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  • richardmitnick 2:17 pm on December 31, 2017 Permalink | Reply
    Tags: Genetics, , HiCRep method to accurately assess the reproducibility of data from Hi-C experiments, New insights into how the genome works inside of a cell, New statistical method for evaluating reproducibility in studies of genome organization" 2017, , Quite often correlation is treated as a proxy of reproducibility in many scientific disciplines but they actually are not the same thing, , With the massive amount of data that is being produced in whole-genome studies it is vital to ensure the quality of the data   

    From Pennsylvania State University: “New statistical method for evaluating reproducibility in studies of genome organization” 2017 

    Penn State Bloc

    Pennsylvania State University

    03 October 2017
    Qunhua Li:
    (814) 863-7395

    Barbara K. Kennedy:
    (814) 863-4682

    Sam Sholtis

    A new, statistical method to evaluate the reproducibility of data from Hi-C — a cutting-edge tool for studying how the genome works in three dimensions inside of a cell — will help ensure that the data in these “big data” studies is reliable.

    Schematic representation of the HiCRep method. HiCRep uses two steps to accurately assess the reproducibility of data from Hi-C experiments. Step 1: Data from Hi-C experiments (represented in triangle graphs) is first smoothed in order to allow researchers to see trends in the data more clearly. Step 2: The data is stratified based on distance to account for the overabundance of nearby interactions in Hi-C data. Credit: Li Laboratory, Penn State University

    “Hi-C captures the physical interactions among different regions of the genome,” said Qunhua Li, assistant professor of statistics at Penn State and lead author of the paper. “These interactions play a role in determining what makes a muscle cell a muscle cell instead of a nerve or cancer cell. However, standard measures to assess data reproducibility often cannot tell if two samples come from the same cell type or from completely unrelated cell types. This makes it difficult to judge if the data is reproducible. We have developed a novel method to accurately evaluate the reproducibility of Hi-C data, which will allow researchers to more confidently interpret the biology from the data.”

    The new method, called HiCRep, developed by a team of researchers at Penn State and the University of Washington, is the first to account for a unique feature of Hi-C data — interactions between regions of the genome that are close together are far more likely to happen by chance and therefore create spurious, or false, similarity between unrelated samples. A paper describing the new method appears in the journal Genome Research.

    “With the massive amount of data that is being produced in whole-genome studies, it is vital to ensure the quality of the data,” said Li. “With high-throughput technologies like Hi-C, we are in a position to gain new insight into how the genome works inside of a cell, but only if the data is reliable and reproducible.”

    Inside the nucleus of a cell there is a massive amount of genetic material in the form of chromosomes — extremely long molecules made of DNA and proteins. The chromosomes, which contain genes and the regulatory DNA sequences that control when and where the genes are used, are organized and packaged into a structure called chromatin. The cell’s fate, whether it becomes a muscle or nerve cell, for example, depends, at least in part, on which parts of the chromatin structure is accessible for genes to be expressed, which parts are closed, and how these regions interact. HiC identifies these interactions by locking the interacting regions of the genome together, isolating them, and then sequencing them to find out where they came from in the genome.

    The HiCRep method is able to accurately reconstruct the biological relationship between different cell types, where other methods fail. Credit: Li Laboratory, Penn State University

    “It’s kind of like a giant bowl of spaghetti in which every place the noodles touch could be a biologically important interaction,” said Li. “Hi-C finds all of these interactions, but the vast majority of them occur between regions of the genome that are very close to each other on the chromosomes and do not have specific biological functions. A consequence of this is that the strength of signals heavily depends on the distance between the interaction regions. This makes it extremely difficult for commonly-used reproducibility measures, such as correlation coefficients, to differentiate Hi-C data because this pattern can look very similar even between very different cell types. Our new method takes this feature of Hi-C into account and allows us to reliably distinguish different cell types.”

    “This reteaches us a basic statistical lesson that is often overlooked in the field,” said Li. “Quite often, correlation is treated as a proxy of reproducibility in many scientific disciplines, but they actually are not the same thing. Correlation is about how strongly two objects are related. Two irrelevant objects can have high correlation by being related to a common factor. This is the case here. Distance is the hidden common factor in the Hi-C data that drives the correlation, making the correlation fail to reflect the information of interest. Ironically, while this phenomenon, known as the confounding effect in statistical terms, is discussed in every elementary statistics course, it is still quite striking to see how often it is overlooked in practice, even among well-trained scientists.“

    The researchers designed HiCRep to systematically account for this distance-dependent feature of Hi-C data. In order to accomplish this, the researchers first smooth the data to allow them to see trends in the data more clearly. They then developed a new measure of similarity that is able to more easily distinguish data from different cell types by stratifying the interactions based on the distance between the two regions. “This is like studying the effect of drug treatment for a population with very different ages. Stratifying by age helps us focus on the drug effect. For our case, stratifying by distance helps us focus on the true relationship between samples.”

    To test their method, the research team evaluated Hi-C data from several different cell types using HiCRep and two traditional methods. Where the traditional methods were tripped up by spurious correlations based on the excess of nearby interactions, HiCRep was able to reliably differentiate the cell types. Additionally, HiCRep could quantify the amount of difference between cell types and accurately reconstruct which cells were more closely related to one another.

    In addition to Li, the research team includes Tao Yang, Feipeng Zhang, Fan Song, Ross C. Hardison, and Feng Yue at Penn State; and Galip Gürkan Yardımcı and William Stafford Noble at the University of Washington. The research was supported by the U.S. National Institutes of Health, a Computation, Bioinformatics, and Statistics (CBIOS) training grant at Penn State, and the Huck Institutes of the Life Sciences at Penn State.

    See the full article here .

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  • richardmitnick 2:27 pm on December 19, 2017 Permalink | Reply
    Tags: 20 “loading” molecules called aminoacyl-tRNA synthetases, , Charles Carter, Genetics, Kurt Gödel's Theorem and the Chemistry of Life, Peter Wills, protein-like molecules rather than RNA may have been the planet’s first self-replicators, ,   

    From Quanta: “The End of the RNA World Is Near, Biochemists Argue” 

    Quanta Magazine
    Quanta Magazine

    December 19, 2017
    Jordana Cepelewicz

    A popular theory holds that life emerged from a rich chemical soup in which RNA was the original self-replicator. But a combination of peptides and RNA might have been more effective.
    Novikov Aleksey

    Four billion years ago, the first molecular precursors to life emerged, swirling about in Earth’s primordial soup of chemicals. Although the identity of these molecules remains a subject of fractious debate, scientists agree that the molecules would have had to perform two major functions: storing information and catalyzing chemical reactions. The modern cell assigns these responsibilities to its DNA and its proteins, respectively — but according to the narrative that dominates origin-of-life research and biology-textbook descriptions today, RNA was the first to play that role, paving the way for DNA and proteins to take over later.

    This hypothesis, proposed in the 1960s and dubbed the “RNA world” two decades later, is usually viewed as the most likely explanation for how life got its start. Alternative “worlds” abound, but they’re often seen as fallback theories, flights of fancy or whimsical thought experiments.

    That’s mainly because, theorizing aside, the RNA world is fortified by much more experimental evidence than any of its competitors have accumulated. Last month, Quanta Magazine reported on an alternative theory suggesting that protein-like molecules, rather than RNA, may have been the planet’s first self-replicators. But its findings were purely computational; the researchers have only just begun experiments to seek support for their claims.

    Now, a pair of researchers has put forth another theory — this time involving the coevolution of RNA and peptides — that they hope will shake the RNA world’s hold.

    Recent papers published in Biosystems and Molecular Biology and Evolution delineated why the RNA world hypothesis does not provide a sufficient foundation for the evolutionary events that followed. Instead, said Charles Carter, a structural biologist at the University of North Carolina, Chapel Hill, who co-authored the papers, the model represents “an expedient proposal.” “There’s no way that a single polymer could carry out all of the necessary processes we now characterize as part of life,” he added.

    And that single polymer certainly couldn’t be RNA, according to his team’s studies. The main objection to the molecule concerns catalysis: Some research has shown that for life to take hold, the mystery polymer would have had to coordinate the rates of chemical reactions that could differ in speed by as much as 20 orders of magnitude. Even if RNA could somehow do this in the prebiotic world, its capabilities as a catalyst would have been adapted to the searing temperatures — around 100 degrees Celsius — that abounded on early Earth. Once the planet started to cool, Carter claims, RNA wouldn’t have been able to evolve and keep up the work of synchronization. Before long, the symphony of chemical reactions would have fallen into disarray.

    Perhaps most importantly, an RNA-only world could not explain the emergence of the genetic code, which nearly all living organisms today use to translate genetic information into proteins. The code takes each of the 64 possible three-nucleotide RNA sequences and maps them to one of the 20 amino acids used to build proteins. Finding a set of rules robust enough to do that would take far too long with RNA alone, said Peter Wills, Carter’s co-author at the University of Auckland in New Zealand — if the RNA world could even reach that point, which he deemed highly unlikely. In Wills’ view, RNA might have been able to catalyze its own formation, making it “chemically reflexive,” but it lacked what he called “computational reflexivity.”

    “A system that uses information the way organisms use genetic information — to synthesize their own components — must contain reflexive information,” Wills said. He defined reflexive information as information that, “when decoded by the system, makes the components that perform exactly that particular decoding.” The RNA of the RNA world hypothesis, he added, is just chemistry because it has no means of controlling its chemistry. “The RNA world doesn’t tell you anything about genetics,” he said.

    Nature had to find a different route, a better shortcut to the genetic code. Carter and Wills think they’ve uncovered that shortcut. It depends on a tight feedback loop — one that would not have developed from RNA alone but instead from a peptide-RNA complex.

    Bringing Peptides Into the Mix

    Carter found hints of that complex in the mid-1970s, when he learned in graduate school that certain structures seen in most proteins are “right-handed.” That is, the atoms in the structures could have two equivalent mirror-image arrangements, but the structures all use just one. Most of the nucleic acids and sugars that make up DNA and RNA are right-handed, too. Carter began to think of RNA and polypeptides as complementary structures, and he modeled a complex in which “they were made for each other, like a hand in a glove.”

    This implied an elementary kind of coding, a basis for the exchange of information between the RNA and the polypeptide. He was on his way to sketching what that might have looked like, working backward from the far more sophisticated modern genetic code. When the RNA world, coined in 1986, rose to prominence, Carter admitted, “I was pretty ticked off.” He felt that his peptide-RNA world, proposed a decade earlier, had been totally ignored.

    Since then, he, Wills and others have collaborated on a theory that circles back to that research. Their main goal was to figure out the very simple genetic code that preceded today’s more specific and complicated one. And so they turned not just to computation but also to genetics.

    At the center of their theory are 20 “loading” molecules called aminoacyl-tRNA synthetases. These catalytic enzymes allow RNA to bond with specific amino acids in keeping with the rules of the genetic code. “In a sense, the genetic code is ‘written’ in the specificity of the active sites” of those enzymes, said Jannie Hofmeyr, a biochemist at Stellenbosch University in South Africa, who was not involved in the study.

    Lucy Reading-Ikkanda/Quanta Magazine

    Previous research showed that the 20 enzymes could be divided evenly into two groups of 10 based on their structure and sequence. These two enzyme classes, it turned out, have certain sequences that code for mutually exclusive amino acids — meaning that the enzymes had to have arisen from complementary strands of the same ancient gene. Carter, Wills and their colleagues found that in this scenario, RNA coded for peptides using a set of just two rules (or, in other words, using just two types of amino acids). The resulting peptide products ended up enforcing the very rules that governed the translation process, thus forming the tight feedback loop the researchers knew would be the linchpin of the theory.

    Gödel’s Theorem and the Chemistry of Life

    Carter sees strong parallels between this kind of loop and the mathematical one described by the philosopher and mathematician Kurt Gödel, whose “incompleteness” theorem states that in any logical system that can represent itself, statements will inevitably arise that cannot be shown to be true or false within that system. “I believe that the analogy to Gödel’s theorem furnishes a quite strong argument for inevitability,” Carter said.

    In their recent papers, Carter and Wills show that their peptide-RNA world solves gaps in origin-of-life history that RNA alone can’t explain. “They provide solid theoretical and experimental evidence that peptides and RNA were jointly involved in the origin of the genetic code right from the start,” Hofmeyr said, “and that metabolism, construction through transcription and translation, and replication must have coevolved.”

    Of course, the Carter-Wills model begins with the genetic code, the existence of which presupposes complex chemical reactions involving molecules like transfer RNA and the loading enzymes. The researchers claim that the events leading up to their proposed scenario involved RNA and peptides interacting (in the complex that Carter described in the 1970s, for example). Yet that suggestion still leaves many open questions about how that chemistry began and what it looked like.

    To answer these questions, theories abound that move far beyond the RNA world. In fact, some scientists take an approach precisely opposite to that of Carter and Wills: They think instead that the earliest stages of life did not need to begin with anything resembling the kind of chemistry seen today. Doron Lancet, a genomics researcher at the Weizmann Institute of Science in Israel, posits an alternative theory that rests on assemblies of lipids that catalyze the entrance and exit of various molecules. Information is carried not by genetic sequences, but rather by the lipid composition of such assemblies.

    Just like the model proposed by Carter and Wills, Lancet’s ideas involve not one type of molecule but a huge variety of them. “More and more bits of evidence are accumulating,” Lancet said, “that can make an alternative hypothesis be right.” The jury is still out on what actually transpired at life’s origins, but the tide seems to be turning away from a story dedicated solely to RNA.

    “We should put only a few of our eggs in the RNA world basket,” Hofmeyr said.

    See the full article here .

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    Formerly known as Simons Science News, Quanta Magazine is an editorially independent online publication launched by the Simons Foundation to enhance public understanding of science. Why Quanta? Albert Einstein called photons “quanta of light.” Our goal is to “illuminate science.” At Quanta Magazine, scientific accuracy is every bit as important as telling a good story. All of our articles are meticulously researched, reported, edited, copy-edited and fact-checked.

    • stewarthoughblog 2:02 am on December 20, 2017 Permalink | Reply

      It is about time that RNA nonsense comes to an end. There is admittedly considerable scientific knowledge that has and can still be gained by studying RNA macromolecules, but the desperation of naturalists proposing it either as the source of first life is intellectually insulting. RNA’s complexity may be assemblable in intelligent design highly managed labs environments, but ridiculous to consider possible in any geochemically relevant primordial environment. RNA is easily mutated, highly reactive, and only an intermediate macromolecule restricted by the protein catch-22.

      Frustratingly, the desperation continues with propositions of lipid collective assembly, but at least science appears to coming to its senses about RNA.


  • richardmitnick 1:03 pm on December 16, 2017 Permalink | Reply
    Tags: , , Experiments with zebrafish, Gdf3, Genetic instructions from mom set the pattern for embryonic development, Genetics, If gdf3 is not supplied to the egg by the mother the fertilized egg cannot produce two of the three major types of cells required for development, mRNAs-messenger RNAs, Ndr1 and Ndr2 are required to form the mesoderm and endoderm, , TGF-beta family of cell-signaling molecules, Vg1   

    From Princeton University Research Blog: “Genetic instructions from mom set the pattern for embryonic development” 

    Princeton University
    Princeton University Research Blog

    December 15, 2017
    Department of Molecular Biology

    No image caption or credit.

    A new study indicates an essential role for a maternally inherited gene in embryonic development. The study found that zebrafish that failed to inherit specific genetic instructions from mom developed fatal defects earlier in development, even if the fish could make their own version of the gene. The study by researchers at Princeton University was published Nov. 15 in the journal eLife.

    When female animals form egg cells inside their ovaries, they deposit messenger RNAs (mRNAs) – a sort of genetic instruction set – in the egg cell cytoplasm. After fertilization, these maternally supplied mRNAs can be translated into proteins required for the early stages of embryonic development, before the embryo is able to produce mRNAs and proteins of its own.

    More than thirty years ago, researchers discovered that mRNAs encoding a protein called Vg1 are deposited in the cytoplasm of frog eggs. “vg1 is famous for being one of the first recognized maternal mRNAs,” said Rebecca Burdine, associate professor of molecular biology at Princeton. “Many papers have been written on how this RNA is localized and regulated, but it was never clear what the Vg1 protein actually does in the developing embryo.”

    Compared to a normal zebrafish embryo (right), an embryo lacking gdf3 (left) inherited from mom shows major defects resulting from its inability to form mesoderm and endoderm cells early in development. Credit: Pelliccia et al., 2017.

    In the study, Burdine and two graduate students Jose Pelliccia and Granton Jindal used CRISPR/Cas9 gene editing to remove Vg1, known as Gdf3 in zebrafish. Embryos that couldn’t produce any Gdf3 of their own–but received a healthy portion of the gdf3 mRNA from their mothers–developed perfectly normally. But embryos that didn’t receive maternal gdf3 mRNA showed major defects early on in their development, dying just three days after fertilization.

    “If gdf3 is not supplied to the egg by the mother, the fertilized egg cannot produce two of the three major types of cells required for development,” Burdine said. “The embryos lack all [cell types known as] mesoderm and endoderm and are left with skin and some neural tissue, [which derive from the third major cell type, the ectoderm].”

    Vg1/Gdf3 is a member of the TGF-beta family of cell-signaling molecules. Two other members of this family, Ndr1 and Ndr2, are required to form the mesoderm and endoderm early in zebrafish development. Embryos lacking maternally supplied gdf3 look very similar to embryos lacking both of these proteins, which are analogous to the Nodal 1 and 2 proteins in mammals.

    The researchers found that maternal gdf3 is required for Ndr1 and Ndr2 to signal at the levels necessary to properly induce the formation of mesoderm and endoderm cells in early zebrafish embryos. In the absence of gdf3, Ndr1 and Ndr2 signaling is dramatically reduced and embryonic development goes awry.

    Nodal signaling is also required later in zebrafish development when it helps to establish differences between the left and right sides of the developing embryo. It does this, in part, by directing the formation of an organ known as Kupffer’s vesicle, whose asymmetric shape helps determine the embryo’s left and right sides. Subsequently, Nodal signaling induces the expression of a third Nodal protein, called southpaw, in a group of mesoderm cells on the left-hand side of the embryo.

    To investigate whether maternally supplied gdf3 mRNA also plays a role in left-right patterning, the researchers used a series of experimental tricks to supply embryos with enough Gdf3 protein to form the mesoderm and endoderm and survive until the later stages of embryonic development.

    As predicted, these embryos showed defects in left-right patterning. Their Kupffer’s vesicles were abnormally symmetric in shape, and southpaw expression was greatly reduced, suggesting that gdf3 is also required for optimal Nodal signaling during later stages of embryonic development. At this stage, however, embryonic gdf3 seems to be capable of doing the job if maternally supplied gdf3 is absent.

    Nodal and Vg1 proteins are known to bind to each other in other species. “Thus, we hypothesize that Gdf3 combines with Ndr1 and Ndr2 to facilitate Nodal signaling during zebrafish development, acting as an essential factor in embryonic patterning,” said Pelliccia, a graduate student in molecular biology. Co-author Jindal earned his Ph.D. in chemical and biological engineering in 2017.

    At the same time as Burdine and colleagues, two other research groups, led by Joe Yost at the University of Utah and Alex Schier at Harvard University, made similar findings on the role of gdf3 during zebrafish development. “All three groups worked together to co-submit and co-publish in eLife, allowing the students involved to all get credit for their hard work,” Burdine said. “It’s a great example of how science should be done.”

    The research was funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant R01HD048584) and the National Science Foundation (graduate research fellowship DGE 1148900).

    See the full article here .

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  • richardmitnick 5:02 pm on December 14, 2017 Permalink | Reply
    Tags: Amino acids are the building blocks of proteins, , Est1 is a subunit of a protein (an enzyme) called telomerase, Genetics, Identifing previously undiscovered activities for a protein, , ,   

    From Salk: “Revealing the best-kept secrets of proteins” 

    Salk Institute bloc

    Salk Institute for Biological Studies

    December 14, 2017
    No writer credit

    From left: John Lubin, Vicki Lundblad and Tim Tucey. Credit: Salk Institute

    Salk scientists develop new approach to identify important undiscovered functions of proteins.

    In the bustling setting of the cell, proteins encounter each other by the thousands. Despite the hubbub, each one manages to selectively interact with just the right partners, thanks to specific contact regions on its surface that are still far more mysterious than might be expected, given decades of research into protein structure and function.

    Now, Salk Institute scientists have developed a new method to discover which surface contacts on proteins are critical for these cellular interactions. The novel approach shows that essential new functions can be uncovered even for well-studied proteins, and has significant implications for therapeutic drug development, which depends heavily on how drugs physically interact with their cellular targets. The paper appeared in the early online version of Genetics in late November, and is slated for publication in the January print edition of the journal.

    “This paper illustrates the power of this methodology,” says senior author Vicki Lundblad, holder of the Ralph S. and Becky O’Conner Chair. “It can not only identify previously undiscovered activities for a protein, but it can also pinpoint the exact amino acids on a protein surface that perform these new functions.”

    Amino acids are the building blocks of proteins. Their specific linear arrangement determines the identity of a protein, and clusters of them on the protein’s surface serve as contacts, regulating how that protein interacts with other proteins and molecules. Lundblad and her colleagues suspected that, despite decades of work deciphering the mysteries of proteins, the extent of this regulatory landscape on the surface of proteins had remained mostly unexplored. Long ago, her group unexpectedly discovered one such regulatory amino acid cluster, while searching one-by-one through 300,000 mutant yeast cells. Although that work opened up a new area of research in the field of telomere biology, Lundblad was determined to figure out a more robust methodology that could rapidly uncover many more of these unexplored protein surfaces.

    Enter John Lubin, now a PhD student in Lundblad’s lab, who began working with her as an undergraduate.

    “My task was to figure out how to search through 30 mutant yeast cells, instead of 300,000, to discover new activities for a protein,” says Lubin, the paper’s co–first author. Timothy Tucey, the other co–first author, was a postdoctoral researcher in Lundblad’s group and is now at Monash University.

    Together they turned to a protein called Est1, which Lundblad had discovered in yeast as a postdoctoral researcher in 1989. Est1 is a subunit of a protein (an enzyme) called telomerase, which keeps the protective caps at the ends of chromosomes (known as telomeres) from getting too short. As the first subunit of telomerase to be discovered, Est1 has been subjected to intensive study by many research groups.

    The Salk team’s approach involved introducing a small, but customized, set of mutations into yeast cells that would selectively disrupt surface contacts on the cells’ Est1 protein. The team then analyzed the cells to see what effect, if any, the various mutations had. Abnormalities resulting from a specific mutation would suggest what the role of the unmutated version was. To do so, they used a genetic trick, by flooding the cells with each mutant protein, and looking for the rare mutant protein that could interfere with cell function, as their previous work had shown that this would preferentially target the protein surface.

    Lundblad’s team discovered four functions for Est1 through this approach. Impairment of any of these four functions by mutations to Est1’s surface amino acids, the scientists found, resulted in cells that had critically short telomeres, indicating specific roles for the Est1 contacts in the telomerase complex.

    “What has us excited about this technique is that it can be applied to numerous proteins,” says Lundblad. “In particular, many therapeutic drugs rely on being able to access a very specific location on a protein surface, which we suspect can be uncovered by this method.”

    Using this approach, her team has already uncovered new functions for a set of proteins that regulate the stability of the genome, and has also applied for grants that fund research into drug targets.

    The work was funded by the National Institutes of Health, the National Science Foundation, the Rose Hills Foundation and the Glenn Center for Aging Research.

    See the full article here .

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    Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

  • richardmitnick 3:28 pm on December 14, 2017 Permalink | Reply
    Tags: , , Genetics, Single-stranded DNA and RNA origami go live,   

    From Wyss: “Single-stranded DNA and RNA origami go live” 

    Harvard bloc tiny
    Wyss Institute bloc
    Wyss Institute

    December 14, 2017
    Benjamin Boettner

    Single-stranded origami technology is based on design rules that can be used to cross DNA strands in and out of single stranded regions to build large nanostructures. Credit: Molgraphics.

    Like genetic DNA (and RNA) in nature, these engineered nanotechnological devices are also made up of strands that are comprised of the four bases known in shorthand as A, C, T, and G. Regions within those strands can spontaneously fold and bind to each other via short complementary base sequences in which As from one sequence specifically bind to Ts from another sequence, and Cs to Gs. Researchers at the Wyss Institute of Biologically Inspired Engineering and elsewhere have used these features to design self-assembling nanostructures such as scaffolded DNA origami and DNA bricks with ever-growing sizes and complexities that are becoming useful for diverse applications. However, the translation of these structures into medical and industrial applications is still challenging, partially because these multi-stranded systems are prone to local defects due to missing stands. In addition, they self-assemble from hundreds to thousands of individual DNA sequences that each need to be verified and tested for high-precision applications, and whose expensive synthesis often produces undesired side products.

    Now, a novel approach published in Science by a collaborative team of researchers from the Wyss Institute, Arizona State University, and Autodesk for the first time enables the design of complex single-stranded DNA and RNA origami that can autonomously fold into diverse, stable, user-defined structures. In contrast to the synthesis of multi-stranded nanostructures, these entirely new types of origami are folded from one single strand, which can be replicated in living cells, allowing their potential low-cost production at large scales and with high purities, opening entirely new opportunities for diverse applications such as drug delivery and nanofabrication.

    Earlier generations of larger-sized origami are composed of a central scaffold strand whose folding and stability requires more than two hundred short staple strands that bridge distant parts of the scaffold and fix them in space. “In contrast to traditional scaffolded origamis, which are assembled from hundreds of components, our new approach allows us to reliably design and synthesize stable single-stranded and self-folding origami,” said Wyss Institute Core Faculty member and corresponding author Peng Yin, Ph.D. “Our fundamentally new approach relies on single-strand folding, rather than multi-component assembly, to produce large nanostructures. This, together with the ability to basically clone and multiply the single component strand in bacteria, presents a game-changing advance in DNA nanotechnology that greatly enhances single-stranded origami’s potential for real-world applications.” Yin is also co-lead of the Wyss Institute’s Molecular Robotics Initiative and Professor of Systems Biology at Harvard Medical School (HMS).

    To first enable the production of single-stranded and stable DNA-based origami with distinct folding patterns, the team had to overcome several challenges. In a large DNA strand that goes through a complex folding process, many sequences need to accurately pair up with sequences that are far away from each other. If this process does not happen in an orderly and precise fashion, the strand gets tangled and forms unspecific knots along the way, rendering it useless. “To avoid this problem, we identified new design rules that we can use to cross DNA strands between different double-stranded regions and developed a web-based automated design tool that allows researchers to integrate many of these events into a folding path leading up to a large knot-free nanocomplex,” said Dongran Han, Ph.D., the study’s first author and a Postdoctoral Fellow on Yin’s team.

    This schematic shows how a single strand of DNA can be programmed to self-fold into a large nanostructure, like, for example, that of a heart. The Wyss Institute researchers used atomic force microscopy to visualize the heart-shaped and a variety of other nanostructures, which can inexpensively and consistently be multiplied using bacteria as nanofactories. Credit: Wyss Institute at Harvard University.

    The largest DNA origami structures created previously were assembled by synthesizing all their constituent sequences individually in vitro and by mixing them together. As a key feature of the new design process, the single-strandedness of the DNA origami allowed the researchers to introduce DNA sequences stably into E. coli bacteria to inexpensively and accurately replicate them with every cell division. “This could greatly facilitate the development of single-stranded origami for high-precision nanotech like drug delivery vehicles, for example, as only a single easy-to-produce molecule needs to be validated and approved,” said Han.

    Finally, the team also adapted single-stranded origami technology to RNA, which as a different nucleic acid material offers certain advantages including, for example, even higher production levels in bacteria, and usefulness for potential intra-cellular and therapeutic RNA applications. Translating the approach to RNA also scales up the size and complexity of synthetic RNA structures 10-fold compared to previous structures made from RNA.

    Their proof-of-concept analysis also proved that protruding DNA loops can be precisely positioned and be used as handles for the attachment of functional proteins. In future developments, single-stranded origami could thus be potentially functionalized by attaching enzymes, fluorescent probes, metal particles, or drugs either to their surfaces or within cavities inside. This could effectively convert single-stranded origami into nanofactories, light-sensing and emitting optical devices, or drug delivery vehicles.

    In this disc-shaped single-stranded DNA origami, visualized with atomic force microscopy, individual protruding hairpins have been introduced at positions that together compose a “smiley face” and that can be functionalized with useful molecules and activities. Credit: Wyss Institute at Harvard University

    “This new advance by the Wyss Institute’s Molecular Robotics Initiative transforms an exciting laboratory research methodology into a potentially transformative technology that can be manufactured at large scale by leveraging the biological machinery of living cells. This work opens a path by which DNA nanotechnology and origami approaches may be translated into products that meet real-world challenges,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at Harvard’s John A. Paulson School of Engineering and Applied Sciences (SEAS).

    The results announced today establish DNA nanotechnology as a viable alternative approach for applications that have the potential to benefit all of us and the Nation as a whole,” said Jim Kurose, Assistant Director of the National Science Foundation’s (NSF) Directorate for Computer and Information Science and Engineering (CISE). “We are delighted this work was supported by NSF’s Expeditions in Computing program, which has, over the last decade funded large teams of researchers to pursue ambitious, fundamental research agendas that help define and shape the future of computer and information science and engineering, and impact our national competitiveness.

    Besides Yin and Han, the study includes corresponding authors Hao Yan, Ph.D., and Fei Zhang, Ph.D., Director and Assistant Professor at the Biodesign Center for Molecular Design and Biomimetics at Arizona State University, Tempe, respectively, and Byoungkwon An, Ph.D., Principle Research Scientist at Autodesk Research, San Francisco; Shuoxing Jiang, Ph.D., Xiaodong Qi, and Yan Liu, Ph.D., Assistant Professor from the Biodesign Institute; Cameron Myhrvold, Ph.D., Bei Wang, and Mingjie Dai, Ph.D., past and present members of Yin’s team at the Wyss Institute; and Maxwell Bates, who worked with An. The study was funded by the Office of Naval Research, the Army Research Office, the National Science Foundation’s Expeditions in Computing program, and the Wyss Institute for Biologically Inspired Engineering.

    See the full article here .

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    The Wyss (pronounced “Veese”) Institute for Biologically Inspired Engineering uses Nature’s design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world.

    Working as an alliance among Harvard’s Schools of Medicine, Engineering, and Arts & Sciences, and in partnership with Beth Israel Deaconess Medical Center, Boston Children’s Hospital, Brigham and Women’s Hospital, Dana Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Tufts University, and Boston University, the Institute crosses disciplinary and institutional barriers to engage in high-risk research that leads to transformative technological breakthroughs.

  • richardmitnick 9:42 am on November 30, 2017 Permalink | Reply
    Tags: An additional pair of bases—X and Y successfully added to DNA, , , , , Genetics, , Scripps   

    From Science Magazine: “Scientists just added two functional letters to the genetic code” 

    Science Magazine

    Nov. 29, 2017
    Giorgia Guglielmi

    James Cavallini/Science Source

    All life forms on Earth use the same genetic alphabet of the bases A, T, C, and G—nitrogen-containing compounds that constitute the building blocks of DNA and spell out the instructions for making proteins. Now, scientists have developed the first bacterium to use extra letters, or unnatural bases, to build proteins. The new research builds on the team’s previous efforts to expand the natural genetic code. In 2014, the scientists engineered Escherichia coli bacteria (pictured) to incorporate an additional pair of bases—X and Y—into their DNA. The bacteria could store the unnatural bases and pass them onto daughter cells. But to be useful, these bases need to be transcribed into RNA molecules and then translated into proteins. So in the new study the researchers slipped the “alien” pair of bases into bacterial genes that also contained traditional bases. The microbes successfully “read” DNA containing the unnatural bases and transcribed it into RNA molecules. What’s more, the bacteria could use these RNA molecules to produce a variant of green fluorescent protein that contains unnatural amino acids, the team reports today in Nature. The traditional four DNA bases code for 20 amino acids, but the addition of X and Y could produce up to 152 amino acids, which might become building blocks for new drugs and novel materials, the scientists say.

    The scientists behind the work at the Scripps Research Institute have already formed a company to try to use the technique to develop new antibiotics, vaccines and other products, though a lot more work needs to be done before this is practical.

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  • richardmitnick 8:37 am on August 17, 2017 Permalink | Reply
    Tags: , , , , Genetics, SynBio FSP-SynBio Future Science Platform, SynBio-synthetic biology   

    From CSIRO blog: “First steps toward a synthetic biology future” 

    CSIRO bloc

    CSIRO blog

    17 August 2017
    Chris McKay

    The Industrial Revolution set off a wave of technological revolutions. Illustration: D O Hill/Wikimedia Commons

    It was steam power in 18th Century Britain that helped set off the Industrial Revolution, an evolution in technology that would change the course of human history. But that turned out to be only the first in a wave of technological revolutions to follow. From the late 1800s, electricity was being harnessed to allow for mass production, and then in the 1980s, electronics and information technology took the world by storm, heralding the third technological revolution and giving us the digital world we know today.

    Now, we’re in the midst of a fourth technological revolution. Building on the digital revolution that came before it, we’re seeing increasing digital connectedness (think Internet of Things) and a fusing of digital technology with biological systems and technologies. And there has been a step change in the speed at which progress is occurring.

    The trend in the cost of sequencing a human-sized genome since 2001. Image: National Human Genome Research Institute.

    Consider the speed of progress during the IT revolution, which saw computing power doubling roughly every two years in accordance with Moore’s Law. Then contrast that with the rate of progress in the field of biotechnology, which has been exponential: the Human Genome Project, starting in 1990, was a $3 billion USD project that sequenced the human genome for the first time over a period of more than 10 years; then as a result of that work, from 2001 a genome could be sequenced for $100 million USD; and today we can sequence a genome for less than $1000.

    It is in this context that the field of synthetic biology (SynBio) has emerged. SynBio is essentially the application of engineering principles to biology. It involves making things from biological components, such as genetic code, to carry out useful activities. These activities could include sustainable production of fuels, treatment and cure of diseases, controlling invasive pests, or sensing toxins in the environment. Indeed, recent advancements in writing DNA code, printing DNA, and gene editing technology have made SynBio one of the fastest growing areas of modern science. It is a rapidly expanding multi-billion dollar industry with significant potential for generating societal benefits and commercial opportunities.

    That’s why SynBio was among the six new Future Science Platforms we announced last year; a program of investment in areas of science that are set to drive innovation and have the potential to help reinvent and create new industries for Australia. The SynBio Future Science Platform (SynBio FSP) is also growing the capability of a new generation of researchers in partnership with some Australian universities—some of the newest recruits, 11 SynBio Future Science Fellows, will be undertaking work on a suite of innovative projects.

    Future Science Fellow Dr Michele Fabris, based at the University of Technology Sydney’s Climate Change Cluster, will be exploring the potential for photosynthetic microalgae to be modified to carry out new functions, like the production of anti-cancer pharmaceutical compounds. Image: Anna Zhu/UTS

    The research projects cover a broad spectrum of activity. There will be environmental and biocontrol applications, such as the development of cell-tissue structures capable of sensing the environment and eliminating toxins, new tools for targeting antibiotic resistant biofilms, and biosensors providing real-time biological monitoring. Some projects will be exploring the potential to use yeast, microalgae or cyanobacteria cells for the production of valuable pharmaceuticals or fuels, driving innovation in chemical and fibre manufacturing. Other projects will be creating new tools and building blocks that will be fundamental in driving progress in SynBio.

    This work will complement other SynBio FSP research being undertaken at CSIRO that will help us position Australia to play a role in the latest technological revolution. It is research that will allow us to better understand global developments and, where appropriate, contribute responsibly to advances in areas as diverse as healthcare, industrial biotechnology, biosecurity, food and agriculture.

    SynBio FSP’s Future Science Fellowships are co-funded partnerships between CSIRO and the host universities, with each partner contributing matching funding. The host universities are Australian National University, Macquarie University, University of Adelaide, University of Queensland, University of the Sunshine Coast, University of Technology Sydney and University of Western Australia.

    See the full article here .

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    CSIRO, the Commonwealth Scientific and Industrial Research Organisation, is Australia’s national science agency and one of the largest and most diverse research agencies in the world.

    The CSIRO blog is designed to entertain, inform and inspire by generally digging around in the work being done by our terrific scientists, and leaving the techie speak and jargon for the experts.

    We aim to bring you stories from across the vast breadth and depth of our organisation: from the wild sea voyages of our Research Vessel Investigator to the mind-blowing astronomy of our Space teams, right through all the different ways our scientists solve national challenges in areas as diverse as Health, Farming, Tech, Manufacturing, Energy, Oceans, and our Environment.

    If you have any questions about anything you find on our blog, we’d love to hear from you. You can reach us at socialmedia@csiro.au.

    And if you’d like to find out more about us, our science, or how to work with us, head over to CSIRO.au

  • richardmitnick 10:26 am on July 20, 2017 Permalink | Reply
    Tags: , Elizabeth Davis spent 21 years trying to receive a correct diagnosis from doctors about her condition which prevented her toes from uncurling causing her to walk with crutches for the most of her life, Genetics, , GTPCH1 impairs her ability to produce dopa, , Mutagenesis, NCGENES project, They were able to treat it — with something as simple as a pill. A pill that has been on the market since 1988 used to treat patients with Parkinson’s disease,   

    From UNC: “The Cure Code” 

    University of North Carolina

    July 18th, 2017
    Alyssa LaFaro

    Davis can now walk fully unsupported and live a relatively normal life thanks to a correct diagnosis from UNC researchers within the NCGENES project. No image credit.

    “Consider this: In 1969, if a disease-linked gene was found in humans, scientists had no simple means to understand the nature of the mutation, no mechanism to compare the altered gene to normal form, and no obvious method to reconstruct the gene mutation in a different organism to study its function. By 1979, that same gene could be shuttled into bacteria, spliced into a viral vector, delivered into the genome of a mammalian cell, cloned, sequenced, and compared to the normal form.” —Siddhartha Mukherjee, “The Gene: An Intimate History”

    “I can move my toes,” Elizabeth Davis says.

    Her 9-year-old son looks at her in awe. The two stand, wide-eyed in the middle of a Verizon Wireless store in Goldsboro, North Carolina. Davis leans hard against her crutches, staring at her feet. She looks up and smiles.

    At age 37 — for the first time in 31 years — Davis can uncurl her toes from a locked position, the symptom of a condition gone misdiagnosed for just as long. Three months later, she sheds her crutches, walking fully unsupported — something she hasn’t done since she was 14 years old.

    In 1975, the same year Davis was born, UNC microbiologists Clyde Hutchison and Marshall Edgell experienced a different kind of life-changing event. They’d been working rigorously to isolate DNA within the smallest-known virus at the time, Phi-X174. More than anything, they wanted to understand how to read the genetic code. Then, later that year and across the pond at St. John’s College in Cambridge, Fred Sanger figured it out. The British biochemist became the first person to develop a relatively rapid method for sequencing DNA, a discovery that won him a Nobel Prize in Chemistry — for the second time.

    In response to Sanger’s discovery, Hutchison took a sabbatical and headed to England to work in his lab. During his first year there, he helped uncover the entire sequence of Phi-X174 — the first time this had been done for any organism. While there, he realized the new ability to read DNA could help him and Edgell solve a different problem they’d been having back in North Carolina: fusing two pieces of DNA code together to create an entirely different sequence.

    After returning to Chapel Hill, Hutchison continued his work with Edgell and also Michael Smith, a researcher at the University of British Columbia who he met while working in Sanger’s lab. Together, the trio successfully fused two differing DNA strands using a more flexible approach to site-directed mutagenesis — a technique that makes gene therapy possible today. They published their results in 1978. Smith would go on to receive the Nobel Prize for this work in 1992.


    The scientific breakthroughs of the 1970s changed the field of genetics forever. In 1980, Sanger received the Nobel Prize for Chemistry for his contributions, along with Walter Gilbert (Harvard), who discovered that individual modules from different genes mix and match to construct entirely new genes; and Paul Berg (Stanford), who developed a technique for splicing recombinant DNA.

    Meanwhile, researchers in Chapel Hill continued to chip away at the mysteries of the gene. Oliver Smithies, who came to UNC in 1987, would later win the Nobel Prize for his work in gene targeting using mouse models. That same year, UNC cancer geneticist Michael Swift and team discover the AT gene, which predisposes women to breast cancer; and George McCoy becomes the first clinical trial participant in the world to receive the genetically engineered Factor VIII gene to treat his hemophilia at the then UNC-Thrombosis and Hemostasis Center.

    Genetics was changing the world. And this was only the beginning.

    An unsolved mystery

    One year after Sanger won the Nobel Prize, Elizabeth Davis turned 6. She soon began walking on her toes, which had suddenly, one day, curled under in pain, making it nearly impossible for her to stride with feet flat on the ground. Her knees knocked together as she struggled to move with the swift pace characteristic of a child her age. Davis continued to walk on her toes for years.

    “I would even brace the school walls when walking down the hallway,” she says. Eventually, the pain became unbearable. By the time she was 12, she’d resigned herself to crutches.

    Doctors believed Davis’ condition could be treated with foot surgery, misdiagnosing her condition for years. By age 14, she had already undergone three procedures — two to lengthen her Achilles tendons and an experimental bone fusion. But each surgery offered little to no relief, and walking only grew more painful for Davis, both physically and emotionally. As her condition worsened, her classmates became cruel — so much so that she dropped out of high school when she was just 16.

    By age 20, Davis grew restless. “The pain was constant,” she remembers. “I could hardly move my legs — they just felt weak. I would drag them behind me as I used my crutches. I couldn’t even lift them.” Doctors suggested she undergo a third Achilles tendon lengthening surgery, the result of which minimally improved her condition.

    “By that age, I just wanted more,” Davis says. “I just wanted to do things, to go places. I wanted the surgery to work. But it didn’t. And the pain continued.”

    It would be another 17 years before doctors realized the problem was hidden in her genome.

    The birth of a department

    In 1990, the start of the Human Genome Project — an international research program to map out the 20,000 genes that define human beings — further fueled new discoveries in the field of genetics. So when Jeff Houpt, then-UNC School of Medicine dean, formed a research advisory committee in 1997 and asked his faculty what the number-one research program the university needed to focus on, they responded: genetics and genome sciences.

    Great minds think alike. At the same time, the College of Arts and Sciences was also hosting its own committee that vied to develop a genetics department. “At this point, I had a vision for a pan-university program,” Houpt shares. “This wasn’t just going to be a program of the medical school.”

    Along with the College, the schools of public health, dentistry, pharmacy, nursing, and information and library science all wanted in, offering financial assistance to the program. Then-Provost Robert Shelton and Chancellor James Moeser both signed off on it as well. “What we wanted from Shelton and Moeser was more money and more positions,” Houpt remembers. “And they agreed to that.”

    By 2000, a hiring committee was ready to interview candidates to chair the new department and genomics center. Terry Magnuson quickly emerged as the lead candidate. He and his team had spent the past 16 years researching developmental abnormalities using genetics and mouse models, successfully changing the genetic background of a mutated gene.

    “It was obvious he was going to have a following,” Houpt remembers. “People were going to listen to him because he’s a good scientist. But more than that, it was pretty clear that Terry was interested in building a program, and this university-wide effort appealed to him.”

    Unanswered pain

    By the time she reached her 30s, Davis’ condition had spread to her arms. She underwent multiple MRIs, nerve and muscle testing, and a spinal tap. She even endured a fifth, unsuccessful surgery on her feet. Physicians misdiagnosed her yet again. A few believed she suffered from hereditary spastic paraplegia, a genetic condition that causes weakness in the legs and hips. Another told her she had cerebral palsy. “But I didn’t want to believe him,” she says — and it’s a good thing she didn’t.

    As Davis continued her search for answers, walking grew more and more painful. “I was always in pain,” she admits. “But some weeks were really, really bad — to the point where I couldn’t even move.” She finally succumbed to the assistance of a wheelchair. “I hated it so much. I barely went anywhere.” And when she did, she needed help.

    Her mother assisted her regularly with everyday tasks like grocery shopping. Her youngest son, Alex, learned to expertly navigate her around high school gyms, baseball fields, and the local YMCA pool so she could watch her other son, Myles, compete in the plethora of sports he participated in.

    “Myles really experienced the worst of it,” Davis says. “I remember one time, in particular. I was taking a shower and knew I was about to fall. I called for him and he came running. He was always there to pick me back up.”

    Sequences and algorithms

    After the Human Genome Project published its results in 2004, genomic sequencing became an option for people with undiagnosed diseases. But analyzing and understanding the 3 billion base pairs that make up a person’s genetic identity was an expensive process. As time progressed and technology improved, though, the technique became more manageable for both physicians and patients.

    Using these new genomic technologies for outpatient care intrigued UNC geneticists James Evans and Jonathan Berg. In 2009, after gathering enough preliminary data, the NIH granted the team the funds to start the North Carolina Clinical Genomic Evaluation by NextGen Exome Sequencing (NCGENES), which uses whole exome sequencing (WES) to uncover the root cause of undiagnosed diseases. Using just two tablespoons of blood, WES tests 1 percent of the genome — a feat that is both miraculous and controversial, creating a whole new wave of ethical questions.

    Simply put: “Some people want information that other people don’t,” Evans explains. Most people want to know about genetic disorders that have treatment options, but when it comes to those that don’t, they’d rather not hear it. “Navigating those different viewpoints can be a challenge,” he says. Privacy and confidentiality also present problems within the insurance world. Although protections exist in the realm of medical insurance, major genetic predispositions could have large implications for life, disability, and long-term care insurance.

    Today, upward of 50 researchers from across Carolina participate in NCGENES to study everything from the protection of data to the delivery of results. More than 750 people with undiagnosed diseases have undergone testing.

    NCGENES wouldn’t exist without the technical infrastructure that tracks, categorizes, and helps analyze genetic material as it makes its way through multiple laboratories — all of which is provided by UNC’s Renaissance Computing Institute (RENCI). A developer of data science cyberinfrastructure, RENCI provides the software programming that helps the team at NCGENES analyze genomes more effectively.

    “You need new computer algorithms to solve new science problems,” RENCI Director Stan Ahalt says. “It takes a multidisciplinary team to understand science problems like genetics — and computer code to make that process go fast.”

    A transformative experience

    By 2013, Davis was in desperate need of a new algorithm. Thankfully, that year, she was referred to Jane Fan, a pediatric neurologist at UNC. After studying Davis’ file, Fan felt sure that the doctors who tried to diagnose her condition failed, making her the perfect candidate for NCGENES.

    Four tubes of blood, 100,000 possible genetic locations, and just over six months later, Fan called Davis. A single gene mutation called GTPCH1 impairs her ability to produce dopa, an amino acid crucial for nervous system function. “I had to hear it in person before I believed it,” Davis admits. “I had been misdiagnosed many times before.”

    Not only were UNC geneticist James Evans and his NCGENES team finally able to accurately diagnose Davis, but they were able to treat it — with something as simple as a pill. A pill that has been on the market since 1988, used to treat patients with Parkinson’s disease.

    And just like that, Davis ‘life was changed forever by genome sequencing.

    Three days after she took one-quarter of a pill, movement returned to her toes while standing in the middle of a Verizon Wireless store in Goldsboro. She began to cry.

    Top-five in the country

    UNC’s genetics department has ranked in the top-five programs for NIH funding across the nation every year since 2012 (and top-10 each year since 2006). “I think we’ve built one of the best genetics departments in the country,” Magnuson says. In 2016 alone, genetics department faculty brought $38 million to Carolina.

    Houpt agrees with Magnuson’s sentiment. “The genetics department is a great example of how universities should run,” he says. “People need to put aside their own interests and see what’s needed. Terry is a leader who’s made each school involved feel like it’s their program and not just a medical school program – which is why he’s now the vice chancellor for research.”

    Today, more than 80 faculty members from across campus conduct world-recognized genetics research in multiple disciplines.

    Ned Sharpless, for example, focuses on cancer. Most recently, the director of the UNC Lineberger Comprehensive Cancer Center lead a study that paired UNCseq — a genetic sequencing protocol that produces volumes of genetic information from a patient’s tumor — with IBM Watson’s ability to quickly pull information from millions of medical papers. A procedure much too intense and time-consuming for the human mind, this data analysis can help physicians make more informed decisions about patient care.

    Another member of Carolina’s Cancer Genetics Program, Charles Perou uses genomics to characterize the diversity of breast cancer tumors — research that helps doctors guarantee patients more individualized care. In 2011, he cofounded GeneCentric, which uses personalized molecular diagnostic assays and targeted drug development to treat cancer.

    In 2015, geneticist Aravind Asokan started StrideBio with University of Florida biochemist Mavis Agbandje-McKenna. The gene therapy company develops novel adeno-associated viral (AAV) vector technologies for treating rare diseases. Although still in its infancy, the company has already partnered with CRISPR Therapeutics and received an initial investment from Hatteras Venture Partners. Asokan has spent nearly a decade studying AAV — and even helped to, previously, cofound Bamboo Therapeutics, acquired by Pfizer for $645 million just last year.

    In 2016, current genetics department Chair Fernando Pardo-Manuel de Villena challenged both Darwin’s theory of natural selection and Mendel’s law of segregation through researching a mouse gene called R2d2. In doing so, he found that a selfish gene can become fixed in a population of organisms while, at the same time, being detrimental to “reproductive fitness” — a discovery that shows the swiftness at which the genome can change, creating implications for an array of fields from basic biology to agriculture and human health.

    A former student of Oliver Smithies, Beverly Koller uses gene targeting in mice to better understand diseases like cystic fibrosis, asthma, and arthritis — research that will ultimately lead to better treatments. Similarly, Mark Heise observes mice to study diseases caused by viruses including infectious arthritis and encephalitis (inflammation of the brain). Both researchers are part of the Collaborative Cross project, a large panel of inbred mouse strains that help map genetic traits — a resource that is UNC lead, according to Magnuson.

    Genetics research stems far beyond the UNC School of Medicine. In 2009, for example, chemist Kevin Weeks and his research team decoded the HIV genome, advancing the development of new therapies and treatments. UNC sociologist Gail Henderson runs the Center for Genomics and Society, which provides research and training on ethical, legal, and social implications of genomic research. In 2015, UNC Eshelman School of Pharmacy Dean Bob Blouin helped the school become the first U.S. hub to join the international Structural Genomics Consortium — focused on discovering selective, small molecules and protein kinases to help speed the creation of new medicines for patients.

    From crutches to a 5K

    After just three months of treatment, Davis walked fully unsupported for the first time since she was 6 years old. She’s since traversed Hershey Park in Pennsylvania, strolled around the World Trade Center in New York, and regularly participated in yoga and spin classes. This past May, she walked her first 5K. “I have crazy endurance,” she says. “When your body feels good, you just want to keep on going.”

    Perhaps, more importantly, Davis is able attend Alex’s sports games without assistance. “When I used to walk into the gym on crutches to watch my oldest son play basketball, everyone would look at my crutches and my legs,” she says. “Now, when I go watch my youngest son play, I have so much more confidence walking in to the gym. People see me.”

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    Carolina’s vibrant people and programs attest to the University’s long-standing place among leaders in higher education since it was chartered in 1789 and opened its doors for students in 1795 as the nation’s first public university. Situated in the beautiful college town of Chapel Hill, N.C., UNC has earned a reputation as one of the best universities in the world. Carolina prides itself on a strong, diverse student body, academic opportunities not found anywhere else, and a value unmatched by any public university in the nation.

  • richardmitnick 8:47 am on July 17, 2017 Permalink | Reply
    Tags: , CRISPR-Cas3, , Genetics, , ,   

    From HMS: “Bringing CRISPR into Focus” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    June 29, 2017

    CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

    Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

    Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detection—a molecular redundancy that prevents unintended genomic damage, the researchers say.

    High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

    “To solve problems of specificity, we need to understand every step of CRISPR complex formation,” said Maofu Liao, assistant professor of cell biology at Harvard Medical School and co-senior author of the study. “Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.”

    Target search

    Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot “enemy” presence.

    Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

    To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

    Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstroms—or roughly three times the diameter of a carbon atom.

    A sample cryo-electron microscope image of CRISPR molecules(left). The research team combined hundreds of thousands of particles into 2D averages (right), before turning them into 3D projections. Image: Xiao et al.

    Seeing is believing

    In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

    Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a “seed bubble.”

    The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an “R-loop” structure.

    Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

    Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

    The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacteria’s own DNA.

    CRISPR forms a “seed bubble” state, which acts as an initial fail-safe mechanism to ensure that CRISPR RNA matches its target DNA. Image: Liao Lab/HMS.

    “To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes,” said Ke, who is co-senior author of the study. “Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.”

    Setting the sights

    Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

    In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

    In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

    “Scientists hypothesized that these states existed but they were lacking the visual proof of their existence,” said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. “The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.”

    “We’ve found that these steps must occur in a precise order,” Luo said. “Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.”

    Additional authors on the study include Yibei Xiao, Robert P. Hayes, Jonathan Kim, Sherwin Ng, and Fang Ding.

    This work is supported by National Institutes of Health grants GM 118174 and GM102543.

    See the full article here .

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    Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

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    Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

    Harvard University has 12 degree-granting Schools in addition to the Radcliffe Institute for Advanced Study. The University has grown from nine students with a single master to an enrollment of more than 20,000 degree candidates including undergraduate, graduate, and professional students. There are more than 360,000 living alumni in the U.S. and over 190 other countries.

  • richardmitnick 1:21 pm on July 8, 2017 Permalink | Reply
    Tags: , , , , , Genetics, , , , UCSD Comet supercomputer   

    From Science Node: “Cracking the CRISPR clock” 

    Science Node bloc
    Science Node

    05 Jul, 2017
    Jan Zverina

    SDSC Dell Comet supercomputer

    Capturing the motion of gyrating proteins at time intervals up to one thousand times greater than previous efforts, a team led by University of California, San Diego (UCSD) researchers has identified the myriad structural changes that activate and drive CRISPR-Cas9, the innovative gene-splicing technology that’s transforming the field of genetic engineering.

    By shedding light on the biophysical details governing the mechanics of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) activity, the study provides a fundamental framework for designing a more efficient and accurate genome-splicing technology that doesn’t yield ‘off-target’ DNA breaks currently frustrating the potential of the CRISPR-Cas9- system, particularly for clinical uses.

    Shake and bake. Gaussian accelerated molecular dynamics simulations and state-of-the-art supercomputing resources reveal the conformational change of the HNH domain (green) from its inactive to active state. Courtesy Giulia Palermo, McCammon Lab, UC San Diego.

    “Although the CRISPR-Cas9 system is rapidly revolutionizing life sciences toward a facile genome editing technology, structural and mechanistic details underlying its function have remained unknown,” says Giulia Palermo, a postdoctoral scholar with the UC San Diego Department of Pharmacology and lead author of the study [PNAS].

    See the full article here

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