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Norton E.S.,Massachusetts Institute of Technology | Beach S.D.,Massachusetts Institute of Technology | Gabrieli J.D.E.,Massachusetts Institute of Technology | Gabrieli J.D.E.,Institute for Medical Engineering and Science
Current Opinion in Neurobiology | Year: 2015

Dyslexia is one of the most common learning disabilities, yet its brain basis and core causes are not yet fully understood. Neuroimaging methods, including structural and functional magnetic resonance imaging, diffusion tensor imaging, and electrophysiology, have significantly contributed to knowledge about the neurobiology of dyslexia. Recent studies have discovered brain differences before formal instruction that likely encourage or discourage learning to read effectively, distinguished between brain differences that likely reflect the etiology of dyslexia versus brain differences that are the consequences of variation in reading experience, and identified distinct neural networks associated with specific psychological factors that are associated with dyslexia. © 2014 Elsevier Ltd. Source


News Article
Site: http://phys.org/biology-news/

MIT researchers have now developed a way to deliver the CRISPR genome repair components more efficiently than previously possible, and they also believe it may be safer for human use. In a study of mice, they found that they could correct the mutated gene that causes a rare liver disorder, in 6 percent of liver cells—enough to cure the mice of the disease, known as tyrosinemia. "This finding really excites us because it makes us think that this is a gene repair system that could be used to treat a range of diseases—not just tyrosinemia but others as well," says Daniel Anderson, associate professor in MIT's Department of Chemical Engineering and a member of MIT's Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES). Anderson is one of the senior authors of a paper describing the findings in the Feb. 1 issue of Nature Biotechnology. Wen Xue, an assistant professor in molecular medicine at the University of Massachusetts Medical School, is also a senior author. The paper's lead author is Hao Yin, a research scientist at the Koch Institute. The CRISPR system relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have previously harnessed this system to create gene-editing complexes composed of a DNA-cutting enzyme called Cas9 and a short RNA that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut. When Cas9 and the short guide RNA targeting a disease gene are delivered into cells, a specific cut is made in the genome, and the cells' DNA repair processes glue the cut back together, often deleting a small portion of the genome. However, if a corrected copy of the gene is also delivered when the cut is made, the DNA repair can lead to correction of the disease gene, permanently repairing the genome. In 2014, Anderson and colleagues described the first use of CRISPR to repair a disease gene in an adult animal. In that study, they were able to cure tyrosinemia in mice. However, delivery of the genetic components required a high-pressure injection, a method that can also cause some damage to the liver. "That was the first demonstration of using CRISPR/Cas9 to do genetic repair in an adult animal," Anderson says. "We were excited by this demonstration but wanted to find a way to develop a drug form of the repair machinery that would be both safer and more efficient." The researchers also wanted to boost the percentage of cells that had the defective gene replaced. In the previous study, about one in 250 liver cells were repaired, which was enough to successfully treat tyrosinemia. However, for many other diseases, a higher percentage of repair would be needed to provide a therapeutic effect. In the new study, Anderson and colleagues developed a combined nanoparticle and viral delivery system to deliver the CRISPR repair machinery. First, they created a nanoparticle from lipids and messenger RNA (mRNA) that encoded the Cas9 enzyme. The other two components—the RNA guide strand and the DNA for the corrected gene—were embedded into a reprogrammed viral particle based on an adeno-associated virus (AAV). The researchers first injected the virus about a week before the lipid nanoparticles, giving the liver cells time to begin producing the RNA guide strand and the DNA template. When the nanoparticles carrying the Cas9 mRNA strand were injected, the cells began producing the Cas9 protein, but only for a few days because the mRNA eventually degraded. This is long enough to perform gene repair, but prevents cas9 from lingering in the cells and potentially disrupting other parts of the cells' genome. "There's some concern that if you had Cas9 in your cells for too long of a period of time, it might cause some genomic instability," Anderson says. "We think the use of the mRNA nanoparticle provides an additional level of safety by making sure the enzyme is not present for too long a period of time." With this method, about one in 16 cells had the gene corrected, a 15-fold improvement over the 2014 study. The researchers also found that this approach generated less off-target DNA cutting than methods in which the Cas9 gene is integrated into a cell's genome. "We did a genome-scale analysis and we have a very high level of on-target effects but almost no off-target effects," Yin says. Anderson's lab has developed similar lipid nanoparticles that are now in clinical development. AAV viral particles are in clinical trials for other purposes, making the researchers optimistic that this CRISPR delivery method could be used in humans, although more studies are needed. The researchers have applied for patents on this technique, which they believe could be used to treat wide range of diseases, especially those of the liver. "There are a range of metabolic diseases and other liver disorders where if you fix a mutated gene you might be really able to have an impact on human health," Anderson says. "It's really exciting to see our team develop this new delivery approach for CRISPR, which I believe has the potential to have far-reaching implications," says Robert Langer, the David H. Koch Institute Professor at MIT and an author of the paper. Explore further: Researchers reverse a liver disorder in mice by correcting a mutated gene More information: Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo, Nature Biotechnology, DOI: 10.1038/nbt.3471


News Article
Site: http://news.mit.edu/topic/mitnanotech-rss.xml

A new paper-based test developed at MIT and other institutions can diagnose Zika virus infection within a few hours. The test, which distinguishes Zika from the very similar dengue virus, can be stored at room temperature and read with a simple electronic reader, making it potentially practical for widespread use. “We have a system that could be widely distributed and used in the field with low cost and very few resources,” says James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Department of Biological Engineering and Institute for Medical Engineering and Science (IMES) and the leader of the research team. An outbreak of the Zika virus that began in Brazil in April 2015 has been linked to a birth defect known as microcephaly. Many infected people experience no symptoms, and when symptoms do appear they are very similar to those of related viruses such as dengue and chikungunya. Currently, patients are diagnosed by testing whether they have antibodies against Zika in their bloodstream, or by looking for pieces of the viral genome in a patient’s blood sample, using a test known as polymerase chain reaction (PCR). However, these tests can take days or weeks to yield results, and the antibody test cannot discriminate accurately between Zika and dengue. “One of the key problems in the field is being able to distinguish what these patients have in areas where these viruses are co-circulating,” says Lee Gehrke, the Hermann L.F. von Helmholtz Professor in IMES and an author of the paper. Collins, Gehrke, and colleagues from Harvard University’s Wyss Institute for Biologically Inspired Engineering and other institutions described the new device in the May 6 online edition of Cell. The paper’s lead authors are Melissa Takahashi, an IMES postdoc; Dana Braff, an MIT graduate student; Keith Pardee, an assistant professor at the University of Toronto and former Wyss Institute research scientist; Alexander Green, an assistant professor at Arizona State University and former Wyss Institute postdoc; and Guillaume Lambert, a visiting scholar at the Wyss Institute. The new device is based on technology that Collins and colleagues previously developed to detect the Ebola virus. In October 2014, the researchers demonstrated that they could create synthetic gene networks and embed them on small discs of paper. These gene networks can be programmed to detect a particular genetic sequence, which causes the paper to change color. Upon learning about the Zika outbreak, the researchers decided to try adapting their device to diagnose Zika, which has spread to other parts of South and North America since the outbreak began in Brazil. “In a small number of weeks, we developed and validated a relatively rapid, inexpensive Zika diagnostic platform,” says Collins, who is also a member of the Wyss Institute. Collins and his colleagues developed sensors, embedded in the paper discs, that can detect 24 different RNA sequences found in the Zika viral genome, which, like that of many viruses, is composed of RNA instead of DNA. When the target RNA sequence is present, it initiates a series of interactions that turns the paper from yellow to purple. This color change can be seen with the naked eye, but the researchers also developed an electronic reader that makes it easier to quantify the change, especially in cases where the sensor is detecting more than one RNA sequence. All of the cellular components necessary for this process — including proteins, nucleic acids, and ribosomes — can be extracted from living cells and freeze-dried onto paper. These paper discs can be stored at room temperature, making it easy to ship them to any location. Once rehydrated, all of the components function just as they would inside a living cell. The researchers also incorporated a step that boosts the amount of viral RNA in the blood sample before exposing it to the sensor, using a system called NASBA (nucleic acid sequence based amplification). This amplification step, which takes one to two hours, increases the test’s sensitivity 1 million-fold. Julius Lucks, an assistant professor of chemical and biomolecular engineering at Cornell University, says that this demonstration of rapidly customizable molecular sensors represents a huge leap for the field of synthetic biology. “What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” says Lucks, who was not involved in the research. The team tested the new device using synthesized RNA sequences corresponding to the Zika genome, which were were then added to human blood serum. The researchers showed that the device could detect very low viral RNA concentrations in those samples and could also distinguish Zika from dengue. The researchers then tested the device with samples taken from monkeys infected with the Zika virus. (Samples from human patients affected by the current Zika outbreak are very difficult to obtain.) They found that in these samples, the device could detect viral RNA concentrations as low as 2 or 3 parts per quadrillion. The researchers envision that this approach could also be adapted to other viruses that may emerge in the future. Collins now hopes to team up with other scientists to further develop the technology for diagnosing Zika. “Here we’ve done a nice proof-of-principle demonstration, but more work and additional testing would be needed to ensure safety and efficacy before actual deployment,” he says. “We’re not far off.” The research was funded by the Wyss Institute for Biologically Inspired Engineering, MIT’s Center for Microbiome Informatics and Therapeutics, the Defense Threat Reduction Agency, and the National Institutes of Health.


This technique, which can track changes in gene expression as cells differentiate, could be particularly useful for studying how stem cells or immune cells mature. It could also shed light on how cancer develops. "Existing methods allow for snapshot measurements of single-cell gene expression, which can provide very in-depth information. What this new approach offers is the ability to track cells over multiple generations and put this information in the context of a cell's lineal history," says Robert Kimmerling, a graduate student in biological engineering and the lead author of a paper describing the technique in the Jan. 6 issue of Nature Communications. The paper's senior authors are Scott Manalis, the Andrew and Erna Viterbi Professor of Biological Engineering and a member of MIT's Koch Institute for Integrative Cancer Research, and Alex Shalek, the Hermann L.F. von Helmholtz Career Development Assistant Professor of Health Sciences and Technology, an assistant professor of chemistry, and a member of MIT's Institute for Medical Engineering and Science. The new method incorporates a recently developed technology called single-cell RNA-seq, which sequences the messenger RNA from a single cell. These RNAs, known collectively as the transcriptome, reveal which genes are being actively transcribed (that is, copied into messenger RNA instructions for building proteins) inside a cell at a given point in time. This helps scientists understand, for example, what makes a skin cell so different from a heart cell even though the cells share the same DNA. "Scientists have well established methods for resolving diverse subsets of a population, but one thing that's not very well worked out is how this diversity is generated. That's the key question we were targeting: how a single founding cell gives rise to very diverse progeny," Kimmerling says. To try to answer that question, the researchers designed a microfluidic device that traps first an individual cell and then all of its descendants. The device has several connected channels, each of which has a trap that can capture a single cell. After the initial cell divides, its daughter cells flow further along the device and get trapped in the next channel. The researchers showed that they can capture up to five generations of cells this way and keep track of their relationships. To get the cells off the chip, the researchers temporarily reverse the direction of the fluid flowing across the chip, allowing them to remove the cells one at a time to perform single-cell RNA-seq. In this study, the researchers captured and sequenced immune cells called T cells. These cells are on constant alert in the body, and when they encounter a cell infected with a virus or bacterium, they leap into action, creating two distinct populations—effector T cells, which seek and destroy infected cells, and memory T cells, which retain a memory of the encounter and circulate in the body indefinitely in case of a subsequent encounter. "A single founding cell can give rise to both effector and memory cell subtypes, but how that diversity is generated isn't very clear," Kimmerling says. The researchers analyzed RNA from recently activated T cells and two subsequent generations. When comparing genes with functions related to T cell activation and differentiation, they found that "sister" cells produced from the same division event are much more similar in their gene expression profiles than two unrelated cells. They also found that "cousin" cells, which have the same "grandmother," are more similar than unrelated cells, which suggests unique, family-specific transcriptional profiles for single T cells. The researchers hope that future studies with this device could help to resolve the long-standing debate over how T cells differentiate into effector cells and memory cells. One theory is that the distinction occurs as early as the first T cell division following activation, while a competing theory suggests that the distinction happens later on, as a result of changes in the cells' microenvironment. To address this question, the researchers believe they would need to analyze the development of T cells taken from a mouse that had been exposed to a foreign pathogen, which would provide a useful model of T cell activation in response to infection. The new device could also be used to link RNA transcriptome information with other cell traits, the researchers say. "It opens up possibilities that have never been there before," Manalis says. "We can further annotate single-cell transcriptome data by applying this strategy to our existing devices for measuring mass, growth rate, density, or deformability." In this study, the researchers also discovered that they could use their new technique to learn which genes are expressed at certain points during the cell division cycle. Because they trap each cell and have a record of when it last divided, they can directly link the "age" of each cell to its transcriptome. They identified a set of about 300 genes that correspond most with time since division (a proxy for cell cycle progression), and found that most of those genes were involved in cell division. Therefore, by measuring the levels of those 300 genes in similar cells, scientists should be able to estimate the ages of those cells. The researchers also found that a leukemia cell line, which proliferates continuously, has a different set of genes that appear to be driving cell division. "In the future, this approach may be able to provide insight into unique transcriptional regulators of cell cycle progression in various cancer models," Kimmerling says. Explore further: New method for analysing RNA sequence data identifies new subtypes of cells


News Article
Site: http://www.biosciencetechnology.com/rss-feeds/all/rss.xml/all

By combining sophisticated RNA sequencing technology with a new device that isolates single cells and their progeny, MIT researchers can now trace detailed family histories for several generations of cells descended from one “ancestor.” This technique, which can track changes in gene expression as cells differentiate, could be particularly useful for studying how stem cells or immune cells mature. It could also shed light on how cancer develops. “Existing methods allow for snapshot measurements of single-cell gene expression, which can provide very in-depth information. What this new approach offers is the ability to track cells over multiple generations and put this information in the context of a cell’s lineal history,” said Robert Kimmerling, a graduate student in biological engineering and the lead author of a paper describing the technique in the Jan. 6 issue of Nature Communications. The paper’s senior authors are Scott Manalis, the Andrew and Erna Viterbi Professor of Biological Engineering and a member of MIT’s Koch Institute for Integrative Cancer Research, and Alex Shalek, the Hermann L.F. von Helmholtz Career Development Assistant Professor of Health Sciences and Technology, an assistant professor of chemistry, and a member of MIT’s Institute for Medical Engineering and Science. The new method incorporates a recently developed technology called single-cell RNA-seq, which sequences the messenger RNA from a single cell. These RNAs, known collectively as the transcriptome, reveal which genes are being actively transcribed (that is, copied into messenger RNA instructions for building proteins) inside a cell at a given point in time. This helps scientists understand, for example, what makes a skin cell so different from a heart cell even though the cells share the same DNA. “Scientists have well established methods for resolving diverse subsets of a population, but one thing that’s not very well worked out is how this diversity is generated. That’s the key question we were targeting: how a single founding cell gives rise to very diverse progeny,” Kimmerling said. To try to answer that question, the researchers designed a microfluidic device that traps first an individual cell and then all of its descendants. The device has several connected channels, each of which has a trap that can capture a single cell. After the initial cell divides, its daughter cells flow further along the device and get trapped in the next channel. The researchers showed that they can capture up to five generations of cells this way and keep track of their relationships. To get the cells off the chip, the researchers temporarily reverse the direction of the fluid flowing across the chip, allowing them to remove the cells one at a time to perform single-cell RNA-seq. In this study, the researchers captured and sequenced immune cells called T cells. These cells are on constant alert in the body, and when they encounter a cell infected with a virus or bacterium, they leap into action, creating two distinct populations — effector T cells, which seek and destroy infected cells, and memory T cells, which retain a memory of the encounter and circulate in the body indefinitely in case of a subsequent encounter. “A single founding cell can give rise to both effector and memory cell subtypes, but how that diversity is generated isn’t very clear,” Kimmerling said. The researchers analyzed RNA from recently activated T cells and two subsequent generations. When comparing genes with functions related to T cell activation and differentiation, they found that “sister” cells produced from the same division event are much more similar in their gene expression profiles than two unrelated cells. They also found that “cousin” cells, which have the same “grandmother,” are more similar than unrelated cells, which suggests unique, family-specific transcriptional profiles for single T cells. The researchers hope that future studies with this device could help to resolve the long-standing debate over how T cells differentiate into effector cells and memory cells. One theory is that the distinction occurs as early as the first T cell division following activation, while a competing theory suggests that the distinction happens later on, as a result of changes in the cells’ microenvironment. To address this question, the researchers believe they would need to analyze the development of T cells taken from a mouse that had been exposed to a foreign pathogen, which would provide a useful model of T cell activation in response to infection. The new device could also be used to link RNA transcriptome information with other cell traits, the researchers say. “It opens up possibilities that have never been there before,” Manalis said. “We can further annotate single-cell transcriptome data by applying this strategy to our existing devices for measuring mass, growth rate, density, or deformability.” “I think this is really beautiful work,” said Dean Felsher, a professor of medicine and pathology at Stanford University School of Medicine. “It builds on what Scott has been doing for a while, which is creating a whole new way of interrogating single-cell measurements. Now he can follow the progeny over multiple generations, which is really hard to do.” In this study, the researchers also discovered that they could use their new technique to learn which genes are expressed at certain points during the cell division cycle. Because they trap each cell and have a record of when it last divided, they can directly link the “age” of each cell to its transcriptome. They identified a set of about 300 genes that correspond most with time since division (a proxy for cell cycle progression), and found that most of those genes were involved in cell division. Therefore, by measuring the levels of those 300 genes in similar cells, scientists should be able to estimate the ages of those cells. The researchers also found that a leukemia cell line, which proliferates continuously, has a different set of genes that appear to be driving cell division. “In the future, this approach may be able to provide insight into unique transcriptional regulators of cell cycle progression in various cancer models,” Kimmerling said.

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