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August 17, 2020
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https://www.sciencedaily.com/releases/2020/08/200817123052.htm
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Bacteria's secret weapon revealed
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Monash Biomedicine Discovery Institute (BDI) scientists have discovered a previously unknown method used by bacteria to evade immune responses.
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The study, published in First author Dr Pankaj Deo said researchers in Dr Thomas Naderer's laboratory took a different approach to understanding the process by which bacteria release toxins that disarm the 'power-house' mitochondria in immune cells.The study showed that immune cells sense that their mitochondria are no longer functional during infections, which triggers apoptosis. "Ironically, it is the activation of host cell death factors that deliver the final blow to mitochondria which induces apoptosis, not the bacterial toxins themselves," Dr Pankaj said.The researchers genetically targeted apoptotic factors and showed that they were able to reduce inflammation in mice, which increased health outcomes.They used the bacterial pathogens Neisseria gonorrhoeae, uropathogenic Escherichia coli and the deadly Pseudomonas aeruginosa, prevalent in hospitals and which can be multi-drug resistant. However, the findings would apply to other species of bacteria too, Dr Deo said.Dr Naderer, who oversaw the research, said that understanding the ways some bacterial infections evade immune response by targeting mitochondria opens new therapeutic possibilities."There's been a lot of effort trying to block endotoxins that kill immune cells but this study really shifts the focus onto different toxins that might be more important," Dr Naderer said."It gives us a few good leads that we can look at as a next step," he said."We've shown in this paper that we can accelerate the immune response," he said. "The other side is that if that response persists and we get constant inflammation -- which is usually associated with bacterial infection and which causes a lot of tissue damage -- we have a new way to shut down that tissue-damaging inflammation.""What scientists have thought before is that when endotoxins are released by bacteria they induce an inflammatory type of programmed cell death called pyroptosis in immune cells," Dr Deo said. Endotoxins are part of the external cell wall of essentially all Gram-negative bacteria."We've found that the pathogenic bacteria use a similar mechanism to release additional toxins," he said. "They kill immune cells by releasing small surface structures called outer membrane vesicles -- packages of toxins that target mitochondria. The mitochondria are disarmed, become dysfunctional then die according to apoptosis or cellular suicide."The scientists will investigate drugs that are now advancing to the clinic, and at re-purposing drugs already in use, perhaps as anti-cancer treatments, to see if they can be used to clear bacterial infections.
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Biotechnology
| 2,020 |
August 14, 2020
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https://www.sciencedaily.com/releases/2020/08/200814142943.htm
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Watching changes in plant metabolism -- live
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Almost all life on Earth, in particular our food and our health, depend on metabolism in plants. In order to understand how these metabolic processes function, researchers at the Institute of the Biology and Biotechnology of Plants at the University of Münster with the participation of the University of Bonn are studying key mechanisms in the regulation of energy metabolism. Now, for the first time, a new method of in vivo biosensor technology has enabled them to monitor in real time what effects environmental changes -- for example, light, temperature, aridity, flooding or pest infestation -- have on the central metabolism of the model plant Arabidopsis thaliana (thale cress). The study has appeared as an advance publication in the journal
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The team of researchers expressed a genetically coded sensor inside the plants in order to make central metabolic process literally 'visible'. "Because plants appear from the outside to be very static, they have to be superfast masters of flexibility and adaptation within their cells," says Dr. Janina Steinbeck, lead author of the study. "We're now able to observe those dynamics live in the living plant." In order to measure the metabolic process in the plant and produce images of it, the researchers used in vivo biosensoring, a method for studying living organisms, tissue or cells in real time. The biosensor consists of a biological recognition element, a protein which specifically binds a molecule to be detected, and a read-out element, a protein which translates the binding to the recognition element into a light signal. The biosensor now being used was originally developed for use in nerve cells. The researchers refined this sensor and developed it so that it could be used in plants.The sensor can directly bind and then release the molecules NAD+ and NADH. The so-called NAD redox system is of paramount importance for electron transfer during metabolism in almost all living things. The sensor consists of a fluorescent blue-green protein and a red one, both of which change their brightness depending on the NAD status in the cell. The sensor read-out in living cells is carried out with a modern confocal laser scanning microscope. The possibility of using NAD in vivo sensing in plants opens up new options for plant researchers. "For us, this new method is an achievement regarding the methodology because now we can gain a direct understanding of metabolic processes precisely where they occur in the plant," explains Prof. Markus Schwarzländer, who heads the Plant Energy Biology working group at the University of Münster. "For example, it was a complete surprise for us to observe that such a key process as NAD metabolism changes so fundamentally during an immune reaction," he adds.Up to now, it had only been possible for the researchers to study this type of metabolic processes by obtaining extracts from the plants and analysing them with biochemical methods. In this approach, however, cells and tissue are destroyed, and it is no longer possible to trace where exactly the metabolic changes occurred. Now, the researchers can track dynamic changes in the redox metabolism -- which, among other functions, serves to provide energy in the cells -- from specific cell compartments, here in the cytosol, in the individual cells, up to complete organs in intact living plants. This approach makes it possible to create a first NAD redox map of the whole plant and to observe redox dynamics in transitions from light to dark as well as changes in the sugar status, cell respiration and oxygen supply. "As a result, it becomes apparent just how directly metabolism and environment are linked," says Markus Schwarzländer. "What was especially exciting was the new connection to the immune response, which we previously had practically no idea about, and which now needs to be studied in more depth."At almost the same time as the publication in
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Biotechnology
| 2,020 |
August 13, 2020
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https://www.sciencedaily.com/releases/2020/08/200813162126.htm
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To understand the machinery of life, this scientist breaks it on purpose
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"I'm fascinated with life, and that's why I want to break it."
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This is how Betül Kaçar, an assistant professor at the University of Arizona with appointments in the Department of Molecular and Cellular Biology, Department of Astronomy and the Lunar and Planetary Laboratory, describes her research. What may sound callous is a legitimate scientific approach in astrobiology. Known as ancestral sequencing, the idea is to "resurrect" genetic sequences from the dawn of life, put them to work in the cellular pathways of modern microbes -- think Jurassic Park but with extinct genes in place of dinosaurs, and study how the organism copes.In a recent paper published in the Kaçar uses ancestral sequencing to find out what makes life tick and how organisms are shaped by evolutionary selection pressure. The insights gained may, in turn, offer clues as to what it takes for organic precursor molecules to give rise to life -- be it on Earth or faraway worlds. In her lab, Kaçar specializes in designing molecules that act like tiny invisible wrenches, wreaking havoc with the delicate cellular machinery that allows organisms to eat, move and multiply -- in short, to live.Kaçar has focused her attention on the translation machinery, a labyrinthine molecular clockwork that translates the information encoded in the bacteria's DNA into proteins. All organisms -- from microbes to algae to trees to humans -- possess this piece of machinery in their cells."We approximate everything about the past based on what we have today," Kaçar said. "All life needs a coding system -- something that takes information and turns it into molecules that can perform tasks -- and the translational machinery does just that. It creates life's alphabet. That's why we think of it as a fossil that has remained largely unchanged, at least at its core. If we ever find life elsewhere, you bet that the first thing we'll look at is its information processing systems, and the translational machinery is just that."So critical is the translational machinery to life on Earth that even over the course of more than 3.5 billion years of evolution, its parts have undergone little substantial change. Scientists have referred to it as "an evolutionary accident frozen in time.""I guess I tend to mess with things I'm not supposed to," Kaçar said. "Locked in time? Let's unlock it. Breaking it would lead the cell to destruction? Let's break it."The researchers took six different strains of Escherichia coli bacteria and genetically engineered the cells with mutated components of their translational machinery. They targeted the step that feeds the unit with genetic information by swapping the shuttle protein with evolutionary cousins taken from other microbes, including a reconstructed ancestor from about 700 million years ago."We get into the heart of the heart of what we think is one of the earliest machineries of life," Kaçar said. "We purposely break it a little, and a lot, to see how the cells deal with this problem. In doing this, we think we create an urgent problem for the cell, and it will fix that."Next, the team mimicked evolution by having the manipulated bacterial strains compete with each other -- like a microbial version of "The Hunger Games." A thousand generations later, some strains fared better than others, as was expected. But when Kaçar's team analyzed exactly how the bacteria responded to perturbations in their translational components, they discovered something unexpected: Initially, natural selection improved the compromised translational machinery, but its focus shifted away to other cellular modules before the machinery's performance was fully restored.To find out why, Kaçar enlisted Sandeep Venkataram, a population genetics expert at the University of California, San Diego.Venkataram likens the process to a game of whack-a-mole, with each mole representing a cellular module. Whenever a module experiences a mutation, it pops up. The hammer smashing it back down is the action of natural selection. Mutations are randomly spread across all modules, so that all moles pop up randomly."We expected that the hammer of natural selection also comes down randomly, but that is not what we found," he said. "Rather, it does not act randomly but has a strong bias, favoring those mutations that provide the largest fitness advantage while it smashes down other less beneficial mutations, even though they also provide a benefit to the organism."In other words, evolution is not a multitasker when it comes to fixing problems."It seems that evolution is myopic," Venkataram said. "It focuses on the most immediate problem, puts a Band-Aid on and then it moves on to the next problem, without thoroughly finishing the problem it was working on before.""It turns out the cells do fix their problems but not in the way we might fix them," Kaçar added. "In a way, it's a bit like organizing a delivery truck as it drives down a bumpy road. You can stack and organize only so many boxes at a time before they inevitably get jumbled around. You never really get the chance to make any large, orderly arrangement."Why natural selection acts in this way remains to be studied, but what the research showed is that, overall, the process results in what the authors call "evolutionary stalling" -- while evolution is busy fixing one problem, it does at the expense of all other issues that need fixing. They conclude that at least in rapidly evolving populations, such as bacteria, adaptation in some modules would stall despite the availability of beneficial mutations. This results in a situation in which organisms can never reach a fully optimized state."The system has to be capable of being less than optimal so that evolution has something to act on in the face of disturbance -- in other words, there needs to be room for improvement," Kaçar said.Kaçar believes this feature of evolution may be a signature of any self-organizing system, and she suspects that this principle has counterparts at all levels of biological hierarchy, going back to life's beginnings, possibly even to prebiotic times when life had not yet materialized.With continued funding from the John Templeton Foundation and NASA, the research group is now working on using ancestral sequencing to go back even further in time, Kaçar said."We want to strip things down even more and create systems that start out as what we would consider pre-life and then transition into what we consider life."
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Biotechnology
| 2,020 |
August 13, 2020
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https://www.sciencedaily.com/releases/2020/08/200813155829.htm
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Busting up the infection cycle of hepatitis b
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Researchers at the University of Delaware, using supercomputing resources and collaborating with scientists at Indiana University, have gained new understanding of the virus that causes hepatitis B and the "spiky ball" that encloses the virus's genetic blueprint.
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The research, which has been published online, ahead of print, by the American Chemical Association journal ACS Chemical Biology, provides insights into how the capsid -- a protein shell that protects the blueprint and also drives the delivery of it to infect a host cell -- assembles itself.Computer simulations performed by the UD scientists investigated the effects of a mutation that impairs the assembly process. Together with collaborators, the researchers revealed that the region of the protein that contains the mutation, the spike, can communicate with the region of the protein that links with other subunits to assemble the capsid. They found evidence that a change in the shape of the capsid protein switches it into an "on" state for assembly.Scientists believe that the capsid is an important target in developing drugs to treat hepatitis B, a life-threatening and incurable infection that afflicts more than 250 million people worldwide."The capsid looks like a spiky ball, with 120 protein dimers that assemble to form it; each dimer contains a spike," said Jodi A. Hadden-Perilla, assistant professor in UD's Department of Chemistry and Biochemistry and a co-author of the new paper. "The capsid is key to the virus infection cycle. If we could disrupt the assembly process, the virus wouldn't be able to produce infectious copies of itself."The Indiana University researchers had been studying the dimers, which are two-part, T-shaped molecular structures, and investigating whether a mutation could activate or deactivate a switch to turn on the capsid's assembly mechanism. They worked with Hadden-Perilla's group, which ran computer simulations to explain how changes in the protein structure induced by the mutation affected the capsid's ability to assemble."What we learned is that this mutation disrupts the structure of the spike at the top of the dimer," Hadden-Perilla said. "This mutation slows down assembly, which actually involves a region of the protein that is far away from the spike. It's clear that these two regions are connected. A change in the shape of the protein, particularly at the spike, may actually activate or deactivate assembly."Her team did its work using the National Science Foundation-supported Blue Waters supercomputer at the University of Illinois at Urbana-Champaign, the largest supercomputer on any university campus in the world, to perform what are known as all-atom molecular dynamics simulations.Molecular dynamics simulations allow researchers to study the way molecules move in order to learn how they carry out their functions in nature. Computer simulations are the only method that can reveal the motion of molecular systems down to the atomic level and are sometimes referred to as the "computational microscope."The paper, titled "The integrity of the intradimer interface of the Hepatitis B Virus capsid protein dimer regulates capsid self-assembly," can be viewed on the journal's website.From Colombia to UD For doctoral student Carolina Pérez Segura, a co-author of the paper, working with data from the supercomputer simulations was the kind of research experience that first brought her to the University of Delaware and then inspired her to stay.She examined numerous simulations and vast amounts of data to investigate the effect of the mutation and "made some important discoveries," Hadden-Perilla said. "We threw her into the deep end in my brand-new research group [last summer], and she did a great job."Pérez Segura came to UD as a participant in the University's Latin American Summer Research Program. A graduate of the Universidad Nacional de Colombia (National University of Colombia), the program marked her first time leaving Colombia and, indeed, her first time traveling by plane. She planned to conduct research under Hadden-Perilla's mentorship for a couple of months and then return home.But, she said, the experience was so meaningful to her that she canceled her plane ticket home and stayed on to work as a visiting scholar with Hadden-Perilla while applying to UD's doctoral program in chemistry. She was accepted and began her studies during spring semester.It was her fascination with computational chemistry that brought her to Delaware, she said, and the work with supercomputers that made her decide to continue that research."While I was an undergraduate, I chose that branch of chemistry as the kind of career I wanted," said Pérez Segura, who worked with a research group in the field, on a smaller scale, in Colombia. "When I was introduced to the idea that math and physics can help you understand biological processes, I knew that was what I wanted to do."I thought it was really amazing to be able to explain biological processes with numbers and computers. I wanted to learn more, and here, there's so much more opportunity to learn it."Although the social and travel restrictions imposed by the coronavirus (COVID-19) pandemic have limited her ability to fully experience American life and culture, she said her experience at UD remains very positive. She's eager to be able to go out more, practice her English and feel a part of American culture, but meanwhile, she's busy with exciting research, she said.She's currently also working on research that Hadden-Perilla is conducting into the virus that causes COVID-19.
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Biotechnology
| 2,020 |
August 13, 2020
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https://www.sciencedaily.com/releases/2020/08/200813155827.htm
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Loss of a specific enzyme boosts fat metabolism and exercise endurance in mice
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Sugars and fats are the primary fuels that power every cell, tissue and organ. For most cells, sugar is the energy source of choice, but when nutrients are scarce, such as during starvation or extreme exertion, cells will switch to breaking down fats instead.
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The mechanisms for how cells rewire their metabolism in response to changes in resource availability are not yet fully understood, but new research reveals a surprising consequence when one such mechanism is turned off: an increased capacity for endurance exercise.In a study published in the Aug. 4 issue of Remarkably, blocking PHD3 production in mice leads to dramatic improvements in certain measures of fitness, the research showed. Compared with their normal littermates, mice lacking the PHD3 enzyme ran 40 percent longer and 50 percent farther on treadmills and had higher VO2 max, a marker of aerobic endurance that measures the maximum oxygen uptake during exercise.The findings shed light on a key mechanism for how cells metabolize fuels and offer clues toward a better understanding of muscle function and fitness, the authors said."Our results suggest that PHD3 inhibition in whole body or skeletal muscle is beneficial for fitness in terms of endurance exercise capacity, running time and running distance," said senior study author Marcia Haigis, professor of cell biology in the Blavatnik Institute at HMS. "Understanding this pathway and how our cells metabolize energy and fuels potentially has broad applications in biology, ranging from cancer control to exercise physiology."However, further studies are needed to elucidate whether this pathway can be manipulated in humans to improve muscle function in disease settings, the authors said.Haigis and colleagues set out to investigate the function of PHD3, an enzyme that they had found to play a role regulating fat metabolism in certain cancers in previous studies. Their work showed that, under normal conditions, PHD3 chemically modifies another enzyme, ACC2, which in turn prevents fatty acids from entering mitochondria to be broken down into energy.In the current study, the researchers' experiments revealed that PHD3 and another enzyme called AMPK simultaneously control the activity of ACC2 to regulate fat metabolism, depending on energy availability.In isolated mouse cells grown in sugar-rich conditions, the team found that PHD3 chemically modifies ACC2 to inhibit fat metabolism. Under low-sugar conditions, however, AMPK activates and places a different, opposing chemical modification on ACC2, which represses PHD3 activity and allows fatty acids to enter the mitochondria to be broken down for energy.These observations were confirmed in live mice that were fasted to induce energy-deficient conditions. In fasted mice, the PHD3-dependent chemical modification to ACC2 was significantly reduced in skeletal and heart muscle, compared to fed mice. By contrast, the AMPK-dependent modification to ACC2 increased.Next, the researchers explored the consequences when PHD3 activity was inhibited, using genetically modified mice that do not express PHD3. Because PHD3 is most highly expressed in skeletal muscle cells and AMPK has previously been shown to increase energy expenditure and exercise tolerance, the team carried out a series of endurance exercise experiments."The question we asked was if we knock out PHD3," Haigis said, "would that increase fat burning capacity and energy production and have a beneficial effect in skeletal muscle, which relies on energy for muscle function and exercise capacity?"To investigate, the team trained young, PHD3-deficient mice to run on an inclined treadmill. They found that these mice ran significantly longer and further before reaching the point of exhaustion, compared to mice with normal PHD3. These PHD3-deficient mice also had higher oxygen consumption rates, as reflected by increased VO2 and VO2 max.After the endurance exercise, the muscles of PHD3-deficient mice had increased rates of fat metabolism and an altered fatty acid composition and metabolic profile. The PHD3-dependent modification to ACC2 was nearly undetectable, but the AMPK-dependent modification increased, suggesting that changes to fat metabolism play a role in improving exercise capacity.These observations held true in mice genetically modified to specifically prevent PHD3 production in skeletal muscle, demonstrating that PHD3 loss in muscle tissues is sufficient to boost exercise capacity, according to the authors."It was exciting to see this big, dramatic effect on exercise capacity, which could be recapitulated with a muscle-specific PHD3 knockout," Haigis said. "The effect of PHD3 loss was very robust and reproducible."The research team also performed a series of molecular analyses to detail the precise molecular interactions that allow PHD3 to modify ACC2, as well as how its activity is repressed by AMPK.The study results suggest a new potential approach for enhancing exercise performance by inhibiting PHD3. While the findings are intriguing, the authors note that further studies are needed to better understand precisely how blocking PHD3 causes a beneficial effect on exercise capacity.In addition, Haigis and colleagues found in previous studies that in certain cancers, such as some forms of leukemia, mutated cells express significantly lower levels of PHD3 and consume fats to fuel aberrant growth and proliferation. Efforts to control this pathway as a potential strategy for treating such cancers may help inform research in other areas, such as muscle disorders.It remains unclear whether there are any negative effects of PHD3 loss. To know whether PHD3 can be manipulated in humans -- for performance enhancement in athletic activities or as a treatment for certain diseases -- will require additional studies in a variety of contexts, the authors said.It also remains unclear if PHD3 loss triggers other changes, such as weight loss, blood sugar and other metabolic markers, which are now being explored by the team."A better understanding of these processes and the mechanisms underlying PHD3 function could someday help unlock new applications in humans, such as novel strategies for treating muscle disorders," Haigis said.Additional authors on the study include Haejin Yoon, Jessica Spinelli, Elma Zaganjor, Samantha Wong, Natalie German, Elizabeth Randall, Afsah Dean, Allen Clermont, Joao Paulo, Daniel Garcia, Hao Li, Olivia Rombold, Nathalie Agar, Laurie Goodyear, Reuben Shaw, Steven Gygi and Johan Auwerx.The study was supported by the National Institutes of Health (grants R01CA213062, P30DK036836, R25 CA-89017 and P41 EB015898), Ludwig Center at Harvard Medical School, Glenn Foundation for Medical Research, Ecole Polytechnique Fédérale de Lausanne and the Fondation Suisse de Recherche sur les Maladies Musculaires.
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Biotechnology
| 2,020 |
August 13, 2020
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https://www.sciencedaily.com/releases/2020/08/200813155825.htm
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Bacterial enzymes hijacked to create complex molecules normally made by plants
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Chemists at Scripps Research have efficiently created three families of complex, oxygen-containing molecules that are normally obtainable only from plants.
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These molecules, called terpenes, are potential starting points for new drugs and other high-value products -- marking an important development for multiple industries. In addition, the new approach could allow chemists to build many other classes of compounds.The chemistry feat is detailed in the Aug. 13 edition of the journal The key to this new method of making molecules is the harnessing, or hijacking, of natural enzymes -- from bacteria, in this case -- to assist in complex chemical transformations that have been impractical or impossible with synthetic chemistry techniques alone, says principal investigator Hans Renata, PhD, an assistant professor in the Department of Chemistry at Scripps Research.Natural enzymes that help build molecules in cells usually perform only one or two highly specific tasks. But the Scripps Research team showed that natural enzymes, even without modification, can be made to perform a wider range of tasks."We think that in general, enzymes are a mostly untapped resource for solving problems in chemical synthesis," Renata says. "Enzymes tend to have some degree of promiscuous activity, in terms of their ability to spur chemical reactions beyond their primary task, and we were able to take advantage of that here."Enzymes help build molecules in all plant, animal and microbial species. Inspired by their efficiency in constructing highly complex molecules, chemists for more than half a century have used enzymes in the lab to help build valuable compounds, including drug compounds -- but usually these compounds are the same molecules the enzymes help build in nature.Harnessing natural enzymes in a broader way, according to their basic biochemical activity, is a new strategy with vast potential."Our view now is that whenever we want to synthesize a complex molecule, the solution probably already exists among nature's enzymes -- we just have to know how to find the enzymes that will work," says senior author Ben Shen, PhD, chair of the Department of Chemistry on the Florida campus and director of Scripps Research's Natural Products Discovery Center.The team succeeded in making nine terpenes known to be produced in Isodon, a family of flowering plants related to mint. The complex compounds belong to three terpene families with related chemical structures: ent-kauranes, ent-atisanes, and ent-trachylobanes. Members of these terpene families have a wide range of biological activities including the suppression of inflammation and tumor growth.The synthesis of each compound, in less than 10 steps for each, was a hybrid process combining current organic synthesis methods with enzyme-mediated synthesis starting from an inexpensive compound called stevioside, the main component of the artificial sweetener Stevia.The chief hurdle was the direct replacement of hydrogen atoms with oxygen atoms in a complex pattern on the carbon-atom skeleton of the starting compound. Current organic synthesis methods have a limited arsenal for such transformations. However, nature has produced many enzymes that can enable these transformations -- each capable of performing its function with a degree of control unmatched by man-made methods."Being an interdisciplinary research group, we were fully aware of the limitations of current organic synthesis methods, but also of the many unique ways that enzymes can overcome these limitations -- and we had the insights to combine traditional synthetic chemistry with enzymatic methods in a synergistic fashion," Renata says.The three enzymes used, which were identified and characterized by Shen, Renata and colleagues only last year, are produced naturally by a bacterium -- one of the 200,000-plus species in the Microbial Strain Collection at Scripps Research's Natural Products Discovery Center."We were able to use these enzymes not only to modify the starting molecules, or scaffolds as we call them, but also to turn one scaffold into another so that we could transform a terpene from one family into a terpene from a different family," says second author Emma King-Smith, a PhD student in the Renata lab.The chemists now intend to use their new approach to make useful quantities of the nine compounds, as well as chemical variants of the compounds, and, with collaborating laboratories, explore their properties as potential drugs or other products."With our strategy, we can make these highly oxidized diterpenes much more easily and in larger quantities than would be possible by isolating them from the plants where they are found naturally," says first author Xiao Zhang, PhD, a postdoctoral research associate in the Renata lab.Just as importantly, the researchers say, they are working to identify reactions and enzymes that will allow them to extend their approach to other classes of molecules.Central to all these efforts is the ongoing development of methods to sift through the DNA of microbes and other organisms to identify the enzymes they encode -- and predict the activities of those enzymes. Billions of distinct enzymes exist in plants, animals, and bacteria on Earth and only a tiny fraction of them have been catalogued to date."We're excited about the potential of discovering new and useful enzymes from our strain library here at Scripps Research," Renata says. "We think that will enable us to solve many other problems in chemical synthesis."The research was funded by the National Institutes of Health (GM134954, GM128895, and GM124461).
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Biotechnology
| 2,020 |
August 13, 2020
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https://www.sciencedaily.com/releases/2020/08/200813142327.htm
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Research gets to the heart of organ shape in nature
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Researchers have shed fresh light on the evolution and function of the shapes we see in nature -- using as a model the heart shaped fruits of the Capsella genus.
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The natural world is full of diverse shapes from organs to whole organisms that are fitted by evolution to perform and reproduce optimally in their environment.The Capsella seed pods with their distinctive heart-shaped shoulders offer an anatomical novelty and an excellent study system for understanding the diversity of shapes.Earlier studies have shown that the expression of key regulatory genes is a primary driver in controlling shape evolution in organs. This new study carried out by John Innes Centre researchers adds another critical step in this pathway by revealing a modification of protein activity that is critical for organ-shape formation.They show that the SUMO-protease HEARTBREAK (HTB) from Capsella rubella controls the activity of the key regulator of fruit development INDEHISCENT via a process called de-SUMOylation.Only via this de-SUMOylation -- a kind of molecular trimming activity -- is a pathway activated which allows biosynthesis of the plant hormone auxin which in turn facilitates anisotropic cell expansion to form the heart-shaped Capsella fruit.Professor Lars Østergaard a programme leader at the John Innes Centre and corresponding author of the paper explains the significance: "We know that the diversity in shape we observe in nature frequently is caused by changes in the position and timing of key regulatory genes: that is how a lot of variation occurs."What we have found is that there is this post translational effect, beyond the gene expression. This protein modification is at the basis of this type of diversity of fruit shape -- and goes a long way to explain the difference for example between the fruits of Capsella and those from the related model plant Arabidopsis. This is about a modification of protein activity at a different stage than we have seen before."Researchers used forward genetic screening -- a technique to study a range of traits -- which identified a mutant with compromised development of the heart-shaped fruit. The mutant was therefore named, heartbreak. They used time-lapse 3D imaging and molecular genetics to characterise the heartbreak phenotype at the cellular and molecular level.First author Dr Yang Dong added: "We now have an entire pathway based on gene expression, hormone dynamics and post translational modification of proteins in such detail that we can test to what extent these kinds of pathways with these components are shared much wider across kingdoms and not just within the plant kingdom."One of the next steps for the researchers is to is to translate this fundamental discovery from the research plant Capsella to the related commercial crop oilseed rape.The research answers a key question about how these shapes appear.But why does nature come up with such an unusual shape as the heart-shaped pods of Capsella? What is the function behind this form? The reason is still debatable, explains Professor Østergaard."Previously we thought these shapes might be a good functional design for seed dispersal because the shape could allow the wind to catch the seed pod walls, but our assays comparing them with Arabidopsis and oilseed rape do not reveal any great advantage of the Capsella fruit in seed dispersal. So, we don't think that can be a major factor."It is possible they could act like solar panels. In other words, maybe they function to capture sunlight and increase photosynthetic capacity. We know that the photosynthetic capacity of the seed pod walls can have a strong effect on seed development inside the pod and therefore on yields. So, by understanding this mechanism it does give us tools to perhaps be able to manipulate the seed pod walls in crops like oilseed rape."
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Biotechnology
| 2,020 |
August 13, 2020
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https://www.sciencedaily.com/releases/2020/08/200813131251.htm
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Single-cell analysis provides new insights into mitochondrial diseases
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Investigators led by a team at Massachusetts General Hospital (MGH) have made discoveries at the single cell level to uncover new details concerning mitochondrial diseases -- inherited disorders that interfere with energy production in the body and currently have no cure. The findings, which are published in the
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Mitochondrial diseases result from failure of mitochondria, specialized compartments within cells that contain their own DNA and produce the energy needed to sustain life. Inherited mutations in mitochondrial DNA (mtDNA) often cause these diseases, and affected patients' cells contain a mixture of mutant and nonmutant mtDNA -- a phenomenon called heteroplasmy. The proportion of mutant mtDNA varies across patients and among tissues within a patient. Also, symptoms range from mild to severe and depend on which cells of the body are affected."It is generally accepted that the fraction of mutant heteroplasmy is what determines whether or not a tissue will exhibit disease. To better understand heteroplasmic dynamics, we applied a brand new genomics technology -- with single cell resolution -- in which we could simultaneously determine the cell type and the fraction of mutant heteroplasmy in thousands of individual blood cells," said senior author Vamsi K. Mootha, MD, investigator in the Department of Molecular Biology at MGH.The researchers examined mtDNA within different blood cell types from 9 individuals with MELAS, one of the most common forms of mtDNA disease associated with brain dysfunction and stroke-like episodes, with a wide range of severity across patients."What makes this study unique is that it is, to our knowledge, the first time anyone has been able to quantify the percentage of disease-causing mitochondrial DNA mutations in thousands of individual cells of different types from the same patient, as well as in multiple patients with inherited mitochondrial disease," said lead author Melissa A. Walker, MD, PhD, an investigator in the Department of Neurology at MGH.The analysis revealed especially low levels of heteroplasmy in T cells, which play important roles in killing infected cells, activating other immune cells, and regulating immune responses."Our observations suggest that certain cell lineages within our body may have a process by which to guard against problematic mtDNA mutations, which is a potentially very exciting finding," said Walker.Additional studies are needed to determine whether differences in heteroplasmy across immune cell types affect the cells' function, and whether assessing such heteroplasmy may help clinicians diagnose and monitor mitochondrial diseases. "Our long-term vision is that single cell genomics may lead to improved blood tests for monitoring the progression of these diseases," said Mootha.In addition, understanding the determinants of reduced T-cell heteroplasmy may motivate new therapeutic strategies for mitochondrial diseases, which currently lack any FDA-approved treatments.Mootha added that mtDNA mutations also occur spontaneously during normal aging. "Although our work focused on rare, inherited diseases, it has potential implications for the heteroplasmic dynamics of aging as well," he said.
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Biotechnology
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August 12, 2020
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https://www.sciencedaily.com/releases/2020/08/200812161331.htm
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Programmed bacteria have something extra
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Rice University chemist Han Xiao and his team have successfully expanded the genetic code of Escherichia coli bacteria to produce a synthetic building block, a "noncanonical amino acid." The result is a living indicator for oxidative stress.
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The work, they say, is a step toward technologies that will allow the generation of novel proteins and organisms with a variety of useful functions.Their study appears in the Cell Press journal Amino acids are the building blocks of DNA. In general, organisms need only 20 of them to program the entire set of proteins necessary for life. But Xiao, with the help of a $1.8 million National Institutes of Health grant, set out to see how a 21st amino acid would enable the design of "unnatural organisms" that serve specific purposes.The new study does just that by engineering bacteria to produce the extra amino acid, called 5-hydroxyl-tryptophan (5HTP), which appears naturally in humans as a precursor to the neurotransmitter serotonin, but not in E. coli. The novel production of 5HTP prompts the bacteria to produce a protein that fluoresces when the organism is under metabolic stress."The process requires a lot of interdisciplinary techniques," Xiao said. "In this study, we combined synthetic chemistry, synthetic biology and metabolic engineering to create a strain that synthesizes and encodes a 21st noncanonical amino acid, and then uses it to produce the desired protein."Xiao said programming the autonomous unnatural bacteria was a three-step process: First, the researchers led by graduate student Yuda Chen created bioorthogonal translational machinery for the amino acid, 5HTP. Second, they found and targeted a blank codon -- a sequence in DNA or RNA that doesn't produce a protein -- and genetically edited it to encode 5HTP. Third, by grafting enzyme clusters from other species into E. coli, they gave the bacteria the ability to produce 5HTP."These 5HTP-containing proteins, isolated from the programmed bacteria, can be further labeled with drugs or other molecules," Xiao said. "Here, we show the strain itself can serve as a living indicator for reactive oxygen species, and the detection limit is really low."While researchers have reported the creation of more than 200 noncanonical amino acids to date, most of them cannot be synthesized by their host organisms. "This has been an ongoing field for decades, but previously people focused on the chemical part," Xiao said. "Our vision is to engineer whole cells with the 21st amino acid that will let us investigate biological or medical problems in living organisms, rather than just dealing with cells in the lab."Moving this technology to the host species eliminates the need to inject artificial building blocks into an organism, because they can synthesize and use it on their own," he said. "That allows us to study noncanonical amino acids at a higher, whole organism level."Ultimately, the researchers hope customized building blocks will allow targeted cells, like those in tumors, to make their own therapeutic drugs. "That's an important future direction for my lab," Xiao said. "We want cells to detect disease, make better medicines and release them in real time. We don't think that's too far away."Co-authors of the paper are Rice postdoctoral fellows Juan Tang, Lushun Wang and Zeru Tian, undergraduate student Adam Cardenas and visiting scholar Xinlei Fang, and Abhishek Chatterjee, an assistant professor of chemistry at Boston College. Xiao is the Norman Hackerman-Welch Young Investigator and an assistant professor of chemistry.The Cancer Prevention and Research Institute of Texas, the Robert A. Welch Foundation, a John S. Dunn Foundation Collaborative Research Award and a Hamill Innovation Award supported the research.
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Biotechnology
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August 12, 2020
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https://www.sciencedaily.com/releases/2020/08/200812153632.htm
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Study provides insights into how Zika virus suppresses the host immune system
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A research team led by scientists at the University of California, Riverside, has outlined how the Zika virus, which constituted an epidemic threat in 2016, suppresses the immune system of its host.
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The Zika virus, or ZIKV, spreads through mosquito bites and sexual intercourse. Currently, no approved vaccine or antivirals against ZIKV exist."Suppressing host immunity is a common strategy employed by viruses to achieve successful infection," said Jikui Song, a professor of biochemistry at UCR, who co-led the study. "Our work provides valuable structural and functional information on the interaction between ZIKV and its host and offers a framework for the development of vaccines and antivirals."The study appears in Nature Structural & Molecular Biology.Song explained the steps involved in suppressing the host immune response. ZIKV encounters the first line of defense by way of a type I interferon, or IFN, response in the host. Secreted by infected cells, IFNs are natural substances that help the host's immune system fight infection. Once ZIKV infects the cell, it presents a nonstructural protein, NS5, which interacts with a key player in the type I IFN pathway: the STAT2 protein. The interaction between ZIKV NS5 and STAT2 degrades STAT2, which inhibits the type I IFN response.The research involved first solving the crystal structure of a complex between a large fragment of ZIKV NS5 and STAT2. This crystal structure guided the researchers in solving the cryo-EM structure of ZIKV NS5 and STAT2, which then led them to come up with a model for how ZIKV NS5 suppresses human STAT2."Understanding the interaction, at the molecular level, between ZIKV NS5 and the host immune factor STAT2, opens up a new window for the rational design of live attenuated vaccines and antivirals" said study co-leader Rong Hai, an assistant professor of virology at UCR. "Targeting the virus-host interaction may also provide an important approach for drug development against SARS-CoV-2, the virus that causes COVID-19."The researchers also generated a panel of mutant ZIKV viruses."To our knowledge, these are the first NS5-based ZIKV mutants, which have the potential to be used as live attenuated ZIKV vaccines," Hai said.Next, the researchers will work on the structure and function of SARS-CoV-2 proteins to identify new targets against COVID-19.
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Biotechnology
| 2,020 |
August 12, 2020
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https://www.sciencedaily.com/releases/2020/08/200812144025.htm
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Spider silk inspires new class of functional synthetic polymers
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Synthetic polymers have changed the world around us, and it would be hard to imagine a world without them. However, they do have their problems. It is for instance hard from a synthetic point of view to precisely control their molecular structure. This makes it harder to finely tune some of their properties, such as the ability to transport ions. To overcome this problem, University of Groningen assistant professor Giuseppe Portale decided to take inspiration from nature. The result was published in
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'I have been working on proton conducting materials on and off since my PhD', says Portale. 'I find it fascinating to know what makes a material transport a proton so I worked a lot on optimizing structures at the nanoscale level to get greater conductivity.' But it was only a few years ago that he considered the possibility of making them from biological, protein-like structures. He came to this idea together with professor Andreas Hermann, a former colleague at the University of Groningen, now working at the DWI -- Leibniz Institute for Interactive Materials in Germany. 'We could immediately see that proton-conducting bio-polymers could be very useful for applications like bio-electronics or sensors', Portale says.But first, they had to see if the idea would work. Portale: 'Our first goal was to prove that we could precisely tune the proton conductivity of the protein-based polymers by tuning the number of ionisable groups per polymer chain'. To do this, the researchers prepared a number of unstructured biopolymers that had different numbers of ionisable groups, in this case, carboxylic acid groups. Their proton conductivity scaled linearly with the number of charged carboxylic acid groups per chain. 'It was not groundbreaking, everybody knows this concept. But we were thrilled that we were able to make something that worked as expected', Portale says.For the next step, Portale relied on his expertise in the field of synthetic polymers: 'I have learned over the years that the nanostructure of a polymer can greatly influence the conductivity. If you have the right nanostructure, it allows the charges to bundle together and increase the local concentration of these ionic groups, which dramatically boosts proton conductivity.' Since the first batch of biopolymers was completely amorphous, the researchers had to switch to a different material. They decided to use a known protein that had the shape of a barrel. 'We engineered this barrel-like protein and added strands containing carbocyclic acid to its surface', Portale explains. 'This increased conductivity greatly.'Unfortunately, the barrel-polymer was not very practical. It had no mechanical strength and it was difficult to process, so Portale and his colleagues had to look for an alternative. They landed on a well-known natural polymer: spider silk. 'This is one of the most fascinating materials in nature, because it is very strong but can also be used in many different ways', says Portale. 'I knew spider silk has a fascinating nanostructure, so we engineered a protein-like polymer that has the main structure of spider silk but was modified to host strands of carbocyclic acid.'The novel material worked like a charm. 'We found that it self-assembles at the nanoscale similarly to spider silk while creating dense clusters of charged groups, which are very beneficial for the proton conductivity', Portale explains. 'And we were able to turn it into a robust centimetre-sized membrane.' The measured proton conductivity was higher than any previously known biomaterials, but they are not there yet according to Portale: 'This was mainly fundamental work. In order to apply this material, we really have to improve it and make it processable.'But even though the work is not yet done, Portale and his co-workers can already dream about applying their polymer: 'We think this material could be useful as a membrane in fuel cells. Maybe not for the large scale fuel cells that you see in cars and factories, but more on a small scale. There is a growing field of implantable bio-electronic devices, for instance, glucose-powered pacemakers. In the coming years, we hope to find out if our polymer can make a difference there, since it is already bio-compatible.'For the short term, Portale mainly thinks about sensors. 'The conductivity we measure in our material is influenced by factors in the environment, like humidity or temperature. So if you want to store something at a certain humidity you can place this polymer between two electrodes and just measure if anything changes.' However, before all these dreams come true, there are a lot of questions to be answered. 'I am very proud that we were able to control these new materials on a molecular scale and build them from scratch. But we still have to learn a lot about their capabilities and see if we can improve them even further.'
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Biotechnology
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August 12, 2020
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https://www.sciencedaily.com/releases/2020/08/200812115328.htm
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Internal differences: A new method for seeing into cells
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Invading cells' private space -- prying into their internal functions, decisions and communications -- could be a powerful tool that may help researchers develop new immunotherapy treatment for cancer. As reported today in
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Amit and his group had previously made significant inroads into seeing into cells when they developed single cell RNA-sequencing -- a means of sequencing all of the RNA in thousands of individual cells at once. The new technique, called INs-seq (intracellular staining and sequencing) -- developed in Amit's lab in a project led by research students Yonatan Katzenelenbogen, Fadi Sheban, Adam Yalin and Dr. Assaf Weiner -- enables scientists to measure, in addition to the RNA, numerous proteins, processes and biochemical pathways occurring inside each of the cells. To do this, they had to develop new methods of getting inside the cell membranes without harming its genetic content. This wealth of "inside information" can help them draw much finer distinctions between different cell subtypes and activities than is possible with existing methods, most of which are able to measure surface proteins only.In fact, Amit compares those existing methods of cell characterization with buying watermelons: They all appear identical from the outside, even though they can taste completely different when you open them up. Distinguishing between subtypes of cells that seem identical from the outside, such as inhibitory- versus effector-immune cells, may be crucial to when it comes to fighting off cancer.Although the principal groups of immune cells had been identified many decades ago, there are hundreds of subtypes with many different functions, which haven't been classified. "Specific immune subtypes, for example, may play a role in promoting cancer or enabling it to evade the immune system, provoke tissue destruction by overreacting to a virus or act mistakenly in autoimmune syndromes, attacking our own body. Until now, there was no sufficiently sensitive means of telling these apart from other subtypes that appear identical from the outside," says Sheban.In order to sort through these different immune functions inside tumors, the Weizmann scientists used their technology to address an issue that researchers had been trying to resolve for decades: Why does the immune system fail to recognize and kill cancer cells, and why does immunotherapy for most tumor types often fail? In searching for an answer, they asked whether cancers might hijack and manipulate particular immune cells to "defend" the cancer cells from the rest of the immune system. "The suspicion that some kind of immune cell might be actively 'collaborating' with cancer is not as strange as it seems," explains Yalin. "Immune responses are often meant to be short-lived, so the immune system has its own mechanisms for shutting them down. Cancers could take advantage of such mechanisms, enhancing the production of the 'shut-down' immune cells, which, in turn, could prevent such immune cells as T lymphocytes that would normally kill them from taking action."Indeed, the team succeeded in identifying T-cell-blocking immune cells, which belonged to a general group known as myeloid cells -- a broad group of innate immune cells that mostly originate in the bone marrow. Although this particular subset of suppressive myeloid cells was new, it was distinguished by a prominent signaling receptor that Amit and his group had seen before, called TREM2. This receptor is critical for the activity of the cells that block the actions of tumor-killing T cells; and normally cells bearing this receptor are crucial for preventing excess tissue damage after injury or calming an inflammatory immune response. But Amit had also come across a version of this receptor in other immune cells involved in Alzheimer's disease, metabolic syndrome and other immune-related pathologies.The group's next step is to develop an immunotherapy treatment using specific antibodies that target this receptor and could prevent these immune-suppressive cells from supporting the tumor. "Because this receptor is only expressed when there is some type of pathology," says Weiner, "targeting it will not damage healthy cells in the body."Preliminary evidence for the TREM2 therapeutics was demonstrated by the scientists in mouse models of cancer with genetically ablated TREM2 receptors. In those mice, tumor-killing T cells "came back to life" and attacked the cancer cells; and the tumors shrank significantly. If treatment based on this finding is, in the future, proven effective for human use, it might be administered on its own or in combination with other forms of immunotherapy.Yeda Research and Development, the technology transfer arm of the Weizmann Institute of Science, is currently working with Amit to develop this immunotherapy antibody for clinical use and there has already been a great deal of interest in INs-seq technology. "Clarifying the mechanisms of autoimmune and neurodegenerative diseases, and answering the question of why the immune system often fails in its fight against cancer or why most patient do not respond to existing immunotherapy -- all of these may come down to specific actions of subsets of immune cells. We believe INs-seq may help researchers identify those particular cells and develop new therapies to treat them," says Katzenelenbogen.
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Biotechnology
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August 12, 2020
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https://www.sciencedaily.com/releases/2020/08/200812094913.htm
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Experimental COVID-19 vaccine prevents severe disease in mice
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An experimental vaccine is effective at preventing pneumonia in mice infected with the COVID-19 virus, according to a study from Washington University School of Medicine in St. Louis. The vaccine, which is made from a mild virus genetically modified to carry a key gene from the COVID-19 virus, is described in the journal
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"Unlike many of the other vaccines under development, this vaccine is made from a virus that is capable of spreading in a limited fashion inside the human body, which means it is likely to generate a strong immune response," said co-senior author Michael S. Diamond, MD, PhD, the Herbert S. Gasser Professor of Medicine and a professor of molecular microbiology, and of pathology and immunology. "Since the virus is capable of replicating, it can be grown to high levels in the lab, so it's easy to scale up and should be more cost-effective than some of the other vaccine candidates. So while what we have shown is just the proof of concept, I think it's very promising. Our vaccine candidate is now being tested in additional animal models with the goal of getting it into clinical trials as soon as possible."Diamond and colleagues -- including co-senior author Sean Whelan, PhD, the Marvin A. Brennecke Distinguished Professor and head of the Department of Molecular Microbiology; and co-first authors Brett Case, PhD, a postdoctoral researcher in Diamond's laboratory, and Paul W. Rothlauf, a graduate student in Whelan's laboratory -- created the experimental vaccine by genetically modifying vesicular stomatitis virus (VSV), a virus of livestock that causes only a mild, short-lived illness in people. They swapped out one gene from VSV for the gene for spike from SARS-CoV-2, the virus that causes COVID-19. The hybrid virus is called VSV-SARS-CoV-2.Spike protein is thought to be one of the keys to immunity against COVID-19. The COVID-19 virus uses spike to latch onto and infect human cells, and the human body defends itself by generating protective antibodies targeting spike. By adding the gene for spike to a fairly harmless virus, the researchers created a hybrid virus that, when given to people, ideally would elicit antibodies against spike that protect against later infection with the COVID-19 virus.The same strategy was used to design the Ebola vaccine that was approved by the U.S. Food and Drug Administration in 2019. That vaccine -- which is made from VSV genetically modified with a gene from Ebola virus -- has been safely administered to thousands of people in Africa, Europe and North America, and helped end the 2018 to 2020 Ebola outbreak in the Democratic Republic of the Congo.As part of this study, the researchers injected mice with VSV-SARS-CoV-2 or a lab strain of VSV for comparison. A subgroup was boosted with a second dose of the experimental vaccine four weeks after the initial injections. Three weeks after each injection, the researchers drew blood from the mice to test for antibodies capable of preventing SARS-CoV-2 from infecting cells. They found high levels of such neutralizing antibodies after one dose, and the levels increased 90-fold after a second dose.Then, the researchers challenged the mice five weeks after their last dose by spraying the COVID-19 virus into their noses. The vaccine completely protected against pneumonia. At four days post infection, there was no infectious virus detectable in the lungs of mice that had been given either one or two doses of the vaccine. In contrast, mice that had received the placebo had high levels of virus in their lungs. In addition, the lungs of vaccinated mice showed fewer signs of inflammation and damage than those of mice that had received the placebo.The experimental vaccine is still in the early stages of development.Mice do not naturally become infected with the COVID-19 virus, so to assess whether the vaccine elicited a protective immune response in them, the researchers used genetically modified mice or, in unmodified mice, employed a complicated technique to induce susceptibility to infection. The researchers are in the process of repeating the experiments in other animal models that are naturally susceptible to the COVID-19 virus. If the vaccine also protects those animals from COVID-19, the next step would be to scale up production under what the Food and Drug Administration refers to as "good manufacturing practice (GMP) conditions" and launch a clinical trial in people.While the data are promising, this vaccine is still months behind in the race to develop a pandemic-ending vaccine. Six vaccines are in the final stage of testing in people, and Anthony Fauci, MD, director of the U.S. National Institute of Allergy and Infectious Diseases, has said he expects a vaccine to be ready for mass distribution early next year."It's really going to depend on how successful the first vaccines that come out for COVID are," Whelan said. "If they don't produce a robust, durable immune response or there are safety issues, there might be the opportunity for a second-generation vaccine that could induce sterilizing immunity and interrupt the cycle of transmission."
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Biotechnology
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August 11, 2020
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https://www.sciencedaily.com/releases/2020/08/200811234951.htm
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'AeroNabs' promise powerful, inhalable protection against COVID-19
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As the world awaits vaccines to bring the COVID-19 pandemic under control, UC San Francisco scientists have devised a novel approach to halting the spread of SARS-CoV-2, the virus that causes the disease.
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Led by UCSF graduate student Michael Schoof, a team of researchers engineered a completely synthetic, production-ready molecule that straitjackets the crucial SARS-CoV-2 machinery that allows the virus to infect our cells. As reported in a new paper, now available on the preprint server bioRxiv, experiments using live virus show that the molecule is among the most potent SARS-CoV-2 antivirals yet discovered.In an aerosol formulation they tested, dubbed "AeroNabs" by the researchers, these molecules could be self-administered with a nasal spray or inhaler. Used once a day, AeroNabs could provide powerful, reliable protection against SARS-CoV-2 until a vaccine becomes available. The research team is in active discussions with commercial partners to ramp up manufacturing and clinical testing of AeroNabs. If these tests are successful, the scientists aim to make AeroNabs widely available as an inexpensive, over-the-counter medication to prevent and treat COVID-19."Far more effective than wearable forms of personal protective equipment, we think of AeroNabs as a molecular form of PPE that could serve as an important stopgap until vaccines provide a more permanent solution to COVID-19," said AeroNabs co-inventor Peter Walter, PhD, professor of biochemistry and biophysics at UCSF and a Howard Hughes Medical Institute Investigator. For those who cannot access or don't respond to SARS-CoV-2 vaccines, Walter added, AeroNabs could be a more permanent line of defense against COVID-19."We assembled an incredible group of talented biochemists, cell biologists, virologists and structural biologists to get the project from start to finish in only a few months," said Schoof, a member of the Walter lab and an AeroNabs co-inventor.Though engineered entirely in the lab, AeroNabs were inspired by nanobodies, antibody-like immune proteins that naturally occur in llamas, camels and related animals. Since their discovery in a Belgian lab in the late 1980s, the distinctive properties of nanobodies have intrigued scientists worldwide."Though they function much like the antibodies found in the human immune system, nanobodies offer a number of unique advantages for effective therapeutics against SARS-CoV-2," explained co-inventor Aashish Manglik, MD, PhD, an assistant professor of pharmaceutical chemistry who frequently employs nanobodies as a tool in his research on the structure and function of proteins that send and receive signals across the cell's membrane.For example, nanobodies are an order of magnitude smaller than human antibodies, which makes them easier to manipulate and modify in the lab. Their small size and relatively simple structure also makes them significantly more stable than the antibodies of other mammals. Plus, unlike human antibodies, nanobodies can be easily and inexpensively mass-produced: scientists insert the genes that contain the molecular blueprints to build nanobodies into E. coli or yeast, and transform these microbes into high-output nanobody factories. The same method has been used safely for decades to mass-produce insulin.But as Manglik noted, "nanobodies were just the starting point for us. Though appealing on their own, we thought we could improve upon them through protein engineering. This eventually led to the development of AeroNabs."SARS-CoV-2 relies on its so-called spike proteins to infect cells. These spikes stud the surface of the virus and impart a crown-like appearance when viewed through an electron microscope -- hence the name "coronavirus" for the viral family that includes SARS-CoV-2. Spikes, however, are more than a mere decoration -- they are the essential key that allows the virus to enter our cells.Like a retractable tool, spikes can switch from a closed, inactive state to an open, active state. When any of a virus particle's approximately 25 spikes become active, that spike's three "receptor-binding domains," or RBDs, become exposed and are primed to attach to ACE2 (pronounced "ace two"), a receptor found on human cells that line the lung and airway.Through a lock-and-key-like interaction between an ACE2 receptor and a spike RBD, the virus gains entry into the cell, where it then transforms its new host into a coronavirus manufacturer. The researchers believed that if they could find nanobodies that impede spike-ACE2 interactions, they could prevent the virus from infecting cells.To find effective candidates, the scientists parsed a recently developed library in Manglik's lab of over 2 billion synthetic nanobodies. After successive rounds of testing, during which they imposed increasingly stringent criteria to eliminate weak or ineffective candidates, the scientists ended up with 21 nanobodies that prevented a modified form of spike from interacting with ACE2.Further experiments, including the use of cryo-electron microscopy to visualize the nanobody-spike interface, showed that the most potent nanobodies blocked spike-ACE2 interactions by strongly attaching themselves directly to the spike RBDs. These nanobodies function a bit like a sheath that covers the RBD "key" and prevents it from being inserted into an ACE2 "lock."With these findings in hand, the researchers still needed to demonstrate that these nanobodies could prevent the real virus from infecting cells. Veronica Rezelj, PhD, a virologist in the lab of Marco Vignuzzi, PhD, at Institut Pasteur in Paris, tested the three most promising nanobodies against live SARS-CoV-2, and found the nanobodies to be extraordinarily potent, preventing infection even at extremely low doses.The most potent of these nanobodies, however, not only acts as a sheath over RBDs, but also like a molecular mousetrap, clamping down on spike in its closed, inactive state, which adds an additional layer of protection against the spike-ACE2 interactions that lead to infection.The scientists then engineered this double-action nanobody in a number of ways to make it into an even more potent antiviral. In one set of experiments, they mutated every one of the amino-acid building blocks of the nanobody that contacts spike to discover two specific changes that yielded a 500-fold increase in potency.In a separate set of experiments, they engineered a molecular chain that could link three nanobodies together. As noted, each spike protein has three RBDs, any of which can attach to ACE2 to grant the virus entry into the cell. The linked triple nanobody devised by the researchers ensured that if one nanobody attaches itself to an RBD, the other two would attach to the remaining RBDs. They found that this triple nanobody is 200,000 times more potent than a single nanobody alone.And when they drew on the results of both modifications, linking three of the powerful mutated nanobodies together, the results were "off the charts," said Walter. "It was so effective that it exceeded our ability to measure its potency."This ultrapotent three-part nanobody construct formed the foundation for AeroNabs.In a final set of experiments, the researchers put the three-part nanobodies through a series of stress tests, subjecting them to high temperatures, turning them into a shelf-stable powder, and making an aerosol. Each of these processes is highly damaging to most proteins, but the scientists confirmed that, thanks to the inherent stability of nanobodies, there was no loss of antiviral potency in the aerosolized form, suggesting that AeroNabs are a potent SARS-CoV-2 antiviral that could be practical to administer via a shelf-stable inhaler or nasal spray."We're not alone in thinking that AeroNabs are a remarkable technology," said Manglik. "Our team is in ongoing discussions with potential commercial partners who are interested in manufacturing and distributing AeroNabs, and we hope to commence human trials soon. If AeroNabs prove as effective as we anticipate, they may help reshape the course of the pandemic worldwide."
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Biotechnology
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August 11, 2020
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https://www.sciencedaily.com/releases/2020/08/200811120139.htm
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Enzyme discovered in the gut could lead to new disease biomarker
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Enzymes used by bacteria to break down mucus in the gut could provide a useful biomarker for intestinal diseases, according to new research published in
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Researchers at the University of Birmingham and Newcastle University have successfully identified and characterised one of the key enzymes involved in this process. They demonstrated how the enzyme enables bacteria to break down and feed off sugars in the layers of mucus lining the gut.The research offers a significant step forward in our understanding of the complex co-dependent relationships at work in the gut, about which little is currently known. Because the mechanism used by the enzyme is particularly distinctive, the researchers anticipate it can be used in the development of new diagnostics for intestinal diseases.The molecules in mucus, called mucin, are constantly produced by the body to generate the layer of mucus in the gut that provides a barrier between the gut's complex populations of bacteria and the rest of the body. Mucin contain chains of sugar molecules called glycans, and these also provide an essential source of nutrients for bacteria.The team investigated how this enzyme sits on the outside of the bacterial cell and clips away parts of the mucin molecule, taking them inside the bacterial cell to be consumed.Because glycans are known to change when certain diseases are present in the body, the researchers anticipate it will be possible to use the enzymes to take a snapshot of the glycans within a biopsy and use that as a biomarker for early detection of the disease.Lead researcher, Dr Lucy Crouch, of the University of Birmingham's School of Biosciences, explains: "Mucus is structured a bit like a tree, with lots of different branches and leaves. Lots of the enzymes discovered so far might clip away some of the leaves to eat, but the enzyme we studied will clip away a whole branch -- that's quite a distinctive mechanism and it gives us a useful biomarker for studying disease."The team have investigated this process in three different diseases. They examined tissue from adults suffering from ulcerative colitis and colorectal cancer, and from preterm infants with necrotising enterocolitis, a serious illness in which the gut becomes inflamed and can start to die. They found that by adding the enzyme to the samples and labelling the glycans with a fluorescent dye, they were able to get useful information about the glycan structure.Dr Crouch adds: "Although we still don't fully understand what the glycan structures are made from and how these vary between different tissue types, we can see that the differences in structure between health and non-healthy tissue is quite distinctive. We hope to be able to use these enzymes to start producing better diagnostics for the very early stages of these diseases."
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Biotechnology
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August 11, 2020
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https://www.sciencedaily.com/releases/2020/08/200811120122.htm
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Study reveals immune-system paralysis in severe COVID-19 cases
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Some people get really sick from COVID-19, and others don't. Nobody knows why.
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Now, a study by investigators at the Stanford University of Medicine and other institutions has turned up immunological deviations and lapses that appear to spell the difference between severe and mild cases of COVID-19.That difference may stem from how our evolutionarily ancient innate immune system responds to SARS-CoV-2, the virus that causes the disease. Found in all creatures from fruit flies to humans, the innate immune system rapidly senses viruses and other pathogens. As soon as it does, it launches an immediate though somewhat indiscriminate attack on them and mobilizes more precisely targeted, but slower-to-get-moving, "sharpshooter" cells belonging to a different branch of the body's pathogen-defense forces, the adaptive immune system."These findings reveal how the immune system goes awry during coronavirus infections, leading to severe disease, and point to potential therapeutic targets," said Bali Pulendran, PhD, professor of pathology and of microbiology and immunology and the senior author of the study, which will be published Aug. 11 in The researchers analyzed the immune response in 76 people with COVID-19 and in 69 healthy people. They found enhanced levels of molecules that promote inflammation in the blood of severely ill COVID-19 patients. Three of the molecules they identified have been shown to be associated with lung inflammation in other diseases but had not been shown previously in COVID-19 infections."These three molecules and their receptors could represent attractive therapeutic targets in combating COVID-19," said Pulendran, who is the Violetta L. Horton Professor. His lab is now testing the therapeutic potential of blocking these molecules in animal models of COVID-19.The scientists also found elevated levels of bacterial debris, such as bacterial DNA and cell-wall materials, in the blood of those COVID-19 patients with severe cases. The more debris, the sicker the patient -- and the more pro-inflammatory substances circulating in his or her blood.The findings suggest that in cases of severe COVID-19, bacterial products ordinarily present only in places such as the gut, lungs and throat may make their way into the bloodstream, kick-starting enhanced inflammation that is conveyed to all points via the circulatory system.But the study also revealed that, paradoxically, key cells of the innate immune system in the blood of COVID-19 patients became increasingly paralyzed as the disease got worse. Instead of being aroused by the presence of viruses or bacteria, these normally vigilant cells remained functionally sluggish.If high blood levels of inflammation-promoting molecules set COVID-19 patients apart from those with milder cases, but blood cells are not producing these molecules, where do they come from? Pulendran believes they originate in tissues somewhere in the body -- most likely patients' lungs, the site of infection."One of the great mysteries of COVID-19 infections has been that some people develop severe disease, while others seem to recover quickly," Pulendran said. "Now we have some insights into why that happens."Pulendran is a member of Stanford Bio-X and a faculty fellow of Stanford ChEM-H.Other Stanford study co-authors are MD/PhD [MSTP] student Madeleine Scott; postdoctoral scholars Thomas Hagan, PhD, and Yupeng Feng, PhD; basic life research scientist Natalia Sigal, PhD; senior research scientist Dhananjay Wagh, PhD; John Coller, PhD, director of Stanford Functional Genomics Facility; Holden Terry Maecker, PhD, professor of microbiology and immunology; and Purvesh Khatri, PhD, associate professor of biomedical informatics and of biomedical data science.Researchers at Emory University, the University of Hong Kong and the Hospital Authority of Hong Kong also participated in the work.The study was sponsored by the National Institutes of Health (grants U19AI090023, U19AI057266, UH2AI132345, U24AI120134, T32AI07290, P51OD011132 and S10OD026799 and contract HHSN272201400006C), the Sean Parker Cancer Institute, the Soffer Endowment and the Violetta Horton Endowment. Stanford's Departments of Pathology and of Microbiology and Immunology also supported the work.
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Biotechnology
| 2,020 |
August 11, 2020
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https://www.sciencedaily.com/releases/2020/08/200811120107.htm
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Recipe for success -- interaction proteomics become a household item
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Proteins in human cells do not function in isolation and their interactions with other proteins define their cellular functions. Therefore, detailed understanding of protein-protein interactions (PPIs) is the key for deciphering regulation of cellular networks and pathways, in both health and disease.
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In a study published in the of September issue (advanced online 10th of August) of The MAC-tag technology allows an easy way to probe the molecular level localisation of protein of interest. The developed MAC-tag and the integrated approach will empower, not only the interaction proteomics community, but also cell/molecular/structural biologists, with an experimentally proven integrated workflow for mapping in detail the physical and functional interactions and the molecular context of proteins."The MAC-tag technology stems from long-term efforts on developing new systems biology tools for systematically studying the molecular interactions of proteins. The identification of protein-protein interactions and their changes in disease settings, such as cancer, has proven in our hands a powerful tool and has allowed us to find exact molecular mechanisms underlying these diseases. In principle, our protocol can be used in so many different ways that we probably have not even envisioned half of them." Dr. Varjosalo states.The MAC-tag technology is currently in use by Dr. Varjosalo and his consortia of virologists, medicinal chemists and other 'omics' researchers in search for novel druggable host proteins as therapeutic targets to inhibit the SARS-CoV-2 infection and therefore to fight Covid-19.
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Biotechnology
| 2,020 |
August 10, 2020
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https://www.sciencedaily.com/releases/2020/08/200810113200.htm
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Imaging method highlights new role for cellular 'skeleton' protein
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While your skeleton helps your body to move, fine skeleton-like filaments within your cells likewise help cellular structures to move. Now, Salk researchers have developed a new imaging method that lets them monitor a small subset of these filaments, called actin.
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"Actin is the most abundant protein in the cell, so when you image it, it's all over the cell," says Uri Manor, director of Salk's Biophotonics Core facility and corresponding author of the paper. "Until now, it's been really hard to tell where individual actin molecules of interest are, because it's difficult to separate the relevant signal from all the background."With the new imaging technique, the Salk team has been able to home in on how actin mediates an important function: helping the cellular "power stations" known as mitochondria divide in two. The work, which appeared in the journal Mitochondrial fission is the process by which these energy-generating structures, or organelles, divide and multiply as part of normal cellular maintenance; the organelles divide not only when a cell itself is dividing, but also when cells are under high amounts of stress or mitochondria are damaged. However, the exact way in which one mitochondrion pinches off into two mitochondria has been poorly understood, particularly how the initial constriction happens. Studies have found that removing actin from a cell entirely, among many other effects, leads to less mitochondrial fission, suggesting a role for actin in the process. But destroying all the actin causes so many cellular defects that it's hard to study the protein's exact role in any one process, the researchers say.So, Manor and his colleagues developed a new way to image actin. Rather than tag all the actin in the cell with fluorescence, they created an actin probe targeted to the outer membrane of mitochondria. Only when actin is within 10 nanometers of the mitochondria does it attach to the sensor, causing the fluorescence signal to increase.Rather than see actin scattered haphazardly around all mitochondrial membranes, as they might if there were no discrete interactions between actin and the organelles, Manor's team saw bright hotspots of actin. And when they looked closely, the hotspots were located at the same locations where another organelle called the endoplasmic reticulum crosses the mitochondria, previously found to be fission sites. Indeed, as the team watched actin hotspots light up and disappear over time, they discovered that 97 percent of mitochondrial fission sites had actin fluorescing around them. (They speculate that there was also actin at the other 3 percent of fission sites, but that it wasn't visible)."This is the clearest evidence I've ever seen that actin is accumulating at fission sites," says Cara Schiavon, co-first author of the paper and a joint postdoctoral fellow in the labs of Uri Manor and Salk Professor Gerald Shadel. "It's much easier to see than when you use any other actin marker."By altering the actin probe so that it attached to the endoplasmic reticulum membrane rather than the mitochondria, the researchers were able to piece together the order in which different components join the mitochondrial fission process. The team's results suggest that the actin attaches to the mitochondria before it reaches the endoplasmic reticulum. This lends important insight towards how the endoplasmic reticulum and mitochondria work together to coordinate mitochondrial fission.In additional experiments described in a pre-print manuscript available on bioRxiv, Manor's team also reports that the same accumulation of endoplasmic reticulum-associated actin is seen at the sites where other cellular organelles -- including endosomes, lysosomes and peroxisomes -- divide. This suggests a broad new role for a subset of actin in organelle dynamics and homeostasis (physiological equilibrium).In the future, the team hopes to look at how genetic mutations known to alter mitochondrial dynamics might also affect actin's interactions with the mitochondria. They also plan to adapt the actin probes to visualize actin that's close to other cellular membranes."This is a universal tool that can now be used for many different applications," says Tong Zhang, a light microscopy specialist at Salk and co-first author of the paper. "By switching out the targeting sequence or the nanobody, you can address other fundamental questions in cell biology.""We're in a golden age of microscopy, where new instruments with ever higher resolution are always being invented; but in spite of that there are still major limitations to what you can see," says Manor. "I think combining these powerful microscopes with new methods that select for exactly what you want to see is the next generation of imaging."
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Biotechnology
| 2,020 |
August 7, 2020
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https://www.sciencedaily.com/releases/2020/08/200807131917.htm
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Sugar-based signature identifies T cells where HIV hides despite antiretroviral therapy
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Scientists at The Wistar Institute may have discovered a new way of identifying and targeting hidden HIV viral reservoirs during treatment with antiretroviral therapy (ART). These findings were published today in
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ART has dramatically increased the health and life expectancy of HIV-infected individuals, suppressing virus replication in the host immune cells and stopping disease progression; however, low yet persistent amounts of virus remain in the blood and tissues despite therapy. Virus persistency limits immune recovery and is associated with chronic levels of inflammation so that treated HIV-infected individuals have higher risk of developing a number of diseases.This persistent infection stems from the ability of HIV to hide in a rare population of CD4 T cells. Finding new markers to identify the virus reservoir is of paramount importance to achieve HIV eradication."With recent advances that we are making in the fields of glycobiology and glycoimmunology, it has become clear that the sugar molecules present on the surface of immune cells play a critical role in regulating their functions and fate," said corresponding author Mohamed Abdel-Mohsen, Ph.D., assistant professor in The Wistar Institute Vaccine & Immunotherapy Center. "However, the relevance of host cell-surface glycosylation in HIV persistence remained largely unexplored, making it a 'dark matter' in our understanding of HIV latency. For the first time, we described a cell-surface glycomic signature that can impact HIV persistence."Persistently infected cells can be divided into two groups: cells where the virus is completely silent and does not produce any RNA (i.e., silent HIV reservoir); and cells where the virus produces low levels of RNA (i.e., active HIV reservoir). Targeting and eliminating both types of reservoirs is the focus of the quest for an HIV cure. A main challenge in this quest is that we do not have a clear understanding of how these two types of infected cells are different from each other and from HIV-uninfected cells. Therefore, identifying markers that can distinguish these cells from each other is critical.For their studies, Abdel-Mohsen and colleagues used a primary cell model of HIV latency to characterize the cell-surface glycomes of HIV-infected cells. They confirmed their results in CD4 cells directly isolated from HIV-infected individuals on ART.They identified a process called fucosylation as a feature of persistently infected T cells in which the viral genome is actively being transcribed. Fucosylation is the attachment of a sugar molecule called fucose to proteins present on the cell surface and is critical for T-cell activation.Researchers also found that the expression of a specific fucosylated antigen called Sialyl-LewisX (SLeX) identifies persistent HIV transcription in vivo and that primary CD4 T cells with high levels of SLeX have higher levels of T-cell pathways and proteins known to drive HIV transcription during ART. Such glycosylation patterns were not found on HIV-infected cells in which the virus is transcriptionally inactive, providing a distinguishing feature between these two cell compartments. Interestingly, researchers also found that HIV itself promotes these cell-surface glycomic changes.Importantly, having a high level of SLeX is a feature of some cancer cells that allow them to metastasize (spread to other sites in the body). Indeed, researchers found that HIV-infected cells with high levels of SLeX are enriched with molecular pathways involved in trafficking between blood and tissues. These differential levels of trafficking might play an important role in the persistence of HIV in tissues, which are the main sites where HIV hides during ART.Based on these findings, the role of fucosylation in HIV persistence warrants further studies to identify how it contributes to HIV persistence and how it could be used to target HIV reservoirs in blood and tissues.
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Biotechnology
| 2,020 |
August 7, 2020
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https://www.sciencedaily.com/releases/2020/08/200807111940.htm
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Measuring electron emission from irradiated biomolecules
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Researchers have successfully determined the characteristics of electron emission when high-velocity ions collide with adenine -- one of the four key nucleobases of DNA.
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When fast-moving ions cross paths with large biomolecules, the resulting collisions produce many low-energy electrons which can go on to ionise the molecules even further. To fully understand how biological structures are affected by this radiation, it is important for physicists to measure how electrons are scattered during collisions. So far, however, researchers' understanding of the process has remained limited. In new research published in EPJ D, researchers in India and Argentina, led by Lokesh Tribedi at the Tata Institute of Fundamental Research, have successfully determined the characteristics of electron emission when high-velocity ions collide with adenine -- one of the four key nucleobases of DNA.Since high-energy ions can break strands of DNA as they collide with them, the team's findings could improve our understanding of how radiation damage increases the risk of cancer developing within cells. In their experiment, they considered the 'double differential cross section' (DDCS) of adenine ionisation. This value defines the probability that electrons with specific energies and scattering angles will be produced when ions and molecules collide head-on, and is critical for understanding the extent to which biomolecules will be ionised by the electrons they emit.To measure the value, Tribedi and colleagues carefully prepared a jet of adenine molecule vapour, which they crossed with a beam of high-energy carbon ions. They then measured the resulting ionisation through the technique of electron spectroscopy, which allowed them to determine the adenine's electron emissions over a wide range of energies and scattering angles. Subsequently, the team could characterise the DDCS of adenine-ion collision; producing a result which largely agreed with predictions made by computer models based on previous theories. Their findings could now lead to important advances in our knowledge of how biomolecules are affected by high-velocity ion radiation; potentially leading to a better understanding of how cancer in cells can arise following radiation damage.
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Biotechnology
| 2,020 |
August 7, 2020
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https://www.sciencedaily.com/releases/2020/08/200807111938.htm
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Pinpointing the cells that keep the body's master circadian clock ticking
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UT Southwestern scientists have developed a genetically engineered mouse and imaging system that lets them visualize fluctuations in the circadian clocks of cell types in mice. The method, described online in the journal
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"This is a really important technical resource for advancing the study of circadian rhythms," says study leader Joseph Takahashi, Ph.D., chair of the department of neuroscience at UT Southwestern Medical Center, a member of UT Southwestern's Peter O'Donnell Jr. Brain Institute, and an investigator with the Howard Hughes Medical Institute (HHMI). "You can use these mice for many different applications."Nearly every cell in humans -- and mice -- has an internal circadian clock that fluctuates on a roughly 24-hour cycle. These cells help dictate not only hunger and sleep cycles, but biological functions such as immunity and metabolism. Defects in the circadian clock have been linked to diseases including cancer, diabetes, and Alzheimer's, as well as sleep disorders. Scientists have long known that a small part of the brain -- called the suprachiasmatic nucleus (SCN) -- integrates information from the eyes about environmental light and dark cycles with the body's master clock. In turn, the SCN helps keep the rest of the cells in the body in sync with each other."What makes the SCN a very special kind of clock is that it's both robust and flexible," says Takahashi. "It's a very strong pacemaker that doesn't lose track of time, but at the same time can shift to adapt to seasons, changing day lengths, or travel between time zones."To study the circadian clock in both the SCN and the rest of the body, Takahashi's research group previously developed a mouse that had a bioluminescent version of PER2 -- one of the key circadian proteins whose levels fluctuate over the course of a day. By watching the bioluminescence levels wax and wane, the researchers could see how PER2 cycled throughout the animals' bodies during the day. But the protein is present in nearly every part of the body, sometimes making it difficult to distinguish the difference in circadian cycles between different cell types mixed together in the same tissue."If you observe a brain slice, for instance, almost every single cell has a PER2 signal, so you can't really distinguish where any particular PER2 signal is coming from," says Takahashi.In the new work, the scientists overcame this problem by turning to a new bioluminescence system that changed color -- from red to green -- only in cells that expressed a particular gene known as Cre. Then, the researchers could engineer mice so that Cre, which is not naturally found in mouse cells, was only present in one cell type at a time.To test the utility of the approach, Takahashi and his colleagues studied two types of cells that make up the brain's SCN -- arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) cells. In the past, scientists have hypothesized that VIP neurons hold the key to keeping the rest of the SCN synchronized.When the research team looked at VIP neurons -- expressing Cre in just those cells, so that PER2 luminesced green in VIP cells, while red elsewhere -- they found that removing circadian genes from the neurons had little overall effect on the circadian rhythms of the VIP neurons, or the rest of the SCN. "Even when VIP neurons no longer had a functioning clock, the rest of the SCN behaved essentially the same," explains Yongli Shan, Ph.D., a UTSW research scientist and lead author of the study. Nearby cells were able to signal to the VIP neurons to keep them in sync with the rest of the SCN, he says.When they repeated the same experiment on AVP neurons, however -- removing key clock genes -- not only did AVP neurons themselves show disrupted rhythms, but the entire SCN stopped synchronously cycling on its usual 24-hour rhythm."What this showed us was that the clock in AVP neurons is really essential for the synchrony of the whole SCN network," says Shan. "That's a surprising result and somewhat counterintuitive, so we hope it leads to more work on AVP neurons going forward."Takahashi says other researchers who study circadian rhythms have already requested the mouse line from his lab to study the daily cycles of other cells. The mice might allow scientists to hone in on the differences in circadian rhythms between cell types within a single organ, or how tumor cells cycle differently than healthy cells, he says."In all sorts of complex or diseased tissues, this can let you see which cells have rhythms and how they might be similar or different from the rhythms of other cell types."This research was supported by funds from the HHMI, the National Institutes of Health (R01 NS106657, R01 GM114424, T32-HLO9701, F32-AG064886), and The Welch Foundation (AU-1971-20180324).
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Biotechnology
| 2,020 |
August 7, 2020
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https://www.sciencedaily.com/releases/2020/08/200807102322.htm
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Oldest enzyme in cellular respiration isolated
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In the first billion years, there was no oxygen on Earth. Life developed in an anoxic environment. Early bacteria probably obtained their energy by breaking down various substances by means of fermentation. However, there also seems to have been a kind of "oxygen-free respiration." This was suggested by studies on primordial microbes that are still found in anoxic habitats today.
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"We already saw ten years ago that there are genes in these microbes that perhaps encode for a primordial respiration enzyme. Since then, we -- as well as other groups worldwide -- have attempted to prove the existence of this respiratory enzyme and to isolate it. For a long time unsuccessfully because the complex was too fragile and fell apart at each attempt to isolate it from the membrane. We found the fragments, but were unable to piece them together again," explains Professor Volker Müller from the Department of Molecular Microbiology and Bioenergetics at Goethe University.Through hard work and perseverance, his doctoral researchers Martin Kuhns and Dragan Trifunovic then achieved a breakthrough in two successive doctoral theses. "In our desperation, we at some point took a heat-loving bacterium, Thermotoga maritima, which grows at temperatures between 60 and 90°C," explains Dragan Trifunovic, who will shortly complete his doctorate. "Thermotoga also contains Rnf genes, and we hoped that the Rnf enzyme in this bacterium would be a bit more stable. Over the years, we then managed to develop a method for isolating the entire Rnf enzyme from the membrane of these bacteria."As the researchers report in their current paper, the enzyme complex functions a bit like a pumped-storage power plant that pumps water into a lake higher up and produces electricity via a turbine from the water flowing back down again.Only in the bacterial cell the Rnf enzyme (biochemical name = ferredoxin:NAD-oxidoreductase) transports sodium ions out of the cell's interior via the cell membrane to the outside and in so doing produces an electric field. This electric field is used to drive a cellular "turbine" (ATP synthase): It allows the sodium ions to flow back along the electric field into the cell's interior and in so doing it obtains energy in the form of the cellular energy currency ATP.The biochemical proof and the bioenergetic characterization of this primordial Rnf enzyme explains how first forms of life produced the central energy currency ATP. The Rnf enzyme evidently functions so well that it is still contained in many bacteria and some archaea today, in some pathogenic bacteria as well where the role of the Rnf enzyme is still entirely unclear."Our studies thus radiate far beyond the organism Thermotoga maritima under investigation and are extremely important for bacterial physiology in general," explains Müller, adding that it is important now to understand exactly how the Rnf enzyme works and what role the individual parts play. "I'm happy to say that we're well on the way here, since we're meanwhile able to produce the Rnf enzyme ourselves using genetic engineering methods," he continues.
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Biotechnology
| 2,020 |
August 7, 2020
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https://www.sciencedaily.com/releases/2020/08/200807093802.htm
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Origins of life: Chemical evolution in a tiny Gulf Stream
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Chemical reactions driven by the geological conditions on the early Earth might have led to the prebiotic evolution of self-replicating molecules. Scientists at Ludwig-Maximilians Universitaet (LMU) in Munich now report on a hydrothermal mechanism that could have promoted the process.
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Life is a product of evolution by natural selection. That's the take-home lesson from Charles Darwin's book "The Origin of Species," published over 150 years ago. But how did the history of life on our planet begin? What kind of process could have led to the formation of the earliest forms of the biomolecules we now know, which subsequently gave rise to the first cell? Scientists believe that, on the (relatively) young Earth, environments must have existed, which were conducive to prebiotic, molecular evolution. A dedicated group of researchers is engaged in attempts to define the conditions under which the first tentative steps in the evolution of complex polymeric molecules from simple chemical precursors could have been feasible. "To get the whole process started, prebiotic chemistry must be embedded in a setting in which an appropriate combination of physical parameters causes a non-equilibrium state to prevail," explains LMU biophysicist Dieter Braun. Together with colleagues based at the Salk Institute in San Diego, he and his team have now taken a big step toward the definition of such a state. Their latest experiments have shown the circulation of warm water (provided by a microscopic version of the Gulf Stream) through pores in volcanic rock can stimulate the replication of RNA strands. The new findings appear in the journal As the carriers of hereditary information in all known lifeforms, RNA and DNA are at the heart of research into the origins of life. Both are linear molecules made up of four types of subunits called bases, and both can be replicated -- and therefore transmitted. The sequence of bases encodes the genetic information. However, the chemical properties of RNA strands differ subtly from those of DNA. While DNA strands pair to form the famous double helix, RNA molecules can fold into three-dimensional structures that are much more varied and functionally versatile. Indeed, specifically folded RNA molecules have been shown to catalyze chemical reactions both in the test-tube and in cells, just as proteins do. These RNAs therefore act like enzymes, and are referred to as 'ribozymes'. The ability to replicate and accelerate chemical transformations motivated the formulation of the 'RNA world' hypothesis. This idea postulates that, during early molecular evolution, RNA molecules served both as stores of information like DNA, and as chemical catalysts. The latter role is performed by proteins in today's organisms, where RNAs are synthesized by enzymes called RNA polymerases.Ribozymes that can link short RNA strands together -- and some that can replicate short RNA templates -- have been created by mutation and Darwinian selection in the laboratory. One of these 'RNA polymerase' ribozymes was used in the new study.Acquisition of the capacity for self-replication of RNA is viewed as the crucial process in prebiotic molecular evolution. In order to simulate conditions under which the process could have become established, Braun and his colleagues set up an experiment in which a 5-mm cylindrical chamber serves as the equivalent of a pore in a volcanic rock. On the early Earth, porous rocks would have been exposed to natural temperature gradients. Hot fluids percolating through rocks below the seafloor would have encountered cooler waters at the sea-bottom, for instance. This explains why submarine hydrothermal vents are the environmental setting for the origin of life most favored by many researchers. In tiny pores, temperature fluctuations can be very considerable, and give rise to heat transfer and convection currents. These conditions can be readily reproduced in the laboratory. In the new study, the LMU team verified that such gradients can greatly stimulate the replication of RNA sequences.One major problem with ribozyme-driven scenario for replication of RNA is that the initial result of the process is a double-stranded RNA. To achieve cyclic replication, the strands must be separated ('melted'), and this requires higher temperatures, which are likely to unfold -- and inactivate -- the ribozyme. Braun and colleagues have now demonstrated how this can be avoided. "In our experiment, local heating of the reaction chamber creates a steep temperature gradient, which sets up a combination of convection, thermophoresis and Brownian motion," says Braun. Convection stirs the system, while thermophoresis transports molecules along the gradient in a size-dependent manner. The result is a microscopic version of an ocean current like the Gulf Stream. This is essential, as it transports short RNA molecules into warmer regions, while the larger, heat-sensitive ribozyme accumulates in the cooler regions, and is protected from melting. Indeed, the researchers were astonished to discover that the ribozyme molecules aggregated to form larger complexes, which further enhances their concentration in the colder region. In this way, the lifetimes of the labile ribozymes could be significantly extended, in spite of the relatively high temperatures. "That was a complete surprise," says Braun.The lengths of the replicated strands obtained are still comparatively limited. The shortest RNA sequences are more efficiently duplicated than the longer, such that the dominant products of replication are reduced to a minimal length. Hence, true Darwinian evolution, which favors synthesis of progressively longer RNA strands, does not occur under these conditions. "However, based on our theoretical calculations, we are confident that further optimization of our temperature traps is feasible," says Braun. A system in which the ribozyme is assembled from shorter RNA strands, which it can replicate separately, is also a possible way forward.
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Biotechnology
| 2,020 |
August 7, 2020
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https://www.sciencedaily.com/releases/2020/08/200807093737.htm
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Success in promoting plant growth for biodiesel
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In JST Strategic Basic Research Programs, a group of Zhongrui Duan (Researcher, Waseda University) and Motoki Tominaga (Associate professor, Waseda University) et al. succeeded in promoting plant growth and increasing seed yield by heterologous expression of protein from Arabidopsis (artificially modified high-speed motor protein(1) ) in Camelina sativa, which is expected as a useful plant for biodiesel.
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Cytoplasmic streaming is seen in any plant cells from algae to higher plants as a phenomenon of active cytoplasmic movement with organelles, such as the endoplasmic reticulum and mitochondria. It is known that cytoplasmic streaming is generated by the sliding of motor protein myosin XI(2), which is binding to organelles, along the cytoskeleton constituting actin filaments. Previously, the research group has achieved the growth promotion and increasing size of the model plant Arabidopsis by the development of high-speed-type myosin. This technology has been expected to apply to other plant species than Arabidopsis.In this study, the research group showed that the increase of seed yield and the growth promotion of stems and leaves in Camelina could be achieved by heterologous expression of high-speed-type myosin XI gene derived from Arabidopsis in Camelina.Considering the increase of seed yield in Camelina enabled by the expression of high-speed-type myosin XI, it is expected to increase the productivity of biodiesel per area unit. In the future, it is aimed to increase the productivity and quality of camelina oil by co-expressing the genes related to fat synthesis and modification of fatty acid composition with high-speed-type myosin XI. Moreover, as the group showed that the promotion of plant growth by the high-speed-type myosin XI is also effective in other plant species than the model plant Arabidopsis, application development, such as the reduction of CO2 and biomass, is also expected by increasing the production of plant resources, such as corn, rice, sugar cane, and jatropha.The protein which converts chemical energy via ATP hydrolysis into physical movement. Myosin moving on actin filaments and kinesin or dynein moving on the microtubules is representative examples.A type of motor protein. There are approximately 80 classes of myosin discovered in animals and plants. In plants, there are two classes of plant-specific myosin: myosin VIII (class 8) and myosin XI (class 11). Cytoplasmic streaming is known to occur by the movement of myosin XI.
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Biotechnology
| 2,020 |
August 6, 2020
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https://www.sciencedaily.com/releases/2020/08/200806153555.htm
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A new tool for modeling the human gut microbiome
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Several thousand strains of bacteria live in the human gut. Some of these are associated with disease, while others have beneficial effects on human health. Figuring out the precise role of each of these bacteria can be difficult, because many of them can't be grown in lab studies using human tissue.
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This difficulty is especially pronounced for species that cannot live in oxygen-rich environments. However, MIT biological and mechanical engineers have now designed a specialized device in which they can grow those oxygen-intolerant bacteria in tissue that replicates the lining of the colon, allowing them to survive for up to four days."We thought it was really important to contribute a tool to the community that could be used for this extreme case," says Linda Griffith, the School of Engineering Professor of Teaching Innovation in MIT's Department of Biological Engineering. "We showed that you can grow these very fastidious organisms, and we were able to study the effects they have on the human colon."Using this system, the researchers showed that they could grow a strain of bacteria called Faecalibacterium prausnitzii, which lives in the human gut and protects against inflammation. They also showed that these bacteria, which are often diminished in patients with Crohn's disease, appear to exert many of their protective effects through the release of a fatty acid called butyrate.Griffith and David Trumper, an MIT professor of mechanical engineering, are the senior authors of the study, which appears today in the journal The human gut's complex microbiome environment is difficult to model using animals such as mice, in part because mice eat a very different diet from humans, Griffith says."We've learned a huge amount from mice and other animal models, but there are a lot of differences, especially when it comes to the gut microbiome," she says.Most of the bacteria that live in the human gut are anaerobic, meaning that they do not require oxygen to survive. Some of these bacteria can tolerate low levels of oxygen, while others, such as To overcome this, the MIT team designed a device that allows them to precisely control oxygen levels in each part of the system. Their device contains a channel that is coated with cells from the human mucosal barrier of the colon. Below these cells, nutrients are pumped in to keep the cells alive. This bottom layer is oxygen-rich, but the concentration of oxygen decreases toward the top of the mucosal cell layer, similarly to what happens in the interior of the human colon.Just as they do in the human colon, the barrier cells in the channel secrete a dense layer of mucus. The MIT team showed that Using this system, the researchers were able to show that "Overall, this pathway has been reduced, which is really similar to what people have seen in humans," Zhang says. "It seems that the bacteria are desensitizing the mammalian cells to not overreact to the dangers in the outside environment, so the inflammation status is being calmed down by the bacteria."Patients with Crohn's disease often have reduced levels of When the researchers added butyrate to the system, without bacteria, it did not generate all of the effects that they saw when the bacteria were present. This suggests that some of the bacteria's effects may be exerted through other mechanisms, which the researchers hope to further investigate.The researchers also plan to use their system to study what happens when they add other species of bacteria that are believed to play a role in Crohn's disease, to try to further explore the effects of each species.They are also planning a study, working with Alessio Fasano, the division chief of pediatric gastroenterology and nutrition at Massachusetts General Hospital, to grow mucosal tissue from patients with celiac disease and other gastrointestinal disorders. This tissue could then be used to study microbe-induced inflammation in cells with different genetic backgrounds."We are hoping to get new data that will show how the microbes and the inflammation work with the genetic background of the host, to see if there could be people who have a genetic susceptibility to having microbes interfere with the mucosal barrier a little more than other people," Griffith says.She also hopes to use the device to study other types of mucosal barriers, including those of the female reproductive tract, such as the cervix and the endometrium.The research was funded by the U.S. National Institutes of Health, the Boehringer Ingelheim SHINE Program, and the National Institute of Environmental Health Sciences.
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Biotechnology
| 2,020 |
August 6, 2020
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https://www.sciencedaily.com/releases/2020/08/200806111850.htm
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Faster rates of evolution are linked to tiny genomes
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Inside every cell lies a genome -- a full set of DNA that contains the instructions for building an organism. Across the biological world, genomes show a staggering diversity in size. For example, the genome of the Japanese white flower,
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Now, in an international collaboration, led by the Okinawa Institute of Science and Technology Graduate University (OIST) and the University of Sydney, and including researchers from the University of the Ryukyus, the Tokyo Institute of Technology, and RIKEN, scientists have found a link between mutation rate -- how quickly the DNA sequence changes -- and genome size. Writing in "This was a really surprising result," said Professor Tom Bourguignon, co-first author of the study and head of the Evolutionary Genomics Unit at OIST. "Currently, the most accepted idea is that population size is the main factor that determines genome size in prokaryotes, particularly in endosymbionts, but our research challenges this view."Endosymbionts are organisms that live inside the bodies or cells of other organisms, and typically have much smaller genomes than their free-living counterparts. The Evolutionary Genomics Unit researches an endosymbiont called "At small population sizes, natural selection is much less effective, and evolution is driven more strongly by chance," said Dr. Yukihiro Kinjo, co-first author and a postdoctoral scholar from the Evolutionary Genomics Unit. "Without enough selection pressure to maintain specific genes, mutations can arise that inactive and erode these genes, eventually leading to their total loss from the genome."While population size as a driving force for genome reduction may be an attractive idea, many free-living prokaryotes that live in larger populations have also evolved smaller genomes, suggesting that it's only part of the story. Additional explanations have also been proposed but, until now, the mutation rate -- or the speed at which evolution occurs -- has been overlooked.In the study, the scientists collected genome data from a diverse range of prokaryotes, including strains from two endosymbiotic lineages and seven free-living lineages.For each lineage, the team constructed an evolutionary tree that showed how the strains had diverged from each other. With the help of the OIST Biological Complexity Unit, led by Professor Simone Pigolotti, the scientists then created models that reconstructed how gene loss had occurred in each strain. They then estimated the mutation rate, population size and selection pressure for each strain and compared it to the amount of gene loss.Surprisingly, the scientists did not find a clear link between estimated population size and rate of gene loss. Instead, they found a relationship between mutation rate and gene loss for seven out of the nine lineages studied, with higher mutation rates associated with faster rates of gene loss, resulting in smaller genomes."Although we haven't established a cause, there is a theoretical prediction that explains this observation; if the rate of mutation outweighs a selection pressure to maintain a gene, the gene will be lost from the genome," said Dr. Kinjo.The scientists also found clues as to how the gene loss occurred, as strains with smaller genomes had lost genes involved in repairing DNA."DNA repair genes fix damaged DNA, so when they are lost the mutation rate of a strain can quickly increase. Most mutations are harmful, so this can quickly inactivate other genes and drive their loss from the genome. If some of these inactivated genes are also involved in DNA repair, this can further accelerate mutation rate and gene loss," explained Professor Gaku Tokuda, from the University of the Ryukyus.Although the answers to how gene loss occurs are becoming clearer, whether there are evolutionary reasons behind why prokaryotes increase their rate of mutation to shrink their genome, and if so, what these reasons are, remains an open question."Figuring out the evolutionary explanation for what we see is really complicated. It could be that an increased rate of mutation occurs to provide an adaptive advantage, such as the removal of unwanted or unnecessary genes. But we still can't rule out the possibility that the increased rate of mutation is non-adaptive and due to chance," said Dr. Kinjo.Overall, their findings shed new light on the evolution of small genomes, prompting a re-think of the current dominant idea of genome reduction being driven by small population sizes."Unlike with population size, our results suggest that mutation rate could drive genome reduction in both free-living and endosymbiotic prokaryotes. This could be the first step in comprehensively understanding what drives changes in genome size across all prokaryotes," said Prof. Bourguignon.
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Biotechnology
| 2,020 |
August 5, 2020
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https://www.sciencedaily.com/releases/2020/08/200805124054.htm
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Autism: How a gene alteration modifies social behavior
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A team of researchers at the Biozentrum, University of Basel, has discovered a new connection between a genetic alteration and social difficulties related to autism: A mutation in the neuroligin-3 gene reduces the effect of the hormone oxytocin. In the journal
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Autism occurs in about one percent of the population and is characterized by alterations in communication, repetitive behavior and social difficulties. There are numerous genetic factors involved in the development of autism. Hundreds of different genes have been identified, including the gene encoding the synaptic adhesion molecule neuroligin-3. The mechanisms by which this large variety of genetic alterations is related to the symptoms of autism are still largely unknown and is one of the major challenges in the development of new treatments.The research team led by Professor Peter Scheiffele at the Biozentrum of the University of Basel has now uncovered an unexpected link between neuroligin-3, a gene that contributes to the likelihood of autism, and the oxytocin signaling pathway in a mouse model. The hormone oxytocin regulates social behavior in mammals, in particular social interactions.Mice with mutations in certain genes which display a typical behavior linked to autism in humans are used as a model system to study autism and help scientists to learn more about the biology of this condition in humans.In such a mouse model, Scheiffele's team has demonstrated for the first time that an autism associated mutation in the neuroligin-3 gene disrupts the oxytocin signaling pathway in the neurons of the brain's reward system in mice and, as a consequence, reduces social interactions between mice. Unexpectedly, loss of neuroligin affects the balance of protein synthesis in these neurons and thus the neuronal responses to oxytocin.It was already speculated that signals mediated by oxytocin could possibly play a role in autism. "However, we were very surprised to discover that mutations in neuroligin-3 impair oxytocin signaling pathways. We have succeeded in putting together two puzzle pieces of the mechanisms underlying autism," says Scheiffele.Furthermore, the research team demonstrated that alterations in the oxytocin system in mice with a neuroligin-3 mutation can be restored by treatment with a pharmacological inhibitor of protein synthesis. This treatment normalized the social behavior of the mice: Like their healthy conspecifics, they reacted differently to familiar mice or mice foreign to them. Importantly, the same inhibitor also improved behavioral symptoms in a second rodent model of autism, indicating that it could be more widely applied in the treatment of autism.The newly discovered convergence between three important elements -- a genetic factor, the changes in neuronal protein synthesis, and the regulation of social behavior by the oxytocin system -sheds some light onto how multiple factors implicated in autism may be connected. In addition, the findings may open new approaches for the treatment of certain aspects of social behavior in some cases of autism, where this is desirable.
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Biotechnology
| 2,020 |
August 5, 2020
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https://www.sciencedaily.com/releases/2020/08/200805124038.htm
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Gut microbes shape our antibodies before we are infected by pathogens
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B cells are white blood cells that develop to produce antibodies. These antibodies, or immunoglobulins, can bind to harmful foreign particles (such as viruses or disease-causing bacteria) to stop them invading and infecting the body's cells. Each B cell carries an individual B cell receptor (BCR) which determines which particles it can bind, rather like each lock accepts a different key. There are many millions of B cells with different receptors in the body. This immense diversity comes from rearranging the genes that code these receptors, so the receptor is slightly different in every B cell resulting in billions of possibilities of different harmful molecules that could be recognized. Intestinal microbes trigger expansion of these B cell populations and antibody production, but until now it was unknown whether this was a random process, or whether the molecules of the intestinal microbes themselves influence the outcome.
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In a research article published in the journal The number of benign microbes living in our intestines is about the same as the number of cells in our body. Mostly these bacteria stay within the intestinal tube rather than penetrate the body tissues. Unfortunately, some penetration is unavoidable, because the intestine only has a single layer of cells that separate the inside of the tube from blood vessels that we need to absorb our food.Dr. Limenitakis used specially designed computer programs to process millions of genetic sequences that compare the antibody repertoire from B cells, depending on whether the microbes stay in the intestine, or whether they reach the bloodstream. In both cases the antibody repertoire is altered, but in rather different ways depending on how the exposure occurs."Interestingly, this is rather predictable depending on the microbe concerned and where it is in the body, indicating that the intestinal microbes direct the development of our antibodies before we get a serious infection and this process is certainly not random," explains Ganal-Vonarburg.There are different sorts of antibodies in the lining of the intestine (IgA) compared with the bloodstream (IgM and IgG). Using the powerful genetic analysis, the researchers showed that the range of different antibodies produced in the intestine was far less that those produced in central body tissues. This means that once microbes get into the body, the immune system has many more possibilities to neutralize and eliminate them, whereas antibodies in the intestine mainly just bind the bacterial molecules that they can see at any one time.Over their life-span mammals face a huge variety of different microbial challenges. It was therefore important to know how once the antibody repertoire could change once had been shaped by a particular microbe when something else came along. The research team answered this question by testing what happened with the same microbe at different sites or with two different microbes on after another.Although intestinal microbes do not directly produce an especially wide range of different antibodies, they sensitize the central immune tissues to produce antibodies if the microbe gets into the bloodstream. When a second microbes comes along, the rather limited intestinal antibody response changes to accommodate this microbe (rather like changing the lock in one's door). This is different from what happens when microbes get into the blood stream to reach the central body tissues when a second set of antibodies is made without compromising the first response to the original microbes (like installing another lock, so the door can be opened with different keys). This shows that central body tissues have the capacity to remember a range of different microbial species and to avoid the dangers of sepsis. It also shows that different B cell immune strategies in different body compartments are important for maintenance of our peaceful existence with our microbial passengers.Dr. Li comments that "Our data show for the first time that not only the composition of our intestinal microbiota, but also the timing and sequence of exposure to certain members of the commensal microbiota, happening predominantly during the first waves of colonisations during early life, have an outcome on the resulting B cell receptor repertoire and subsequent immunity to pathogens."
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Biotechnology
| 2,020 |
August 5, 2020
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https://www.sciencedaily.com/releases/2020/08/200805110115.htm
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Discovery could lead to more potent garlic, boosting flavor and bad breath
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For centuries, people around the world have used garlic as a spice, natural remedy, and pest deterrent -- but they didn't know how powerful or pungent the heads of garlic were until they tasted them.
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But what if farmers were able to grow garlic and know exactly how potent it would be? What if buyers could pick their garlic based on its might?A team of Virginia Tech researchers recently discovered a new step in the metabolic process that produces the enzyme allicin, which leads to garlic's delectable flavor and aroma, a finding that upends decades of previous scientific belief. Their work could boost the malodorous -- yet delicious -- characteristics that garlic-lovers the world over savor."This information changes the whole story about how garlic could be improved or we could make the compounds responsible of its unique flavor," said Hannah Valentino, a College of Agriculture and Life Sciences Ph.D. candidate. "This could lead to a new strain of garlic that would produce more flavor."The discovery of this pathway opens the door for better control of production and more consistent crops, which would help farmers. Garlic could be sold as strong or weak, depending on consumer preferences.The research was recently published in the Journal of Biological Chemistry.When Valentino, an Institute for Critical Technology and Applied Science doctoral fellow, and her team set out to test the generally accepted biological process that creates allicin, they found it just didn't happen.That's when the team of researchers set out to discover what was really happening in garlic.As they peeled back the layers, they realized there was no fuel to power the previous accepted biological process that creates allicin."By using rational design, Hannah found a potential substrate," said Pablo Sobrado, professor of biochemistry in the College of Agriculture and Life Sciences and a member of the research team. "This is significant because by finding the metabolic pathway and understanding how the enzyme actually works and its structure gives us a blueprint of how allicin is created during biosynthesis."Valentino and the team -- which included undergraduate students -- worked in the Sobrado Lab in the Fralin Life Sciences Institute directly with the substrates that comprise garlic, doing their work solely in vitro.Hannah Valentino, left, and Pablo Sobrado, right, are conducting research that is laying the foundation for a future in which buyers can choose garlic based on its strength and flavor profile.The researchers found that allicin, the component that gives garlic its smell and flavor, was produced by an entirely different biosynthetic process. Allyl-mercaptan reacts with flavin-containing monooxygenase, which then becomes allyl-sulfenic acid.Importantly, the allicin levels can be tested, allowing farmers to know the strength of their crops without the need for genetic engineering. Greater flavor can simply be predicted, meaning powerful garlic could simply be bred or engineered."We have a basic understanding of the biosynthesis of allicin that it is involved in flavor and smell, but we also now understand an enzyme that we can try to modulate, or a modify, to increase or decrease the level of the flavor molecules based on these biological processes," Sobrado said.Because of their work, the future awaits for fields of garlic harsh enough to keep even the most terrifying vampires at bay.
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Biotechnology
| 2,020 |
August 5, 2020
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https://www.sciencedaily.com/releases/2020/08/200805110108.htm
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Taste bud cells might not be a target of SARS-CoV-2
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An intriguing early symptom among some COVID-19 patients is the loss of the sense of smell and/or taste, which has led to the suspicion that the virus that causes the illness, SARS-CoV-2, could be targeting taste buds. But as researchers report in
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Viruses cause infection by invading specific cells in the body and reproducing, often damaging or killing those cells in the process. Research has shown that SARS-CoV-2 enters human cells through angiotensin-converting enzyme 2 (ACE2), a receptor on the surface of some cells, including those of the human tongue. Hong-Xiang Liu and colleagues wanted to find out whether ACE2 was expressed specifically in taste bud cells, as well as when this receptor first emerges on tongue cells during fetal development, by studying mice as a model organism. Although the mouse version of ACE2 isn't susceptible to SARS-CoV-2, studying where it's expressed in mice could help clarify what happens when people become infected and lose the sense of taste.By analyzing data from oral cells of adult mice, the researchers found that ACE2 was enriched in cells that give the tongue its rough surface, but couldn't be found in most taste bud cells. That means the virus probably does not cause taste loss through direct infection of these cells, the researchers say. Instead, taste buds might be damaged by inflammation caused by the infection. The team also showed that other viruses that affect taste, including the flu virus, might affect different tongue cell types. Further, the researchers analyzed data from oral cells of mice at three developmental stages and found ACE2 in newborn mice but not in fetuses. Previous studies in humans that were not focused on oral cells suggest ACE2 could be expressed at an early fetal stage and then again at a later stage. Therefore, the team states that fetuses could have distinct susceptibilities to SARS-CoV-2 infection at different stages and more work is needed to determine the timing and location of human ACE2 expression.The authors acknowledge funding from the National Institutes of Health and the University of Georgia Research Foundation.
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Biotechnology
| 2,020 |
August 5, 2020
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https://www.sciencedaily.com/releases/2020/08/200805102012.htm
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Molecular forces: The surprising stretching behavior of DNA
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When large forces, for example in bridge construction, act on a heavy beam, the beam will be slightly deformed. Calculating the relationship between forces, internal stresses and deformations is one of the standard tasks in civil engineering. But what happens when you apply these considerations to tiny objects -- for example, to a single DNA double helix?
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Experiments with DNA molecules show that their mechanical properties are completely different from what those of macroscopic objects -- and this has important consequences for biology and medicine. Scientists at TU Wien (Vienna) has now succeeded in explaining these properties in detail by combining ideas from civil engineering and physics.At first glance, you might think of the DNA double helix as a tiny little spring that you can simply stretch and compress just like you would an ordinary spring. But it is not quite that simple: "If you stretch a piece of DNA, you would actually expect the number of turns to decrease. But in certain cases the opposite is true: "When the helix gets longer, it sometimes twists even more," says civil engineer Johannes Kalliauer from the Institute of Mechanics of Materials and Structures at TU Wien. "Apart from that, DNA molecules are much more ductile than the materials we usually deal with in civil engineering: They can become 70% longer under tensile stress."These strange mechanical properties of DNA are of great importance for biology and medicine: "When the genetic information is read from the DNA molecule in a living cell, the details of the geometry can determine whether a reading error occurs, which in the worst case can even cause cancer," says Johannes Kalliauer. "Until now, molecular biology has had to be satisfied with empirical methods to explain the relationship between forces and the geometry of DNA."In his dissertation, Johannes Kalliauer got to the bottom of this issue -- and he did so in the form of a rather unusual combination of subjects: His work was supervised on the one hand by the civil engineer Prof. Christian Hellmich, and on the other hand by Prof. Gerhard Kahl from the Institute of Theoretical Physics."We used molecular dynamics methods to reproduce the DNA molecule on an atomic scale on the computer," explains Kalliauer. "You determine how the DNA helices are compressed, stretched or twisted -- and then you calculate the forces that occur and the final position of the atoms." Such calculations are very complex and only possible with the help of large supercomputers -- Johannes Kalliauer used the Vienna Scientific Cluster (VSC) for this purpose.That way, the strange experimental findings could be explaned -- such as the counterintuitive result that in certain cases the DNA twists even more when stretched. "It's hard to imagine on a large scale, but at the atomic level it all makes sense," says Johannes Kalliauer.Within the atomic models of theoretical physics, interatomic forces and distances can be determined. Using certain rules developed by the team based on principles from civil engineering, the relevant force quantities required to describe the DNA strand as a whole can then be determined -- similar to the way the statics of a beam in civil engineering can be described using some important cross-sectional properties."We are working in an interesting intermediate world here, between the microscopic and the macroscopic," says Johannes Kalliauer. "The special thing about this research project is that you really need both perspectives and you have to combine them."This combination of significantly different size scales plays a central role at the Institute for Mechanics of Materials and Structures time and again. After all, the material properties that we feel every day on a large scale are always determined by behaviour at the micro level. The current work, which has now been published in the "
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Biotechnology
| 2,020 |
August 4, 2020
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https://www.sciencedaily.com/releases/2020/08/200804122214.htm
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Artificial organelles created to control cellular behavior
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Biomedical engineers at Duke University have demonstrated a method for controlling the phase separation of an emerging class of proteins to create artificial membrane-less organelles within human cells. The advance, similar to controlling how vinegar forms droplets within oil, creates opportunities for engineering synthetic structures to modulate existing cell functions or create entirely new behaviors within cells.
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The results appear online on August 3 in the journal Proteins function by folding into specific 3-D shapes that interact with different biomolecular structures. Researchers previously believed that proteins needed these fixed shapes to function. But in the last two decades, a large new class of intrinsically disordered proteins (IDPs) have been discovered that have large regions that are "floppy" -- that is, they do not fold into a defined 3-D shape. It is now understood these regions play an important, previously unrecognized role in controlling various cellular functions.IDPs are also useful for biomedical applications because they can undergo phase transitions -- changing from a liquid to a gel, for example, or from a soluble to an insoluble state, and back again -- in response to environmental triggers, like changes in temperature. These features also dictate their phase behavior in cellular environments and are controlled by adjusting characteristics of the IDPs such as their molecular weight or the sequence in which the amino acids are linked together."Although there are many natural IDPs that show phase behavior in cells, they come in many different flavors, and it has been difficult to discern the rules that govern this behavior," said Ashutosh Chilkoti, the Alan L. Kaganov Distinguished Professor of Biomedical Engineering at Duke. "This paper provides very simple engineering principles to program this behavior within a cell.""Others in the field have taken a top-down approach where they'll make a change to a natural IDP and see how its behavior changes within a cell," said Michael Dzuricky, a research scientist working in the Chilkoti laboratory and first author of the study. "We're taking the opposite approach and building our own artificial IDPs from simple thermodynamic principles. This enables us and others to precisely tune a single property -- the shape of the IDPs phase diagram -- to better understand how this parameter affects biological behavior"In the new paper, the researchers begin by looking to nature for examples of IDPs that come together to form "biomolecular condensates" within cells. These weakly-held-together structures allow cells to create compartments without also building a membrane to encapsulate it. Using one such IDP from the common fruit fly as a basis, the researchers draw from their extensive history of working with IDPs to engineer a molecularly simpler artificial version that retains the same behavior.This simpler version allowed the researchers to make precise changes to the molecular weight of the IDP and amino acids of the IDPs. The researchers show that, depending on how these two variables are tweaked, the IDPs come together to form these compartments at different temperatures in a test tube. And by consistently trying various tweaks and temperatures, the researchers gained a solid understanding of which design parameters are most important to control the IDP's behavior.A test tube, however, is not the same as a living cell, so the researchers then went one step further to demonstrate how their engineered IDPs behave within E. coli. As predicted, their artificial IDPs grouped together to form a tiny droplet within the cell's cytoplasm. And because the IDP's behavior was now so well understood, the researchers showed they could predictably control how they coalesced using their test tube principles as a guide."We were able to change temperatures in cells to develop a complete description of their phase behavior, which mirrored our test tube predictions," said Dzuricky. "At this point, we were able to design different artificial IDP systems where the droplets that are formed have different material properties."Put another way, because the researchers understood how to manipulate the size and composition of the IDPs to respond to temperature, they could program the IDPs to form droplets or compartments of varying densities within cells. To show how this ability might be useful to biomedical engineers, the researchers then used their newfound knowledge, as nature often does, to create an organelle that performs a specific function within a cell.The researchers showed that they could use the IDPs to encapsulate an enzyme to control its activity level. By varying the molecular weight of the IDPs, the IDPs hold on the enzyme either increased or decreased, which in turn affected how much it could interact with the rest of the cell.To demonstrate this ability, the researchers chose an enzyme used by E. coli to convert lactose into usable sugars. However, in this case, the researchers tracked this enzyme's activity with a fluorescent reporter in real-time to determine how the engineered IDP organelle was affecting enzyme activity.In the future, the researchers believe they could use their new IDP organelles to control the activity levels of biomolecules important to disease states. Or to learn how natural IDPs fill similar cellular roles and understand how and why they sometimes malfunction."This is the first time anybody has been able to precisely define how the protein sequence controls phase separation behavior inside cells," said Dzuricky. "We used an artificial system, but we think that the same rules apply to natural IDPs and are excited to begin testing this theory.""We can also now start to program this type of phase behavior with any protein in a cell by fusing them to these artificial IDPs," said Chilkoti. "We hope that these artificial IDPs will provide new tool for synthetic biology to control cell behavior."This research was supported by the National Institutes of Health (R35GM127042) and the National Science Foundation (DMR-17-29671, CHE-1709735).
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Biotechnology
| 2,020 |
August 4, 2020
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https://www.sciencedaily.com/releases/2020/08/200804122213.htm
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AI may offer a better way to ID drug-resistant superbugs
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Biomedical engineers at Duke University have shown that different strains of the same bacterial pathogen can be distinguished by a machine learning analysis of their growth dynamics alone, which can then also accurately predict other traits such as resistance to antibiotics. The demonstration could point to methods for identifying diseases and predicting their behaviors that are faster, simpler, less expensive and more accurate than current standard techniques.
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The results appear online on August 3 in the For most of the history of microbiology, bacteria identification has relied on growing cultures and analyzing the physical traits and behaviors of the resulting bacterial colony. It wasn't until recently that scientists could simply run a genetic test.Genetic sequencing, however, isn't universally available and can often take a long time. And even with the ability to sequence entire genomes, it can be difficult to tie specific genetic variations to different behaviors in the real world.For example, even though researchers know the genetic mutations that help shield/protect bacteria from beta-lactam antibiotics -- the most commonly used antibiotic in the world -- sometimes the DNA isn't the whole story. While a single resistant bacteria usually can't survive a dose of antibiotics on its own, large populations often can.Lingchong You, professor of biomedical engineering at Duke, and his graduate student, Carolyn Zhang, wondered if a new twist on older methods might work better. Maybe they could amplify one specific physical characteristic and use it to not only identify the pathogen, but to make an educated guess about other traits such as antibiotic resistance."We thought that the slight variance in the genes between strains of bacteria might have a subtle effect on their metabolism," You said. "But because bacterial growth is exponential, that subtle effect could be amplified enough for us to take advantage of it. To me, that notion is somewhat intuitive, but I was surprised at how well it actually worked."How quickly a bacterial culture grows in a laboratory depends on the richness of the media it is growing in and its chemical environment. But as the population grows, the culture consumes nutrients and produces chemical byproducts. Even if different strains start with the exact same environmental conditions, subtle differences in how they grow and influence their surroundings accumulate over time.In the study, You and Zhang took more than 200 strains of bacterial pathogens, most of which were variations of E. coli, put them into identical growth environments, and carefully measured their population density as it increased. Because of their slight genetic differences, the cultures grew in fits and starts, each possessing a unique temporal fluctuation pattern. The researchers then fed the growth dynamics data into a machine learning program, which taught itself to identify and match the growth profiles to the different strains.To their surprise, it worked really well."Using growth data from only one initial condition, the model was able to identify a particular strain with more than 92 percent accuracy," You said. "And when we used four different starting environments instead of one, that accuracy rose to about 98 percent."Taking this idea one step further, You and Zhang then looked to see if they could use growth dynamic profiles to predict another phenotype -- antibiotic resistance.The researchers once again loaded a machine learning program with the growth dynamic profiles from all but one of the various strains, along with data about their resilience to four different antibiotics. They then tested to see if the resulting model could predict the final strain's antibiotic resistances from its growth profile. To bulk up their dataset, they repeated this process for all of the other strains.The results showed that the growth dynamic profile alone could successfully predict a strain's resistance to antibiotics 60 to 75 percent of the time."This is actually on par or better than some of the current techniques in the literature, including many that use genetic sequencing data," said You. "And this was just a proof of principle. We believe that with higher-resolution data of the growth dynamics, we could do an even better job in the long term."The researchers also looked to see if the strains exhibiting similar growth curves also had similar genetic profiles. As it turns out, the two are completely uncorrelated, demonstrating once again how difficult it can be to map cellular traits and behaviors to specific stretches of DNA.Moving forward, You plans to optimize the growth curve procedure to reduce the time it takes to identify a strain from 2 to 3 days to perhaps 12 hours. He's also planning on using high-definition cameras to see if mapping how bacterial colonies grow in space in a Petri dish can help make the process even more accurate.
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Biotechnology
| 2,020 |
August 4, 2020
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https://www.sciencedaily.com/releases/2020/08/200804111532.htm
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Cell diversity in the embryo
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A research team at the Max Planck Institute for Molecular Genetics in Berlin has explored the role of factors in embryonic development that do not alter the sequence of DNA, but only epigenetically modify its "packaging." In the scientific journal
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A fertilized egg cell develops into a complete organism with a multitude of different tissues and organs, although the genetic information is exactly the same in every cell. A complex clockwork of molecules regulates which cell in the body fulfills each task and determines the proper time and place to activate each gene.Epigenetic regulator factors are part of this molecular mechanism and act to modify the "packaging" of the DNA molecule without altering the underlying genetic information. Specifically, they act to bookmark the DNA and control what parts can be accessed in each cell.Most of these regulators are essential, and embryos lacking them tend to die during the time of development when organs begin to emerge. However, these regulators may have specific functions that differ in every cell, making them difficult to study. This has also been a major hindrance for studying these proteins, which are not only relevant for the development of embryos, but also involved in the formation of cancer."The same regulator is present in all cells, but can have very different tasks, depending on cell type and time of development," says Stefanie Grosswendt, one of the first authors of a new study in the scientific journal Nature.Grosswendt and her colleague Helene Kretzmer from Alexander Meissner's lab at the Max Planck Institute for Molecular Genetics (MPIMG) in Berlin together with Zachary Smith from Harvard University, MA, have now succeeded in elucidating the significance of epigenetic regulators for embryonic development with unprecedented precision.The researchers analyzed ten of the most important epigenetic regulators. Using the CRISPR-Cas9 system, they first specifically removed the genes coding for the regulatory factors in fertilized oocytes and then observed the effects on embryo development days later.After the embryos had developed for about six to nine days, the team examined the anatomical and molecular changes that resulted from the absence of the respective regulator. They found that the cellular composition of many of the embryos was substantially altered. Cells of certain types existed in excessive numbers, while others were not produced at all.In order to make sense of these changes on a molecular level, researchers examined hundreds to thousands of individual cells from embryos, from which single epigenetic regulators had been systematically removed. They sequenced the RNA molecules of almost 280,000 individual cells to investigate the consequences of the loss of function. RNA relays information encoded on the DNA, allowing researchers to understand the identity and behavior of cells using sequencing technologies.In their analysis, the scientists focused on a phase of development, in which epigenetic regulators are particularly important. When they compared the data of altered and unaltered embryos, they identified genes that were dysregulated, and cell types that are abnormally over- or underproduced. From this overall picture, they deduced previously unknown functions of many epigenetic regulators.An eight-day-old mouse embryo looks a bit like a seahorse and does not have any organs yet. "From the outer appearance of an early embryo, one can often only guess which structures and organs will form and which will not," say bioinformatician Helene Kretzmer and biologist Zachary Smith, who are also both first authors of the publication. "Our sequencing allows for a much more precise and high resolution view."The single-cell analysis gave them a highly detailed view over the first nine days of mouse development. Often, switching off a single regulator led to ripple effects throughout the network of interacting genes, with many differentially activated or inactivated genes over the course of development.Removing the epigenetic regulator Polycomb (PRC2) had a particularly striking impact. "Without PRC2, the embryo looks egg-shaped and very small after eight and a half days, which is very unusual," says Kretzmer. "We see vast changes to how DNA is packaged that happens much earlier, long before the embryo develops morphological abnormalities."The researchers found that PRC2 is responsible for limiting the amount of germline progenitor cells -- the cells that later become sperm and eggs. Without PRC2, the embryo develops an excessive number of these cells, loses its shape, and dies after a short time."With the combination of new technologies we addressed issues that have been up in the air for 25 years," says Alexander Meissner, who headed the study. "We now understand better how epigenetic regulators arrange for the many different types of cells in the body."The work is only the first step for even more detailed investigations, says Meissner. "Our method lets us investigate other factors such as transcription or growth factors or even a combination of these. We are now able to observe very early developmental stages in a level of detail that was previously unthinkable."
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Biotechnology
| 2,020 |
August 4, 2020
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https://www.sciencedaily.com/releases/2020/08/200804111526.htm
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The wrong track: How papillomaviruses trick the immune system
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Specific antibodies protect us against viral infections -- or do they not? Researchers at the German Cancer Research Center (DKFZ) studied the immune response to papillomaviruses in mice and discovered a hitherto unknown mechanism by which the pathogens outwit the immune system: At the beginning of the infection cycle, they produce a longer version of a protein that surrounds the viral genome. The body produces antibodies against this protein, but they are not effective in fighting the pathogen.
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The human immune system has a wide variety of defense strategies to protect the body against pathogens, one of which involves producing antibodies to fight viruses and bacteria. Over time, however, these pathogens have developed elaborate ways of escaping the immune system.Scientists are already aware of some of these strategies. In human papillomaviruses (HPV), however, up until now they have only known about such strategies in innate, already present immunity and not in adaptive immunity, which does not develop until pathogens enter the body and is associated with the production of antibodies.Frank Rösl and his co-workers from DKFZ under the supervision of Daniel Hasche have now discovered a new mechanism by which cutaneous papillomaviruses (specific to the skin) trick the immune system.Certain cutaneous HPV, such as HPV5 and HPV8, occur as natural infections on the skin. They are not sexually transmitted, but are passed on from the mother to the newborn child. Thus, family members are usually colonized with the same HPV types. An infection normally goes unnoticed, because the body is able to overcome it. Depending on the individual status of a person's immune system, their genetic predisposition, age, and other external factors such as UV radiation, however, certain cutaneous HPV types are able to stimulate cell division in their host cells. This leads to skin changes and in rare cases to development of a squamous cell carcinoma, also known as fair-skin cancer.The experiments were conducted in a particular mouse species, Mastomys coucha, which, like humans, can become infected with cutaneous papillomaviruses shortly after birth and produce specific antibodies against the virus. In combination with UV radiation, infected animals are more likely to develop squamous cell cancer.The animals' immune system produces antibodies against the two viral proteins L1 and L2 that make up the virus particles, also called capsids. These antibodies can prevent the viruses from entering the host cells and thus neutralize the virus. However, the experiments carried out by the DKFZ scientists showed that besides the normal L1 protein, the viruses also produce a longer version. The latter is not able to actually take part in forming the viral capsid. Instead, it acts as a kind of bait against which the immune system directs its response and produces specific antibodies.However, the scientists were able to demonstrate that these antibodies are not effective in fighting the papillomavirus. Instead of neutralizing the infectious pathogen through binding to L1, the antibodies merely bind the nonfunctional protein used as bait. While the immune system is busy producing these non-neutralizing antibodies, the virus can continue to replicate and spread throughout the body. It take several more months before neutralizing antibodies are produced that target the normal L1 protein and ultimately the infectious viruses themselves."In both rodents and humans, in almost all HPV types that can cause cancer, the L1 gene is designed such that a longer version of the protein can be produced. This is also true for high-risk HPV types such as HPV16 and HPV18, which can cause cervical cancer. It therefore appears to be a common mechanism that enables the viruses to replicate and spread efficiently during the early stage of infection," Daniel Hasche explained. "The fact that antibodies against papillomaviruses can be detected is therefore not necessarily associated with protection against infection. This will need to be taken into account in future when evaluating and interpreting epidemiological studies," Frank Rösl added.
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Biotechnology
| 2,020 |
August 3, 2020
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https://www.sciencedaily.com/releases/2020/08/200803160500.htm
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Hydrogel paves way for biomedical breakthrough
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Published in
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To function optimally in the body, a manufactured implant -- whether it be an artificial hip, a fabricated spinal disc or engineered tissue -- must bond and interact with appropriate surrounding tissues and living cells.When that doesn't happen an implant may fail or, worse still, be rejected by the body. Worldwide, implant failures and rejections are a significant cost to health systems, placing large financial and health burdens on patients.The team, which was led by School of Biomedical Engineering, Dr Behnam Akhavan and Professor Marcela Bilek, successfully combined hydrogels including those made from silk with Teflon and polystyrene polymers."Despite being similar to the natural tissue of the body; in medical science hydrogels are notoriously difficult to work with as they are inherently weak and structurally unstable. They do not easily attach to solids which means they often cannot be used in mechanically demanding applications such as in cartilage and bone tissue engineering," said Dr Akhavan.Hydrogels are highly attractive for tissue engineering because of their functional and structural similarity to human body soft tissue," said Biomedical Engineering PhD student Ms Rashi Walia, who carried out the research in collaboration with the University of Sydney's School of Physics and School of Chemical and Biomolecular Engineering, as well as Tufts University in Massachusetts, USA."Our group's unique plasma process, recently reported in ACS Applied Materials and Interfaces, enables us to activate all surfaces of complex, porous structures, such as scaffolds, to covalently attach biomolecules and hydrogels," said ARC Laureate and Biomedical Engineering academic, Professor Marcela Bilek."These advances enable the creation of mechanically robust complex-shaped polymeric scaffolds infused with hydrogel, bringing us a step closer to mimicking the characteristics of natural tissues within the body," said Professor Bilek."The plasma process is carried out in a single step, generates zero waste, and does not require additional chemicals that can be harmful to the environment."Biomedical devices, organ implants, biosensors and tissue engineering scaffolds that are set to benefit from the new hydrogel technology."There are several scenarios in which this technology can be used. The gel could be loaded with a drug to release slowly over time, or it can be used to mimic structures such as bone-cartilage," said Dr Akhavan."These materials are also excellent candidates for applications such as lab-on-a-chip platforms, bioreactors that mimic organs, and biomimetic constructs for tissue repair as well as antifouling coatings for surfaces submerged in marine environments."The research tested the material using biomolecules found in the body, which demonstrated a positive cellular response.Dr Akhavan and the team will be progressing their area of research and will further develop the technology to combine hydrogels with non-polymeric solid materials, such as ceramics and metals.
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Biotechnology
| 2,020 |
August 3, 2020
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https://www.sciencedaily.com/releases/2020/08/200803140009.htm
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AI and single-cell genomics
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Traditional single-cell sequencing methods help to reveal insights about cellular differences and functions -- but they do this with static snapshots only rather than time-lapse films. This limitation makes it difficult to draw conclusions about the dynamics of cell development and gene activity. The recently introduced method "RNA velocity" aims to reconstruct the developmental trajectory of a cell on a computational basis (leveraging ratios of unspliced and spliced transcripts). This method, however, is applicable to steady-state populations only. Researchers were therefore looking for ways to extend the concept of RNA velocity to dynamic populations which are of crucial importance to understand cell development and disease response.
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Researchers from the Institute of Computational Biology at Helmholtz Zentrum München and the Department of Mathematics at TUM developed "scVelo" (single-cell velocity). The method estimates RNA velocity with an AI-based model by solving the full gene-wise transcriptional dynamics. This allows them to generalize the concept of RNA velocity to a wide variety of biological systems including dynamic populations."We have used scVelo to reveal cell development in the endocrine pancreas, in the hippocampus, and to study dynamic processes in lung regeneration -- and this is just the beginning," says Volker Bergen, main creator of scVelo and first author of the corresponding study in Nature Biotechnology.With scVelo researchers can estimate reaction rates of RNA transcription, splicing and degradation without the need of any experimental data. These rates can help to better understand the cell identity and phenotypic heterogeneity. Their introduction of a latent time reconstructs the unknown developmental time to position the cells along the trajectory of the underlying biological process. That is particularly useful to better understand cellular decision making. Moreover, scVelo reveals regulatory changes and putative driver genes therein. This helps to understand not only how but also why cells are developing the way they do.AI-based tools like scVelo give rise to personalized treatments. Going from static snapshots to full dynamics allows researchers to move from descriptive towards predictive models. In the future, this might help to better understand disease progression such as tumor formation, or to unravel cell signaling in response to cancer treatment."scVelo has been downloaded almost 60,000 times since its release last year. It has become a stepping-stone tooltowards the kinetic foundation for single-cell transcriptomics," adds Prof. Fabian Theis, who conceived the study and serves as Director at the Institute for Computational Biology at Helmholtz Zentrums München and Chair for Mathematical Modeling of Biological Systems at TUM.
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Biotechnology
| 2,020 |
August 3, 2020
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https://www.sciencedaily.com/releases/2020/08/200803120158.htm
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Cannabinoids may affect activity of other pharmaceuticals
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Cannabinoid-containing products may alter the effects of some prescription drugs, according to Penn State College of Medicine researchers. They published information that could help medical professionals make safe prescribing choices for their patients who use prescription, over-the-counter or illicit cannabinoid products.
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Kent Vrana, professor and chair of pharmacology at the College of Medicine, and Paul Kocis, a pharmacist at Penn State Health Milton S. Hershey Medical Center, compiled a list of 57 medications that may not function as intended when used with medical cannabinoids, CBD oil (hemp oil) and medical or recreational marijuana. The list was published in the journal The medications on the list have a narrow therapeutic index, meaning they are prescribed at specific doses -- enough to be effective, but not enough to cause harm. Vrana says it's important for medical professionals to consider the list when prescribing medical cannabinoids and how it may affect other medications a patient is taking.To develop the list, the researchers looked at the prescribing information for four prescription cannabinoid medications. This information included a list of enzymes in the body that process the active ingredients in those medications, which can include delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). They compared this information against prescribing information from common medications using information available from regulatory agencies like the U.S. Food and Drug Administration to identify where there may be overlap, called a drug-drug interaction.The list contains a variety of drugs from heart medications to antibiotics and antifungals. As one example, researchers identified warfarin, a common anticoagulant that prevents harmful blood clots from forming, as having a potential drug-drug interaction with cannabinoid products. Often prescribed for patients with atrial fibrillation or following cardiac valve replacement, the drug has a narrow therapeutic index, and Vrana cautions that medical professionals consider this potential drug-drug interaction both when prescribing warfarin to patients on prescription cannabinoids or prescribing cannabinoids to a patient taking warfarin.The researchers say that medical professionals should also consider patient use of CBD oil products and medical and recreational marijuana when using or prescribing drugs on the identified list. Most of those products lack government regulation and there is little to no prescribing or drug-drug interaction information for those products."Unregulated products often contain the same active ingredients as medical cannabinoids, though they may be present in different concentrations," Vrana said. "The drug-drug interaction information from medical cannabinoids may be useful as medical professionals consider the potential impact of over-the-counter or illicit cannabinoid products."Vrana advises that patients be honest with their health care providers about their use of cannabinoid products -- from over-the-counter products to recreational marijuana. He says that doing so can help ensure the safe and effective use of prescribed medications.In addition to the identified list of 57 prescription medications with a narrow therapeutic index that is potentially impacted by concomitant cannabinoid use, a comprehensive list of 139 medications that could have a potential drug-drug interaction with a cannabinoid is available online. Vrana and Kocis plan to routinely update this drug-drug interaction list as newer medications are approved and real-world evidence accumulates.Kent Vrana received a sponsored research agreement from PA Options for Wellness, a medical cannabis provider and clinical registrant in Pennsylvania, and this research was supported in part by the agreement. The College of Medicine and PA Options for Wellness have a 10-year research agreement designed to help physicians and patients make better informed clinical decisions related to cannabinoids.
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Biotechnology
| 2,020 |
August 3, 2020
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https://www.sciencedaily.com/releases/2020/08/200803120150.htm
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Cells relax their membrane to control protein sorting
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The tension in the outer membrane of cells plays an important role in a number of biological processes. A localised drop in tension, for example, makes it easier for the surface to be bending inward and form invaginations that will become free vesicles inside the cell. These are delimited by a membrane that contains all proteins originally present in the invaginations. A fundamental function of these so-called endosomes is to sort proteins to their cellular destination, e.g. reutilization or degradation. Are the functions of endosomes modulated by variations in tension? Scientists from the University of Geneva (UNIGE) and the Chemical Biology National Centre of Competence in Research have answered in the affirmative thanks to their high-precision research published in the journal
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How can chemical tools be used in real-time to measure the physical properties of a cell's internals structures while still alive? This is one of the challenges that the National Centre of Competence in Research (NCCR) Chemical Biology -- directed at the UNIGE -- has been trying to meet since it was launched in 2010. To help reach this goal, a team from the NCCR developed molecular probes that have the capacity to penetrate cells and bind selectively the membrane of some organelles (mitochondria, endoplasmic reticulum, lysosomes, etc.) that function in the depth of the cytoplasm. One of the special features of these microscopic tools is that they change their fluorescence when they are distorted by a variation in the tension of the membrane in which they are inserted.The first physiological process that the research team decided to study using this new tool was the formation of intraluminal vesicles (ILVs) inside an organelle, the endosomes. These ILVs can be compared to tiny garbage bags that contain the proteins and other compounds that need to be processed. Endosomes then transport ILVs to the lysosomes, which are the cellular power plants for destroying and recycling waste from the cells. The aim of the Geneva study was to determine whether a drop in the tension of the organelle's membrane may be responsible for the formation of the ILVs, and thus whether protein sorting in the cell is regulated by the membrane physical properties."We submitted our cells to a hypertonic shock, meaning we increased the concentration of the solutes (compounds in solution) in their environment," begins Vincent Mercier, a researcher in the Department of Biochemistry in UNIGE's Faculty of Science and the article's first author. "In response, cells expelled water to equilibrate solutes concentrations in and out of the cell. Their volume decreased as a result, as did the tension of the membrane. Using our probes, we observed that the membrane of the endosomes relaxed in the same way as the membrane of the entire cell."Better still, this drop in tension was accompanied by the mobilisation on the surface of the organelles of the compounds needed to form a complex (called ESCRT-III) that is exactly the main molecular machine required to produce ILVs. A different experiment was carried out to correlate these findings with actual ILV production."We also exposed our cells to epidermal growth factors (EGFs), which we know trigger the production of ILV after a cascade of reactions," says Aurélien Roux, Professor at UNIGE's Department of Biochemistry. "Using the same probes, we were able to calculate that this process is also accompanied by a drop in the tension of the organelle membranes. These results, obtained thanks to a multidisciplinary collaboration combining skills in biology, chemistry and physics, leads us to conclude that the tension of the membrane controls the functions of the organelles."It is an important conclusion since the formation of ILV from the membrane of the endosomes is a process that is essential for the proper functioning of the cells. In the specific case of this study, this biomechanical apparatus can be used to trap and quickly destroy the EGFs, thereby interrupt the signal delivered by this growth factor before it gets carried away. A disturbance in this control mechanism is often associated with the onset of cancer or degenerative diseases.
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Biotechnology
| 2,020 |
August 3, 2020
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https://www.sciencedaily.com/releases/2020/08/200803105237.htm
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How a gooey slime helps bacteria survive
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Bacteria have the ability to adapt to their environment to survive the host's immune defense. One such survival strategy includes the formation of a biofilm that prevents the immune system or antibiotics from reaching the bacteria. In a new study, researchers from the University of Tsukuba revealed that modulations to biofilm structure as a result of temperature changes are regulated by the production of a novel extracellular protein called BsaA in the bacterium
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"We have previously shown that temperature is an environmental cue that influences To achieve their goal, the researchers constructed a library of 1,360 mutant (gene knockout) cells in "Our results show that BsaA is necessary for pellicle-like biofilm formation at 25°C and conferral of tolerance to antibiotics," says lead author of the study Professor Nozomu Obana. "We know that biofilms contain heterogeneous cell populations, which leads to multicellular behaviors. We therefore wanted to know whether cellular heterogeneity affects the production of BsaA and thus the formation of a pellicle-like biofilm."The researchers found that the protein SipW controls the polymerization of BsaA to a biofilm, and used this to study biofilm formation. By constructing "At 25°C,
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Biotechnology
| 2,020 |
August 3, 2020
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https://www.sciencedaily.com/releases/2020/08/200803105228.htm
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Cold-sensitive staphylococci reveal a weakness
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"
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Golden staph ("My laboratory studies a protein, RNA helicase, which plays an important role in golden staph's ability to adapt to very different environments," begins Patrick Linder, a professor in the Department of Microbiology and Molecular Medicine in UNIGE's Faculty of Medicine. "When the environment changes, the bacterium has to be able to synthesise new proteins that are more suitable and stop the production of others that are no longer useful. The helicase we're interested in -- called CshA -- is involved in the degradation of the RNA, those molecules derived from the DNA and used in protein synthesis."Oddly enough, when the helicase is absent (due, for example, to a genetic mutation), the researchers found that the cultured bacteria could no longer form colonies if the temperature dropped below a certain threshold (around 25°C).The Geneva-based biologists undertook a series of experiments designed to improve our understanding of the link between golden staph's sensitivity to cold, the degradation of the RNA and the adaptation capacity. They discovered that the same helicase is probably also required in another physiological process, namely the synthesis of fatty acids, which are the constituents of bacterial membranes."Using cultured golden staph stripped of helicases, we succeeded in isolating 82 gene mutations (appearing spontaneously in many different bacteria), which meant that their holders regained the ability to form colonies at 25°C," continues Vanessa Khemici, a researcher in Patrick Linder's laboratory and the article's first author. "We identified almost all the affected genes, and no less than two thirds of them are involved in the fatty acid synthesis."The findings also helped the researchers understand that the lack of the helicase has the effect of deregulating the fatty acid synthesis and decreasing the flexibility of the membrane when the temperature drops. This prevents the membrane from fulfilling its functions properly and the bacterium from growing. In a second step, each of the 82 mutations succeeded in its own way in restoring the initial balance by acting on the different genetic levers involved in fatty acid synthesis."A section of the scientific community supports the idea that a future treatment against staphylococcus will involve a drug capable of inhibiting fatty acid synthesis," notes Professor Linder, "but there is a controversy about it because some studies contradict this point of view."The results of the Geneva scientists do not provide a clear-cut answer or make it possible to directly develop a drug against these bacteria. Nevertheless, they fit into this context and provide a better understanding of golden staph's fundamental mechanisms. The discovery of this unprecedented link between the fluidity of the membrane and adaptation to environmental change represents an important step in the fight against the bacterium. It is undoubtedly for these reasons that the journal decided to publish an overview in parallel with the article.
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Biotechnology
| 2,020 |
July 31, 2020
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https://www.sciencedaily.com/releases/2020/07/200731102641.htm
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Tinkering with roundworm proteins offers hope for anti-aging drugs
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KAIST researchers have been able to dial up and down creatures' lifespans by altering the activity of proteins found in roundworm cells that tell them to convert sugar into energy when their cellular energy is running low. Humans also have these proteins, offering up the intriguing possibilities for developing longevity-promoting drugs. These new findings were published on July 1 in Science Advances.
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The roundworm The proteins VRK-1 and AMPK work in tandem in roundworm cells, with the former telling the latter to get to work by sticking a phosphate molecule, composed of one phosphorus and four oxygen atoms, on it. In turn, AMPK's role is to monitor energy levels in cells, when cellular energy is running low. In essence, VRK-1 regulates AMPK, and AMPK regulates the cellular energy status.Using a range of different biological research tools, including introducing foreign genes into the worm, a group of researchers led by Professor Seung-Jae V. Lee from the Department of Biological Sciences at KAIST were able to dial up and down the activity of the gene that tells cells to produce the VRK-1 protein. This gene has remained pretty much unchanged throughout evolution. Most complex organisms have this same gene, including humans.Lead author of the study Sangsoon Park and his colleagues confirmed that the overexpression, or increased production, of the VRK-1 protein boosted the lifespan of the The research team found that the activity of the VRK-1-to-AMPK cellular-energy monitoring process is increased in low cellular energy status by reduced mitochondrial respiration, the set of metabolic chemical reactions that make use of the oxygen the worm breathes to convert macronutrients from food into the energy "currency" that cells spend to do everything they need to do.It is already known that mitochondria, the energy-producing engine rooms in cells, play a crucial role in aging, and declines in the functioning of mitochondria are associated with age-related diseases. At the same time, the mild inhibition of mitochondrial respiration has been shown to promote longevity in a range of species, including flies and mammals.When the research team performed similar tinkering with cultured human cells, they found they could also replicate this ramping up and down of the VRK-1-to-AMPK process that occurs in roundworms."This raises the intriguing possibility that VRK-1 also functions as a factor in governing human longevity, and so perhaps we can start developing longevity-promoting drugs that alter the activity of VRK-1," explained Professor Lee.At the very least, the research points us in an interesting direction for investigating new therapeutic strategies to combat metabolic disorders by targeting the modulation of VRK-1. Metabolic disorders involve the disruption of chemical reactions in the body, including diseases of the mitochondria.But before metabolic disorder therapeutics or longevity drugs can be contemplated by scientists, further research still needs to be carried out to better understand how VRK-1 works to activate AMPK, as well as figure out the precise mechanics of how AMPK controls cellular energy.This work was supported by the National Research Foundation (NRF), and the Ministry of Science and ICT (MSIT) of Korea.
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Biotechnology
| 2,020 |
July 30, 2020
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https://www.sciencedaily.com/releases/2020/07/200730141353.htm
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First gene knockout in a cephalopod achieved
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A team at the Marine Biological Laboratory (MBL) has achieved the first gene knockout in a cephalopod using the squid
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The team used CRISPR-Cas9 genome editing to knock out a pigmentation gene in squid embryos, which eliminated pigmentation in the eye and in skin cells (chromatophores) with high efficiency."This is a critical first step toward the ability to knock out -- and knock in -- genes in cephalopods to address a host of biological questions," Rosenthal says.Cephalopods (squid, octopus and cuttlefish) have the largest brain of all invertebrates, a distributed nervous system capable of instantaneous camouflage and sophisticated behaviors, a unique body plan, and the ability to extensively recode their own genetic information within messenger RNA, along with other distinctive features. These open many avenues for study and have applications in a wide range of fields, from evolution and development, to medicine, robotics, materials science, and artificial intelligence.The ability to knock out a gene to test its function is an important step in developing cephalopods as genetically tractable organisms for biological research, augmenting the handful of species that currently dominate genetic studies, such as fruit flies, zebrafish, and mice.It is also a necessary step toward having the capacity to knock in genes that facilitate research, such as genes that encode fluorescent proteins that can be imaged to track neural activity or other dynamic processes."CRISPR-Cas9 worked really well in Studies with Recently, Rosenthal and colleagues discovered extensive recoding of mRNA in the nervous system of For these reasons, the MBL Cephalopod Program's next goal is to transfer the new knockout technology to a smaller cephalopod species, Euprymna berryi (the hummingbird bobtail squid), which is relatively easy to culture to make genetic strains.
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Biotechnology
| 2,020 |
July 30, 2020
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https://www.sciencedaily.com/releases/2020/07/200730141327.htm
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Stay or leave? A tale of two virus strategies revealed by math
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As small and relatively simple as they may be, even viruses have strategies. Now, researchers in Japan report that they can evaluate two of these strategies through a combination of biology and math, providing a new tool for insight into viruses that could be used to develop better treatments.
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Unable to reproduce on their own, viruses replicate by infecting a living organism's cells and getting the cells to make copies of them. Two main options exist for copies of a virus's genetic structure made in the cell: stay in the cell as a template for making even more copies or get packaged as a new virus and leave in an attempt to infect other cells.Each option comes with trade-offs, so an individual virus's strategy of how much weight to place on each one should directly influence the progression of an infection and any health problems it may cause."While such strategies are expected to be in play, showing the existence of the strategy itself has been difficult," says Shingo Iwami, associate professor of the Faculty of Science at Kyushu University and associate investigator of the Institute for the Advanced Study of Human Biology (WPI-ASHBi) at Kyoto University.However, as reported in the journal While one of the studied virus strains causes severe and sudden symptoms, the other is a genetically modified version developed in the laboratory to increase virus production, which is important for creating stocks of viruses for the development of treatments and vaccines.As an experimental base for the modeling, Watashi's group measured characteristics of each virus's behavior -- such as number of infected cells and the amount of viral genetic code inside and outside of the cells -- over several days for cells grown and infected in the lab.Iwami and his group then developed a mathematical model with parameters to take into account key processes like the replication and release rates of the viral genetic information to explain the experimental data.By finding the range of model parameters that reasonably reproduce the experimentally observed results, they could quantify differences in behavior between the two strains. In particular, they estimated that the fraction of replicated genetic code packaged by the lab-developed strain to make new viruses was three times that for the other strain, indicating the preference of a leave strategy for the former and a stay strategy for the latter."The stay strategy initially produces copies of the genetic code faster, while the leave strategy emphasizes newly infecting cells," explains Iwami. "Though other mathematical models exist, ours is the first to evaluate these opposing evolutionary strategies."The current model does have some limitations, such as assuming some processes are constant and excluding some of the detailed biological processes, but for now, it provides a relatively simple way to gain an overall insight into two virus strategies."Such strategies may be common in other chronic virus infections, and understanding them could help us develop effective therapeutic methods to counter individual virus strategies," Iwami comments.
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Biotechnology
| 2,020 |
July 30, 2020
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https://www.sciencedaily.com/releases/2020/07/200730141315.htm
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New understanding of CRISPR-Cas9 tool could improve gene editing
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Within a mere eight years, CRISPR-Cas9 has become the go-to genome editor for both basic research and gene therapy. But CRISPR-Cas9 also has spawned other potentially powerful DNA manipulation tools that could help fix genetic mutations responsible for hereditary diseases.
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Researchers at the University of California, Berkeley, have now obtained the first 3D structure of one of the most promising of these tools: base editors, which bind to DNA and, instead of cutting, precisely replace one nucleotide with another.First created four years ago, base editors are already being used in attempts to correct single-nucleotide mutations in the human genome. Base editors now available could address about 60% of all known genetic diseases -- potentially more than 15,000 inherited disorders -- caused by a mutation in only one nucleotide.The detailed 3D structure, reported in the July 31 issue of the journal "We were able to observe for the first time a base editor in action," said UC Berkeley postdoctoral fellow Gavin Knott. "Now we can understand not only when it works and when it doesn't, but also design the next generation of base editors to make them even better and more clinically appropriate."A base editor is a type of Cas9 fusion protein that employs a partially deactivated Cas9 -- its snipping shears are disabled so that it cuts only one strand of DNA -- and an enzyme that, for example, activates or silences a gene, or modifies adjacent areas of DNA. Because the new study reports the first structure of a Cas9 fusion protein, it could help guide the invention of myriad other Cas9-based gene-editing tools."We actually see for the first time that base editors behave as two independent modules: You have the Cas9 module that gives you specificity, and then you have a catalytic module that provides you with the activity," said Audrone Lapinaite, a former UC Berkeley postdoctoral fellow who is now an assistant professor at Arizona State University in Tempe. "The structures we got of this base editor bound to its target really give us a way to think about Cas9 fusion proteins, in general, giving us ideas which region of Cas9 is more beneficial for fusing other proteins."Lapinaite and Knott, who recently accepted a position as a research fellow at Monash University in Australia, are co-first authors of the paper.In 2012, researchers first showed how to reengineer a bacterial enzyme, Cas9, and turn it into a gene-editing tool in all types of cells, from bacterial to human. The brainchild of UC Berkeley biochemist Jennifer Doudna and her French colleague, Emmanuelle Charpentier, CRISPR-Cas9 has transformed biological research and brought gene therapy into the clinic for the first time in decades.Scientists quickly co-opted Cas9 to produce a slew of other tools. Basically a mash-up of protein and RNA, Cas9 precisely targets a specific stretch of DNA and then precisely snips it, like a pair of scissors. The scissors function can be broken, however, allowing Cas9 to target and bind DNA without cutting. In this way, Cas9 can ferry different enzymes to targeted regions of DNA, allowing the enzymes to manipulate genes.In 2016, David Liu of Harvard University combined a Cas9 with another bacterial protein to allow the surgically precise replacement of one nucleotide with another: the first base editor.While the early adenine base editor was slow, the newest version, called ABE8e, is blindingly fast: It completes nearly 100% of intended base edits in 15 minutes. Yet, ABE8e may be more prone to edit unintended pieces of DNA in a test tube, potentially creating what are known as off-target effects.The newly revealed structure was obtained with a high-powered imaging technique called cryo-electron microscopy (cryoEM). Activity assays showed why ABE8e is prone to create more off-target edits: The deaminase protein fused to Cas9 is always active. As Cas9 hops around the nucleus, it binds and releases hundreds or thousands of DNA segments before it finds its intended target. The attached deaminase, like a loose cannon, doesn't wait for a perfect match and often edits a base before Cas9 comes to rest on its final target.Knowing how the effector domain and Cas9 are linked can lead to a redesign that makes the enzyme active only when Cas9 has found its target."If you really want to design truly specific fusion protein, you have to find a way to make the catalytic domain more a part of Cas9, so that it would sense when Cas9 is on the correct target and only then get activated, instead of being active all the time," Lapinaite said.The structure of ABE8e also pinpoints two specific changes in the deaminase protein that make it work faster than the early version of the base editor, ABE7.10. Those two point mutations allow the protein to grip the DNA tighter and more efficiently replace A with G."As a structural biologist, I really want to look at a molecule and think about ways to rationally improve it. This structure and accompanying biochemistry really give us that power," Knott added. "We can now make rational predications for how this system will behave in a cell, because we can see it and predict how it's going to break or predict ways to make it better."
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Biotechnology
| 2,020 |
July 30, 2020
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https://www.sciencedaily.com/releases/2020/07/200730092610.htm
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Compounds show promise in search for tuberculosis antibiotics
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Compounds tested for their potential as antibiotics have demonstrated promising activity against one of the deadliest infectious diseases -- tuberculosis (TB).
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Researchers from the John Innes Centre evaluated two compounds with antibacterial properties, which had been produced by the company Redx Pharma as antibiotic candidates, particularly against TB.TB, which is caused by the bacterium Mycobacterium tuberculosis, is often thought of as a disease of the past. But in recent years it has been increasing due, in part, to rising resistance to treatments and decreasing efficacy of vaccines.One strategy in the search for new treatments is to find compounds that exploit well-known existing targets for drugs such as the bacterial enzyme DNA gyrase. This member of the DNA topoisomerase family of enzymes is required for bacterial DNA functionality, so compounds that inhibit its activity are much sought after as antibiotic candidates.Using X-ray crystallography, the team elucidated the molecular details of the action of the compounds against their target.Surprisingly, a very common mutation in DNA gyrase that causes bacteria to be resistant to a related group of antibiotics, the aminocoumarins, did not lead to resistance to the compounds under scrutiny here."We hope that companies and academic groups working to develop new antibiotics will find this study useful. It opens the way for further synthesis and investigation of compounds that interact with this target," says Professor Tony Maxwell one of the authors of the study which appears in the To date, efforts to develop new treatments for TB have been unsuccessful, with current treatments having been used for over 50 years.World Health Organisation (WHO) figures reveal that each day over 4000 people die from TB and 300,000 people fall ill from the disease. Nearly 500,000 people fell ill with drug-resistant TB in 2018.Professor Maxwell says: "Antimicrobial resistance (AMR) is now well-recognised as one of the biggest problems facing us in the in the 21st Century. If we don't find solutions to it soon, we could be looking at epidemics of bacterial disease going forward. People know about the bubonic plague and other such terrible pestilence through studying history but it's not overstating the case that bacterial diseases of this sort could reemerge if we do not have effective antibiotics. We must find a way of bringing together expertise from the academic and business sector towards decisive action."
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Biotechnology
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July 30, 2020
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https://www.sciencedaily.com/releases/2020/07/200730092608.htm
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New set of channels connecting malaria parasite and blood cells
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Researchers at the National Institutes of Health and other institutions have discovered another set of pore-like holes, or channels, traversing the membrane-bound sac that encloses the deadliest malaria parasite as it infects red blood cells. The channels enable the transport of lipids -- fat-like molecules -- between the blood cell and parasite, Plasmodium falciparum. The parasite draws lipids from the cell to sustain its growth and may also secrete other types of lipids to hijack cell functions to meet its needs.
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The finding follows an earlier discovery of another set of channels through the membrane enabling the two-way flow of proteins and non-fatty nutrients between the parasite and red blood cells. Together, the discoveries raise the possibility of treatments that block the flow of nutrients to starve the parasite.The research team was led by Joshua Zimmerberg, M.D., Ph.D., a senior investigator in the Section on Integrative Biophysics at NIH's Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). The study appears in In 2018, there were 228 million cases of malaria worldwide, leading to more than 400,000 deaths, 67% of which were among children under 5, according to the World Health Organization. In the current study, researchers determined that the channels through the sac, or vacuole, that encloses the parasite are made of Niemann-Pick C1-related protein (PfNCR1). The PfNCR1 channels are restricted to locations where the vacuole membrane touches the parasite's membrane. The channels the team discovered in the previous study are formed by exported protein 2 (EXP2). Areas of the vacuole membrane containing EXP2 are located far from the parasite's membrane, at an average distance of 20 to 40 nanometers. The researchers believe that the parasite may use this variation in distance to separate the two transport systems.
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Biotechnology
| 2,020 |
July 30, 2020
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https://www.sciencedaily.com/releases/2020/07/200730110130.htm
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Peering into the secrets of phages to see how they kill bacterial superbugs
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A research collaboration involving Monash University has made an exciting discovery that may eventually lead to targeted treatments to combat drug-resistant bacterial infections, one of the greatest threats to global health.
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The study, led by Monash Biomedicine Discovery Institute's Associate Professor Fasséli Coulibaly and Professor Trevor Lithgow is published in The study was a collaboration between researchers at the Monash Biomedicine Discovery Institute (BDI), the Monash University Centre to Impact AMR and the University of Cambridge.What they saw was an incredible "choreography" by the phages as they assembled the main components of their particles: a head filled with the viral DNA and a tail used to infect the bacteria."We saw how the building blocks of the particle interlock in an intricate choreography. At a molecular level, arms swing out and curl around each other forming a continuous chain that braces the head of the phage," Associate Professor Coulibaly said."This rigid chainmail provides further protection to the DNA of the phage. Surprisingly, the tail on the other hand remains flexible. It's able to bend and not break as it captures the bacteria and ultimately injects them with the phage DNA."Phages are a class of viruses that infect bacteria, and each phage is specific for the species of bacteria it can kill. Phages can be purified to a point of being FDA-approved for treatment of people with bacterial infections, and documented success has been had in the USA, Europe and, recently, Australia.At Monash University, the Centre to Impact AMR is grappling with these issues and is looking at the types of phages needed for new, "phage therapies" to treat bacterial infections."This finding will help us overcome one of the most critical hurdles in phage therapies which is a precise understanding of how phage work, in order to predict in advance and select with accuracy the best phage for each patient infection," Professor Lithgow said."It could help move phage therapies from compassionate use, where all other treatment options have been exhausted, to more widespread clinical use."Antimicrobial resistance (AMR) is one of the biggest threats to global health, food security and economic development. It is a pressing health and humanitarian crisis in Asia, that is increasing in severity globally.AMR affects all aspects of society and is driven by many interconnected factors including antibiotic overuse, and the rapidly adaptive nature of bacteria to evolve into drug-resistant forms. At-risk groups for AMR infections are many, and include COVID-19 patients on respirators, mothers and children during childbirth, surgery patients, people with cancer and chronic disease and the elderly.The first authors of this study Dr Joshua Hardy and Dr Rhys Dunstan used the Ramaciotti centre for cryo-electron microscopy at Monash University, the Monash molecular crystallisation facility, and the Australian Synchrotron for the structure determination.
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Biotechnology
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July 29, 2020
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https://www.sciencedaily.com/releases/2020/07/200729124418.htm
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Butterfly genomics: Monarchs migrate and fly differently, but meet up and mate
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Each year, millions of monarch butterflies migrate across eastern North America to fly from as far north as the U.S.-Canadian border to overwinter in central Mexico -- covering as much as 3,000 miles. Meanwhile, on the other side of the Rocky Mountains, western monarchs generally fly 300 miles down to the Pacific Coast to spend the winter in California. It was long believed that the eastern and western monarchs were genetically distinct populations.
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A new study, however, confirms that while the eastern and western butterflies fly differently, they are genetically the same. The journal "It was surprising," says Jaap de Roode, Emory professor of biology and senior author of the study. His lab is one of a handful in the world that studies monarch butterflies."You would expect that organisms with different behaviors and ecologies would show some genetic differences," de Roode says. "But we found that you cannot distinguish genetically between the western and eastern butterflies."The current paper builds on previous work by the de Roode lab that found similarities between 11 genetic markers of the eastern and western monarchs, as well as other more limited genetic studies and observational and tracking data."This is the first genome-wide comparison of eastern and western monarchs to try to understand their behavioral differences better," says Venkat Talla, first author of the current study and an Emory post-doctoral fellow in the lab.Talla analyzed more than 20 million DNA mutations in 43 monarch genomes and found no evidence for genomic differentiation between eastern and western monarchs. Instead, he found identical levels of genetic diversity."Our work shows that the eastern and western monarchs are mating together and exchanging genetic material to a much greater extent than was previously realized," Talla says. "And it adds to the evidence that it is likely differences in their environments that shapes the differences in their patterns of migration."Co-author Amanda Pierce, who led the earlier study on 11 genetic markers, launched the project while she was a graduate student in the De Roode Lab."Monarch butterflies are so fragile and so lightweight, and yet they are able to travel thousands of miles," Pierce says. "They are beautiful creatures and a great model system to understand unique, innate behaviors. We know that migration is ingrained in their genetic wiring in some way."After monarchs leave their overwintering sites, they fly north and lay eggs. The caterpillars turn into butterflies and then fly further, mating and laying another generation of eggs. The process repeats for several generations until finally, as the days grow shorter and the temperatures cooler, monarchs emerge from their chrysalises and start to fly south. This migratory generation does not expend any energy on breeding or laying eggs, saving it all for the long journey."For every butterfly that makes it to California or to Mexico, that's its first journey there," Pierce marvels.Previous work had identified a propensity for the eastern and western monarchs to have slight differences in their wing shapes. For the current paper, the researchers wanted to identify any variations in their flight styles.They collected eastern monarchs from a migratory stopover site in Saint Marks, Florida, and western monarchs from one of their overwintering sites near Oceano, California. Pierce ran flight trials with the butterflies by tethering them to a mill that restricted their flight patterns to circles with a circumference of about 25 feet. The trials were performed in a laboratory under controlled light and temperature conditions that mimicked overwintering sites. Artificial flowers were arranged around the circumference of the flight mills."The idea was to try to give them some semblance of a 'natural' environment to help motivate them and to orient them," Pierce explains.Butterflies were released unharmed from the flight mills after performing short trials.The results showed that the eastern monarchs would choose to fly for longer distances while the western monarchs flew shorter distances but with stronger bursts of speed. "The more powerful flight trait of the western monarch is like a sprinter, essentially," Pierce says, "while the eastern monarchs show a flight trait more like marathoners."Pierce has since graduated from Emory and now works as a geneticist for the Environmental Protection Agency in Washington, D.C.Talla, who specializes in bioinformatics, grew up in India where the rich diversity of wildlife inspired him to become an evolutionary biologist. He moved to Sweden to get his PhD, where he studied the genomics of the European wood white butterfly. Although all wood whites appear identical visually, they are actually three different species."One of the big questions I'm interested in answering is how does an individual species wind up becoming multiple species?" Talla says. "I want to understand all the processes involved in that evolution."He jumped at the chance to join the De Roode Lab. "Monarchs have always been at the top of my list of butterflies I wanted to study because of their incredible migrations," Talla says. "They are a fascinating species."Last November, he joined de Roode on a lab field trip to the eastern monarch overwintering site, inside and adjacent to the Monarch Butterfly Biosphere Reserve in central Mexico. Tens to hundreds of millions of monarchs blanket the trees and landscape through the winter. "It's a mind-blowing sight," Talla says. "It makes you wonder how they all know how to get there."Previous tracking and observational studies had shown that at least some western monarchs fly south to Mexico instead of west to California. The full-genome analysis suggests that more than just a few of the western monarchs may be making the trip to Mexico where they mix with the eastern monarchs. And when the butterflies depart Mexico, some may fly west instead of east."Evidence from multiple directions is coming together to support the same view," de Roode says.The findings may help in the conservation of monarchs. Due to a combination of habitat loss, climate change and lack of nectaring flowers, numbers of both eastern and western monarchs have declined in recent decades, with the western ones showing the most precipitous drop. The U.S. Fish and Wildlife Service is currently considering whether the butterflies need special protections."If environmental factors are all that drives the differences between the eastern and western monarchs, it's possible that we could help the western population by transplanting some of the eastern ones to the west," de Roode says.The De Roode lab now plans to investigate what exactly in the environments of the butterflies triggers different expressions of their genes.The work was funded by Emory University, the National Science Foundation and the National Institutes of Health.
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Biotechnology
| 2,020 |
July 29, 2020
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https://www.sciencedaily.com/releases/2020/07/200729124355.htm
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New insights into wound healing
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When we get a wound on our skin, the cells in our bodies quickly mobilize to repair it. While it has been known how cells heal wounds and how scars form, a team led by researchers from Washington University in St. Louis has determined for the first time how the process begins, which may provide new insight into wound healing, fibrosis and cancer metastasis.
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The team, led by Delaram Shakiba, a postdoctoral fellow from the NSF Science and Technology Center for Engineering Mechanobiology (CEMB) at the McKelvey School of Engineering, discovered the way fibroblasts, or common cells in connective tissue, interact with the extracellular matrix, which provides structural support as well as biochemical and biomechanical cues to cells. The team uncovered a recursive process that goes on between the cells and their environment as well as structures in the cells that were previously unknown.Results of the research were published in "Clinical efforts to prevent the progression of fibrocontractile diseases, such as scarring and fibrosis, have been largely unsuccessful, in part because the mechanisms that cells use to interact with the protein fibers around them are unclear," Shakiba said. "We found that fibroblasts use completely different mechanisms in the early -- and I think the most treatable -- stages of these interactions, and that their responses to drugs can therefore be the opposite of what they would be in the later stages."Genin, who is the co-director of the CEMB, said the process has stymied mechanobiology researchers for some time."Researchers in the field of mechanobiology thought that cells pulled in collagen from the extracellular matrix by reaching out with long protrusions, grabbing it and pulling it back," Genin said. "We discovered that this wasn't the case. A cell has to push its way out through collagen first, then instead of grabbing on, it essentially shoots tiny hairs, or filopodia, out of the sides of its arms, pulls in collagen that way, then retracts."Now that they understand this process, Genin said, they can control the shape that a cell takes."With our colleagues at CEMB at the University of Pennsylvania, we were able to validate some mathematical models to go through the engineering process, and we now have the basic rules that cells follow," he said. "We can now begin to design specific stimuli to direct a cell to behave in a certain way in building a tissue-engineered structure."The researchers learned they could control the cell shape in two ways: First, by controlling the boundaries around it, and second, by inhibiting or upregulating particular proteins involved in the remodeling of the collagen.Fibroblasts pull the edges of a wound together, causing it to contract or close up. Collagen in the cells then remodels the extracellular matrix to fully close the wound. This is where mechanobiology comes into play."There's a balance between tension and compression inside a cell that is newly exposed to fibrous proteins," Genin said. "There is tension in actin cables, and by playing with that balance, we can make these protrusions grow extremely long," Genin said. "We can stop the remodeling from occurring or we can increase it."The team used a 3D-mapping technique -- the first time it has been applied to collagen -- along with a computational model to calculate the 3D strain and stress fields created by the protrusions from the cells. As cells accumulated collagen, tension-driven remodeling and alignment of collagen fibers led to the formation of collagen tracts. This requires cooperative interactions among cells, through which cells can interact mechanically."New methods of microscopy, tissue engineering and biomechanical modeling greatly enhance our understanding of the mechanisms by which cells modify and repair the tissues they populate," Elson said. "Fibrous cellular structures generate and guide forces that compress and reorient their extracellular fibrous environment. This raises new questions about the molecular mechanisms of these functions and how cells regulate the forces they exert and how they govern the extent of matrix deformation.""Wound healing is a great example of how these processes are important in a physiologic way," Genin said. "We'll be able to come up with insight in how to train cells not to excessively compact the collagen around them."
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Biotechnology
| 2,020 |
July 29, 2020
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https://www.sciencedaily.com/releases/2020/07/200729114837.htm
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Researchers map mechanisms in the largest CRISPR system
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The largest and most complex CRISPR system has been visualized by researchers from the University of Copenhagen in a new study. The system may have potential applications in biomedicine and biotechnology, the researchers believe.
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CRISPR technology can be used to edit genes and revolutionised the scientific world when it was first introduced. CRISPR-Cas9 is likely the most known CRISPR-system and popularly known as the gene scissor.That is just one out of the many various CRISPR systems that exist. Now researchers from the University of Copenhagen (UCPH) have mapped and analysed the atomic structure one of the most complex CRISPR systems so far."We have solved the largest and most complicated CRISPR-Cas complex seen so far. We now understand how this system works on a molecular level," says co-author Guillermo Montoya, who is Professor at the Novo Nordisk Foundation Center for Protein Research (NNF CPR), UCPH.The researchers have studied a complex called Cmr-β, which belongs to the subgroup of so-called type III-B CRISPR-Cas complexes. The new results have been published in the scientific journal CRISPR is a system found in bacteria, among other organisms, and it is involved in bacteria's immune system. Here it plays a main role in the constant fight against invading phages, a virus that attacks bacteria.In the new study, the researchers have studied Cmr's role in the immune system and delved into the mechanisms behind its immune response against phages and how it is regulated."Our findings, in collaboration with the She group at the Faculty of Sciences, highlight the diverse defence strategies of type III complexes. We have also identified a unique subunit called Cmr7, which seems to control the complex activity, and we further believe that it may defend against prospective viral anti-CRISPR proteins," says co-author Nicholas Heelund Sofos, postdoc at NNF CPR.The Cmr system mapped by the researchers in the new study can among other things remove single-stranded RNA and DNA. Though it will be very difficult to use for gene editing like CRISPR-Cas9.It is too big and complex. But in the future, it may still be key to understand the immune response of bacteria and it could have some use in the fight against antibiotic resistance."This complex plays an important role in the fight between bacteria and phages. Antibiotic resistance comes from this type of fight. Therefore, our results may constitute an important knowledge for fighting antibiotic resistance.""The complex may also have therapeutic potential. In the future, we may be able to use this for diagnostics or a health problem we may not even have seen yet. Now, our goal is to look for an application for this system," says Guillermo Montoya.The researchers used the advanced technology cryo electron microscopy -- also called CryoEM -- to outline the system. All research and data collection was conducted at the University of Copenhagen.CRISPR-Cas* CRISPR stands for clustered regularly interspaced short palindromic repeats. Cas stands for CRISPR-associated protein.* CRISPR-Cas9 is probably the most well-known of the CRISPR systems and can be used for gene editing. One protein is associated with the system: Cas9.* Many small proteins are associated with the Cmr-β system outlined by the researchers in the new study.
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Biotechnology
| 2,020 |
July 29, 2020
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https://www.sciencedaily.com/releases/2020/07/200729114811.htm
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RNA sequences involved in regulating gene expression identified
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The human genome contains about 20,000 protein-coding genes, but the coding parts of our genes account for only about 2 percent of the entire genome. For the past two decades, scientists have been trying to find out what the other 98 percent is doing.
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A research consortium known as ENCODE (Encyclopedia of DNA Elements) has made significant progress toward that goal, identifying many genome locations that bind to regulatory proteins, helping to control which genes get turned on or off. In a new study that is also part of ENCODE, researchers have now identified many additional sites that code for RNA molecules that are likely to influence gene expression.These RNA sequences do not get translated into proteins, but act in a variety of ways to control how much protein is made from protein-coding genes. The research team, which includes scientists from MIT and several other institutions, made use of RNA-binding proteins to help them locate and assign possible functions to tens of thousands of sequences of the genome."This is the first large-scale functional genomic analysis of RNA-binding proteins with multiple different techniques," says Christopher Burge, an MIT professor of biology. "With the technologies for studying RNA-binding proteins now approaching the level of those that have been available for studying DNA-binding proteins, we hope to bring RNA function more fully into the genomic world."Burge is one of the senior authors of the study, along with Xiang-Dong Fu and Gene Yeo of the University of California at San Diego, Eric Lecuyer of the University of Montreal, and Brenton Graveley of UConn Health.The lead authors of the study, which appears today in Much of the ENCODE project has thus far relied on detecting regulatory sequences of DNA using a technique called ChIP-seq. This technique allows researchers to identify DNA sites that are bound to DNA-binding proteins such as transcription factors, helping to determine the functions of those DNA sequences.However, Burge points out, this technique won't detect genomic elements that must be copied into RNA before getting involved in gene regulation. Instead, the RNA team relied on a technique known as eCLIP, which uses ultraviolet light to cross-link RNA molecules with RNA-binding proteins (RBPs) inside cells. Researchers then isolate specific RBPs using antibodies and sequence the RNAs they were bound to.RBPs have many different functions -- some are splicing factors, which help to cut out sections of protein-coding messenger RNA, while others terminate transcription, enhance protein translation, break down RNA after translation, or guide RNA to a specific location in the cell. Determining the RNA sequences that are bound to RBPs can help to reveal information about the function of those RNA molecules."RBP binding sites are candidate functional elements in the transcriptome," Burge says. "However, not all sites of binding have a function, so then you need to complement that with other types of assays to assess function."The researchers performed eCLIP on about 150 RBPs and integrated those results with data from another set of experiments in which they knocked down the expression of about 260 RBPs, one at a time, in human cells. They then measured the effects of this knockdown on the RNA molecules that interact with the protein.Using a technique developed by Burge's lab, the researchers were also able to narrow down more precisely where the RBPs bind to RNA. This technique, known as RNA Bind-N-Seq, reveals very short sequences, sometimes containing structural motifs such as bulges or hairpins, that RBPs bind to.Overall, the researchers were able to study about 350 of the 1,500 known human RBPs, using one or more of these techniques per protein. RNA splicing factors often have different activity depending on where they bind in a transcript, for example activating splicing when they bind at one end of an intron and repressing it when they bind the other end. Combining the data from these techniques allowed the researchers to produce an "atlas" of maps describing how each RBP's activity depends on its binding location."Why they activate in one location and repress when they bind to another location is a longstanding puzzle," Burge says. "But having this set of maps may help researchers to figure out what protein features are associated with each pattern of activity."Additionally, Lecuyer's group at the University of Montreal used green fluorescent protein to tag more than 300 RBPs and pinpoint their locations within cells, such as the nucleus, the cytoplasm, or the mitochondria. This location information can also help scientists to learn more about the functions of each RBP and the RNA it binds to.Many research labs around the world are now using these data in an effort to uncover links between some of the RNA sequences identified and human diseases. For many diseases, researchers have identified genetic variants called single nucleotide polymorphisms (SNPs) that are more common in people with a particular disease."If those occur in a protein-coding region, you can predict the effects on protein structure and function, which is done all the time. But if they occur in a noncoding region, it's harder to figure out what they may be doing," Burge says. "If they hit a noncoding region that we identified as binding to an RBP, and disrupt the RBP's motif, then we could predict that the SNP may alter the splicing or stability of the gene."Burge and his colleagues now plan to use their RNA-based techniques to generate data on additional RNA-binding proteins."This work provides a resource that the human genetics community can use to help identify genetic variants that function at the RNA level," he says.The research was funded by the National Human Genome Research Institute ENCODE Project, as well as a grant from the Fonds de Recherche de Québec-Santé.
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Biotechnology
| 2,020 |
July 28, 2020
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https://www.sciencedaily.com/releases/2020/07/200728182549.htm
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Researchers discover 'Marie Kondo' protein which aids in organizing fruit fly embryos
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Researchers at the University of Colorado School of Medicine have discovered a protein in fruit fly embryos, dubbed Marie Kondo, that destroys maternal proteins. Much like namesake, author and clutter consultant Marie Kondo, this gene removes unnecessary molecules, keeping embryos organized.
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Fertilized egg cells are loaded with maternal molecules that control the earliest steps of embryonic development. A critical stage of development is when the embryo destroys these inherited molecules and begins to make its own. These molecules include proteins and messenger RNAs (which encode instructions for making proteins). Existing research had identified how messenger RNAs are destroyed, but how maternal proteins are discarded, however, has been unknown.According to the study, published in the journal "Ordinarily, when we talk about getting rid of maternal gene products, we tend to focus on mRNA, or the coded information for making a protein," says Olivia Rissland, assistant professor of biochemistry and molecular genetics at the University of Colorado School of Medicine and study co-author. "However, we don't often talk about destruction of the proteins themselves. One implication of our study is that, during early stages of development, destruction of maternal proteins might be more tightly controlled than we had thought."Rissland says this discovery opens the door to more research into embryonic protein destruction. "The reason why we started looking at these proteins is because they control RNA. Now, we want to see what other proteins are destroyed and how protein destruction affects early development, not just in fruit flies, but in other animals too."Further information:
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Biotechnology
| 2,020 |
July 28, 2020
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https://www.sciencedaily.com/releases/2020/07/200728130835.htm
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New soil models may ease atmospheric CO2, climate change
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To remove carbon dioxide from the Earth's atmosphere in an effort to slow climate change, scientists must get their hands dirty and peek underground.
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In an article published July 27 in Carbon's journey into the soil is akin to a busy New York City rush hour. "Everything in the soil is bustling and changing all the time on a daily or hourly basis," said Lehmann, professor of soil biogeochemistry and the lead author on the piece."Microorganisms are on the street, but carbon quickly disappears around the corner or hides in nooks and crannies," he said. "Microorganisms in the soils that consume carbon can never be sure what tomorrow looks like."Think of it this way: Sometimes soil microorganisms see a lot of carbon but still cannot devour it.Lehmann and an international, interdisciplinary group of scientists propose the creation of new soil carbon-persistence models through the lens of "functional complexity" -- the interplay between time and space in soil carbon's changing molecular structure.Functional complexity drives carbon sequestration, and scientists must know specifically how carbon stays in the ground, according to Lehmann."Even if soil microorganisms have a full smorgasbord in front of them, they don't know what to eat if there is very little of each kind of carbon," said Lehmann, a fellow at Cornell Atkinson Center for Sustainability. "Although there is plenty of carbon, microorganisms starve, especially if they have to adjust to ever-changing conditions in a crazy maze."With new models, scientists believe they can find out exactly how sequestration works. It could then be properly reflected in the next assessment of the United Nations Intergovernmental Panel on Climate Change (IPCC) -- which likely will address drawing down atmospheric carbon.Lehmann said that with modeling techniques gleaned from the field of engineering, for example, soil scientists can find better management methods to reduce atmospheric carbon."Collaboration in a stellar group of thinkers from diverse disciplines was key for us to come up with a new view on this old conundrum," he said. "We seem to be building climate models based on an erroneous understanding of why organic carbon stays in soil and how microbes are eating it. We need a new thinking to incorporate the best models for IPCC and other climate prediction efforts."
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Biotechnology
| 2,020 |
July 28, 2020
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https://www.sciencedaily.com/releases/2020/07/200728130830.htm
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Exposure to enzymes causes peculiar response in liquid droplets formed by DNA
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"A watched pot never boils," as the saying goes, but that was not the case for UC Santa Barbara researchers watching a "pot" of liquids formed from DNA. In fact, the opposite happened.
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With research partners at the Ludwig-Maximilians University (LMU), in Munich, Germany, the team's findings appear in the Recent advances in cellular biology have enabled scientists to learn that the molecular components of living cells (such as DNA and proteins) can bind to each other and form liquid droplets that appear similar to oil droplets in shaken salad dressing. These cellular droplets interact with other components to carry out basic processes that are critical to life, yet little is known about how the interactions function. To gain insight into these fundamental processes, the researchers used modern methods of nanotechnology to engineer a model system -- a liquid droplet formed from particles of DNA -- and then watched those droplets as they interacted with a DNA-cleaving enzyme.Surprisingly, they found that, in certain cases, adding the enzyme caused the DNA droplets to suddenly start bubbling, like boiling water."The bizarre thing about the bubbling DNA is that we didn't heat the system; it's as if a pot of water started boiling even though you forgot to turn on the stove," said project co-leader Omar Saleh, a UC Santa Barbara assistant professor of materials and bioengineering. However, the bubbling behavior didn't always occur; sometimes adding the enzyme would cause the droplets to shrink away smoothly, and it was unclear why one response or the other would occur.To get to the bottom of this mystery, the team carried out a rigorous set of precision experiments to quantify the shrinking and bubbling behaviors. They identified two types of shrinking behavior: the first caused by enzymes cutting the DNA only on the droplet surface, and the second caused by enzymes penetrating inside the droplet. "This observation was critical to unraveling the behavior, as it put into our heads the idea that the enzyme could start nibbling away at the droplets from the inside," said co-leader Tim Liedl, a professor at the LMU, where the experiments were conducted.By comparing the droplet response to the DNA particle design, the team cracked the case: they found that bubbling and penetration-based shrinking occurred together, and happened only when the DNA particles were lightly bound together, whereas strongly bound DNA particles would keep the enzyme on the outside. As Saleh noted, "It's like trying to walk through a crowd -- if the crowd is tightly holding hands, you wouldn't be able to get through."The bubbles, then, happen only in the lightly bound systems, when the enzyme can get through the crowded DNA particles to the interior of the droplet, and begin to eat away at the droplet from the inside. The chemical fragments created by the enzyme lead to an osmotic effect in which water is drawn in from the outside, causing a swelling phenomenon that produces the bubbles. The bubbles grow, reach the droplet surface, and then release the fragments in a burp-like gaseous outburst. "It is quite striking to watch, as the bubbles swell and pop over and over," said Liedl.The work demonstrates a complex relationship between the basic material properties of a biomolecular liquid and its interactions with external components. The team believes that the insight gained from studying the bubbling process will lead both to better models of living processes and to enhanced abilities to engineer liquid droplets for use as synthetic bioreactors.The research was made possible by an award to Saleh from the Alexander von Humboldt Foundation, which enabled him to visit Munich and work directly with Liedl on this project. "These types of international collaborations are extremely productive," Saleh said.
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Biotechnology
| 2,020 |
July 28, 2020
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https://www.sciencedaily.com/releases/2020/07/200728130826.htm
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Biologists zero in on cells' environmental sensing mechanism
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Evolutionary and developmental biologist Craig Albertson and colleagues at the University of Massachusetts Amherst report that they have identified a molecular mechanism that allows an organism to change the way it looks depending on the environment it is exposed to, a process known as phenotypic plasticity.
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In addition to lead investigators Albertson and Rolf Karlstrom, the team includes recently graduated doctoral students Dina Navon and Ira Male, current Ph.D. candidate Emily Tetrault and undergraduate Benjamin Aaronson. Their paper appears now in Albertson explains that the project stems from a desire to better understand how genes and the environment interact to direct anatomical shape. "We know that our features are determined by genes, but we also know that many physical features are shaped by the environment as well. In identical twins, for example, if one becomes a long-distance runner and the other a body builder, they are going to end up with very different physiques. The skeleton is especially sensitive to such environmental inputs."Albertson works with a system -- cichlid fishes -- known throughout the scientific world as champions of phenotypic plasticity that can alter, in a single season, jawbone hardness or shape to match feeding conditions. They are also well known for their rapid evolution and diversity in jaw shapes, which has enabled cichlids to adapt to many different food sources, including algae, plankton, fish, snails and even the scales of other fishes.Albertson has spent much of the past two decades trying to reveal the genetic differences that underlie differences in jaw shape between species. Now he and colleagues identify the well-studied chemical/molecular system known as the Hedgehog (Hh) signaling pathway as an important player. More recently he explored whether the same pathway might also contribute to differences in jaw shape that arise within species through phenotypic plasticity.An important clue came as Albertson learned more about how this molecular pathway works. He explains, "There is a well-known mechano-sensor on most cells, including those that make the skeleton, called the primary cilium. Cells that lack this organelle are unable to sense or respond to environmental input, including mechanical load. It turns out that several key protein components of the Hedgehog pathway are physically associated with this structure, making it an obvious candidate for an environmentally sensitive signal."In the current study, the research team first showed that plasticity in the rate of bone deposition in cichlids forced to feed using different foraging modes was associated with different Hh levels. Greater levels of the signal were detected in fish from the environment where more bone was laid down and vice versa. To really nail the question, Albertson teamed up with Karlstrom, who had previously developed sophisticated tools to study Hh signaling in zebrafish.He explains, "Rolf has a bunch of really slick transgenic systems for manipulating that molecular signal in real time. It is sort of like a volume knob on your stereo -- you can turn it up or turn it down, and then see how it influences your trait of interest." In this case, they wanted to see whether Hh levels influenced plasticity in bone deposition rates.They found that unmanipulated zebrafish deposited different amounts of bone in different foraging environments. When Hh levels were reduced, these differences went away, but when Hh levels were increased, differences in bone deposition rates were dramatically increased.Albertson, explains, "Bone cells in these fish are innately sensitive to different mechanical environments. But we were able to play with this system using a single molecular switch -- you turn up the Hh signal and the cells become more sensitive to the environment, or you turn the molecular sensor down and the cells become almost deaf to the environment.""That the same molecular machinery underlies both the evolutionary divergence and plasticity of the jaw is notable," Albertson explains. "It is consistent with long-held theory that suggests short-term plasticity might bias the direction of long-term evolution, which explains why evolution can be predictable in lineages that have repeatedly evolved to similar habitats." Albertson adds, "The Hh signal has also been shown to regulate plasticity in beetle horns, so there may be something special that positions it to be an environmental sensor across tissues and animals."Such intriguing questions will be the topic for future investigations, the authors add.
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Biotechnology
| 2,020 |
July 28, 2020
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https://www.sciencedaily.com/releases/2020/07/200728113625.htm
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Multiomics investigation revealing the characteristics of HIV-1-infected cells in vivo
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For eradication of HIV-1 infection, it is important to elucidate the detailed features and heterogeneity of HIV-1-infected cells in vivo. In this study, a hematopoietic stem cell-transplanted humanized mouse model infected with a gene-modified HIV-1 was used to reveal multiple characteristics of HIV-1-producing cells in vivo.
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A research group at The Institute of Medical Science, The University of Tokyo (IMSUT) using HIV-1-infected cells performed "multiomics" analyses, which are technologies recently developed to comprehensively investigate the features of biological samples."Our findings describe multiple characteristics of HIV-1-producing cells in vivo, which could provide clues for the development of an HIV-1 cure.," said the lead scientist, Kei Sato, Associate Professor (Principal Investigator) in the Division of Systems Virology, Department of Infectious Disease Control, IMSUT.The results of this research were published in For eradication of HIV-1 infection, it is important to gain an in-depth understanding of the wide-ranging characteristics of HIV-1-infected cells in vivo.Recently developed 'omics' analyses (*1) can be a powerful tool to identify the characteristics of HIV-1-infected cells. However, it should be noted that a large majority of the CD4+ T cells(*2) in infected individuals are uninfected, and therefore, the transcriptional profiles of "bulk" CD4+ T cells in vivo do not reflect those of "pure" HIV-1-producing cells.Multiomics analysis to comprehensively reveal the features of HIV-1-infected cells in vivoIn this study, the research group used a human hematopoietic stem cell-transplanted humanized mouse model that maintains human leukopoiesis under relatively stable immunological conditions in vivo and a replication-competent reporter HIV-1, and used four recently developed techniques to investigate viral genomics and transcriptomics.According to the research group, this study consisted of the four following analyses:First, droplet digital PCR revealed the presence of potential reservoirs in infected humanized mice. Second, ligation mediated PCR showed the preference of HIV-1 to integrate into open chromatin regions, as suggested by the association of the epigenetic modifications of integration sites with viral production. Third, digital RNA-sequencing quantified the absolute copy number of viral transcripts in the HIV-1-producing cells in vivo and further identified the differentially expressed genes between virus-infected and uninfected cells. Finally, single-cell RNA-sequencing revealed and characterized the heterogeneity of the HIV-1-producing cells in vivo.Associate Professor Sato emphasized "To our knowledge, this study is the first investigation to describe multiple aspects of HIV-1-producing cells and also the first comprehensive investigation of the characteristics of HIV-1-infected cells in vivo" .A field of scientific study that aims to comprehensively characterize and quantify the feature of biological samples. The combination of multiple biological "-ome" information categories (e.g., genome and transcriptome).An immune cell subset that orchestrates the acquired immune response.This study was supported in part by AMED J-PRIDE (19fm0208006h0003 to Kei Sato), KAKENHI Scientific Research B (18H02662 to Kei Sato), KAKENHI Scientific Research on Innovative Areas "Neovirology" (16H06429, 16K21723, 17H05813, 19H04826 to Kei Sato), JST CREST (to Kei Sato), JSPS KAKENHI PAGS 16H06279 (to K.Sato), AMED Research Program on HIV/AIDS 19fk0410014 (19fk0410014, 19fk0410019 to Yoshio Koyanagi and Kei Sato), and JSPS Research Fellow (PD 19J01713 to Jumpei Ito).
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Biotechnology
| 2,020 |
July 28, 2020
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https://www.sciencedaily.com/releases/2020/07/200728113546.htm
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Researchers discover cell communication mechanism that drives cancer adaptation
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Collaborative Cancer Research UK-funded studies from University of Oxford researchers have uncovered a new mechanism by which cancer cells adapt to the stresses they encounter as they grow and respond to therapies. This mechanism involves cells releasing small vesicles, known as exosomes. These contain complex mixtures of proteins, RNAs and other molecules, which can re-programme surrounding cells. Exosomes are thought to be released by all cells within the body, and play important roles in many processes in healthy individuals such as immunity and reproduction. But, in cancer they can turn bad and drive pathological changes such as tumour growth and metastasis.
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Up until now, research has suggested that exosomes are made in compartments in cells known as late endosomes, which are also used to keep cells healthy by clearing out damaged proteins and structures in the cell. By combining complementary analysis in fruit flies and human cancer cells, the collaborative teams have shown that exosomes are also made in the cell's recycling system, which diverts reusable proteins away from the waste disposal system. They are called Rab11a-exosomes and carry a different set of cargos that may help cancers to grow and survive current treatments.As a tumour grows bigger, the cells within it are starved of key nutrients such as amino acids, and these stressed cells produce Rab11a-exosomes loaded with molecules made by the cancer cells. According to Associate Professor Deborah Goberdhan, who led the research: "These 'bad exosomes' can then give other cells around them a growth-promoting boost and can potentially lead to selection of more aggressive cell types and a worse outcome. The production of Rab11a-exosomes may explain why some patients don't respond to certain treatments and why others frequently develop resistance to therapies.""It's becoming increasingly clear that anti-cancer therapies that block growth may need to be given in combination with drugs that prevent tumour cells adapting to the therapy, and reducing the production of these exosomes might be one important way to do this.""A key step will be to work out how the bad exosomes that drive cancer progression are made, so that therapies can be designed to block them. This is likely to take some time. However, developing ways to detect these exosomes in patient blood is an important shorter-term goal. Such an approach might detect cancer at early stages or predict how patients will respond to drugs, both of which could have a major impact on cancer survival and the design of more personalised treatments for patients."Dr Emily Farthing, Senior Research Information Manager at Cancer Research UK said: "This exciting research has discovered that exosomes can act in a way we weren't previously aware of, which could be helping tumours to grow and become resistant to anti-cancer treatments. This lab-based work is still a long way off benefitting people with cancer, but provides helpful clues to how we might be able to tackle the disease in new ways in future."The newly published research has already attracted further funding to start screening for these alternative exosomes in patients, and a major current focus of the team is to identify ways of blocking their production, so that their role in cancer pathology can be fully assessed.Professor Goberdhan said: "By continuing to combine analysis in human cancer cell lines and flies, we have started to highlight genetic manipulations that appear to specifically block the production of Rab11a-exosomes, which we are now following up."
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Biotechnology
| 2,020 |
July 28, 2020
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https://www.sciencedaily.com/releases/2020/07/200728113516.htm
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The amazing travels of small RNAs
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In most organisms, small bits of RNA play a key role in gene regulation by silencing gene expression. They do this by targeting and docking onto complementary sequences of gene transcripts (also RNA molecules), which stops the cell machinery from using them to make proteins. This mechanism is called RNA interference (RNAi), and it is critically important in biology.
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Remarkably, the RNAi phenomenon is not necessarily confined to single cells; it can also manifest in other tissues and organs far away from the cell of origin. Researchers have been able to observe it mostly in plants, but also in "lower" animals such as the nematode worm Still, one key question had so far gone unanswered: which messenger substance traverses cells and tissues? "We were able to rule out proteins 20 years ago, once it was discovered that RNAi can travel in plants," says Olivier Voinnet, Professor of RNA Biology at ETH Zurich. RNAi requires that the messenger docks to a complementary sequence of the gene transcript to be silenced. "Proteins alone don't have this capability. DNA leaving the cell nucleus is also unlikely," Voinnet continues. "The most likely candidate has always been an RNA molecule." What has been unclear until now is which precise type and form of RNA -- long, short, single- or double-stranded, bound to proteins or not.But now, the ETH researchers are shedding light on this process in a new study. They are the first to demonstrate unequivocally that these distant messengers in plants are short double?stranded RNA molecules. These consist of pairs (or double-strands) of just 21 to 24 nucleotides (the building blocks of RNA) called small interfering RNAs, or siRNAs for short. The team's paper was recently published in the journal siRNAs usually emerge as large and complex populations from the genomes of viruses that have infected a cell. But a cell's own genes can also serve as blueprints for these molecules. As a result, cells can use RNAi to silence not only invading viruses but also their own genes.Because RNAi moves, plants have the amazing capacity to modulate gene expression at a distance. This might be particularly important for them to constantly adapt their new growth, enabling what is called "phenotypic plasticity."In their new study, the researchers ruled out the possibility that other types of nucleic acids or complexes composed of RNA and proteins move across plant cells. "We can definitively show that double?stranded siRNAs are necessary and sufficient to induce RNAi in distant cells and tissues of plants," Voinnet says.Not only did the ETH researchers identify the elusive long-distance messengers, they also show, in their study, how siRNAs move and carry out their function. They found that, as long as an siRNA molecule exists as a free double-strand, it is mobile because it cannot bind to a matching RNA transcript. To bind, it first has to be "uploaded" to a specific Argonaute (AGO) effector protein. Only once bound to the correct AGO protein can the siRNA silence the target transcript; the process eventually destroys the fragment itself. The model plant used for the study has ten different AGO proteins, several of which recognise matching siRNA fragments with specific signatures; these signatures are not homogeneous among the large cohorts of mobile siRNAs produced from viruses or the plant's own genes.Different AGO proteins occur in distinct cells and tissues. The ETH researchers found that as part of the uploading process, matching AGO proteins "consume" a fraction of siRNAs in the cell of origin, but the non-loaded fraction can exit the cell.Depending on the presence or absence of certain AGO proteins within the cells traversed by the mobile siRNAs, the molecules, again, will be consumed or not. For example, if there are a plethora of AGO proteins on hand, they will trap plenty of siRNAs with various signatures, essentially stopping movement. If a cell contains hardly any AGO, on the other hand, then most siRNAs will leave and travel greater distances. And finally, if a cell contains large quantities of only one specific AGO, then only those siRNAs with the matching signature will be consumed, while the others will move. In other words, siRNAs are selectively filtered and consumed as they make their way through the plant tissue.Until now, the plant RNAi community had thought that RNAi moves along linear gradients. However, this does not take into account that AGO proteins selectively use up some siRNAs -- but not others -- as they move. The new study points out that this consumption process is, in fact, anything but linear."The amount and diversity of AGO proteins in traversed cells coupled to the siRNA-intrinsic signatures function together as a kind of molecular sieve, the form of which may differ from cell type to cell type along the siRNA path. Depending on the spatial configuration of this sieve, a wide variety of siRNA movement patterns can be produced," Voinnet explains. He adds: "Even more interestingly, some AGOs can be induced by stress or developmental signals such that the spatial shape of the sieve can change and evolve at any given time."The countless movement patterns thus lend the mobile RNAi system almost boundless flexibility and versatility in shaping gene expression across distances. Now that they have understood the process, the team of researchers is trying to engineer artificial sieves in plants as a way to control, with high precision, when and where specific siRNAs can move, a method which could have applications in agriculture.
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Biotechnology
| 2,020 |
July 27, 2020
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https://www.sciencedaily.com/releases/2020/07/200727154206.htm
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The big gulp: Inside-out protection of parasitic worms against host defenses
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A team of developmental biologists at the Morgridge Institute for Research has discovered a means by which schistosomes, parasitic worms that infect more than 200 million people in tropical climates, are able to outfox the host's immune system.
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Morgridge postdoctoral fellow Jayhun Lee and colleagues reported in today's (July 27) issue of Schistosomiasis, the neglected tropical disease caused by schistosome infection, remains one of the major parasitic diseases affecting developing countries, according to the World Health Organization. It is especially impactful in children, resulting in anemia, stunting, and learning disorders. Despite its enormous impact on human health and the resulting socioeconomic losses, it remains an understudied and neglected disease.Schistosomes have a complex life cycle, which begins in freshwater that is contaminated with human excrement. The parasites hatch from eggs released via human waste and infect a specific species of snail. In the snail, the parasite produces massive numbers of larval offspring, called cercariae. Once released from the snail, these fast-swimming, fork-tailed larvae burrow through human skin and cause infection.After penetrating the host's skin, the parasites migrate into blood vessels and find their way to the vein that supplies the liver. Here, they pair with a mate and grow into mature adults, living for over a decade while releasing hundreds of eggs daily. Many of these eggs get lodged in host organs, such as the liver, resulting in chronic tissue damage.Currently, only a single drug, praziquantel, is used to fight schistosomiasis, but it only works on adult worms, does not protect from reinfection, and some strains have developed resistance to the drug. Thus, it is critical to devise new strategies for targeting these parasites."One big question we're interested in is how these parasites can thrive for decades in the bloodstream, while avoiding the host immune system," says Lee.Lee works in the lab of Phillip Newmark, a Morgridge investigator, Professor of Integrative Biology at the University of Wisconsin-Madison and investigator of the Howard Hughes Medical Institute (HHMI). The Newmark lab has primarily studied planarians, flatworms with an almost limitless capacity for regeneration. About 10 years ago, the lab began applying their knowledge of planarian biology to understand the planarian's parasitic cousin, the schistosome. By understanding how schistosomes develop inside the host, the lab hopes to find new ways to combat this disease.In the new study, the team investigated a handful of stem cells that are inherited from the larval stage of the parasite. Stem cells in the parasite are necessary for their survival and reproduction, but their role during the early stages inside the mammalian host has been unclear. They found that the stem cells generate a specialized gland associated with the parasite's digestive tract called the esophageal gland -- weeks before the animals start feeding on blood.Why would the stem cells need to make this gland so early?Suspecting that the esophageal gland might be important for the survival of the parasites, the team disrupted a gene critical for making the esophageal gland and cultured the parasites in a dish. Despite now lacking an esophageal gland, the viability and behavior of the parasites were not affected when cultured outside the host."I think this is normally where you would consider dropping the project," Lee says, since the esophageal gland appeared to have no function in the parasites cultured in vitro.However, since current culture conditions do not fully reflect the in vivo environment of the host vasculature (such as the lack of host immune cells and blood flow), the team decided to take the project further by examining the function of the gland when the parasite is living inside the mammalian host.The next experiments were made possible by a technique pioneered by Donato Cioli in the 1970s in which schistosomes are surgically transplanted into the mesenteric veins of rodent hosts."This technically challenging experiment is the only way to introduce experimentally manipulated adult schistosomes back into the mammalian host, as only the larval stage of the parasite is able to penetrate the host skin," says HHMI research specialist Tracy Chong, who performed the surgical transplantations.Chong transplanted parasites lacking the esophageal gland into mice. In contrast to parasites cultured in a dish, lack of the esophageal gland led to lethality in the mammalian host."Based on clues from previous studies, we hypothesized that the esophageal gland of the parasite was acting as a barrier to prevent host immune cells from infiltrating the parasite," says Lee.To test this idea, the team surgically transplanted parasites lacking the esophageal gland into immunocompromised mice. The gland-lacking parasites were able to survive in immunocompromised mice, just like they did in culture dishes. Follow-up experiments in which the parasites were fed fluorescent immune cells showed that gland-lacking parasites were unable to destroy immune cells before they entered the parasite's gut."Our results show that the esophageal gland is an important barrier that needs to be in place before these parasites start feeding and ingesting immune cells," Lee adds. "We are hopeful this research will lead to new targets to fight these parasites."Lee says the next chapter in this ongoing work will be to define the hundreds of different proteins that make up the esophageal gland."When we characterize these proteins, we might be able to find a way to block or disable their function, which would then allow immune cells to get inside the parasites and kill them," he says.
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Biotechnology
| 2,020 |
July 27, 2020
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https://www.sciencedaily.com/releases/2020/07/200727114751.htm
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Epstein-Barr virus rewires host epigenomes to drive stomach cancer
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The Epstein-Barr virus (EBV), one of the most common human viruses, is associated with about 8-10 per cent of stomach -- or gastric -- cancers, the third leading cause of cancer death globally. Researchers from Chiba University in Japan, Duke-NUS Medical School, Singapore, and the Agency for Science, Technology and Research (A*STAR)'s Genome Institute of Singapore (GIS) have revealed a novel paradigm in EBV-associated gastric cancer, whereby the EBV viral genome directly alters the host epigenetic landscape to promote the activation of proto-oncogenes (genes involved in normal cell growth that can mutate into cancer-causing genes) and tumorigenesis.
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The human genome is the complete set of human genetic information, and the epigenome describes modifications to the genome that determine whether genes are turned on or off when and where they are needed. Unlike genetic information, the epigenome is dynamic and responsive to external stimuli; certain external stimuli can cause abnormal DNA modifications which, in turn, can disrupt normal gene expression and contribute to cancer development.The research group, led by senior and co-corresponding authors, Dr Atsushi Kaneda, Professor at the Graduate School of Medicine, Chiba University, and Dr Patrick Tan, Professor at the Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, and Executive Director of GIS, conducted a comprehensive analysis of three-dimensional genomic structures in human cells. These ranged from gastric cancer cell lines, patient samples, normal gastric epithelial cells, and EBV-associated gastric cancer. Combined with virus infection analyses, the researchers found abnormally activated genomic regions specific to EBV-positive stomach cancer. Experimental EBV infection of cultured stomach cells reproduced the phenomena of EBV binding to these inactive and closed genomic regions and their abnormal activation.?Cells put active marks on genomic regions necessary for their behaviours and utilise them, and inactive marks on unnecessary genomic regions that are tightly closed and not to be utilised," explained Prof Kaneda. "We made the striking observation that strong inactive marks were lost in specific genomic regions when we infected stomach cells with EBV."The researchers further found that genetic enhancers (short pieces of DNA that help encourage genes to make proteins) 'silenced' in the closed regions were activated by the virus to upregulate nearby cancer-related genes, leading to the proliferation of cancerous cells. This 'enhancer infestation' model, as the researchers termed it, reveals a novel mechanism of tumorigenesis that does not require genetic alterations, and instead works by reprograming the epigenetic landscape of human cells to convert latent enhancers from a silenced to an active state.Prof Patrick Tan, who is also a member of the Singapore Gastric Cancer Consortium, remarked, ?In all EBV-positive stomach cancer cells and primary stomach cancer patient samples studied, EBV DNA bound to largely the same genomic regions that also showed abnormal activation. These same regions also changed from inactive to active states by experimental EBV infection."This mechanism of 'enhancer infestation' led to the activation of neighbouring proto-oncogenes in human cells and it is likely to contribute to EBV-associated oncogenesis in multiple cancer cell types. Notably, the researchers also found that, even after eliminating EBV genomes, epigenetic modifications that were induced continued to persist, suggesting a 'hit-and-run' mechanism in which, once an EBV episome alters the chromatin topology of human cells, these altered topologies are stable and persist even after removal of the EBV episome.Prof Kaneda reiterated, "While 8-10 per cent of stomach cancer is associated with EBV, we believe our enhancer infestation model provides a new mechanism of cancer involving epigenomic alterations and viral infection that may be relevant to a broader range of cancers and associated diseases."Prof Tan added, "Infections by EBV are estimated to cause over 200,000 cancers per year worldwide, including certain stomach cancers. Our study highlights new potential drug targets in EBV-positive malignancies, revealed by epigenetics and previously invisible using more conventional genetic sequencing studies."
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Biotechnology
| 2,020 |
July 27, 2020
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https://www.sciencedaily.com/releases/2020/07/200727114718.htm
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Cancer mutations caused by bacterial toxin preventable
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Studying DNA mutations in cancers can help clarify how cancers develop and what makes cancer cells different from normal, healthy cells. The team of scientists from Duke-NUS' Cancer and Stem Cell Biology (CSCB) programme, specifically wanted to look more closely at Asian cancers to expand the list of 65 currently known mutation patterns found in cancers. Scientists expect there are still some rare mutation patterns that have yet to be discovered.
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The researchers examined 872 colorectal lesions from 201 patients with unexplained, non-inherited polyposis. This condition involves the formation of intestinal polyps that can predispose people to cancer. It is usually caused by a mutation in the Adenomatous polyposis coli (APC) gene. This gene is critical for several cellular processes and acts as a tumour suppressor by preventing cells from growing and dividing uncontrollably. The team found that almost 20 per cent of the patients had tumors with mutations in the APC gene with characteristics similar to those caused by a bacterial toxin known as colibactin, suggesting the toxin's involvement in initiating polyp formation in these individuals.This study was built upon an earlier study where the team analysed DNA mutations in 36 Asian patients who were treated in Singapore for a mouth cancer called oral squamous cell carcinoma. "We found a very specific pattern of DNA mutations in the oral cancer of patients who also had severe bacterial infection in their mouth. We found that these DNA mutations had been caused by a toxin called colibactin, which is produced by these bacteria," said Dr Arnoud Boot, Senior Research Fellow at the CSCB programme and the lead author of this study.Colibactin, is produced by a specific group of Escherichia coli bacteria, which normally live in the gut. While exposure to colibactin is difficult to prevent, the research team suggests that regular brushing of teeth might have prevented the bacterial infection that appears to have triggered the cancer-causing DNA mutation in the oral cancer patient who was examined."There are bacteria that sometimes live in the human body, making a toxin that contributes to cancer formation. In addition to understanding what causes cancer, our results also indicate that some cancers that are caused by colibactin might be preventable," said Professor Steve Rozen from Duke-NUS' CSCB programme, who is the corresponding author of one of these studies."These kinds of investigations by our researchers from the CSCB programme are crucial in better understanding how to prevent cancer by targeting cancer-specific changes," said Prof Patrick Casey, Senior Vice Dean for Research at Duke-NUS.The team next aims to analyse a larger dataset of cancers that could be associated with colibactin.
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Biotechnology
| 2,020 |
July 24, 2020
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https://www.sciencedaily.com/releases/2020/07/200724143013.htm
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Genetic mutations help MRSA to become highly resistant to antibiotics
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Scientists from the University of Sheffield have found that genetic mutations in MRSA allow it to evolve and become more resistant to antibiotics such as penicillin.
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The research, published in Most clinical MRSA exhibits a low level of antibiotic resistance, due to the cells acquiring a new gene encoding a protein (MecA) that makes its cell wall, some strains can evolve high-level resistance and pose a serious threat.Antibiotics, such as penicillin and methicillin, do not bind well to the new protein (MecA) meaning they cannot 'kill' the bacteria. The next phase of this research is to understand how this protein works with other factors within the bacteria to allow a higher level of antibiotic resistance.Findings from the research pave the way for more understanding of the cause and evolution of antibiotic resistance, and will help researchers develop new treatments and drugs for MRSA.Simon Foster, Professor of Molecular Microbiology at the University and Principal Investigator of the research, said: "Antibiotics have been a mainstay of human healthcare for over 70 years, but the emergence of antimicrobial resistance is now a global catastrophe. In order to combat antimicrobial resistant organisms, we have to understand them. Our work uncovers the complex mechanisms that underpin resistance, giving insight into how we might tackle this global challenge."The research is part of a collaborative project called the Physics of Antimicrobial Resistance which involves the Universities of Sheffield, Newcastle, Edinburgh and Cambridge, funded by UK Research and Innovation (UKRI).Dr Viralkumar Panchal, Postdoctoral Researcher at the University of Sheffield and leader of the research, said: "The research provides a new outlook into the process of evolution of resistance and reveals important details of how MRSA is so resistant. We can now exploit these findings to develop new cures."Globally, the effectiveness of antimicrobial compounds is decreasing as infectious species become increasingly resistant. The University of Sheffield's Florey Institute for Host-Pathogen Interactions aims to create a world-leading focus on antimicrobial resistance from fundamental science to translation and brings together scientists and clinicians to tackle this problem.
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Biotechnology
| 2,020 |
July 24, 2020
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https://www.sciencedaily.com/releases/2020/07/200724141347.htm
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Antiviral method against herpes paves the way for combating incurable viral infections
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Researchers at Lund University in Sweden have discovered a new method to treat human herpes viruses. The new broad-spectrum method targets physical properties in the genome of the virus rather than viral proteins, which have previously been targeted. The treatment consists of new molecules that penetrate the protein shell of the virus and prevent genes from leaving the virus to infect the cell. It does not lead to resistance and acts independently of mutations in the genome of the virus. The results are published in the journal
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Herpes virus infections are lifelong, with latency periods between recurring reactivations, making treatment difficult. The major challenge lies in the fact that all existing antiviral drugs to treat herpes viruses lead to rapid development of resistance in patients with compromised immune systems where the need for herpes treatment is the greatest (e.g. newborn children, patients with HIV, cancer or who have undergone organ transplantation). Both the molecular and physical properties of a virus determine the course of infection. However, the physical properties have so far received little attention, according to researcher Alex Evilevitch."We have a new and unique approach to studying viruses based on their specific physical properties. Our discovery marks a breakthrough in the development of antiviral drugs as it does not target specific viral proteins that can rapidly mutate, causing the development of drug resistance -- something that remains unresolved by current antiviral drugs against herpes and other viruses. We hope that our research will contribute to the fight against viral infections that have so far been incurable," says Alex Evilevitch, Associate Professor and senior lecturer at Lund University who, together with his research team, Virus Biophysics, has published the new findings.The virus consists of a thin protein shell, a capsid, and inside it lies its genome, the genes. Alex Evilevitch has previously discovered that the herpes virus has high internal pressure because it is tightly packed with genetic material."The pressure is 20 atmospheres, which is four times higher than in a champagne bottle and this allows herpes viruses to infect a cell by ejecting its genes at high speed into the cell nucleus after the virus has entered the cell. The cell is then tricked into becoming a small virus factory that produces new viruses that can infect and kill other cells in the tissue, leading to different disease states," explains Alex Evilevitch.He, with the help of preclinical studies at the National Institutes of Health in the United States, has identified small molecules that are able to penetrate the virus and "turn off" the pressure in the genome of the virus without damaging the cell. These molecules proved to have a strong antiviral effect that was several times higher than the standard treatment against certain herpes types with the drug Aciclovir, as well as against resistant herpesvirus strains where Aciclovir does not work. The approach prevented viral infection.Since all types of herpes viruses have similar structure and physical properties, this antiviral treatment works on all types of viruses within the herpes family."The drugs available today for combatting viral infections are highly specialised against the viral proteins, and if the virus mutates, which regularly occurs, the drug is rendered ineffective. However, if you succeed in developing a treatment that attacks the physical properties of a virus, such as lowering the pressure inside the herpes virus shell, it should be possible to counter many different types of viral infections within the same virus family using the same drug. In addition, it would work even if the virus mutates because the mutations do not affect the internal pressure of the herpes virus."The result of the present study is a first step towards the goal of developing a drug and we already have positive preliminary data showing that a herpes infection can be stopped for all types of herpes virus including the resistant strains."
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Biotechnology
| 2,020 |
July 24, 2020
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https://www.sciencedaily.com/releases/2020/07/200724104228.htm
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In cell studies, seaweed extract outperforms remdesivir in blocking COVID-19 virus
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In a test of antiviral effectiveness against the virus that causes COVID-19, an extract from edible seaweeds substantially outperformed remdesivir, the current standard antiviral used to combat the disease. Heparin, a common blood thinner, and a heparin variant stripped of its anticoagulant properties, performed on par with remdesivir in inhibiting SARS-CoV-2 infection in mammalian cells.
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Published online today in The spike protein on the surface of SARS-CoV-2 latches onto the ACE-2 receptor, a molecule on the surface of human cells. Once secured, the virus inserts its own genetic material into the cell, hijacking the cellular machinery to produce replica viruses. But the virus could just as easily be persuaded to lock onto a decoy molecule that offers a similar fit. The neutralized virus would be trapped and eventually degrade naturally.Previous research has shown this decoy technique works in trapping other viruses, including dengue, Zika, and influenza A."We're learning how to block viral infection, and that is knowledge we are going to need if we want to rapidly confront pandemics," said Jonathan Dordick, the lead researcher and a professor of chemical and biological engineering at Rensselaer Polytechnic Institute. "The reality is that we don't have great antivirals. To protect ourselves against future pandemics, we are going to need an arsenal of approaches that we can quickly adapt to emerging viruses."The The researchers performed a dose response study known as an EC50 -- shorthand for the effective concentration of the compound that inhibits 50% of viral infectivity -- with each of the five compounds on mammalian cells. For the results of an EC50, which are given in a molar concentration, a lower value signals a more potent compound.RPI-27 yielded an EC50 value of approximately 83 nanomolar, while a similar previously published and independent in vitro test of remdesivir on the same mammalian cells yielded an EC50 of 770 nanomolar. Heparin yielded an EC50 of 2.1 micromolar, or about one-third as active as remdesivir, and a non-anticoagulant analog of heparin yielded an EC50 of 5.0 micromolar, about one-fifth as active as remdesivir.A separate test found no cellular toxicity in any of the compounds, even at the highest concentrations tested."What interests us is a new way of getting at infection," said Robert Linhardt, a Rensselaer professor of chemistry and chemical biology who is collaborating with Dordick to develop the decoy strategy. "The current thinking is that the COVID-19 infection starts in the nose, and either of these substances could be the basis for a nasal spray. If you could simply treat the infection early, or even treat before you have the infection, you would have a way of blocking it before it enters the body."Dordick added that compounds from seaweed "could serve as a basis for an oral delivery approach to address potential gastrointestinal infection."In studying SARS-CoV-2 sequencing data, Dordick and Linhardt recognized several motifs on the structure of the spike protein that promised a fit compatible with heparin, a result borne out in the binding study. The spike protein is heavily encrusted in glycans, an adaptation that protects it from human enzymes which could degrade it, and prepares it to bind with a specific receptor on the cell surface."It's a very complicated mechanism that we quite frankly don't know all the details about, but we're getting more information," said Dordick. "One thing that's become clear with this study is that the larger the molecule, the better the fit. The more successful compounds are the larger sulfated polysaccharides that offer a greater number of sites on the molecules to trap the virus."Molecular modeling based on the binding study revealed sites on the spike protein where the heparin was able to interact, raising the prospects for similar sulfated polysaccharides."This exciting research by Professors Dordick and Linhardt is among several ongoing research efforts at CBIS, as well as elsewhere at Rensselaer, to tackle the challenges of the COVID-19 pandemic through novel therapeutic approaches and the repurposing of existing drugs," said CBIS Director Deepak Vashishth."Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro" was published in
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Biotechnology
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July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723172009.htm
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Biologists shed light on how cells move resources
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Florida State University researchers have new insight into the tiny packages that cells use to move molecules, a structure that is key to cellular metabolism, drug delivery and more.
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Their research uncovered more about the proteins that form the outer structure of those cellular packages. The work was published in the journal "Just like human mail carriers have to transport packages of different shapes and sizes, cells also have to transport a variety of materials to the right compartments within them," said Scott Stagg, associate professor of chemistry and biochemistry and a study co-author. "They need to bring in molecules from outside the cell and transport them between the different cellular compartments, and they have little molecular machines called vesicles that function like postal carriers moving microscopic packages from one compartment to another."Scientists have previously observed cells create vesicles -- fluid-filled sacks that move materials within a cell or from one cell to another. They have also observed a protein called clathrin form a cage-like arrangement that made up the outside structure of vesicles.But there were still questions about how exactly clathrin forms those structures and what determines the shapes it can take.Using high-powered microscopes, the FSU researchers discovered that another protein, known as an adaptor protein, ties multiple clathrin molecules together in a way that allows those structures to take on different sizes.They also showed that the clathrin coat could make a so-called "basket" shape, and one that scientists had thought the protein could not form, showing that clathrin assembly is more complicated than previously thought."We learned a lot about clathrin-coated vesicles by looking at the ones that were made by cells themselves," said Mohammadreza Paraan, a researcher at FSU's Institute of Molecular Biophysics and the study's lead author. "We found new structures and patterns that really surprised us."The researchers found that the clathrin structures that other researchers formed in a test tube were different from the ones they saw from cells."This shows that there are things we don't understand about how clathrin coat assembly is regulated and progresses in cells," Stagg said. "Our hypothesis is that the cargo that vesicles carry has a role in dictating how the coats are made and that explains why we see different structures."The ability for cells to form vesicles is essential. It is the main route by which molecules like hormones, proteins and viruses enter cells and move within them. If it stops working, cells can die, or disease can take hold in an organism.Understanding cellular transport is also important because the process is often hijacked by viruses like influenza or the virus that causes COVID-19 to gain entry to the cell."Understanding the molecular mechanisms of clathrin-based transport is important because it is such a fundamental process," Stagg said. "It touches on so many cellular processes. The better we understand it, the more likely it is that we can manipulate it to do things like stop virus entry, enhance drug delivery inside cells or modulate neurotransmitter levels in the brain, just to mention a few. It's a really exciting time for clathrin research."This work was supported by the National Institutes of Health.
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Biotechnology
| 2,020 |
July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723152336.htm
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New CRISPR C-to-G DNA base editor expands the landscape of precision genome editing
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New genome-editing technologies developed by researchers in J. Keith Joung's laboratory at the Massachusetts General Hospital (MGH) have the potential to help understand disease-associated genetic mutations that are based on C-to-G (cytosine to guanine) single base changes. The new base editors are also designed to minimize unintended ("off-target") mutations that could potentially cause undesirable side effects.
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The new CRISPR-guided DNA base editing technologies are designed to efficiently induce "transversion" alterations of DNA bases while minimizing levels of unwanted "bystander" mutations.The proof-of-concept C-to-G base editor, called CGBE1, and a smaller version, miniCGBE1, are described in a paper by co-first authors Ibrahim C. Kurt and Ronghao Zhou that was published online in the journal CRISPR (clustered regularly interspaced short palindromic repeats) is a gene-editing technology first discovered as a defense mechanism in bacteria and then harnessed by scientists as a tool for snipping out and/or repairing DNA sequences. The first CRISPR techniques relied on creating and repairing double-strand DNA breaks."Base editing is a new form of CRISPR gene editing that was developed by David Liu's lab at Harvard University and the Broad Institute. It is not based on introducing a double-stranded break in DNA, but is rather focused on directly changing a single base in DNA," explains co-corresponding author Julian Grünewald, MD, of the MGH Molecular Pathology Unit and Harvard Medical School (HMS).Base editors are fusion proteins that use a modified form of CRISPR-Cas that is targeted to a specific target site with the help of a guide RNA, where it then deploys an enzyme called a deaminase to modify a specific base to create a desired DNA change. For example, the technique can be used to convert a cytosine (C) base to a thymine (T) base, both bases within the pyrimidine class (performed with a cytosine base editor, or CBE). Similarly, an adenine base editor (ABE) is capable of converting an adenine (A) to a guanine (G), both being purine bases.CGBE1 leverages a CBE variant that was published in 2019 by J. Keith Joung, MD, PhD and colleagues in The new CGBE1 tool incorporates the deaminase from this SECURE-CBE variant, which together with other components enables the technically challenging swapping of bases from one class to another while still minimizing the risk of unwanted changes."There are known disease-associated mutations or pathogenic mutations that could be fixed by this type of editing," Grünewald says.However, the exact number of diseases that might be correctable with CGBE1 or a similar editing platform is unclear."We're still at an early stage with this new class of transversion base editors; CGBE1 still requires additional optimization and it would be premature to say this is ready for the clinic. But we envision that CGBE1 could be useful for research applications, enabling the introduction of specific C-to-G mutations," he says.
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Biotechnology
| 2,020 |
July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723143803.htm
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Two distinct circuits drive inhibition in the sensory thalamus of the brain
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The thalamus is a "Grand Central Station" for sensory information coming to our brains. Almost every sight, sound, taste and touch we perceive travels to our brain's cortex via the thalamus. It is theorized that the thalamus plays a major role in consciousness itself. Not only does sensory information pass through the thalamus, it is also processed and transformed by the thalamus so our cortex can better understand and interpret these signals from the world around us.
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One powerful type of transformation comes from interactions between excitatory neurons that carry data to the neocortex and inhibitory neurons of the thalamic reticular nucleus, or TRN, that regulate flow of that data. Although the TRN has long been recognized as important, much less has been known about what kinds of cells are in the TRN, how they are organized and how they function.Now a paper published in the journal "These results provide fundamental insights about how subnetworks of TRN neurons may differentially process distinct classes of thalamic information," Cruikshank said. "The genetic distinctions add some excitement to our main findings because they will enable powerful new optogenetic and chemogenetic strategies for probing behavioral and perceptual functions of these TRN sub-circuits. A long-term goal for many of us working in this area is to learn how the TRN orchestrates information flow to and from the neocortex, guiding attention to important stimuli and suppressing distractions. If such an understanding is eventually achieved, it could help clarify how conscious awareness is lost during a form of epilepsy -- absence epilepsy -- that critically involves the TRN."Cruikshank is an assistant professor in the University of Alabama at Birmingham Department of Neurobiology. The experimental work was done at Brown University, Providence, Rhode Island, where Cruikshank was a research track professor prior to joining UAB last November.In some of the study details, the researchers first found that the somatosensory TRN has two sets of neurons. In a central core of the TRN are neurons that express calbindin protein and mRNA. This core is surrounded by a shell of neurons that express somatostatin protein and mRNA.There are also two somatosensory thalamocortical nuclei -- that is, nuclei that transmit sensory information from the thalamus to the neocortex. One is the first-order ventral posterior nucleus, or VP, and the other is the higher-order posterior medial thalamic nucleus, or POM. These two nuclei send distinct information to different neocortical targets, and also send collateral axons to the TRN. So, the researchers sought to clarify the organization of those circuits, focusing on how first-order and higher-order thalamocortical nuclei communicate with the two subtypes of TRN neurons. "This is essential to understand thalamic information processing," Cruikshank said.Cruikshank and colleagues used a channelrhodopsin-yellow fluorescent protein anterograde tracing method from either the VP or POM to map their inputs to the TRN. They found a stark anatomical segregation of projections that aligned with the segregation of TRN cell types -- VP axons made strong synaptic connections with cells in the calbindin-rich central zone of the TRN; POM axons synapsed with cells along the somatostatin-dense edges. The segregated projections were largely reciprocal -- that is, the two TRN cell types predominantly inhibited the same thalamocortical nuclei that drove them.The researchers further showed that the TRN sub-circuits had functionally different physiological mechanisms that lead to distinct processing. "Our experiments revealed that central and edge cells differentially transform their native excitatory thalamic inputs into distinct spiking outputs through differences in both dynamics of their synaptic inputs and their intrinsic burstiness," Cruikshank said. "We were intrigued that the TRN response patterns seemed to match the types of information carried in the two sub-circuits. The primary central cells had strong but transient spiking -- ideal for processing discrete sensory events. The edge cell responses were more stable and sustained -- consistent with temporally distributed higher-order signals integrated from multiple sources.When the researchers looked at the visual TRN, they found sub-circuits similar to the somatosensory TRN. This, in turn, suggests, the researchers say, that a primary central core -- flanked by higher-order edge neurons -- may be a widespread TRN motif.The findings challenge a longstanding hypothesis about the way TRN implements its role as a gatekeeper of information flow. "It has been proposed that inhibitory cross-talk between distinct thalamic circuits may allow them to regulate one another locally," Cruikshank said. "However, the sharp and reciprocal segregation of sub-circuits we observed suggests that intrathalamic cross-talk may play a minor role, and perhaps we should look to other mechanisms for cross-system regulation.""This work by Scott Cruikshank separates an otherwise jumbled bag of nerve cells into elegant sub-circuits serving distinct functions with distinct properties and projections," said Craig Powell, M.D., Ph.D., chair of Neurobiology at UAB. "The results help us better understand how different types of sensory inputs are processed by the brain. The TRN is a key brain region responsible for childhood onset seizures called absence seizures, so this work may help identify novel therapies for this childhood epilepsy that is common in neurodevelopmental disorders and is a focus of the Civitan International Research Center at UAB."Support came from National Institutes of Health grants NS100016 and GM103645, and National Science Foundation grants 1738633, 1058262 and 1632738.
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Biotechnology
| 2,020 |
July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723143801.htm
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PolyA-miner assesses the effect of alternative polyadenylation on gene expression
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Researchers with an interest in unraveling gene regulation in human health and disease are expanding their horizons by closely looking at alternative polyadenylation (APA), an under-charted mechanism that regulates gene expression.
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"APA is about modifying one of the ends, called the 3-prime end (3′end), of RNA strands that are transcribed from DNA. The modification consists of changing the length of a tail of adenosines, one of the RNA building blocks, at the 3′end before RNA is translated into proteins," said first author Dr. Hari Krishna Yalamanchili, a postdoctoral associate in the lab of Dr. Zhandong Liu at Baylor College of Medicine. "This adenosine chain helps to determine how long the messenger RNA lasts in the cell, influencing how much protein is produced from it."The interest in APA has resulted in the development of several 3′ sequencing (3′Seq) techniques that allow for precise identification on APA sites on RNA strands. But what researchers are missing is a robust computational tool that is specifically designed to analyze the wealth of 3′Seq data that has been generated."Until now, researchers have been using traditional RNA sequencing computational tools to analyze the 3′Seq datasets. Although this approach produces results, it does not maximize the potential amount of information that can be extracted from that data," Yalamanchili said. "Here we developed a computational tool that precisely analyzes 3′Seq data. We call it PolyA-miner."Yalamanchili and his colleagues used their new computational tool to analyze existing 3′Seq datasets. PolyA-miner not only reproduced the analyses achieved with traditional computational tools, but also identified novel APA sites that were not detected with the other analytical approaches."We were surprised when the PolyA-miner analysis of a glioblastoma cell line dataset identified more than twice the number of genes with APA changes than were initially reported," Yalamanchili said."I think that the most exciting part of this new tool is that it enables us to precisely reflect gene-level 3′ changes and to identify many more APA events than before. With other analytical approaches, we underestimate the effect and number of poly-adenylation events," said Liu, associate professor of pediatrics and neurology at Baylor and the Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital.This development has tremendous implications for basic research and for the potential translation of scientific findings into the clinic. APA is considered a major mechanism for RNA regulation that has strong relevance both in cancer and neurological diseases. PolyA-miner can assist scientists looking to identify the genetic causes of these diseases by determining whether there are differences in APA between diseased and normal cells. With this new analysis, scientists can take a fresh look at existing genomic datasets that may provide an answer to the cause of human conditions, as well as studying newly developed datasets."Previously, people knew about APA changes, but did not consider them to be major contributors to gene regulation mainly because we lacked the computational tools to determine APA's overall influence on gene expression," Yalamanchili said. "PolyA-miner has shown that APA seems to play a larger role in gene regulation than we had previously thought."
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Biotechnology
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July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723143722.htm
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'Self-eating' process of stem cells may be the key to new regenerative therapies
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The self-eating process in embryonic stem cells known as chaperone-mediated autophagy (CMA) and a related metabolite may serve as promising new therapeutic targets to repair or regenerate damaged cells and organs, Penn Medicine researchers show in a new study published online in
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Human bodies contain over 200 different types of specialized cells. All of them can be derived from embryonic stem (ES) cells, which relentlessly self-renew while retaining the ability to differentiate into any cell type in adult animals, a state known as pluripotency. Researchers have known that the cells' metabolism plays a role in this process; however, it wasn't clear exactly how the cells' internal wiring works to keep that state and ultimately decide stem cell fate.The new preclinical study, for the first time, shows how the stem cells keeps CMA at low levels to promote that self-renewal, and when the stem cell is ready, it switches that suppression off to enhance CMA, among other activities, and differentiate into specialized cells."It's an intriguing discovery in the field of stem cell biology and for researchers looking to develop therapies for tissue or organ regeneration," said senior author Xiaolu Yang, PhD, a professor of Cancer Biology at the Abramson Family Cancer Research Institute in the Perelman School of Medicine at the University of Pennsylvania. "We reveal two novel ways to potentially manipulate the self-renewal and differentiation of stem cells: CMA and a metabolite, known as alpha-ketoglutarate, that is regulated by CMA. Rationally intervening or guiding these functions could be a powerful way to increase the efficiency of regenerative medicine approaches."Autophagy is a cell-eating mechanism necessary for survival and function of most living organisms. When cells self-eat, the intracellular materials are delivered to lysosomes, which are organelles that help break down these materials. There are a few forms of autophagy. However, unlike the other forms, which are present in all eukaryotic cells, CMA is unique to mammals. To date, the physiological role of CMA remains unclear.Using metabolomic and genetic laboratory techniques on the embryonic stem cells of mice, the researchers sought to better understand significant changes that took place during their pluripotent state and subsequent differentiation.They found that CMA activity is kept at a minimum due to two cellular factors critical for pluripotency -- Oct4 and Sox2 -- that suppresses a gene known as LAMP2A, which provides instructions for making a protein called lysosomal associated membrane protein-2 necessary in CMA. The minimal CMA activity allows stem cells to maintain high levels of alpha-ketoglutarate, a metabolite that is crucial to reinforce a cell's pluripotent state, the researchers found.When it's time for differentiation, the cells begin to upregulate CMA due to the reduction in Oct4 and Sox2. Augmented CMA activity leads to the degradation of key enzymes responsible for the production of alpha-ketoglutarate. This leads to a reduction in alpha-ketoglutarate levels as well as an increases in other cellular activities to promote differentiation. These findings reveal that CMA and alpha-ketoglutarate dictate the fate of embryonic stem cells.Embryonic stem cells are often called pluripotent due to their remarkable ability to give rise to every cell type in the body, except the placenta and umbilical cord. Embryonic stem cells not only provide a superb system to study early mammalian development, but also hold great promise for regenerative therapies to treat various human disorders. The development of stem-cell based regenerative medicine therapies has rapidly increased in the last decade, with several approaches in studies shown to repair damaged heart tissue, replace cells in solid organ transplantation, and in some cases address neurological disorders."This newly discovered role of autophagy in the stem cell is the beginning of further investigations that could lead to researchers and physician-scientists to better therapies to treat various disorders," Yang said.
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Biotechnology
| 2,020 |
July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723143704.htm
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Driving immunometabolism to control lung infection
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When drugs to kill microbes are ineffective, host-directed therapy uses the body's own immune system to deal with the infection. This approach is being tested in patients with COVID-19, and now a team of researchers at Trinity College Dublin has published a study showing how it might also work in the fight against tuberculosis (TB). The findings are published in the journal
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Although the bacteria that causes TB (called Mtb) has scourged humankind for millennia, we do not fully understand the complexities and interplay of the human immune response to this ancient bug. Worryingly, there are increasing numbers of people with antibiotic resistant TB, which is hard to treat and is becoming a global threat to public health.Scientists at the Trinity Translational Medicine Institute (TTMI) at St. James's Hospital are dedicated to understanding the intricacies of the human immune response to Mtb with the aim of finding ways to target and promote the immune response to overcome the infection. Scientists already know that the human immune response can both under or over respond to the bacteria resulting in a difficulty to treat the disease. This complex immune response is analogous to driving with both the accelerator and the brakes fully engaged at the same time.The research team led by Health Research Board Emerging Investigator, Dr Sharee Basdeo, and Professor Joseph Keane, Clinical Medicine, Trinity College has recently discovered a way to manipulate human immune responses to Mtb to tip the balance in favour of the patient. All changes in immune cell responses to an infection are governed by changes in what genes are active and 'open' for business. Because our DNA stretches out to nearly 2 meters but needs to fit inside every tiny cell in our body, it needs to be very tightly packed up. Its packaging, and how easy it is to open and close, very often dictates the activity of the genes. This is known as "epigenetics."The research team used a drug approved for cancer treatment called suberoylanilide hydroxamic acid (SAHA for short, also known as Vorinostat). This drug is an epigenetic inhibitor, meaning it can block the machinery that closes up genes. Using this drug on human immune cells that are infected with the bacteria that causes TB, they discovered that SAHA releases the brakes on the immune system by stopping the production of an anti-inflammatory signal while at the same time promoting more appropriate pro-inflammatory signals that may help the patient to clear the infection. Importantly, the team discovered that this fine-tuning of the immune response early in the reaction to infection also benefits later immune responses, which may also aid in the design of future vaccine strategies.Dr Donal Cox, Research Fellow, Clinical Medicine, Trinity College and senior author on the paper, suggests that this may be a new and exciting addition to our arsenal of antibiotic therapies. He said:"Understanding and being able to manipulate the immune system is a crucial component of treating infectious diseases. Having host directed therapies targeting the human immune response will be key in addressing the likely pandemics that will arise in the future due to increasing antibiotic resistance, particularly TB.""We would also like to thank and highlight the important contributions that patients made in this study by providing blood and lung cells without which this research would not be possible."This work was funded by grants from the Irish Research Council, The Royal City of Dublin Hospital Trust and the Health Research Board.
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Biotechnology
| 2,020 |
July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723143651.htm
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New cell profiling method could speed TB drug discovery
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A new technology that combines high throughput imaging and machine learning could speed discovery of drugs to fight tuberculosis, which for generations has killed more people worldwide than any other disease caused by a single agent -- 4,000 people every day.
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Current treatment requires multiple drugs for at least six months and sometimes years, and antibiotic resistance is growing, increasing urgency for finding new treatments.However, drug discovery typically requires production of hundreds of derivatives of an original compound in order to find the most effective version. The new technology -- dubbed MorphEUS (Morphological Evaluation and Understanding of drug Stress) -- provides a rapid, efficient, cost-effective way to determine how specific compounds act to destroy Mycobcterium tuberculosis (M. tb), the bacterium that causes tuberculosis."We urgently need shorter, more effective TB therapies, and MorphEUS enables us to screen through drug candidates, see how they actually affect the cell, and learn which drugs have unique ways to kill the M. tb," said Bree Aldridge, associate professor of molecular biology and microbiology at Tufts University School of Medicine and senior author on the associated paper about the new platform published online in the Aldridge and her colleagues applied MorphEUS to 34 currently available antibiotics for which modes of action were already established and three non-commercial compounds. MorphEUS categorized the drugs correctly 94 percent of the time. In the remaining instances, MorphEUS identified previously unknown target pathways.The search for new TB treatments has been stymied by difficulties in identifying the biological activity of compounds early in the drug discovery process and the need to clarify the mechanism of action of existing therapies. Antibacterials kill pathogens via specific molecular actions, for example, by destroying the microbe's cell wall or inhibiting protein synthesis. The drugs leave clues to their particular modus operandi: characteristic physical unraveling of the bacterial cells, which may affect length, width, shape of structures like the chromosome, staining ability, and other properties. Morphological profiling to categorize drugs by these changes is well-established with pathogens such as E. coli, but Aldridge's team was the first to test it with M. tb."We found that conventional morphological profiling approaches didn't work with M. tb, because the bacterium's inherent response to treatment was extremely variable, and changes in morphology were much less obvious than in bacteria like E. coli," said Trever C. Smith II, co-first author on the paper and a postdoctoral researcher in the Aldridge laboratory.MorphEUS harnesses this variation by incorporating measurements of heterogeneity itself into morphological profiles and combining this enhanced feature set with machine learning and other complex analytical tools. Network webs and matrices visualize the data analysis. For example, much of the heterogeneity in staining patterns in M. tb is due to its thick, complex cell wall. There is increased staining and less variation in staining patterns when M. tb is treated with cell-wall targeting antibiotics compared with other classes of antibiotics. "With MorphEUS, we used the distribution of staining across a large number of bacilli to learn how each drug acts on M. tb," said Aldridge. "Similarly, we looked at staining intensity and the spread of that brightness across thousands of cells to identify more subtle patterns."MorphEUS can also determine if drugs have off-target or secondary effects that are otherwise hard to identify. Such complex mechanisms of drug action can be key in designing multidrug therapies."We expect that the success of MorphEUS in profiling drug action in an organism like M. tb with significant inherent heterogeneity and subtle cytological responsiveness will make it useful in other pathogens and cell types," said Aldridge, who is also a core faculty member of Tufts Center for Integrated Management of Antimicrobial Resistance, member of the immunology and molecular microbiology program faculties at Tufts Graduate School of Biomedical Sciences, and an adjunct associate professor at Tufts University's School of Engineering.MorphEUS, like all cytological profiling techniques, is data-driven and based on classification among a pool of other profiles. It requires multiple representative profiles from M. tb treated with compounds known to target the same broad cellular target. As the drug set expands, the accuracy and resolution of MorphEUS will improve. MorphEUS is also limited in its ability to identify target pathways of compounds with novel mechanisms of action that are unlike the other profiled drugs in the reference set.
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Biotechnology
| 2,020 |
July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723143638.htm
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Battling harmful algae blooms
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Throughout the world's oceans in global nutrient cycles, food chains, and climate, as well as increasingly in human-made industrial processes, a diverse set of planktonic microbes, such as algae, play an integral role. For nearly all of these planktonic microbes, however, little is known about them genetically beyond a few marker sequences, while their morphology, biological interactions, metabolism, and ecological significance remain a mystery.
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Algae produce half of the oxygen in earth's atmosphere and some forms of algae are used in industrial applications -- such as producing high omega-3 fatty acids for baby formula or being used for biofuels -- so there are many reasons a better understanding of algae could be beneficial. There is another side to algae, however, as some species can create harmful algal blooms (HABs), and those have been the focus of the research of University of Delaware's Kathryn Coyne.To help advance the understanding of the cellular instructions that underpin microbial life in the sea, Coyne joined more than 100 scientists from institutions around the globe to publish a compilation of methods, or protocols, for laboratory experiments that will help scientists gain a better understanding of the genetic underpinnings of marine algae as a resource article in the journal The work was funded by the Gordon and Betty Moore Foundation's Marine Microbiology Initiative. For her contribution to the collaboration, Coyne worked specifically with Heterosigma akashiwo, a species of algae that can produce HABs.One of the mysteries about H. akashiwo is that while some strains produce toxins that can kill fish, other strains are non-toxic."We don't have a clear understanding of what kind of toxin they produce. We just know that when there are blooms of this algae in some areas of the world, they are associated with massive fish kills," said Coyne, an associate professor of marine biosciences in UD's College of Earth, Ocean, and Environment (CEOE), and director of the Delaware Sea Grant program. "We also don't know why some strains produce toxins, or what stimulates this toxin production."Scientists often use genome manipulation to better understand how microbes respond to the environment or to identify genes that may be involved in a specific response, like production of toxins. Unlike other algal species that serve as models for genome manipulations, however, H. akashiwo doesn't have a cell wall, instead having only a thin membrane that holds the cell shape. Coyne explained that having a cell wall can be an impediment to genome manipulations and that these kinds of experiments usually entail some effort initially just to remove the cell wall or make it more porous.Because H. akashiwo lacked a cell wall, Coyne and her research team proposed that genome manipulation might be more straightforward with this species, and were able to demonstrate that using a couple of gene manipulation methods that have been successful on other model species."We created a piece of genetic material that could be introduced into Heterosigma cells that would make them resistant to a specific antibiotic," said Coyne. "If we were successful, we would be able to grow them in this antibiotic and cells that had incorporated the resistance gene would survive."Coyne worked with Deepak Nanjappa, a postdoctoral researcher in her lab who is also an author on the paper, as well as Pam Green and her lab members, Vinay Nagarajan, a postdoctoral researcher, and Monica Accerbi, a research associate in Green's lab at the Delaware Biotechnology Institute (DBI).Together, they tried a handful of methods and optimized those that were successful for Heterosigma. One method in particular was replicated successfully several times, showing that they were able to produce a genetically modified strain of Heterosigma. Using this approach, scientists can now probe the genome of Heterosigma akashiwo to gain a better understanding of how this species responds to environmental cues, or what genes are responsible for its toxicity.One of the aims of the project was to make all of the methods developed freely available so that scientists can take that information and use it in their own research."The Moore Foundation funded this project with the expectation that all of the methods developed during this research would be published," said Coyne. "Nothing is proprietary for this project, so we can share any of the protocols that we developed for Heterosigma."In addition, Coyne had another paper published in the scientific journal, Harmful Algae, that detailed her work with Yanfei Wang, a doctoral student in CEOE, studying the algicidal bacterium Shewanella and how it could be used to remediate HABs.Shewanella, which is an algicidal bacterium that has been isolated from the Delaware Inland Bays, is being developed as a biological control for HABs. It secretes water-soluble compounds that inhibit the growth of dinoflagellates, single-celled organisms that often produce toxins and contribute to HABs. Other research on this species of Shewanella shows that it has no negative effects on the growth of other species of algae, or on fish or shellfish. Since it was isolated from local waters, it may be considered an "environmentally neutral" solution to controlling HABs.In order to use Shewanella in the natural environment to control HABs, there first needs to be a method to safely deploy the bacterium in areas that are at risk for HABs.To move this HAB control solution closer to reality, Coyne and Wang immobilized Shewanella into several porous materials. Funded by Delaware Sea Grant, this research determined how well each material retained the bacteria over time, and whether the immobilized form of Shewanella was effective at controlling the growth of dinoflagellates.Unlike other HAB control approaches, such as application of toxic chemicals like copper sulfate, the advantage of using immobilized algicidal bacteria is the potential for continuous control of HABs without the need for frequent reapplication. The immobilized bacteria can also be removed when it is no longer needed.This research found that an alginate hydrogel was the most successful of the porous materials used in the study, and had the best retention of Shewanella cells.This research also showed that Shewanella cells immobilized in alginate beads were as effective as free bacteria in controlling the growth of the harmful species while at the same time having no negative impacts on a non-harmful control species.Overall, the study suggests that immobilized Shewanella may be used as an environmentally friendly approach to prevent or mitigate the blooms of harmful dinoflagellates and provides insight and directions for future studies.
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Biotechnology
| 2,020 |
July 23, 2020
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https://www.sciencedaily.com/releases/2020/07/200723143635.htm
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A never-before-seen cell state may explain cancer's ability to resist drugs
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Cancer's knack for developing resistance to chemotherapy has long been a major obstacle to achieving lasting remissions or cures. While tumors may shrink soon after chemotherapy, many times they eventually grow back.
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Scientists once thought that unique genetic mutations in tumors underlay this drug resistance. But more and more, they are casting their eyes on other, nongenetic changes in cancer cells to explain their adaptability.For example, one way that cancer cells can develop resistance is by changing their identity. A prostate cancer cell that is sensitive to hormone-blocking therapy might morph into a cell type that does not require the hormone for its growth.Rather than specific mutations driving them, identity changes like these come about through changes in gene expression -- cells turning specific genes on or off. As a result of these changes, a single tumor can become very different in its cellular makeup. This heterogeneity creates challenges for treatment, since a single drug is unlikely to work against so many different cell types.A new study from a team of researchers at the Sloan Kettering Institute, the Koch Institute for Integrative Cancer Research at MIT, and the Klarman Cell Observatory at the Broad Institute finds that this tumor heterogeneity can be traced to a common source: a particularly flexible cell state that is characteristic of a subset of cells in a tumor and can generate many other diverse cell types."The high-plasticity cell state is the starting point for much of the heterogeneity we see in tumors," says Tuomas Tammela, an Assistant Member in the Cancer Biology and Genetics Program at SKI and the corresponding author on the new paper, published July 23 in the journal Because this cell state produces nearly all the cellular heterogeneity that emerges in tumors, it is an attractive target for potential therapies.The particular tumors the researchers examined were lung cancer tumors growing in mice. Jason Chan, a physician-scientist doing a fellowship in the Tammela lab and one of the paper's lead authors, says finding this unusual cell state was a surprise."This highly plastic cell state is something completely new," he says. "When we saw it, we didn't know what it was because it was so different. It didn't look like normal lung cells where the cancer came from, and it didn't really look like lung cancer either. It had features of embryonic germ layer stem cells, cartilage stem cells, and even kidney cells, all mixed together."Nevertheless, he and his colleagues found these cells in every tumor they examined, which suggested that the cells were doing something biologically very important.The researchers identified these highly plastic cells by employing a relatively new laboratory technique called single cell RNA sequencing (scRNA-Seq). This technique allows researchers to take "snap shots" of individual cells' gene expression profiles -- revealing which genes are on or off. By performing scRNA-Seq on tumors as they grew over time, they were able to watch when and how different cell types emerged over the course of a tumor's evolution. From these data, the researchers were able to create a kind of map of which cells came from which other cells."The map contains major highways and little dirt roads," Dr. Tammela says. "The high-plasticity cell state that we identified sits right in the middle of the map. It has a lot of paths coming in, and it has even more paths coming out."This high-plasticity cell state emerged consistently in a tumor's evolution and persisted throughout its growth. In fact, Dr. Tammela says, "it was the only cell state that we found to be present in every single tumor."Plasticity -- the ability of a cell to give rise to other cells that take on different identities -- is a well-known feature of stem cells. Stem cells play important roles in embryonic development and in tissue repair. Many scientists think that cancers arise from specific cancer stem cells.But Dr. Tammela and colleagues do not think these high-plasticity cells are stem cells."When we compare the gene expression signature of these highly plastic cells to normal stems cells or known cancer stem cells, the signatures don't match at all. They look completely different," he says.And unlike stem cells, they're not there at the very beginning of a tumor's growth. They only emerge later.Many prior studies have looked for possible "resistance mutations" -- genetic changes that account for a tumor's ability to resist the effects of cancer drugs. While some have been found, more often the basis of resistance remains a mysterious. The new findings offer a potential solution to the mystery."Our model could explain why certain cancer cells are resistant to therapy and don't have a genetic basis for that resistance that we can identify," Dr. Chan says.Importantly, it's not all the cells in the tumor that are adapting, he explains. It's a subset of the cancer cells that are just more plastic, more malleable.By combining chemotherapy drugs with new medications that target these highly plastic cells, the researchers think it might be possible to avert the emergence of resistance and provide longer lasting remissions.
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Biotechnology
| 2,020 |
July 22, 2020
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https://www.sciencedaily.com/releases/2020/07/200722142727.htm
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Novel 'on-off' switch discovered in plant defenses
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To ensure survival, living organisms are equipped with defensive systems that detect threats and respond with effective counter measures.
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Plants are known to mount quick defenses against a variety of threats -- from attacking insects to invading pathogens. These intricate immune response mechanisms operate through a complex network that plant biologists have sought to untangle.Crucial to these defenses is the timing and duration of immune responses. Humans are equipped with a strong and rapid inflammation response that is essential to ward off disease, but chronic and persistent inflammation can be harmful to our health. Similarly, plants feature defenses that are timed for rapid and effective responses against pathogens, yet tightly controlled to avoid threatening the host organism.Keini Dressano, Alisa Huffaker and their colleagues at the University of California San Diego's Division of Biological Sciences have discovered a critical "on-off" switch in the plant immune response system. As described July 20 in their report published in "These findings have provided new insights into how the complex intricacies of plant immune responses are orchestrated to successfully fight off pathogens, and lay a path forward for improving plant disease resistance to ensure future food stability," said Huffaker, an assistant professor in the Section of Cell and Developmental Biology.The novel switch was found in Arabidopsis plants to control splicing of mRNA transcripts that encode signaling protein regulators of the plant immune response. To turn immune defenses on, the researchers say, a simple chemical modification of the RNA-binding protein reverses mRNA splicing that normally keeps immune responses deactivated. To turn the immune response back off, a second chemical modification of the RNA-binding protein returns mRNA splicing to "normal," and the immune response is back to being held in check."This work went beyond simply identifying a new regulator of plant immunity," said Huffaker, of the detailed mechanisms uncovered. "We discovered specific chemical modifications that control regulatory function, transcriptional targets of the regulator, differential splicing of the targets and precise effects of splicing on both target function and overall plant immune responses and disease resistance."
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Biotechnology
| 2,020 |
July 22, 2020
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https://www.sciencedaily.com/releases/2020/07/200722134922.htm
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How infectious bacteria can produce genetic variants among sibling cells
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In human reproduction, the genes of the mother and father are combined and mixed in countless variations. Their offspring can differ significantly from one another. However, bacteria multiply by simple cell division, so that the two daughter cells carry the same genetic material as the mother cell. A research team led by Dr. Simon Heilbronner from the Interfaculty Institute for Microbiology and Infection Medicine at the University of Tübingen and the German Center for Infection Research has recently discovered how infectious bacteria can produce genetic variants among sibling cells. Certain sections of the genetic material are doubled or multiplied. This gives the bacteria new capabilities that make it possible for them to influence the immune system of the host in their favour. The results of this study, published in the journal
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If bacteria multiply by simple division, clones are created. The cells all have the same genetic composition and the same properties. "However, the bacteria must remain flexible, because their environmental conditions are constantly changing. This is particularly true of pathogens that are struggling with the human immune system and need to deal with any antibiotics that may be administered if they are to survive," says Dr. Heilbronner. His team has shown how the bacterial pathogen Staphylococcus aureus causes inflammation, and how variants develop if gene exchange with other bacterial communities is not possible."We found that in Staphylococcus aureus, some parts of the genetic material may be available in the form of several exact copies. The number of such copies varies greatly between closely related bacteria," according to Dr. Heilbronner. Genetic mechanisms during cell division result in duplicates being able to multiply in the genetic material of the bacteria. "They can expand and shorten again, like an accordion. This results in a variety of daughter cells with different properties in the course of a few generations." Expanded genetic material leads to stronger protein production by the bacterial cell. "For example, if these proteins transport antibiotics out of the cell or influence the immune system, the bacteria may improve their chances of survival," according to the researcher.The Tübingen researchers have now shown that such genetic processes occur frequently in Staphylococcus aureus. "Administration of antibiotics can strengthen them. The pathogens now have better ways to respond to human immune cells." The team believes that these processes are important in the evolution of pathogens that are successful and therefore dangerous for humans. The team's findings will be used in the development of new forms of treatment by the Tübingen Cluster of Excellence "Controlling Microbes to Fight Infections."
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Biotechnology
| 2,020 |
July 22, 2020
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https://www.sciencedaily.com/releases/2020/07/200722134905.htm
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Researchers discover new pathways that could help treat RNA viruses
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Researchers at the University of New Hampshire have identified new pathways in an RNA-based virus where inhibitors, like medical treatments, unbind. The finding could be beneficial in understanding how these inhibitors react and potentially help develop a new generation of drugs to target viruses with high death rates, like HIV-1, Zika, Ebola and SARS-CoV2, the virus that causes COVID-19.
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"When we first started this research, we never anticipated that we'd be in the midst of a pandemic caused by an RNA virus," said Harish Vashisth, associate professor of chemical engineering. "But as these types of viruses emerge our findings will hopefully offer an enhanced understanding of how viral RNAs interact with inhibitors and be used to design better treatments."Similar to how humans are made up of a series of different chromosomes, known as DNA, many viruses have a genetic makeup of RNA molecules. These RNA-based genomes contain potential sites where inhibitors can attach and deactivate the virus. Part of the challenge in drug development can be fluctuations in the viral genome that may prevent the inhibitors from attaching.In their paper, recently published in the "We observed what are called rare base-flipping events involved in the inhibitor binding/unbinding process that provided the new details of the underlying mechanism of this process," said Vashisth. "Our hope is that this adds new possibilities to a field traditionally focused on static biomolecular structures and lead to new medications."
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Biotechnology
| 2,020 |
July 22, 2020
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https://www.sciencedaily.com/releases/2020/07/200722112659.htm
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New role for white blood cells in the developing brain
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Whether white blood cells can be found in the brain has been controversial, and their role there a complete mystery. In a study published in
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Like a highly fortified headquarters, our brain enjoys special protection from what is circulating in the rest of our body through the blood-brain barrier. This highly selective border makes sure that passage from the blood to the brain is tightly regulated.The blood-brain barrier also separates the brain from our body's immune system, which is why it has its own resident immune cells, called microglia, which trigger inflammation and tissue repair. Microglia arrive in the brain during embryonic development, and later on, the population becomes self-renewing.Yet, white blood cells -- which are part of our immune system -- have been found to play a role in different brain diseases, including multiple sclerosis, Alzheimer's and Parkinson's disease or stroke. Whether or not white blood cells can be found in healthy brains as well, and what they might be doing there, has been subject of intense debate. An interdisciplinary team of scientists led by Prof. Adrian Liston (Babraham Institute and VIB-KU Leuven) set out to find the answers."A misconception about white blood cells comes from their name," explains Dr Oliver Burton (Babraham Institute). "These 'immune cells' are not just present in the blood. They are constantly circulating around our body and enter all of our organs, including -- as it turns out -- the brain. We are only just starting to discover what white blood cells do when they leave the blood. This research indicates that they act as a go-between, transferring information from the rest of the body to the brain environment"The team quantified and characterised a small but distinct population of brain-resident T helper cells present in mouse and human brain tissue. T cells are a specific type of white blood cells specialized for scanning cell surfaces for evidence of infection and triggering an appropriate immune response. New technologies allowed the researchers to study the cells in great detail, including the processes by which circulating T cells entered the brain and began to develop the features of brain-resident T cells.Dr Carlos Roca (Babraham Institute): "Science is becoming increasingly multidisciplinary. Here, we didn't just bring in expertise from immunology, neuroscience and microbiology, but also from computer science and applied mathematics. New approaches for data analysis allow us to reach a much deeper level of understanding of the biology of the white blood cells we found in the brain."When T helper cells are absent from the brain, the scientists found that the resident immune cells -- microglia -- in the mouse brain remained suspended between a fetal and adult developmental state. Observationally, mice lacking brain T cells showed multiple changes in their behavior. The analysis points to an important role for brain-resident T cells in brain development. If T cells participate in normal brain development in mice, could the same be true in humans?"In mice, the wave of entry of immune cells at birth triggers a switch in brain development," says Liston. "Humans have a much longer gestation than mice though, and we don't know about the timing of immune cell entry into the brain. Does this occur before birth? Is it delayed until after birth? Did a change in timing of entry contribute to the evolution of enhanced cognitive capacity in humans?"The findings open up a whole new range of questions about how the brain and our immune system interact. "It has been really exciting to work on this project. We are learning so much about how our immune system can alter our brain, and how our brain modifies our immune system. The two are far more interconnected than we previously thought," says Dr Emanuela Pasciuto (VIB-KU Leuven).The study also brings in a connection with the gut microbiome, says Liston: "There are now multiple links between the bacteria in our gut and different neurological conditions, but without any convincing explanations for what connects them. We show that white blood cells are modified by gut bacteria, and then take that information with them into the brain. This could be the route by which our gut microbiome influences the brain."Taken together, the results contribute towards the increasing recognition of the role of immune cells in the brain and shed new light on its involvement in a range of neurological diseases.
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Biotechnology
| 2,020 |
July 22, 2020
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https://www.sciencedaily.com/releases/2020/07/200722093450.htm
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Cells communicate by doing the 'wave'
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Cells work around the clock to deliver, maintain, and control every aspect of life. And just as with humans, communication is a key to their success.
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Every essential biological process requires some form of communication among cells, not only with their immediate neighbors but also to those significantly farther away. Current understanding is that this information exchange relies on the diffusion of signaling molecules or on cell-to-cell relays.Publishing in the journal "Mechanical and biochemical signals in cells fundamentally control everything from homeostasis, development, to diseases," explains Tsuyoshi Hirashima, leader of the study."We knew from past experiments how vital the ERK pathway is in cell activity, but the mechanism of how it can propagate in a collection of cells was incomplete."MAPK/ERK is so fundamental that it exists in all cells, controlling a wide range of actions from growth and development to eventual cell death. The pathway is activated when a receptor protein on the cell surface binds with a signaling molecule, resulting in a cascade of proteins and reactions spreading throughout the cell's interior.Employing a live imaging technique that can visualize an individual cell's active ERK pathway, the team began observing the effects of cell movement. What they found was unexpected: when a cell began to extend itself, ERK activity increased, causing the cell to contract."Cells are tightly connected and packed together, so when one starts contracting from ERK activation, it pulls in its neighbors," elaborates Hirashima. This then caused surrounding cells to extend, activating their ERK, resulting in contractions that lead to a kind of tug-of-war propagating into colony movement."Researchers had previously proposed that cells extend when ERK is activated, so our results came as quite a surprise."The team incorporated these observations into a mathematical model, combining mechano-chemical regulations with quantitative parameters. The output demonstrated consistency with experimental data."Our work clearly shows that the ERK-mediated mechano-chemical feedback system generates complicated multicellular patterns," concludes Hirashima."This will provide a new basis for understanding many biological processes, including tissue repair and tumor metastasis."
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Biotechnology
| 2,020 |
July 21, 2020
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https://www.sciencedaily.com/releases/2020/07/200721162446.htm
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Lab-made virus mimics COVID-19 virus
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Airborne and potentially deadly, the virus that causes COVID-19 can only be studied safely under high-level biosafety conditions. Scientists handling the infectious virus must wear full-body biohazard suits with pressurized respirators, and work inside laboratories with multiple containment levels and specialized ventilation systems. While necessary to protect laboratory workers, these safety precautions slow down efforts to find drugs and vaccines for COVID-19 since many scientists lack access to the required biosafety facilities.
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To help remedy that, researchers at Washington University School of Medicine in St. Louis have developed a hybrid virus that will enable more scientists to enter the fight against the pandemic. The researchers genetically modified a mild virus by swapping one of its genes for one from SARS-CoV-2, the virus that causes COVID-19. The resulting hybrid virus infects cells and is recognized by antibodies just like SARS-CoV-2, but can be handled under ordinary laboratory safety conditions.The study is available online in "I've never had this many requests for a scientific material in such a short period of time," said co-senior author Sean Whelan, PhD, the Marvin A. Brennecke Distinguished Professor and head of the Department of Molecular Microbiology. "We've distributed the virus to researchers in Argentina, Brazil, Mexico, Canada and, of course, all over the U.S. We have requests pending from the U.K. and Germany. Even before we published, people heard that we were working on this and started requesting the material."To create a model of SARS-CoV-2 that would be safer to handle, Whelan and colleagues -- including co-senior author Michael S. Diamond, MD, PhD, the Herbert S. Gasser Professor of Medicine, and co-first authors Brett Case, PhD, a postdoctoral researcher in Diamond's laboratory, and Paul W. Rothlauf, a graduate student in Whelan's laboratory -- started with vesicular stomatitis virus (VSV). This virus is a workhorse of virology labs because it is fairly innocuous and easy to manipulate genetically. Primarily a virus of cattle, horses and pigs, VSV occasionally infects people, causing a mild flu-like illness that lasts three to five days.Viruses have proteins on their surfaces that they use to latch onto and infect cells. The researchers removed VSV's surface-protein gene and replaced it with the one from SARS-CoV-2, known as spike. The switch created a new virus that targets cells like SARS-CoV-2 but lacks the other genes needed to cause severe disease. They dubbed the hybrid virus VSV-SARS-CoV-2.Using serum from COVID-19 survivors and purified antibodies, the researchers showed that the hybrid virus was recognized by antibodies very much like a real SARS-CoV-2 virus that came from a COVID-19 patient. Antibodies or sera that prevented the hybrid virus from infecting cells also blocked the real SARS-CoV-2 virus from doing so; antibodies or sera that failed to stop the hybrid virus also failed to deter the real SARS-CoV-2. In addition, a decoy molecule was equally effective at misdirecting both viruses and preventing them from infecting cells."Humans certainly develop antibodies against other SARS-CoV-2 proteins, but it's the antibodies against spike that seem to be most important for protection," Whelan said. "So as long as a virus has the spike protein, it looks to the human immune system like SARS-CoV-2, for all intents and purposes."The hybrid virus could help scientists evaluate a range of antibody-based preventives and treatments for COVID-19. The virus could be used to assess whether an experimental vaccine elicits neutralizing antibodies, to measure whether a COVID-19 survivor carries enough neutralizing antibodies to donate plasma to COVID-19 patients, or to identify antibodies with the potential to be developed into antiviral drugs."One of the problems in evaluating neutralizing antibodies is that a lot of these tests require a BSL-3 facility, and most clinical labs and companies don't have BSL-3 facilities," said Diamond, who is also a professor of molecular microbiology, and of pathology and immunology. "With this surrogate virus, you can take serum, plasma or antibodies and do high-throughput analyses at BSL-2 levels, which every lab has, without a risk of getting infected. And we know that it correlates almost perfectly with the data we get from bona fide infectious SARS-CoV-2."Since the hybrid virus looks like SARS-CoV-2 to the immune system but does not cause severe disease, it is a potential vaccine candidate, Diamond added. He, Whelan and colleagues are conducting animal studies to evaluate the possibility.
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Biotechnology
| 2,020 |
July 21, 2020
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https://www.sciencedaily.com/releases/2020/07/200721132731.htm
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Silver-plated gold nanostars detect early cancer biomarkers
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Biomedical engineers at Duke University have engineered a method for simultaneously detecting the presence of multiple specific microRNAs in RNA extracted from tissue samples without the need for labeling or target amplification. The technique could be used to identify early biomarkers of cancer and other diseases without the need for the elaborate, time-consuming, expensive processes and special laboratory equipment required by current technologies.
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The results appeared online on May 4 in the journal "The general research focus in my lab has been on the early detection of diseases in people before they even know they're sick," said Tuan Vo-Dinh, director of the Fitzpatrick Institute for Photonics and the R. Eugene and Susie E. Goodson Distinguished Professor of Biomedical Engineering at Duke. "And to do that, you need to be able to go upstream, at the genomic level, to look at biomarkers like microRNA."MicroRNAs are short RNA molecules that bind to messenger RNA and stop them from delivering their instructions to the body's protein-producing machines. This could effectively silence certain sections of DNA or regulate gene expression, thus altering the behaviors of certain biological functions. More than 2000 microRNAs have been discovered in humans that affect development, differentiation, growth and metabolism.As researchers have discovered and learned more about these tiny genetic packages, many microRNAs have been linked to the misregulation of biological functions, resulting in diseases ranging from brain tumors to Alzheimer's. These discoveries have led to an increasing interest in using microRNAs as disease biomarkers and therapeutic targets. Due to the very small amounts of miRNAs present in bodily samples, traditional methods of studying them require genetic-amplification processes such as quantitative reverse transcription PCR (qRT-PCR) and RNA sequencing.While these technologies perform admirably in well-equipped laboratories and research studies that can take months or years, they aren't as well-suited for fast diagnostic results at the clinic or out in the field. To try to bridge this gap in applicability, Vo-Dinh and his colleagues are turning to silver-plated gold nanostars."Gold nanostars have multiple spikes that can act as lighting rods for enhancing electromagnetic waves, which is a unique feature of the particle's shape," said Vo-Dinh, who also holds a faculty appointment in Duke chemistry. "Our tiny nanosensors, called 'inverse molecular sentinels,' take advantage of this ability to create clear signals of the presence of multiple microRNAs."While the name is a mouthful, the basic idea of the nanosensor design is to get a label molecule to move very close to the star's spikes when a specific stretch of target RNA is recognized and captured. When a laser is then shined on the triggered sensor, the lightning rod effect of the nanostar tips causes the label molecule to shine extremely brightly, signaling the capture of the target RNA.The researchers set this trigger by tethering a label molecule to one of the nanostar's points with a stretch of DNA. Although it's built to curl in on itself in a loop, the DNA is held open by an RNA "spacer" that is tailored to bind with the target microRNA being tested for. When that microRNA comes by, it sticks to and removes the spacer, allowing the DNA to curl in on itself in a loop and bring the label molecule in close contact with the nanostar.Under laser excitation, that label emits a light called a Raman signal, which is generally very weak. But the shape of the nanostars -- and a coupling effect of separate reactions caused by the gold nanostars and silver coating -- amplifies Raman signals several million-folds, making them easier to detect."The Raman signals of label molecules exhibit sharp peaks with very specific colors like spectral fingerprints that make them easily distinguished from one another when detected," said Vo-Dinh. "Thus we can actually design different sensors for different microRNAs on nanostars, each with label molecules exhibiting their own specific spectral fingerprints. And because the signal is so strong, we can detect each one of these fingerprints independently of each other."In this clinical study, Vo-Dinh and this team collaborated with Katherine Garman, associate professor of medicine, and colleagues at the Duke Cancer Institute to use the new nanosensor platform to demonstrate that they can detect miR-21, a specific microRNA associated with very early stages of esophageal cancer, just as well as other more elaborate state-of-the-art methods. In this case, the use of miR-21 alone is enough to distinguish healthy tissue samples from cancerous samples. For other diseases, however, it might be necessary to detect several other microRNAs to get a reliable diagnosis, which is exactly why the researchers are so excited by the general applicability of their inverse molecular sentinel nanobiosensors."Usually three or four genetic biomarkers might be sufficient to get a good diagnosis, and these types of biomarkers can unmistakably identify each disease," said Vo-Dinh. "That's why we're encouraged by just how strong of a signal our nanostars create without the need of time-consuming target amplification. Our method could provide a diagnostic alternative to histopathology and PCR, thus simplifying the testing process for cancer diagnostics."For more than three years, Vo-Dinh has worked with his colleagues and Duke's Office of Licensing and Ventures to patent his nanostar-based biosensors. With that patent recently awarded, the researchers are excited to begin testing the limits of their technology's abilities and exploring technology transfer possibilities with the private sector."Following these encouraging results, we are now very excited to apply this technology to detect colon cancer directly from blood samples in a new NIH-funded project," said Vo-Dinh. "It's very challenging to detect early biomarkers of cancer directly in the blood before a tumor even forms, but we have high hopes."
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Biotechnology
| 2,020 |
July 21, 2020
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https://www.sciencedaily.com/releases/2020/07/200721102209.htm
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Skin stem cells shuffle sugars as they age
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Age shows nowhere better than on the skin. The ravages of time on skin and the epidermal stem cells that differentiate to replenish its outer layer have been hypothesized, but there has been no method to evaluate their aging at the molecular level. Now, researchers at the University of Tsukuba and the National Institute of Advanced Industrial Science and Technology (AIST) have revealed that changes in the complex sugars called glycans that coat the surface of epidermal stem cells can serve as a potential biological marker of aging.
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Skin is the largest human organ and a vital barrier against infection and fluid loss. Aging impairs environmental defenses and wound healing, while increasing hair loss and cancer risk. A key process underlying epidermal function in health and disease is cellular glycosylation that mediates cell-cell interactions and cell-matrix adhesions. Glycosylation involves attaching glycans to proteins; the profile of all glycans on and in a cell -- collectively 'the cell glycome' -- could reflect its functional scope and serve as an index of its age.The researchers first isolated epidermal stem cells from the skin of young and old laboratory mice, including both hair follicle cells and interfollicular epidermal cells. These cells underwent glycan profiling using the lectin microarray platform; this technique uses lectins -- proteins that bind specific glycans -- and enables glycome analysis even for cells sparsely dispersed in tissues."Our results clearly showed that high mannose-type N-glycans are replaced by a2-3/6 sialylated complex type N-glycans in older epidermal stem cells," senior author, Professor Hiromi Yanagisawa, explains. "We followed this with gene expression analysis; this revealed up-regulation of a glycosylation-related mannosidase and two sialyltransferase genes, suggesting that this 'glycome shift' may be mediated by age-modulated glycosyltransferase and glycosidase expression."Finally, to check whether the glycan changes were the cause or merely the result of aging, the research team overexpressed the up-regulated glycogenes in primary epidermal mouse keratinocytes Professor Aiko Sada, currently Principal Investigator at Kumamoto University, and Professor Hiroaki Tateno at AIST, co-corresponding authors, explain the implications of their results. "Our work is broadly targeted at investigating stem cell dysfunction specifically in aging skin. Future advances may help manage skin disorders at the stem cell level, including age-related degenerative changes, impaired wound healing and cancer."
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Biotechnology
| 2,020 |
July 21, 2020
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https://www.sciencedaily.com/releases/2020/07/200721102156.htm
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Artificial cells produce parts of viruses for safe studies
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Scientists searching for better diagnostic tests, drugs or vaccines against a virus must all begin by deciphering the structure of that virus. And when the virus in question is highly pathogenic, investigating, testing or developing these can be quite dangerous. Prof. Roy Bar-Ziv, Staff Scientist Dr. Shirley Shulman Daube, Dr. Ohad Vonshak, a former research student in Bar-Ziv's lab, and current research student Yiftach Divon have an original solution to this obstacle. They demonstrated the production of viral parts within artificial cells.
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The cells are micrometer-sized compartments etched into a silicon chip. At the bottom of each compartment, the scientists affixed DNA strands, packing them densely. The edges of the artificial cells were carpeted with receptors that can capture the proteins produced within the cells. To begin with, the scientists flooded their cells with everything needed to make proteins -- molecules and enzymes needed to read the DNA information and translate it into proteins. Then, with no further human intervention, the receptor carpet trapped one of the proteins produced in the bottoms of the cells, with the rest of the viral proteins binding to one another, producing structures that the scientists had earlier "programmed" into the system. In this case, they created assorted small parts of a virus that infects bacteria (a bacteriophage)."We discovered," says Bar-Ziv, "that we can control the assembly process -- both the efficiency and the final products -- through the design of the artificial cells. This included the cells' geometric structure, and the placement and organization of the genes. These all determine which proteins will be produced and, down the line, what will be made from these proteins once they are assembled."Vonshak adds: "Since these are miniaturized artificial cells, we can place a great many of them on a single chip. We can alter the design of various cells, so that diverse tasks are performed at different locations on the same chip."The features of the system developed at the Weizmann Institute -- including the ability to produce different small parts of a single virus at once, could give scientists around the globe a new tool for evaluating tests, drugs and vaccines against that virus. Adds Divon: "And because the artificial parts -- even if they faithfully reproduced parts of the virus -- do not include the use of actual viruses, they would be especially safe from beginning to end." "Another possible application," says Shulman Daube, "might be the development of a chip that could rapidly and efficiently conduct thousands of medical tests all at once."Participating in this research were Stefanie Förste, Dr. Sophia Rudorf and Prof. Reinhard Lipowsky from the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany, and David Garenne and Prof. Vincent Noireaux from the University of Minnesota. The research was published today in
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Biotechnology
| 2,020 |
July 20, 2020
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https://www.sciencedaily.com/releases/2020/07/200720145911.htm
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COVID-19 replicating RNA vaccine has robust response in nonhuman primates
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A replicating RNA vaccine, formulated with a lipid-based nanoparticle emulsion that goes by the acronym LION, produces antibodies against the COVID-19 coronavirus in mice and primates with a single immunization. These antibodies potently neutralize the virus.
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The effects occurred within two weeks after administration through injection into a muscle. The level of antibodies generated was comparable to those in people who are recovering from COVID-19.The vaccine induced coronavirus-neutralizing antibodies robustly in both younger and older mice. This hopeful finding was well-received by the researchers, because of the concern that the elderly are less likely to respond to vaccination due to their aging immune systems.Vulnerability to severe COVID-19 in older people increases with age; a vaccination suitable for this high-risk population is a key goal of the scientists.This vaccine design, as shown in lab studies, is designed to avoid immune responses that could enhance a respiratory disease induced by the coronavirus. Instead, it directs the immune response toward more protective antiviral measures. In addition to antibody production that can block the infection, the vaccine induces T cells, a type of white blood cell that provides a second line of defense if antibodies don't completely block the infection.The methods and results of animal tests of the replicating RNA coronavirus vaccine candidate vaccine are published July 20 in The lead author of the paper is Jesse H. Erasmus, a Washington Research Foundation Postdoctoral Fellow in the laboratory of Deborah Heydenberg Fuller. She is a professor of microbiology at the University of Washington School of Medicine and division chief of Infectious Diseases and Translational Medicine at the Washington National Primate Research Center.As COVID-19 continues to spread, the discovery and widespread distribution of safe and effective vaccines are essential for slamming down the pandemic. Scores of vaccine candidates are in various stages of testing around the world, from preclinical studies to human trials."A vaccine that can stop COVID-19," Fuller wrote, "will ideally induce protective immunity after only a single immunization, avoid immune responses that could exacerbate virus-induced pathology, be amenable to rapid and cost-effective scale-up and manufacturing, and be capable of inducing immunity in all populations including the elderly who typically respond poorly to vaccines.""That's a tall order," she added. She sees conventional nucleic acid vaccines as promising, but at least two immunizations are needed to instill immunity in people.Most DNA vaccines require high doses to achieve protective levels of immunity in humans. Traditional messenger RNA vaccines formulated with lipid nanoparticles to increase their effectiveness may face obstacles of mass-production and shelf life.To try to overcome these limitations, the labs of Fuller and her collaborators at the National Institutes of Health Rocky Mountain Laboratories and HDT Bio Corp. have developed a replicating RNA version of a coronavirus vaccine.Replicating RNA vaccines for other infectious diseases and cancers are in the pipeline at several institutions.Replicating RNA expresses a greater amount of protein, and also triggers a virus-sensing stress response that encourages other immune activation.In the case of the COVID-19 vaccine candidate, the RNA enters cells and instructs them to produce proteins that teach the body to recognize coronaviruses and attack them with antibodies and T cells.This blockade might keep the viruses from fusing to cells and injecting their genetic code for commandeering cellular activities.These antibodies induced by the vaccine provide protection by interfering with the protein machinery on the spikes of the coronavirus.This replicating RNA vaccine contains the novel Lipid InOrganic Nanoparticle (LION) developed by Seattle-based biotechnology company HDT Bio Corp."We are pleased with the collaboration with UW to move our RNA vaccine platform forward," said the company's CEO, Steve Reed.Amit P. Khandhar, the lead formulation developer, added, "RNA molecules are highly susceptible to degradation by enzymes. LION is a next-generation nanoparticle formulation that protects the RNA molecule and enables in vivo delivery of the vaccine after a simple mixing step at the pharmacy."The nanoparticle enhances the vaccine's ability to provoke the desired immune reaction, and also its stability. This vaccine is stable at room temperature for at least one week. Its components would allow it to be rapidly manufactured in large quantities, should it prove safe and effective in human trials.The scientists anticipate that lower and fewer doses would need to be made to immunize a population.A key differentiating factor between LION and the lipid nanoparticle delivery vehicle used in other mRNA COVID-19 vaccines is its ability to be formulated with mRNA by simple mixing at the bedside.The two-vial approach enabled by LION allows for manufacturing of the formulation independently from the mRNA component.The research team is working to advance the vaccine to Phase 1 testing in people, in which it would be introduced into a small group of healthy volunteers to gather preliminary data on whether it is safe and generates the desired immune response.HDT is advancing the replicon RNA with LION vaccine toward clinical development under the name HDT-301.
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Biotechnology
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July 20, 2020
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https://www.sciencedaily.com/releases/2020/07/200720112222.htm
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Cheese making relies on milk proteins to form structure
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Cheese production relies on coagulation of milk proteins into a gel matrix after addition of rennet. Milk that does not coagulate (NC) under optimal conditions affects the manufacturing process, requiring a longer processing time and lowering the cheese yield, which, in turn, has economic impact. In an article appearing in the
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The authors of this study analyzed protein composition in NC and coagulating milk samples from 616 Swedish Red cows. They reported that the relative concentrations, genetic variants, and posttranslational modifications of the proteins all contribute to whether rennet could induce coagulation in each sample. The NC milk had higher relative concentrations of ?-lactalbumin and ß-casein and lower relative concentrations of ß-lactoglobulin and ?-casein when compared with coagulating milk."The non-coagulating characteristics of milk relate to protein composition and genetic variants of the milk proteins," said first author Kajsa Nilsson, PhD, Lund University, Lund, Sweden. "Roughly 18 percent of Swedish Red cows produce noncoagulating milk, which is a high prevalence. Cheese-producing dairies would benefit from eliminating the NC milk from their processes, and breeding could reduce or remove this milk trait," said Nilsson.These results can be used to further understand the mechanisms behind NC milk, develop breeding strategies to reduce this milk trait, and limit use of NC milk for cheese processing.
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Biotechnology
| 2,020 |
July 20, 2020
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https://www.sciencedaily.com/releases/2020/07/200720103324.htm
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Specialized cellular compartments discovered in bacteria
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Researchers at McGill University have discovered bacterial organelles involved in gene expression, suggesting that bacteria may not be as simple as once thought. This finding could offer new targets for the development of new antibiotics.
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The study, published in Just like the human body is made up of organs that perform specialized functions, individual cells contain specialized compartments -- such as energy-producing mitochondria -- called organelles. Complex cells contain many different organelles, most of which are enclosed by a membrane that holds them together. Because bacteria do not have membrane-bound organelles, they were assumed to lack them altogether.Stephanie Weber, an assistant professor in McGill's Department of Biology, and her team are the first to show that bacteria do in fact have such specialized compartments."Our paper provides evidence for a bacterial organelle that is held together by "sticky" proteins rather than a membrane," says Weber, who is the study's senior author.The bacterial organelles described in the study are formed in a similar fashion to membraneless cellular compartments found in more complex eukaryotic cells (cells with a nucleus) through a process called phase separation, the same phenomenon that causes oil and vinegar to separate in salad dressing."This is the first direct evidence of phase separation in bacteria, so it may be a universal process in all cell types, and could even have been involved in the origin of life," explains Weber.Because of the small size of the bacterial cells they were studying, Weber's team used an imaging technique -- photo activated localization microscopy -- to track the organelle-forming proteins.Weber is now trying to understand exactly how the proteins assemble into organelles. Because they're involved in the first steps of gene expression -- transcription -- she believes they might also be an interesting target for the development of a new generation of antibiotic drugs, which are urgently needed to combat drug resistance.
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Biotechnology
| 2,020 |
July 20, 2020
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https://www.sciencedaily.com/releases/2020/07/200720102050.htm
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How smart, ultrathin nanosheets go fishing for proteins
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An interdisciplinary team from Frankfurt and Jena has developed a kind of bait with which to fish protein complexes out of mixtures. Thanks to this "bait," the desired protein is available much faster for further examination in the electron microscope. The research team has christened this innovative layer of ultrathin molecular carbon the "smart nanosheet." With the help of this new development, diseases and their treatment with drugs can be better understood, for example.
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"With our process, new types of proteins can be isolated from mixtures and characterized within a week," explains Daniel Rhinow from the Max Planck Institute of Biophysics in Frankfurt. "To date, just the isolation of the proteins was often part of a doctorate lasting several years." Together with Andreas Terfort (Goethe University) and Andrey Turchanin (Friedrich Schiller University Jena), the idea evolved a few years ago of fishing the desired proteins directly out of mixtures by equipping a nanosheet with recognition sites onto which the target protein bonds. The researchers have now succeeded in making proteins directly available for examination using electron cryo-microscopy through a "smart nanosheet."Electron cryo-microscopy is based on the shock-freezing of a sample at temperatures under -150 °C. In this process, the protein maintains its structure, no interfering fixing and coloring agents are needed, and the electrons can easily irradiate the frozen object. The result is high-resolution, three-dimensional images of the tiniest structures -- for example of viruses and DNA, almost down to the scale of a hydrogen atom.In preparation, the proteins are shock-frozen in an extremely thin layer of water on a minute metal grid. Previously, samples had to be cleaned in a complex procedure -- often involving an extensive loss of material -- prior to their examination in an electron microscope. The electron microscopy procedure is only successful if just one type of protein is bound in the water layer.The research group led by Turchanin is now using nanosheets that are merely one nanometer thick and composed of a cross-linked molecular self-assembled monolayer. Terfort's group coats this nanosheet with a gelling agent as the basis for the thin film of water needed for freezing. The researchers then attach recognition sites (a special nitrilotriacetic acid group with nickel ions) to it. The team led by Rhinow uses the "smart nanosheets" treated in this way to fish proteins out of a mixture. These were marked beforehand with a histidine chain with which they bond to the recognition sites; all other interfering particles can be rinsed off. The nanosheet with the bound protein can then be examined directly with the electron microscope."Our smart nanosheets are particularly efficient because the hydrogel layer stabilizes the thin film of water required and at the same time suppresses the non-specific binding of interfering particles," explains Julian Scherr of Goethe University. "In this way, molecular structural biology can now examine protein structures and functions much faster." The knowledge gained from this can be used, for example, to better understand diseases and their treatment with drugs.The team has patented the new nanosheets and additionally already found a manufacturer who will bring this useful tool onto the market.
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Biotechnology
| 2,020 |
July 20, 2020
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https://www.sciencedaily.com/releases/2020/07/200720093234.htm
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Portable DNA device can detect tree pests in under two hours
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Asian gypsy moths feed on a wide range of important plants and trees. White pine blister rust can kill young trees in only a couple of years. But it's not always easy to detect the presence of these destructive species just by looking at spots and bumps on a tree, or on the exterior of a cargo ship.
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Now a new rapid DNA detection method developed at the University of British Columbia can identify these pests and pathogens in less than two hours, without using complicated processes or chemicals -- a substantial time savings compared to the several days it currently takes to send samples to a lab for testing."Sometimes, a spot is just a spot," explains forestry professor Richard Hamelin, who designed the system with collaborators from UBC, Natural Resources Canada and the Canadian Food Inspection Agency. "Other times, it's a deadly fungus or an exotic bug that has hitched a ride on a shipping container and has the potential to decimate local parks, forests and farms. So you want to know as soon as possible what you're looking at, so that you can collect more samples to assess the extent of the invasion or begin to formulate a plan of action."Hamelin's research focuses on using genomics to design better detection and monitoring methods for invasive pests and pathogens that threaten forests. For almost 25 years, he's been looking for a fast, accurate, inexpensive DNA test that can be performed even in places, like forests, without fast Internet or steady power supply.He may have found it. The method, demonstrated in a preview last year for forestry policymakers in Ottawa, is straightforward. Tiny samples like parts of leaves or branches, or insect parts like wings and antennae, are dropped into a tube and popped into a small, battery-powered device (the Franklin thermo cycler, made by Philadelphia-based Biomeme). The device checks to see if these DNA fragments match the genomic material of the target species and generates a signal that can be visualized on a paired smartphone."With this system, we can tell with nearly 100 per cent accuracy if it is a match or not, if we're looking at a threatening invasive species or one that's benign," said Hamelin. "We can analyze up to nine samples from the same or different species at a time, and it's all lightweight enough -- the thermocycler weighs only 1.3 kilos -- to fit into your backpack with room to spare."The method relies on PCR testing, the method that is currently also the gold standard for COVID-19. PCR testing effectively analyzes even tiny amounts of DNA by amplifying (through applying heating and cooling cycles) a portion of the genetic material to a level where it can be detected.Hamelin's research was supported by Genome Canada, Genome BC and Genome Quebec and published in "Our forestry, agriculture and horticulture are vital industries contributing billions of dollars to Canada's economy so it's essential that we protect them from their enemies," added Hamelin. "With early detection and steady surveillance, we can ensure that potential problems are nipped, so to speak, in the bud."
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Biotechnology
| 2,020 |
July 16, 2020
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https://www.sciencedaily.com/releases/2020/07/200716220933.htm
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Pioneering method reveals dynamic structure in HIV
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Viruses are scary. They invade our cells like invisible armies, and each type brings its own strategy of attack. While viruses devastate communities of humans and animals, scientists scramble to fight back. Many utilize electron microscopy, a tool that can "see" what individual molecules in the virus are doing. Yet even the most sophisticated technology requires that the sample be frozen and immobilized to get the highest resolution.
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Now, physicists from the University of Utah have pioneered a way of imaging virus-like particles in real time, at room temperature, with impressive resolution. In a new study, the method reveals that the lattice, which forms the major structural component of the human immunodeficiency virus (HIV), is dynamic. The discovery of a diffusing lattice made from Gag and GagPol proteins, long considered to be completely static, opens up potential new therapies.When HIV particles bud from an infected cell, the viruses experience a lag time before they become infectious. Protease, an enzyme that is embedded as a half-molecule in GagPol proteins, must bond to other similar molecules in a process called dimerization. This triggers the viral maturation that leads to infectious particles. No one knows how these half protease molecules find each other and dimerize, but it may have to do with the rearrangement of the lattice formed by Gag and GagPol proteins that lay just inside of the viral envelope. Gag is the major structural protein and has been shown to be enough to assemble virus-like particles. Gag molecules form a lattice hexagonal structure that intertwines with itself with miniscule gaps interspersed. The new method showed that the Gag protein lattice is not a static one."This method is one step ahead by using microscopy that traditionally only gives static information. In addition to new microscopy methods, we used a mathematical model and biochemical experiments to verify the lattice dynamics," said lead author Ipsita Saha, graduate research assistant at the U's Department of Physics & Astronomy. "Apart from the virus, a major implication of the method is that you can see how molecules move around in a cell. You can study any biomedical structure with this."The paper published in The scientists weren't looking for dynamic structures at first -- they just wanted to study the Gag protein lattice. Saha led the two year effort to "hack" microscopy techniques to be able to study virus particles at room temperature to observe their behavior in real life. The scale of the virus is miniscule -- about 120 nanometers in diameter -- so Saha used interferometric photoactivated localization microscopy (iPALM).First, Saha tagged the Gag with a fluorescent protein called Dendra2 and produced virus-like particles of the resulting Gag-Dendra2 proteins. These virus-like particles are the same as HIV particles, but made only of the Gag-Dendra2 protein lattice structure. Saha showed that the resulting Gag-Dendra2 proteins assembled the virus-like particles the same way as virus-like particle made up regular Gag proteins. The fluorescent attachment allowed iPALM to image the particle with a 10 nanometer resolution. The scientists found that each immobilized virus-like particle incorporated 1400 to 2400 Gag-Dendra2 proteins arranged in a hexagonal lattice. When they used the iPALM data to reconstruct a time-lapse image of the lattice, it appeared that the lattice of Gag-Dendra2 were not static over time. To make sure, they independently verified it in two ways: mathematically and biochemically.First, they divided up the protein lattice into uniform separate segments. Using a correlation analysis, they tested how each segment correlated with itself over time, from 10 to 100 seconds. If each segment continued to correlate with itself, the proteins were stationary. If they lost correlation, the proteins had diffused. They found that over time, the proteins were quite dynamic.The second way they verified the dynamic lattice was biochemically. For this experiment, they created virus-like particles whose lattice consisted of 80% of Gag wild type proteins, 10% of Gag tagged with SNAP, and 10% of gag tagged with Halo. SNAP and Halo are proteins that can bind a linker which binds them together forever. The idea was to identify whether the molecules in the protein lattice stayed stationary, or if they migrated positions."The Gag-proteins assemble themselves randomly. The SNAP and Halo molecules could be anywhere within the lattice -- some may be close to one another, and some will be far away," Saha said. "If the lattice changes, there's a chance that the molecules come close to one another."Saha introduced a molecule called Haxs8 into the virus-like particles. Haxs8 is a dimerizer -- a molecule that covalently binds SNAP and Halo proteins when they are within binding radius of one another. If SNAP or Halo molecules move next to each other, they'll produce a dimerized complex. She tracked these dimerized complex concentrations over time. If the concentration changed, it would indicate that new pairs of molecules found each other. If the concentration decreased, it would indicate the proteins broke apart. Either way, it would indicate that movement had taken place. They found that over time, the percentage of the dimerized complex increased; HALO and SNAP Gag proteins were moving all over the lattice and coming together over time.This is the first study to show that the protein lattice structure of an enveloped virus is dynamic. This new tool will be important to better understand the changes that occur within the lattice as new virus particles go from immaturity to dangerously infectious."What are the molecular mechanisms that lead to infection? It opens up a new line of study," said Saha. "If you can figure out that process, maybe you can do something to prevent them from finding each other, like a type of drug that would stop the virus in its tracks."
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Biotechnology
| 2,020 |
July 16, 2020
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https://www.sciencedaily.com/releases/2020/07/200716163035.htm
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Dangerous parasite controls host cell to spread around body
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Researchers at Indiana University School of Medicine have discovered new information about how a dangerous parasite takes control of a patient's cells as it spreads throughout their body, an important finding that could help in the development of new drugs to treat this infection.
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"The parasite essentially hijacks these cells, using them as vehicles to get to various organ systems, including the brain," said Leonardo Augusto, PhD, a postdoctoral fellow in the Department of Pharmacology and Toxicology and lead author on the National Institutes of Health-funded study, which was recently published in Toxoplasma gondii infects up to one-third of the world's population. People typically become infected with it through exposure to cat feces, which is where it goes through its reproductive phases, or consumption of contaminated food and water. The parasite causes life-threatening issues in some patients because of its ability to disseminate to the brain. In the brain and other tissues, the parasite persists as a latent cyst, waiting to reactivate if immunity should wane, such as what happens in HIV/AIDS patients."One of the key problems in battling an infection like Toxoplasma is controlling its spread to other parts of the body," Augusto said. "Upon ingestion of the parasite, it makes its way into immune cells and causes them to move -- a behavior called hypermigratory activity. How these parasites cause their infected cells to start migrating is largely unknown."The team's new research is shedding light on this important clinical question, discovering that the parasite trips an alarm system in its host cell that leads to the activation of a protein called IRE1. IRE1 helps the cell cope with stress, which can involve getting it to move to a different location. In cells infected with Toxoplasma, IRE1 connects to the cytoskeleton, a network of structural proteins that gives the cell its shape and coordinates movement. By engaging this network through IRE1, Toxoplasma takes the wheel and causes hypermigration."When we infected host cells that were depleted of IRE1, they could no longer move," Augusto said. "These cells were greatly impaired at disseminating Toxoplasma to the brains of infected mice."These findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection. A better understanding of this pathogen dissemination is helpful in the development of new drugs to curtail the spread of a Toxoplasma gondii infection throughout the body.
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Biotechnology
| 2,020 |
July 16, 2020
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https://www.sciencedaily.com/releases/2020/07/200716144732.htm
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Two paths of aging: New insights on promoting healthspan
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Molecular biologists and bioengineers at the University of California San Diego have unraveled key mechanisms behind the mysteries of aging. They isolated two distinct paths that cells travel during aging and engineered a new way to genetically program these processes to extend lifespan.
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The research is described July 17 in the journal Our lifespans as humans are determined by the aging of our individual cells. To understand whether different cells age at the same rate and by the same cause, the researchers studied aging in the budding yeast Saccharomyces cerevisiae, a tractable model for investigating mechanisms of aging, including the aging paths of skin and stem cells.The scientists discovered that cells of the same genetic material and within the same environment can age in strikingly distinct ways, their fates unfolding through different molecular and cellular trajectories. Using microfluidics, computer modeling and other techniques, they found that about half of the cells age through a gradual decline in the stability of the nucleolus, a region of nuclear DNA where key components of protein-producing "factories" are synthesized. In contrast, the other half age due to dysfunction of their mitochondria, the energy production units of cells.The cells embark upon either the nucleolar or mitochondrial path early in life, and follow this "aging route" throughout their entire lifespan through decline and death. At the heart of the controls the researchers found a master circuit that guides these aging processes."To understand how cells make these decisions, we identified the molecular processes underlying each aging route and the connections among them, revealing a molecular circuit that controls cell aging, analogous to electric circuits that control home appliances," said Nan Hao, senior author of the study and an associate professor in the Section of Molecular Biology, Division of Biological Sciences.Having developed a new model of the aging landscape, Hao and his coauthors found they could manipulate and ultimately optimize the aging process. Computer simulations helped the researchers reprogram the master molecular circuit by modifying its DNA, allowing them to genetically create a novel aging route that features a dramatically extended lifespan."Our study raises the possibility of rationally designing gene or chemical-based therapies to reprogram how human cells age, with a goal of effectively delaying human aging and extending human healthspan," said Hao.The researchers will now test their new model in more complex cells and organisms and eventually in human cells to seek similar aging routes. They also plan to test chemical techniques and evaluate how combinations of therapeutics and drug "cocktails" might guide pathways to longevity."Much of the work featured in this paper benefits from a strong interdisciplinary team that was assembled," said Biological Sciences Professor of Molecular Biology Lorraine Pillus, one of the study's coauthors. "One great aspect of the team is that we not only do the modeling but we then do the experimentation to determine whether the model is correct or not. These iterative processes are critical for the work that we are doing."
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Biotechnology
| 2,020 |
July 16, 2020
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https://www.sciencedaily.com/releases/2020/07/200716144728.htm
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Megaphages harbor mini-Cas proteins ideal for gene editing
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The DNA-cutting proteins central to CRISPR-Cas9 and related gene-editing tools originally came from bacteria, but a newfound variety of Cas proteins apparently evolved in viruses that infect bacteria.
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The new Cas proteins were found in the largest known bacteria-infecting viruses, called bacteriophages, and are the most compact working Cas variants yet discovered -- half the size of today's workhorse, Cas9.Smaller and more compact Cas proteins are easier to ferry into cells to do genome editing, since they can be packed into small delivery vehicles, including one of the most popular: a deactivated virus called adeno-associated virus (AAV). Hypercompact Cas proteins also leave space inside AAV for additional cargo.As one of the smallest Cas proteins known to date, the newly discovered CasΦ (Cas-phi) has advantages over current genome-editing tools when they must be delivered into cells to manipulate crop genes or cure human disease."Adenoviruses are the perfect Trojan horse for delivering gene editors: You can easily program the viruses to reach almost any part in the body," said Patrick Pausch, a postdoctoral fellow at the University of California, Berkeley, and in UC Berkeley's Innovative Genomics Institute (IGI), a joint UC Berkeley/UCSF research group devoted to discovering and studying novel tools for gene editing in agriculture and human diseases. "But you can only pack a really small Cas9 into such a virus to deliver it. If you would have other CRISPR-Cas systems that are really compact, compared to Cas9, that gives you enough space for additional elements: different proteins fused to the Cas protein, DNA repair templates or other factors that regulate the Cas protein and control the gene editing outcome."Apparently these "megaphages" use the CasΦ protein -- the Greek letter Φ, or phi, is used as shorthand for bacteriophages -- to trick bacteria into fighting off rival viruses, instead of itself."The thing that actually made me interested in studying this protein specifically is that all the known CRISPR-Cas systems were originally discovered in bacteria and Archaea to fend off viruses, but this was the only time where a completely new type of CRISPR-Cas system was first found, and so far only found, in viral genomes," said Basem Al-Shayeb, a doctoral student in the IGI. "That made us think about what could be different about this protein, and with that came a lot of interesting properties that we then found in the lab."Among these properties: CasΦ evolved to be streamlined, combining several functions in one protein, so that it can dispense with half the protein segments of Cas9. It is as selective in targeting specific regions of DNA as the original Cas9 enzyme from bacteria, and just as efficient, and it works in bacteria, animal and plants cells, making it a promising, broadly applicable gene editor."This study shows that this virus-encoded CRISPR-Cas protein is actually very good at what it does, but it is a lot smaller, about half the size of Cas9," said IGI executive director Jennifer Doudna, a UC Berkeley professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator. "That matters, because it might make it a lot easier to deliver it into cells than what we are finding with Cas9. When we think about how CRISPR will be applied in the future, that is really one of the most important bottlenecks to the field right now: delivery. We think this very tiny virus-encoded CRISPR-Cas system may be one way to break through that barrier."Pausch and Al-Shayeb are first authors of a paper describing CasΦ that will appear this week in the journal The CasΦ protein was first discovered last year by Al-Shayeb in the laboratory of Jill Banfield, a a UC Berkeley professor of earth and planetary science and environment science, policy and management. The megaphages containing CasΦ were part of a group they dubbed Biggiephage and were found in a variety of environments, from vernal pools and water-saturated forest floors to cow manure lagoons."We use metagenomic sequencing to discover the Bacteria, Archaea and viruses in many different environments and then explore their gene inventories to understand how the organisms function independently and in combination within their communities," Banfield said. "CRISPR-Cas systems on phage are a particularly interesting aspect of the interplay between viruses and their hosts."While metagenomics allowed the researchers to isolate the gene coding for CasΦ, its sequence told them only that it was a Cas protein in the Type V family, though evolutionarily distant from other Type V Cas proteins, such as Cas12a, CasX (Cas12e) and Cas14. They had no idea whether it was functional as an immune system against foreign DNA. The current study showed that, similar to Cas9, CasΦ targets and cleaves foreign genomes in bacterial cells, as well as double-stranded DNA in human embryonic kidney cells and cells of the plant Arabidopsis thaliana. It also can target a broader range of DNA sequences than can Cas9.The ability of CasΦ to cut double-stranded DNA is a big plus. All other compact Cas proteins preferentially cut single-stranded DNA. So, while they may fit neatly into compact delivery systems like AAV, they are much less useful when editing DNA, which is double-stranded, inside cells.As was the case after Cas9's gene-editing prowess was first recognized in 2012, there is a lot of room for optimizing CasΦ for gene editing and discovering the best rules for designing guide RNAs to target specific genes, Pausch said.Other co-authors of the paper are Ezra Bisom-Rapp, Connor Tsuchida, Brady Cress and Gavin Knott of UC Berkeley and Zheng Li and Steven E. Jacobsen of UCLA. The researchers were funded, in part, by the Paul G. Allen Frontiers Group, National Institutes of Health Somatic Cell Genome Editing consortium (U01AI142817-02) and National Science Foundation (DGE 1752814).
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Biotechnology
| 2,020 |
July 16, 2020
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https://www.sciencedaily.com/releases/2020/07/200716123002.htm
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Genome guardians stop and reel in DNA to correct replication errors
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On the DNA assembly line, two proofreading proteins work together as an emergency stop button to prevent replication errors. New research from North Carolina State University and the University of North Carolina at Chapel Hill shows how these proteins -- MutL and MutS -- prevent DNA replication errors by creating an immobile structure that calls more proteins to the site to repair the error. This structure could also prevent the mismatched region from being "packed" back into the cell during division.
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When a cell prepares to divide, the DNA splits, with the double helix "unzipping" into two separate backbones. New nucleotides -- adenine, cytosine, guanine or thymine -- are filled into the gaps on the other side of the backbone, pairing with their counterparts (adenine with thymine and cytosine with guanine) and replicating the DNA to make a copy for both the old and the new cells. The nucleotides are a correct match most of the time, but occasionally -- about one time in 10 million -- there is a mismatch."Although mismatches are rare, the human genome contains approximately six billion nucleotides in every cell, resulting in approximately 600 errors per cell, and the human body consists of more than 37 trillion cells," says Dorothy Erie, chemistry professor at UNC-Chapel Hill, member of UNC's Lineberger Comprehensive Cancer Center and co-corresponding author of the work. "Consequently, if these errors go unchecked they can result in a vast array of mutations, which in turn can result in a variety of cancers, collectively known as Lynch Syndrome."A pair of proteins known as MutS and MutL work together to initiate repair of these mismatches. MutS slides along the newly created side of the DNA strand after it's replicated, proofreading it. When it finds a mismatch, it locks into place at the site of the error and recruits MutL to come and join it. MutL marks the newly formed DNA strand as defective and signals a different protein to gobble up the portion of the DNA containing the error. Then the nucleotide matching starts over, filling the gap again. The entire process reduces replication errors around a thousand-fold, serving as one of our body's best defenses against genetic mutations that can lead to cancer."We know that MutS and MutL find, bind, and recruit repair proteins to DNA," says biophysicist Keith Weninger, university faculty scholar at NC State and co-corresponding author of the work. "But one question remained -- do MutS and MutL move from the mismatch during the repair recruiting process, or stay where they are?"In two separate papers appearing in Using both fluorescent and non-fluorescent imaging techniques, including atomic force microscopy, optical spectroscopy and tethered particle motion, the researchers found that MutL "freezes" MutS in place at the site of the mismatch, forming a stable complex that stays in that vicinity until repair can take place. The complex appears to reel in the DNA around the mismatch as well, marking and protecting the DNA region until repair can occur."Due to the mobility of these proteins, current thinking envisioned MutS and MutL sliding freely along the mismatched strand, rather than stopping," Weninger says. "This work demonstrates that the process is different than previously thought."Additionally, the complex's interaction with the strand effectively stops any other processes until repair takes place. So the defective DNA strand cannot be repacked into a chromosome and then carried forward through cell division."
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Biotechnology
| 2,020 |
July 16, 2020
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https://www.sciencedaily.com/releases/2020/07/200716111623.htm
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CBD may help avert lung destruction in COVID-19, research suggests
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Cannabidiol, or CBD, may help reduce the cytokine storm and excessive lung inflammation that is killing many patients with COVID-19, researchers say.
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While more work, including clinical trials to determine optimal dosage and timing, is needed before CBD becomes part of the treatment for COVID-19, researchers at the Dental College of Georgia and Medical College of Georgia have early evidence it could help patients showing signs of respiratory distress avoid extreme interventions like mechanical ventilation as well as death from acute respiratory distress syndrome."ARDS is a major killer in severe cases of some respiratory viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and we have an urgent need for better intervention and treatment strategies," says Dr. Babak Baban, immunologist and interim associate dean for research at DCG and corresponding author of the study in the journal Our laboratory studies indicate pure CBD can help the lungs recover from the overwhelming inflammation, or cytokine storm, caused by the COVID-19 virus, and restore healthier oxygen levels in the body, says co-author Dr. Jack Yu, physician-scientist and chief of pediatric plastic surgery at MCG.Their CBD findings were enabled by their additional finding of a safe and relatively inexpensive model to duplicate the lung damage caused by ARDS. Work on the virus itself is limited to a handful of labs in the nation that can safely manage the highly contagious virus, and their newly reported approach opens more doors for studying SARS-CoV-2, COVID-19 and similar virus-induced conditions, they say.Their model, which takes advantage of the large, unique genetic structure of the novel coronavirus, produced classic symptoms of ARDS like the overwhelming, destructive immune response, then CBD significantly downregulated classic indicators of the excess, like inflammation-promoting cytokines as it improved oxygen levels in the blood and enabled the lungs to recover from the structural damage.A major problem with SARS-CoV-2 is instead of just killing the virus, the over-the-top immune response can quickly disable the lungs, transforming them to a place where virus is replicated, rather than a place that makes oxygen available for our bodies and eliminates potentially harmful gases like carbon dioxide.Mechanical ventilators can take over these vital functions for a while, and enable critically ill people to use less energy to just breathe and have more energy to fight infection, while ideally the lungs recover from the assault. However evidence suggests 30-50% of patients who get to the point of mechanical ventilation, don't survive.The cytokines in these now famous "storms" are a class of molecules like interferon and interleukin, secreted by immune cells and other cells like endothelial cells that line blood vessels, which impact cell communication and can both promote and deter inflammation. In the case of COVID-19, there is excessive production of inflammation-promoting molecules like the interleukins IL-6 and IL-1?, as well as immune cells like neutrophils and monocytes, the researchers say.They looked at objective measures of lung function in mice like levels of proinflammatory cytokines, oxygen levels in the blood before and after treatment, as well as temperature, an indicator of inflammation. Oxygen levels went up, while temperatures and cytokine levels went down with CBD therapy. Days later, a more detailed analysis of the lungs, reinforced reduction of key indicators of destructive inflammation, which their model, like the virus, drove way up including reduced levels of IL-6 and infiltrating neutrophils.In fact, both clinical symptoms and physical lung changes resulting from ARDS were reversed with CBD treatment, they say.Their model was created with the help of a synthetic analog of double-stranded RNA called POLY (I:C). In humans, our double-stranded DNA contains our genetic information and our single-stranded RNA carries out the instruction of our DNA to make certain proteins. In the family of coronaviruses, the double-stranded RNA carries the genetic material needed to reproduce the viruses and hijacks the cell machinery of our body to do that, Baban says."The natural instinct of the virus is to make more of itself," Baban says. "It weaves with our DNA to make the cell produce food and everything it needs." Viruses also tend to have a tissue or tissues they prefer -- some can and do go anywhere -- and for SARS-CoV-2, the lungs are high on the list, he says.Our bodies aren't used to this double-stranded RNA so, like the virus, POLY (I:C) gets the immediate and extreme attention of toll-like receptor 3, a family of receptors that help our body recognize invaders like a virus and activate our frontline, innate immune response."The toll-like receptors 3 see this and just go nuts," Yu says. The fact that the coronaviruses are literally big and have the largest known viral RNA genome make such a vigorous cytokine and immune response both plausible and probable, adds Baban.Mice received three, once-a-day doses of POLY (I:C) in the nasal passageway. CBD was given by a shot in the abdomen, the first dose two hours after the second POLY (I:C) treatment, then every other day for a total of three days in a process that sought to mimic mice getting treatment about the time a human would begin to experience trouble breathing and likely seek medical care. Given too early, CBD might actually interfere with a proper immune response against the virus, Yu says.CBD quickly improved the clinical symptoms, then later detailed studies of the lungs showed damage to their structure, like tissue overgrowth, scarring and swelling, also had totally or partially resolved. Their next steps include doing similar studies on other organs impacted by COVID-19 including the gut, heart and brain, Baban says.At least one way CBD is thought to calm the immune response is because it looks similar to endocannabinoids, a natural cell signaling system in our bodies believed to be involved in a wide variety of functions from sleep to reproduction to inflammation and immune response. CB1 and CB2, the main receptors for this system, are found extensively throughout the body including the brain and respiratory system, where we breathe in humanmade and natural irritants in the air -- as well as viruses and bacteria -- that might inflame. While understanding the workings of the natural endocannabinoid system is still very much a work in progress, it's thought that one way CBD works to reduce seizures, for example, is indirectly through the large number of CB1 receptors in the brain, says Yu.CBD is available without a prescription, and is used to treat problems like seizures as well as Parkinson's, Crohn's and other conditions where pain and/or inflammation are a major factor. It's derived from the hemp and cannabis plant, which are essentially the same although hemp has a much lower concentration of the "high" producing THC. Other investigators have shown the calming effect of CBD, for example, can block IL-6 in other models of inflammatory disease.ARDS is a rapid, severe infection of the lungs that results in widespread inflammation, shortness of breath, rapid breathing and the inability to sustain adequate oxygen levels to the body and brain. Shortness of breath or difficulty breathing are some of the early signs of COVID-19. ARDS is a major cause of death in patients who are critically ill for a variety of reasons, including common sepsis.Hesam Khodadadi, a PhD student in The Graduate School at AU, is first author of the study; Dr. Évila Lopes Salles, a postdoctoral fellow in Baban's lab, is second author. Coauthors also include Dr. Kumar Vaibhav, toxicologist in the MCG Department of Neurosurgery; Dr. Krishnan M. Dhandapani, neuroscientist in the MCG Department of Neurosurgery; and Dr. David C. Hess, neurologist and MCG dean.The work was supported in part by the National Institutes of Health.
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Biotechnology
| 2,020 |
July 16, 2020
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https://www.sciencedaily.com/releases/2020/07/200716101534.htm
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Un-natural mRNAs modified with sulfur atoms boost efficient protein synthesis
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Since mRNAs play a key role in protein synthesis in vivo, the use of mRNAs as medicines and for in vitro protein synthesis has been desired. In particular, mRNA therapeutics hold the potential for application to vaccine therapy(1) against coronaviruses and are being developed. However, the efficiency of protein production with mRNAs in the natural form is not sufficient enough for certain purposes, including application to mRNA therapeutics. Therefore, mRNA molecules allowing for efficient protein production have been required to be developed.
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A ribosome(2) repeats the following three steps to synthesize a protein in vivo using an mRNA as a template (translation reaction): 1) Initiation step: A ribosome binds to an mRNA to form a translation initiation complex; 2) Elongation step: The ribosome moves on the mRNA and links amino acids to synthesize a protein; and 3) Termination step: The protein synthesis process concludes, and the ribosome is liberated. In the translation reaction cycle, the initiation step takes the longest time.Collaborative research by a group of Nagoya University consisting of Professor Hiroshi Abe, Research Assistant Professor Naoko Abe, and graduate student Daisuke Kawaguchi with Yoshihiro Shimizu, a team leader at RIKEN, has succeeded in the development of modified messenger RNAs (mRNAs). The modified mRNA contains sulfur atoms in the place of oxygen atoms of phosphate moieties of natural mRNAs. It is capable of supporting protein synthesis at increased efficiency. They discovered that modified mRNAs accelerated the initiation step of the translation reactions and improved efficiency of protein synthesis by at least 20 times compared with that using natural-form mRNAs."This method is expected to be used for large-scale synthesis of proteins as raw materials for the production of biomaterials. Moreover, the application of the results obtained in this study to eukaryotic translation systems enables the efficient production of mRNA therapeutics for protein replacement therapy(3) to contribute to medical treatments. Furthermore, there are virtually no previous reports on the molecular design of highly functional mRNAs; therefore, the successful design achieved in this study can guide a future direction of the molecular design of modified mRNAs.This study was supported by the Strategic Basic Research Program CREST of the Japan Science and Technology Agency (JST).(1) Vaccine therapyA method of administering a protein antigen to individuals to elicit antibodies that can reduce the susceptibility to infectious diseases. In the case of an mRNA vaccine, an mRNA for in vivo expression of an antigen protein is administered, and then antibodies are produced against the expressed antigen protein.(2) RibosomeMulticomponent machinery providing a place where sequence information of an mRNA is read, and a protein is synthesized based on the sequence information while migrating on the mRNA. A ribosome is composed of ribosomal proteins and ribosomal RNAs.(3) Protein replacement therapyA treatment method that aims at improvement by supplementing protein from the outside when the deficiency of proteins (enzymes, etc.) is a cause of an illness.
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Biotechnology
| 2,020 |
July 15, 2020
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https://www.sciencedaily.com/releases/2020/07/200715142344.htm
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Scientists uncover key process in the manufacture of ribosomes and proteins
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Researchers at the University of Toronto have shown that an enzyme called RNA polymerase (Pol) II drives generation of the building blocks of ribosomes, the molecular machines that manufacture all proteins in cells based on the genetic code.
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The discovery reveals a previously unknown function for the enzyme in the nucleolus, the site of ribosome manufacture inside of human cells, where the enzyme had not been seen before. Pol II is one of three RNA polymerases that together enable cells to transfer genetic information from DNA to RNA and then proteins."Our study redefines the division of labour among the three main RNA polymerases, by identifying Pol II as a major factor in the control of nucleolar organizations underlying protein synthesis," said Karim Mekhail, a professor of laboratory medicine and pathobiology at U of T. "It also provides a tool for other researchers to interrogate the function of certain nucleic acid structures more precisely across the genome."The journal Mekhail and his colleagues found that inside the nucleolus, Pol II enables the expression of ribosomal RNA genes -- a key step in the creation of ribosomes, essential molecular complexes that make proteins in all cells. Pol II, they showed, generates R-loops -- hybrid DNA-RNA structures -- that directly shield ribosomal RNA genes from molecular disruptors called sense intergenic non-coding RNAs (or sincRNAs).Those disruptors are produced by Pol I in intergenic, non-protein-coding sequences of DNA between genes, and they become more active in various conditions: disruption of Pol II, under environmental stress, and in Ewing sarcoma."Pol II puts the brakes on Pol I and prevents sincRNAs from 'sinking' the nucleolus," said Mekhail, who holds the Canada Research Chair in Spatial Genome Organization. "That's how we united the name and action of the disruptors in our discussions of this work."Mekhail and his team developed a new technology to test the function of R-loops at specific locations on chromosomes, which they dubbed the 'red laser' system. "The existing tool in the field would obliterate R-loops across the whole genome, but we wanted to test the function of R-loops associated with a given genetic locus," said Mekhail. "We were able to turn an old technology into a modern laser-guided missile, which we are still working to further improve."Two U of T students were co-lead authors on the study -- Karan (Josh) Abraham and Negin Khosraviani -- and Mekhail said they made exceptional and complementary contributions to the research.Abraham is an MD/PhD student who began work on the project in 2014. "I pursued this work having observed enrichment of Pol II at ribosomal DNA genes in the nucleolus, which was compelling," said Abraham, who will finish his medical training next year. "It's incumbent upon every scientist to challenge existing models should the evidence support an alternate one."Khosraviani is a doctoral student who joined the lab in 2018, and she said teamwork and time management were critical. "We could not have completed this research without the help and dedication of our entire lab. Coordination with local and international collaborators was also essential," she said.Mekhail's team worked with colleagues across U of T and affiliated hospitals on the study, and with international collaborators at the University of Texas at San Antonio and University of Miami.Next steps based on this research could include exploration of sincRNAs and nucleolar disorganization as biomarkers for various cancers, and whether tumours with those features respond to drugs that target intergenic Pol I or II."COVID-19 has been devastating, but other diseases have not stopped," said Mekhail, who temporarily closed his physical lab space during the pandemic but has continued working with his team to analyze and publish results. "For example, cancer is still rampant and affecting people's lives. We have to do what we can and look forward to building on the progress we've made as soon as possible."
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Biotechnology
| 2,020 |
July 15, 2020
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https://www.sciencedaily.com/releases/2020/07/200715131236.htm
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Designing DNA from scratch: Engineering the functions of micrometer-sized DNA droplets
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In living organisms, DNA is the storage unit of all genetic information. It is with this information that proteins are encoded, which then enable biological systems to function as needed for the organism to survive. DNA's functioning is enabled by its structure: a double-stranded helix formed via the joining of specific pairs of molecules called 'nucleotides' in specific orders, called 'sequences'. In recent decades, scientists in the fields of DNA nanotechnology have been able to design DNA sequences to construct desired nanostructures and microstructures, which can be used to investigate biomolecular functions or create artificial cell systems.
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The customization of the designs of sequences in DNA nanotechnology also enables the interactions among DNA molecules to be controlled and programmed. The inter-molecular interactions in cells cause various phenomena. A phenomenon called "liquid-liquid phase separation (LLPS)" -- the separation of a liquid into a denser phase of droplets within a more dilute phase -- plays an important role in many biological processes. LLPS artificially induced via DNA nanotechnology can help deepen our understanding of the applicability of LLPS and provide a methodology for controlling bio-macromolecular droplets.Therefore, a team of scientists from Tokyo Tech, led by Professor Masahiro Takinoue, designed specific DNA-nanostructures to understand the influence of DNA sequences and demonstrate controllability on LLPS -- into DNA-rich and DNA-poor phases -- in artificially designed DNA nanostructures.Their study, published in When they added another set of constructed Y-motifs with sticky ends that are incompatible with the previous set, two sets of droplets were formed for each type of Y-motif. This demonstrated that DNA sequences can be tailored to fuse exclusively with similar ones.Prof Takinuoe and team then created a special DNA structure that can bridge together the incompatible Y-motifs. Upon adding this to the mixture of Y-motifs, droplets composed of both motifs were formed. Further construction of a cleavable variant of the special bridge DNA structure and subsequent addition of a certain cleaving enzyme caused the fission of droplets and the mixed droplets to separate into Janus-shaped droplets with unmixable halves containing the two types of Y-motif. By conjugating cargo molecules with DNA strands compatible with either one type of Y-motif, the scientists were able to localize the cargo molecules exclusively on one half of the droplet.Thus, the scientists were able to 'program' DNA and 'control' their behavior, opening doors to a new technique for creating artificial reaction environments to study biological systems and drug delivery. Prof Takinoue explains: "Living systems are well-organized dynamic structures whose behavior is regulated by the information encoded in biopolymers (such as DNA). Our DNA-based liquid-liquid phase separation system could provide a new basis for the development of artificial cell engineering."Because precise DNA sequences can be readily produced using available bioengineering techniques, the potential applications of manipulating material behaviors through DNA sequences are far-reaching. Prof Takinoue concludes: "The phase behavior shown in this study could be expanded to other materials that can be modified with DNA, which may enable us to design phases and create droplets for various materials. Moreover, we envision that the observed autonomous behavior of macromolecular structures could one day serve for the development of robotic molecular systems comparable to those of living cells."
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Biotechnology
| 2,020 |
July 15, 2020
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https://www.sciencedaily.com/releases/2020/07/200715131226.htm
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Botany: Slow growth the key to long term cold sensing
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Plants have to interpret temperature fluctuations over timescales ranging from hours to months to align their growth and development with the seasons.
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Much is known about how plants respond to temperature but the mechanisms that allow them to measure the temperature signal are less well understood.In this study which appears in "We have found a new temperature sensing mechanism that holds a long-term memory of the cold, integrating over fluctuating temperatures to measure cold duration. This is a new type of physical mechanism for temperature-sensing and can guide further studies in this area" explains first author Dr Yusheng Zhao.Using a forward genetic screen -- looking at the genetics of plants showing a particular trait -- they found a dysfunctional response. These plants showed high levels of a protein called VIN3 in warm temperatures. This protein is well known as being upregulated during periods of cold and interacts with the epigenetic molecular memory system that allows plants to remember cold.Dr Yusheng Zhao found these plants had one of two versions of mutated NTL8, a transcription factor or regulator protein that activated VIN3 even without cold.To understand the role of NTL8 they tagged it with a fluorescent protein (GFP) and looked with help from the Bioimaging platform at the John Innes Centre to show where this protein is present compared to VIN3. This showed that the mutated version was found everywhere in the plant and the wild type protein was mostly observed in the growing tips of roots. It also showed that it accumulates slowly over time in the cold.Using a theoretical approach to explore the problem further, the team reasoned that understanding how fast the NTL8 protein degrades may offer insight into how the slow dynamics of NTL8 and VIN3 operate. They discovered the NTL8 protein is long-lasting, as predicted by the theory.Mathematical modelling showed that the main factor determining the amount of NTL8 protein is growth dependent dilution. If the weather gets warmer, the plants grow quicker and as cells multiply, the amount of NTL8 becomes diluted. In contrast, in cooler temperatures plants grow more slowly and NTL8 is more concentrated, being able to accumulate over time. The mathematical model can reproduce the observations of NTL8 protein levels seen in the warm and cold.To further test the model, they added chemicals and hormones to change plant growth to see if this changed the levels of NTL8 as predicted by the model, which it did. In the roots they added the plant growth hormone Gibberellin, which causes plants to grow faster and NTL8 levels were lower, as expected. When they added an inhibitor of growth, NTL8 protein levels were higher in the whole plant. The team did similar experiments on the roots, and these predictions were confirmed too.Dr Rea Antoniou-Kourounioti joint first author adds: "We were surprised by the simplicity of the new temperature mechanism we discovered, which recycles temperature information from one process [growth] to create a completely new temperature sensing mechanism for another [vernalization -- the acceleration of flowering by cold]. We could reproduce most of the temperature-dependent changes in our experimental observations with our model by just changing the growth rate between warm and cold.""This study revolutionises our understanding of how temperature is sensed by plants, and particularly how fluctuating long-term environmental conditions are integrated," says Professor Martin Howard."This study shows the fantastic synergy when experimental approaches are combined with computational modelling. We would never have figured out this mechanism by doing either separately," says Caroline Dean.The findings will be useful for understanding how plants as well as other organisms sense the long-term fluctuating environmental signals and could apply to crops.
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Biotechnology
| 2,020 |
July 15, 2020
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https://www.sciencedaily.com/releases/2020/07/200715123145.htm
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Exploring how a scorpion toxin might help treat heart attacks
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Scientists are discovering potential life-saving medicines from an unlikely source: the venom of creatures like snakes, spiders and scorpions. Scorpion venom, in particular, contains a peptide that has beneficial effects on the cardiovascular system of rats with high blood pressure. Now, researchers reporting in ACS'
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Scorpion venom is a complex mixture of biologically active molecules, including neurotoxins, vasodilators and antimicrobial compounds, among many others. Although the venom is painful for those unlucky enough to be stung by a scorpion, individual venom compounds, if isolated and administered at the proper dose, could have surprising health benefits. One promising compound is the tripeptide KPP (Lys-Pro-Pro), which is a piece of a larger scorpion toxin. KPP was shown to cause blood vessels to dilate and blood pressure to decline in hypertensive rats. Thiago Verano-Braga, Adriano Pimenta and colleagues wanted to find out what exactly KPP does to heart muscle cells. The answer could explain the peptide's beneficial effects.The researchers treated mouse cardiac muscle cells in a petri dish with KPP and measured the levels of proteins expressed by the cells at different times using mass spectrometry. They found that KPP regulated proteins associated with cell death, energy production, muscle contraction and protein turnover. In addition, the scorpion peptide triggered the phosphorylation of a mouse protein called AKT, which activated it and another protein involved in the production of nitric oxide, a vasodilator. KPP treatment, however, caused dephosphorylation of a protein called phospholamban, leading to reduced contraction of cardiac muscle cells. Both AKT and phospholamban are already known to protect cardiac tissue from injuries caused by lack of oxygen. These results suggest that KPP should be further investigated as a drug lead for heart attacks and other cardiovascular problems, the researchers say.
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Biotechnology
| 2,020 |
July 15, 2020
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https://www.sciencedaily.com/releases/2020/07/200715142358.htm
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Molecular 'tails' are secret ingredient for gene activation in humans, yeast, and other organisms
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It might seem as though humans have little in common with the lowly yeast cell. Humans have hair, skin, muscles, and bones, among other attributes. Yeast have, well, none of those things.
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But besides their obvious differences, yeast and humans, and much of life for that matter, have a great deal in common, especially at the cellular level. One of these commonalities is the enzyme our cells use to make RNA copies of sections of our DNA. The enzyme slides along a strand of DNA that has been unpacked from the chromosome in which it resides, to "read" the genetic code, and then assembles an RNA strand that contains the same code. This copying process, known as transcription, is what happens at a molecular level when a gene is activated in an organism. The enzyme responsible for it, RNA polymerase, is found in all eukaryotic cells (cells with a nucleus) and it is essentially the same in all of them, whether the cells are from a redwood, an earthworm, a caribou, or a mushroom.That fact has presented a mystery for scientists, though: Although the DNA in a yeast cell is different in many ways from the DNA in a human cell, the same enzyme is able to work with both. Now, a team of Caltech researchers has discovered one way this happens.In a paper appearing in the July 15 issue of "An interesting question has been how the wide, molecularly diverse range of species on Earth can all use the same mechanism of gene activation," says Quintero Cadena. "Specifically, because this mechanism requires that two parts of a DNA molecule come together, it should be more difficult for species with long DNA molecules to transcribe genes."To envision how the amino acid tails help the enzymes work with two pieces of a long DNA molecule, it helps to imagine the tails and DNA like pieces of Velcro, with the enzyme consisting of two Velcro halves that each latch onto a complementary section of DNA. To begin transcribing the DNA into RNA, the two halves need to "find" each other and link up. This process of linking up is actually rather arbitrary. The two pieces of DNA move around randomly inside the cell until they happen to bump into each other.Longer amino acid tails do not increase the chances of those random encounters, but they do make the enzymes more "sticky," so when they bump into each other, it is more likely that they will stay together.That is not the only way the amino acid tails assist in the transcription of DNA, however. Quintero Cadena says by bringing more enzymes together, the tails may also sometimes create a membraneless organelle, essentially a zone within the cell where DNA transcription is localized. Generally, each organelle of a cell can be thought of as a discrete object that is surrounded by its own membrane -- one that holds in its contents. However, the team's research shows that when it comes to polymerase, their tails help the enzymes to gather themselves into a locale without needing to be contained by a membrane. This is because the amino acid tails attached to the polymerase enzyme have a greater affinity for other amino acid tails than they do for the fluid that fills the cell. It is not unlike how oil that has been mixed into water will separate itself and gather into its own droplets.However, Quintero Cadena adds, unlike oil, which has no affinity for water, the amino acid tails can be chemically tailored by the cell to have as much or as little affinity for other cellular contents as necessary. This allows the cell to adjust how strongly the enzymes gather around each other.Quintero Cadena says these discoveries provide a clearer idea of how genes are activated in a cell, and how the same cellular machinery has adapted through evolution to function in very different organisms."In the short term, this subtly but importantly changes the cartoon in our heads of how molecules interact to turn on a gene," he says. "In the long term, a better understanding of gene activation paints a more complete picture of the inner workings of a cell, which may help us understand how things that go wrong in a cell can contribute to diseases, and more generally to understand how cells change over time to adapt in different environments."
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Biotechnology
| 2,020 |
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