surprisingly vital role in maintaining blood glucose levels.

To learn what different cells do, scientists switch them on and off and observe what the effects are. There are many methods that do this, but they all have problems: too invasive, or too slow, or not precise enough. Now, a new method to control the activity of neurons in mice, devised by scientists at Rockefeller University and Rensselaer Polytechnic Institute, avoids these downfalls by using magnetic forces to remotely control the flow of ions into specifically targeted cells.

Jeffrey Friedman, Marilyn M. Simpson Professor and head of the Laboratory of Molecular Genetics, and colleagues successfully employed this system to study the role of the central nervous system in glucose metabolism. Published online today in Nature, the findings suggest a group of neurons in the hypothalamus play a vital role in maintaining blood glucose levels.

“These results are exciting because they provide a broader view of how blood glucose is regulated–they emphasize how crucial the brain is in this process,” says Friedman. “And having a new means for controlling neural activity, one that doesn’t require an implant and allows you to elicit rapid responses, fills a useful niche between the methods that are already available.”

It may also be possible to adapt this method for clinical applications, says Jonathan Dordick of Rensselaer. “Depending on the type of cell we target, and the activity we enhance or decrease within that cell, this approach holds potential in development of therapeutic modalities, for example, in metabolic and neurologic diseases.”

Previous work led by Friedman and Dordick tested a similar method to turn on insulin production in diabetic mice. The system couples a natural iron storage particle, ferritin, and a fluorescent tag to an ion channel called TRPV1, also known as the capsaicin chili pepper receptor. Ferritin can be affected by forces such as radio waves or magnetic fields, and its presence tethered to TRPV1 can change the conformation of the ion channel.

“Normally radio waves or magnetic fields, at these strengths, will pass through tissue without having any effect,” says first author Sarah Stanley (now Assistant Professor of Medicine, Endocrinology, Diabetes and Bone Disease at Icahn School of Medicine at Mount Sinai). “But when this modified ferritin is present, it responds and absorbs the energy of the radiofrequency or magnetic fields, producing motion. This motion opens the channel and allows ions into the cell. Depending on the ions flowing through the channel, this can either activate or inhibit the cells’ activity.”

This study is the first to turn neurons on and off remotely with radio waves and magnetic fields. TRPV1 normally allows positive ions — such as calcium or sodium–to flow in, which activates neurons and transmits neuronal signals. The researchers were also able to achieve the opposite effect, neuronal inhibition, by mutating the TRPV1 channel to only allow negative chloride ions to flow through.

“The modified TRPV1 channel was targeted specifically to glucose sensing neurons using a genetic technique known as Cre-dependent expression,” says Stanley. “To test whether a magnetic field could remotely modulate these neurons, we simply placed the mice near the electromagnetic coil of an MRI machine.”

Using this novel method, the researchers investigated the role these glucose sensing neurons play in blood glucose metabolism. Hormones released by the pancreas, including insulin, maintain stable levels of glucose in the blood. A region of the brain called the ventromedial hypothalamus was thought to play a role in regulating blood glucose, but it was not possible with previous methods to decipher which cells were actually involved.

Friedman and colleagues found that when they switched these neurons on with magnetic forces, blood glucose increased, insulin levels decreased, and behaviorally, the mice ate more. When they inhibited the neurons, on the other hand, the opposite occurred, and blood glucose decreased.

“We tend to think about blood glucose being under the control of the pancreas, so it was surprising that the brain can affect blood glucose in either direction to the extent that it can,” says Friedman. “It’s been clear for a while that blood glucose can increase if the brain senses that it’s low, but the robustness of the decrease we saw when these neurons were inhibited was unexpected.”

The researchers’ system has several advantages that make it ideal for studies on other circuits in the brain, or elsewhere. It can be applied to any circuit, including dispersed cells like those involved in the immune system. It has a faster time scale than similar chemogenetic tools, and it doesn’t require an implant as is the case with so-called optogenetic techniques.

In addition to its utility as a research tool, the technique may also have clinical applications. “Although it is a long ways off, this technique may offer an alternative to deep brain stimulation or trans-magnetic stimulation,” says Friedman. “We’d like to explore the possibility that this could provide some of the benefits of these without such an invasive procedure or cumbersome device.”

https://www.sciencedaily.com/  Science Daily

https://www.sciencedaily.com/releases/2016/03/160323152021.htm  Original web page at Science Daily

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Researchers work on lowering greenhouse gas emissions from poultry houses

The University of Delaware’s Hong Li is part of a research team looking at how adding alum as an amendment to poultry litter reduces ammonia and greenhouse gas concentrations and emissions, specifically carbon dioxide, in poultry houses.

Li partnered with researchers at the United States Department of Agriculture (USDA), the University of Tennessee and Oklahoma State University for the project and the results of the research were recently published in the Journal of Environmental Quality.

Li, assistant professor in the Department of Animal and Food Sciences (ANFS) in UD’s College of Agriculture and Natural Resources, said that the project is ongoing and that the main challenge for the poultry industry is controlling nutrient emissions from poultry houses and conserving energy while also providing for the welfare of the birds inside the houses.

Acid-based chemical compounds, alum and PLT — another amendment — that are added to the bedding material in poultry houses prior to the birds entering have proven to be a very effective tool in controlling ammonia emissions.

“In the poultry industry, ammonia is a major concern. Ammonia during the growth period is high, especially during the wintertime. Ammonia can do a lot of damage to the animal, especially the respiratory system, and can effect overall animal health and welfare,” said Li.

Also, if ammonia is emitted to the air from the poultry house, it is a precursor of fine particles and there are national Clean Air Act regulations from the Environmental Protection Agency that have strict guidelines for controlling emissions.

“We need to control the ammonia, not only for the animal health but also for the public health. That’s why I’m doing the research, to reduce the ammonia emissions and improve the animal health and the public air quality, especially for the rural areas, to make sure our agriculture is sustainable,” said Li.

Li said that there are several products on the market to control ammonia in poultry houses and alum is the preferred product for growers in Arkansas, where the study was conducted.

While adding alum to poultry litter is known to reduce ammonia concentration in poultry houses, its effects on greenhouse gas emissions had been unknown.

Li’s role in the study was on the engineering side and he helped Philip Moore, one of the authors of the paper and a pioneer researcher on alum in poultry production with the USDA, develop an automatic air sampling system to evaluate the emissions reduction by using alum in the broiler house.

“We not only looked at ammonia reduction, we also looked at the whole environmental footprint — how the alum could potentially impact the greenhouse emissions — and the results showed that we reduced quite a bit of carbon dioxide emissions,” said Li.

The carbon dioxide was reduced in two ways. First, because alum is an acidic product, it reduces microbial activity in the litter and reduces the ammonia emissions.

Ammonia comes from uric acid being broken down by bacteria and enzymes. Once the uric acid is broken down, two products are created — one is ammonia and one is carbon dioxide.

“By reducing the bacterial activity, we reduce ammonia and also we reduce the carbon dioxide; that’s one aspect of how we reduce carbon dioxide,” said Li.

Second, by using acid-based litter amendments in poultry litter, growers can reduce the ventilation rate and reduce fuel used for heating the poultry houses, especially during the winter.

“In the broiler industry, we want to control ammonia to improve animal health and welfare. They have to keep the bird comfortable with optimum temperatures. However, if you want to have lower ammonia, you have to bring in more fresh air, remove more of the ammonia-laden air. As a result, you have to over ventilate the house,” Li said.

“That means you have to burn more fuel to keep the house warm. By using the acid-based litter amendments, we can reduce the ventilation rate and the fuel use, which reduces the carbon dioxide emission from the house through the heating process. Basically, if we reduce the microbial activity and also reduce the heating, we can generate lower carbon dioxide emissions.”

http://www.sciencedaily.com/  Science Daily

http://www.sciencedaily.com/releases/2016/01/160112125514.htm  Original web page at Science Daily

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Devising an inexpensive, quick tuberculosis test for developing areas

Tuberculosis (TB) is a highly infectious disease and a major global health problem, especially in countries with developing health care systems. Because there is no fast, easy way to detect TB, the deadly infection can spread quickly through communities. Now, a team reports in ACS Sensors the development of a rapid, sensitive and low-cost method for detecting the disease in resource-limited areas.

The typical way that physicians screen for TB, which is caused by the bacterium Mycobacterium tuberculosis (Mtb), is with a tuberculin skin test or an examination of a patient’s sputum under a microscope. To weed out false positives, a more reliable test that involves growing Mtb cultures can be performed, but that requires weeks to complete. For all of these methods, experienced personnel are needed. Another approach that is both quick and accurate is a nucleic acid amplification test, which makes many copies of the Mtb DNA in a sample. However, it is expensive and requires a lab setting. So, Matt Trau, Nicholas P. West and colleagues set out to create a simple, inexpensive and reliable way to quickly test for TB.

The researchers began with a newly created nucleic acid amplification test that does not require expensive lab equipment to detect Mtb. Still, this modified test typically uses costly fluorescence technology to read the results. So the team substituted the fluorescence detector with a colorimetric assay that changes to a blue hue if the infection is present, allowing health care workers to identify positive test results right away with the naked eye. They demonstrated how the modified diagnostic could be put on cheap, disposable electrochemical sensors for increased sensitivity, even in the field. Because the assay is inexpensive, quick and highly specific for the Mtb bacterium, the researchers say it could have a big impact in low-resource communities.

http://www.sciencedaily.com/  Science Daily

http://www.sciencedaily.com/releases/2015/12/151216135828.htm  Original web page at Science Daily

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New class of DNA repair enzyme discovered

This year’s Nobel Prize in chemistry was given to three scientists who each focused on one piece of the DNA repair puzzle. Now a new study, reported online Oct. 28 in the journal Nature, reports the discovery of a new class of DNA repair enzyme.

When the structure of DNA was first discovered, scientists imagined it to be extremely chemically stable, which allowed it to act as a blueprint for passing the basic traits of parents along to their offspring. Although this view has remained prevalent among the public, biologists have since learned that the double helix is in fact a highly reactive molecule that is constantly being damaged and that cells must make unceasing repair efforts to protect the genetic information that it contains.

“It’s a double-edged sword,” said Brandt Eichman, associate professor of biological sciences and biochemistry at Vanderbilt University, who headed the research team that made the new discovery. “If DNA were too reactive then it wouldn’t be capable of storing genetic information. But, if it were too stable, then it wouldn’t allow organisms to evolve.”

The DNA double-helix has a spiral staircase structure with the outer edges made from sugar and phosphate molecules joined by stair steps composed of pairs of four nucleotide bases (adenine, cytosine, guanine and thymine) that serve as the basic letters in the genetic code.

There are two basic sources of DNA damage or lesions: environmental sources including ultraviolet light, toxic chemicals and ionizing radiation and internal sources, including a number of the cell’s own metabolites (the chemicals it produces during normal metabolism), reactive oxygen species and even water.

“More than 10,000 DNA damage events occur each day in every cell in the human body that must be repaired for DNA to function properly,” said first author Elwood Mullins, a postdoctoral research associate in the Eichman lab.

The newly discovered DNA repair enzyme is a DNA glycosylase, a family of enzymes discovered by Tomas Lindahl, who received this year’s Nobel prize for recognizing that these enzymes removed damaged DNA bases through a process called base-excision repair. It was the first of about 10 different DNA repair pathways that biologists have identified to date.

In base-excision repair, a specific glycosylase molecule binds to DNA at the location of a lesion and bends the double-helix in a way that causes the damaged base to flip from the inside of the helix to the outside. The enzyme fits around the flipped out base and holds it in a position that exposes its link to the DNA’s sugar backbone, allowing the enzyme to detach it. After the damaged base has been removed, additional DNA-repair proteins move in to replace it with a pristine base.

Eichman and his collaborators discovered that a glycosylase called AlkD found in Bacillus cereus — a soil-dwelling bacterium responsible for a type of food poisoning called the “fried rice syndrome” — works in a totally different fashion. It does not require base flipping to recognize damaged DNA or repair it.

Seven years ago, Eichman’s group discovered that AlkD had a structure unlike any of the other glycosylases. The researchers determined that the enzyme was able to locate damaged DNA that has a positive electrical charge. This is the signature of alkylation, attaching chains of carbon and hydrogen atoms of varying lengths (methyl, ethyl etc.), to specific positions on the damaged base. Positively charged alkylated bases are among the most abundant and detrimental forms of DNA damage. However, they are highly unstable, which has made them very difficult to study.

Now the researchers have captured crystallographic snapshots of AlkD in the act of excising alkylation damage and have shown that the enzyme doesn’t use base flipping. Instead, they have determined that AlkD forms a series of interactions with the DNA backbone at and around the lesion while the lesion is still stacked in the double helix. Several of these interactions are contributed by three amino acids in the enzyme that catalyze excision of the damaged base.

According to the researchers, the AlkD mechanism has some remarkable properties:

  • It can recognize damaged bases indirectly. AlkD identifies lesions by interacting with the DNA backbone without contacting the damaged base itself.
  • It can repair many different types of lesions as long as they are positively charged. By contrast, the base-flipping mechanism used by other glycosylases relies on a relatively tight binding pocket in the enzyme, so each glycosylase is designed to work with a limited number of lesions. AlkD doesn’t have the same type of pocket so it isn’t restricted in the same way. Instead, the catalytic mechanism that AlkD uses is limited to removing positively charged lesions.
  • It can excise much bulkier lesions than other glycosylases. Base excision repair is generally limited to relatively small lesions. A different pathway, called nucleotide excision repair, handles larger lesions like those caused by UV radiation damage. However, Eichman’s team discovered that AlkD could excise extremely bulky lesions, such as the one caused by the antibiotic yatakemycin, which is beyond the capability of other glycosylases.

“Our discovery shows that we still have a lot to learn about DNA repair, and that there may be alternative repair pathways yet to be discovered. It certainly shows us that a much broader range of DNA damage can be removed in ways that we didn’t think were possible,” said Eichman. “Bacteria are using this to their advantage to protect themselves against the antibacterial agents they produce. Humans may even have DNA-repair enzymes that operate in a similar fashion to remove complex types of DNA damage. This could have clinical relevance because these enzymes, if they exist, could be reducing the effectiveness of drugs designed to kill cancer cells by shutting down their ability to replicate.”

http://www.sciencedaily.com/   Science Daily

http://www.sciencedaily.com/releases/2015/10/151029190840.htm  Original web page at Science Daily

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* Imitating viruses to deliver drugs to cells

Viruses are able to redirect the functioning of cells in order to infect them. Inspired by their mode of action, scientists from the CNRS and Université de Strasbourg have designed a “chemical virus” that can cross the double lipid layer that surrounds cells, and then disintegrate in the intracellular medium in order to release active compounds. To achieve this, the team used two polymers they had designed, which notably can self-assemble or dissociate, depending on the conditions. This work, the result of collaborative efforts by chemists, biologists and biophysicists, is published in the 1st September issue of Angewandte Chemie International Edition.

Biotechnological advances have offered access to a wealth of compounds with therapeutic potential. Many of these compounds are only active inside human cells but remain unusable because the lipid membrane surrounding these cells is a barrier they cannot cross. The challenge is therefore to find transfer solutions that can cross this barrier.

By imitating the ability of viruses to penetrate into cells, chemists in the Laboratoire de Conception et Application de Molécules Bioactives (CNRS/Université de Strasbourg) sought to design particles capable of releasing macromolecules that are only active inside cells. To achieve this, these particles must comply with several, often contradictory, constraints. They must remain stable in the extracellular medium, they must be able to bind to the cells so that they be internalized, but they must be more fragile inside the cells so that they can release their content. Using two polymers designed by the team, the scientists succeeded in creating a “chemical virus” that meets the conditions necessary for the direct delivery of active proteins into cells.

In practice, the first polymer (pGi-Ni2+) serves as a substrate for the proteins that bind to it. The second, recently patented polymer (πPEI), encapsulates this assembly thanks to its positive charges, which bind to the negative charges of pGi-Ni2+. The particles obtained (30-40 nanometers in diameter) are able to recognize the cell membrane and bind to it. This binding activates a cellular response: the nanoparticle is surrounded by a membrane fragment and enters the intracellular compartment, called the endosome. Although they remain stable outside the cell, the assemblies are attacked by the acidity that prevails within this new environment. Furthermore, this drop in pH allows the πPEI to burst the endosome, releasing its content of active compounds.

Thanks to this assembly, the scientists were able to concentrate enough active proteins within the cells to achieve a notable biological effect. Thus by delivering a protein called caspase 3 into cancer cell lines, they succeeded in inducing 80% cell death.

The in vitro results are encouraging, particularly since this “chemical virus” only becomes toxic at a dose ten times higher than that used during the study. Furthermore, preliminary results in the mouse have not revealed any excess mortality. However, elimination by the body of the two polymers remains an open question. The next stage will consist in testing this method in-depth and in vivo, in animals. In the short term, this system will serve as a research tool to vectorize recombinant and/or chemically modified proteins into cells. In the longer term, this work could make it possible to apply pharmaceutical proteins to intracellular targets and contribute to the development of innovative drugs.

To achieve maximum efficacy, the scientists are hoping to combine siRNA (small nucleic acids that specifically target the expression of certain genes) with the proteins, and these could also be delivered using the same particles. Vectorization consists in controlling the distribution of an active substance to a given target by combining it with a vector. This enables delivery of a protein inside a cell via the intermediary of a biocompatible vehicle.

http://www.sciencedaily.com/  Science Daily

http://www.sciencedaily.com/releases/2015/08/150828135244.htm  Original web page at Science Daily

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Activated neurons produce protective protein against neurodegenerative conditions

Activated neurons produce a protein that protects against nerve cell death. Prof. Dr. Hilmar. When nerve cells die, e.g. as a result of a stroke, Alzheimer’s disease or through age-related processes, the result may be considerable impairments of memory. Earlier studies led by Prof. Bading have shown that brain activity counteracts the death of nerve cells. The NMDA receptor plays a major role at the molecular level. This type of receptor is a molecule set in motion by biochemical neurotransmitters. Due to neuronal activity, the NMDA receptor causes calcium to enter the cell. The calcium signal is spreading within the cell, invades the cell nucleus and switches on a genetic protection programme. Prof. Bading’s group identified this nuclear calcium-induced gene programme a few years ago. “However, it was not clear to us how it leads to a protective shield,” Hilmar Bading explains

The scientists have now discovered the explanation for this — again by studying NMDA receptors. If these receptors are not located at the neuronal junctions, i.e. the synapses, they do not contribute to the protection of cells. On the contrary, they severely damage nerve cells and cause them to die. “Life and death are only a few thousandths of a millimetre away from one another. Outside the synapse the NMDA receptor is no longer Dr. Jekyll, it becomes Mr. Hyde,” Hilmar Bading comments. The current research results show that toxic extrasynaptic NMDA receptors are suppressed through brain activity. The Heidelberg research team has identified activin A as the protein activating this process.

Activin A plays an important role e.g. in the menstruation cycle and in healing wounds. It is produced in the nervous system thanks to neuronal activity. This leads to a reduction in extrasynaptic NMDA receptors and builds up a protective shield, according to Prof. Bading. Activin A also mediates the well-known protective properties of brain-derived neurotrophic factor (BDNF), a signalling molecule that protects existing neurons and synapses, and helps developing new ones. “Activin A can therefore be regarded as a crucial activator of a common neuroprotective mechanism in the brain.”

The discoveries made by the Heidelberg neurobiologists open up new prospects for treating degenerative diseases of the nervous system. In their study they showed that activin A in mice was able to significantly reduce brain damage after a stroke. “Our research results also indicate that activin A may possibly be used to treat Alzheimer’s disease or Huntington’s disease. The characteristic degeneration of nerve cells associated with these two diseases seems to be due to an increased activity of toxic extrasynaptic NMDA receptors,” says Prof. Bading. “In everyday terms the new findings mean: An active brain produces activin A thereby protecting itself from neurodegeneration.”

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http://www.sciencedaily.com/releases/2015/08/150824065021.htm  Original web page at Science Daily

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Molecular inhibitor breaks cycle that leads to Alzheimer’s

A molecular chaperone has been found to inhibit a key stage in the development of Alzheimer’s disease and break the toxic chain reaction that leads to the death of brain cells, a new study shows. The research provides an effective basis for searching for candidate molecules that could be used to treat the condition. It may not actually be too difficult to find other molecules that do this, it’s just that it hasn’t been clear what to look for until recently. A good tactic now is to search for other molecules that have this same highly targeted effect and to see if these can be used as the starting point for developing a future therapy.

A molecule that can block the progress of Alzheimer’s disease at a crucial stage in its development has been identified by researchers in a new study, raising the prospect that more such molecules may now be found. The report shows that a molecular chaperone, a type of molecule that occurs naturally in humans, can play the role of an “inhibitor” part-way through the molecular process that is thought to cause Alzheimer’s, breaking the cycle of events that scientists believe leads to the disease. Specifically, the molecule, called Brichos, sticks to threads made up of malfunctioning proteins, called amyloid fibrils, which are the hallmark of the disease. By doing so, it stops these threads from coming into contact with other proteins, thereby helping to avoid the formation of highly toxic clusters that enable the condition to proliferate in the brain. This step — where fibrils made up of malfunctioning proteins assist in the formation of toxic clusters — is considered to be one of the most critical stages in the development of Alzheimer’s in sufferers. By finding a molecule that prevents it from occurring, scientists have moved closer to identifying a substance that could eventually be used to treat the disease. The discovery was made possible by an overall strategy that could now be applied to find other molecules with similar capabilities, extending the range of options for future drug development.

The research was carried out by an international team comprising academics from the Department of Chemistry at the University of Cambridge, the Karolinska Institute in Stockholm, Lund University, the Swedish University of Agricultural Sciences, and Tallinn University. Their findings are reported in the journal Nature Structural & Molecular Biology. Dr Samuel Cohen, a Research Fellow at St John’s College, Cambridge, and lead author of the report, said: “A great deal of work in this field has gone into understanding which microscopic processes are important in the development of Alzheimer’s disease; now we are starting to reap the rewards of this hard work. Our study shows, for the first time, one of these critical processes being specifically inhibited, and reveals that by doing so we can prevent the toxic effects of protein aggregation that are associated with this terrible condition.”

Alzheimer’s disease is one of a number of conditions caused by naturally occurring protein molecules folding into the wrong shape and then sticking together — or nucleating — with other proteins to create thin filamentous structures called amyloid fibrils. Proteins perform important functions in the body by folding into a particular shape, but sometimes they can misfold, potentially kick-starting this deadly process. Recent research, much of it by the academics behind the latest study, has however suggested a second critical step in the disease’s development. After amyloid fibrils first form from misfolded proteins, they help other proteins which come into contact with them to misfold and form small clusters, called oligomers. These oligomers are highly toxic to nerve cells and are now thought to be responsible for the devastating effects of Alzheimer’s disease. This second stage, known as secondary nucleation, sets off a chain reaction which creates many more toxic oligomers, and ultimately amyloid fibrils, generating the toxic effects that eventually manifest themselves as Alzheimer’s. Without the secondary nucleation process, single molecules would have to misfold and form toxic clusters unaided, which is a much slower and far less devastating process. By studying the molecular processes by which each of these steps takes effect, the research team assembled a wealth of data that enabled them to model not only what happens during the progression of Alzheimer’s disease, but also what might happen if one stage in the process was somehow switched off. “We had reached a stage where we knew what the data should look like if we inhibited any given step in the process, including secondary nucleation,” Cohen said. “Working closely with our collaborators in Sweden — who had developed groundbreaking experimental methods to monitor the process — we were able to identify a molecule that produced exactly the results that we were hoping to see in experiments.”

The results indicated that the molecule, Brichos, effectively inhibits secondary nucleation. Typically, Brichos functions as a “molecular chaperone” in humans; a term given to “housekeeping” molecules that help proteins to avoid misfolding and aggregation. Lab tests, however, revealed that when this molecular chaperone encounters an amyloid fibril, it binds itself to catalytic sites on its surface. This essentially forms a coating that prevents the fibrils from assisting other proteins in misfolding and nucleating into toxic oligomers. The research team then carried out further tests in which living mouse brain tissue was exposed to amyloid-beta, the specific protein that forms the amyloid fibrils in Alzheimer’s disease. Allowing the amyloid-beta to misfold and form amyloids increased toxicity in the tissue significantly. When this happened in the presence of the molecular chaperone, however, amyloid fibrils still formed but the toxicity did not develop in the brain tissue, confirming that the molecule had suppressed the chain reaction from secondary nucleation that feeds the catastrophic production of oligomers leading to Alzheimer’s disease. By modelling what might happen if secondary nucleation is switched off and then finding a molecule that performs that function, the research team suggests that they have discovered a strategy that may lead to the identification of other molecules that could have a similar effect. “It may not actually be too difficult to find other molecules that do this, it’s just that it hasn’t been clear what to look for until recently,” Cohen said. “It’s striking that nature — through molecular chaperones — has evolved a similar approach to our own by focusing on very specifically inhibiting the key steps leading to Alzheimer’s. A good tactic now is to search for other molecules that have this same highly targeted effect and to see if these can be used as the starting point for developing a future therapy.

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http://www.sciencedaily.com/releases/2015/02/150217114547.htm Original web page at Science Daily

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* Nasal test developed for to diagnose Creutzfeldt-Jakob disease

A nasal brush test can rapidly and accurately diagnose Creutzfeldt-Jakob disease (CJD), an incurable and ultimately fatal neurodegenerative disorder, according to a study by National Institutes of Health (NIH) scientists and their Italian colleagues. Up to now, a definitive CJD diagnosis requires testing brain tissue obtained after death or by biopsy in living patients. The study describing the less invasive nasal test appears in the Aug. 7 issue of the New England Journal of Medicine. CJD is a prion disease. These diseases originate when, for reasons not fully understood, normally harmless prion protein molecules become abnormal and gather in clusters. Prion diseases affect animals and people. Human prion diseases include variant, familial and sporadic CJD. The most common form, sporadic CJD, affects an estimated 1 in one million people annually worldwide. Other prion diseases include scrapie in sheep; chronic wasting disease in deer, elk and moose; and bovine spongiform encephalopathy (BSE), or mad cow disease, in cattle. Scientists have associated the accumulation of these clusters with tissue damage that leaves sponge-like holes in the brain. “This exciting advance, the culmination of decades of studies on prion diseases, markedly improves on available diagnostic tests for CJD that are less reliable, more difficult for patients to tolerate, and require more time to obtain results,” said Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases (NIAID), a component of NIH. “With additional validation, this test has potential for use in clinical and agricultural settings.” An easy-to-use diagnostic test would let doctors clearly differentiate prion diseases from other brain diseases, according to Byron Caughey, Ph.D., the lead NIAID scientist involved in the study. Although specific CJD treatments are not available, prospects for their development and effectiveness could be enhanced by early and accurate diagnoses. Further, a test that identifies people with various forms of prion diseases could help to prevent the spread of prion diseases among and between species. For instance, it is known that human prion diseases can be transmitted via medical procedures such as blood transfusions, transplants and the contamination of surgical instruments. People also have contracted variant CJD after exposure to BSE-infected cattle.

The NIAID study involved 31 nasal samples from patients with CJD and 43 nasal samples from patients who had other neurologic diseases or no neurologic disease at all. These samples were collected primarily by Gianluigi Zanusso, M.D., Ph.D., and colleagues at the University of Verona in Italy, who developed the technique of brushing the inside of the nose to collect olfactory neurons connected to the brain. Testing in Dr. Caughey’s lab in Montana then correctly identified 30 of the 31 CJD patients (97 percent sensitivity) and correctly showed negative results for all 43 of the non-CJD patients (100 percent specificity). By comparison, tests using cerebral spinal fluid — currently used to detect sporadic CJD — were 77 percent sensitive and 100 percent specific, and the results took twice as long to obtain.

http://www.sciencedaily.com/  Science Daily

http://www.sciencedaily.com/releases/2014/08/14080710554.htm  Original web page at Science Daily

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Potential ‘universal’ blood test for cancer discovered

Researchers from the University of Bradford have devised a simple blood test that can be used to diagnose whether people have cancer or not. The test will enable doctors to rule out cancer in patients presenting with certain symptoms, saving time and preventing costly and unnecessary invasive procedures such as colonoscopies and biopsies being carried out. Alternatively, it could be a useful aid for investigating patients who are suspected of having a cancer that is currently hard to diagnose. Early results have shown the method gives a high degree of accuracy diagnosing cancer and pre-cancerous conditions from the blood of patients with melanoma, colon cancer and lung cancer. The research is published online in FASEB Journal, the US journal of the Federation of American Societies for Experimental Biology. The Lymphocyte Genome Sensitivity (LGS) test looks at white blood cells and measures the damage caused to their DNA when subjected to different intensities of ultraviolet light (UVA), which is known to damage DNA. The results of the empirical study show a clear distinction between the damage to the white blood cells from patients with cancer, with pre-cancerous conditions and from healthy patients. Professor Diana Anderson, from the University’s School of Life Sciences led the research. She said: “White blood cells are part of the body’s natural defence system. We know that they are under stress when they are fighting cancer or other diseases, so I wondered whether anything measureable could be seen if we put them under further stress with UVA light.We found that people with cancer have DNA which is more easily damaged by ultraviolet light than other people, so the test shows the sensitivity to damage of all the DNA — the genome — in a cell.” The study looked at blood samples taken from 208 individuals. Ninety-four healthy individuals were recruited from staff and students at the University of Bradford and 114 blood samples were collected from patients referred to specialist clinics within Bradford Royal Infirmary prior to diagnosis and treatment. The samples were coded, anonymised, randomised and then exposed to UVA light through five different depths of agar. The UVA damage was observed in the form of pieces of DNA being pulled in an electric field towards the positive end of the field, causing a comet-like tail. In the LGS test, the longer the tail the more DNA damage, and the measurements correlated to those patients who were ultimately diagnosed with cancer (58), those with pre-cancerous conditions (56) and those who were healthy (94).

“These are early results completed on three different types of cancer and we accept that more research needs to be done; but these results so far are remarkable,” said Professor Anderson. “Whilst the numbers of people we tested are, in epidemiological terms, quite small, in molecular epidemiological terms, the results are powerful. We’ve identified significant differences between the healthy volunteers, suspected cancer patients and confirmed cancer patients of mixed ages at a statistically significant level of P<0.001. This means that the possibility of these results happening by chance is 1 in 1000. We believe that this confirms the test’s potential as a diagnostic tool.” Professor Anderson believes that if the LGS proves to be a useful cancer diagnostic test, it would be a highly valuable addition to the more traditional investigative procedures for detecting cancer. A clinical trial is currently underway at Bradford Royal Infirmary. This will investigate the effectiveness of the LGS test in correctly predicting which patients referred by their GPs with suspected colorectal cancer would, or would not, benefit from a colonoscopy — currently the preferred investigation method.

http://www.sciencedaily.com/  Science Daily

http://www.sciencedaily.com/releases/2014/07/140728094410.htm  Original web page at Science Daily

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* Scientists reproduce evolutionary changes by manipulating embryonic development of mice

A group of researchers from the University of Helsinki and the Universitat Autònoma de Barcelona have been able to experimentally reproduce morphological changes in mice which took millions of years to occur. in nature Through small and gradual modifications in the embryonic development of mice teeth, induced in the laboratory, scientists have obtained teeth which morphologically are very similar to those observed in the fossil registry of rodent species which separated from mice millions of years ago. To modify the development of their teeth, the team from the Institute of Biotechnology of the University of Helsinki worked with embryonic teeth cultures from mice not coded by the ectodysplasin A (EDA) protein, which regulates the formation of structures and differentiation of organs in the embryo throughout its development. The teeth obtained with these cultures which present this mutation develop into very basic forms, with very uniform crowns. Scientists gradually added different amounts of the EDA protein to the embryonic cells and let them develop. The researchers observed that the teeth formed with different degrees of complexity in their crown. The more primitive changes observed coincide with those which took place in animals of the Triassic period, some two hundred million years ago. The development of more posterior patterns coincides with the different stages of evolution found in rodents which became extinct already in the Palaeocene Epoch, some 60 million years ago. Researchers have thus achieved experimentally to reproduce the transitions observed in the fossil registry of mammal teeth. The team of scientists were able to contrast the shape of these teeth with a computer-generated prediction model created by Isaac Salazar-Ciudad, researcher at the UAB and at the University of Helsinki, which reproduces how the tooth changes from a group of equal cells to a complex three-dimensional structure, with the full shape of a molar tooth, calculating the position of space of each cell. The model is capable of predicting the changes in the morphology of the tooth when a gene is modified, and therefore offers an explanation of the mechanisms that cause these specific changes to occur in the shape of teeth throughout evolution. The research appears in Nature.

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http://www.sciencedaily.com/releases/2014/07/140730133255.htm  Original web page at Science Daily

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Functioning of aged brains and muscles in mice made younger: More progress with GDF 11, anti-aging protein

In two separate papers given early online release today by the journal Science — which is publishing the papers this coming Friday, Professors Amy Wagers and Lee Rubin, of Harvard’s Department of Stem Cell and Regenerative Biology (HSCRB), report that injections of a protein known as GDF11, which is found in humans as well as mice, improved the exercise capability of mice equivalent in age to that of about a 70-year-old human, and also improved the function of the olfactory region of the brains of the older mice — they could detect smell as younger mice do. Rubin and Wagers each said that, baring unexpected developments, they expect to have GDF11 in initial human clinical trials within three to five years. Postdoctoral fellow Lida Katsimpardi is the lead author on the Rubin group’s paper, and postdocs Manisha Sinha and Young Jang are the lead authors on the paper from the Wagers group. Both studies examined the effect of GDF11 in two ways. First, by using what is called a parabiotic system, in which two mice are surgically joined and the blood of the younger mouse circulates through the older mouse. And second, by injecting the older mice with GDF11, which in an earlier study by Wagers and Richard Lee, of Brigham and Women’s Hospital who is also an author on the two papers released today, was shown to be sufficient to reverse characteristics of aging in the heart. Doug Melton, co-chair of HSCRB and co-director of HSCI, reacted to the two papers by saying that he couldn’t “recall a more exciting finding to come from stem cell science and clever experiments. This should give us all hope for a healthier future. We all wonder why we were stronger and mentally more agile when young, and these two unusually exciting papers actually point to a possible answer: the higher levels of the protein GDF11 we have when young. There seems to be little question that, at least in animals, GDF11 has an amazing capacity to restore aging muscle and brain function,” he said. Melton, Harvard’s Xander University Professor continued, saying that the ongoing collaboration between Wagers, a stem cell biologist whose focus has been on muscle, Rubin, whose focus is on neurodegenerative diseases and using patient generated stem cells as targets for drug discover, and Lee, a practicing cardiologist and researcher, “is a perfect example of the power of the Harvard Stem Cell Institute as an engine of truly collaborative efforts and discovery, bringing together people with big, unique ideas and expertise in different biological areas.”

As Melton noted, GDF11 is naturally found in much higher concentration in young mice than in older mice, and raising its levels in the older mice has improved the function of every organ system thus far studied. Wagers first began using the parabiotic system in mice 14 years ago as a post doctoral fellow at Stanford University, when she and colleagues Thomas Rando, of Stanford, Irina Conboy, of UC Berkley, and Irving Weissman, of Stanford, observed that the blood of young mice circulating in old mice seemed to have some rejuvenating effects on muscle repair after injury. Last year she and Richard Lee published a paper in which they reported that when exposed to the blood of young mice, the enlarged, weakened hearts of older mice returned to a more youthful size, and their function improved. And then working with a Colorado firm, the pair reported that GDF11 was the factor in the blood apparently responsible for the rejuvenating effect. That finding has raised hopes that GDF11 may prove, in some form, to be a possible treatment for diastolic heart failure, a fatal condition in the elderly that now is irreversible, and fatal. “From the previous work it could have seemed that GD11 was heart specific,” said Wagers, “but this shows that it is active in multiple organs and cell types… Prior studies of skeletal muscle and the parabiotic effect really focused on regenerative biology. Muscle was damaged and assayed on how well it could recover,” Wagers explained. She continued: “The additional piece is that while prior studies of young blood factors have shown that we achieve restoration of muscle stem cell function and they repair the muscle better, in this study, we also saw repair of DNA damage associated with aging, and we got it in association with recovery of function, and we saw improvements in unmanipulated muscle. Based on other studies, we think that the accumulation DNA damage in muscle stem cells might be reflect an inability of the cells to properly differentiate to make mature muscle cells, which is needed for adequate muscle repair.

Read more: http://www.sciencedaily.com/releases/2014/05/140504133205.htm

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May 27, 2014

http://www.sciencedaily.com/releases/2014/05/140504133205.htm Original web page at Science Daily

 

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* Cutting cancer to pieces: New research on bleomycin

A variety of cancers are treated with the anti-tumor agent bleomycin, though its disease-fighting properties remain poorly understood. In a new study, lead author Basab Roy — a researcher at Arizona State University’s Biodesign Institute — describes bleomycin’s ability to cut through double-stranded DNA in cancerous cells, like a pair of scissors. Such DNA cleavage often leads to cell death in particular types of cancer cells. The paper is co-authored by professor Sidney Hecht, director of Biodesign’s Center for BioEnergetics. The study presents, for the first time, alternative biochemical mechanisms for DNA cleavage by bleomycin. The new research will help inform efforts to fine-tune the drug, improving its cancer-killing properties, while limiting toxicity to healthy cells. Results of the study recently appeared in the Journal of the American Chemical Society. Bleomycin is part of a family of structurally related antibiotics produced by the bacterium, Streptomyces verticillus. Three potent versions of the drug, labeled A2 , A5 and B2 are the primary forms in clinical use against cancer. Bleomycin’s cancer-fighting capacity was first observed in 1966 by Japanese researcher Hamao Umezawa. The drug gained FDA approval in 1973 and has been in use since then, particularly for the treatment of Hodgkin’s lymphoma, squamous cell carcinomas, and testicular cancer. One of the attractive properties of bleomycin is the fact that it can be administered in fairly low doses, relative to many other cancer therapies. Previous research has shown that bleomycin can cause death in aberrant cells by migrating to the cell nucleus, binding with DNA and subsequently causing breaks in the DNA sequence. Following a binding event, a molecule of bleomycin can effectively slice through one or both strands of DNA.

Cleavage of DNA is believed to be the primary mechanism by which bleomycin kills cancer cells, particularly through double-strand cleavages, which are more challenging for the cellular machinery to repair. “There are several mechanisms for repairing both single-strand and double-strand breaks in DNA, but double-strand breaks are a more potent form of DNA lesion,” Roy says. The Center for BioEnergetics has been studying several forms of bleomycin, developing a sizeable library of variants, with the goal of engineering the best bleomycin analog. Roy is particularly interested in the subtle biochemistry of bleomycin, including the specificity of its binding regions along the DNA strand and the drug’s detailed mechanisms of DNA cleavage. For the new study, bleomycin A5 was used. Bleomycin A5 has similar DNA binding and cleaving properties as bleomycin A2 and B2. Previous research has revealed that bleomycin binds with highly specific regions of the DNA strand, typically G-C sites, where a guanosine base pairs with a cytidine. Further, the strength of this binding is closely associated with the degree of double-strand DNA cleavage. From a pool of random DNA sequences, a library of 10 hairpin DNAs was selected, based on their strong binding affinity for bleomycin A5. Hairpin DNAs are looped structures, which form when a segment of a DNA strand base-pairs with another portion of the same strand. These hairpin DNAs were used to investigate double-strand cleavage by bleomycin. Each of the 10 DNA samples underwent double-strand cleavage at more than one site. Further, all of the observed cleavage sites were found within or in close proximity to an 8 base pair variable region, which had been randomized to create the original library. Examination of the 10 DNA samples exposed to bleomycin revealed a total of 31 double-strand cleavage sites. Earlier research had described the form of double-strand DNA cleavage by bleomycin which occurred at 14 of these sites; however the remaining 17 cases of double-stranded cleavage occurred through a different mechanism, described for the first time in the present study. As in earlier studies, iron (FeII) was used as a cofactor for bleomycin in the binding events. Two types of bleomycin binding and cleavage activity are detailed in the paper. In the first, bleomycin and its iron cofactor (Fe.BLM) bind with hairpin DNA at a primary site. Typically, this is a site with a particular sequence: 5´-G-Py-B-3´. (Here, 5´ refers to one end of the DNA hairpin, G refers to the base guanosine, Py refers to a pyrimidinic base — either cytidine or thymidine, B refers to any nucleobase and 3´ refers to the other DNA end. The result of this binding is the abstraction of a hydrogen atom at the primary site. Two results are possible following the primary binding event, one causing a single-strand break in the primary site, the other, failing to produce full cleavage of the strand, producing instead a site lacking either a purine or pyrimidine base. This is known as an AP site. In the first case — where bleomycin achieves single strand cleavage — the bleomycin molecule can then become reactivated, once more abstracting a hydrogen atom from the opposing DNA strand. The opposite stand can again follow one of two pathways, a) full cleavage of the opposing strand, yielding a double strand cleavage or b) formation of an AP site. The authors note that this AP site can lead to strand cleavage through the opposing DNA strand with the addition of a mild base like n-butylamine.

Results of the study emphasized the correlation between the strength of bleomycin binding to DNA and the frequency of double strand cleavage. Of the 10 sample hairpin DNAs, the two most tightly bound to bleomycin each showed 5 double strand cleavages whereas the least tightly bound samples exhibited just two double strand cleavages. This important study proposes, for the first time, a plausible mechanism for DNA cleavage by bleomycin that may lead to tumor cell killing as well as identifying the most common sequences involved in DNA site binding and subsequent strand breakage. Roy stresses that a great deal of work remains, to elucidate the biochemical causes of tight binding by bleomycin. Further, bleomycin’s specificity for cancer cells remains enigmatic. New work in the Hecht lab however has identified the carbohydrate moiety of the molecule as being responsible for tumor cell targeting. “Cancer is still a black hole,” says Roy. “We’re trying to make this particular molecule (bleomycin) better. There is still so much to learn.”

http://www.sciencedaily.com/  Science Daily

May 27, 2014

http://www.sciencedaily.com/releases/2014/04/140430192749.htm  Original web page at Science Daily

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* What bank voles can teach us about prion disease transmission and neurodegeneration

When cannibals ate brains of people who died from prion disease, many of them fell ill with the fatal neurodegenerative disease as well. Likewise, when cows were fed protein contaminated with bovine prions, many of them developed mad cow disease. On the other hand, transmission of prions between species, for example from cows, sheep, or deer to humans, is — fortunately — inefficient, and only a small proportion of exposed recipients become sick within their lifetimes. A study published on April 3rd in PLOS Pathogens takes a close look at one exception to this rule: bank voles appear to lack a species barrier for prion transmission, and their universal susceptibility turns out to be both informative and useful for the development of strategies to prevent prion transmission. Prions are misfolded, toxic versions of a protein called PrP, which in its normal form is present in all mammalian species that have been examined. Toxic prions are “infectious”; they can induce existing, properly folded PrP proteins to convert into the disease-associated prion form. Prion diseases are rare, but they share features with more common neurodegenerative diseases like Alzheimer’s disease.

Trying to understand the unusual susceptibility of bank voles to prions from other species, Stanley Prusiner, Joel Watts, Kurt Giles and colleagues, from the University of California in San Francisco, USA, first tested whether the susceptibility is an intrinsic property of the voles’ PrP, or whether other factors present in these rodents make them vulnerable. The scientists introduced into mice the gene that codes for the normal bank vole prion protein, thereby generating mice that express bank vole PrP, but not mouse PrP. When these mice get older, some of them spontaneously develop neurologic illness, but in the younger ones the bank vole PrP is in its normal, benign folded state. The scientists then exposed young mice to toxic misfolded prions from 8 different species, including human, cattle, elk, sheep, and hamster. They found that all of these foreign-species prions can cause prion disease in the transgenic mice, and that the disease develops often more rapidly than it does in bank voles. The latter is likely because the transgenic mice express higher levels of bank vole PrP than are naturally present in the voles. The results show that the universal susceptibility of bank voles to cross-species prion transmission is an intrinsic property of bank vole PrP. Because the transgenic mice develop prion disease rapidly, the scientists propose that the mice will be useful tools in studying the processes by which toxic prions “convert” healthy PrP and thereby destroy the brain. And because that process is similar across many neurodegenerative diseases, better understanding prion disease development might have broader implications.

http://www.sciencedaily.com/  Science Daily

April 29, 2014

http://www.sciencedaily.com/releases/2014/04/140403212519.htm  Original web page at Science Daily

 

 

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It slices, it dices, and it protects the body from harm

The discovery of the structure of this enzyme, a first-responder in the body’s “innate immune system,” could enable new strategies for fighting infectious agents and possibly prostate cancer and obesity. The work was published Feb. 27 in the journal Science. Until now, the research community has lacked a structural model of the human form of this enzyme, known as RNase L, said Alexei Korennykh, an assistant professor of molecular biology and leader of the team that made the discovery. “Now that we have the human RNase L structure, we can begin to understand the effects of carcinogenic mutations in the RNase L gene. For example, families with hereditary prostate cancers often carry genetic mutations in the region, or locus, encoding RNase L,” Korennykh said. The connection is so strong that the RNase L locus also goes by the name “hereditary prostate cancer.” The newly found structure reveals the positions of these mutations and explains why some of these mutations could be detrimental, perhaps leading to cancer, Korennykh said. RNase L is also essential for insulin function and has been implicated in obesity. The Princeton team’s work has also led to new insights on the enzyme’s function. The enzyme is an important player in the innate immune system, a rapid and broad response to invaders that includes the production of a molecule called interferon. Interferon relays distress signals from infected cells to neighboring healthy cells, thereby activating RNase L to turn on its ability to slice through RNA, a type of genetic material that is similar to DNA. The result is new cells armed for destruction of the foreign RNA.

The 3D structure uncovered by Korennykh and his team consists of two nearly identical subunits called protomers. The researchers found that one protomer finds and attaches to the RNA, while the other protomer snips it. The initial protomer latches onto one of the four “letters” that make up the RNA code, in particular, the “U,” which stands for a component of RNA called uridine. The other protomer “counts” RNA letters starting from the U, skips exactly one letter, then cuts the RNA. Although the enzyme can slice any RNA, even that of the body’s own cells, it only does so when activated by interferon. “We were surprised to find that the two protomers were identical but have different roles, one binding and one slicing,” Korennykh said. “Enzymes usually have distinct sites that bind the substrate and catalyze reactions. In the case of RNase L, it appears that the same exact protein surface can do both binding and catalysis. One RNase L subunit randomly adopts a binding role, whereas the other identical subunit has no other choice but to do catalysis.” To discover the enzyme’s structure, the researchers first created a crystal of the RNase L enzyme. The main challenge was finding the right combination of chemical treatments that would force the enzyme to crystallize without destroying it.

After much trial and error and with the help of an automated system, postdoctoral research associate Jesse Donovan and graduate student Yuchen Han succeeded in making the crystals. Next, the crystals were bombarded with powerful X-rays, which diffract when they hit the atoms in the crystal and form patterns indicative of the crystal’s structure. The diffraction patterns revealed how the atoms of RNase L are arranged in 3D space. At the same time Sneha Rath, a graduate student in Korennykh’s laboratory, worked on understanding the RNA cleavage mechanism of RNase L using synthetic RNA fragments. Rath’s results matched the structural findings of Han and Donovan, and the two pieces of data ultimately revealed how RNase L cleaves its RNA targets. Han, Donovan and Rath contributed equally to the paper and are listed as co-first authors. Finally, senior research specialist Gena Whitney and graduate student Alisha Chitrakar conducted additional studies of RNase L in human cells, confirming the 3D structure. Now that the human structure has been solved, researchers can explore ways to either enhance or dampen RNase L activity for medical and therapeutic uses, Korennykh said. “This work illustrates the wonderful usefulness of doing both crystallography and careful kinetic and enzymatic studies at the same time,” said Peter Walter, professor of biochemistry and biophysics at the University of California-San Francisco School of Medicine. “Crystallography gives a static picture which becomes vastly enhanced by studies of the kinetics.”

http://www.eurekalert.org/bysubject/medicine.php  Eurek Alert! Medicine

March 18, 2024

http://www.eurekalert.org/pub_releases/2014-02/pu-isi022814.php  Original web page at Eurek Alert! Medicine

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The shape of infectious prions

Prions are unique infective agents — unlike viruses, bacteria, fungi and other parasites, prions do not contain either DNA or RNA. Despite their seemingly simple structure, they can propagate their pathological effects like wildfire, by “infecting” normal proteins. PrPSc (the pathological form of the prion protein) can induce normal prion proteins (PrPC) to acquire the wrong conformation and convert into further disease-causing agents. “When they are healthy, they look like tiny spheres; when they are malignant, they appear as cubes” stated Giuseppe Legname, principal investigator of the Prion Biology Laboratory at the Scuola Internazionale Superiore di Studi Avanzati (SISSA) in Trieste, when describing prion proteins. Prions are “misfolded” proteins that cause a group of incurable neurodegenerative diseases, including spongiform encephalopathies (for example, mad cow diseases) and Creutzfeldt-Jakob disease. Legname and coworkers have recently published a detailed analysis of the early mechanisms of misfolding. Their research has just been published in the Journal of the American Chemical Society, the most authoritative scientific journal in the field.

“For the first time, our experimental study has investigated the structural elements leading to the disease-causing conversion” explains Legname. “With the help of X-rays, we observed some synthetic prion proteins engineered in our lab by applying a new approach — we used nanobodies, i.e. small proteins that act as a scaffolding and induce prions to stabilize their structure.” Legname and colleagues reported that misfolding originates in a specific part of the protein named “N-terminal.” “The prion protein consists of two subunits. The C-terminal has a clearly defined and well-known structure, whereas the unstructured N-terminal is disordered, and still largely unknown. This is the very area where the early prion pathological misfolding occurs” adds Legname. “The looser conformation of the N-terminal likely determines a dynamic structure, which can thus change the protein shape.” “Works like ours are the first, important steps to understand the mechanisms underlying the pathogenic effect of prions” concludes Legname. “Elucidating the misfolding process is essential to the future development of drugs and therapeutic strategies against incurable neurodegenerative diseases.”

http://www.sciencedaily.com/ Science Daily
February 18, 2014

http://www.sciencedaily.com/releases/2014/01/140124082602.htm Original web page at Science Daily

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Symphony of life, revealed: New imaging technique captures vibrations of proteins, tiny motions critical to human life

Like the strings on a violin or the pipes of an organ, the proteins in the human body vibrate in different patterns, scientists have long suspected. Now, a new study provides what researchers say is the first conclusive evidence that this is true. Using a technique they developed based on terahertz near-field microscopy, scientists from the University at Buffalo and Hauptman-Woodward Medical Research Institute (HWI) have for the first time observed in detail the vibrations of lysozyme, an antibacterial protein found in many animals. The team found that the vibrations, which were previously thought to dissipate quickly, actually persist in molecules like the “ringing of a bell,” said UB physics professor Andrea Markelz, PhD, who led the study. These tiny motions enable proteins to change shape quickly so they can readily bind to other proteins, a process that is necessary for the body to perform critical biological functions like absorbing oxygen, repairing cells and replicating DNA, Markelz said. The research opens the door to a whole new way of studying the basic cellular processes that enable life. “People have been trying to measure these vibrations in proteins for many, many years, since the 1960s,” Markelz said. “In the past, to look at these large-scale, correlated motions in proteins was a challenge that required extremely dry and cold environments and expensive facilities.” “Our technique is easier and much faster,” she said. “You don’t need to cool the proteins to below freezing or use a synchrotron light source or a nuclear reactor — all things people have used previously to try and examine these vibrations.” The findings will appear in Nature Communications on Jan. 16.

To observe the protein vibrations, Markelz’ team relied on an interesting characteristic of proteins: The fact that they vibrate at the same frequency as the light they absorb. This is analogous to the way wine glasses tremble and shatter when a singer hits exactly the right note. Markelz explained: Wine glasses vibrate because they are absorbing the energy of sound waves, and the shape of a glass determines what pitches of sound it can absorb. Similarly, proteins with different structures will absorb and vibrate in response to light of different frequencies. So, to study vibrations in lysozyme, Markelz and her colleagues exposed a sample to light of different frequencies and polarizations, and measured the types of light the protein absorbed. This technique, developed with Edward Snell, a senior research scientist at HWI and assistant professor of structural biology at UB, allowed the team to identify which sections of the protein vibrated under normal biological conditions. The researchers were also able to see that the vibrations endured over time, challenging existing assumptions.

“If you tap on a bell, it rings for some time, and with a sound that is specific to the bell. This is how the proteins behave,” Markelz said. “Many scientists have previously thought a protein is more like a wet sponge than a bell: If you tap on a wet sponge, you don’t get any sustained sound.” Markelz said the team’s technique for studying vibrations could be used in the future to document how natural and artificial inhibitors stop proteins from performing vital functions by blocking desired vibrations. “We can now try to understand the actual structural mechanisms behind these biological processes and how they are controlled,” Markelz said. “The cellular system is just amazing,” she said. “You can think of a cell as a little machine that does lots of different things — it senses, it makes more of itself, it reads and replicates DNA, and for all of these things to occur, proteins have to vibrate and interact with one another.”

http://www.sciencedaily.com/  Science Daily February 4, 2014
 
http://www.sciencedaily.com/releases/2014/01/140116084838.htm  Original web page at Science Daily

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Wrong molecular turn leads down path to type 2 diabetes

Computing resources at the U.S. Department of Energy’s (DOE) Argonne National Laboratory have helped researchers better grasp how proteins misfold to create the tissue-damaging structures that lead to type 2 diabetes. The structures, called amyloid fibrils, are also implicated in neurodegenerative conditions such as Alzheimer’s and Parkinson’s, and in prion diseases like Creutzfeldt-Jacob and mad cow disease. The results pinpoint a critical intermediate step in the chemical pathway that leads to amyloid fibril formation. With the new culprit in view, future work could target a possible treatment, such as designing an inhibitor to interfere with the harmful pathway. The results also helped reconcile earlier data from other labs that until now appeared contradictory. An amyloid fibril is a large structure consisting of misfolded proteins. Such fibrils form plaques, or areas of tissue damage, that researchers can observe with microscopes. Fibrils are believed to arise when proteins deviate from their normal 3D structures and instead adopt misfolded states that tend to clump together.

Like puzzle pieces, proteins are only useful when they have the correct shape. And since the fibrils they form when misfolded are strong, scientists believe that hope primarily lies not in dismantling them, but in heading off the folding errors. The researchers used two main approaches to identify the intermediate step and understand the pathway. University of Wisconsin-Madison professor Martin Zanni used a sophisticated technique that relies on 2-D infrared spectroscopy to follow the sequence of events in the chemical reactions leading to fibril formation. His technique can measure extremely fast processes using very small samples. Then Juan de Pablo and Chi-Cheng Chiu from the University of Chicago’s Institute for Molecular Engineering interpreted Zanni’s measurements with data from molecular simulations to arrive at a complete picture of the early events leading to amyloid formation. De Pablo and Chiu used Intrepid, an IBM Blue Gene/P computer system at the Argonne Leadership Computing Facility (ALCF), and resources at the University of Chicago Research Computing Center. De Pablo and Chiu composed, ran and interpreted large-scale computer simulations of the pathway in action, and the results supplied an essential model of the molecular steps involved in the reaction.

“Using only one of the two methods would have been like running a race with only one leg,” de Pablo said. “By combining both computation and experiment, we can get to our answers faster and more dependably.” Together, researchers located an entire step that had been missing, and whose absence had been fueling confusion. An earlier study indicated that the intermediate step was likely a floppy loop area formed by proteins, which didn’t seem compatible with the tough, damaging fibril as an end result. Researchers believed that the fibrils should come from a rigid structure called a β-sheet. The new data show, however, that both structures occur as the reaction changes over time. Transient rigid β-sheets form, then morph into floppy protein loops, which finally take the form of more β-sheets. The final β-sheets bind together and stack up to form the damaging fibrils. The focus now will be to target the new intermediate step. With more data, researchers could design an inhibitor drug to bind to the offending protein, block the molecule and halt the pathway’s progression. Next, de Pablo intends to learn more about the particular protein intermediate that is implicated in type 2 diabetes. He has examined the basic units and small aggregates consisting of two or at most three molecules. “Now we need to understand how these small aggregates disrupt cell membranes,” he said. “We also want to decipher how the fibril grows from a small nucleus.”

To do so, he is pushing forward with plans to investigate bigger systems by using more supercomputing clout. He was recently awarded computing time on Argonne’s IBM Blue Gene/Q, called Mira, the newest resource available to users at the ALCF. Mira is a 10-petaflops computer that packs 10 quadrillion calculations into each second of computing time. De Pablo, Zanni and their collaborators will also apply the method from this publication to determine the intermediate steps in diseases other than type 2 diabetes, including neurodegenerative diseases like Alzheimer’s. Scientists attribute more than 20 human diseases to the formation of amyloid fibrils. In each disease, the misfolding of a specific protein — a different one in every disease — is what triggers the problematic intermediate β-sheet. “We want to understand the broader origins of the misfolding and aggregation problem,” de Pablo said, “which we can do by looking at a wide range of molecules associated with different diseases. The eventual goal is to answer a few vital questions: What are the early stage misfolding events and small aggregates that form? How do they form? And how can we design inhibitors to stop them from forming?”

Science Daily
January 21, 2014

Original web page at Science Daily

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‘Designer sperm’ inserts custom genes into offspring

Get ready: The “new genetics” promises to change faulty genes of future generations by introducing new, functioning genes using “designer sperm.” A new research report appearing online in The FASEB Journal, shows that introducing new genetic material via a viral vector into the sperm of mice leads to the presence and activity of those genes in the resulting embryos. This new genetic material is actually inherited, present and functioning through three generations of the mice tested. This discovery — if successful in humans — could lead to a new frontier in genetic medicine in which diseases and disorders are effectively cured, and new human attributes, such as organ regeneration, may be possible. “Transgenic technology is a most important tool for researching all kinds of disease in humans and animals, and for understanding crucial problems in biology,” said Anil Chandrashekran, Ph.D., study author from the Department of Veterinary Clinical Sciences at The Royal Veterinary College in North Mimms, United Kingdom. To achieve these results, Chandrashekran and colleagues used lentiviruses to generate transgenic animals via the male germ line. When pseudotyped lentiviral vectors encoding green fluorescent protein (GFP) were incubated with mouse spermatozoa, these sperm were highly successful in producing transgenics. Lentivirally-transduced mouse spermatozoa were used in vitro fertilization studies and when followed by embryo transfer, at least 42 percent of founders were transgenic for GFP. GFP expression was detected in a wide range of murine tissues, including testis and the transgene was stably transmitted to a third generation of transgenic animals.

“Using modified sperm to insert genetic material has the potential to be a major breakthrough not only in future research, but also in human medicine,” said Gerald Weissmann, M.D., Editor-in-Chief of The FASEB Journal. “It facilitates the development of transgenic animal models, and may lead to therapeutic benefits for people as well. For years we have chased effective gene therapies and have hit numerous speed bumps and dead ends. If we are able to alter sperm to improve the health of future generations, it would completely change our notions of ‘preventative medicine.'”

Science Daily
January 7, 2014

Original we page at Science Daily

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Molecule controls blood sugar effectively in humans and also promotes weight loss in rodents

A peptide similar to the one pictured was effective at treating type-2 diabetes in a small clinical trial. Joe Chabenne, Faming Zhang, Richard DiMarchi/Indiana University An experimental diabetes treatment that packs the action of two natural hormones into a single injectable agent has been shown to successfully lower blood sugar in humans, monkeys and rodents. Marking a new approach in the treatment of the disease, the currently unnamed molecule also seems likely to cause fewer gastrointestinal side effects in humans than did other diabetes medicines. “We aimed for achieving the best glycaemic control with as little effect on the gut as possible,” says Richard DiMarchi, a biomolecular scientist at Indiana University in Bloomington, and a member of the international team that publishes the results today in Science Translational Medicine. The molecule, which targets receptors for the two hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), was developed in DiMarchi’s lab. Swiss pharmaceutical company Roche, based in Basel, supported the research and has licensed the agent.

As part of the study, 44 patients with type 2 diabetes received once-weekly injection of the dual-action molecule at various doses for six weeks while nine others received placebo injections. Blood tests showed a dose-dependent response; at the highest doses, a standard marker of blood glucose levels dropped an average of 1.1 percentage points from the baseline (which ranged from 7.4% to 7.9%; normal levels are below 5.7% in non-diabetic patients). In the placebo group, the marker dropped by just 0.16 points. There were no significant changes in body weight in the human trial, but the animal studies suggest that a long-term treatment at higher doses could also treat obesity. Obese mice receiving the highest doses of the molecule lost nearly 19% of their body weight in just one week, compared with about 9% for mice treated with equivalent amounts of a commonly prescribed diabetes drug called liraglutide. Both GLP-1 and GIP naturally respond to spikes in blood sugar by stimulating insulin production. GLP-1 also decreases appetite and suppresses glucagon, a hormone that raises blood sugar. Several current diabetes drugs, including exenatide and liraglutide, work by mimicking GLP-1. But as DiMarchi notes, about 10–30% of people taking such drugs develop gastrointestinal distress, including nausea, flatulence and sometimes vomiting. In the latest trial, just two people in the treatment group complained of mild nausea.

DiMarchi and his colleagues have previously shown that combining GLP-1 and oestrogen could reverse risk factors for diabetes in mice. But there have been few attempts to create drugs that harness the power of GIP. The hormone clearly has a natural role in controlling blood sugar, says DiMarchi, so it should not be ignored in drug development. “The best pharmacology replicates physiology,” he says. “This a key that works on both locks.” By combining human trials with thorough rodent and monkey studies in a single paper, the researchers have “delivered a complete package”, says Philipp Scherer, chair of diabetes research at the University of Texas Southwestern Medical Center in Dallas. “Usually, if you just have complete rodent data, you can make quite a splash.” However, the suggestion that the treatment causes relatively few side effects needs to be confirmed in a larger clinical trial, he says. DiMarchi says that the next step will be a long-term study that compares the latest approach to an established GLP-1 mimic. “This is still years away from being an approved drug,” he adds.

Nature
November 12, 2013

Original web page at Nature

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One in 2000 people may carry infectious prions in the UK

The prion protein that causes variant Creutzfeldt–Jakob disease destroys neurons in the brain. As a new study in the British Medical Journal reveals that 1 in 2000 people in the UK may harbour the infectious prion protein which causes variant Creutzfeldt–Jakob disease (vCJD), Nature explains what this means. The usually fatal condition is the human form of bovine spongiform encephalopathy (BSE) — dubbed ‘mad cow disease’ in the UK after an outbreak of the disease in the 1980s. Both diseases are caused by misfolded proteins called prions, which induce other proteins in the brain to clump, eventually destroying neurons. Humans are thought to contract the disease by consuming beef containing infected bovine brain or other central nervous system tissue. But it also spreads through blood transfusions, and some worry that the prion disease is transmitted via contaminated surgical instruments. The BSE outbreak in the 1980s and 1990s led to a surge in British vCJD cases, and a total of 177 have been detected in the UK to date, with just one in the last two years. Cases of vCJD peaked in 2000, leading some scientists to speculate that the disease takes about a decade to develop. Yet other studies of different forms of CJD suggest its incubation time could be much longer, indicating that many people in Britain could be carrying the infection without symptoms.

Studies have come to varying conclusions as to just how many people harbour the abnormal prion protein (PrP) that causes vCJD. Surveys of tens of thousands of appendices and tonsils, discarded after surgery, have shown PrP prevalence rates ranging from 1 in 40,002 to 1 in 10,0003 or even zero4. Because of the uncertainty over just how many people have the abnormal prion, a body that advises the government on the disease called for a comprehensive prevalence survey in 2008. The study was led by Sebastian Bradner, a neuropathologist at University College London, and it tested for Prp in 32,441 appendices from anonymized donors, collected from 41 UK hospitals. The researchers found 16 positive cases, translating to a prevalence rate of about 1 in 2,0001. Not especially, according to Roland Salmon, a retired consultant epidemiologist who wrote an editorial in the British Medical Journal to accompany Bradner’s paper. The number reported by Bradner’s team is of the same magnitude of the prevalence rate found in a previous survey that found 3 positives in 12,674 patients.

Does this mean that 1 in 2000 people in United Kingdom will develop vCJD? This is unlikely. Animal studies have suggested that the lymphoid system, which includes the spleen and tonsils, is more easily infected with PrP than the brain. But the relationship between infection in the lymphoid system and in the brain is not clear. These cases of detected prions could represent silent carriers, who will not go on to develop clinical vCJD.

“We have no idea if none or a proportion of these people will develop clinical disease, that remains to be seen,” says Graham Jackson, at the MRC Prion Unit at University College London. “Whatever the reasons for infected individuals entering a long term asymptomatic state, much depends on whether this could be expected to last indefinitely, or eventually lead to clinical symptoms,” said the UK government’s advisory committee on dangerous pathogens when the raw numbers were released in 2012. The committee’s predecessor, which commissioned the report said, “The precautionary assumption is that further clinical cases may appear after much longer incubation periods than those seen so far, though their number could be significantly reduced by intervening deaths from other causes.” That’s still an open question. The United Kingdom has taken extraordinary measures to protect its blood supply from vCJD, such as banning blood donation from people who received transfusions after 1980. It is not yet clear whether people with lymphoid-system infections can transmit PrP through their blood. Jackson says the latest study underscores the need for better diagnostics to detect prion infection in blood. “Despite the merciful lack of clinical cases it tells us this problem has not gone away, far from it.”

Nature
October 29, 2013

Original web page at Nature

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Molecule that triggers septic shock identified

The body’s immune system is set up much like a home security system; it has sensors on the outside of cells that act like motion detectors — floodlights — that click on when there’s an intruder rustling in the bushes, bacteria that seem suspect. For over a decade researchers have known about one group of external sensors called Toll-like receptors that detect when bacteria are nearby. Now, researchers at the University of North Carolina School of Medicine have identified a sensor pathway inside cells. These internal sensors are like motion detectors inside a house; they trigger an alarm that signals for help — a response from the immune system. This research, published in the Sept. 13, 2013 issue of the journal Science, indicates that both exterior and interior sensors work together to detect the same component of bacterial cell membranes, a molecule called lipopolysaccharide or LPS. By showing how the immune system distinguishes between suspicious activity and real threats, the study could lead to new therapies for septic shock — when the immune system overreacts to a bacterial infection to such an extent that it causes more harm than good.

“During the defense against an infection you want to be able to differentiate between the bacteria that stay on the outside of the cell and the ones that get inside,” said senior study author Edward A. Miao, MD, PhD, assistant professor of microbiology and immunology. “You can think of the exterior sensors as a yellow alert; they tell us that bacteria are present. But these bacteria could either be simple ones in the wrong place, or very dangerous ones that could cause a serious infection. The interior sensors act as a red alert; they warn us that there are bacteria with ill intent that have the genetic capacity to invade and manipulate our cells.” The body responds to a bacterial infection by increasing blood vessel permeability near the area under attack, which allows immune system cells to leave the bloodstream and seek and destroy the bacteria. Fluid also leaks into the area surrounding the infection, causing characteristic swelling. This is beneficial in fighting infection, but when the infection gets out of hand and these immune response occur throughout the body, blood pressure plummets, overtaxing the heart and leading to organ failure and often death. This increasingly prevalent syndrome, known as septic shock, afflicts over 750,000 people each year in the United States at a cost of nearly $17 billion. About half of the cases of septic shock are caused by bacteria that produce LPS, also known as endotoxin. In fact, much of what is known about endotoxic shock comes from studying animals injected with high doses of LPS. For example, previous studies pinpointed the role of the Toll-like receptor 4 gene (TLR4) as a sensor on the outside of cells; mice without that gene resisted endotoxic shock.

In a study published in January 2013, also in the journal Science, Miao and his colleagues showed that a sensor called caspase-11 sounds an alert when bacteria enter a cell. However, it wasn’t clear which of the thousands of molecules that make up a bacterial cell triggers that new sensor. In the current study, Miao and his colleagues investigated which bits of foreign material were being detected. They took apart and delivered different chunks of bacteria into the cytoplasmic compartment inside the cell. To their surprise, they found that the caspase-11 sensor inside the cell was detecting the same molecule, LPS, as the TLR4 sensor outside the cell. The researchers wondered whether there was a link between these two sensors. Through a number of experiments in animal models of sepsis, Miao’s team showed that the exterior and interior alarms work together through a two-step defense mechanism: LPS is first seen on the outside of the cell by TLR4, which sets the interior caspase-11 alarm into a watchful state. At very high doses, the LPS crosses into the cell, tripping the caspase-11 alarm. The end result is the generation of the red alert signal, which causes the cell to explode, a form of cell death called pyroptosis. During an infection, the immune system essentially burns the house down around the invading bacteria, depriving it of a place to replicate, and exposing it to more potent immune defenses. During sepsis, however, too much fire leads to the onset of shock. Miao says that figuring out how these two sensors get activated in response to a bacterial infection could help researchers develop new ways of preventing or treating septic shock, a condition that kills about half its victims. “The septic shock we see in patients is probably a lot more complicated than what we see in this experimental system,” said Miao. “The next question we need to ask is whether these same sensors are going off in people with septic shock, and if so, is there a way to block them so we can keep patients from dying.”

Science Daily
October 1, 2013

Original web page at Science Daily

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Birds appear to lack important anti-inflammatory protein

From bird flu to the West Nile virus, bird diseases can have a vast impact on humans. Thus, understanding bird immune systems can help people in a variety of ways, including protecting ourselves from disease and protecting our interests in birds as food animals. An important element in the immune system of many animals’ immune systems — including mammals, reptiles, amphibians, and most animals with a backbone — is a protein called tristetraprolin, or TTP. TTP plays an anti-inflammatory role, largely through keeping another protein, called tumor necrosis factor alpha (TNF), in check. Studies have shown that mice bred without TTP develop chronic inflammation that affects their entire bodies. Even animals missing TTP in just one immune cell type develop a catastrophic and deadly inflammation when they’re exposed to tiny amounts of a molecule from bacteria, underlying the importance of this protein. And yet, researchers have not been able to find TTP in birds. Could birds really be that different from the vast majority of their closest animal relatives? To answer that question, researcher Perry Blackshear and his colleagues at the National Institute of Environmental Health Sciences conducted a comprehensive examination, using several different methods to look for TTP in birds. Their results suggest that birds are truly an anomaly, having no analog for TTP in their immune systems.

Their article is entitled “Life Without TTP: Apparent Absence of an Important Anti-Inflammatory Protein in Birds.” It appears in the Articles in Press section of the American Journal of Physiology: Regulatory, Integrative, and Comparative Physiology, published by the American Physiological Society. Methodology The researchers performed a comprehensive search to look for TTP in birds using several different approaches, to include databases of bird genomes in order to look for sequences of bird genes that were similar to other animals’ genes for TTP. They also exposed chicken cells to protein from other animals and a molecule from bacteria, both scenarios that stimulate TTP production in other animals, looking directly for TTP production. Finally, they looked at which genes were active when these chicken cells were exposed to the foreign proteins and bacteria molecule to see if any genes were the chicken version of TTP. Results Despite this wide-ranging search, the study authors didn’t find any bird analog to TTP. Though their genomic searches turned up sequences for close relatives in the same protein family as TTP in birds and all the other animals they compared, they couldn’t find any bird version of TTP in bird genetic databases. Tests showed that the chicken cells they studied didn’t produce TTP when exposed to foreign animal proteins or a bacterial molecule. Additionally, these cells had no evidence of active genes during these tests that appeared to be for TTP production.

These findings suggest that, surprisingly, birds are missing this protein that is so pivotal to their closest relatives’ immune systems. To better understand how birds combat disease, the authors say, researchers will need to figure out how birds handle the same immune challenges differently compared to mammals, reptiles, and other animals that make TTP. “From an immunological standpoint,” the authors say, “it will be both interesting and important to determine how birds cope differently with the environmental and microbiological assaults that stimulate the acute innate immune response in mammals. This will be important to understand, both to protect birds from infections, and to protect man from bird-transmitted” viruses.

Science Daily
October 1, 2013

Original web page at Science Daily

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Hormone receptors may regulate effect of nutrition on life expectancy not only in roundworms, but perhaps also in humans

A reduced caloric intake increases life expectancy in many species. But how diet prolongs the lives of model organisms such as fruit flies and roundworms has remained a mystery until recently. Scientists at the Max Planck Institute for Biology of Ageing in Cologne discovered that a hormone receptor is one of the links between nutrition and life expectancy in the roundworms. The receptor protein NHR-62 increases the lifespan of the animals by twenty per cent if their calorie intake is reduced. Furthermore, another study showed that the hormone receptor NHR-8 affects development into adulthood as well as the maximum lifespan of the worms. It may be possible that receptors related to these are also responsible for regulating life expectancy in human beings. The roundworm Caenorhabditis elegans lives only about 20 days. This makes it an ideal research subject, as the complete lifecycle of the worm can be studied in a short time. Also, the worm consists of less than a thousand cells, and its genetic make-up has been extensively analysed, and contains many genes similar to humans. The scientists in Adam Antebi’s team at the Max Planck Institute for Biology of Ageing use Caenorhabditis elegans to find out how hormones influence ageing. They are particularly interested in hormone receptors that reside in the cell nucleus, which regulate the activity of metabolic genes.

Their results indicate that the receptor NHR-62 must be active for reduced dietary intake to fully prolong the life of worms. If NHR-62 is inactive, Caenorhabditis elegans will live 25% longer under dietary restriction than if this receptor is inactive. “It seems that there is an as yet unknown hormone which regulates lifespan using NHR-62. If we can identify this hormone and administer it to the worm, we may prolong its life without having to change its calorie intake,” Antebi explains. A restricted diet also affects the expression of genes dramatically: out of the approximate 20,000 worm genes, 3,000 change their activity, and 600 of these show a dependence on NHR-62. It follows that there are many other candidates for improving life expectancy. Since humans have receptors similar to NHR-62, so-called HNF-4α, the Max Planck scientists suspect that the hormone receptors may not only control the maximum lifespan of roundworms, but might affect human beings as well. However, nutrition also affects lifespan in several other ways. Another study by the scientists has shown that worms lacking the hormone receptor NHR-8 will remain longer in a pre-pubertal stage before they reach adulthood. They also die earlier than animals with this receptor. NHR-8 is a nuclear receptor, responsible for the animal’s cholesterol balance. “Without it, the worm cannot produce enough steroid hormones from the cholesterol and therefore reaches sexual maturity later on. In addition, its fatty acid metabolism changes and its life expectancy drops,” explains Antebi. Receptors similar to NHR-8 can be found in human beings too. Conceivably cholesterol metabolism could therefore regulate physical development and affect life expectancy in humans as well.

Science Daily
August 20, 2013

Original web page at Science Daily

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Memory-boosting chemical identified in mice: Cell biologists find molecule targets a key biological pathway

Memory improved in mice injected with a small, drug-like molecule discovered by UCSF San Francisco researchers studying how cells respond to biological stress. The same biochemical pathway the molecule acts on might one day be targeted in humans to improve memory, according to the senior author of the study, Peter Walter, PhD, UCSF professor of biochemistry and biophysics and a Howard Hughes Investigator. The discovery of the molecule and the results of the subsequent memory tests in mice were published in eLife, an online scientific open-access journal, on May 28, 2013. In one memory test included in the study, normal mice were able to relocate a submerged platform about three times faster after receiving injections of the potent chemical than mice that received sham injections. The mice that received the chemical also better remembered cues associated with unpleasant stimuli — the sort of fear conditioning that could help a mouse avoid being preyed upon. Notably, the findings suggest that despite what would seem to be the importance of having the best biochemical mechanisms to maximize the power of memory, evolution does not seem to have provided them, Walter said. “It appears that the process of evolution has not optimized memory consolidation; otherwise I don’t think we could have improved upon it the way we did in our study with normal, healthy mice,” Walter said.

The memory-boosting chemical was singled out from among 100,000 chemicals screened at the Small Molecule Discovery Center at UCSF for their potential to perturb a protective biochemical pathway within cells that is activated when cells are unable to keep up with the need to fold proteins into their working forms. However, UCSF postdoctoral fellow Carmela Sidrauski, PhD, discovered that the chemical acts within the cell beyond the biochemical pathway that activates this unfolded protein response, to more broadly impact what’s known as the integrated stress response. In this response, several biochemical pathways converge on a single molecular lynchpin, a protein called eIF2 alpha. Scientists have known that in organisms ranging in complexity from yeast to humans different kinds of cellular stress — a backlog of unfolded proteins, DNA-damaging UV light, a shortage of the amino acid building blocks needed to make protein, viral infection, iron deficiency — trigger different enzymes to act downstream to switch off eIF2 alpha. “Among other things, the inactivation of eIF2 alpha is a brake on memory consolidation,” Walter said, perhaps an evolutionary consequence of a cell or organism becoming better able to adapt in other ways.

Turning off eIF2 alpha dials down production of most proteins, some of which may be needed for memory formation, Walter said. But eIF2 alpha inactivation also ramps up production of a few key proteins that help cells cope with stress. Study co-author Nahum Sonenberg, PhD, of McGill University previously linked memory and eIF2 alpha in genetic studies of mice, and his lab group also conducted the memory tests for the current study. The chemical identified by the UCSF researchers is called ISRIB, which stands for integrated stress response inhibitor. ISRIB counters the effects of eIF2 alpha inactivation inside cells, the researchers found. “ISRIB shows good pharmacokinetic properties how a drug is absorbed, distributed and eliminated, readily crosses the blood-brain barrier, and exhibits no overt toxicity in mice, which makes it very useful for studies in mice,” Walter said. These properties also indicate that ISRIB might serve as a good starting point for human drug development, according to Walter. Walter said he is looking for scientists to collaborate with in new studies of cognition and memory in mouse models of neurodegenerative diseases and aging, using ISRIB or related molecules. In addition, chemicals such as ISRIB could play a role in fighting cancers, which take advantage of stress responses to fuel their own growth, Walter said. Walter already is exploring ways to manipulate the unfolded protein response to inhibit tumor growth, based on his earlier discoveries. At a more basic level, Walter said, he and other scientists can now use ISRIB to learn more about the role of the unfolded protein response and the integrated stress response in disease and normal physiology.

Science Daily
July 9, 2013

Original web page at Science Daily

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Hitting ‘reset’ in protein synthesis restores myelination: suggests new treatment for misfolded protein diseases

A potential new treatment strategy for patients with Charcot-Marie-Tooth disease is on the horizon, thanks to research by neuroscientists now at the University at Buffalo’s Hunter James Kelly Research Institute and their colleagues in Italy and England. The institute is the research arm of the Hunter’s Hope Foundation, established in 1997 by Jim Kelly, Buffalo Bills Hall of Fame quarterback, and his wife, Jill, after their infant son Hunter was diagnosed with Krabbe leukodystrophy, an inherited fatal disorder of the nervous system. Hunter died in 2005 at the age of eight. The institute conducts research on myelin and its related diseases with the goal of developing new ways of understanding and treating conditions such as Krabbe disease and other leukodystrophies. Charcot-Marie-Tooth or CMT disease, which affects the peripheral nerves, is among the most common of hereditary neurological disorders; it is a disease of myelin and it results from misfolded proteins in cells that produce myelin. The new findings sere published online earlier this month in The Journal of Experimental Medicine.

They may have relevance for other diseases that result from misfolded proteins, including Alzheimer’s disease, Parkinson’s, multiple sclerosis, Type 1 diabetes, cancer and mad cow disease. The paper shows that missteps in translational homeostasis, the process of regulating new protein production so that cells maintain a precise balance between lipids and proteins, may be how some genetic mutations in CMT cause neuropathy. CMT neuropathies are common, hereditary and progressive; in severe cases, patients end up in wheelchairs. These diseases significantly affect quality of life but not longevity, taking a major toll on patients, families and society, the researchers note. “It’s possible that our finding could lead to the development of an effective treatment not just for CMT neuropathies but also for other diseases related to misfolded proteins,” says Lawrence Wrabetz, MD, director of the institute and professor of neurology and biochemistry in UB’s School of Medicine and Biomedical Sciences and senior author on the paper. Maurizio D’Antonio, of the Division of Genetics and Cell Biology of the San Raffaele Scientific Institute in Milan is first author; Wrabetz did most of this research while he was at San Raffaele, prior to coming to UB.

The research finding centers around the synthesis of misfolded proteins in Schwann cells, which make myelin in nerves. Myelin is the crucial fatty material that wraps the axons of neurons and allows them to signal effectively. Many CMT neuropathies are associated with mutations in a gene known as P0, which glues the wraps of myelin together. Wrabetz has previously shown in experiments with transgenic mice that those mutations cause the myelin to break down, which in turn, causes degeneration of peripheral nerves and wasting of muscles. When cells recognize that the misfolded proteins are being synthesized, cells respond by severely reducing protein production in an effort to correct the problem, Wrabetz explains. The cells commence protein synthesis again when a protein called Gadd34 gets involved. “After cells have reacted to, and corrected, misfolding of proteins, the job of Gadd34 is to turn protein synthesis back on,” says Wrabetz. “What we have shown is that once Gadd34 is turned back on, it activates synthesis of proteins at a level that’s too high — that’s what causes more problems in myelination. “We have provided proof of principle that Gadd34 causes a problem with translational homeostasis and that’s what causes some neuropathies,” says Wrabetz. “We’ve shown that if we just reduce Gadd34, we actually get better myelination. So, leaving protein synthesis turned partially off is better than turning it back on, completely.”

In both cultures and a transgenic mouse model of CMT neuropathies, the researchers improved myelin by reducing Gadd34 with salubrinal, a small molecule research drug. While salubrinal is not appropriate for human use, Wrabetz and colleagues at UB and elsewhere are working to develop derivatives that are appropriate. “If we can demonstrate that a new version of this molecule is safe and effective, then it could be part of a new therapeutic strategy for CMT and possibly other misfolded protein diseases as well,” says Wrabetz. And while CMT is the focus of this particular research, the work is helping scientists at the Hunter James Kelly Research Institute enrich their understanding of myelin disorders in general. “What we learn in one disease, such as CMT, may inform how we think about toxins for others, such as Krabbe’s,” Wrabetz says. “We’d like to build a foundation and answer basic questions about where and when toxicity in diseases begin.” The misfolded protein diseases are an interesting and challenging group of diseases to study, he continues. “CMT, for example, is caused by mutations in more than 40 different genes,” he says. “When there are so many different genes involved and so many different mechanisms, you have to find a unifying mechanism: this problem of Gadd34 turning protein synthesis on at too high a level could be one unifying mechanism. The hope is that this proof of principle applies to more than just CMT and may lead to improved treatments for Alzheimer’s, Parkinson’s, Type 1 diabetes and the other diseases caused by misfolded proteins.”

Science Daily
May 14, 2013

Original web page at Science Daily

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Hydrogen peroxide vapor enhances hospital disinfection of superbugs

Infection control experts at The Johns Hopkins Hospital have found that a combination of robot-like devices that disperse a bleaching agent into the air and then detoxify the disinfecting chemical are highly effective at killing and preventing the spread of multiple-drug-resistant bacteria, or so-called hospital superbugs. A study report on the use of hydrogen peroxide vaporizers — first deployed in several Singapore hospitals during the 2002 outbreak of severe acute respiratory syndrome, or SARS, and later stocked by several U.S. government agencies in case of an anthrax attack – has been published Jan. 1 in the journal Clinical Infectious Diseases. In the study, the Johns Hopkins team placed the devices in single hospital rooms after routine cleaning to disperse a thin film of the bleaching hydrogen peroxide across all exposed hospital equipment surfaces, as well as on room floors and walls. Results showed that the enhanced cleaning reduced by 64 percent the number of patients who later became contaminated with any of the most common drug-resistant organisms. Moreover, researchers found that protection from infection was conferred on patients regardless of whether the previous room occupant was infected with drug-resistant bacteria or not.

“Hydrogen peroxide vapor, as spread around patients’ rooms by these devices, represents a major technological advance in preventing the spread of dangerous bacteria inside hospitals and, especially, from one patient occupant to the next, even though sick patients were never in the same room at the same time,” says infectious disease specialist and study senior investigator Trish Perl, M.D., M.Sc. Of special note, researchers say, was that enhanced cleaning with the vapor reduced by 80 percent a patient’s chances of becoming colonized by a particularly aggressive and hard-to-treat bacterium, vancomycin-resistant enterococci (VRE). In what is believed to be the first head-to-head comparison between traditional hand-cleaning and mopping with bleaching agents and robotic vaporizers, researchers routinely tested patients and their surroundings not only for VRE, but also for the more common methicillin-resistant Staphylococcus aureus, or MRSA, and lesser-known bacteria, including Clostridium difficile and Acinetobacter baumannii. Some 6,350 patient admissions to JHH were closely tracked as part of the two-and-a-half-year analysis, as patients moved into and out of 180 private hospital rooms. Almost half the rooms received enhanced cleaning with hydrogen peroxide vapor in between patients, while the rest did not. Overall, multiple-drug-resistant organisms were found on room surfaces in 21 percent of rooms tested, but mostly in rooms that did not undergo enhanced cleaning.

Perl says that patients bringing in or picking up drug-resistant organisms while undergoing treatment in hospitals is a persistent and growing problem, and previous research has shown that patients who stay in a hospital room previously occupied by an infected patient are at greater risk of becoming infected. “Our study results are evidence that technological solutions, when combined with standard cleaning, can effectively and systematically decontaminate patients’ rooms and augment other behavioral practices, such as strict hospital staff compliance with hand-washing and bathing patients in disinfecting chlorhexidine when they are first admitted to the hospital,” says Perl, senior hospital epidemiologist for the Johns Hopkins Health System and a professor at the Johns Hopkins University School of Medicine. “Our goal is to improve all hospital infection control practices, including cleaning and disinfection, as well as behavioral and environmental practices, to the point where preventing the spread of these multiple-drug-resistant organisms also minimizes the chances of patients becoming infected and improves their chances of recovery,” says Perl.

The paired robot-like devices, each about the size of a washing machine and weighing nearly 60 pounds, as well as supplies used in the study, were provided by their manufacturer, Bioquell Inc. of Horsham, Pa. After the room has been cleaned, the vents are covered and the two devices are placed inside. The sliding door is closed, and the room is sealed. Then, the larger of the two devices disperses hydrogen peroxide into the room, leaving a very tiny, almost invisible layer (only 2 microns to 6 microns in thickness) on all exposed surfaces, including keyboards and monitors, as well as tables and chairs. Because hydrogen peroxide can be toxic to humans if ingested or corrosive if left on the skin for too long, the second, smaller device is activated to break down the bleach into its component water and oxygen parts. The combined operation takes the devices about an hour and a half to complete. “What is so exciting about this new method of infection control is that the devices are easy to use and hospital staff embrace it very quickly,” says surgeon and study co-investigator Pamela Lipsett, M.D., M.H.P.E. Lipsett, a professor and director of surgical and critical care fellowship training at Johns Hopkins, says that during the study and before room cleanings, staff were “wheeling in” other pieces of equipment so these, too, could be decontaminated by the hydrogen peroxide vapor.

As a result of the study and the researchers’ recommendation, JHH has purchased two of the Bioquell decontaminating units, which cost more than $40,000 per pair. The devices, already in use at some 20 other hospitals across the country, will be used at Johns Hopkins to decontaminate rooms typically housing high-risk patients under strict isolation precautions because of severe infection with a multiple-drug-resistant organism. Researchers say they next plan to study the devices’ effectiveness at decontaminating the outside packaging of unused but potentially exposed hospital supplies, which are typically discarded even though their seals remain intact. The research team also wants to coordinate study testing among other hospitals to validate their Johns Hopkins findings. Larger and longer studies may also be planned, to precisely measure and determine how well the devices work against the spread of each hospital superbug.

Science Daily
January 22, 2013

Original web page at Science Daily