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    Displaying items by tag: mRNA

    cervical-smear-test1Researchers have to develop a test with the aid of which will be able to determine the presence of cancer in the body, regardless of the type. Initially, the scientific team of Anderson Cancer Center at the University of Texas working on a quest to discover genetic mutation, which can be confirmed pancreatic cancer without the need for biopsy. The researchers found that cancer cells, like other healthy individual specific small particles known as exosomes, 1983, having "footprint" of the respective tumor.

    Exosomes are small bubbles that form in the cytoplasm and secreted by cells into the extracellular environment. They can be found in a study of various body fluids, such as blood plasma, cerebrospinal fluid, urine, saliva, breast milk even. Their size is in the range of the virus, they are larger than the low density lipoprotein (bad cholesterol molecule), but smaller than the red blood cells. Their diameter is between 30 and 100 nanometers.

    Exosomes carrying the proteins, RNA and lipids. Participate in the regulation of immune responses and are an important component of intercellular communication. Relatively recently it was found that the transferred microRNA and mRNA to specific target cells and in particular, that provide for horizontal transport of mRNA between the cells, i.e. they are carried out by means of differentiation of the cell recipient. It is believed that the nucleic acids are transferred, are involved in the epigenetic inheritance. There is evidence that protein exosomes to create favorable changes in tumor growth cell-around environment.

    Researchers from the University of Texas believes it can develop a test that decipher coded in the excuzemes. This can not only determine the presence of cancerous processes in the body, but he caught at the beginning of tumorigenesis, which will be of immense value in medical practice for early detection, diagnosis and treatment of patients.

    At this stage there is no such medical text with the help of which you can find out whether a person suffers from some kind of tumor. Medicine use multiple tests "recognize" one or another gene mutation, pointing respectively to one or another type of tumor and whether it is malignant or benign.

    To be diagnosed with a tumor disease, it is first necessary to determine if it exists, to reach it, if it is available, and finally there are always risks and costs of surgical interventions, said Dr. Raghu Kaluri team .

    According to him, the genetic analysis of exosomes will help to determine not only the presence of a tumor process in the organism, but also its identification without biopsy. Different types of cancer produce different chromosomal mutations explains it with the test will be possible to know whether the cancer, pancreatic or brain, for example.

    Such a tool will undoubtedly enhance the ability of physicians to detect cancer in its early stages and effectively treating oncological diseases are written in the Journal of Biological Chemistry. Still a lot of work on the development of the test, which is not an easy task, given that the very exosomes is still studied by science.

    Published in News

    madetoorderaProtein synthesis in the extensions of nerve cells, called dendrites, underlies long-term memory formation in the brain, among other functions. "Thousands of messenger RNAs reside in dendrites, yet the dynamics of how multiple dendrite messenger RNAs translate into their final proteins remain elusive," says James Eberwine, PhD, professor of Pharmacology, Perelman School of Medicine at the University of Pennsylvania, and co-director of the Penn Genome Frontiers Institute.
    Dendrites, which branch from the cell body of the neuron, play a key role in the communication between cells of the nervous system, allowing for many neurons to connect with each other. Dendrites detect the electrical and chemical signals transmitted to the neuron by the axons of other neurons. The synapse is the neuronal structure where this chemical connection is formed, and investigators surmise that it is here where learning and memory occur.
    Previous studies in the Eberwine lab have shown that translation of messenger RNAs (mRNAs) into proteins occurs in dendrites at focal points called translational hotspots. Local protein synthesis in dendrites, not in the cell body of nerves, provides the ability to respond rapidly and selectively to external stimuli. This ability is especially important in neurons that have highly polarized cell morphology, meaning one end of the cell has a very different shape from the other end.
    In dendrites and axons these rapid structural and functional changes occur concurrently – their length, size, shape, and number change to suit the needs of neuronal cell body communication.
    These structural and chemical changes – called synaptic plasticity—require rapid, new synthesis of proteins. Cells may use different rates of translation in different types of mRNA to produce the right amounts and ratios of required proteins.
    Knowing how proteins are made to order – as it were - at the synapse can help researchers better understand how memories are made. Nevertheless, the role of this "local" environment in regulating which messenger RNAs are translated into proteins in a neuron's periphery is still a mystery.
    Eberwine, first author Tae Kyung Kim, PhD, a postdoc in the Eberwine lab, and colleagues including Jai Yoon Sul, PhD, assistant professor in Pharmacology, showed that protein translation of two dendrite mRNAs is complex in space and time, as reported online in Cell Reports this week.
    "We needed to look at more than one RNA at the same time to get a better handle on real- world processes, and this is the first study to do that in a live neuron," Eberwine explains.
    At Home in the Hippocampus
    The team looked at two RNAs that make proteins that bind to glutamate, the dominant neurotransmitter in the brain. Using rat hippocampus neurons the researchers found a heterogeneous distribution of translational hotspots along dendrites for the two mRNAs.
    This finding indicates that RNA translation is dictated by translational hotspots, not solely when RNA is present. A translational hot spot is characterized by where translation is occurring in a ribosome at any one time in a discrete spot. Since hotspots are not uniform, understanding individual hotspot dynamics is important to understanding learning and memory.
    "It's not always one particular RNA that dominates at a translation hotspot versus another type of RNA," says Eberwine. "Since there are 1,000 to 3,000 different mRNA types present in the dendrite overall, but not 1,000 to 3,000 different translational hot spots, do the mRNAs 'take turns' being translated in space and time at the ribosomes at the hotspots?"
    The researchers engineered the glutamate receptor RNAs to contain different fluorescent proteins that are independently detectable, as well as a photo-switchable protein to determine when new proteins were being made. In the case of the photo-switchable protein studies, when an mRNA for the glutamate receptor protein is marked green, it means it has already been translated.
    When a laser is passed over the green protein, it changes to red as a way of tagging when it has been been translated, and new proteins synthesized at that hotspot would be green, which is visible by the appearance of yellow fluorescence (green + red, as measured by light on the visible spectrum). These tricks of the light allow the team to keep track of newly made proteins over time and space.
    "This is the first time this method of protein labeling has been used to measure the act of translation of multiple proteins over space and time in a quantitative way," says Eberwine. "We call it quantitative functional genomics of live cell translation."
    "Our results suggest that the location of the translational hotspot is a regulator of the simultaneous translation of multiple messenger RNAs in nerve cell dendrites and therefore synaptic plasticity," says Sul.
    Laying the Groundwork
    Almost 10 years ago, the Eberwine lab discovered that nerve-cell dendrites have the capacity to splice messenger RNA, a process once believed to take place only in the nucleus of cells. Here, a gene is copied into mRNA, which possesses both exons (mature mRNA regions that code for proteins) and introns (non-coding regions). mRNA splicing works by cutting out introns and merging the remaining exon pieces, resulting in an mRNA capable of being translated into a specific protein.
    The vast array of proteins within the human body arises in part from the many ways that mRNAs can be spliced and reconnected. Specifically, splicing removes pieces of intron and exon regions from the RNA. The resulting spliced RNA is made into protein.
    If the RNA has different exons spliced in and out of it, then different proteins can be made from this RNA. The Eberwine lab was successful in showing that splicing can occur in dendrites because they used sensitive technologies developed in their lab, which permits them to detect and quantify RNA splicing, as well as the translated protein in single isolated dendrites.
    Understanding the dynamics of RNA biology and protein translation in dendrites promises to provide insight into regulatory mechanisms that may be modulated for therapeutic purposes in neurological and psychiatric illnesses. The directed development of therapeutics requires this detailed knowledge, says Eberwine.

    Published in News

    ahiddengenetScientists routinely seek to reprogram bacteria to produce proteins for drugs, biofuels and more, but they have struggled to get those bugs to follow orders. But a hidden feature of the genetic code, it turns out, could get bugs with the program. The feature controls how much of the desired protein bacteria produce, a team from the Wyss Institute for Biologically Inspired Engineering at Harvard University reported in the September 26 online issue of Science.
    The findings could be a boon for biotechnologists, and they could help synthetic biologists reprogram bacteria to make new drugs and biological devices.
    By combining high-speed "next-generation" DNA sequencing and DNA synthesis technologies, Sri Kosuri, Ph.D., a Wyss Institute staff scientist, George Church, Ph.D., a core faculty member at the Wyss Institute and professor of genetics at Harvard Medical School, and Daniel Goodman, a Wyss Institute graduate research fellow, found that using more rare words, or codons, near the start of a gene removes roadblocks to protein production.
    "Now that we understand how rare codons control gene expression, we can better predict how to synthesize genes that make enzymes, drugs, or whatever you want to make in a cell," Kosuri said.
    To produce a protein, a cell must first make working copies of the gene encoding it. These copies, called messenger RNA (mRNA), consist of a specific string of words, or codons. Each codon represents one of the 20 different amino acids that cells use to assemble proteins. But since the cell uses 61 codons to represent 20 amino acids, many codons have synonyms that represent the same amino acid.
    In bacteria, as in books, some words are used more often than others, and molecular biologists have noticed over the last few years that rare codons appear more frequently near the start of a gene. What's more, genes whose opening sequences have more rare codons produce more protein than genes whose opening sequences do not.
    No one knew for sure why rare codons had these effects, but many biologists suspected that they function as a highway on-ramp for ribosomes, the molecular machines that build proteins. According to this idea, called the codon ramp hypothesis, ribosomes wait on the on-ramp, then accelerate slowly along the mRNA highway, allowing the cell to make proteins with all deliberate speed. But without the on-ramp, the ribosomes gun it down the mRNA highway, then collide like bumper cars, causing traffic accidents that slow protein production. Other biologists suspected rare codons acted via different mechanisms. These include mRNA folding, which could create roadblocks for ribosomes that block the highway and slow protein production.
    To see which ideas were correct, the three researchers used a high-speed, multiplexed method that they'd reported in August in The Proceedings of the National Academy of Sciences.
    First, they tested how well rare codons activated genes by mass-producing 14,000 snippets of DNA with either common or rare codons; splicing them near the start of a gene that makes cells glow green, and inserting each of those hybrid genes into different bacteria. Then they grew those bugs, sorted them into bins based on how intensely they glowed, and sequenced the snippets to look for rare codons.
    They found that genes that opened with rare codons consistently made more protein, and a single codon change could spur cells to make 60 times more protein.
    "That's a big deal for the cell, especially if you want to pump out a lot of the protein you're making," Goodman said.
    The results were also consistent with the codon-ramp hypothesis, which predicts that rare codons themselves, rather than folded mRNA, slow protein production. But the researchers also found that the more mRNA folded, the less of the corresponding protein it produced—a result that undermined the hypothesis.
    To put the hypothesis to a definitive test, the Wyss team made and tested more than 14,000 mRNAs – including some with rare codons that didn't fold well, and others that folded well but had no rare codons. By quickly measuring protein production from each mRNA and analyzing the results statistically, they could separate the two effects.
    The results showed clearly that RNA folding, not rare codons, controlled protein production, and that scientists can increase protein production by altering folding, Goodman said.
    The new method could help resolve other thorny debates in molecular biology. "The combination of high-throughput synthesis and next-gen sequencing allows us to answer big, complicated questions that were previously impossible to tease apart," Church said.
    "These findings on codon use could help scientists engineer bacteria more precisely than ever before, which is tremendous in itself, and they provide a way to greatly increase the efficiency of microbial manufacturing, which could have huge commercial value as well," said Wyss Institute Founding Director Don Ingber, M.D., Ph.D. "They also underscore the incredible value of the new automated technologies that have emerged from the Synthetic Biology Platform that George leads, which enable us to synthesize and analyze genes more rapidly than ever before."

    Published in News

    CRISPR-mice-virus-detection-teratoma-teratoma-service-ipsc-transgenic-mouseMIT researchers have shown that genes on or off in yeast and human cells by controlling, when DNA into RNA - bypokrok that will allow scientists to better understand the function of these genes copy.
    This technique can also be easier to control the environment of the cell produkujídrogy or disease realize engineer, says Timothy Lu, assistant professor of electrical engineering and computer science and bio - engineering and lead author of a paper describing the new approach in the journal ACS Synthetic Biology.
    "I think it will be much easier to build synthetic circuits" says Lu, a member of the Synthetic Biology Center at MIT. "It should increase the extent and speed with which we can build a number of synthetic circuits in yeast and mammalian cells."
    The new method is based on a system of viral proteins have been used recently for the treatment of genomes of bacteria and human cells. Initial system called CRISPR consists of two parts: a protein which binds to DNA and washers and a short strand of RNA, leaders of the protein into the correct position in the genome.
    "CRISPR system is so powerful that it can be used for a variety of DNA - binding regions of a simple conversion of this handbook RNA targeted basis" says Lu. "By simply reprogramming the RNA sequence of this protein can be any place you want to call the genome or synthetic circuit."
    The main author of the article is Farzadfard Fahim, an MIT graduate student of biology. Samuel Perl, student of Electrical Engineering and Computer Science, is takéautorem.

    Targeting transcription

    In previous studies CRISPR was used to cut pieces of the gene is switched off or replace it with another gene. Lu and his colleagues decided to use the CRISPR system for a different purpose : control of gene transcription is the process by which DNA sequence into RNA (mRNA ), which goes beyond genes copied instructions.
    Transcription is strictly regulated by proteins called transcription factors. These proteins bind to specific DNA sequences in the promoter region of the gene, and either modify or block the enzymes needed for the copy of the gene into mRNA.
    Which acts as a transcription factor for this study, the researchers adjust CRISPR system. Firstly, the modified CRISPR normal protein known as time9 so that it no longer cut the DNA bound thereto. They are also added to the protein, the segment that is activated or suppressed by modulating gene expression machinery of cellular transcription.
    Time9 to get to the right place, the researchers also corresponds delivered to the target cells, the genes for RNA guides who chtějísekvence DNA activates the promoter of the gene.
    Researchers have shown that when the guide RNA and protein time9 connection in the target cell, the target gene přesněpravý atranskripce. To their surprise, the same complex time9 also used to block transcription to be found in other parts of the gene.
    "It's nice that allows you to make a positive and negative regulation of the same protein, but with different RNA targeted to various managerial positions in the promoter" says Lu.

    "A lot of elasticity"

    The new system should be much simpler than other newly developed two systems for the transcription of DNA - binding proteins such as zinc finger transcriptional activator and effector Nucleases ( Talens ) is known, says Lu. Although effective, the construction and assembly of proteins is time- consuming and expensive.
    "There is a lot of flexibility with CRISPR, and it really comes from the fact that you do not spend more time in protein engineering. You can only change the sequence of the nucleic acid RNA, " says Lu.
    "The fact that this can be used for effective regulation of transcription in both yeast and mammalian cells, it is very encouraging," says Kobi Benenson, Professor of Biosystems Science and Engineering at ETH Zurich, which is not part of the research team." This technology may be used in the very near future genetic engineering and synthetic biology applications for biopharmaceuticals - "Tissue engineering and gene therapy, among other things"
    Researchers also transcriptional control designed so that it can be triggered by specific small molecules, which may be included in the cell, such as sugars. For this, the guide RNA genes constructed so that they are only produced Primal molecule is available. No small molecules, there are no guidelines aRNA gene is targeted at rest.
    This type of control could be used to explore the role of naturally occurring genes on and off at specific times during development or progression useful, says Lu.
    Lu is now working on the development of advanced synthetic circuits for applications such decisions are based on multiple inputs made ​​by the mobile environment. " We want to be able to scale- up the most complex circuits and show that anyone is ever built in yeast and mammalian cells" he says.

    Published in News

    human-retina-genes-antibodies-gentaurInvestigators at Massachusetts Eye and Ear and Harvard Medical School have published the most thorough description of gene expression in the human retina reported to date. In a study published today in the journal BMC Genomics, Drs. Michael Farkas, Eric Pierce and colleagues in the Ocular Genomics Institute (OGI) at Mass. Eye and Ear reported a complete catalog of the genes expressed in the retina.

    The retina is the neural tissue in the back of the eye that initiates vision.  It is responsible to receiving light signals, converting them into neurologic signals and sending those signals to the brain so that we can see.  If one thinks of the eye as a camera, the retina in the “film” in the camera. For these studies, the investigators used a technique called RNA sequencing (RNA-seq) to identify all of the messenger RNAs (mRNAs) produced in the human retina.  The resulting catalog of expressed genes, or transcriptome, demonstrates that the majority of the 20,000+ genes in the human body are expressed in the retina.  This in itself is not surprising, because the retina is a complex tissue comprised of 60 cell types.

    In a more surprising result, Dr. Farkas and colleagues identified almost 30,000 novel exons and over 100 potential novel genes that had not been identified previously. Exons are the portions of the genome that are used to encode proteins or other genetic elements.  The investigators validated almost 15,000 of these novel transcript features and found that more than 99 percent of them could be reproducibly detected. Several thousand of the novel exons appear to be used specifically in the retina.  In total, the newly detected mRNA sequence increased the number of exons identified in the human genome by 3 percent. 

    “While this may not sound like a lot, it shows that there is more to discover about the human genome, and that each tissue may use distinct parts of the genome,” said Dr. Pierce, Director of the OGI and the Solman and Libe Friedman Associate Professor of Ophthalmology, Harvard Medical School.

    This work is valuable to help scientists understand how the retina worksand how it is affected by disease. For example, Dr. Pierce and colleagues in the OGI study inherited retinal degenerations, which are common causes of vision loss. These diseases are caused by misspellings or mutations in genes that are needed for vision. To date, investigators have identified more than 200 retinal degeneration disease genes, but still can’t find the cause of disease for up to half of the patients affected by these disorders. Identification of new exons used in the retina may help find the cause of disease in these patients. 

    Identifying the genetic cause of patients’ retinal degeneration has become especially important with the recent success of clinical trials of gene therapy for RPE65 Leber congenital amaurosis (LCA). As a follow-up to these initial proof-of-concept trials, clinical trials of gene therapy for four other genetic forms of inherited retinal degeneration are currently in progress. Further, studies in animal models have reported successful gene therapy for multiple additional genetic types of IRD. There is thus an unprecedented opportunity to translate research progress into provide sight preserving and/or restoring treatment to patients with retinal degenerative disorders.

    About Massachusetts Eye and Ear 
    Mass. Eye and Ear clinicians and scientists are driven by a mission to find cures for blindness, deafness and diseases of the head and neck.  After uniting with Schepens Eye Research Institute in 2011, Mass. Eye and Ear in Boston became the world's largest vision and hearing research center, offering hope and healing to patients everywhere through discovery and innovation.  Mass. Eye and Ear is a Harvard Medical School teaching hospital and trains future medical leaders in ophthalmology and otolaryngology, through residency as well as clinical and research fellowships.  Internationally acclaimed since its founding in 1824, Mass. Eye and Ear employs full-time, board-certified physicians who offer high-quality and affordable specialty care that ranges from the routine to the very complex.  U.S. News & World Report’s “Best Hospitals Survey” has consistently ranked the Mass. Eye and Ear Departments of Otolaryngology and Ophthalmology as among the top hospitals in the nation. Mass. Eye and Ear is home to the Ocular Genomics Institute which aims to translate the promise of personalized genomic medicine into clinical care for ophthalmic disorders. 

    Published in News