A magnetic separation technique developed by researchers at Duke University's Pratt School of Engineering and Purdue University makes it relatively simple to sort through beads hundreds of times smaller than the period at the end of this sentence.
The method could lead to new technologies for medical or environmental testing, according to the researchers. For example, specially coated magnetic particles designed to attract particular viruses or bacteria might be used in tailored combinations to simultaneously test for multiple infectious pathogens in a blood or water sample.
Benjamin Yellen, assistant professor of mechanical engineering and materials science at Duke, and Gil Lee, associate professor of chemical engineering at Purdue, report their findings in the December issue of the journal Lab on a Chip.
"If there were five viruses that a patient might have been exposed to, you could potentially develop a technique to look for those five viruses all at the same time," Yellen said. In principle, such a test could be done with just a single drop of blood, as long as there was virus in the sample.
As an initial demonstration of the concept, the researchers attached two "model pathogens," a baker's yeast and a soil bacterium, to magnetic beads, and used their technique to selectively isolate them.
The magnetic separation method, which the researchers call magnetophoresis, uses a rotating magnetic field and a microchip containing an array of miniature magnets to separate tiny magnetic beads based on their size within a matter of minutes.
The physics behind the technique is as interesting as its potential applications, Yellen added. "The method causes certain particles to become essentially immobile -- just jittering back and forth -- while others move off the chip where they can be isolated. It implies that we could achieve effectively infinite separation between particle types. We thought our technique would work well for bioseparation, but we hadn't predicted it would be this good."
While the researchers know how to precisely control which particles move and which stay put, by varying the frequency of the magnetic field they apply, the underlying physics responsible for the behavior remains partly unexplained and will be the subject of future investigation, Yellen said.
Micrometer and nanometer sized "superparamagnetic" beads already are used widely to magnetically separate biological molecules and cells from complex fluid mixtures, Yellen said. Superparamagnetism is a form of magnetic behavior which occurs primarily in materials composed of very small magnetic grains. Such materials are commonly used for drug delivery and imaging applications and in biomedical devices because they become magnetized only in the presence of an externally applied magnetic field, which helps prevent clumping.
Over the past few decades, however, there have been few new developments in the field of magnetic separation, according to the researchers, with most of the efforts focused on using stronger and stronger magnetic fields and field gradients.
"Now, we've demonstrated a fundamentally new and different approach to magnetic separation, which can dramatically increase the separation efficiency, not by exploiting stronger fields and field gradients, but rather by precisely tuning the mobility of beads and exploiting the non-linear dynamics of particles moving in a traveling wave," Yellen said.
Collaborators on the study include Duke graduate students Randall Erb and Hui Son; Rodward Hewlin, Jr., an undergraduate at the North Carolina Agricultural and Technical State University who worked in Yellen's laboratory; and Hao Shang, a postdoctoral fellow at Purdue. The work was funded by the National Science Foundation and the NASA Institute for Nanoelectronics and Computing at Purdue.
Source: Kendall Morgan
Duke University
четверг, 29 сентября 2011 г.
понедельник, 26 сентября 2011 г.
T-Ray Breakthrough Could Make Detecting Disease Far Easier
A breakthrough in the harnessing of 'T-rays' - electromagnetic terahertz waves - which could dramatically improve the detecting and sensing of objects as varied as biological cell abnormalities and explosives has been announced.
Researchers at the University of Bath, UK, and in Spain have said they have found a way to control the flow of terahertz radiation down a metal wire. Their findings are set out in a letter published in the current journal Physical Review Letters.
Terahertz radiation, whose frequency is around one thousand billion cycles a second, bridges the gap between the microwave and infrared parts of the electromagnetic spectrum.
Materials interact with radiation at T-ray frequencies in different ways than with radiation in other parts of the spectrum, making T-rays potentially important in detecting and analysing chemicals by analysing how they absorb T-rays fired at them.
This would allow quality control of prescribed drugs and detection of explosives to be carried out more easily, as many complex molecules have distinctive signatures in this part of the electromagnetic spectrum.
T-ray applications are presently limited by the relatively poor ability to focus the rays, which is achieved using the conventional means of lenses and mirrors to focus the radiation. This limits the spot size of focused T-rays to a substantial fraction of a millimetre and this has made studies of small objects such as biological cells with high resolution are virtually impossible.
But in their work the researchers found that although ordinary metal wire would not guide T-rays very well, if a series of tiny grooves was cut into the wire, it would do so much more effectively. If such a corrugated metal wire is then tapered to a point it becomes possible to very efficiently transport radiation to a point as small as a few millionths of a metre across.
This might, for example, lead to breakthroughs in examining very small objects such as the interior of biological cells where it might be possible to detect diseases or abnormalities. T-rays could also be directed to the interior of objects which could be useful in applications like endoscopic probing for cancerous cells or explosive detection.
"This is a significant development that would allow unprecedented accuracy in studying tiny objects and sensing chemicals using T-rays" said Dr Stefan Maier, of the University of Bath's Department of Physics, who leads the research.
"Metal wire ordinarily has a limited ability to allow T-rays to flow along it, but our idea was to overcome this by corrugating its surface with a series of grooves, in effect creating an artificial material or 'metamaterial' as far as the T-rays are concerned."
"In this way, the T-rays can be focused to the tip of the wire and guided into confined spaces or used to detect small objects, with important implications for disease detection or finding explosive that are hidden."
Dr Maier is working with Dr Steve Andrews at Bath, and with Professor Francisco GarcAa-Vidal, of the Universidad AutAnoma de Madrid, and Luis MartAn-Moreno, of the Universidad de Zaragoza-CSIC.
The project, which is funded by the Royal Society in the UK, the EU and the US Airforce, is one year into its three-year term. The researchers hope to produce a working model within a year.
Contact: Tony Trueman
University of Bath
Researchers at the University of Bath, UK, and in Spain have said they have found a way to control the flow of terahertz radiation down a metal wire. Their findings are set out in a letter published in the current journal Physical Review Letters.
Terahertz radiation, whose frequency is around one thousand billion cycles a second, bridges the gap between the microwave and infrared parts of the electromagnetic spectrum.
Materials interact with radiation at T-ray frequencies in different ways than with radiation in other parts of the spectrum, making T-rays potentially important in detecting and analysing chemicals by analysing how they absorb T-rays fired at them.
This would allow quality control of prescribed drugs and detection of explosives to be carried out more easily, as many complex molecules have distinctive signatures in this part of the electromagnetic spectrum.
T-ray applications are presently limited by the relatively poor ability to focus the rays, which is achieved using the conventional means of lenses and mirrors to focus the radiation. This limits the spot size of focused T-rays to a substantial fraction of a millimetre and this has made studies of small objects such as biological cells with high resolution are virtually impossible.
But in their work the researchers found that although ordinary metal wire would not guide T-rays very well, if a series of tiny grooves was cut into the wire, it would do so much more effectively. If such a corrugated metal wire is then tapered to a point it becomes possible to very efficiently transport radiation to a point as small as a few millionths of a metre across.
This might, for example, lead to breakthroughs in examining very small objects such as the interior of biological cells where it might be possible to detect diseases or abnormalities. T-rays could also be directed to the interior of objects which could be useful in applications like endoscopic probing for cancerous cells or explosive detection.
"This is a significant development that would allow unprecedented accuracy in studying tiny objects and sensing chemicals using T-rays" said Dr Stefan Maier, of the University of Bath's Department of Physics, who leads the research.
"Metal wire ordinarily has a limited ability to allow T-rays to flow along it, but our idea was to overcome this by corrugating its surface with a series of grooves, in effect creating an artificial material or 'metamaterial' as far as the T-rays are concerned."
"In this way, the T-rays can be focused to the tip of the wire and guided into confined spaces or used to detect small objects, with important implications for disease detection or finding explosive that are hidden."
Dr Maier is working with Dr Steve Andrews at Bath, and with Professor Francisco GarcAa-Vidal, of the Universidad AutAnoma de Madrid, and Luis MartAn-Moreno, of the Universidad de Zaragoza-CSIC.
The project, which is funded by the Royal Society in the UK, the EU and the US Airforce, is one year into its three-year term. The researchers hope to produce a working model within a year.
Contact: Tony Trueman
University of Bath
пятница, 23 сентября 2011 г.
'Marathon Mice' Elucidate Little-Known Muscle Type
Researchers report in the January issue of the journal Cell Metabolism, published by Cell Press, the discovery of a genetic "switch" that drives the formation of a poorly understood type of muscle. Moreover, they found, animals whose muscles were full of the so-called IIX fibers were able to run farther and at higher work loads than normal mice could.
The findings could ultimately lead to novel drugs designed to change the composition of muscle, the researchers said. Such treatments might have the potential to boost physical strength and endurance in patients with a variety of muscle wasting conditions.
The research team, led by Bruce Spiegelman of Harvard Medical School, found that increasing activity of the gene known as PGC-1Гў in the skeletal muscles of mice caused them to become crowded with IIX muscle fibers, which are normally much less prevalent.
"One reason why less is known about IIX fibers is that no one muscle group is packed with them," Spiegelman said. "For the first time, we have a mouse model very enriched in IIX fibers. These mice show a greatly increased capacity to sustain physical activity."
Skeletal muscle converts chemical energy into motion and force, ranging from rapid and sudden bursts of intense activity to continuous low-intensity work, the researchers said. At one functional extreme, muscles such as the soleus--a broad, flat muscle found in the calf of the leg--perform slow but steady activities such as maintaining posture. At the other extreme, muscles such as the quadriceps typically perform intense and rapid activities.
To fulfill these varied roles, muscles vary in their proportion of "slow-twitch" muscle fibers (types I and IIA), ideal for slow and constant roles, and "fast-twitch" fibers (type IIB), better suited to rapid and sudden activity of shorter duration. The fiber types are defined by which "myosin heavy chains" (MHCs) they contain and by their metabolic capacity, a feature largely determined by the number of energy-producing mitochondria they house. Myosins are motor proteins that consist of both "heavy" and "light" amino acid chains.
While most muscles in mammals contain a mixture of slow- and fast-twitch fiber types, some muscle beds are enriched for one type or the other, Spiegelman said. However, adult skeletal muscles also contain fibers with an abundance of a fourth MHC, type IIX, about which much less is known.
IIX fibers seem to have the oxidative metabolism of slow-twitch fibers mixed with the biophysical properties of fast-twitch fibers. Oxidative metabolism is by far the most efficient way of generating energy, Spiegelman said.
In the current study, the researchers produced mice with higher than normal levels of the transcriptional coactivator PGC-1Гў in their skeletal muscles. Transcriptional coactivators work with other cellular factors and machinery to control the activity of other genes. While earlier studies had found that the related coactivator PGC-1Гў plays a role in determining muscle type, the role of PGC-1Гў wasn't known.
"The muscle from the PGC-1Гў transgenic mice was strikingly redder in appearance than wild-type controls," indicative of their increased mitochondrial content, the researchers now report. Upon further examination, the researchers were surprised to find that the fibers showed a reduction in I, IIA, and IIB MHCs and as much as a 5-fold increase in IIX MHC.
Nearly 100% of muscle fibers in the transgenic animals contained abundant MHC IIX mRNA and protein, they found, as compared to only 15%-20% in normal animals. PGC-1Гў also changed the muscles' metabolic characteristics by driving the activity of genes that spark proliferation of mitochondria.
The PGC-1Гў animals with more IIX muscle fibers showed a greater capacity for aerobic exercise, they found. Transgenic mice were able to run, on average, for 32.5 min to exhaustion, compared to 26 min for their normal littermates, Spiegelman's group reported.
"These data have potential importance for the therapy of a number of muscular and neuromuscular diseases in humans," Spiegelman's group concluded.
"Many conditions accompanied by loss of physical mobility, including paraplegia, prolonged bed rest, and muscular dystrophies, involve a loss of oxidative fibers and their replacement with glycolytic fibers. This, in turn, results in a further loss of resistance to fatigue, exacerbating the patient's condition in a downward spiral. The identification of PGC-1Гў as a potential mediator of the development of oxidative type IIX fibers suggests new ways to modulate muscle fiber type in health and disease."
The researchers include Zoltan Arany, Eric Smith, Wenli Yang, Yanhong Ma, Sherry Chin, and Bruce M. Spiegelman of Dana-Farber Cancer Institute and Harvard Medical School in Boston, MA; Nathan Lebrasseur and Carl Morris of Boston University School of Medicine in Boston, MA
This work was supported by NIH grants HL079172 to Z.A. and DK54477 and DK61562 to B.M.S.
Arany et al.: "The Transcriptional Coactivator PGC-1b Drives the Formation of Oxidative Type IIX Fibers in Skeletal Muscle." Publishing in Cell Metabolism 5, 35-46, January 2007 DOI 10.1016/j.cmet.2006.12.003 cellmetabolism/
Contact: Erin Doonan
Cell Press
The findings could ultimately lead to novel drugs designed to change the composition of muscle, the researchers said. Such treatments might have the potential to boost physical strength and endurance in patients with a variety of muscle wasting conditions.
The research team, led by Bruce Spiegelman of Harvard Medical School, found that increasing activity of the gene known as PGC-1Гў in the skeletal muscles of mice caused them to become crowded with IIX muscle fibers, which are normally much less prevalent.
"One reason why less is known about IIX fibers is that no one muscle group is packed with them," Spiegelman said. "For the first time, we have a mouse model very enriched in IIX fibers. These mice show a greatly increased capacity to sustain physical activity."
Skeletal muscle converts chemical energy into motion and force, ranging from rapid and sudden bursts of intense activity to continuous low-intensity work, the researchers said. At one functional extreme, muscles such as the soleus--a broad, flat muscle found in the calf of the leg--perform slow but steady activities such as maintaining posture. At the other extreme, muscles such as the quadriceps typically perform intense and rapid activities.
To fulfill these varied roles, muscles vary in their proportion of "slow-twitch" muscle fibers (types I and IIA), ideal for slow and constant roles, and "fast-twitch" fibers (type IIB), better suited to rapid and sudden activity of shorter duration. The fiber types are defined by which "myosin heavy chains" (MHCs) they contain and by their metabolic capacity, a feature largely determined by the number of energy-producing mitochondria they house. Myosins are motor proteins that consist of both "heavy" and "light" amino acid chains.
While most muscles in mammals contain a mixture of slow- and fast-twitch fiber types, some muscle beds are enriched for one type or the other, Spiegelman said. However, adult skeletal muscles also contain fibers with an abundance of a fourth MHC, type IIX, about which much less is known.
IIX fibers seem to have the oxidative metabolism of slow-twitch fibers mixed with the biophysical properties of fast-twitch fibers. Oxidative metabolism is by far the most efficient way of generating energy, Spiegelman said.
In the current study, the researchers produced mice with higher than normal levels of the transcriptional coactivator PGC-1Гў in their skeletal muscles. Transcriptional coactivators work with other cellular factors and machinery to control the activity of other genes. While earlier studies had found that the related coactivator PGC-1Гў plays a role in determining muscle type, the role of PGC-1Гў wasn't known.
"The muscle from the PGC-1Гў transgenic mice was strikingly redder in appearance than wild-type controls," indicative of their increased mitochondrial content, the researchers now report. Upon further examination, the researchers were surprised to find that the fibers showed a reduction in I, IIA, and IIB MHCs and as much as a 5-fold increase in IIX MHC.
Nearly 100% of muscle fibers in the transgenic animals contained abundant MHC IIX mRNA and protein, they found, as compared to only 15%-20% in normal animals. PGC-1Гў also changed the muscles' metabolic characteristics by driving the activity of genes that spark proliferation of mitochondria.
The PGC-1Гў animals with more IIX muscle fibers showed a greater capacity for aerobic exercise, they found. Transgenic mice were able to run, on average, for 32.5 min to exhaustion, compared to 26 min for their normal littermates, Spiegelman's group reported.
"These data have potential importance for the therapy of a number of muscular and neuromuscular diseases in humans," Spiegelman's group concluded.
"Many conditions accompanied by loss of physical mobility, including paraplegia, prolonged bed rest, and muscular dystrophies, involve a loss of oxidative fibers and their replacement with glycolytic fibers. This, in turn, results in a further loss of resistance to fatigue, exacerbating the patient's condition in a downward spiral. The identification of PGC-1Гў as a potential mediator of the development of oxidative type IIX fibers suggests new ways to modulate muscle fiber type in health and disease."
The researchers include Zoltan Arany, Eric Smith, Wenli Yang, Yanhong Ma, Sherry Chin, and Bruce M. Spiegelman of Dana-Farber Cancer Institute and Harvard Medical School in Boston, MA; Nathan Lebrasseur and Carl Morris of Boston University School of Medicine in Boston, MA
This work was supported by NIH grants HL079172 to Z.A. and DK54477 and DK61562 to B.M.S.
Arany et al.: "The Transcriptional Coactivator PGC-1b Drives the Formation of Oxidative Type IIX Fibers in Skeletal Muscle." Publishing in Cell Metabolism 5, 35-46, January 2007 DOI 10.1016/j.cmet.2006.12.003 cellmetabolism/
Contact: Erin Doonan
Cell Press
вторник, 20 сентября 2011 г.
Blood Pressure Drug May Help Stall Parkinson's
Gloria E. Meredith, Ph.D., collaborated with D. James Surmeier, Ph.D. and other scientists at Northwestern University to study the drug, Isradipine, and its possible effects on Parkinson's disease. The findings of this study were published this week in an article in Nature. Dr. Meredith, Professor and Chair of the Department of Cellular and Molecular Pharmacology at Rosalind Franklin University of Medicine and Science, and an expert in Parkinson's disease, co-authored the article. She commented, "Parkinson's disease is a motor disorder caused by the death of dopamine-producing nerve cells in our brains. There is a big race to protect these neurons from dying. Currently available drugs only treat the symptoms."
Dr. Meredith, who studies a chronic mouse model that mimics the signs and symptoms of Parkinson's disease, said, "Isradipine is a common drug used to treat hypertension and stroke. Dr. Surmeier had developed the idea that this drug, a calcium channel blocker, may help protect dopamine neurons in humans. He also designed the basic study. We joined together to see if the drug could stop cells from dying in the mouse model. Our findings indicated that isradipine slowed the disease process and destruction of the dopamine-producing neurons. Results from the mouse model indicate that if the drug works in humans, then it could be used as a means to prevent the onset of this disease or slow its progression."
The next phase will be to develop clinical trials in humans. Dr. Meredith said, "It's exciting to think that if we can protect the dopamine neurons from dying, we may prevent the disease, and improve the quality of life for patients."
Dr. Meredith has been studying Parkinson's disease for over 15 years. Her research is currently funded by the National Institute of Neurological Disorders and Stroke (NINDS), a division of the National Institutes of Health (NIH), and the Department of Defense.
Rosalind Franklin University of Medicine and Science educates medical doctors, health professionals and biomedical scientists in a personalized atmosphere. The University is located at 3333 Green Bay Road, North Chicago, IL 60064 and encompasses Chicago Medical School, College of Health professions, Dr. William M. Scholl College of Podiatric Medicine, and the School of Graduate and Postdoctoral Studies. Visit at rosalindfranklin/ and lifeindiscovery/.
Contact: Priscilla Khoury
Rosalind Franklin University of Medicine and Science
Dr. Meredith, who studies a chronic mouse model that mimics the signs and symptoms of Parkinson's disease, said, "Isradipine is a common drug used to treat hypertension and stroke. Dr. Surmeier had developed the idea that this drug, a calcium channel blocker, may help protect dopamine neurons in humans. He also designed the basic study. We joined together to see if the drug could stop cells from dying in the mouse model. Our findings indicated that isradipine slowed the disease process and destruction of the dopamine-producing neurons. Results from the mouse model indicate that if the drug works in humans, then it could be used as a means to prevent the onset of this disease or slow its progression."
The next phase will be to develop clinical trials in humans. Dr. Meredith said, "It's exciting to think that if we can protect the dopamine neurons from dying, we may prevent the disease, and improve the quality of life for patients."
Dr. Meredith has been studying Parkinson's disease for over 15 years. Her research is currently funded by the National Institute of Neurological Disorders and Stroke (NINDS), a division of the National Institutes of Health (NIH), and the Department of Defense.
Rosalind Franklin University of Medicine and Science educates medical doctors, health professionals and biomedical scientists in a personalized atmosphere. The University is located at 3333 Green Bay Road, North Chicago, IL 60064 and encompasses Chicago Medical School, College of Health professions, Dr. William M. Scholl College of Podiatric Medicine, and the School of Graduate and Postdoctoral Studies. Visit at rosalindfranklin/ and lifeindiscovery/.
Contact: Priscilla Khoury
Rosalind Franklin University of Medicine and Science
суббота, 17 сентября 2011 г.
The Vasculature Emerges As A Potential Therapeutic Target In Treating ADPKD Liver Cysts
As part of an effort to develop effective medical therapies that block the progression of liver cyst growth in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD), researchers at the University of Colorado Anschutz Medical Center have found that the liver cyst walls develop and maintain a vasculature as they grow out from the body of the liver and that factors released by epithelial cells that line the liver cyst wall lumen can drive the proliferation and development of vascular endothelial cells.
The findings, which appear in the October 2009 issue of Experimental Biology and Medicine, are the result of a multi-disciplinary team assembled by Dr. Brian Doctor. Dr. Nick Barry, a biophysicist, and Dr. Ryan McWilliams, a medical resident, employed complimentary imaging techniques to visualize and characterize the vasculature within native liver cyst walls of human ADPKD patients and pkd2(WS25/-) mice, an orthologous mouse model of ADPKD. Kelley Brodsky, a senior research associate, and Dr. Claudia Amura, a cell biologist, then used in vitro assays of endothelial cell proliferation and vascular development to demonstrate that human liver cyst fluids, which contain a variety of cytokines and growth factors secreted by the liver cyst lining epithelium, are capable of driving the angiogenic phenotype of endothelial cells. Further, inhibition of VEGF receptor signaling dramatically impeded this angiogenic phenotype. Dr. Doctor noted that "by establishing the presence of the vasculature within the enlarging liver cyst walls and defining the putative signaling pathways that induce angiogenesis within them, this study opens up an exciting new direction in the quest to develop medical therapies that can block the often devastating growth of liver cysts in patients with ADPKD".
In summary, while there are differences in their vascular density and distribution, both human ADPKD and pkd2(WS25/-) mouse liver cyst walls develop vascular structures as they grow out from the liver. In vitro studies demonstrate that angiogenic factors secreted by the liver cyst wall epithelium, including VEGF-A and IL-8, can drive angiogenic development of human endothelial cells. This development is blocked by inhibition of VEGF receptor signaling. Dr. Steven R. Goodman, Editor-in-Chief of Experimental Biology and Medicine said "The article by Brodsky and colleagues provides the important insight that the liver cyst walls maintain a vasculature as they grow out from the liver and that VEGF receptor signaling plays a key role in inducing angiogenesis. These multidisciplinary studies lay a framework for the development of new therapies aimed at preventing the growth of liver cysts in patients with ADPKD".
Notes:
Dr. R. Brian Doctor
Society for Experimental Biology and Medicine
The findings, which appear in the October 2009 issue of Experimental Biology and Medicine, are the result of a multi-disciplinary team assembled by Dr. Brian Doctor. Dr. Nick Barry, a biophysicist, and Dr. Ryan McWilliams, a medical resident, employed complimentary imaging techniques to visualize and characterize the vasculature within native liver cyst walls of human ADPKD patients and pkd2(WS25/-) mice, an orthologous mouse model of ADPKD. Kelley Brodsky, a senior research associate, and Dr. Claudia Amura, a cell biologist, then used in vitro assays of endothelial cell proliferation and vascular development to demonstrate that human liver cyst fluids, which contain a variety of cytokines and growth factors secreted by the liver cyst lining epithelium, are capable of driving the angiogenic phenotype of endothelial cells. Further, inhibition of VEGF receptor signaling dramatically impeded this angiogenic phenotype. Dr. Doctor noted that "by establishing the presence of the vasculature within the enlarging liver cyst walls and defining the putative signaling pathways that induce angiogenesis within them, this study opens up an exciting new direction in the quest to develop medical therapies that can block the often devastating growth of liver cysts in patients with ADPKD".
In summary, while there are differences in their vascular density and distribution, both human ADPKD and pkd2(WS25/-) mouse liver cyst walls develop vascular structures as they grow out from the liver. In vitro studies demonstrate that angiogenic factors secreted by the liver cyst wall epithelium, including VEGF-A and IL-8, can drive angiogenic development of human endothelial cells. This development is blocked by inhibition of VEGF receptor signaling. Dr. Steven R. Goodman, Editor-in-Chief of Experimental Biology and Medicine said "The article by Brodsky and colleagues provides the important insight that the liver cyst walls maintain a vasculature as they grow out from the liver and that VEGF receptor signaling plays a key role in inducing angiogenesis. These multidisciplinary studies lay a framework for the development of new therapies aimed at preventing the growth of liver cysts in patients with ADPKD".
Notes:
Dr. R. Brian Doctor
Society for Experimental Biology and Medicine
среда, 14 сентября 2011 г.
Protein Center's Success Nets $5.4 Million More
The University of Arkansas Center for Protein Structure and Function will receive more than $5.4 million over the next five years from the National Institutes of Health to continue the biomedical research it has established during the last decade.
The new funds will help expand the five already established core facilities within the center that support biomedical research. The money also will provide start-up funds to scientists with innovative ideas for new research projects.
"These grants will allow researchers to get some experimental evidence for their ideas so that they can write a major grant proposal," said Frank Millett, Distinguished Professor of chemistry and biochemistry and director of the center. "You have to have significant preliminary results to get funding from NIH these days."
Center projects emphasize developing a detailed understanding of the structure and function of proteins that could lead to improved treatments of human disease. Center scientists study proteins involved in cancer, heart disease, osteoporosis, the flu and other diseases and conditions.
The researchers examining these proteins use five core facilities to do their work. These include the nuclear magnetic resonance spectroscopy facility directed by James Hinton; the X-ray crystallography facility headed by Joshua Sakon; the mass spectrometry facility headed by Jackson Lay and Alan Tackett; the chemical synthesis facility headed by Robert Gawley; and the large-scale protein production facility directed by Ralph Henry. Scientists often use many or all of these facilities as they progress through different stages of a research project.
"These facilities have helped everyone involved in biomedical research on campus become competitive in getting research grants," Millett said. Researchers in the center have brought in more than $60 million in grant support since it was founded in 2000.
In addition to building the core facilities, the university has hired 12 new tenure-track faculty members since 2000, in chemistry and biochemistry and in biological sciences as well as at the University of Arkansas for Medical Sciences. The university provided substantial start-up funds for these faculty members, which helped the center succeed and get continued funding.
"The reviewers noted very positively the university commitment here," Millett said. "It was a major factor in getting all of these grants."
Millett himself is no stranger to NIH funding: He has had continuous support from the organization for the past 36 years for his research into how energy is produced in animals at the molecular level. Defects in the process of energy production lead to degenerative processes, including aging, as well as problems linked to heart disease and other medical conditions.
The Center's original senior investigators include: center director Millett, program coordinator Roger Koeppe, Bill Durham, James Hinton, Peter Pulay, Jackson Lay, Lothar Schafer and Charles Wilkins in chemistry and biochemistry. New senior investigators brought in as part of the NIH funding include Robert Gawley, Distinguished Professor of chemistry and biochemistry and Julie Stenken, professor and Twenty-First Century Chair in chemistry and biochemistry.
Mid-career investigators who started out as junior investigators include Denise Greathouse, T.K.S. Kumar, Matt McIntosh, Joshua Sakon and Wesley Stites in chemistry and biochemistry; Robyn Goforth, Ralph Henry, Michael Lehmann and Kathryn Curtin in biological sciences; and Paul Grover Miller, Kevin D. Raney and Alan Tackett in biochemistry and molecular biology at the University of Arkansas for Medical Sciences. These scientists "graduated" and got their own major research funding after receiving support from the center.
Current junior investigators include Paul Adams, Colin Heyes, Daniel Lessner and Nan Zheng in chemistry and biochemistry and Yu-Chun Du in biological sciences. All junior investigators have reduced teaching loads and can devote at least half of their time to research.
"To get faculty who are competitive for national grants, you need to be able to give them support for the first few years," Millett said. The NIH funding for major instrumentation and the university commitment to start-up funds for laboratories created an environment where these junior researchers and the entire center could succeed, he said.
The grant is funded by the Centers of Biomedical Research Excellence program of the National Center for Research Resources, a part of the National Institutes of Health.
Source: University of Arkansas
The new funds will help expand the five already established core facilities within the center that support biomedical research. The money also will provide start-up funds to scientists with innovative ideas for new research projects.
"These grants will allow researchers to get some experimental evidence for their ideas so that they can write a major grant proposal," said Frank Millett, Distinguished Professor of chemistry and biochemistry and director of the center. "You have to have significant preliminary results to get funding from NIH these days."
Center projects emphasize developing a detailed understanding of the structure and function of proteins that could lead to improved treatments of human disease. Center scientists study proteins involved in cancer, heart disease, osteoporosis, the flu and other diseases and conditions.
The researchers examining these proteins use five core facilities to do their work. These include the nuclear magnetic resonance spectroscopy facility directed by James Hinton; the X-ray crystallography facility headed by Joshua Sakon; the mass spectrometry facility headed by Jackson Lay and Alan Tackett; the chemical synthesis facility headed by Robert Gawley; and the large-scale protein production facility directed by Ralph Henry. Scientists often use many or all of these facilities as they progress through different stages of a research project.
"These facilities have helped everyone involved in biomedical research on campus become competitive in getting research grants," Millett said. Researchers in the center have brought in more than $60 million in grant support since it was founded in 2000.
In addition to building the core facilities, the university has hired 12 new tenure-track faculty members since 2000, in chemistry and biochemistry and in biological sciences as well as at the University of Arkansas for Medical Sciences. The university provided substantial start-up funds for these faculty members, which helped the center succeed and get continued funding.
"The reviewers noted very positively the university commitment here," Millett said. "It was a major factor in getting all of these grants."
Millett himself is no stranger to NIH funding: He has had continuous support from the organization for the past 36 years for his research into how energy is produced in animals at the molecular level. Defects in the process of energy production lead to degenerative processes, including aging, as well as problems linked to heart disease and other medical conditions.
The Center's original senior investigators include: center director Millett, program coordinator Roger Koeppe, Bill Durham, James Hinton, Peter Pulay, Jackson Lay, Lothar Schafer and Charles Wilkins in chemistry and biochemistry. New senior investigators brought in as part of the NIH funding include Robert Gawley, Distinguished Professor of chemistry and biochemistry and Julie Stenken, professor and Twenty-First Century Chair in chemistry and biochemistry.
Mid-career investigators who started out as junior investigators include Denise Greathouse, T.K.S. Kumar, Matt McIntosh, Joshua Sakon and Wesley Stites in chemistry and biochemistry; Robyn Goforth, Ralph Henry, Michael Lehmann and Kathryn Curtin in biological sciences; and Paul Grover Miller, Kevin D. Raney and Alan Tackett in biochemistry and molecular biology at the University of Arkansas for Medical Sciences. These scientists "graduated" and got their own major research funding after receiving support from the center.
Current junior investigators include Paul Adams, Colin Heyes, Daniel Lessner and Nan Zheng in chemistry and biochemistry and Yu-Chun Du in biological sciences. All junior investigators have reduced teaching loads and can devote at least half of their time to research.
"To get faculty who are competitive for national grants, you need to be able to give them support for the first few years," Millett said. The NIH funding for major instrumentation and the university commitment to start-up funds for laboratories created an environment where these junior researchers and the entire center could succeed, he said.
The grant is funded by the Centers of Biomedical Research Excellence program of the National Center for Research Resources, a part of the National Institutes of Health.
Source: University of Arkansas
воскресенье, 11 сентября 2011 г.
Supposed Help Against Tumors - How Glioblastoma Tumor Cells Use The Body's Protection
Glioblastoma is one of the most common but also most aggressive brain tumors, almost invariably leading to death in a short time. It consists of different cell types and their precursors, complicating successful treatment. To fight the driving force of the tumor - the tumor stem cells - scientists have been trying to initiate apoptosis in these cells. However, Dr. Ana Martin-Villalba (German Cancer Research Center, DKFZ, Heidelberg, Germany) suspects that the activated apoptosis program accelerates the progress of the disease. "The tumor growth declines when apoptosis is blocked," she reported at the conference "Brain Tumor 2008" at the Max DelbrГјck Center for Molecular Medicine (MDC) Berlin-Buch, Germany.
Glioblastomas grow like corals and form filigran branches into nearby, healthy brain tissue. For that reason it is very difficult for neurosurgeons to remove the tumor entirely because the risk of damaging healthy tissue is too high. Moreover, glioblastomas are resistant to conventional therapies which normally activate the body's apoptosis program.
This programmed cell death is a vital process. It plays an important role during development but also in the adult organism. Together with its partner CD95L, the molecular switch CD95 ensures that sick or abnormal cells are removed. Once activated, CD95 triggers a chain of different signals which in the end lead to the death of the damaged cell. Until recently, scientists were convinced that triggering apoptosis in brain tumors was a useful tool for not only killing the tumor but also the cells of its origin - the tumor stem cells.
The scientist from Heidelberg could show that CD95 as well as its partner CD95L is active in the tumor cells. However, the cells do not die. "Instead, the signal stimulates the tumor cells to migrate into neighboring, healthy brain regions," Dr. Martin-Villalba explained. For instance, it activates the protein MMP which "drills" its way into the brain tissue. "Contrary to our expectations," the neuroscientist said, "what we find when we activate apoptosis in the tumor cells is that we help them spread into healthy nerve tissue."
In experiments with mice, the researchers could already show that the tumor proliferates less aggressively when they block CD95L with an antibody, thus inhibiting the activation of programmed cell death. "With this changed perspective, we hope to develop new ideas for tumor therapy in the future," Dr. Martin-Villalba said.
Altogether, about 180 scientists and clinicians from Europe and the USA came to the two-day conference, which ended this Friday afternoon. The organizers were the MDC, the Charité - Universitätsmedizin Berlin, and HELIOS Kliniken GmbH, Berlin, a private clinic in Berlin-Buch.
Barbara Bachtler
Press and Public Affairs
Max DelbrГјck Center for Molecular Medicine (MDC) Berlin-Buch
Robert-Rössle-Straße 10; 13125 Berlin; Germany
mdc-berlin
Further information:
dkfz-heidelberg/en/molekulare-neurobiologie/index.html
cell/cancer-cell/retrieve/pii/S1535610808000433
Glioblastomas grow like corals and form filigran branches into nearby, healthy brain tissue. For that reason it is very difficult for neurosurgeons to remove the tumor entirely because the risk of damaging healthy tissue is too high. Moreover, glioblastomas are resistant to conventional therapies which normally activate the body's apoptosis program.
This programmed cell death is a vital process. It plays an important role during development but also in the adult organism. Together with its partner CD95L, the molecular switch CD95 ensures that sick or abnormal cells are removed. Once activated, CD95 triggers a chain of different signals which in the end lead to the death of the damaged cell. Until recently, scientists were convinced that triggering apoptosis in brain tumors was a useful tool for not only killing the tumor but also the cells of its origin - the tumor stem cells.
The scientist from Heidelberg could show that CD95 as well as its partner CD95L is active in the tumor cells. However, the cells do not die. "Instead, the signal stimulates the tumor cells to migrate into neighboring, healthy brain regions," Dr. Martin-Villalba explained. For instance, it activates the protein MMP which "drills" its way into the brain tissue. "Contrary to our expectations," the neuroscientist said, "what we find when we activate apoptosis in the tumor cells is that we help them spread into healthy nerve tissue."
In experiments with mice, the researchers could already show that the tumor proliferates less aggressively when they block CD95L with an antibody, thus inhibiting the activation of programmed cell death. "With this changed perspective, we hope to develop new ideas for tumor therapy in the future," Dr. Martin-Villalba said.
Altogether, about 180 scientists and clinicians from Europe and the USA came to the two-day conference, which ended this Friday afternoon. The organizers were the MDC, the Charité - Universitätsmedizin Berlin, and HELIOS Kliniken GmbH, Berlin, a private clinic in Berlin-Buch.
Barbara Bachtler
Press and Public Affairs
Max DelbrГјck Center for Molecular Medicine (MDC) Berlin-Buch
Robert-Rössle-Straße 10; 13125 Berlin; Germany
mdc-berlin
Further information:
dkfz-heidelberg/en/molekulare-neurobiologie/index.html
cell/cancer-cell/retrieve/pii/S1535610808000433
четверг, 8 сентября 2011 г.
Molecular Clues To Wilson Disease Discovered By Rice Lab
Using a combination of computer simulations and cutting-edge lab experiments, physical biochemists at Rice University have discovered how a small genetic mutation -- which is known to cause Wilson disease -- subtly changes the structure of a large, complex protein that the body uses to keep copper from building up to toxic levels.
"The protein we study is like a big puzzle," said lead author Agustina Rodriguez-Granillo, the Rice doctoral student in biochemistry and cell biology who carried out the mathematical simulations and laboratory research. "The mutation that causes most cases of Wilson disease is well-known, but our study looks at the overall puzzle to see how such a small mutation can alter the shape and function of such a large and complex protein."
The protein in question is called ATP7B, which is a multidomain protein that sits in an internal membrane and regulates the movement of copper atoms inside human cells. Though large quantities of copper can be toxic, our bodies need a small amount for key enzymes involved in, for example, respiration and brain functions. ATP7B acts something like a warehouse manager, locking up bulk quantities of copper and handing it out for use in these proteins.
Wilson disease is a genetic disorder that alters the ATP7B protein's ability to work, causing copper to build up to toxic levels in the liver, brain, eyes and other organs. Over time the disease can cause life-threatening organ damage. Wilson disease affects as many as 150,000 people worldwide.
The new study is available online from the Journal of Molecular Biology. It focused on the genetic flaw that causes most cases of Wilson disease. That flaw, known as H1069Q, is caused when just one out of the more than 1,400 amino acids in ATP7B is changed. That amino acid is a histidine located at position 1069. In the disease-causing form of the protein, this histidine is replaced with a glutamic acid.
"This mutation occurs at a crucial location where the protein typically binds with a molecule called ATP that provides the energy the protein needs to move copper from place to place," said study co-author Pernilla Wittung-Stafshede, an adjunct professor of biochemistry and cell biology at Rice and Rodriguez-Granillo's adviser. Wittung-Stafshede, professor in chemistry at Umea University in Sweden, said, "Past studies have compared the behavior of the mutant protein with that of the nonmutant and found very little difference, so it was unclear how this small change led to the devastating effects that are seen in Wilson disease."
Using a combination of experimental data and computer simulations that looked specifically at a portion of the protein called the N-domain, where the H1069Q mutation occurs, Wittung-Stafshede, Rodriguez-Granillo and postdoctoral researcher Erik Sedlak (now at the University of Texas at San Antonio) confirmed that ATP's function was significantly reduced in the mutant form of the protein. They also found that the mutation caused structural changes in other sections of the protein that were far away from the mutation site. For example, the healthy form of the protein is capped with a large, flexible loop. The purpose of the loop is unknown, but its shape is altered and more compact in the diseased form of the protein.
"This implies that the loop has some importance, perhaps in regulation of ATP7B's activities, and we intend to follow up on this in our future studies," Rodriguez-Granillo said.
The research was supported by the Robert A. Welch Foundation.
Source: Jade Boyd
Rice University
"The protein we study is like a big puzzle," said lead author Agustina Rodriguez-Granillo, the Rice doctoral student in biochemistry and cell biology who carried out the mathematical simulations and laboratory research. "The mutation that causes most cases of Wilson disease is well-known, but our study looks at the overall puzzle to see how such a small mutation can alter the shape and function of such a large and complex protein."
The protein in question is called ATP7B, which is a multidomain protein that sits in an internal membrane and regulates the movement of copper atoms inside human cells. Though large quantities of copper can be toxic, our bodies need a small amount for key enzymes involved in, for example, respiration and brain functions. ATP7B acts something like a warehouse manager, locking up bulk quantities of copper and handing it out for use in these proteins.
Wilson disease is a genetic disorder that alters the ATP7B protein's ability to work, causing copper to build up to toxic levels in the liver, brain, eyes and other organs. Over time the disease can cause life-threatening organ damage. Wilson disease affects as many as 150,000 people worldwide.
The new study is available online from the Journal of Molecular Biology. It focused on the genetic flaw that causes most cases of Wilson disease. That flaw, known as H1069Q, is caused when just one out of the more than 1,400 amino acids in ATP7B is changed. That amino acid is a histidine located at position 1069. In the disease-causing form of the protein, this histidine is replaced with a glutamic acid.
"This mutation occurs at a crucial location where the protein typically binds with a molecule called ATP that provides the energy the protein needs to move copper from place to place," said study co-author Pernilla Wittung-Stafshede, an adjunct professor of biochemistry and cell biology at Rice and Rodriguez-Granillo's adviser. Wittung-Stafshede, professor in chemistry at Umea University in Sweden, said, "Past studies have compared the behavior of the mutant protein with that of the nonmutant and found very little difference, so it was unclear how this small change led to the devastating effects that are seen in Wilson disease."
Using a combination of experimental data and computer simulations that looked specifically at a portion of the protein called the N-domain, where the H1069Q mutation occurs, Wittung-Stafshede, Rodriguez-Granillo and postdoctoral researcher Erik Sedlak (now at the University of Texas at San Antonio) confirmed that ATP's function was significantly reduced in the mutant form of the protein. They also found that the mutation caused structural changes in other sections of the protein that were far away from the mutation site. For example, the healthy form of the protein is capped with a large, flexible loop. The purpose of the loop is unknown, but its shape is altered and more compact in the diseased form of the protein.
"This implies that the loop has some importance, perhaps in regulation of ATP7B's activities, and we intend to follow up on this in our future studies," Rodriguez-Granillo said.
The research was supported by the Robert A. Welch Foundation.
Source: Jade Boyd
Rice University
понедельник, 5 сентября 2011 г.
Kinexus Announces The Launch Of A New Protein Kinase Microarray
Kinexus Bioinformatics Corporation announced the commercial release of its novel Protein Kinase Microarray with 200 recombinant human protein kinases for screening. The microarray has wide utility including applications for drug target counter screening, to identify novel kinase substrates, establish kinase antibody specificities, and for the discovery and testing of protein kinase-protein and protein kinase-compound interactions.
With these new and unique kinase microarray services, clients can now inexpensively assay the abilities of their lead compounds to inhibit any of the 200 different protein kinases for as low as $1.65 per kinase with triplicate measurements. Industry standards for these types of measurements typically cost $4-5 per kinase tracked. This counter screening of kinases can be used to establish the specificity of promising therapeutic inhibitors or target kinases in much more cost effective manner than previously available. All compounds can be further validated for direct activity effects in vitro with the Kinase-Inhibitor Profiling Services offered by Kinexus. This complements the Kinex(TM) 800 Antibody Microarray Services, in which endogenous physiological substrates of target kinases can also be defined to measure the effects of drug leads in vivo in cultured cells and tissues from treated animals. The specificity of suspected protein kinase-protein interactions can also be investigated with the new kinase microarray.
"Protein kinases are well recognized by the pharmaceutical and biotech industry as highly productive targets for drugs with the potentially treat over 400 human diseases, commented Dr. Steven Pelech, President and Chief Scientific Officer of Kinexus. "With 516 human protein kinases and only about 75 that have been seriously targeted by the pharmaceutical industry so far, there are exciting possibilities for the identification of new kinase drug targets and new applications for existing drugs with this type of technology".
Source: Kinexus Bioinformatics Corporation
With these new and unique kinase microarray services, clients can now inexpensively assay the abilities of their lead compounds to inhibit any of the 200 different protein kinases for as low as $1.65 per kinase with triplicate measurements. Industry standards for these types of measurements typically cost $4-5 per kinase tracked. This counter screening of kinases can be used to establish the specificity of promising therapeutic inhibitors or target kinases in much more cost effective manner than previously available. All compounds can be further validated for direct activity effects in vitro with the Kinase-Inhibitor Profiling Services offered by Kinexus. This complements the Kinex(TM) 800 Antibody Microarray Services, in which endogenous physiological substrates of target kinases can also be defined to measure the effects of drug leads in vivo in cultured cells and tissues from treated animals. The specificity of suspected protein kinase-protein interactions can also be investigated with the new kinase microarray.
"Protein kinases are well recognized by the pharmaceutical and biotech industry as highly productive targets for drugs with the potentially treat over 400 human diseases, commented Dr. Steven Pelech, President and Chief Scientific Officer of Kinexus. "With 516 human protein kinases and only about 75 that have been seriously targeted by the pharmaceutical industry so far, there are exciting possibilities for the identification of new kinase drug targets and new applications for existing drugs with this type of technology".
Source: Kinexus Bioinformatics Corporation
пятница, 2 сентября 2011 г.
Researchers Hot On The Trail Of Brain Cell Degeneration
A research team headed by Academy Research Fellow Michael Courtney has identified a new molecular pathway in neurons. The pathway is a factor in the degeneration of brain cells, which in turn plays an important role in neurological conditions and diseases, such as Alzheimer's disease, epilepsy and stroke. Courtney and his team, based at the A. I. Virtanen Institute of the University of Kuopio, joined forces with Docent Eleanor Coffey's team at the Turku Centre for Biotechnology to carry out the study as part of a series of successful collaborations between the two teams. The results of their study are published in the latest issue of Nature Neuroscience.
In a number of neurodegenerative diseases, neurons in the brain are over-stimulated, which triggers programmed cell death, or apoptosis. The study shows that the Rho protein, which has long been recognised as an important player in cancer formation, also plays a key role in the destruction of neurons in disease.
"These surprising findings add an entire pathway to the map of neurodegenerative signalling processes," says Courtney. "This area of investigation could therefore offer novel therapeutic strategies for neurodegenerative diseases".
Targeting molecular signals
How neurons actually die has been unclear. It is likely that it is associated with a variety of different mechanisms. Research has shown that the destruction of cells be over-stimulation depends on excess entry of calcium into the cells. Researchers have long been trying to map how cells generate destruction signals in response to the calcium, in the hope of finding new targets for drug design.
The object of the study, the Rho protein, belongs to a family of proteins able to influence signals that had been linked to cell degeneration. The two teams' analysis demonstrated that over-stimulation causes activation of Rho as well as cell destruction signals. Blocking Rho activity by genetic modification keeps the protein in an inactive state, and the nerve cells thus survive a previously toxic level of over-stimulation.
The study identifies a new factor provoking cell degeneration. It is more than likely that future research will uncover more such factors interacting with each other. Investigating these will benefit new forms of treatment and advance research that aims to alleviate symptoms. The researchers behind the study hope that the results can be used in planning new targets for drugs to reduce the cell destruction signals caused by calcium entry. Finding new targets for medicine development is also significant in terms of the economy, owing to the costly treatment of these diseases, both in Finland and globally.
Cooperation between biocentres gets research going
The teams' study is a perfect example of the cooperation between biocentres in Finland (Biocenter Finland) and international networking. The research was funded mainly by the Academy of Finland and the European Union. The two research teams are part of a Europe-wide consortium, STRESSPROTECT, within the EU Sixth Framework Programme. The consortium aims at generating the basis for novel neuroprotective drugs for neurodegenerative conditions involving over-stimulation of neurons (neuroprotect.eu).
SUOMEN AKATEMIA (ACADEMY OF FINLAND)
Vilhonvuorenkatu 6
PO Box 99
00 501 Helsinki
aka.fi/
In a number of neurodegenerative diseases, neurons in the brain are over-stimulated, which triggers programmed cell death, or apoptosis. The study shows that the Rho protein, which has long been recognised as an important player in cancer formation, also plays a key role in the destruction of neurons in disease.
"These surprising findings add an entire pathway to the map of neurodegenerative signalling processes," says Courtney. "This area of investigation could therefore offer novel therapeutic strategies for neurodegenerative diseases".
Targeting molecular signals
How neurons actually die has been unclear. It is likely that it is associated with a variety of different mechanisms. Research has shown that the destruction of cells be over-stimulation depends on excess entry of calcium into the cells. Researchers have long been trying to map how cells generate destruction signals in response to the calcium, in the hope of finding new targets for drug design.
The object of the study, the Rho protein, belongs to a family of proteins able to influence signals that had been linked to cell degeneration. The two teams' analysis demonstrated that over-stimulation causes activation of Rho as well as cell destruction signals. Blocking Rho activity by genetic modification keeps the protein in an inactive state, and the nerve cells thus survive a previously toxic level of over-stimulation.
The study identifies a new factor provoking cell degeneration. It is more than likely that future research will uncover more such factors interacting with each other. Investigating these will benefit new forms of treatment and advance research that aims to alleviate symptoms. The researchers behind the study hope that the results can be used in planning new targets for drugs to reduce the cell destruction signals caused by calcium entry. Finding new targets for medicine development is also significant in terms of the economy, owing to the costly treatment of these diseases, both in Finland and globally.
Cooperation between biocentres gets research going
The teams' study is a perfect example of the cooperation between biocentres in Finland (Biocenter Finland) and international networking. The research was funded mainly by the Academy of Finland and the European Union. The two research teams are part of a Europe-wide consortium, STRESSPROTECT, within the EU Sixth Framework Programme. The consortium aims at generating the basis for novel neuroprotective drugs for neurodegenerative conditions involving over-stimulation of neurons (neuroprotect.eu).
SUOMEN AKATEMIA (ACADEMY OF FINLAND)
Vilhonvuorenkatu 6
PO Box 99
00 501 Helsinki
aka.fi/
Подписаться на:
Комментарии (Atom)