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Monday, January 10, 2011
Faster Approach Hastens Creation of Powerful DNA Targeting Tool
A team of researchers has developed a less labor-intensive way to create synthetic enzymes that target specific DNA sequences for inactivation, repair, or alteration.
The study, performed by researchers from the molecular pathology unit of Massachusetts General Hospital (MGH; Boston, MA, USA), was published December 2010 online in the journal Nature Methods, and described a highly effective but less labor-intensive way to generate powerful tools called zinc-finger nucleases (ZFNs).
“With our approach, called context-dependent assembly, any scientist can use either standard molecular biology techniques or commercial DNA synthesis to design ZFNs for their target gene of interest,” said J. Keith Joung, MD, PhD, associate chief for research in MGH pathology, the study’s senior author. “ZFNs are broadly applicable, powerful tools for manipulating the genomes of cells from various organisms--including humans--and may provide a way to efficiently correct gene mutations responsible for human disease, avoiding problems resulting from the imprecise nature of current gene therapy approaches using viral vectors.”
Most human transcription factors that control whether a genetic signal is converted into a protein bind to specific DNA sequences using peptides called zinc fingers. Zinc-finger nucleases are synthetic “designer” proteins combining a zinc-finger domain, engineered to bind a specific DNA sequence, with an enzyme that breaks both DNA strands at the targeted site. While ZFNs have great potential, creating the customized proteins has been challenging.
In the simplest approach, called modular assembly, individual peptides are linked together similar to beads on a string to create a multifinger protein hypothetically able to recognize long DNA segments. Dr. Joung and others have shown that, in practice, modular assembly has a very low success rate for creating multifinger proteins. This high failure rate is most likely caused by “context-dependent” effects that individual zinc fingers can have on the DNA-binding activities of their neighboring fingers. Assembling peptides that do not work well together would be like trying to put together jigsaw puzzle pieces that do not fit.
In 2008, Dr. Joung and colleagues at the University of Minnesota (Twin Cities, USA) and other institutions, members of the Zinc Finger Consortium, reported developing a method called OPEN (Oligomerized Pool ENgineering), which takes these context-dependent effects into account. But although OPEN works well, it can be labor-intensive and very time consuming--requiring up to one year for a lab to establish the technology and two months of work to create desired ZFNs. To address these limitations, the MGH research team has assembled an extensive archive of zinc fingers known to work well when positioned together--basically, puzzle pieces that already have been put together. Using this context-dependent technique, the investigators were able to assemble dozens of ZFNs in as little as four days.
“With this archive in hand, any researcher can easily generate their own ZFNs in less than a week, and no special expertise is needed,” Dr. Joung explained. “In addition to being much faster, context-dependent assembly can generate large numbers of ZFNs simultaneously, which is hard to do with OPEN because it is more labor intensive.”
As was the case with OPEN, the Joung lab and the Zinc Finger Consortium will make the software and reagents required to practice context-dependent assembly available to all academic laboratories.
“One of the holy grails of genetics is the ability to make targeted changes to individual genes,” stated Laurie Tompkins, PhD, who over sees genetics grants at the National Institute of General Medical Sciences, one of the US National Institutes of Health (Bethesda, MD, USA) and a major supporter of this study. “Dr. Joung and his colleagues have developed an extraordinarily simple, efficient strategy for using zinc finger technology to swap out altered versions of genes for normal ones--or vice versa--providing basic scientists and clinicians alike with a broadly applicable research tool.”
Added Dr. Joung, an associate professor of pathology at Harvard Medical School (Boston, MA, USA), “At this point, I believe that context-dependent assembly will have the biggest impact on researchers using ZFNs to genetically manipulate model organisms, possibly even models developed from pluripotent stem cells. Other big impacts should be enabling researchers to create knockout mutations in a large series of genes involved in a common pathway or related to a specific disease and to use ZFNs to create comprehensive collections of mutants for every gene in an organism.”
Dr. Joung is also a member of the MGH Center for Computational and Integrative Biology and Center for Cancer Research. The challenges presented to scientists interested in using ZFNs in their investigations were described in an article in the Fall 2010 issue of the MGH-sponsored magazine Proto.
The study, performed by researchers from the molecular pathology unit of Massachusetts General Hospital (MGH; Boston, MA, USA), was published December 2010 online in the journal Nature Methods, and described a highly effective but less labor-intensive way to generate powerful tools called zinc-finger nucleases (ZFNs).
“With our approach, called context-dependent assembly, any scientist can use either standard molecular biology techniques or commercial DNA synthesis to design ZFNs for their target gene of interest,” said J. Keith Joung, MD, PhD, associate chief for research in MGH pathology, the study’s senior author. “ZFNs are broadly applicable, powerful tools for manipulating the genomes of cells from various organisms--including humans--and may provide a way to efficiently correct gene mutations responsible for human disease, avoiding problems resulting from the imprecise nature of current gene therapy approaches using viral vectors.”
Most human transcription factors that control whether a genetic signal is converted into a protein bind to specific DNA sequences using peptides called zinc fingers. Zinc-finger nucleases are synthetic “designer” proteins combining a zinc-finger domain, engineered to bind a specific DNA sequence, with an enzyme that breaks both DNA strands at the targeted site. While ZFNs have great potential, creating the customized proteins has been challenging.
In the simplest approach, called modular assembly, individual peptides are linked together similar to beads on a string to create a multifinger protein hypothetically able to recognize long DNA segments. Dr. Joung and others have shown that, in practice, modular assembly has a very low success rate for creating multifinger proteins. This high failure rate is most likely caused by “context-dependent” effects that individual zinc fingers can have on the DNA-binding activities of their neighboring fingers. Assembling peptides that do not work well together would be like trying to put together jigsaw puzzle pieces that do not fit.
In 2008, Dr. Joung and colleagues at the University of Minnesota (Twin Cities, USA) and other institutions, members of the Zinc Finger Consortium, reported developing a method called OPEN (Oligomerized Pool ENgineering), which takes these context-dependent effects into account. But although OPEN works well, it can be labor-intensive and very time consuming--requiring up to one year for a lab to establish the technology and two months of work to create desired ZFNs. To address these limitations, the MGH research team has assembled an extensive archive of zinc fingers known to work well when positioned together--basically, puzzle pieces that already have been put together. Using this context-dependent technique, the investigators were able to assemble dozens of ZFNs in as little as four days.
“With this archive in hand, any researcher can easily generate their own ZFNs in less than a week, and no special expertise is needed,” Dr. Joung explained. “In addition to being much faster, context-dependent assembly can generate large numbers of ZFNs simultaneously, which is hard to do with OPEN because it is more labor intensive.”
As was the case with OPEN, the Joung lab and the Zinc Finger Consortium will make the software and reagents required to practice context-dependent assembly available to all academic laboratories.
“One of the holy grails of genetics is the ability to make targeted changes to individual genes,” stated Laurie Tompkins, PhD, who over sees genetics grants at the National Institute of General Medical Sciences, one of the US National Institutes of Health (Bethesda, MD, USA) and a major supporter of this study. “Dr. Joung and his colleagues have developed an extraordinarily simple, efficient strategy for using zinc finger technology to swap out altered versions of genes for normal ones--or vice versa--providing basic scientists and clinicians alike with a broadly applicable research tool.”
Added Dr. Joung, an associate professor of pathology at Harvard Medical School (Boston, MA, USA), “At this point, I believe that context-dependent assembly will have the biggest impact on researchers using ZFNs to genetically manipulate model organisms, possibly even models developed from pluripotent stem cells. Other big impacts should be enabling researchers to create knockout mutations in a large series of genes involved in a common pathway or related to a specific disease and to use ZFNs to create comprehensive collections of mutants for every gene in an organism.”
Dr. Joung is also a member of the MGH Center for Computational and Integrative Biology and Center for Cancer Research. The challenges presented to scientists interested in using ZFNs in their investigations were described in an article in the Fall 2010 issue of the MGH-sponsored magazine Proto.
Sunday, January 9, 2011
Natural Plant Compound Fights Inflammation
Researchers have discovered how abscisic acid, a natural plant hormone with known beneficial properties for the treatment of disease, helps combat inflammation. The findings reveal significant new drug targets for the development of treatments for inflammatory and immune-mediated diseases.
The scientists, from Virginia Bioinformatics Institute at Virginia Tech (Blacksburg, USA), published their results in the November 2010 issue of the Journal of Biological Chemistry. They had reported some of the major molecular events in the immune system of mice that contribute to inflammation-related disease, including the involvement of a specific molecule found on the surface of immune cells involved in the body’s fight against infection. They have now gone a step further and revealed the process by which the natural drug abscisic acid interacts with this protein, known as peroxisome proliferator-activated receptor-gamma, to block inflammation and the consequent onset of disease.
"In previous work, our research group demonstrated that abscisic acid has beneficial effects on several conditions and diseases including obesity-related inflammation, diabetes, atherosclerosis, and inflammatory bowel disease,” said Dr. Josep Bassaganya-Riera, associate professor of immunology at the Virginia Bioinformatics Institute, leader of the Nutritional Immunology and Molecular Medicine Group in the institute’s cyberInfrastructure division, and lead investigator of the study. “One idea for how abscisic acid reduces inflammation in these instances is that it binds to a special region of peroxisome proliferator-activated receptor-gamma, a binding site known as the ligand-binding domain where the drug would be expected to latch on to and exert its effect. Our results show that this is not the case and, for the first time, we have demonstrated that abscisic acid works independently of this ligand-binding domain of the receptor.”
“The outcomes of this research illustrate the synergism that can result from combining computational and experimental approaches to characterize therapeutic targets,” said Dr. David Bevan, associate professor of biochemistry at Virginia Tech. “By using molecular modeling approaches we were able to identify a potential binding site for abscisic acid on the lanthionine synthetase C-like 2 protein, a protein required for the beneficial health effects of abscisic acid. We were also able, again using docking studies, to reveal reasons for the lack of direct association of abscisic acid with peroxisome proliferator-activated receptor-gamma, which was experimentally validated by ligand-binding assays.”
“This information is significant because it suggests the existence of new therapeutic targets or alternative modes of action that account for the effects of abscisic acid in the immune system,” added Dr. Bassaganya-Riera. “Drugs that bind to the ligand-binding domain of peroxisome proliferator-activated receptor-gamma such as Avandia are associated with severe cardiovascular side effects. In contrast, the newly discovered alternative mechanism of peroxisome proliferator-activated receptor-gamma activation by abscisic acid does not appear to be linked to any known adverse side effects, thereby representing a promising new therapeutic avenue."
“Lanthionine synthetase C-like 2 represents the first step in a pathway leading to activation of peroxisome proliferator-activated receptor-gamma in immune cells by abscisic acid,” said Dr. Raquel Hontecillas, assistant professor of immunology at the Virginia Bioinformatics Institute and one of the lead investigators of the study. “We have also shown that abscisic acid affects the expression of several genes involved in inflammation, metabolism and cell signaling, which provides further clues for possible intervention points in the treatment of inflammatory and immune-mediated diseases.”
The researchers plan to isolate more closely some of the new drug targets in the molecular network of the immune response as they continue to dissect the way that the naturally occurring drug abscisic acid reduces damage due to inflammation. Moreover, this new understanding on how abscisic acid works will be used to develop new classes of drugs that target the same alternative pathway of peroxisome proliferator-activated receptor-gamma activation, a potentially safer method than the use of drugs that target direct binding to the receptor.
The scientists, from Virginia Bioinformatics Institute at Virginia Tech (Blacksburg, USA), published their results in the November 2010 issue of the Journal of Biological Chemistry. They had reported some of the major molecular events in the immune system of mice that contribute to inflammation-related disease, including the involvement of a specific molecule found on the surface of immune cells involved in the body’s fight against infection. They have now gone a step further and revealed the process by which the natural drug abscisic acid interacts with this protein, known as peroxisome proliferator-activated receptor-gamma, to block inflammation and the consequent onset of disease.
"In previous work, our research group demonstrated that abscisic acid has beneficial effects on several conditions and diseases including obesity-related inflammation, diabetes, atherosclerosis, and inflammatory bowel disease,” said Dr. Josep Bassaganya-Riera, associate professor of immunology at the Virginia Bioinformatics Institute, leader of the Nutritional Immunology and Molecular Medicine Group in the institute’s cyberInfrastructure division, and lead investigator of the study. “One idea for how abscisic acid reduces inflammation in these instances is that it binds to a special region of peroxisome proliferator-activated receptor-gamma, a binding site known as the ligand-binding domain where the drug would be expected to latch on to and exert its effect. Our results show that this is not the case and, for the first time, we have demonstrated that abscisic acid works independently of this ligand-binding domain of the receptor.”
“The outcomes of this research illustrate the synergism that can result from combining computational and experimental approaches to characterize therapeutic targets,” said Dr. David Bevan, associate professor of biochemistry at Virginia Tech. “By using molecular modeling approaches we were able to identify a potential binding site for abscisic acid on the lanthionine synthetase C-like 2 protein, a protein required for the beneficial health effects of abscisic acid. We were also able, again using docking studies, to reveal reasons for the lack of direct association of abscisic acid with peroxisome proliferator-activated receptor-gamma, which was experimentally validated by ligand-binding assays.”
“This information is significant because it suggests the existence of new therapeutic targets or alternative modes of action that account for the effects of abscisic acid in the immune system,” added Dr. Bassaganya-Riera. “Drugs that bind to the ligand-binding domain of peroxisome proliferator-activated receptor-gamma such as Avandia are associated with severe cardiovascular side effects. In contrast, the newly discovered alternative mechanism of peroxisome proliferator-activated receptor-gamma activation by abscisic acid does not appear to be linked to any known adverse side effects, thereby representing a promising new therapeutic avenue."
“Lanthionine synthetase C-like 2 represents the first step in a pathway leading to activation of peroxisome proliferator-activated receptor-gamma in immune cells by abscisic acid,” said Dr. Raquel Hontecillas, assistant professor of immunology at the Virginia Bioinformatics Institute and one of the lead investigators of the study. “We have also shown that abscisic acid affects the expression of several genes involved in inflammation, metabolism and cell signaling, which provides further clues for possible intervention points in the treatment of inflammatory and immune-mediated diseases.”
The researchers plan to isolate more closely some of the new drug targets in the molecular network of the immune response as they continue to dissect the way that the naturally occurring drug abscisic acid reduces damage due to inflammation. Moreover, this new understanding on how abscisic acid works will be used to develop new classes of drugs that target the same alternative pathway of peroxisome proliferator-activated receptor-gamma activation, a potentially safer method than the use of drugs that target direct binding to the receptor.
Wednesday, December 29, 2010
SLU Research Fuels Hope for Hard-To-Treat Hepatitis C Patients
ST. LOUIS - The outlook for patients with hepatitis C continues to improve as results from a clinical trial led by a Saint Louis University researcher found that the drug boceprevir helped cure hard-to-treat patients. The findings were reported at the 61st annual meeting of the American Association for the Study of Liver Disease's earlier in November.
Bruce R. Bacon, M.D., professor of internal medicine at Saint Louis University School of Medicine and co-principal investigator of the HCV RESPOND-2 study, studied the protease inhibitor, boceprevir, and found that it significantly increased the number of patients whose blood had undetectable levels of the virus.
"These findings are especially significant for patients who don't respond to initial treatment," said Bacon. "When the hepatitis C virus is not eliminated, debilitating fatigue and more serious problems can follow."
Hepatitis C is caused by a virus that is transmitted by contact with blood. The infection may initially be asymptomatic, but for patients who develop chronic hepatitis C infection, inflammation of the liver may develop, leading to fibrosis and cirrhosis (scarring of the liver), as well as other complications including liver cancer and death.
The prognosis varies for patients with chronic hepatitis C. With the current standard therapy, about half fully recover after an initial course of peginterferon and ribavirin anti-viral therapy that may last from six months to a year.
The remaining patients, known as non-responders, may improve with initial treatment but the virus is not eliminated, or may not respond to treatment at all. For this group, the only current option is to retreat patients with the same or similar drugs, which increases the likelihood of severe treatment side-effects. In addition, researchers have found that the success of treatment depends on the major strain, or genotype, of hepatitis C that a patient has.
The HCV RESPOND-2 study looked at 403 patients with chronic hepatitis C infections with genotype one, the most difficult strain of the virus to treat, who still had significant levels of the virus after being treated with peginterferon and ribavirin, the standard hepatitis C treatment.
"These results are very exciting," Bacon said. "In this study, boceprevir helped cure significantly more patients in 36 weeks of therapy than did treatment with peginterferon and ribavirin alone."
A second study, HCV SPRINT-2, examined patients with hepatitis C with genotype one who had not yet been treated with the standard treatment. They, too, responded well to the drug.
Bacon calls the progress made in treating hepatitis C remarkable.
"We've gone from the discovery of the virus in 1989 to where we are now, 22 years later, when we have the ability to cure a large majority of those with hepatitis C," Bacon said. "It's a true success story."
"Drugs like boceprevir are going to revolutionize care of those with hepatitis C."
The clinical trial was funded by Merck, which expects to begin seeking FDA approval this year.
Established in 1836, Saint Louis University School of Medicine has the distinction of awarding the first medical degree west of the Mississippi River. The school educates physicians and biomedical scientists, conducts medical research, and provides health care on a local, national and international level. Research at the school seeks new cures and treatments in five key areas: cancer, liver disease, heart/lung disease, aging and brain disease, and infectious disease.
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| Bruce Bacon, M.D. |
"These findings are especially significant for patients who don't respond to initial treatment," said Bacon. "When the hepatitis C virus is not eliminated, debilitating fatigue and more serious problems can follow."
Hepatitis C is caused by a virus that is transmitted by contact with blood. The infection may initially be asymptomatic, but for patients who develop chronic hepatitis C infection, inflammation of the liver may develop, leading to fibrosis and cirrhosis (scarring of the liver), as well as other complications including liver cancer and death.
The prognosis varies for patients with chronic hepatitis C. With the current standard therapy, about half fully recover after an initial course of peginterferon and ribavirin anti-viral therapy that may last from six months to a year.
The remaining patients, known as non-responders, may improve with initial treatment but the virus is not eliminated, or may not respond to treatment at all. For this group, the only current option is to retreat patients with the same or similar drugs, which increases the likelihood of severe treatment side-effects. In addition, researchers have found that the success of treatment depends on the major strain, or genotype, of hepatitis C that a patient has.
The HCV RESPOND-2 study looked at 403 patients with chronic hepatitis C infections with genotype one, the most difficult strain of the virus to treat, who still had significant levels of the virus after being treated with peginterferon and ribavirin, the standard hepatitis C treatment.
"These results are very exciting," Bacon said. "In this study, boceprevir helped cure significantly more patients in 36 weeks of therapy than did treatment with peginterferon and ribavirin alone."
A second study, HCV SPRINT-2, examined patients with hepatitis C with genotype one who had not yet been treated with the standard treatment. They, too, responded well to the drug.
Bacon calls the progress made in treating hepatitis C remarkable.
"We've gone from the discovery of the virus in 1989 to where we are now, 22 years later, when we have the ability to cure a large majority of those with hepatitis C," Bacon said. "It's a true success story."
"Drugs like boceprevir are going to revolutionize care of those with hepatitis C."
The clinical trial was funded by Merck, which expects to begin seeking FDA approval this year.
Established in 1836, Saint Louis University School of Medicine has the distinction of awarding the first medical degree west of the Mississippi River. The school educates physicians and biomedical scientists, conducts medical research, and provides health care on a local, national and international level. Research at the school seeks new cures and treatments in five key areas: cancer, liver disease, heart/lung disease, aging and brain disease, and infectious disease.
Saturday, October 16, 2010
Calculations for Molecular Biology and Biotechnology
This book is the first comprehensive guide devoted exclusively to calculations encountered in the genetic engineering laboratory. Mathematics, as a vital component of the successful design and interpretation of basic research, is used daily in laboratory work. This guide, written for students, technicians, and scientists, provides example calculations for the most frequently confronted problems encountered in gene discovery and analysis. The text and sample calculations are written in an easy-to-follow format. It is the perfect laboratory companion for anyone working in DNA manipulation and analysis.
Molecular Diagnostics: Fundamentals, Methods, And Clinical Applications Ebook Download
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Thursday, September 16, 2010
UK scientists devise 'one-hour test' for TB
Scientists in the UK say they have devised a new ultra-sensitive test which can diagnose the presence of the tuberculosis bacterium in one hour.
Its developers claim the test can spot all strains of the disease and could reduce both the incidence and the consequences of the disease worldwide.
According to the World Health Organization, in 2008, an estimated 1.3 million people died from TB worldwide.
Genetic signature The standard identification test for TB involves taking mucus coughed up from the lungs and growing a bacterial culture in the laboratory.
But it can take up to eight weeks to reach a diagnosis, by which time the individual might have infected many more people.
Other more rapid tests exist which scan for an antigen found in many TB strains, but they may not detect all infections, say the HPA.
The new test focuses on a particular DNA region within the bacterium which the researchers says is present in all strains of the disease.
Once a sample is taken, a scientific technique know as a "polymerase chain reaction" is used to amplify the volume of DNA available so that the genetic signature can be identified.
Tuberculosis
- Tuberculosis is an infectious disease, which usually affects the lungs
- It is transmitted via droplets from the lungs of people with the active form of the disease
- In healthy people, infection often causes no symptoms
- Symptoms of active TB include coughing, chest pains, weakness, weight loss, fever and night sweats
- Tuberculosis is treatable with a course of antibiotics
- In the UK, around 9,000 cases of TB are reported each year, mainly in big cities like London
"This is a new test," says the HPA's Dr Cath Arnold, who led the study. "We're looking for a genetic marker which is present in all strains of TB we've seen so far."
We're confident that it will pick up very small amounts and tests so far have show that it seems to be as sensitive as the gold standard of using culture, but there are various aspects which we need to develop further before we can offer it as an off-the-shelf product."Details of the work are being presented at the HPA's annual conference at the University of Warwick.
The HPA test comes just weeks after details of a rival project were published in the New England Journal of Medicine.
The rival test is called "Xpert MTB/RIF" and its developers claim it can deliver a diagnosis in under two hours. They say their automated cartridge machine can also identify resistance to drugs used to treat TB.
Difficult diagnosis Dr Mario Raviglione, director of the World Heath Organization's Stop TB department, says these new generation tests could potentially revolutionise TB treatment.
The WHO estimates that a third of the world's population carry TB bacteria. Only 5-10% of people who are infected become sick or infectious at some time during their life.
People with HIV and who carry TB bacteria are much more likely to develop the disease.
Recent years have seen a resurgence in TB infections in developed countries, and have seen the rise of strains resistant to medication.
Last year in the UK, the number of cases rose by more than 5% to 9,153, according to provisional figures from the HPA. More than a third of the cases were in London.
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