Bar Harbor, ME, United States
Bar Harbor, ME, United States

The Howard Hughes Medical Institute is a United States non-profit medical research organization based in Chevy Chase, Maryland. It was founded by the American businessman Howard Hughes in 1953. It is one of the largest private funding organizations for biological and medical research in the United States. HHMI spends about $1 million per HHMI Investigator per year, which amounts to annual investment in biomedical research of about $825 million. The institute has an endowment of $16.9 billion, making it the second-wealthiest philanthropic organization in the United States and the second best endowed medical research foundation in the world. HHMI is the former owner of the Hughes Aircraft Company - an American aerospace firm which was divested to various firms over time. Wikipedia.

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Howard Hughes Medical Institute | Date: 2015-04-01

The presently-disclosed subject matter includes azetidine-substituted fluorescent compounds, where the compounds may be used as probes, dyes, tags, and the like. The presently-disclosed subject matter also includes kits comprising the same as well as methods for using the same to detect a target substance.

Buskirk A.R.,Howard Hughes Medical Institute | Green R.,Howard Hughes Medical Institute
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2017

Ribosomes translate genetic information into polypeptides in several basic steps: initiation, elongation, termination and recycling. When ribosomes are arrested during elongation or termination, the cell’s capacity for protein synthesis is reduced. There are numerous quality control systems in place to distinguish between paused ribosomes that need some extra input to proceed and terminally stalled ribosomes that need to be rescued. Here, we discuss similarities and differences in the systems for resolution of pauses and rescue of arrested ribosomes in bacteria and eukaryotes, and how ribosome profiling has transformed our ability to decipher these molecular events. This article is part of the themed issue ‘Perspectives on the ribosome’. © 2017 The Author(s) Published by the Royal Society. All rights reserved.

Frank J.,Howard Hughes Medical Institute | Frank J.,Columbia University
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2017

During the work cycle of elongation, the ribosome, a molecular machine of vast complexity, exists in a large number of states distinguished by constellation of its subunits, its subunit domains and binding partners. Single-particle cryogenic electron microscopy (cryo-EM), developed over the past 40 years, is uniquely suited to determine the structure of molecular machines in their native states. With the emergence, 10 years ago, of unsupervised clustering techniques in the analysis of single-particle data, it has been possible to determine multiple structures from a sample containing ribosomes equilibrating in different thermally accessible states. In addition, recent advances in detector technology have made it possible to reach near-atomic resolution for some of these states. With these capabilities, single-particle cryo-EM has been at the forefront of exploring ribosome dynamics during its functional cycle, along with single-molecule fluorescence resonance energy transfer and molecular dynamics computations, offering insights into molecular architecture uniquely honed by evolution to capitalize on thermal energy in the ambient environment. This article is part of the themed issue ‘Perspectives on the ribosome’. © 2017 The Author(s) Published by the Royal Society. All rights reserved.

Park E.,Howard Hughes Medical Institute | Campbell E.B.,Howard Hughes Medical Institute | MacKinnon R.,Howard Hughes Medical Institute
Nature | Year: 2017

CLC proteins transport chloride (Cl-) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl- ions passively, whereas others are secondary active transporters that exchange two Cl- ions for one H+. The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture on the basis of sequence homology. Here we determined the structure of a bovine CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl- transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl- passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl-/H+ transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl- down its electrochemical gradient. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Hite R.K.,Howard Hughes Medical Institute | Tao X.,Howard Hughes Medical Institute | MacKinnon R.,Howard Hughes Medical Institute
Nature | Year: 2017

The precise control of an ion channel gate by environmental stimuli is crucial for the fulfilment of its biological role. The gate in Slo1 K+ channels is regulated by two separate stimuli, intracellular Ca2+ concentration and membrane voltage. Slo1 is thus central to understanding the relationship between intracellular Ca2+ and membrane excitability. Here we present the Slo1 structure from Aplysia californica in the absence of Ca2+ and compare it with the Ca2+ -bound channel. We show that Ca2+ binding at two unique binding sites per subunit stabilizes an expanded conformation of the Ca2+ sensor gating ring. These conformational changes are propagated from the gating ring to the pore through covalent linkers and through protein interfaces formed between the gating ring and the voltage sensors. The gating ring and the voltage sensors are directly connected through these interfaces, which allow membrane voltage to regulate gating of the pore by influencing the Ca2+ sensors. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Tao X.,Howard Hughes Medical Institute | Hite R.K.,Howard Hughes Medical Institute | MacKinnon R.,Howard Hughes Medical Institute
Nature | Year: 2017

The Ca2+ -activated K+ channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca2+ renders Slo1 central to processes that couple electrical signalling to Ca2+ -mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca2+ and Mg2+ at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage-sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca2+ -binding domain from a neighbouring subunit. Unique structural features of the Slo1 voltage sensor suggest that it undergoes different conformational changes than other known voltage sensors. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels.

Sieber M.H.,Howard Hughes Medical Institute | Spradling A.C.,Howard Hughes Medical Institute
Current Opinion in Genetics and Development | Year: 2017

During development, cells adopt distinct metabolic strategies to support growth, produce energy, and meet the demands of a mature tissue. Some of these metabolic states maintain a constrained program of nutrient utilization, while others providing metabolic flexibility as a means to couple developmental progression with nutrient availability. Here we discuss our understanding of metabolic programs, and how they support specific aspects of animal development. During phases of rapid proliferation a subset of metabolic programs provide the building blocks to support growth. During differentiation, metabolic programs shift to support the unique demands of each tissue. Finally, we discuss how a model system, such as Drosophila egg development, can provide a versatile platform to discover novel mechanisms controlling programmed shift in metabolism. © 2017 Elsevier Ltd

Tomasetti C.,Johns Hopkins University | Vogelstein B.,Howard Hughes Medical Institute
Science | Year: 2017

Cancers are caused by mutations that may be inherited, induced by environmental factors, or result from DNA replication errors (R).We studied the relationship between the number of normal stem cell divisions and the risk of 17 cancer types in 69 countries throughout the world. The data revealed a strong correlation (median = 0.80) between cancer incidence and normal stem cell divisions in all countries, regardless of their environment. The major role of R mutations in cancer etiology was supported by an independent approach, based solely on cancer genome sequencing and epidemiological data, which suggested that R mutations are responsible for two-thirds of the mutations in human cancers. All of these results are consistent with epidemiological estimates of the fraction of cancers that can be prevented by changes in the environment. Moreover, they accentuate the importance of early detection and intervention to reduce deaths from the many cancers arising from unavoidable R mutations.

Chen X.,Howard Hughes Medical Institute | Rosbash M.,Howard Hughes Medical Institute
Nature Communications | Year: 2017

Many biological and behavioural processes of animals are governed by an endogenous circadian clock, which is dependent on transcriptional regulation. Here we address post-transcriptional regulation and the role of miRNAs in Drosophila circadian rhythms. At least six miRNAs show cycling expression levels within the pigment dispersing factor (PDF) cell-pacemaker neurons; only mir-92a peaks during the night. In vivo calcium monitoring, dynamics of PDF projections, ArcLight, GCaMP6 imaging and sleep assays indicate that mir-92a suppresses neuronal excitability. In addition, mir-92a levels within PDF cells respond to light pulses and also affect the phase shift response. Translating ribosome affinity purification (TRAP) and in vitro luciferase reporter assay indicate that mir-92a suppresses expression of sirt2, which is homologous to human sir2 and sirt3. sirt2 RNAi also phenocopies mir-92a overexpression. These experiments indicate that sirt2 is a functional mir-92a target and that mir-92a modulates PDF neuronal excitability via suppressing SIRT2 levels in a rhythmic manner. © The Author(s) 2017.

Taylor J.P.,Howard Hughes Medical Institute | Brown R.H.,University of Massachusetts Medical School | Cleveland D.W.,Ludwig Institute for Cancer Research | Cleveland D.W.,University of California at San Diego
Nature | Year: 2016

Amyotrophic lateral sclerosis (ALS) is a progressive and uniformly fatal neurodegenerative disease. A plethora of genetic factors have been identified that drive the degeneration of motor neurons in ALS, increase susceptibility to the disease or influence the rate of its progression. Emerging themes include dysfunction in RNA metabolism and protein homeostasis, with specific defects in nucleocytoplasmic trafficking, the induction of stress at the endoplasmic reticulum and impaired dynamics of ribonucleoprotein bodies such as RNA granules that assemble through liquid-liquid phase separation. Extraordinary progress in understanding the biology of ALS provides new reasons for optimism that meaningful therapies will be identified. © 2016 Macmilan publishers Limited, part of springer Nature. All Rights reserved.

Ticau S.,Howard Hughes Medical Institute | Correa I.R.,Jr
Nature Structural and Molecular Biology | Year: 2017

The opening and closing of two ring-shaped Mcm2–7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2–7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2–7 during origin licensing. The two Mcm2–7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2–7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2–7 prevented recruitment of the second Mcm2–7. Our findings identify key mechanisms controlling the Mcm2–7 DNA-entry gate during origin licensing, and reveal that the two Mcm2–7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control. © 2017 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

de la Cruz M.J.,Howard Hughes Medical Institute
Nature Methods | Year: 2017

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography. © 2017 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

Burton N.O.,Howard Hughes Medical Institute
Nature Cell Biology | Year: 2017

In 1893 August Weismann proposed that information about the environment could not pass from somatic cells to germ cells, a hypothesis now known as the Weismann barrier. However, recent studies have indicated that parental exposure to environmental stress can modify progeny physiology and that parental stress can contribute to progeny disorders. The mechanisms regulating these phenomena are poorly understood. We report that the nematode Caenorhabditis elegans can protect itself from osmotic stress by entering a state of arrested development and can protect its progeny from osmotic stress by increasing the expression of the glycerol biosynthetic enzyme GPDH-2 in progeny. Both of these protective mechanisms are regulated by insulin-like signalling: insulin-like signalling to the intestine regulates developmental arrest, while insulin-like signalling to the maternal germline regulates glycerol metabolism in progeny. Thus, there is a heritable link between insulin-like signalling to the maternal germline and progeny metabolism and gene expression. We speculate that analogous modulation of insulin-like signalling to the germline is responsible for effects of the maternal environment on human diseases that involve insulin signalling, such as obesity and type-2 diabetes. © 2017 Nature Publishing Group

Kruglyak L.,Howard Hughes Medical Institute
Genetics | Year: 2016

The Genetics Society of America’s Edward Novitski Prize recognizes an extraordinary level of creativity and intellectual ingenuity in the solution of significant problems in genetics research. The 2016 winner, Leonid Kruglyak, has made innovative contributions to the fields of linkage analysis, population genetics, and genomics, while drawing on a combination of mathematical, computational, and experimental approaches. Among other achievements, his work on statistical standards for genome-wide linkage studies has transformed their experimental design, and the linkage analysis program GENEHUNTER has been used to identify hundreds of human disease loci. Kruglyak’s group also pioneered expression quantitative trait locus studies, which enabled variation in global gene expression to shed light on the genetics of complex human diseases. In recent years, his laboratory has focused on using genomic technology to establish Saccharomyces cerevisiae and Caenorhabditis elegans as model organisms for studies of complex genetic variation. © 2016 by the Genetics Society of America.

Johnson Z.L.,Howard Hughes Medical Institute | Chen J.,Howard Hughes Medical Institute
Cell | Year: 2017

The multidrug resistance protein MRP1 is an ATP-binding cassette (ABC) transporter that confers resistance to many anticancer drugs and plays a role in the disposition and efficacy of several opiates, antidepressants, statins, and antibiotics. In addition, MRP1 regulates redox homeostasis, inflammation, and hormone secretion. Using electron cryomicroscopy, we determined the molecular structures of bovine MRP1 in two conformations: an apo form at 3.5 Å without any added substrate and a complex form at 3.3 Å with one of its physiological substrates, leukotriene C4. These structures show that by forming a single bipartite binding site, MRP1 can recognize a spectrum of substrates with different chemical structures. We also observed large conformational changes induced by leukotriene C4, explaining how substrate binding primes the transporter for ATP hydrolysis. Structural comparison of MRP1 and P-glycoprotein advances our understanding of the common and unique properties of these two important molecules in multidrug resistance to chemotherapy. Structural analysis of an ABC transporter contributing to drug efflux and resistance to cancer therapy shows how substrate binding promotes the ATP-dependent transport cycle. © 2017 Elsevier Inc.

Verkoczy L.,Duke University | Alt F.W.,Howard Hughes Medical Institute | Tian M.,Howard Hughes Medical Institute
Immunological Reviews | Year: 2017

A major challenge for HIV-1 vaccine research is developing a successful immunization approach for inducing broadly neutralizing antibodies (bnAbs). A key shortcoming in meeting this challenge has been the lack of animal models capable of identifying impediments limiting bnAb induction and ranking vaccine strategies for their ability to promote bnAb development. Since 2010, immunoglobulin knockin (KI) technology, involving inserting functional rearranged human variable exons into the mouse IgH and IgL loci has been used to express bnAbs in mice. This approach has allowed immune tolerance mechanisms limiting bnAb production to be elucidated and strategies to overcome such limitations to be evaluated. From these studies, along with the wealth of knowledge afforded by analyses of recombinant Ig-based bnAb structures, it became apparent that key functional features of bnAbs often are problematic for their elicitation in mice by classic vaccine paradigms, necessitating more iterative testing of new vaccine concepts. In this regard, bnAb KI models expressing deduced precursor V(D)J rearrangements of mature bnAbs or unrearranged germline V, D, J segments (that can be assembled into variable region exons that encode bnAb precursors), have been engineered to evaluate novel immunogens/regimens for effectiveness in driving bnAb responses. One promising approach emerging from such studies is the ability of sequentially administered, modified immunogens (designed to bind progressively more mature bnAb precursors) to initiate affinity maturation. Here, we review insights gained from bnAb KI studies regarding the regulation and induction of bnAbs, and discuss new Ig KI methodologies to manipulate the production and/or expression of bnAbs in vivo, to further facilitate vaccine-guided bnAb induction studies. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Bell S.P.,Howard Hughes Medical Institute
eLife | Year: 2017

Human cells that lack a subunit in their origin recognition complex are viable, which suggests the existence of alternative mechanisms to initiate DNA replication. © 2017, eLife Sciences Publications Ltd. All rights reserved.

Iwasaki A.,Howard Hughes Medical Institute
Trends in Molecular Medicine | Year: 2017

This review highlights recent advances in how the innate and adaptive immune systems control the blood-brain barrier (BBB) and the blood-nerve barrier (BNB). Interferons and TAM receptors play key roles in innate immune control of the BBB. Cells of the adaptive immune system, particularly CD4+ T cells, take distinct routes to enter neural tissues and mediate immune surveillance. Furthermore, T cell-mediated opening of the BBB and the BNB is crucial to allow antibody access and thereby block the replication of neurotropic viruses. Such novel insights gained from basic research provide key foundations for future design of therapeutic strategies - enabling antibody access to the brain may be key to cancer immunotherapy and to the use of vaccines against neurodegenerative conditions such as Alzheimer's disease. The BBB and BNB limit antibody access to the brain and the peripheral nervous system.The integrity of the BBB and the BNB is enforced by type I and type III interferons, and is reduced by TNF-α, IL-1, IL-6, type II interferon (IFN-γ) and IL-17A.CD4+ T cells of the type 1 and type 17 T helper cell subtypes can open the BBB and BNB upon entry into neuronal tissues.CD4+ T cell opening of the BBB is necessary for antibody-mediated block of neurotropic virus replication.CD4+ T cell-mediated regulation of the BBB has important implications for cancer immunotherapy and for vaccine design to treat conditions such as Alzheimer's disease. © 2017 Elsevier Ltd.

Wang X.,Howard Hughes Medical Institute | Moazed D.,Howard Hughes Medical Institute
Science | Year: 2017

Epigenetic inheritance mechanisms play fundamental roles in maintaining cellular memory of gene expression states. In fission yeast, histone H3 lysine 9 (H3K9me) is methylated at heterochromatic domains. These domains can be epigenetically inherited when epi1+, encoding an enzyme that promotes H3K9 demethylation, is deleted. How native epigenetic states are stably maintained in epe1+ cells remains unknown. Here, we developed a system to examine the role of DNA sequence and genomic context in propagation of a cis-heritable H3K9me-dependent silenced state. We show that in epe1+ cells, in addition to sequence-independent mechanisms that propagate H3K9me, epigenetic inheritance of silencing requires binding sites for sequence-dependent ATF/CREB family transcription factors within their native chromosomal context. Thus, specific DNA sequences contribute to cis inheritance of H3K9me and silent epigenetic states. © 2017 American Association for the Advancement of Science.

Lim W.A.,Howard Hughes Medical Institute | June C.H.,University of Pennsylvania
Cell | Year: 2017

Chimeric antigen receptor (CAR) T cells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Clinical experience has helped to define the major challenges that must be met to make engineered T cells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools for programming immune cells. We discuss how these tools could be used to design the next generation of smart T cell precision therapeutics. © 2017

News Article | April 27, 2017

A protein derived from ticks enhances the effectiveness of antibiotic treatment for methicillin-resistant Staphylococcus aureus, or MRSA, according to a Yale-led study. The strategy of using the protein in combination with existing treatments can help address the growing challenge of antibiotic-resistant MRSA and other staph infections, the researchers said. Resistance to antibiotic treatment is a widespread problem in medicine, and MRSA is prime example of a resistant bacterium that can cause deadly infections. Staph bacteria like MRSA are able to resist treatment in part because they secrete a protective outer layer — a biofilm — that prevents the immune system and antibiotics from gaining access to them. Researchers have identified a tick protein, IAFGP, that alters the biofilm. In the study, the Yale-led team combined the protein, and a molecule derived from it, with antibiotics currently used to treat MRSA as well as other antibiotics that are not standard treatment. The researchers tested the combination of agents — IAFGP with three different antibiotics — in culture and also in MRSA-infected mice and flies. They found that in each case, the combination improved the ability of the antibiotic to combat the bacteria. “If you take this protein and you add it to the current treatment, it makes the treatment much more potent,” said Dr. Erol Fikrig, chief of the Infectious Diseases Section at Yale School of Medicine and the study’s lead author. “If you take the protein and add it to drugs not used in current treatment, it makes them potent as well.” While the study was not done in humans, it provides a novel strategy for tackling antibiotic-resistance bacterial infections. MRSA, which has become resistant to several different antibiotics, can cause severe infections affecting the skin, lungs, and blood. “Our hope is it expands the group of antibiotics that can be used to treat methicillin,” said Fikrig. The findings were published in the journal Antimicrobial Agents and Chemotherapy. Other authors on the study Nabil M. Abraham, Lei Liu, Brandon L. Jutras, Kristen Murfin, Ali Acar, Timur O. Yarovinsky, Erica Sutton, Martin Heisig, and Christine Jacobs-Wagner. The study was supported, in part, by a gift from the John Monsky and Jennifer Weis Monsky Lyme Disease Research Fund and the Howard Hughes Medical Institute.

News Article | April 20, 2017

Proper nutrition can unleash amazing powers, moms have always assured us, frequently citing Popeye the Sailor Man as evidence. Now, two University of Colorado Boulder scientists have confirmed just how potent some nutrients can be. In findings published today in the journal Cell, postdoctoral fellow Hongyun Tang and Professor Min Han, both of CU Boulder's Department of Molecular, Cellular and Developmental Biology and the Howard Hughes Medical Institute, detail how fat levels in a tiny soil-dwelling roundworm (C. elegans) can tip the balance between whether the worm makes eggs or sperm. Although the researchers discovered this phenomenon in worms, the research could have implications for future studies into human fertility and reproductive development. Scientists have long recognized a connection between dietary fat and reproductive development in mammals, including humans. "Studies in humans and rats have suggested that a high-fat diet is a major cause of early puberty in girls," said Han, the senior author of the paper. "It makes sense from an evolutionary standpoint that reproductive success would be coupled to food availability." However, Han said, scientists understand surprisingly little about just how fat levels might be translated into fertility. C. elegans comes in two sexes: males and hermaphrodites. Males produce sperm throughout their reproductive lives while hermaphrodites produce sperm only during a brief period, and later switch gears to making eggs. Through a meticulous series of experiments, Tang homed in on one nutrient, a fatty acid called myristic acid, whose abundance, it turns out, worms can "monitor" using an enzyme called acyl-CoA synthase 4 (ACS-4). Fatty acids are chemical building blocks of dietary fats, so their level in a worm's body is one measure of the general nutritional quality of their food. Tang found that the levels of certain fatty acids, including myristic acid, can influence the switch from sperm to egg production. When Tang depleted a particular myristic acid derivative from the germ cells in hermaphrodite worms by blocking the action of ACS-4, the worms never made the switch to making eggs. Typically, a worm's sex-determination -- in this case, making eggs or sperm -- comes down to counting chromosomes. Hermaphrodites' cells have two sex chromosomes while males have only one. Tang, however, discovered that the worm's sex-determination system could also tap into information gathered by monitoring fatty acid levels. But before the fatty acid can become tethered to the protein, it must be temporarily hitched to a molecule called CoA. ACS-4 is the enzyme that links myristic acid to CoA. Tang found that the worms' sex-determination system can be overruled by the levels of myristoyl-CoA and myristoylated proteins. He determined that one component of the sex-determination system, a protein called MAP kinase, proved to be the link between the fatty acid monitoring and the switch from production of sperm to eggs. This fatty-acid sensing phenomenon does not appear to be particular to hermaphroditic nematode species like C. elegans. When Tang partially blocked ACS-4 in females of a conventional male/female nematode species, many of the females made sperm instead of eggs. The researchers call this newly-discovered ACS-mediated mechanism a "lipid sensor" and believe it could be a widespread strategy by which animals translate cues from the environment into physiological responses. The key proteins at work in the nematodes have highly similar counterparts in humans, suggesting that similar regulatory pathways may operate in people. The work was supported by the Howard Hughes Medical Institute.

News Article | April 17, 2017

Just as paleontologists study dinosaur bones to learn when these prehistoric creatures lived and died, biologists are examining DNA to find out when ancient viruses were at their peak, and how they met their demise. New research from The Rockefeller University has uncovered how our ancestors may have wiped out a primordial virus around 11 million years ago. Published in the journal eLife, the findings suggest that our primate ancestors evolved a defense mechanism that involved manipulating the function of one of the virus's genes, turning the virus against itself. "Analyzing viral fossils can provide a wealth of insight into events that occurred in the distant past," says senior author Paul Bieniasz, head of the Laboratory of Retrovirology and a Howard Hughes Medical Institute investigator. "In particular, this study is an example of how viruses themselves can provide the genetic material that animals use to combat them, sometimes leading to viral extinction." Retroviruses, a class of viruses that include the human immunodeficiency virus (HIV), are abundant in nature and can leave lasting traces of their existence if they infect cells of the germ line. Unlike other viruses, they include a step in their life cycle where their genetic material is integrated into the genome of their host -- creating a genetic fossil record that can be preserved in the genomes of the host and its evolutionary descendants. To examine how extinct viral lineages could once have been eliminated, Bieniasz and colleagues analyzed retroviral fossils left by human endogenous retrovirus T (HERV-T), which replicated in our primate ancestors for approximately 25 million years before it was eradicated about 11 million years ago. Working with Robert Gifford from the University of Glasgow, the team first compiled a near-complete catalog of HERV-T fossils in old-world monkey and ape genomes. They then reconstructed the HERV-T retrovirus' outer envelope protein--a molecule that allows a virus particle to bind to cells and begin the viral replication cycle. "Our analyses suggested that HERV-T likely used a cell-surface protein called MCT-1 to bind to cells and infect ancient old-world primates," says first author Daniel Blanco-Melo, a former graduate student in the Bieniasz lab. The researches also identified a fossilized HERV-T gene in the genomes of contemporary humans that was absent in more distantly-related primate genomes. They found that this gene encodes a well-preserved envelope protein that can block retroviral infection by depleting MCT-1 from cell surfaces. "It appears this gene was integrated into the ancestral primate genome around 13 to 19 million years ago, and we believe it was around this time that the function of this gene switched," says Blanco-Melo. "Ancestral hominids evolved a defense mechanism against HERV-T, using the virus's own gene against itself, eventually leading to its extinction."

News Article | May 2, 2017

When opioids act on the brain, they trigger the same processes that give people feelings of pleasure from activities like eating, but they do it far more intensely. Opioids also make some brain cells pump out a chemical messenger called dopamine, which encourages more drug use. Over time, that can produce craving that continues even long after someone stops using opioids, which can lead to relapse. In other brain circuits, opioids initially produce drowsiness and slower breathing. With repeated exposure, these circuits adapt so that a person feels relatively normal while using the drugs. But that adaptation also means that when a person is not using, they feel jittery and anxious — some of the symptoms of withdrawal. Opioids can also impair people's self-control if taken over time, so it's harder to stop using them even if people want to and even if the drugs no longer give them pleasure. Dr. Nora Volkow, director of the National Institute on Drug Abuse, compared the effect of the drugs to driving with bad steering. "Your steering wheel does not work properly. So not only are you actually accelerating with intense desire and motivation to get the drug, you are not able to self-regulate and say, 'If I take the drug, I will end up in jail.'" This Associated Press series was produced in partnership with the Howard Hughes Medical Institute's Department of Science Education. The AP is solely responsible for all content.

Researchers from UT Southwestern Medical Center have developed a first-of-its-kind nanoparticle vaccine immunotherapy that targets several different cancer types. The nanovaccine consists of tumor antigens – tumor proteins that can be recognized by the immune system – inside a synthetic polymer nanoparticle. Nanoparticle vaccines deliver minuscule particulates that stimulate the immune system to mount an immune response. The goal is to help people’s own bodies fight cancer. “What is unique about our design is the simplicity of the single-polymer composition that can precisely deliver tumor antigens to immune cells while stimulating innate immunity. These actions result in safe and robust production of tumor-specific T cells that kill cancer cells,” said Dr. Jinming Gao, a Professor of Pharmacology and Otolaryngology in UT Southwestern’s Harold C. Simmons Comprehensive Cancer Center. A study outlining this research, published online in Nature Nanotechnology, reported that the nanovaccine had anti-tumor efficacy in multiple tumor types in mice. The research was a collaboration between the laboratories of study senior authors Dr. Gao and Dr. Zhijian “James” Chen, Professor of Molecular Biology and Director of the Center for Inflammation Research. The Center was established in 2015 to study how the body senses infection and to develop approaches to exploit this knowledge to create new treatments for infection, immune disorders, and autoimmunity. Typical vaccines require immune cells to pick up tumor antigens in a “depot system” and then travel to the lymphoid organs for T cell activation, Dr. Gao said. Instead, nanoparticle vaccines can travel directly to the body’s lymph nodes to activate tumor-specific immune responses. “For nanoparticle vaccines to work, they must deliver antigens to proper cellular compartments within specialized immune cells called antigen-presenting cells and stimulate innate immunity,” said Dr. Chen, also a Howard Hughes Medical Institute Investigator and holder of the George L. MacGregor Distinguished Chair in Biomedical Science. “Our nanovaccine did all of those things.” In this case, the experimental UTSW nanovaccine works by activating an adaptor protein called STING, which in turn stimulates the body’s immune defense system to ward off cancer. The scientists examined a variety of tumor models in mice: melanoma, colorectal cancer, and HPV-related cancers of the cervix, head, neck, and anogenital regions. In most cases, the nanovaccine slowed tumor growth and extended the animals’ lives. Other vaccine technologies have been used in cancer immunotherapy. However, they are usually complex – consisting of live bacteria or multiplex biological stimulants, Dr. Gao said. This complexity can make production costly and, in some cases, lead to immune-related toxicities in patients. With the emergence of new nanotechnology tools and increased understanding of polymeric drug delivery, Dr. Gao said, the field of nanoparticle vaccines has grown and attracted intense interest from academia and industry in the past decade. “Recent advances in understanding innate and adaptive immunity have also led to more collaborations between immunologists and nanotechnologists,” said Dr. Chen. “These partnerships are critical in propelling the rapid development of new generations of nanovaccines.” The investigative team is now working with physicians at UT Southwestern to explore clinical testing of the STING-activating nanovaccines for a variety of cancer indications. Combining nanovaccines with radiation or other immunotherapy strategies such as “checkpoint inhibition” can further augment their anti-tumor effectiveness. Study lead authors from UT Southwestern were Dr. Min Luo, research scientist; Dr. Hua Wang, Instructor of Molecular Biology; and Dr. Zhaohui Wang, postdoctoral fellow. Other UTSW researchers involved included graduate students Yang Li, Chensu Wang, Haocheng Cai, and Mingjian Du; Dr. Gang Huang, Instructor of Pharmacology and in the Simmons Comprehensive Cancer Center; Dr. Xiang Chen, research specialist; Dr. Zhigang Lu, Instructor of Physiology; Dr. Matthew Porembka, Assistant Professor of Surgery and a Dedman Family Scholar in Clinical Care; Dr. Jayanthi Lea, Associate Professor of Obstetrics and Gynecology and holder of the Patricia Duniven Fletcher Distinguished Professorship in Gynecological Oncology; Dr. Arthur Frankel, Professor of Internal Medicine and in the Simmons Comprehensive Cancer Center; and Dr. Yang-Xin Fu, Professor of Pathology and Immunology, and holder of the Mary Nell and Ralph B. Rogers Professorship in Immunology. Their work was supported by the National Institutes of Health, the Cancer Prevention and Research Institute of Texas, a UTSW Small Animal Imaging Resource grant and a Simmons Comprehensive Cancer Center support grant.

Artist's depiction of a type IV secretion system nestled within a bacterial cell membrane. This structure shoots out thousands of toxic molecules (purple) into human cells during an infection. Credit: Caltech Experts predict that by 2050, antibiotic-resistant bacteria will cause as many deaths as cancer. Now, for the first time, Caltech scientists have created a 3-D image of a molecular structure that many different bacteria use to pump toxins into human cells and spread antibiotic-resistance genes to other bacteria. Understanding the architecture of this structure is a first step toward combating its effects. The study was conducted in the laboratory of Grant Jensen, professor of biophysics and biology and Howard Hughes Medical Institute Investigator. A paper describing the work first appeared online in the March 23 issue of EMBO Reports. The researchers looked specifically at Legionella, the bacteria that causes Legionnaires' disease, a severe and often lethal form of pneumonia. When Legionella invades a human cell, it wraps itself in a protective vesicle and opens the molecular structure, known as a type IV secretion system. The molecular "machine" sits in the cell membrane of the bacterium and proceeds to shoot tens of thousands of toxic molecules—hundreds of different types—into the human cell, hijacking cellular pathways and overwhelming the cell's defenses. Some type IV secretion systems are thought to be instrumental in spreading antibiotic-resistance genes throughout the bacterial population. "Understanding the structure of the type IV system is crucial to developing new antibiotics to disable it," says first author and postdoctoral scholar Debnath Ghosal. "While this study focuses only on the secretion system of Legionella, a very similar machine is used by many bacteria—such as the pathogens that cause stomach cancer, Q fever, and whooping cough." To image the structure—which, at 40 nanometers in diameter, is about 1,000 times too small to be seen by the human eye—the researchers employed a technique called electron cryotomography. In this method, bacteria are frozen alive and then rotated under a powerful electron microscope to create a series of 2-D images that can be digitally reconstructed into a 3-D picture. This was the first-ever image of a type IV machine within a bacterium. The imaging revealed that the structure is shaped into concentric arches, like the symbol for Wi-Fi. Understanding the structure should eventually aid efforts to design drugs that can block the machine. Developing a drug that would disable even one core protein component of the secretion system, Ghosal says, would enable human cells to fight back against the bacterial infection. "Most current antibiotics focus on destroying the cellular envelope that encompasses a bacterial cell, preventing it from replicating," says Jensen. "Developing new antibiotics that target different aspects of the bacterial cell, such as the type IV secretion system, would enable us to block infections in additional ways." The paper is titled "In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography." In addition to Ghosal and Jensen, coauthors are Caltech research scientist Yi Wei Chang, Kwang Cheol Jeong of the Washington University School of Medicine and the University of Florida, and Joseph Vogel of the Washington University School of Medicine. Funding was provided by the National Institutes of Health and the National Institute of Allergy and Infectious Diseases. Explore further: Spread of antibiotic resistance understood by unravelling bacterial secretion system

News Article | April 26, 2017

A protein derived from ticks enhances the effectiveness of antibiotic treatment for methicillin-resistant Staphylococcus aureus, or MRSA, a new study shows. Using the protein in combination with existing treatments might help address the growing challenge of MRSA and other staph infections, researchers say. Resistance to antibiotic treatment is a widespread problem in medicine—and MRSA can cause deadly infections. Some staph bacteria are able to resist treatment in part because they secrete a protective outer layer—a biofilm—that prevents the immune system and antibiotics from gaining access to them. For the new study, published in Antimicrobial Agents and Chemotherapy, researchers identified IAFGP, a tick protein that alters the biofilm. Scientists combined the protein, and a molecule derived from it, with antibiotics currently used to treat MRSA as well as other antibiotics that are not standard treatment. When they tested the combination of agents—IAFGP with three different antibiotics—in culture and in MRSA-infected mice and flies, each case showed that the combination improved the ability of the antibiotic to combat the bacteria. “If you take this protein and you add it to the current treatment, it makes the treatment much more potent,” says lead author Erol Fikrig, chief of the Infectious Diseases Section at Yale School of Medicine. “If you take the protein and add it to drugs not used in current treatment, it makes them potent as well.” While the study was not done in humans, it provides a new way to tackle antibiotic-resistance bacterial infections, scientists say. MRSA, which has become resistant to several different antibiotics, can cause severe infections affecting the skin, lungs, and blood. “Our hope is it expands the group of antibiotics that can be used to treat methicillin,” Fikrig says. Support for the study came, in part, from the John Monsky and Jennifer Weis Monsky Lyme Disease Research Fund and the Howard Hughes Medical Institute.

News Article | May 1, 2017

CAMBRIDGE, MA - Using the gene-editing system known as CRISPR, MIT researchers have shown in mice that they can generate colon tumors that very closely resemble human tumors. This advance should help scientists learn more about how the disease progresses and allow them to test new therapies. Once formed, many of these experimental tumors spread to the liver, just like human colon cancers often do. These metastases are the most common cause of death from colon cancer. "That's been a missing piece in the study of colon cancer. There is really no reliable method for recapitulating the metastatic progression from a primary tumor in the colon to the liver," says Omer Yilmaz, an MIT assistant professor of biology, a member of MIT's Koch Institute for Integrative Cancer Research, and the lead senior author of the study, which appears in the May 1 issue of Nature Biotechnology. The study builds on recent work by Tyler Jacks, the director of the Koch Institute, who has also used CRISPR to generate lung and liver tumors in mice. "CRISPR-based technologies have begun to revolutionize many aspects of cancer research, including building mouse models of the disease with greater speed and greater precision. This study is a good example of both," says Jacks, who is also an author of the Nature Biotechnology paper. The paper's lead authors are Jatin Roper, a research affiliate at the Koch Institute and a gastroenterologist at Tufts Medical Center, and Tuomas Tammela, a research scientist at the Koch Institute. For many years, cancer biologists have taken two distinct approaches to modeling cancer. One is to grow immortalized human cancer cells known as cancer cell lines in a lab dish. "We've learned a lot by studying these two-dimensional cell lines, but they have limitations," Yilmaz says. "They don't really reproduce the complex in vivo environment of a tumor." Another widely used technique is genetically engineering mice with mutations that predispose them to develop cancer. However, it can take years to breed such mice, especially if they have more than one cancer-linked mutation. Recently, researchers have begun using CRISPR to generate cancer models. CRISPR, originally discovered by biologists studying the bacterial immune system, consists of a DNA-cutting enzyme called Cas9 and short RNA guide strands that target specific sequences of the genome, telling Cas9 where to make its cuts. Using this process, scientists can make targeted mutations in the genomes of living animals, either deleting genes or inserting new ones. To induce cancer mutations, the investigators package the genes for Cas9 and the RNA guide strand into viruses called lentiviruses, which are then injected into the target organs of adult mice. Yilmaz, who studies colon cancer and how it is influenced by genes, diet, and aging, decided to adapt this approach to generate colon tumors in mice. He and members of his lab were already working on a technique for growing miniature tissues known as organoids -- three-dimensional growths that, in this case, accurately replicate the structure of the colon. In the new paper, the researchers used CRISPR to introduce cancer-causing mutations into the organoids and then delivered them via colonoscopy to the colon, where they attached to the lining and formed tumors. "We were able to transplant these 3-D mini-intestinal tumors into the colon of recipient mice and recapitulate many aspects of human disease," Yilmaz says. Once the tumors are established in the mice, the researchers can introduce additional mutations at any time, allowing them to study the influence of each mutation on tumor initiation, progression, and metastasis. Almost 30 years ago, scientists discovered that colon tumors in humans usually acquire cancerous mutations in a particular order, but they haven't been able to accurately model this in mice until now. "In human patients, mutations never occur all at once," Tammela says. "Mutations are acquired over time as the tumor progresses and becomes more aggressive, more invasive, and more metastatic. Now we can model this in mice." To demonstrate that ability, the MIT team delivered organoids with a mutated form of the APC gene, which is the cancer-initiating mutation in 80 percent of colon cancer patients. Once the tumors were established, they introduced a mutated form of KRAS, which is commonly found in colon and many other cancers. The scientists also delivered components of the CRISPR system directly into the colon wall to quickly model colon cancer by editing the APC gene. They then added CRISPR components to also edit the gene for P53, which is commonly mutated in colon and other cancers. "These new approaches reduce the time frame to develop genetically engineered mice from two years to just a few months, and involve very basic gene engineering with CRISPR," Roper says. "We used P53 and KRAS to demonstrate the principle that the CRISPR editing approach and the organoid transplantation approach can be used to very quickly model any possible cancer-associated gene." In this study, the researchers also showed that they could grow tumor cells from patients into organoids that could be transplanted into mice. This could give doctors a way to perform "personalized medicine" in which they test various treatment options against a patient's own tumor cells. Yilmaz' lab is now using these techniques to study how other factors such as metabolism, diet, and aging affect colon cancer development. The researchers are also using this approach to test potential new colon cancer drugs. The research was funded by the Howard Hughes Medical Institute, the National Institutes of Health, the Department of Defense, the V Foundation V Scholar Award, the Sidney Kimmel Scholar Award, the Pew-Stewart Trust Scholar Award, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the American Federation of Aging Research, and the Hope Funds for Cancer Research.

News Article | April 17, 2017

University of Utah professors Bradley R. Cairns, professor and chair of Oncological Sciences and senior director of Basic Science; Dana Carroll, distinguished professor of Biochemistry; and Christopher D. Hacon, distinguished professor of Mathematics, were raised to a high honor in science today with their election to the American Academy of Arts and Sciences. The three scientists join 225 U.S. scholars, scientists, writers, artists, as well as civic, business and philanthropic leaders, elected by the Academy, which is headquartered in Cambridge, Mass. Members of the 2017 class include winners of the Pulitzer Prize and the Wolf Prize, MacArthur Fellows, Fields Medalists, Presidential Medal of Freedom and National Medal of Arts recipients, and Academy Award, Grammy Award, Emmy Award and Tony Award winners. Bradley R. Cairns was honored for his work examining how chromatin, the structures that package chromosomal DNA, switch genes on or off. He is working to understand how changes in chromatin affect cellular mechanisms that can lead to cancer development. As an investigator at the Huntsman Cancer Institute (HCI) at the University of Utah, Cairns is using zebrafish to study genes associated with many types of cancers. Along with this latest honor, Cairns has been a Howard Hughes Medical Institute Investigator since 2000. "Dr. Cairns has made fundamental discoveries in the areas of DNA remodeling and regulation of gene expression that are influencing how we think about human development and disease," said Mary Beckerle, CEO and director of HCI. "In addition to his innovative and high-impact scientific work, Dr. Cairns is also an exceptional leader who has built a culture of excellence and collaboration at Huntsman Cancer Institute and the University of Utah. The American Academy of Arts and Sciences couldn't have chosen a better person to honor with membership in this distinguished society." Dana Carroll has been on the faculty at the U of U Health for 42 years. Starting 21 years ago, he developed the earliest of the precise genome editing platforms, zinc-finger nucleases. He has worked with ZFNs and the successor technologies, TALENs and CRISPR-Cas, all of which are being used around the world to learn the consequences of specific mutations, to improve agricultural plant and animals, and to develop treatments for human diseases. Carroll received both the 2012 Edward Novitski Prize from the Genetics Society of America and the 2014 Herbert Sober Lectureship from the American Society of Biochemistry and Molecular Biology. He also received the 2016 Distinguished Innovation and Impact Award from the University of Utah. "I am ecstatic to hear that Dr. Dana Carroll has been elected into the American Academy of Arts and Sciences," said U of U Vice President for Research Andrew Weyrich. "Dr. Carroll is a pioneer in the research community for his groundwork in genome editing platforms, which have been used effectively to modify the genomes of over 80 organisms. This is a great honor for his continuous dedication and contribution to research and science." Christopher Hacon is a scholar of algebraic geometry, the field of mathematics that studies geometric objects defined by polynomial equations. He is particularly interested in objects that exist in more than three dimensions, and he and his colleagues have applied studies of these objects to extend the "minimal model program," a foundational principle of algebraic geometry, into higher dimensions. The American Mathematical Society has lauded the work of Hacon and his colleagues as "a watershed in algebraic geometry." Hacon is a fellow of the American Mathematical Society and recipient of the 2016 EH Moore Research Article Prize, the 2015 Distinguished Scholarly and Creative Research Award from the University of Utah, the 2011 Antonio Feltrinelli Prize in Mathematics Mechanics and Applications, the 2009 Frank Nelson Cole Prize in Algebra and the 2007 Clay Research Award. "Hacon's election as a member of the AAAS stands in recognition of his towering stature as a research mathematician and his deep contributions not only to the discipline of mathematics, but also to the University of Utah," said Peter Trapa, chair of the Department of Mathematics. "It is an honor to welcome this new class of exceptional women and men as part of our distinguished membership," said Don Randel, Chair of the Academy's Board of Directors. "Their talents and expertise will enrich the life of the Academy and strengthen our capacity to spread knowledge and understanding in service to the nation." This release and associated images can be found here.

News Article | April 17, 2017

Scientists have uncovered how our ancestors may have wiped out an ancient retrovirus around 11 million years ago. Retroviruses, which include human immunodeficiency virus (HIV), are abundant in nature. Unlike other viruses, which do not usually leave a physical trace of their existence, retroviruses include a step in their life cycle where their genetic material is integrated into the genome of their host. This integration has created a genetic fossil record of extinct retroviruses that is preserved in the genomes of modern organisms. Writing in the journal eLife, researchers from the Rockefeller University and the Howard Hughes Medical Institute (HHMI), US, set out to discover how extinct viral lineages could have been eliminated. To do this, they analysed retroviral fossils left by human endogenous retrovirus T (HERV-T), which replicated in our primate ancestors for approximately 25 million years before it was eradicated about 11 million years ago. Working with Robert Gifford from the University of Glasgow, the team first compiled a near-complete catalog of HERV-T fossils in old-world monkey and ape genomes. They then reconstructed the HERV-T retrovirus' outer envelope protein - a type of protein that allows a virus particle to bind to cells and begin the viral replication cycle. "Our analyses first suggested that HERV-T likely used a cell-surface protein called MCT-1 to bind to cells and infect ancient old-world primates," says first author Daniel Blanco-Melo, who carried out the study at the Rockefeller University but is now a postdoctoral researcher at the Icahn School of Medicine at Mount Sinai, New York. "Next, we identified one particular fossilised HERV-T gene in the human genome that encodes an unexpectedly well-preserved envelope protein. This gene was absent in non-hominid primate genomes, but was integrated into an ancestral hominid genome around 13 to 19 million years ago. We believe its function may have been switched around this time so that it could block infection by causing MCT-1 depletion from cell surfaces." Taken together, these findings suggest a scenario in which HERV-T began to infiltrate primate germlines (series of cells that are seen as continuing through successive generations of an organism) using MCT-1 as a receptor. Ancestral hominids later evolved a defence mechanism whereby they switched a HERV-T gene to serve as an antiviral gene against itself. "Broadly speaking, this study shows how analysing viral fossils can provide a wealth of insight into events that occurred in the distant past," says senior author Paul Bieniasz, HHMI Investigator and Professor of Retrovirology at the Rockefeller University. "In particular, it represents an example of how viruses themselves can provide the genetic material that animals use to combat them, sometimes leading to their own extinction." The paper 'Co-option of an endogenous retrovirus envelope for host defense in hominid ancestors' can be freely accessed online at http://dx. . Contents, including text, figures and data, are free to reuse under a CC BY 4.0 license. eLife is a unique collaboration between the funders and practitioners of research to improve the way important research is selected, presented, and shared. eLife publishes outstanding works across the life sciences and biomedicine -- from basic biological research to applied, translational, and clinical studies. All papers are selected by active scientists in the research community. Decisions and responses are agreed by the reviewers and consolidated by the Reviewing Editor into a single, clear set of instructions for authors, removing the need for laborious cycles of revision and allowing authors to publish their findings quickly. eLife is supported by the Howard Hughes Medical Institute, the Max Planck Society, and the Wellcome Trust. Learn more at

News Article | April 10, 2017

Biomedical engineers have developed a way to deliver drugs to specific types of neurons in the brain, providing an unprecedented ability to study neurological diseases while also promising a more targeted way to treat them. Drugs are the tool of choice for studying the connections between neurons, and continue to be the mainstream treatment for neurological disease. But a major drawback in both endeavors is that the drugs affect all types of neurons, complicating the study of how cell receptors in the synapse - the gap between neurons - work in an intact brain, and how their manipulation can lead to clinical benefits and side effects. A new method named DART (Drugs Acutely Restricted by Tethering) may overcome these limitations. Developed by researchers at Duke University and the Howard Hughes Medical Institute, DART offers researchers the first opportunity to test what happens when a drug is targeted exclusively to one cell type. In its inaugural study, DART reveals how movement difficulties in a mouse model of Parkinson's Disease are controlled by the AMPA receptor (AMPAR) - a synaptic protein that enables neurons to receive fast incoming signals from other neurons in the brain. The results reveal why a recent clinical trial of an AMPAR-blocking drug failed, and offer a new approach to using the pharmaceutical. The paper appeared online in the journal Science. "This study marks a major milestone in behavioral neuropharmacology," said Michael Tadross, assistant professor of biomedical engineering, who is in the process of moving his laboratory from the HHMI Janelia Research Campus to Duke. "The insights we gained in studying Parkinson's mice were unexpected and could not have been obtained with any previous method." DART works by genetically programming a specific cell type to express a sort of GPS beacon. The "beacon" is an enzyme borrowed from bacteria that is inert - it does nothing more than sit on the cell surface. Nothing, that is, until researchers deliver drugs loaded with a special homing device. Researchers administer these drugs at such low doses that they do not affect other cells. Because the homing system is so efficient, however, the drug is captured by the tagged cells' surface, accumulating within minutes to concentrations that are 100 to 1,000 times higher than anywhere else. In an experiment using a mouse model of Parkinson's disease, Tadross and colleagues attached the homing signal beacon to two types of neurons found in the basal ganglia - the region of the brain responsible for motor control. One type, referred to as D1 neurons, are believed to give a "go" command. The other, referred to as D2 neurons, are thought to do just the opposite, providing commands to stop movements. Using DART, Tadross delivered an AMPAR-blocking pharmaceutical to only D1-neurons, only D2-neurons, or both. When delivered to both cell types simultaneously, the drugs improved only one of several components of motor dysfunction - mirroring the lackluster results of recent human clinical trials. The team then found that delivering the drug to only the D1/"go" neurons did absolutely nothing. Surprisingly, however, by targeting the same drug to D2/"stop" neurons, the mice's movements became more frequent, faster, fluid and linear - in other words, much closer to normal. While the drug stops neurons from receiving certain incoming signals, it does not completely shut them down. This nuance is particularly important for a subset of the D2 neurons that have two prominent forms of firing. With DART, these components could be separately manipulated, providing the first evidence that Parkinson's motor deficits are attributable to the AMPAR-based component of firing in these cells. Tadross said this level of nuance could not have been obtained with prior cell type-specific methods that completely shut neurons down. "Already in our first use of DART, we've learned something new about the synaptic basis of circuit dysfunction in Parkinson's disease," said Tadross. "We've discovered that targeting a specific receptor on specific types of neurons can lead to surprisingly potent improvements." Tadross is already looking into how this discovery might translate into a new therapy by delivering drugs to these neurons through an emerging viral technique. He is also beginning work to develop a version of DART that does not need the genetically added homing beacon to work. Both efforts will require years of research before seeing fruition - but that's not stopping Tadross. "All too often in basic science, approaches are developed that may 'one day' make a difference to human health," he said. "At Duke, there's a palpable emphasis on providing new treatments to people as quickly as possible. I'm very excited that in this environment, my lab can work collaboratively with scientists, physicians, and biotech to solve the real-world challenges involved." This research was funded by the Howard Hughes Medical Institute.

News Article | April 26, 2017

Regenerative medicine using human pluripotent stem cells to grow transplantable tissue outside the body carries the promise to treat a range of intractable disorders, such as diabetes and Parkinson's disease. However, a research team from the Harvard Stem Cell Institute (HSCI), Harvard Medical School (HMS), and the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard has found that as stem cell lines grow in a lab dish, they often acquire mutations in the TP53 (p53) gene, an important tumor suppressor responsible for controlling cell growth and division. Their research suggests that genetic sequencing technologies should be used to screen for mutated cells in stem cell cultures, so that cultures with mutated cells can be excluded from scientific experiments and clinical therapies. If such methods are not employed it could lead to an elevated cancer risk in those receiving transplants. The paper, published online in the journal Nature on April, 26, comes at just the right time, the researchers said, as experimental treatments using human pluripotent stem cells are ramping up across the country. "Our results underscore the need for the field of regenerative medicine to proceed with care," said the study's co-corresponding author Kevin Eggan, an HSCI Principal Faculty member and the director of stem cell biology for the Stanley Center. Eggan's lab in Harvard University's Department of Stem Cell and Regenerative Biology uses human stem cells to study the mechanisms of brain disorders, including amyotrophic lateral sclerosis, intellectual disability, and schizophrenia. The research, the team said, should not discourage the pursuit of experimental treatments but instead be heeded as a call to screen rigorously all cell lines for mutations at various stages of development as well as immediately before transplantation. "Our findings indicate that an additional series of quality control checks should be implemented during the production of stem cells and their downstream use in developing therapies," Eggan said. "Fortunately, these genetic checks can be readily performed with precise, sensitive, and increasingly inexpensive sequencing methods." With human stem cells, researchers can recreate human tissue in the lab. This enables them to study the mechanisms by which certain genes can predispose an individual to a particular disease. Eggan has been working with Steve McCarroll, associate professor of genetics at Harvard Medical School and director of genetics at the Stanley Center, to study how genes shape the biology of neurons, which can be derived from these stem cells. McCarroll's lab recently discovered a common, precancerous condition in which a blood stem cell in the body acquires a pro-growth mutation and then outcompetes a person's normal stem cells, becoming the dominant generator of his or her blood cells. People in whom this condition has appeared are 12 times more likely to develop blood cancer later in life. The study's lead authors, Florian Merkle and Sulagna Ghosh, collaborated with Eggan and McCarroll to test whether laboratory-grown stem cells might be vulnerable to an analogous process. "Cells in the lab, like cells in the body, acquire mutations all the time," said McCarroll, co-corresponding author. "Mutations in most genes have little impact on the larger tissue or cell line. But cells with a pro-growth mutation can outcompete other cells, become very numerous, and 'take over' a tissue. We found that this process of clonal selection - the basis of cancer formation in the body - is also routinely happening in laboratories." To find acquired mutations, the researchers performed genetic analyses on 140 stem cell lines--26 of which were developed for therapeutic purposes using Good Manufacturing Practices, a quality control standard set by regulatory agencies in multiple countries. The remaining 114 were listed on the NIH registry of human pluripotent stem cells. "While we expected to find some mutations in stem cell lines, we were surprised to find that about five percent of the stem cell lines we analyzed had acquired mutations in a tumor-suppressing gene called p53," said Merkle. Nicknamed the "guardian of the genome," p53 controls cell growth and cell death. People who inherit p53 mutations develop a rare disorder called Li-Fraumeni Syndrome, which confers a near 100 percent risk of developing cancer in a wide range of tissue types. The specific mutations that the researchers observed are "dominant negative" mutations, meaning, when present on even one copy of P53, they are able to compromise the function of the normal protein, whose components are made from both gene copies. The exact same dominant-negative mutations are among the most commonly observed mutations in human cancers. "These precise mutations are very familiar to cancer scientists. They are among the worst P53 mutations to have," said Sulagna Ghosh, a co-lead author of the study. The researchers performed a sophisticated set of DNA analyses to rule out the possibility that these mutations had been inherited rather than acquired as the cells grew in the lab. In subsequent experiments, the Harvard scientists found that p53 mutant cells outperformed and outcompeted non-mutant cells in the lab dish. In other words, a culture with a million healthy cells and one p53 mutant cell, said Eggan, could quickly become a culture of only mutant cells. "The spectrum of tissues at risk for transformation when harboring a p53 mutation include many of those that we would like to target for repair with regenerative medicine using human pluripotent stem cells," said Eggan. Those organs include the pancreas, brain, blood, bone, skin, liver and lungs. However, Eggan and McCarroll emphasized that now that this phenomenon has been found, inexpensive gene-sequencing tests will allow researchers to identify and remove from the production line cell cultures with concerning mutations that might prove dangerous after transplantation. The researchers point out in their paper that screening approaches to identify these p53 mutations and others that confer cancer risk already exist and are used in cancer diagnostics. In fact, in an ongoing clinical trial that is transplanting cells derived from induced pluripotent stem cells (iPSCs), gene sequencing is used to ensure the transplanted cell products are free of dangerous mutations. This work was supported by the Harvard Stem Cell Institute, the Stanley Center for Psychiatric Research, The Rosetrees Trust and The Azrieli Foundation, Howard Hughes Medical Institute, the Wellcome Trust, the Medical Research Council, and the Academy of Medical Sciences, and by grants from the NIH (HL109525, 5P01GM099117, 5K99NS08371, HG006855, MH105641).

News Article | May 4, 2017

University of California, Berkeley, researchers have described 10 new CRISPR enzymes that, once activated, behave like Pac-Man to chew up RNA in a way that could be used as sensitive detectors of infectious viruses. The new CRISPR enzymes are variants of a CRISPR protein, Cas13a, which the UC Berkeley researchers reported last September in Nature could be used to detect specific sequences of RNA, such as from a virus. They showed that once CRISPR-Cas13a binds to its target RNA, it begins to indiscriminately cut up all RNA, easily cutting RNA linked to a reporter molecule, making it fluoresce to allow signal detection. Two teams of researchers at the Broad Institute subsequently paired CRISPR-Cas13a with RNA amplification, and showed that the system, which they dubbed SHERLOCK, could detect viral RNA at extremely low concentrations, detecting the presence of dengue and Zika viral RNA, for example. Such a system could be used to detect any type of RNA, including RNA distinctive of cancer cells. While the original Cas13a enzyme used by the UC Berkeley and Broad teams cuts RNA at one specific nucleic acid, uracil, three of the new Cas13a variants cut RNA at adenine. This difference allows simultaneous detection of two different RNA molecules, such as from two different viruses. "We have taken our foundational research a step further in finding other homologs of the Cas13a family that have different nucleotide preferences, enabling concurrent detection of different reporters with, say, a red and a green fluorescent signal, allowing a multiplexed enzymatic detection system," said first author Alexandra East-Seletsky, a UC Berkeley graduate student in the laboratory of Jennifer Doudna, one of the inventors of the CRISPR-Cas9 gene-editing tool. East-Seletsky was also a co-first author of the September Nature paper. East-Seletsky, Doudna and their UC Berkeley colleagues will report their findings May 4 in the journal Molecular Cell. The CRISPR-Cas13a family, formerly referred to as CRISPR-C2c2, is related to CRISPR-Cas9, which is already revolutionizing biomedical research and treatment because of the ease of targeting it to unique DNA sequences to cut or edit. While the Cas9 protein cuts double-stranded DNA at specific sequences, the Cas13a protein - a nucleic acid-cutting enzyme referred to as a nuclease - latches onto specific RNA sequences, and not only cuts that specific RNA, but runs amok to cut and destroy all RNA present. "Think of binding between Cas13a and its RNA target as an on-off switch -- target binding turns on the enzyme to go be a Pac-Man in the cell, chewing up all RNA nearby," East-Seletsky said. This RNA killing spree can kill the cell. In their September Nature paper, the UC Berkeley researchers argued that the Pac-Man activity of CRISPR-Cas13a is its main role in bacteria, aimed at killing infectious viruses or phages. As part of the immune system of some bacteria, it allows infected cells to commit suicide to save their sister microbes from infection. Similar non-CRISPR suicide systems exist in other bacteria. The UC Berkeley researchers subsequently searched databases of bacterial genomes and found 10 other Cas13a-like proteins, which they synthesized and studied to assess their ability to find and cut RNA. Of those, seven resembled the original Cas13a, while three differed in where they cut RNA. RNA, which serves many functions inside the cell, including as messenger RNA - working copies of DNA - consists of four different nucleotides: adenine, cytosine, guanine and uracil. "Building on our original work, we now show that it is possible to multiplex these enzymes together, extending the scope of the technology," East-Seletsky said. "There is so much diversity within the CRISPR-Cas13a family that can be utilized for many applications, including RNA detection." Doudna, a professor of molecular biology and of chemistry and a Howard Hughes Medical Institute investigator, noted that detection of infectious RNA may or may not require amplification, which is a complicated step. "Our intention is to develop the Cas13a family of enzymes for point-of-care diagnostics that are robust and simple to deploy", Doudna said. Co-authors with East-Seletsky and Doudna are former UC Berkeley postdoctoral fellow Mitchell O'Connell, now an assistant professor at the University of Rochester, and UC Berkeley postdocs David Burstein and Gavin Knott. The work was supported in part by a Frontiers Science award from the Paul Allen Institute and by the National Science Foundation (MCB-1244557).

News Article | May 4, 2017

Already extolled for their health benefits as a food compound, omega-3 fatty acids now appear to also play a critical role in preserving the integrity of the blood-brain barrier, which protects the central nervous system from blood-borne bacteria, toxins and other pathogens, according to new research from Harvard Medical School. Reporting in the May 3 issue of Neuron, a team led by Chenghua Gu, associate professor of neurobiology at Harvard Medical School, describes the first molecular explanation for how the barrier remains closed by suppressing transcytosis--a process for transporting molecules across cells in vesicles, or small bubbles. They found that the formation of these vesicles is inhibited by the lipid composition of blood vessel cells in the central nervous system, which involves a balance between omega-3 fatty acids and other lipids maintained by the lipid transport protein Mfsd2a. While the blood-brain barrier is a critical evolutionary mechanism that protects the central nervous system from harm, it also represents a major hurdle for delivering therapeutic compounds into the brain. Blocking the activity of Mfsd2a may be a strategy for getting drugs across the barrier and into the brain to treat a range of disorders such as brain cancer, stroke and Alzheimer's. "This study presents the first clear molecular mechanism for how low rates of transcytosis are achieved in central nervous system blood vessels to ensure the impermeable nature of the blood-brain barrier," Gu said. "There is still a lot we do not know about how the barrier is regulated. A better understanding of the mechanisms will allow us to begin to manipulate it, with the goal of getting therapeutics into the brain safely and effectively." The blood-brain barrier is composed of a network of endothelial cells that line blood vessels in the central nervous system. These cells are connected by tight junctions that prevent most molecules from passing between them, including many drugs that target brain diseases. In a 2014 study published in Nature, Gu and colleagues discovered that a gene and the protein it encodes, Mfsd2a, inhibits transcytosis and is critical for maintaining the blood-brain barrier. Mice that lacked Mfsd2a, which is found only in endothelial cells in the central nervous system, had higher rates of vesicle formation and leaky barriers, despite having normal tight junctions. In the current study, Gu, Benjamin Andreone, a neurology student at Harvard Medical School, and their colleagues examined how Mfsd2a maintains the blood-brain barrier. Mfsd2a is a transporter protein that moves lipids containing DHA, an omega-3 fatty acid found in fish oil and nuts, into the cell membrane. To test the importance of this function to the barrier, the team created mice with a mutated form of Mfsd2a, in which a single amino acid substitution shut down its ability to transport DHA. They injected these mice with a fluorescent dye and observed leaky blood-brain barriers and higher rates of vesicle formation and transcytosis--mirroring mice that completely lacked Mfsd2a. A comparison of the lipid composition of endothelial cells in brain capillaries against those in lung capillaries--which do not have barrier properties and do not express Mfsd2a--revealed that brain endothelial cells had around two- to five-fold higher levels of DHA-containing lipids. Additional experiments revealed that Mfsd2a suppresses transcytosis by inhibiting the formation of caveolae--a type of vesicle that forms when a small segment of the cell membrane pinches in on itself. As expected, mice with normal Cav-1, a protein required for caveolae formation, and that lacked Mfsd2a exhibited higher transcytosis and leaky barriers. Mice that lacked both Mfsd2a and Cav-1, however, had low transcytosis and impermeable blood-brain barriers. "We think that by incorporating DHA into the membrane, Mfsd2a is fundamentally changing the composition of the membrane and making it unfavorable for the formation of these specific type of caveolae," Andreone said. "Even though we observed low rates of vesicle formation and transcytosis in blood-brain barrier cells decades ago, this is the first time that a cellular mechanism can explain this phenomenon." By revealing the role of Mfsd2a and how it controls transcytosis in the central nervous system, Gu and her colleagues hope to shed light on new strategies to open the barrier and allow drugs to enter and remain in the brain. They are currently testing the efficacy of an antibody that potentially can temporarily block the function of Msfd2a, and whether caveolae-mediated transcytosis can be leveraged to shuttle therapeutics across the barrier. "Many of the drugs that could be effective against diseases of the brain have a hard time crossing the blood-brain barrier," Gu said. "Suppressing Mfsd2a may be an additional strategy that allows us to increase transcytosis, and deliver cargo such as antibodies against beta-amyloid or compounds that selectively attack tumor cells. If we can find a way across the barrier, the impact would be enormous." This work was supported by The National Institutes of Health (grants F31NS090669, NS092473), the Mahoney postdoctoral fellowship, the Howard Hughes Medical Institute, the Kaneb Fellowship, Fidelity Biosciences Research Initiative and the Harvard Blavatnik Biomedical Accelerator. Harvard Medical School has more than 11,000 faculty working in 10 academic departments located at the School's Boston campus or in hospital-based clinical departments at 15 Harvard-affiliated teaching hospitals and research institutes: Beth Israel Deaconess Medical Center, Boston Children's Hospital, Brigham and Women's Hospital, Cambridge Health Alliance, Dana-Farber Cancer Institute, Harvard Pilgrim Health Care Institute, Hebrew SeniorLife, Joslin Diabetes Center, Judge Baker Children's Center, Massachusetts Eye and Ear/Schepens Eye Research Institute, Massachusetts General Hospital, McLean Hospital, Mount Auburn Hospital, Spaulding Rehabilitation Network and VA Boston Healthcare System.

News Article | April 17, 2017

Zika virus is a mosquito-borne infectious disease linked to certain birth defects in infants in South and Central America and the United States. A Lawrence Berkeley National Laboratory (Berkeley Lab) researcher, Banumathi Sankaran, worked as part of a multi-institutional team to map a key viral protein called NS5. Necessary to virus reproduction, NS5 contains two enzyme activities: one reduces the body's ability to mount an immune response against infection and the other helps start the genetic replication process. The work was led by Indiana University's Cheng Kao and Pingwei Li at Texas A&M University (TAMU). In a study published March 27 in Nature Communications, the team described the structure and function of these two enzyme active sites. They also showed comparisons between this protein and those from other related viruses that cause dengue fever, West Nile virus, Japanese encephalitis virus, and hepatitis C. These comparisons will help researchers as they search for possible compounds to halt the ability of the virus to reproduce. Working with researchers from TAMU, Sankaran, a research scientist in the Molecular Biophysics and Integrated Bioimaging Division at Berkeley Lab, used X-ray crystallography to solve the atomic structure of NS5 in the Berkeley Center for Structural Biology at the Advanced Light Source (ALS). "The ALS was critical to the success of this project," said Li. "The powerful beam and the sensitive detector on beamline 5.0.2 made it possible for us to obtain data on our poor quality crystals." Sankaran is in charge of the Collaborative Crystallography (CC) program at the ALS. Funded by the NIH, this program is a fast, reliable and transparent mail-in crystallographic service for the structural biology community. Since the mosquitos carrying the vector have been spreading into the Southern U.S., interest in studying the virus has increased and TAMU's Li indicated that several groups currently are working on similar structural determination efforts. "Having access to state-of-the-art facilities provided by our Collaborative Crystallography program resulted in a rapid turnaround of this project," according to Sankaran. While the researchers have gathered a lot of information about how to target this protein, there are still puzzles remaining. "One of the most important unresolved questions about Zika NS5 is how it catalyzes the synthesis of RNA," said Li. "We look forward to further studies and future collaborations, which will be invaluable for therapeutics discovery." Funding for this work was provided by the National Institutes of Health and the Howard Hughes Medical Institute. The ALS is a DOE Office of Science User Facility. Lawrence Berkeley National Laboratory addresses the world's most urgent scientific challenges by advancing sustainable energy, protecting human health, creating new materials, and revealing the origin and fate of the universe. Founded in 1931, Berkeley Lab's scientific expertise has been recognized with 13 Nobel Prizes. The University of California manages Berkeley Lab for the U.S. Department of Energy's Office of Science. For more, visit http://www. . DOE's Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit

News Article | May 4, 2017

Scientists have shown why fruit flies don’t get lost. Their brains contain cells that act like a compass, marking the direction of flight. It may seem like a small matter, but all animals — even Siri-dependent humans — have some kind of internal navigation system. It’s so vital to survival that it is probably linked to many brain functions, including thought, memory and mood. “Everyone can recall a moment of panic when they took a wrong turn and lost their sense of direction,” says Sung Soo Kim of the Howard Hughes Medical Institute’s Janelia Research Campus in Ashburn, Va. “This sense is central to our lives.” But it’s a complex system that is still not well understood. Human nerve cells involved in the process are spread throughout the brain. In fruit flies, the circuitry is much more straightforward. Two years ago, Janelia researchers reported that the flies appear to have a group of about 50 cells connected in a sort of ring in the center of their brains that serve as an internal compass. But the scientists could only theorize how the system worked. In a series of experiments published online May 4 in Science, Kim and his Janelia colleagues describe how nerve cell activity in the circle changes when the insects fly. The scientists tethered Drosophila melanogaster flies to tiny metal rods that kept them from wriggling under a microscope. Each fly was then surrounded with virtual reality cues — like a passing landscape — that made it think it was moving. As a fly flapped its wings, the scientists recorded which nerve cells, or neurons, were active, and when. The experiments clusters of about four to five neurons would fire on the side of the ring corresponding to the direction of flight: one part of the ring for forward, another next to it for left, and so on. The researchers then tested whether artificially activating cells at different points along the ring, essentially moving the compass needle, would disrupt the flies’ sense of direction. If the insects thought they were heading north, the scientists used a laser to stimulate neurons on the south side of the ring. When that happened, the flies attempted to make random turns. “It seemed very confusing for the fly,” Kim says. When this same experiment was conducted in darkness, which by itself is disorienting, the results were difficult to interpret, the scientists say, because it was impossible to know how much the inability to see was responsible for the haphazard flight. Mammals don’t have the same convenient ring of navigation cells. Instead, neurons known as “head direction” cells are found in many brain regions. Those cells turn on as you move, noting the direction you’re facing, helping you find your way around even without a GPS device announcing the next turn. “We know that head direction cells exist in mammals, but we don’t know the architecture of the system,” says Hervé Rouault, a coauthor of the new study. While internal navigation is more complicated in mammals, it’s important to understand a basic system like the one found in fruit flies, says Adrien Peyrache of McGill University in Montreal, who studies the neural basis of direction in rodents. In mammals, head direction cells don’t form a ring, but they do act in concert, he says. “One of the core questions is how the systems are wired.”

News Article | May 7, 2017

Once activated, 10 newly described CRISPR enzymes behave like Pac-Man to run amok and destroy RNA. The findings could be useful for detecting infectious viruses. The new enzymes are variants of a CRISPR protein, Cas13a. In September, scientists reported in the journal Nature that Cas13a could be used to detect specific sequences of RNA, such as those from a virus. They showed that once it binds to its target RNA, it begins to indiscriminately cut up all RNA linked to a reporter molecule, making it fluoresce to allow signal detection. Researchers subsequently paired CRISPR-Cas13a with RNA amplification, and showed that the system—which they dubbed Sherlock—could see viral RNA at extremely low concentrations to detect the presence of dengue and Zika, for example. Such a system could be used to detect any type of RNA, including RNA distinctive of cancer cells, researchers say. While the original Cas13a enzyme cuts RNA at one specific nucleic acid, uracil, three of the new Cas13a variants cut RNA at adenine. This difference allows simultaneous detection of two different RNA molecules, such as from two different viruses. “We have taken our foundational research a step further in finding other homologs of the Cas13a family that have different nucleotide preferences, enabling concurrent detection of different reporters with, say, a red and a green fluorescent signal, allowing a multiplexed enzymatic detection system,” says first author Alexandra East-Seletsky, a graduate student in the laboratory of Jennifer Doudna at the University of California, Berkeley. Doudna is one of the inventors of the CRISPR-Cas9 gene-editing tool. The new findings appear in Molecular Cell. The CRISPR-Cas13a family, formerly referred to as CRISPR-C2c2, is related to CRISPR-Cas9, which is already revolutionizing biomedical research and treatment because of the ease of targeting it to unique DNA sequences to cut or edit. While the Cas9 protein cuts double-stranded DNA at specific sequences, the Cas13a protein—a nucleic acid-cutting enzyme referred to as a nuclease—latches onto specific RNA sequences, and not only cuts that specific RNA, but runs amok to cut and destroy all RNA present. “Think of binding between Cas13a and its RNA target as an on-off switch—target binding turns on the enzyme to go be a Pac-Man in the cell, chewing up all RNA nearby,” East-Seletsky says. This RNA killing spree can kill the cell. In the Nature paper, researchers argued that the Pac-Man activity of CRISPR-Cas13a is its main role in bacteria, aimed at killing infectious viruses or phages. As part of the immune system of some bacteria, it allows infected cells to commit suicide to save their sister microbes from infection. Similar non-CRISPR suicide systems exist in other bacteria. The researchers subsequently searched databases of bacterial genomes and found 10 other Cas13a-like proteins, which they synthesized and studied to assess their ability to find and cut RNA. Of those, seven resembled the original Cas13a, while three differed in where they cut RNA. RNA, which serves many functions inside the cell, including as messenger RNA—working copies of DNA—consists of four different nucleotides: adenine, cytosine, guanine, and uracil. “Building on our original work, we now show that it is possible to multiplex these enzymes together, extending the scope of the technology,” East-Seletsky says. “There is so much diversity within the CRISPR-Cas13a family that can be utilized for many applications, including RNA detection.” Doudna, a professor of molecular biology and of chemistry and a Howard Hughes Medical Institute investigator, says detection of infectious RNA may or may not require amplification, which is a complicated step. “Our intention is to develop the Cas13a family of enzymes for point-of-care diagnostics that are robust and simple to deploy.” Former UC Berkeley postdoctoral fellow Mitchell O’Connell, now an assistant professor at the University of Rochester, and UC Berkeley postdocs David Burstein and Gavin Knott are coauthors of the study. The work was supported in part by a Frontiers Science award from the Paul Allen Institute and by the National Science Foundation.

News Article | May 4, 2017

The new CRISPR enzymes are variants of a CRISPR protein, Cas13a, which the UC Berkeley researchers reported last September in Nature could be used to detect specific sequences of RNA, such as from a virus. They showed that once CRISPR-Cas13a binds to its target RNA, it begins to indiscriminately cut up all RNA, easily cutting RNA linked to a reporter molecule, making it fluoresce to allow signal detection. Two teams of researchers at the Broad Institute subsequently paired CRISPR-Cas13a with RNA amplification, and showed that the system, which they dubbed SHERLOCK, could detect viral RNA at extremely low concentrations, detecting the presence of dengue and Zika viral RNA, for example. Such a system could be used to detect any type of RNA, including RNA distinctive of cancer cells. While the original Cas13a enzyme used by the UC Berkeley and Broad teams cuts RNA at one specific nucleic acid, uracil, three of the new Cas13a variants cut RNA at adenine. This difference allows simultaneous detection of two different RNA molecules, such as from two different viruses. "We have taken our foundational research a step further in finding other homologs of the Cas13a family that have different nucleotide preferences, enabling concurrent detection of different reporters with, say, a red and a green fluorescent signal, allowing a multiplexed enzymatic detection system," said first author Alexandra East-Seletsky, a UC Berkeley graduate student in the laboratory of Jennifer Doudna, one of the inventors of the CRISPR-Cas9 gene-editing tool. East-Seletsky was also a co-first author of the September Nature paper. East-Seletsky, Doudna and their UC Berkeley colleagues will report their findings May 4 in the journal Molecular Cell. The CRISPR-Cas13a family, formerly referred to as CRISPR-C2c2, is related to CRISPR-Cas9, which is already revolutionizing biomedical research and treatment because of the ease of targeting it to unique DNA sequences to cut or edit. While the Cas9 protein cuts double-stranded DNA at specific sequences, the Cas13a protein - a nucleic acid-cutting enzyme referred to as a nuclease - latches onto specific RNA sequences, and not only cuts that specific RNA, but runs amok to cut and destroy all RNA present. "Think of binding between Cas13a and its RNA target as an on-off switch—target binding turns on the enzyme to go be a Pac-Man in the cell, chewing up all RNA nearby," East-Seletsky said. This RNA killing spree can kill the cell. In their September Nature paper, the UC Berkeley researchers argued that the Pac-Man activity of CRISPR-Cas13a is its main role in bacteria, aimed at killing infectious viruses or phages. As part of the immune system of some bacteria, it allows infected cells to commit suicide to save their sister microbes from infection. Similar non-CRISPR suicide systems exist in other bacteria. The UC Berkeley researchers subsequently searched databases of bacterial genomes and found 10 other Cas13a-like proteins, which they synthesized and studied to assess their ability to find and cut RNA. Of those, seven resembled the original Cas13a, while three differed in where they cut RNA. RNA, which serves many functions inside the cell, including as messenger RNA - working copies of DNA - consists of four different nucleotides: adenine, cytosine, guanine and uracil. "Building on our original work, we now show that it is possible to multiplex these enzymes together, extending the scope of the technology," East-Seletsky said. "There is so much diversity within the CRISPR-Cas13a family that can be utilized for many applications, including RNA detection." Doudna, a professor of molecular biology and of chemistry and a Howard Hughes Medical Institute investigator, noted that detection of infectious RNA may or may not require amplification, which is a complicated step. "Our intention is to develop the Cas13a family of enzymes for point-of-care diagnostics that are robust and simple to deploy", Doudna said.

News Article | May 8, 2017

UT Southwestern Medical Center researchers have identified the cells that directly give rise to hair as well as the mechanism that causes hair to turn gray – findings that could one day help identify possible treatments for balding and hair graying. “Although this project was started in an effort to understand how certain kinds of tumors form, we ended up learning why hair turns gray and discovering the identity of the cell that directly gives rise to hair,” said Dr. Lu Le, Associate Professor of Dermatology with the Harold C. Simmons Comprehensive Cancer Center at UT Southwestern. “With this knowledge, we hope in the future to create a topical compound or to safely deliver the necessary gene to hair follicles to correct these cosmetic problems.” The researchers found that a protein called KROX20, more commonly associated with nerve development, in this case turns on in skin cells that become the hair shaft. These hair precursor, or progenitor, cells then produce a protein called stem cell factor (SCF) that the researchers showed is essential for hair pigmentation. When they deleted the SCF gene in the hair progenitor cells in mouse models, the animal’s hair turned white. When they deleted the KROX20-producing cells, no hair grew and the mice became bald, according to the study. Dr. Le, who holds the Thomas L. Shields, M.D. Professorship in Dermatology, said he and his researchers serendipitously uncovered this explanation for balding and hair graying while studying a disorder called Neurofibromatosis Type 1, a rare genetic disease that causes tumors to grow on nerves. Scientists already knew that stem cells contained in a bulge area of hair follicles are involved in making hair and that SCF is important for pigmented cells, said Dr. Le, a member of the Hamon Center for Regenerative Science and Medicine. What they did not know in detail is what happens after those stem cells move down to the base, or bulb, of hair follicles and which cells in the hair follicles produce SCF – or that cells involved in hair shaft creation make the KROX20 protein, he said. If cells with functioning KROX20 and SCF are present, they move up from the bulb, interact with pigment-producing melanocyte cells, and grow into pigmented hairs. But without SCF, the hair in mouse models was gray, and then turned white with age, according to the study. Without KROX20-producing cells, no hair grew, the study said. UT Southwestern researchers will now try to find out if the KROX20 in cells and the SCF gene stop working properly as people age, leading to the graying and hair thinning seen in older people – as well as in male pattern baldness, Dr. Le said. The research also could provide answers about why we age in general as hair graying and hair loss are among the first signs of aging. Other researchers include first author Dr. Chung-Ping Liao, Assistant Instructor; Dr. Sean Morrison, Professor and Director of the Children’s Medical Center Research Institute at UT Southwestern and of Pediatrics, and Howard Hughes Medical Institute Investigator, who holds the Kathryne and Gene Bishop Distinguished Chair in Pediatric Research at Children’s Research Institute at UT Southwestern and the Mary McDermott Cook Chair in Pediatric Genetics; and Reid Booker, a former UT Southwestern researcher. The research was supported by the National Cancer Institute, Specialized Programs of Research Excellence (SPORE) grant, National Institutes of Health, the Dermatology Foundation, the Children’s Tumor Foundation, and the Burroughs Wellcome Fund.

News Article | May 8, 2017

New research from Boston Children's Hospital and Beth Israel Deaconess Medical Center (BIDMC) shows that chronic sleep loss increases pain sensitivity. It suggests that chronic pain sufferers can get relief by getting more sleep, or, short of that, taking medications to promote wakefulness such as caffeine. Both approaches performed better than standard analgesics in a rigorous study in mice, described in the May 8, 2017 issue of Nature Medicine. Pain physiologist Alban Latremoliere, PhD, of Boston Children's and sleep physiologist Chloe Alexandre, PhD, of BIDMC precisely measured the effects of acute or chronic sleep loss on sleepiness and sensitivity to both painful and non-painful stimuli. They then tested standard pain medications, like ibuprofen and morphine, as well as wakefulness-promoting agents like caffeine and modafinil. Their findings reveal an unexpected role for alertness in setting pain sensitivity. The team started by measuring normal sleep cycles, using tiny headsets that took electroencephalography (EEG) and electromyography (EMG) readings. "For each mouse, we have exact baseline data on how much they sleep and what their sensory sensitivity is," says Latremoliere, who works in the lab of Clifford Woolf, PhD, in the F.M. Kirby Neurobiology Center at Boston Children's. Next, unlike other sleep studies that force mice to stay awake walking treadmills or falling from platforms, Alexandre, Latremoliere and colleagues deprived mice of sleep in a way that mimics what happens with people: They entertained them. "We developed a protocol to chronically sleep-deprive mice in a non-stressful manner, by providing them with toys and activities at the time they were supposed to go to sleep, thereby extending the wake period," says Alexandre, who works in the lab of Thomas Scammell, MD, at BIDMC. "This is similar to what most of us do when we stay awake a little bit too much watching late-night TV each weekday." To keep the mice awake, researchers kept vigil, providing the mice with custom-made toys as interest flagged while being careful not to overstimulate them. "Mice love nesting, so when they started to get sleepy (as seen by their EEG/EMG pattern) we would give them nesting materials like a wipe or cotton ball," says Latremoliere. "Rodents also like chewing, so we introduced a lot of activities based around chewing, for example, having to chew through something to get to a cotton ball." In this way, they kept groups of six to 12 mice awake for as long as 12 hours in one session, or six hours for five consecutive days, monitoring sleepiness and stress hormones (to make sure they weren't stressed) and testing for pain along the way. Pain sensitivity was measured in a blinded fashion by exposing mice to controlled amounts of heat, cold, pressure or capsaicin (the agent in hot chili peppers) and then measuring how long it took the animal to move away (or lick away the discomfort caused by capsaicin). The researchers also tested responses to non-painful stimuli, such as jumping when startled by a sudden loud sound. "We found that five consecutive days of moderate sleep deprivation can significantly exacerbate pain sensitivity over time in otherwise healthy mice," says Alexandre. "The response was specific to pain, and was not due to a state of general hyperexcitability to any stimuli." Surprisingly, common analgesics like ibuprofen did not block sleep-loss-induced pain hypersensitivity. Even morphine lost most of its efficacy in sleep-deprived mice. These observations suggest that patients using these drugs for pain relief might have to increase their dose to compensate for lost efficacy due to sleep loss, thereby increasing their risk for side effects. In contrast, both caffeine and modafinil, drugs used to promote wakefulness, successfully blocked the pain hypersensitivity caused by both acute and chronic sleep loss. Interestingly, in non-sleep-deprived mice, these compounds had no analgesic properties. "This represents a new kind of analgesic that hadn't been considered before, one that depends on the biological state of the animal," says Woolf, director of the Kirby Center at Boston Children's. "Such drugs could help disrupt the chronic pain cycle, in which pain disrupts sleep, which then promotes pain, which further disrupts sleep." The researchers conclude that rather than just taking painkillers, patients with chronic pain might benefit from better sleep habits or sleep-promoting medications at night, coupled with daytime alertness-promoting agents to try to break the pain cycle. Some painkillers already include caffeine as an ingredient, although its mechanism of action isn't yet known. Both caffeine and modafinil boost dopamine circuits in the brain, so that may provide a clue. "This work was supported by a novel NIH program that required a pain scientist to join a non-pain scientist to tackle a completely new area of research," notes Scammel, professor of neurology at BIDMC. "This cross-disciplinary collaboration enabled our labs to discover unsuspected links between sleep and pain with actionable clinical implications for improving pain management." "Many patients with chronic pain suffer from poor sleep and daytime fatigue, and some pain medications themselves can contribute to these co-morbidities," notes Kiran Maski, MD, a specialist in sleep disorders at Boston Children's. "This study suggests a novel approach to pain management that would be relatively easy to implement in clinical care. Clinical research is needed to understand what sleep duration is required and to test the efficacy of wake-promoting medications in chronic pain patients." Alexandre (BIDMC) and Latremoliere (Boston Children's) were co-first authors on the paper. Woolf (Boston Children's) and Scammell (BIDMC) were co-senior authors. Ashley Ferreira and Giulia Miracca of Boston Children's and Mihoko Yamamoto of BIDMC were coauthors. This work was supported by grants from the NIH (R01DE022912, R01NS038253). The Neurodevelopmental Behavior and Pharmacokinetics Cores at Boston Children's Hospital, the metabolic Physiology Core at BIDMC (P30DK057521) and P01HL09491 also supported this study. Boston Children's Hospital is home to the world's largest research enterprise based at a pediatric medical center, where its discoveries have benefited both children and adults since 1869. More than 1,100 scientists, including seven members of the National Academy of Sciences, 11 members of the Institute of Medicine and 10 members of the Howard Hughes Medical Institute comprise Boston Children's research community. Founded as a 20-bed hospital for children, Boston Children's today is a 404-bed comprehensive center for pediatric and adolescent health care. Boston Children's is also the primary pediatric teaching affiliate of Harvard Medical School. For more, visit our Vector and Thriving blogs and follow us on our social media channels @BostonChildrens, @BCH_Innovation, Facebook and YouTube. Beth Israel Deaconess Medical Center is a patient care, teaching and research affiliate of Harvard Medical School and consistently ranks as a national leader among independent hospitals in National Institutes of Health funding. BIDMC is in the community with Beth Israel Deaconess Hospital-Milton, Beth Israel Deaconess Hospital-Needham, Beth Israel Deaconess Hospital-Plymouth, Anna Jaques Hospital, Cambridge Health Alliance, Lawrence General Hospital, MetroWest Medical Center, Signature Healthcare, Beth Israel Deaconess HealthCare, Community Care Alliance and Atrius Health. BIDMC is also clinically affiliated with the Joslin Diabetes Center and Hebrew Rehabilitation Center and is a research partner of Dana-Farber/Harvard Cancer Center and the Jackson Laboratory. BIDMC is the official hospital of the Boston Red Sox. For more information, visit http://www. .

News Article | May 5, 2017

(Boston)--Joseph Park, a second-year student at Boston University School of Medicine (BUSM), was recently awarded his third Howard Hughes Medical Institute's (HHMI) Medical Research Fellowship. He is one of 79 medical students selected to conduct in-depth, mentored biomedical research. Park graduated from Princeton University in 2013 with a degree in molecular biology. His interest in the field of infectious diseases began during his second year at BUSM under the direction of assistant professor of microbiology Stephanie Oberhaus, PhD. Park was awarded his first HHMI fellowship in 2015 and worked with Matthew Waldor, MD, PhD, at Brigham and Women's Hospital to explore the interaction between bacterial pathogens and human cells. Much of his worked has focused on Vibrio parahaemolyticus, a seafood-borne bacterial pathogen that causes gastrointestinal infection. A second HHMI fellowship in 2016 allowed him to continue with this work. For his third HHMI fellowship, Park plans to delve into the cell biology of chlamydia in hopes of advancing knowledge of the disease. "I am grateful to HHMI for the opportunity to train to become a physician-scientist who advances basic science knowledge and translates expertise to clinical care," said Park. Each fellow receives $43,000 in grant support and spends a year pursuing basic, translational, or applied biomedical research at one of 32 academic or nonprofit research institutions across the U.S. The Howard Hughes Medical Institute plays an important role in advancing scientific research and education in the United States. Its scientists, located across the country and around the world, have made important discoveries that advance both human health and our fundamental understanding of biology. The Institute aims to transform science education into a creative, interdisciplinary endeavor that reflects the excitement of real research. Originally established in 1848 as the New England Female Medical College, and incorporated into Boston University in 1873, Boston University School of Medicine (BUSM) today is a leading academic medical center with an enrollment of more than 700 medical students and 950 students pursuing degrees in graduate medical sciences. BUSM faculty contribute to more than 950 active grants and contracts, with total anticipated awards valued at more than $693 million in amyloidosis, arthritis, cardiovascular disease, cancer, infectious diseases, pulmonary disease and dermatology, among other areas. The School's teaching affiliates include Boston Medical Center, its primary teaching hospital, the Boston VA Healthcare System, Kaiser Permanente in northern California, as well as Boston HealthNet, a network of 15 community health centers. For more information, please visit http://www.

News Article | May 2, 2017

New research shows how two drastically different organisms--a green alga and the spotted salamander--get along as cellular roommates. Scientists at the American Museum of Natural History and Gettysburg College found that this symbiosis, the only known example that includes a vertebrate species, puts stress on algal cells, changing the way they make energy, but does not seem to negatively impact salamander cells. The work is published today in the journal eLife. "Science shows us the many ways that life is interconnected, especially on the microscopic level, where we see how many organisms depend on close contact with or internalization of other species for food, defense, or reproduction," said lead author John Burns, a postdoctoral researcher in the Museum's Division of Invertebrate Zoology. "But the relationship between this particular alga and salamander is very unusual." Scientists have known for more than a century that a green alga (Oophila amblystomatis) grows in the egg cases of the spotted salamander (Ambystoma maculatum)--the strange pairing is visible to the naked eye in the green hue of salamanders' eggs. The symbiosis was originally thought to occur only between the salamander embryo and the algae living outside it. The embryos produce nitrogen-rich waste that is useful to algae, and the algae increases the oxygen content of the fluid around the respiring embryos. But recent research has revealed that the algae are actually located inside cells all over the spotted salamander's body. This cell-within-a-cell relationship can also be found in corals and in the guts of cicadas, but the green alga-spotted salamander interaction is the only known example of a symbiont entering the cells of a vertebrate species. "This is really such a strange arrangement to think about, that the salamanders allow the algae to live in their egg cases. It would be like having a bunch of green algae in a womb," said study co-author Ryan Kerney, an assistant professor at Gettysburg College. "What we set out to look at now is the kind of molecular change that happens when the salamander cells and green algae cells are together." In the new eLife study, the researchers compared RNA from the cells of five different groups: salamander cells with algae, salamander cells without algae, the algal cells living in salamander cells, the algae living in the egg capsules, and algae cultured in the laboratory. They found that algae inside salamander cells are stressed and change the way they make energy. Instead of using light energy to produce food to support the salamander host, as happens in coral-algae interactions, the algae in salamander cells struggle to adapt to their new environment. Whether the algae benefits from this cell-within-a-cell interaction remains unclear. In stark contrast, affected salamander cells appear to recognize the alga as foreign but show no signs of stress during the interaction. The researchers found that the salamanders overexpress several genes that might suppress an immune response, suggesting that the host cell experience is neutral or beneficial. "We are learning that these two fundamentally different cells are changing each other dramatically, and this might be relevant for other symbiotic systems, including human and parasitic microbe relationships," said study co-author Eunsoo Kim, an assistant curator in the Museum's Division of Invertebrate Zoology. Other authors on this study include Huanjia Zhang and Elizabeth Hill, undergraduate students at Gettysburg College. This work was supported, in part, by the Howard Hughes Medical Institute and the National Science Foundation, grant #s 1428065 and #1453639. For more information about this project, see this recent episode of the Museum's Shelf Life series: http://www. The American Museum of Natural History, founded in 1869, is one of the world's preeminent scientific, educational, and cultural institutions. The Museum encompasses 45 permanent exhibition halls, including the Rose Center for Earth and Space and the Hayden Planetarium, as well as galleries for temporary exhibitions. It is home to the Theodore Roosevelt Memorial, New York State's official memorial to its 33rd governor and the nation's 26th president, and a tribute to Roosevelt's enduring legacy of conservation. The Museum's five active research divisions and three cross-disciplinary centers support approximately 200 scientists, whose work draws on a world-class permanent collection of more than 34 million specimens and artifacts, as well as specialized collections for frozen tissue and genomic and astrophysical data, and one of the largest natural history libraries in the world. Through its Richard Gilder Graduate School, it is the only American museum authorized to grant the Ph.D. degree and the Master of Arts in Teaching degree. Annual attendance has grown to approximately 5 million, and the Museum's exhibitions and Space Shows can be seen in venues on five continents. The Museum's website and collection of apps for mobile devices extend its collections, exhibitions, and educational programs to millions more beyond its walls. Visit for more information.

News Article | April 19, 2017

Researchers at Johns Hopkins and George Washington universities report new evidence that proteins created by defective forms of HIV long previously believed to be harmless actually interact with our immune systems and are actively monitored by a specific type of immune cell, called cytotoxic T cells. In a report on the study, conducted on laboratory-grown human cells and published April 12 in the journal Cell Host and Microbe, the investigators say their experiments show that while defective HIV proviruses -- the viral genetic material -- cannot create functional infectious HIVs, a specific subset called "hypermutated" HIV proviruses creates proteins that cytotoxic T cells recognize as HIV. HIV proviruses can outnumber functional HIV 1000 copies to one and the faulty proteins they create can complicate efforts to measure a patient's viral load, exhaust immune systems, shield functional HIV from attack by natural means or drugs, and seriously complicate the development of a cure. Researchers believe that if they can exploit the "hypermutated" form of these proviruses, it could help them eliminate more of the defective HIV proviruses and develop a cure for HIV infection. "The virus has a lot of ways, even in its defective forms, to distract our immune systems, and understanding how they do this is essential in finding a cure," says Ya Chi Ho, M.D., Ph.D., instructor of medicine at the Johns Hopkins University School of Medicine, and the lead study investigator. In the study, the scientists collected nine different defective HIV proviruses from six people infected with HIV, then transfected cultures of human immune cells with them in the laboratory. They grew and tested the transfected cells for markers of HIV proliferation -- such as RNA and proteins -- and found that all of them were capable of creating these components despite their mutations. "The fact that defective proviruses can contribute to viral RNA and protein production is concerning, because it means that the measurements of HIV load in infected patients may not be as accurate as we thought. Part of the count is coming from defective viruses," says Ho. After verifying that defective HIV proviruses created HIV proteins, the researchers then tested whether human immune system cells could biologically recognize and interact with those proteins. The group again transfected cells in the lab with 6 different types of defective HIV provirus taken from patients. In collaboration with Dr. R. Brad Jones, Ph.D., co-first author of the paper and assistant professor of microbiology, immunology and tropical medicine at the George Washington School of Medicine and Health Sciences, Ho's team matched cytotoxic T lymphocytes, the immune cells responsible for recognizing and destroying HIV, from the corresponding patient to the infected cells. The researchers observed that cells containing a the "hypermutated" HIV can be recognized by an infected patient's cytotoxic T cells. "If we identify and find a way to use the right protein, perhaps one of those expressed by the "hypermutated" HIV we found in this study, we could create a potent vaccine which could boost the immune system enough to eliminate HIV altogether," says Ho. However, defective HIV proviruses can distract the immune cells from attacking fully infectious normal HIV. "The cytotoxic T lymphocytes' ability to identify and target the real threat appears to be greatly impaired, because they may attack proteins from defective proviruses instead of the real thing," says Ho. Ho believes that further information about the mutant proviruses could give scientists the tools to target them, get around them, and create a cure for HIV -- a long elusive goal for virologists. Other researchers involved in this study include Ross A. Pollack, Mihaela Pertea, Katherine M. Bruner, Alyssa R. Martin, Adam A. Capoferri, Subul A. Beg and Robert F. Siliciano from the Johns Hopkins University School of Medicine; R. Brad Jones, Allison S. Thomas, Szu-Han Huang and Sara Karandish of the George Washington University; Eitan Halper-Stromberg of the University of Colorado; Patrick C. Young of the Icahn School of Medicine at Mount Sinai; Colin Kovacs of the University of Toronto & The Maple Leaf Medical Clinic; and Erika Benko of the Maple Leaf Medical Clinic. This work was supported by the National Institute of Allergy and Infectious Diseases Extramural Activities (1R21AI118402-01, AI096114, 1U1AI096109), The Martin Dulaney CARE and DARE Collaboratories, the ARCHE Collaborative Research Grant from the Foundations for AIDS Research Generature Cure initiative, the Johns Hopkins Center for AIDS Research, the W. Smith Charitable Trust AIDS Research Grant, Gilead Science HIV Cure Research Grant, the Howard Hughes Medical Institute and the Bill and Melinda Gates Foundation.

(—A team of researchers with the Howard Hughes Medical Institute has found that a ring of cells in the middle of the fruit fly brain acts as a compass, helping the insect understand where it is, where it has been and where it is going. In their paper published in the journal Science, the team explains how they expanded on research they began two years ago and what their findings may mean for mammal internal navigation. As the researchers note, two years ago, they discovered a group of approximately 50 neurons forming a ring in the center of the fruit fly brain that appeared to serve a navigational purpose. Since that time, they have studied how the ring might help the tiny insects make their way around in their environment. To find out, the researchers affixed fruit flies to a metal rod that held them in place and then played virtual reality scenes around them, simulating movement in their natural environment. As a fly moved its wings attempting to fly in the simulated surroundings, the researchers recorded neural activity in the ring. They found that individual clusters in the ring would fire corresponding to the direction in which the fly was trying to move. The researchers then genetically modified some of the neurons in the ring to activate when exposed to light. This allowed the team to manipulate the information the fly was receiving regarding its flight path. Firing light at the cells caused the fly to lose track of itself in its surroundings, strongly suggesting that the team was correct in their belief that the neural ring was similar to a compass. The team also ran similar experiments in which the flies were induced to fly in the dark, and found that though the fly appeared disoriented, it was not clear if it was due to interference by the team or just poor navigational skills in the dark in general. As the researchers point out, their study offers evidence of the purpose of the neural ring, but does not explain how its nerves are activated, or how the fly receives information from the ring and uses it as a navigational aid. They plan to continue their research to see if they can find answers to such questions. More information: Sung Soo Kim et al. Ring attractor dynamics in the Drosophila central brain, Science (2017). DOI: 10.1126/science.aal4835 Abstract Ring attractors are a class of recurrent networks hypothesized to underlie the representation of heading direction. Such network structures, schematized as a ring of neurons whose connectivity depends on their heading preferences, can sustain a bump-like activity pattern whose location can be updated by continuous shifts along either turn direction. We recently reported that a population of fly neurons represents the animal's heading via bump-like activity dynamics. We combined two-photon calcium imaging in head-fixed flying flies with optogenetics to overwrite the existing population representation with an artificial one, which was then maintained by the circuit with naturalistic dynamics. A network with local excitation and global inhibition enforces this unique and persistent heading representation. Ring attractor networks have long been invoked in theoretical work; our study provides physiological evidence of their existence and functional architecture.

News Article | May 4, 2017

Runners, swimmers, and cyclists are familiar with the phenomenon of "hitting the wall" when the connection between brain and body feels like it's been lost: You know that you're still trying to move, but doing it feels more conceptual than physical. In Cell Metabolism, researchers show in mice the physiological basis for why this phenomenon occurs. Their research also found that training is not the only way to enhance endurance - it can also be achieved using a small molecule to stimulate a pathway that was already known to be activated by training. "It turns out that 'hitting the wall' happens when your brain can no longer get enough glucose. At that point, you're toast," says co-corresponding author Ronald Evans, a Howard Hughes Medical Institute Investigator and Director of the Gene Expression Laboratory at the Salk Institute. "We previously believed that training improves endurance because it allows the muscles to more effectively burn fat as an energy source." But in this study, they show that it's the other side of this dual metabolic program that may be more important: training progressively reprograms muscle to burn less glucose, thereby preserving it as an energy source for your brain. Muscle can use either fat or glucose as its energy source, but the brain relies solely on glucose. Research over the past two decades from Evans and the study's co-corresponding author, Michael Downes, a Senior Staff Scientist in the lab, has focused on a transcription factor called PPARδ, which activates pathways involved when athletes train to increase their endurance. Here, the researchers demonstrate that this metabolic adaptation both is dependent on PPARδ and can be stimulated by molecularly activating PPARδ. In the first set of experiments, they genetically knocked out PPARδ in the muscles of mice and studied the effects. "When we did this and then ran those animals on a treadmill, we found that the genes that are normally induced by exercise failed to be induced," Downes says. "This indicates that PPARδ plays a central role in exercise, and that it's an important molecular switch gating energy entry into the muscle." In the next part, they used a small-molecule drug to activate PPARδ in the muscles of sedentary mice. They found that the drug not only increased fat oxidation in muscle, but it also forestalled the effects of hypoglycemia, or loss of blood glucose, on the brain. As a result, the mice who had been given the drug were able to increase the length of time they could run before "hitting the wall" - from 160 to 270 minutes - despite having no training to improve their endurance. "What we illustrate in this paper is that if you want to move the wall, there is more than one way to do so," Evans says. "The standard method is to train; you will improve a bit with each run. But we've shown improvement can happen without expending the energy that otherwise would be needed to get to this point." While the researchers recognize that these discoveries could be exploited by athletes wanting to gain a competitive advantage, the greatest promise lies in improving endurance in people who are unable to exercise due to health problems. This could include the frail, the elderly, and people who are confined to bed after injuries or surgery, as well as those affected by conditions like Duchenne muscular dystrophy, cystic fibrosis, cachexia (wasting syndrome), and chronic obstructive pulmonary disease. "Exercise is valuable for many different kinds of problems," Evans says. "With this research, you can begin to think about how a therapeutic that confers the advantages of fitness could help people gain health benefits. The greater potential is essentially unlimited." This research was primarily funded by the National Institutes of Health and the National Health and Medical Research Council of Australia. Article: PPARδ Promotes Running Endurance by Preserving Glucose, Fan et al, Cell Metabolism, doi: 10.1016/j.cmet.2017.04.006, published 2 May 2017.

News Article | April 17, 2017

Researchers at the Rockefeller University have untangled how a molecule called DND1 enables the proper formation of eggs and sperm—essential parts of any species that reproduces sexually. Published in Nature, the findings suggest that a pool of stem cells, which will ultimately give rise to eggs and sperm, can only survive if DND1 is around. The protein prevents a host of factors related to cell death and inflammation from killing these stem cells off. "We already knew that mutations in the DND1 gene can cause a substantial loss of germline stem cells and male sterility—and now we know why," says Thomas Tuschl, head of the Laboratory of RNA Molecular Biology and Howard Hughes Medical Institute Investigator. Tuschl led the study with Markus Hafner, a former postdoctoral fellow in the Tuschl lab who is now at the National Institutes of Health, and research associate Masashi Yamaji. For a gene to be expressed, it must be copied from DNA to so-called messenger RNA (mRNA), which brings it outside of the nucleus. The mRNA then recruits the necessary building blocks to make a protein. There are many places along a gene's journey to becoming a protein where regulators can step in to either ramp up or tone down the resulting level of protein in a cell. DND1 is one of these regulators, and scientists used to think its function is to increase the stability of mRNA. However, Tuschl and colleagues found that it does just the opposite: DND1 binds to sites made up of a specific code on mRNA, and attracts a complex responsible for destabilizing the targeted mRNAs, thereby halting further protein production. That code can be repeated throughout an mRNA sequence, and the researchers found that more repeats of this code meant more of a chance that the mRNA would be eliminated. The researchers also identified all of the mRNAs that DND1 targets, which included genes related to inflammation, differentiation, and cell death—genes whose activity is supposed to be turned off at this point in development. When a cell shuts down these genes, it stops producing their mRNAs. However, mRNAs that were copied earlier may still be floating around, ready to build a protein. "We think that DND1 helps to sharpen the transition from one developmental stage to the next by targeting mRNAs that should have already been turned off, and clearing them from the cell," says Yamaji. "By halting the production of proteins that otherwise promote cell death, DND1 allows germline stem cells to grow and be maintained in proper numbers." Explore further: Protein production in differentiating stem cells is more complex than previously thought More information: Masashi Yamaji et al. DND1 maintains germline stem cells via recruitment of the CCR4–NOT complex to target mRNAs, Nature (2017). DOI: 10.1038/nature21690

News Article | May 3, 2017

The researchers were led by Michael Dyer, Ph.D., a Howard Hughes Medical Institute Investigator and St. Jude Department of Developmental Neurobiology chair. The work appears in the May 3 issue of the journal Neuron. Epigenetic controls are molecular switches that turn genes on or off to orchestrate a cell's development from a generic cell to a specialized cell like a neuron. While the "genome" of thousands of individual genes is like data stored on a computer disk, the "epigenome" is like a computer program that controls how stored data are read. Researchers know that epigenetic malfunctions can drive cancers and degenerative diseases, but they have not cracked the "epigenetic code"—the specific changes in the organization of the nucleus that guide each type of cell to differentiate from a progenitor cell to a specialized cell. The researchers used tools of epigenomic analysis to trace the specific epigenetic switches controlling each of thousands of genes in both mouse and human retinal cells as the cells progressed through development. Analyzing the data revealed surprises about the epigenetic processes of retinal neuron development, Dyer said. One such surprise was the relative importance of two types of epigenetic control switches for retinal development. One control is DNA methylation, which is a chemical alteration of a gene that switches it on or off. The other control switch is histone modification. Histones are proteins that serve as a scaffold for coiling up the DNA into the tight space of the nucleus. "The perception of the research community was that DNA methylation was the major epigenetic controller," Dyer said. "But to our surprise, only a small percentage of the changes in gene expression during development had any correlation with DNA methylation. It's at the histone level that we saw the really profound changes during differentiation." Another unexpected discovery, Dyer said, was the point during development when the immature cells transition from making new tissue by dividing rapidly, to differentiating into a mature retinal neuron. "It's like flipping a giant switch," Dyer said. "Early in development, all the cells are immature progenitors that are rapidly growing and dividing. Then, when those cells stop growing and start becoming neurons, there was a dramatic shift in the epigenome. "We thought cells would actively shut down those progenitor growth genes, because it would not want them to reactivate and lead to a tumor," he said. "But instead, many of those genes just went from a very active state into what we call an 'empty' state. The cell didn't make any particular effort to shut them down. On the flip side, those genes needed for differentiation, which were repressed in the progenitor cells, had their epigenetic repression removed." Mapping the epigenetic changes in developing retinoblastoma mouse and human cells yielded similar important insights,Dyer said. "We still don't know which type of cell gives rise to retinoblastoma," he said. "The tumor cells have a mixed program of progenitor cells and neurons. It appears as though they are stuck at the stage when the epigenetic switch is normally flipped to transition from progenitors to neurons. "While this study can't answer the origin question, it did narrow down the developmental window when the normal cell becomes a tumor cell," he said. "I would have guessed that it would be very early, when a progenitor was rapidly dividing. But we found that the decision point was during a period when the cells were transitioning from rapid growth to differentiation. This insight will allow us to focus on that stage to better understand how retinoblastomas originate." Added Robert Fulton, director of technology development at Washington University's McDonnell Genome Institute, which contributed to the sequencing and analysis of the data: "This research is a great example of the value of comprehensive genomic analyses and the insights that can be gained from thorough, well-designed studies. To really understand the origins of retinoblastoma, we need to look beyond genes to understand how epigenetic changes drive cancer." The new epigenomic data will also enable scientists to search for epigenetic abnormalities underlying adult retinal disease, Dyer said. "There are some patients with retinal disease who don't show gene mutations that we know are responsible for disease," he said. "Instead, those people might have mutations in epigenetic controls called 'enhancers.' We've provided the first map of these enhancers in the retina, so researchers can discover such mutations." Dyer and his colleagues also mapped the three-dimensional organization of the retinal epigenome to discover how retinal cells package their genes in concentric regions of the cell nucleus. The organization makes some genes more available than others to be turned on and off. "It's like packing a suitcase for a trip," he said. "You put the clothes you need in a suitcase to take with you; but those you don't need, you leave in the closet. In our studies, we're trying to decipher the functional significance of why the retinal cell packs some genes away and makes others more accessible. "All our data will serve as a key resource for investigators exploring specific questions about retinal development and disease," Dyer said. To make the data readily available to other researchers, he has uploaded it to ProteinPaint, a St. Jude web portal that gives scientists worldwide access to masses of cancer genomic data. Besides senior author Dyer, the paper's co-first authors were Issam Aldiri  and Beisi Xu, both of St. Jude. The other authors were Lu Wang, Daniel Hiler, Lyra Griffiths, Marie-Elizabeth Barabas,Jiakun Zhang, Xiang Chen, Xin Zhou, John Easton,Jinghui Zhang, Marc Valentine, Abbas Shirinifard, Suresh Thiagarajan, Andras Sablauer,Sharon Frase, and James R. Downing, all of St. Jude; Dianna Johnson, of the University of Tennessee Health Science Center; and Elaine Mardis and Richard Wilson of the Washington University School of Medicine in St. Louis. This work was supported in part by funding from Howard Hughes Medical Institute, the National Cancer Institute (CA21765), the National Institutes of Health (EY014867, EY018599 and CA168875), Alex's Lemonade Stand Foundation for Childhood Cancer, the Tully Family Foundation, the Peterson Foundation and ALSAC, the fundraising and awareness organization of St. Jude. St. Jude Children's Research Hospital is leading the way the world understands, treats and cures childhood cancer and other life-threatening diseases. It is the only National Cancer Institute-designated Comprehensive Cancer Center devoted solely to children. Treatments developed at St. Jude have helped push the overall childhood cancer survival rate from 20 percent to 80 percent since the hospital opened more than 50 years ago. St. Jude freely shares the breakthroughs it makes, and every child saved at St. Jude means doctors and scientists worldwide can use that knowledge to save thousands more children. Families never receive a bill from St. Jude for treatment, travel, housing and food — because all a family should worry about is helping their child live. To learn more, visit or follow St. Jude on social media at @stjuderesearch. Washington University School of Medicine's 2,100 employed and volunteer faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Children's hospitals. The School of Medicine is one of the leading medical research, teaching and patient-care institutions in the nation, currently ranked seventh in the nation by U.S. News & World Report. Through its affiliations with Barnes-Jewish and St. Louis Children's hospitals, the School of Medicine is linked to BJC HealthCare. To view the original version on PR Newswire, visit:

News Article | April 25, 2017

A new technique developed at the University of Virginia School of Medicine will let a single cancer research lab do the work of dozens, dramatically accelerating the search for new treatments and cures. And the technique will benefit not just cancer research but research into every disease driven by gene mutations, from cystic fibrosis to Alzheimer's disease - ultimately enabling customized treatments for patients in a way never before possible. The new technique lets scientists analyze the effects of gene mutations at an unprecedented scale and speed, and at a fraction of the cost of traditional methods. For patients, this means that rather than thinking about the right drug for a certain disease, doctors will think about the right drug to treat the patient's specific gene mutation. "Every patient shouldn't receive the same treatment. No way. Not even if they have the same syndrome, the same disease," said UVA researcher J. Julius Zhu, PhD, who led the team that created the new technique. "It's very individual in the patient, and they have to be treated in different ways." Understanding the effect of gene mutations has, traditionally, been much like trying to figure out what an unseen elephant looks like just by touching it. Touch enough places and you might get a rough idea, but the process will be long and slow and frustrating. "The way we have had to do this is so slow," said Zhu, of UVA's Department of Pharmacology and the UVA Cancer Center. "You can do one gene and one mutation at a time. Now, hopefully, we can do like 40 or 100 of them simultaneously." Zhu's approach uses an HIV-like virus to replace genes with mutant genes, so that scientists can understand the effects caused by the mutation. He developed the approach, requiring years of effort, out of a desire to both speed up research and also make it possible for more labs to participate. "Even with the CRISPR [gene editing] technology we have now, it still costs a huge amount of money and time and most labs cannot do it, so we wanted to develop something simple every lab can do," he said. "No other approach is so efficient and fast right now. You'd need to spend 10 years to do what we are doing in three months, so it's an entirely different scale." To demonstrate the effectiveness of his new technique, Zhu already has analyzed approximately 50 mutations of the BRaf gene, mutations that have been linked to tumors and to a neurodevelopmental disorder known as cardio-facio-cutaneous syndrome. The work sheds important light on the role of the mutations in disease. Zhu's new technique may even let researchers revisit failed experimental treatments, determine why they failed and identify patients in which they will be effective. It may be that a treatment didn't work because the patient didn't have the right mutation, or because the treatment didn't affect the gene in the right way. It's not as simple as turning a gene on or off, Zhu noted; instead, a treatment must prompt the right amount of gene activity, and that may require prodding a gene to do more or pulling on the reins so that it does less. "The problem in the cancer field is that they have many high-profile papers of clinical trials [that] all failed in some way," he said. "We wondered why in these patients sometimes it doesn't work, that with the same drug some patients are getting better and some are getting worse. The reason is that you don't know which drugs are going to help with their particular mutation. So that would be true precision medicine: You have the same condition, the same syndrome, but a different mutation, so you have to use different drugs." Zhu and his team have described the technique in an article published in the scientific journal Genes & Development, making it available to scientists around the world. The paper was written by Chae-Seok Lim, Xi Kang, Vincent Mirabella, Huaye Zhang, Qian Bu, Yoichi Araki, Elizabeth T. Hoang, Shiqiang Wang, Ying Shen, Sukwoo Choi, Bong-Kiun Kaang, Qiang Chang, Zhiping P. Pang, Richard L. Huganir and Zhu. The work was supported by the National Natural Science Foundation of China, the Robert Wood Johnson Foundation, the National Honor Scientist Program of Korea, the Howard Hughes Medical Institute and the National Institutes of Health, grants MH108321, NS065183, NS089578, HD064743, AA023797, MH64856, NS036715, NS053570, NS091452 and NS092548.

News Article | May 2, 2017

The National Academy of Sciences announced today the election of 84 new members and 21 foreign associates in recognition of their distinguished and continuing achievements in original research. The National Academy of Sciences announced today the election of 84 new members and 21 foreign associates in recognition of their distinguished and continuing achievements in original research. Those elected today bring the total number of active members to 2,290 and the total number of foreign associates to 475. Foreign associates are nonvoting members of the Academy, with citizenship outside the United States. Newly elected members and their affiliations at the time of election are: Bates, Frank S.; Regents Professor, department of chemical engineering and materials science, University of Minnesota, Minneapolis Beilinson, Alexander; David and Mary Winton Green University Professor, department of mathematics, The University of Chicago, Chicago Bell, Stephen P.; investigator, Howard Hughes Medical Institute; and professor of biology, department of biology, Massachusetts Institute of Technology, Cambridge Bhatia, Sangeeta N.; John J. (1929) and Dorothy Wilson Professor, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge Buzsáki, György; professor, Neuroscience Institute, departments of physiology and neuroscience, New York University Langone Medical Center, New York City Carroll, Dana; distinguished professor, department of biochemistry, University of Utah School of Medicine, Salt Lake City Cohen, Judith G.; Kate Van Nuys Page Professor of Astronomy, department of astronomy, California Institute of Technology, Pasadena Crabtree, Robert H.; Conkey P. Whitehead Professor of Chemistry, department of chemistry, Yale University, New Haven, Conn. Cronan, John E.; professor and head of microbiology, professor of biochemistry, and Microbiology Alumni Professor, department of microbiology, University of Illinois, Urbana-Champaign Cummins, Christopher C.; Henry Dreyfus Professor of Chemistry, Massachusetts Institute of Technology, Cambridge Darensbourg, Marcetta Y.; distinguished professor of chemistry, department of chemistry, Texas A&M University, College Station DeVore, Ronald A.; The Walter E. Koss Professor and distinguished professor, department of mathematics, Texas A&M University, College Station Diamond, Douglas W.; Merton H. Miller Distinguished Service Professor of Finance, The University of Chicago, Chicago Doe, Chris Q.; investigator, Howard Hughes Medical Institute; and professor of biology, Institute of Molecular Biology, University of Oregon, Eugene Duflo, Esther; Co-founder and co-Director of the Abdul Latif Jameel Poverty Action Lab, and Professor of Poverty Alleviation and Development Economics, Massachusetts Institute of Technology, Cambridge Edwards, Robert Haas; professor of neurology and physiology, University of California, San Francisco Firestone, Mary K.; professor and associate dean of instruction and student affairs, department of environmental science policy and management, University of California, Berkeley Fischhoff, Baruch; Howard Heinz University Professor, department of social and decision sciences and department of engineering and public policy, Carnegie Mellon University, Pittsburgh Ginty, David D.; investigator, Howard Hughes Medical Institute; and Edward R. and Anne G. Lefler Professor of Neurobiology, department of neurobiology, Harvard Medical School, Boston Glass, Christopher K.; professor of cellular and molecular medicine and professor of medicine, University of California, San Diego Goldman, Yale E.; professor, department of physiology, Pennsylvania Muscle Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia González, Gabriela; spokesperson, LIGO Scientific Collaboration; and professor, department of physics and astronomy, Louisiana State University, Baton Rouge Hagan, John L.; John D. MacArthur Professor of Sociology and Law, department of sociology, Northwestern University, Evanston, Ill. Hatten, Mary E.; Frederick P. Rose Professor, laboratory of developmental neurobiology, The Rockefeller University, New York City Hebard, Arthur F.; distinguished professor of physics, department of physics, University of Florida, Gainesville Jensen, Klavs F.; Warren K. Lewis Professor of Chemical Engineering and professor of materials science and engineering, Massachusetts Institute of Technology, Cambridge Kahn, Barbara B.; vice chair for research strategy and George R. Minot Professor of Medicine at Harvard Medical School, Beth Israel Deaconess Medical Center, Boston Kinder, Donald R.; Philip E. Converse Collegiate Professor of Political Science and Psychology and research scientist, department of political science, Center for Political Studies, Institute for Social Research, University of Michigan, Ann Arbor Lazar, Mitchell A.; Willard and Rhoda Ware Professor in Diabetes and Metabolic Diseases, and director, Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia Locksley, Richard M.; investigator, Howard Hughes Medical Institute; and professor, department of medicine (infectious diseases), and Marion and Herbert Sandler Distinguished Professorship in Asthma Research, University of California, San Francisco Lozano, Guillermina; professor and chair, department of genetics, The University of Texas M.D. Anderson Cancer Center, Houston Mavalvala, Nergis; Curtis and Kathleen Marble Professor of Astrophysics and associate head, department of physics, Massachusetts Institute of Technology, Cambridge Moore, Jeffrey Scott; Murchison-Mallory Professor of Chemistry, department of chemistry, University of Illinois, Urbana-Champaign Moore, Melissa J.; chief scientific officer, mRNA Research Platform, Moderna Therapeutics, Cambridge, Mass.; and Eleanor Eustis Farrington Chair of Cancer Research Professor, RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester Nunnari, Jodi M.; professor, department of molecular and cellular biology, University of California, Davis O'Farrell, Patrick H.; professor of biochemistry and biophysics, department of biochemistry and biophysics, University of California, San Francisco Ort, Donald R.; research leader and Robert Emerson Professor, USDA/ARS Global Change and Photosynthesis Research Unit, departments of plant biology and crop sciences, University of Illinois, Urbana-Champaign Parker, Gary; professor, department of civil and environmental engineering and department of geology, University of Illinois, Urbana-Champaign Patapoutian, Ardem; investigator, Howard Hughes Medical Institute; and professor, department of molecular and cellular neuroscience, The Scripps Research Institute, La Jolla, Calif. Pellegrini, Claudio; distinguished professor emeritus, department of physics and astronomy, University of California, Los Angeles Pikaard, Craig, S.; investigator, Howard Hughes Medical Institute and Gordon and Betty Moore Foundation; and distinguished professor of biology and molecular and cellular biochemistry, department of biology, Indiana University, Bloomington Read, Nicholas; Henry Ford II Professor of Physics and professor of applied physics and mathematics, Yale University, New Haven, Conn. Roediger, Henry L.; James S. McDonnell Distinguished and University Professor of Psychology, department of psychology and brain sciences, Washington University, St. Louis Rosenzweig, Amy C.; Weinberg Family Distinguished Professor of Life Sciences, and professor, departments of molecular biosciences and of chemistry, Northwestern University, Evanston, Ill. Seto, Karen C.; professor, Yale School of Forestry and Environmental Studies, New Haven, Conn. Seyfarth, Robert M.; professor of psychology and member of the graduate groups in anthropology and biology, University of Pennsylvania, Philadelphia Sibley, L. David; Alan A. and Edith L. Wolff Distinguished Professor in Molecular Microbiology, department of molecular microbiology, Washington University School of Medicine, St. Louis Spielman, Daniel A.; Henry Ford II Professor of Computer Science and Mathematics, departments of computer science and mathematics, Yale University, New Haven, Conn. Sudan, Madhu; Gordon McKay Professor of Computer Science, John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Mass. Tishkoff, Sarah; David and Lyn Silfen University Professor, departments of genetics and biology, University of Pennsylvania, Philadelphia Van Essen, David C.; Alumni Professor of Neurobiology, department of anatomy and neurobiology, Washington University School of Medicine, St. Louis Vidale, John E.; professor, department of earth and space sciences, University of Washington, Seattle Wennberg, Paul O.; R. Stanton Avery Professor of Atmospheric Chemistry and Environmental Science and Engineering, California Institute of Technology, Pasadena Wilson, Rachel I.; Martin Family Professor of Basic Research in the Field of Neurobiology, department of neurobiology, Harvard Medical School, Boston Zachos, James C.; professor, department of earth and planetary sciences, University of California, Santa Cruz, Santa Cruz Newly elected foreign associates, their affiliations at the time of election, and their country of citizenship are: Addadi, Lia; professor and Dorothy and Patrick E. Gorman Chair of Biological Ultrastructure, department of structural science, Weizmann Institute of Science, Rehovot, Israel (Israel/Italy) Folke, Carl; director and professor, The Beijer Institute of Ecological Economics, Royal Swedish Academy of Sciences, Stockholm, Sweden (Sweden) Freeman, Kenneth C.; Duffield Professor of Astronomy, Mount Stromlo and Siding Spring Observatories, Research School of Astronomy and Astrophysics, Australian National University, Weston Creek (Australia) Lee, Sang Yup; distinguished professor, dean, and director, department of chemical and biomolecular engineering, Korea Advanced Institute of Science and Technology, Daejeon, South Korea (South Korea) Levitzki, Alexander; professor of biochemistry, unit of cellular signaling, department of biological chemistry, The Hebrew University of Jerusalem, Jerusalem (Israel) Peiris, Joseph Sriyal Malik; Tam Wah-Ching Professorship in Medical Science, School of Public Health, The University of Hong Kong, Pokfulam, Hong Kong, People's Republic of China (Sri Lanka) Robinson, Carol Vivien; Dr. Lee's Professor of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, England (United Kingdom) Thesleff, Irma; academician of science, professor, and research director, developmental biology program, Institute of Biotechnology, University of Helsinki, Helsinki (Finland) Underdal, Arild; professor of political science, department of political science, University of Oslo, Oslo, Norway (Norway) The National Academy of Sciences is a private, nonprofit institution that was established under a congressional charter signed by President Abraham Lincoln in 1863. It recognizes achievement in science by election to membership, and -- with the National Academy of Engineering and the National Academy of Medicine -- provides science, engineering, and health policy advice to the federal government and other organizations.

News Article | April 13, 2017

The 2017 Experimental Biology conference is almost here! Here are some of this year’s highlights: Dr. Michael Welsh from the Howard Hughes Medical Institute and University of Iowa will be presenting the Walter B. Cannon Award Lecture. He will be speaking about Cystic Fibrosis. This year’s Nobel lecture in Physiology or Medicine will be given by Laureate Dr. Louis J Ignarro who, along with Drs. Robert Furchgott and Ferid Murad, won the prize in 1998 for discovering how nitric oxide works in the cardiovascular system. I am also looking forward to the many comparative physiology sessions at this year’s meeting, which include sessions on how stress impacts the cardiovascular system and how hypoxia affects development and health. I am especially looking forward to this year’s August Krogh lecture that will be given by Dr. Warren Burggren from the University of North Texas who will speak about developmental plasticity, epigenetics and evolution. This should be a great year for comparative physiology! I’ll keep you posted!

News Article | May 2, 2017

Science Says: Why are opioids so addictive? By The Associated Press When opioids act on the brain, they trigger the same processes that give people feelings of pleasure from activities like eating, but they do it far more intensely. Opioids also make some brain cells pump out a chemical messenger called dopamine, which encourages more drug use. Over time, that can produce craving that continues even long after someone stops using opioids, which can lead to relapse. In other brain circuits, opioids initially produce drowsiness and slower breathing. With repeated exposure, these circuits adapt so that a person feels relatively normal while using the drugs. But that adaptation also means that when a person is not using, they feel jittery and anxious — some of the symptoms of withdrawal. Opioids can also impair people's self-control if taken over time, so it's harder to stop using them even if people want to and even if the drugs no longer give them pleasure. Dr. Nora Volkow, director of the National Institute on Drug Abuse, compared the effect of the drugs to driving with bad steering. "Your steering wheel does not work properly. So not only are you actually accelerating with intense desire and motivation to get the drug, you are not able to self-regulate and say, 'If I take the drug, I will end up in jail.'" This Associated Press series was produced in partnership with the Howard Hughes Medical Institute's Department of Science Education. The AP is solely responsible for all content.

News Article | May 2, 2017

Science Says: Why are opioids so addictive? By The Associated Press When opioids act on the brain, they trigger the same processes that give people feelings of pleasure from activities like eating, but they do it far more intensely. Opioids also make some brain cells pump out a chemical messenger called dopamine, which encourages more drug use. Over time, that can produce craving that continues even long after someone stops using opioids, which can lead to relapse. In other brain circuits, opioids initially produce drowsiness and slower breathing. With repeated exposure, these circuits adapt so that a person feels relatively normal while using the drugs. But that adaptation also means that when a person is not using, they feel jittery and anxious — some of the symptoms of withdrawal. Opioids can also impair people's self-control if taken over time, so it's harder to stop using them even if people want to and even if the drugs no longer give them pleasure. Dr. Nora Volkow, director of the National Institute on Drug Abuse, compared the effect of the drugs to driving with bad steering. "Your steering wheel does not work properly. So not only are you actually accelerating with intense desire and motivation to get the drug, you are not able to self-regulate and say, 'If I take the drug, I will end up in jail.'" This Associated Press series was produced in partnership with the Howard Hughes Medical Institute's Department of Science Education. The AP is solely responsible for all content.

Bhandari D.,Howard Hughes Medical Institute | Reinisch K.,Yale University | Ferro-Novick S.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2010

Transport protein particle (TRAPP; also known as trafficking protein particle), a multimeric guanine nucleotide-exchange factor for the yeast GTPase Ypt1 and its mammalian homologue, RAB1, regulates multiple membrane trafficking pathways. TRAPP complexes exist in three forms, each of which activates Ypt1 or RAB1 through a common core of subunits and regulates complex localization through distinct subunits. Whereas TRAPPI and TRAPPII tether coated vesicles during endoplasmic reticulum to Golgi and intra-Golgi traffic, respectively, TRAPPIII has recently been shown to be reqiured for autophagy. These advances illustrate how the TRAPP complexes link Ypt1 and RAB1 activation to distinct membrane-tethering events. © 2010 Macmillan Publishers Limited. All rights reserved.

Stein A.,Howard Hughes Medical Institute | Rapoport T.A.,Howard Hughes Medical Institute
Cell | Year: 2014

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we use purified components from Saccharomyces cerevisiae to analyze the mechanism of retrotranslocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process in which the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain and discriminates misfolded from folded polypeptides. Both Hrd1p and substrate are polyubiquitinated, resulting in the binding of Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the deubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of polyubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retrotranslocation in which Hrd1p forms a membrane conduit for misfolded proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Davis R.J.,Howard Hughes Medical Institute
Seminars in Immunology | Year: 2014

The binding of tumour necrosis factor α (TNFα) to cell surface receptors engages multiple signal transduction pathways, including three groups of mitogen-activated protein (MAP) kinases: extracellular-signal-regulated kinases (ERKs); the cJun NH2-terminal kinases (JNKs); and the p38 MAP kinases. These MAP kinase signalling pathways induce a secondary response by increasing the expression of several inflammatory cytokines (including TNFα) that contribute to the biological activity of TNFα. MAP kinases therefore function both upstream and down-stream of signalling by TNFα receptors. Here we review mechanisms that mediate these actions of MAP kinases during the response to TNFα. © 2014 Elsevier Ltd.

Araci I.E.,Howard Hughes Medical Institute | Brisk P.,University of California at Riverside
Current Opinion in Biotechnology | Year: 2014

In 2002, Thorsen et al. integrated thousands of micromechanical valves on a single microfluidic chip and demonstrated that the control of the fluidic networks can be simplified through multiplexors [. 1]. This enabled realization of highly parallel and automated fluidic processes with substantial sample economy advantage. Moreover, the fabrication of these devices by multilayer soft lithography was easy and reliable hence contributed to the power of the technology; microfluidic large scale integration (mLSI). Since then, mLSI has found use in wide variety of applications in biology and chemistry. In the meantime, efforts to improve the technology have been ongoing. These efforts mostly focus on; novel materials, components, micromechanical valve actuation methods, and chip architectures for mLSI. In this review, these technological advances are discussed and, recent examples of the mLSI applications are summarized. © 2013 Elsevier Ltd.

Molkentin J.D.,Howard Hughes Medical Institute
Circulation Research | Year: 2011

Transient receptor potential (TRP) channels of multiple subclasses are expressed in the heart, although their functions are only now beginning to emerge, especially for the TRPC subclass that appears to regulate the cardiac hypertrophic response. Although TRP channels permeate many different cations, they are most often ascribed a specific biological function because of Ca 2+ influx, either for microdomain signaling or to reload internal Ca2+ stores in the endoplasmic reticulum through a store-operated mechanism. However, adult cardiac myocytes arguably do not require store-operated Ca2+ entry to regulate sarcoplasmic reticulum Ca 2+ levels and excitation-contraction coupling; hence, TRP channels expressed in the heart most likely coordinate signaling within local domains or through direct interaction with Ca-dependent regulatory proteins. Here, we review the emerging evidence that TRP channels, especially TRPCs, are critical regulators of microdomain signaling in the heart to control pathological hypertrophy in coordination with signaling through effectors such as calcineurin and NFAT (nuclear factor of activated T cells). © 2011 American Heart Association, Inc.

Chen X.,University of California at Riverside | Chen X.,Howard Hughes Medical Institute
Current Opinion in Genetics and Development | Year: 2012

microRNAs (miRNAs) and small interfering RNAs (siRNAs), which constitute two major classes of endogenous small RNAs in plants, impact a multitude of developmental and physiological processes by imparting sequence specificity to gene and genome regulation. Although lacking the third major class of small RNAs found in animals, Piwi-interacting RNAs (piRNAs), plants have expanded their repertoire of endogenous siRNAs, some of which fulfill similar molecular and developmental functions as piRNAs in animals. Research on plant miRNAs and siRNAs has contributed invaluable insights into small RNA biology, thanks to the highly conserved molecular logic behind the biogenesis and actions of small RNAs. Here, I review progress in the plant small RNA field in the past two years, with an emphasis on recent findings related to plant development. I do not recount the numerous developmental processes regulated by small RNAs; instead, I focus on major principles that have been derived from recent studies and draw parallels, when applicable, between plants and animals. © 2012 Elsevier Ltd.

Rozhkov N.V.,Howard Hughes Medical Institute | Hannon G.J.,Howard Hughes Medical Institute
Genes and Development | Year: 2013

Silencing of transposons in the Drosophila ovary relies on three Piwi family proteins-Piwi, Aubergine (Aub), and Ago3-acting in concert with their small RNA guides, the Piwi-interacting RNAs (piRNAs). Aub and Ago3 are found in the germcell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell typespecific knockdowns with measurements of steady-state transposon mRNA levels, nascent RNA synthesis, chromatin state, and small RNA abundance. In somatic cells, Piwi loss led to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady-state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on local chromatin states and transcription but also participating in germ cell piRNA biogenesis. © 2013 by Cold Spring Harbor Laboratory Press.

Doudna J.A.,Howard Hughes Medical Institute | Doudna J.A.,University of California at Berkeley | Doudna J.A.,Lawrence Berkeley National Laboratory
Annual Review of Biophysics | Year: 2013

Small RNA molecules regulate eukaryotic gene expression during development and in response to stresses including viral ection. Specialized ribonucleases and RNA-binding proteins govern the production and action of small regulatory RNAs. After initial processing in the nucleus by Drosha, precursor microRNAs (pre-miRNAs) are transported to the cytoplasm, where Dicer cleavage generates mature microRNAs (miRNAs) and short interfering RNAs (siRNAs). These double-stranded products assemble with Argonaute proteins such that one strand is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs by endonucleolytic cleavage or translational repression. Molecular structures of Dicer and Argonaute proteins, and of RNA-bound complexes, have offered exciting insights into the mechanisms operating at the heart of RNA-silencing pathways. Copyright © 2013 by Annual Reviews.

Ji L.,University of California at Riverside | Chen X.,University of California at Riverside | Chen X.,Howard Hughes Medical Institute
Cell Research | Year: 2012

As central components of RNA silencing, small RNAs play diverse and important roles in many biological processes in eukaryotes. Aberrant reduction or elevation in the levels of small RNAs is associated with many developmental and physiological defects. The in vivo levels of small RNAs are precisely regulated through modulating the rates of their biogenesis and turnover. 2′-O-methylation on the 3′ terminal ribose is a major mechanism that increases the stability of small RNAs. The small RNA methyltransferase HUA ENHANCER1 (HEN1) and its homologs methylate microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs (piRNAs) in animals, and siRNAs in Drosophila. 3′ nucleotide addition, especially uridylation, and 3′-5′ exonucleolytic degradation are major mechanisms that turnover small RNAs. Other mechanisms impacting small RNA stability include complementary RNAs, cis-elements in small RNA sequences and RNA-binding proteins. Investigations are ongoing to further understand how small RNA stability impacts their accumulation in vivo in order to improve the utilization of RNA silencing in biotechnology and therapeutic applications. © 2012 IBCB, SIBS, CAS All rights reserved.

Karginov F.V.,Howard Hughes Medical Institute | Karginov F.V.,University of California at Riverside | Hannon G.J.,Howard Hughes Medical Institute
Genes and Development | Year: 2013

When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Argonaute 2 (Ago2)/microRNA (miRNA) machinery has been shown to participate in stress-induced translational up-regulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by cross-linking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq). Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3′ untranslated region (UTR) and coding sequence (CDS) sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2-binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA sequencing (RNA-seq). Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian miRNAs in mediating the translational component of the stress response. © 2013 by Cold Spring Harbor Laboratory Press.

Baker T.A.,Howard Hughes Medical Institute
Annual Review of Biochemistry | Year: 2011

AAA+ family proteolytic machines (ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and the 26S proteasome) perform protein quality control and are used in regulatory circuits in all cells. These machines contain a compartmental protease, with active sites sequestered in an interior chamber, and a hexameric ring of AAA+ ATPases. Substrate proteins are tethered to the ring, either directly or via adaptor proteins. An unstructured region of the substrate is engaged in the axial pore of the AAA+ ring, and cycles of ATP binding/hydrolysis drive conformational changes that create pulses of pulling that denature the substrate and translocate the unfolded polypeptide through the pore and into the degradation chamber. Here, we review our current understanding of the molecular mechanisms of substrate recognition, adaptor function, and ATP-fueled unfolding and translocation. The unfolding activities of these and related AAA+ machines can also be used to disassemble or remodel macromolecular complexes and to resolubilize aggregates. © 2011 by Annual Reviews. All rights reserved.

Rogers K.,University of California at Riverside | Chen X.,University of California at Riverside | Chen X.,Howard Hughes Medical Institute
Plant Cell | Year: 2013

MicroRNAs (miRNAs) are small RNAs that control gene expression through silencing of target mRNAs. Mature miRNAs are processed from primary miRNA transcripts by the endonuclease activity of the DICER-LIKE1 (DCL1) protein complex. Mechanisms exist that allow the DCL1 complex to precisely excise the miRNA from its precursor. Our understanding of miRNA biogenesis, particularly its intersection with transcription and other aspects of RNA metabolism such as splicing, is still evolving. Mature miRNAs are incorporated into an ARGONAUTE (AGO) effector complex competent for target gene silencing but are also subjected to turnover through a degradation mechanism that is beginning to be understood. The mechanisms of miRNA target silencing in plants are no longer limited to AGO-catalyzed slicing, and the contribution of translational inhibition is increasingly appreciated. Here, we review the mechanisms underlying the biogenesis, turnover, and activities of plant miRNAs. © 2013 American Society of Plant Biologists. All rights reserved.

Penmatsa A.,University of Oregon | Penmatsa A.,Indian Institute of Science | Wang K.H.,Amgen | Gouaux E.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2015

Most antidepressants elicit their therapeutic benefits through selective blockade of Na + /Cl â -coupled neurotransmitter transporters. Here we report X-ray structures of the Drosophila melanogaster dopamine transporter in complexes with the polycyclic antidepressants nisoxetine or reboxetine. The inhibitors stabilize the transporter in an outward-open conformation by occupying the substrate-binding site. These structures explain how interactions between the binding pocket and substituents on the aromatic rings of antidepressants modulate drug-transporter selectivity. © 2015 Nature America, Inc. All rights reserved.

Riccomagno M.M.,University of California at Riverside | Kolodkin A.L.,Howard Hughes Medical Institute
Annual Review of Cell and Developmental Biology | Year: 2015

The assembly of functional neural circuits requires the combined action of progressive and regressive events. Regressive events encompass a variety of inhibitory developmental processes, including axon and dendrite pruning, which facilitate the removal of exuberant neuronal connections. Most axon pruning involves the removal of axons that had already made synaptic connections; thus, axon pruning is tightly associated with synapse elimination. In many instances, these developmental processes are regulated by the interplay between neurons and glial cells that act instructively during neural remodeling. Owing to the importance of axon and dendritic pruning, these remodeling events require precise spatial and temporal control, and this is achieved by a range of distinct molecular mechanisms. Disruption of these mechanisms results in abnormal pruning, which has been linked to brain dysfunction. Therefore, understanding the mechanisms of axon and dendritic pruning will be instrumental in advancing our knowledge of neural disease and mental disorders. © 2015 by Annual Reviews. All rights reserved.

Root C.M.,Howard Hughes Medical Institute | Denny C.A.,New York State Psychiatric Institute | Hen R.,New York State Psychiatric Institute | Hen R.,Columbia University | Axel R.,Howard Hughes Medical Institute
Nature | Year: 2014

Innate behaviours are observed in naive animals without prior learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically determined and stereotyped. The neural circuits that convey olfactory information from the sense organ to the cortical and subcortical olfactory centres have been anatomically defined, but the specific pathways responsible for innate responses to volatile odours have not been identified. Here we devise genetic strategies that demonstrate that a stereotyped neural circuit that transmits information from the olfactory bulb to cortical amygdala is necessary for innate aversive and appetitive behaviours. Moreover, we use the promoter of the activity-dependent gene arc to express the photosensitive ion channel, channelrhodopsin, in neurons of the cortical amygdala activated by odours that elicit innate behaviours. Optical activation of these neurons leads to appropriate behaviours that recapitulate the responses to innate odours. These data indicate that the cortical amygdala plays a critical role in generating innate odour-driven behaviours but do not preclude its participation in learned olfactory behaviours. © 2014 Macmillan Publishers Limited. All rights reserved.

Rando T.A.,Stanford University | Chang H.Y.,Stanford University | Chang H.Y.,Howard Hughes Medical Institute
Cell | Year: 2012

The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges. © 2012 Elsevier Inc.

Hughes J.F.,Howard Hughes Medical Institute | Rozen S.,National University of Singapore
Annual Review of Genomics and Human Genetics | Year: 2012

In mammals, the Y chromosome plays the pivotal role in male sex determination and is essential for normal sperm production. Yet only three Y chromosomes have been completely sequenced to date-those of human, chimpanzee, and rhesus macaque. While Y chromosomes are notoriously difficult to sequence owing to their highly repetitive genomic landscapes, these dedicated sequencing efforts have generated tremendous yields in medical, biological, and evolutionary insight. Knowledge of the complex structural organization of the human Y chromosome and a complete catalog of its gene content have provided a deeper understanding of the mechanisms that generate disease-causing mutations and large-scale rearrangements. Variation among human Y-chromosome sequences has been an invaluable tool for understanding relationships among human populations. Comprehensive comparisons of the human Y-chromosome sequence with those of other primates have illuminated aspects of Y-chromosome evolutionary dynamics over much longer timescales (>25 million years compared with 100,000 years). The future sequencing of additional Y chromosomes will provide a basis for a more comprehensive understanding of the evolution of Y chromosomes and their roles in reproductive biology. © 2012 by Annual Reviews. All rights reserved.

Birkenfeld A.L.,Howard Hughes Medical Institute | Shulman G.I.,Howard Hughes Medical Institute | Shulman G.I.,Novo Nordisk AS
Hepatology | Year: 2014

Nonalcoholic fatty liver disease (NAFLD), hepatic insulin resistance, and type 2 diabetes are all strongly associated and are all reaching epidemic proportions. Whether there is a causal link between NAFLD and hepatic insulin resistance is controversial. This review will discuss recent studies in both humans and animal models of NAFLD that have implicated increases in hepatic diacylglycerol (DAG) content leading to activation of novel protein kinase Cε{lunate} (PKCε{lunate}) resulting in decreased insulin signaling in the pathogenesis of NAFLD-associated hepatic insulin resistance and type 2 diabetes. The DAG-PKCε{lunate} hypothesis can explain the occurrence of hepatic insulin resistance observed in most cases of NAFLD associated with obesity, lipodystrophy, and type 2 diabetes. © 2013 by the American Association for the Study of Liver Diseases.

Rowitch D.H.,Howard Hughes Medical Institute | Rowitch D.H.,University of California at San Francisco | Kriegstein A.R.,University of California at San Francisco
Nature | Year: 2010

Oligodendrocytes and astrocytes are macroglial cells of the vertebrate central nervous system. These cells have diverse roles in the maintenance of neurological function. In the embryo, the genetic mechanisms that underlie the specification of macroglial precursors in vivo appear strikingly similar to those that regulate the development of the diverse neuron types. The switch from producing neuronal to glial subtype-specific precursors can be modelled as an interplay between region-restricted components and temporal regulators that determine neurogenic or gliogenic phases of development, contributing to glial diversity. Gaining insight into the developmental genetics of macroglia has great potential to improve our understanding of a variety of neurological disorders in humans. © 2010 Macmillan Publishers Limited. All rights reserved.

Green D.R.,St Judes Childrens Research Hospital | Levine B.,Howard Hughes Medical Institute
Cell | Year: 2014

The health of metazoan organisms requires an effective response to organellar and cellular damage either by repair of such damage and/or by elimination of the damaged parts of the cells or the damaged cell in its entirety. Here, we consider the progress that has been made in the last few decades in determining the fates of damaged organelles and damaged cells through discrete, but genetically overlapping, pathways involving the selective autophagy and cell death machinery. We further discuss the ways in which the autophagy machinery may impact the clearance and consequences of dying cells for host physiology. Failure in the proper removal of damaged organelles and/or damaged cells by selective autophagy and cell death processes is likely to contribute to developmental abnormalities, cancer, aging, inflammation, and other diseases. © 2014 Elsevier Inc.

Prinz W.A.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | Rapoport T.A.,Howard Hughes Medical Institute
Cell | Year: 2011

How is the characteristic shape of an organelle generated? Recent work has provided insight into how the tubular network of the endoplasmic reticulum (ER) is formed. The tubules themselves are shaped by the reticulons and DP1/Yop1p, whereas their fusion into a network is brought about by membrane-bound GTPases that include the atlastins, Sey1p, and RHD3. © 2011 Elsevier Inc.

News Article | February 28, 2017

By inhibiting the proteasome -- the cell's garbage disposal -- in a novel way, a new treatment causes cancer cells to fill up with 'trash' and self-destruct The genomes of cancer cells--cells that do not obey signals to stop reproducing--are riddled with genetic mutations, causing them inadvertently to make many dysfunctional proteins. Like all other cells, cancer cells need to be vigilant about cleaning themselves up in order to survive. Now, biologists in the laboratory of Ray Deshaies, Caltech professor of biology and Howard Hughes Medical Institute Investigator, have developed a new way to inhibit the cancer cell cleanup mechanism, causing the cells to fill up with defective proteins and thus self-destruct. The findings appear online in a paper in the February 27 issue of Nature Chemical Biology. The proteasome is a hollow cylindrical structure that serves as a kind of cellular garbage disposal. It lets in proteins through small openings on each end, chops them up, and spits out the remains. When a bad protein is made by a cell, the protein gets tagged with chains composed of at least four copies of a small protein called ubiquitin. The tags signal to the proteasome that the bad protein needs to be destroyed. One part of the proteasome, called Rpn11, cuts off the ubiquitin chain as the defective protein is being stuffed into the garbage disposal. This step is necessary because the ubiquitin chain is too big to fit inside the proteasome. A new compound developed by the Deshaies group, in collaboration with researchers from UC San Diego, inhibits Rpn11 activity, making it impossible for the proteasome to fully destroy bad proteins. Massive accumulation of these bad proteins causes catastrophic stress to the cell that results in cell death. While the compound affects the proteasomes in all cells, normal cells are thought to produce fewer dysfunctional proteins than cancer cells. Some types of cancer cells are therefore more sensitive than normal cells to proteasome inhibition and thus even a small dose of the drug can be fatal to them. "All current cancer drugs that target the proteasome work by inhibiting the protein-chopping enzymes on the inside of the proteasome; therefore they all have similar drawbacks and tend to lose efficacy over time," says Jing Li, a postdoctoral scholar in biology and biological engineering and first author on the paper. "Our research offers an alternative path to disabling proteasome function, including in cells that no longer respond to the existing drugs." The compound was tested in human cancer cells in the laboratory, but more work needs to be done to further improve its potency and to evaluate its potential as a therapeutic drug through testing in animals. The paper is titled "Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11." In addition to Li and Deshaies, other Caltech coauthors are postdoctoral fellow Tanya Yakushi and Sonja Hess, director of the Proteome Exploration Laboratory. The work was funded by grants from the Caltech Gates Grubstake Fund, Amgen, the National Institutes of Health, the Gordon and Betty Moore Foundation, the Beckman Institute, and the Howard Hughes Medical Institute.

News Article | February 15, 2017

WASHINGTON - Clinical trials for genome editing of the human germline - adding, removing, or replacing DNA base pairs in gametes or early embryos - could be permitted in the future, but only for serious conditions under stringent oversight, says a new report from the National Academy of Sciences and the National Academy of Medicine. The report outlines several criteria that should be met before allowing germline editing clinical trials to go forward. Genome editing has already entered clinical trials for non-heritable applications, but should be allowed only for treating or preventing diseases or disabilities at this time. Genome editing is not new. But new powerful, precise, and less costly genome editing tools, such as CRISPR/Cas9, have led to an explosion of new research opportunities and potential clinical applications, both heritable and non-heritable, to address a wide range of human health issues. Recognizing the promise and the concerns related to this technology, NAS and NAM appointed a study committee of international experts to examine the scientific, ethical, and governance issues surrounding human genome editing. Human genome editing is already widely used in basic research and is in the early stages of development and trials for clinical applications that involve non-heritable (somatic) cells. These therapies affect only the patient, not any offspring, and should continue for treatment and prevention of disease and disability, using the existing ethical norms and regulatory framework for development of gene therapy. Oversight authorities should evaluate safety and efficacy of proposed somatic applications in the context of the risks and benefits of intended use. However, there is significant public concern about the prospect of using these same techniques for so-called "enhancement" of human traits and capacities such as physical strength, or even for uses that are not possible, such as improving intelligence. The report recommends that genome editing for enhancement should not be allowed at this time, and that broad public input and discussion should be solicited before allowing clinical trials for somatic genome editing for any purpose other than treating or preventing disease or disability. "Human genome editing holds tremendous promise for understanding, treating, or preventing many devastating genetic diseases, and for improving treatment of many other illnesses," said Alta Charo, co-chair of the study committee and Sheldon B. Lubar Distinguished Chair and Warren P. Knowles Professor of Law and Bioethics, University of Wisconsin-Madison. "However, genome editing to enhance traits or abilities beyond ordinary health raises concerns about whether the benefits can outweigh the risks, and about fairness if available only to some people." Germline genome editing, in contrast, is contentious because genetic changes would be inherited by the next generation. Many view germline editing as crossing an "ethically inviolable" line, the report says. Concerns raised include spiritual objections to interfering with human reproduction to speculation about effects on social attitudes toward people with disabilities to possible risks to the health and safety of future children. But germline genome editing could provide some parents who are carriers of genetic diseases with their best or most acceptable option for having genetically related children who are born free of these diseases. Heritable germline editing is not ready to be tried in humans. Much more research is needed before it could meet the appropriate risk and benefit standards for clinical trials. The technology is advancing very rapidly, though, making heritable genome editing of early embryos, eggs, sperm, or precursor cells in the foreseeable future "a realistic possibility that deserves serious consideration," the report says. Although heritable germline genome editing trials must be approached with caution, the committee said, caution does not mean prohibition. At present, heritable germline editing is not permissible in the United States, due to an ongoing prohibition on the U.S. Food and Drug Administration's ability to use federal funds to review "research in which a human embryo is intentionally created or modified to include a heritable genetic modification." A number of other countries have signed an international convention that prohibits germline modification. If current restrictions are removed, and for countries where germline editing would already be permitted, the committee recommended stringent criteria that would need to be met before going forward with clinical trials. They include: (1) absence of reasonable alternatives; (2) restriction to editing genes that have been convincingly demonstrated to cause or strongly predispose to a serious disease or condition; (3) credible pre-clinical and/or clinical data on risks and potential health benefits; (4) ongoing, rigorous oversight during clinical trials; (5) comprehensive plans for long-term multigenerational follow-up; and (6) continued reassessment of both health and societal benefits and risks, with wide-ranging, ongoing input from the public. Policymaking surrounding human genome editing applications should incorporate public participation, and funding of genome editing research should include support to study the socio-political, ethical, and legal aspects and evaluate efforts to build public communication and engagement on these issues. "Genome editing research is very much an international endeavor, and all nations should ensure that any potential clinical applications reflect societal values and be subject to appropriate oversight and regulation," said committee co-chair Richard Hynes, Howard Hughes Medical Institute Investigator and Daniel K. Ludwig Professor for Cancer Research, Massachusetts Institute of Technology. "These overarching principles and the responsibilities that flow from them should be reflected in each nation's scientific community and regulatory processes. Such international coordination would enhance consistency of regulation." The study was funded by the Defense Advanced Research Projects Agency, the Greenwall Foundation, the John D. and Catherine T. MacArthur Foundation, U.S. Department of Health and Human Services, U.S. Food and Drug Administration, and the Wellcome Trust, with additional support from the National Academies' Presidents' Circle Fund and the National Academy of Sciences W.K. Kellogg Foundation Fund. The National Academy of Sciences and the National Academy of Medicine are private, nonprofit institutions that, along with the National Academy of Engineering, provide independent, objective analysis and advice to the nation to solve complex problems and inform public policy decisions related to science, technology, and medicine. The Academies operate under an 1863 congressional charter to the National Academy of Sciences, signed by President Lincoln. For more information, visit http://www. . Copies of Human Genome Editing: Science, Ethics, and Governance are available at http://www. or by calling 202-334-3313 or 1-800-624-6242. Reporters may obtain a copy from the Office of News and Public Information (contacts listed above). R. Alta Charo1 (co-chair) Sheldon B. Lubar Distinguished Chair and Warren P. Knowles Professor of Law and Bioethics University of Wisconsin Madison Richard O. Hynes1,2 (co-chair) Investigator Howard Hughes Medical Institute, and Daniel K. Ludwig Professor for Cancer Research Massachusetts Institute of Technology Cambridge Ellen Wright Clayton1 Craig Weaver Professor of Pediatrics, and Professor of Law Vanderbilt University Nashville, Tenn. Barry S. Coller1,2 David Rockefeller Professor of Medicine, Physician in Chief, and Head Allen and Frances Adler Laboratory of Blood and Vascular Biology Rockefeller University New York City Ephrat Levy-Lahad Director Fuld Family Department of Medical Genetics Shaare Zedek Medical Center Faculty of Medicine Hebrew University of Jerusalem Jerusalem Luigi Naldini Professor of Cell and Tissue Biology and of Gene and Cell Therapy San Raffaele University, and Director San Raffaele Telethon Institute for Gene Therapy Milan Duanqing Pei Professor and Director General Guangzhou Institute of Biomedicine and Health Chinese Academy of Sciences Guangzhou, China Janet Rossant2 Senior Scientist and Chief of Research Emeritus Hospital for Sick Children University of Toronto Toronto Dietram A. Scheufele John E. Ross Professor in Science Communication and Vilas Distinguished Achievement Professor University of Wisconsin Madison Jonathan Weissman2 Professor Department of Cellular and Molecular Pharmacology University of California San Francisco Keith R. Yamamoto1,2 Vice Chancellor for Science Policy and Strategy University of California San Francisco

Gemberling M.,Howard Hughes Medical Institute | Bailey T.J.,University of Notre Dame | Hyde D.R.,University of Notre Dame | Poss K.D.,Howard Hughes Medical Institute
Trends in Genetics | Year: 2013

For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues and, in some cases, have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. © 2013 Elsevier Ltd.

Kolodkin A.L.,Howard Hughes Medical Institute | Tessier-Lavigne M.,Genentech | Tessier-Lavigne M.,Rockefeller University
Cold Spring Harbor Perspectives in Biology | Year: 2011

The complex patterns of neuronal wiring in the adult nervous system depend on a series of guidance events during neural development that establish a framework on which functional circuits can be built. In this subject collection the cellular and molecular mechanisms that underlie neuronal guidance are considered from several perspectives ranging from how cytoskeletal dynamics within extending neuronal growth cones steer axons to howguidance cues influence synaptogenesis. We introduce here some basic topics to frame the more detailed reviews in following articles including the cellular strategies that define basic themes governing neuronal wiring throughout life an enumeration of the molecular cues and receptors knownto play key guidance roles during neural development and an overview of the signaling mechanisms that transduce guidance information into growth-cone steering. © 2011 Cold Spring Harbor Laboratory Press.

Korennykh A.,Princeton University | Walter P.,Howard Hughes Medical Institute | Walter P.,University of California at San Francisco
Annual Review of Cell and Developmental Biology | Year: 2012

The unfolded protein response (UPR) is a network of intracellular signaling pathways that maintain the protein-folding capacity of the endoplasmic reticulum (ER) in eukaryotic cells. Dedicated molecular sensors embedded in the ER membrane detect incompletely folded or unfolded proteins in the ER lumen and activate a transcriptional program that increases the abundance of the ER according to need. In metazoans the UPR additionally regulates translation and thus relieves unfolded protein load by globally reducing protein synthesis. If homeostasis in the ER cannot be reestablished, the metazoan UPR switches from the prosurvival to the apoptotic mode. The UPR involves a complex, coordinated action of many genes that is controlled by one ER-embedded sensor, Ire1, in yeasts, and three sensors, Ire1, PERK, and ATF6, in higher eukaryotes, including human. We discuss the emerging molecular understanding of the UPR and focus on the structural biology of Ire1 and PERK, the two recently crystallized UPR sensors. Copyright © 2012 by Annual Reviews. All rights reserved.

Pikaard C.S.,Howard Hughes Medical Institute | Scheid O.M.,Gregor Mendel Institute of Molecular Plant Biology
Cold Spring Harbor Perspectives in Biology | Year: 2014

The study of epigenetics in plants has a long and rich history, from initial descriptions of non-Mendelian gene behaviors to seminal discoveries of chromatin-modifying proteins and RNAs that mediate gene silencing in most eukaryotes, including humans. Genetic screens in the model plant Arabidopsis have been particularly rewarding, identifying more than 130 epigenetic regulators thus far. The diversity of epigenetic pathways in plants is remarkable, presumably contributing to the phenotypic plasticity of plant postembryonic development and the ability to survive and reproduce in unpredictable environments. © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Lutz J.-F.,Charles Sadron Institute | Ouchi M.,Kyoto University | Liu D.R.,Howard Hughes Medical Institute | Sawamoto M.,Kyoto University
Science | Year: 2013

Sequence-controlled polymers are macromolecules in which monomer units of different chemical nature are arranged in an ordered fashion. The most prominent examples are biological and have been studied and used primarily by molecular biologists and biochemists. However, recent progress in protein- and DNA-based nanotechnologies has shown the relevance of sequence-controlled polymers to nonbiological applications, including data storage, nanoelectronics, and catalysis. In addition, synthetic polymer chemistry has provided interesting routes for preparing nonnatural sequence-controlled polymers. Although these synthetic macromolecules do not yet compare in functional scope with their natural counterparts, they open up opportunities for controlling the structure, self-assembly, and macroscopic properties of polymer materials.

Devireddy L.R.,Case Western Reserve University | Hart D.O.,Howard Hughes Medical Institute | Goetz D.H.,University of California at San Francisco | Green M.R.,Howard Hughes Medical Institute
Cell | Year: 2010

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans. © 2010 Elsevier Inc.

Sabio G.,CSIC - National Center for Biotechnology | Davis R.J.,Howard Hughes Medical Institute
Trends in Biochemical Sciences | Year: 2010

The cJun NH2-terminal kinase isoform JNK1 is implicated in the mechanism of obesity-induced insulin resistance. Feeding a high-fat diet causes activation of the JNK1 signaling pathway, insulin resistance, and obesity in mice. Germ-line ablation of Jnk1 prevents both diet-induced obesity and insulin resistance. Genetic analysis indicates that the effects of JNK1 on insulin resistance can be separated from effects of JNK1 on obesity. Emerging research indicates that JNK1 plays multiple roles in the regulation of insulin resistance, including altered gene expression, hormone/cytokine production, and lipid metabolism. Together, these studies establish JNK1 as a potential pharmacological target for the development of drugs that might be useful for the treatment of insulin resistance, metabolic syndrome, and type 2 diabetes. © 2010 Elsevier Ltd.

Toettcher J.E.,University of California at San Francisco | Weiner O.D.,University of California at San Francisco | Lim W.A.,University of California at San Francisco | Lim W.A.,Howard Hughes Medical Institute
Cell | Year: 2013

The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk. Moreover, we elucidate how a single pathway can specify distinct physiological outcomes: by combining distinct temporal patterns of stimulation with proteomic profiling, we identify signaling programs that differentially respond to Ras dynamics, including a paracrine circuit that activates STAT3 only after persistent (>1 hr) Ras activation. Optogenetic stimulation provides a powerful tool for analyzing the intrinsic transmission properties of pathway modules and identifying how they dynamically encode distinct outcomes. © 2013 Elsevier Inc.

Yang Z.,University of California at San Francisco | Sullivan B.M.,Howard Hughes Medical Institute | Sullivan B.M.,University of California at San Francisco | Allen C.D.C.,University of California at San Francisco
Immunity | Year: 2012

IgE antibodies may be protective in parasite immunity, but their aberrant production can lead to allergic disease and life-threatening anaphylaxis. Despite the importance of IgE regulation, few studies have directly examined the B cells that express IgE, because these cells are rare and difficult to detect. Here, we describe fluorescent IgE reporter mice and validate a flow cytometry procedure to allow sensitive and specific identification of IgE-expressing B cells in vivo. Similar to IgG1+ cells, IgE+ B cells differentiated into germinal center (GC) B cells and plasma cells (PCs) during primary immune responses to a T cell-dependent hapten-protein conjugate and the helminth Nippostrongylus brasiliensis. However, the participation of IgE+ B cells in GCs was transient. IgE+ B cells had an atypical propensity to upregulate the transcription factor Blimp-1 and undergo PC differentiation. Most IgE+ PCs were short lived and showed reduced affinity maturation, revealing intrinsic mechanisms that restrict the IgE antibody response. © 2012 Elsevier Inc.

Sheng M.,Genentech | Sabatini B.L.,Howard Hughes Medical Institute | Sudhof T.C.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

Alzheimer's disease (AD) is a major cause of dementia in the elderly. Pathologically, AD is characterized by the accumulation of insoluble aggregates of Aβ-peptides that are proteolytic cleavage products of the amyloid-β precursor protein ("plaques") and by insoluble filaments composed of hyperphosphorylated tau protein ("tangles"). Familial forms of AD often display increased production of Aβ peptides and/or altered activity of presenilins, the catalytic subunits of γ-secretase that produce Aβ peptides. Although the pathogenesis of AD remains unclear, recent studies have highlighted two major themes that are likely important. First, oligomeric Aβ species have strong detrimental effects on synapse function and structure, particularly on the postsynaptic side. Second, decreased presenilin function impairs synaptic transmission and promotes neurodegeneration. The mechanisms underlying these processes are beginning to be elucidated, and, although their relevance to AD remains debated, understanding these processes will likely allow new therapeutic avenues to AD. © 2012 Cold Spring Harbor Laboratory Press; all rights reserved.

Metzger M.J.,Howard Hughes Medical Institute | Reinisch C.,Environment Canada | Sherry J.,Environment Canada | Goff S.P.,Howard Hughes Medical Institute
Cell | Year: 2015

Summary Outbreaks of fatal leukemia-like cancers of marine bivalves throughout the world have led to massive population loss. The cause of the disease is unknown. We recently identified a retrotransposon, Steamer, that is highly expressed and amplified to high copy number in neoplastic cells of soft-shell clams (Mya arenaria). Through analysis of Steamer integration sites, mitochondrial DNA single-nucleotide polymorphisms (SNPs), and polymorphic microsatellite alleles, we show that the genotypes of neoplastic cells do not match those of the host animal. Instead, neoplastic cells from dispersed locations in New York, Maine, and Prince Edward Island (PEI), Canada, all have nearly identical genotypes that differ from those of the host. These results indicate that the cancer is spreading between animals in the marine environment as a clonal transmissible cell derived from a single original clam. Our findings suggest that horizontal transmission of cancer cells is more widespread in nature than previously supposed. PaperClip © 2015 Elsevier Inc.

Molofsky A.B.,University of California at San Francisco | Savage A.K.,Howard Hughes Medical Institute | Savage A.K.,University of California at San Francisco | Locksley R.M.,Howard Hughes Medical Institute | Locksley R.M.,University of California at San Francisco
Immunity | Year: 2015

Interleukin-33 (IL-33) is a nuclear-associated cytokine of the IL-1 family originally described as a potent inducer of allergic type 2 immunity. IL-33 signals via the receptor ST2, which is highly expressed on group 2 innate lymphoid cells (ILC2s) and T helper 2 (Th2) cells, thus underpinning its association with helminth infection and allergic pathology. Recent studies have revealed ST2 expression on subsets of regulatory Tcells, and for a role for IL-33 in tissue homeostasis and repair that suggests previously unrecognized interactions within these cellular networks. IL-33 can participate in pathologic fibrotic reactions, or, in the setting of microbial invasion, can cooperate with inflammatory cytokines to promote responses by cytotoxic NK cells, Th1 cells, and CD8+ Tcells. Here, we highlight the regulation and function of IL-33 and ST2 and review their roles in homeostasis, damage, and inflammation, suggesting a conceptual framework for future studies. © 2015 Elsevier Inc.

Southworth D.,Howard Hughes Medical Institute | Southworth D.,University of California at San Francisco | Agard D.,Howard Hughes Medical Institute | Agard D.,University of California at San Francisco
Molecular Cell | Year: 2011

Hsp90 is an essential molecular chaperone required for the folding and activation of many hundreds of cellular " client" proteins. The ATP-dependent chaperone cycle involves significant conformational rearrangements of the Hsp90 dimer and interaction with a network of cochaperone proteins. Little is known about the mechanism of client protein binding or how cochaperone interactions modulate Hsp90 conformational states. We have determined the cryo-EM structure of the human Hsp90:Hop complex that receives client proteins from the Hsp70 chaperone. Hop stabilizes an alternate Hsp90 open state, where hydrophobic client-binding surfaces have converged and the N-terminal domains have rotated and match the closed, ATP conformation. Hsp90 is thus simultaneously poised for client loading by Hsp70 and subsequent N-terminal dimerization and ATP hydrolysis. Upon binding of a single Hsp70, the Hsp90:Hop conformation remains essentially unchanged. These results identify distinct functions for the Hop cochaperone, revealing an asymmetric mechanism for Hsp90 regulation and client loading. © 2011 Elsevier Inc.

Yeh E.,Stanford University | DeRisi J.L.,University of California at San Francisco | DeRisi J.L.,Howard Hughes Medical Institute
PLoS Biology | Year: 2011

Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development. © 2011 Yeh, DeRisi.

Lim W.A.,University of California at San Francisco | Lim W.A.,Howard Hughes Medical Institute | Lee C.M.,University of California at San Francisco | Tang C.,Tsinghua University
Molecular Cell | Year: 2013

A challenge in biology is to understand how complex molecular networks in the cell execute sophisticated regulatory functions. Here we explore the idea that there are common and general principles that link network structures to biological functions, principles that constrain the design solutions that evolution can converge upon for accomplishing a given cellular task. We describe approaches for classifying networks based on abstract architectures and functions, rather than on the specific molecular components of the networks. For any common regulatory task, can we define the space of all possible molecular solutions? Such inverse approaches might ultimately allow the assembly of a design table of core molecular algorithms that could serve as a guide for building synthetic networks and modulating disease networks. © 2013 Elsevier Inc.

Li G.-W.,Howard Hughes Medical Institute | Li G.-W.,University of California at San Francisco | Burkhardt D.,University of California at San Francisco | Gross C.,University of California at San Francisco | And 2 more authors.
Cell | Year: 2014

Quantitative views of cellular functions require precise measures of rates of biomolecule production, especially proteins - the direct effectors of biological processes. Here, we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E coli data set transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. Members of multiprotein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles for optimizing design. These include how the level of different types of transcription factors is optimized for rapid response and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. Our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from quantitative analyses of protein synthesis. © 2014 Elsevier Inc.

Richardson J.L.,University of Connecticut | Urban M.C.,University of Connecticut | Bolnick D.I.,Howard Hughes Medical Institute | Skelly D.K.,Yale University
Trends in Ecology and Evolution | Year: 2014

Local adaptation has been a major focus of evolutionary ecologists working across diverse systems for decades. However, little of this research has explored variation at microgeographic scales because it has often been assumed that high rates of gene flow will prevent adaptive divergence at fine spatial scales. Here, we establish a quantitative definition of microgeographic adaptation based on Wright's dispersal neighborhood that standardizes dispersal abilities, enabling this measure to be compared across species. We use this definition to evaluate growing evidence of evolutionary divergence at fine spatial scales. We identify the main mechanisms known to facilitate this adaptation and highlight illustrative examples of microgeographic evolution in nature. Collectively, this evidence requires that we revisit our understanding of the spatial scale of adaptation and consider how microgeographic adaptation and its driving mechanisms can fundamentally alter ecological and evolutionary dynamics in nature. © 2014 Elsevier Ltd.

Fischbach M.A.,University of California at San Francisco | Bluestone J.A.,University of California at San Francisco | Lim W.A.,University of California at San Francisco | Lim W.A.,Howard Hughes Medical Institute
Science Translational Medicine | Year: 2013

Two decades ago, the pharmaceutical industry-long dominated by small-molecule drugs-was revolutionized by the the advent of biologics. Today, biomedicine sits on the cusp of a new revolution: the use of microbial and human cells as versatile therapeutic engines. Here, we discuss the promise of this "third pillar" of therapeutics in the context of current scientif c, regulatory, economic, and perceptual challenges. History suggests that the advent of cellular medicines will require the development of a foundational cellular engineering science that provides a systematic framework for safely and predictably altering and regulating cellular behaviors.

Malim M.H.,King's College London | Bieniasz P.D.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Medicine | Year: 2012

Retroviruses have long been a fertile model for discovering host-pathogen interactions and their associated biological principles and processes. These advances have not only informed fundamental concepts of viral replication and pathogenesis but have also provided novel insights into host cell biology. This is illustrated by the recent descriptions of host-encoded restriction factors that can serve as effective inhibitors of retroviral replication. Here, we review our understanding of the three restriction factors that have been widely shown to be potent inhibitors of HIV-1: namely, APOBEC3G, TRIM5α, and tetherin. In each case, we discuss how these unrelated proteins were identified, the mechanisms by which they inhibit replication, the means used by HIV-1 to evade their action, and their potential contributions to viral pathogenesis as well as inter- and intraspecies transmission. © 2012 Cold Spring Harbor Laboratory Press.all rights reserved.

Qiu Y.,University of California at San Francisco | Nguyen K.D.,University of California at San Francisco | Odegaard J.I.,University of California at San Francisco | Cui X.,University of California at San Francisco | And 4 more authors.
Cell | Year: 2014

Beige fat, which expresses the thermogenic protein UCP1, provides a defense against cold and obesity. Although a cold environment is the physiologic stimulus for inducing beige fat in mice and humans, the events that lead from the sensing of cold to the development of beige fat remain poorly understood. Here, we identify the efferent beige fat thermogenic circuit, consisting of eosinophils, type 2 cytokines interleukin (IL)-4/13, and alternatively activated macrophages. Genetic loss of eosinophils or IL-4/13 signaling impairs cold-induced biogenesis of beige fat. Mechanistically, macrophages recruited to cold-stressed subcutaneous white adipose tissue (scWAT) undergo alternative activation to induce tyrosine hydroxylase expression and catecholamine production, factors required for browning of scWAT. Conversely, administration of IL-4 to thermoneutral mice increases beige fat mass and thermogenic capacity to ameliorate pre-established obesity. Together, our findings have uncovered the efferent circuit controlling biogenesis of beige fat and provide support for its targeting to treat obesity. © 2014 Elsevier Inc.

Cheng Y.,University of California at San Francisco | Grigorieff N.,Janelia Research Campus | Penczek P.A.,University of Houston | Walz T.,Howard Hughes Medical Institute
Cell | Year: 2015

Cryo-electron microscopy (cryo-EM) of single-particle specimens is used to determine the structure of proteins and macromolecular complexes without the need for crystals. Recent advances in detector technology and software algorithms now allow images of unprecedented quality to be recorded and structures to be determined at near-atomic resolution. However, compared with X-ray crystallography, cryo-EM is a young technique with distinct challenges. This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners. © 2015 Elsevier Inc.

Ngo T.T.M.,University of Illinois at Urbana - Champaign | Zhang Q.,University of Illinois at Urbana - Champaign | Zhou R.,University of Illinois at Urbana - Champaign | Yodh J.G.,University of Illinois at Urbana - Champaign | And 2 more authors.
Cell | Year: 2015

Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications. © 2015 Elsevier Inc.

Von Moltke J.,University of California at San Francisco | Ji M.,University of California at San Francisco | Ji M.,Howard Hughes Medical Institute | Liang H.-E.,University of California at San Francisco | And 2 more authors.
Nature | Year: 2016

Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells and smooth muscle hypercontractility. This response, known as 'weep and sweep', requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (TH2 cells). Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages, including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells. © 2016 Macmillan Publishers Limited.

Street T.,University of California at San Francisco | Lavery L.,University of California at San Francisco | Agard D.,University of California at San Francisco | Agard D.,Howard Hughes Medical Institute
Molecular Cell | Year: 2011

Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations; however, the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (Δ131. Δ), a well-studied fragment of staphylococcal nuclease. SAXS measurements reveal that under apo conditions, Hsp90 partially closes around Δ131. Δ, and in the presence of AMPPNP, Δ131. Δ binds with increased affinity to Hsp90's fully closed state. FRET measurements show that Δ131. Δ accelerates the nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90. NMR measurements reveal that Hsp90 binds to a specific, highly structured region of Δ131. Δ. These results suggest that Hsp90 preferentially binds a locally structured region in a globally unfolded protein, and this binding drives functional changes in the chaperone by lowering a rate-limiting conformational barrier. © 2011 Elsevier Inc.

Cho C.,University of California at San Francisco | Vale R.D.,Howard Hughes Medical Institute
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2012

Dynein is a large cytoskeletal motor protein that belongs to the AAA. + (ATPases associated with diverse cellular activities) superfamily. While dynein has had a rich history of cellular research, its molecular mechanism of motility remains poorly understood. Here we describe recent X-ray crystallographic studies that reveal the architecture of dynein's catalytic ring, mechanical linker element, and microtubule binding domain. This structural information has given rise to new hypotheses on how the dynein motor domain might change its conformation in order to produce motility along microtubules. This article is part of a Special Issue entitled: AAA ATPases: structure and function. © 2011 Elsevier B.V.

Wood A.J.,Howard Hughes Medical Institute | Wood A.J.,King's College London | Severson A.F.,Howard Hughes Medical Institute | Meyer B.J.,Howard Hughes Medical Institute
Nature Reviews Genetics | Year: 2010

Condensin and cohesin complexes act in diverse nuclear processes in addition to their widely known roles in chromosome compaction and sister chromatid cohesion. Recent work has elucidated the contribution of condensin and cohesin to interphase genome organization, control of gene expression, metazoan development and meiosis. Despite these wide-ranging functions, several themes have come to light: both complexes establish higher-order chromosome structure by inhibiting or promoting interactions between distant genomic regions, both complexes influence the chromosomal association of other proteins, and both complexes achieve functional specialization by swapping homologous subunits. Emerging data are expanding the range of processes in which condensin and cohesin are known to participate and are enhancing our knowledge of how chromosome architecture is regulated to influence numerous cellular functions. © 2010 Macmillan Publishers Limited. All rights reserved.

Liu Q.,Brookhaven National Laboratory | Liu Q.,Virginia Commonwealth University | Hendrickson W.A.,Brookhaven National Laboratory | Hendrickson W.A.,Howard Hughes Medical Institute | Hendrickson W.A.,Columbia University
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single-or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Z max ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f′′ scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.

Wolff S.,Howard Hughes Medical Institute | Weissman J.S.,University of California at San Francisco | Dillin A.,Howard Hughes Medical Institute
Cell | Year: 2014

Proteins are notorious for their unpleasant behavior - continually at risk of misfolding, collecting damage, aggregating, and causing toxicity and disease. To counter these challenges, cells have evolved elaborate chaperone and quality control networks that can resolve damage at the level of the protein, organelle, cell, or tissue. On the smallest scale, the integrity of individual proteins is monitored during their synthesis. On a larger scale, cells use compartmentalized defenses and networks of communication, capable sometimes of signaling between cells, to respond to changes in the proteome's health. Together, these layered defenses help protect cells from damaged proteins. © 2014 Elsevier Inc.

Siliciano R.F.,Howard Hughes Medical Institute | Greene W.C.,University of California at San Francisco
Cold Spring Harbor Perspectives in Medicine | Year: 2011

HIV-1 can establish a state of latent infection at the level of individual T cells. Latently infected cells are rare in vivo and appear to arise when activated CD4+ T cells, the major targets cells for HIV-1, become infected and survive long enough to revert back to a resting memory state, which is nonpermissive for viral gene expression. Because latent virus resides in memory T cells, it persists indefinitely even in patients on potent antiretroviral therapy. This latent reservoir is recognized as a major barrier to curing HIV-1 infection. The molecular mechanisms of latency are complex and include the absence in resting CD4+ T cells of nuclear forms of key host transcription factors (e.g., NFκB and NFAT), the absence of Tat and associated host factors that promote efficient transcriptional elongation, epigenetic changes inhibiting HIV-1 gene expression, and transcriptional interference. The presence of a latent reservoir for HIV-1 helps explain the presence of very low levels of viremia in patients on antiretroviral therapy. These viruses are released from latently infected cells that have become activated and perhaps from other stable reservoirs but are blocked from additional rounds of replication by the drugs. Several approaches are under exploration for reactivating latent virus with the hope that this will allow elimination of the latent reservoir. © 2011 Cold Spring Harbor Laboratory Press; all rights reserved.

Wang B.,Howard Hughes Medical Institute | Wang B.,University of California at San Francisco | Zhao L.,Howard Hughes Medical Institute | Fish M.,Howard Hughes Medical Institute | And 2 more authors.
Nature | Year: 2015

The source of new hepatocytes in the uninjured liver has remained an open question. By lineage tracing using the Wnt-responsive gene Axin2 in mice, we identify a population of proliferating and self-renewing cells adjacent to the central vein in the liver lobule. These pericentral cells express the early liver progenitor marker Tbx3, are diploid, and thereby differ from mature hepatocytes, which are mostly polyploid. The descendants of pericentral cells differentiate into Tbx3-negative, polyploid hepatocytes, and can replace all hepatocytes along the liver lobule during homeostatic renewal. Adjacent central vein endothelial cells provide Wnt signals that maintain the pericentral cells, thereby constituting the niche. Thus, we identify a cell population in the liver that subserves homeostatic hepatocyte renewal, characterize its anatomical niche, and identify molecular signals that regulate its activity. © 2015 Macmillan Publishers Limited. All rights reserved.

Huang B.,University of California at San Francisco | Babcock H.,Howard Hughes Medical Institute | Zhuang X.,Howard Hughes Medical Institute
Cell | Year: 2010

Anyone who has used a light microscope has wished that its resolution could be a little better. Now, after centuries of gradual improvements, fluorescence microscopy has made a quantum leap in its resolving power due, in large part, to advancements over the past several years in a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM. Then, we describe recent applications of super-resolution microscopy in cells, which demonstrate how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology. © 2010 Elsevier Inc.

Ha T.,University of Illinois at Urbana - Champaign | Ha T.,Howard Hughes Medical Institute | Tinnefeld P.,TU Braunschweig
Annual Review of Physical Chemistry | Year: 2012

Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own " personalities" regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments. © Copyright ©2012 by Annual Reviews. All rights reserved.

News Article | February 23, 2017

February 23, 2017 (Cambridge, MA) -- Proteins do most of the work inside the human body, supporting the structure, function, regulation, and repair of organs, tissues and cells. Proteins are synthesized as extended chains of amino acids that must fold into intricate three-dimensional shapes to perform their work. However, protein folding is a very delicate process: crowded conditions within a cell or environmental changes around the cell can drive proteins to misfold and clump together or aggregate--causing disease. To support effective folding, cells deploy protein-folding chaperones to protect them from misfolding and aggregation. The heat-shock proteins 90 and 70 (HSP90 and HSP70) are protein-folding chaperones that together assist a large fraction of the proteins in cells to fold and function. In so doing, these major chaperones have been shown to alter the biological effects of genetic mutations in model organisms such as in plants, yeast, flies and fish. However, it has been unclear whether and how chaperones influence the consequences of genetic mutation in humans. Researchers at the Whitehead Institute have now uncovered a role for HSP90 in humans, not only as a modifier of the effects of mutations, but as a mediator of the impact of the environment on the function of mutant proteins. And these effects of HSP90 can alter the course of human diseases. Their work is discussed in the paper, HSP90 shapes the consequences of human genetic variation, which appears today in Cell. "By helping proteins fold and function, HSP90 influences a range of characteristics in simple organisms on which natural selection can act, and thus alters the course of evolution," says Georgios Karras, lead author of the study and a post-doctoral researcher at Whitehead Institute. "Our work demonstrates that HSP90 plays a similar role in humans, and in doing so it influences how disease-causing mutations manifest within cells and in the clinic. We found that HSP90 exerts a protective role on the functions of mutant proteins it directly binds, buffering the detrimental effects of mutations they carry." While HSP90 may buffer the effect of a mutation, its ability to do so is highly susceptible to environment changes. "HSP90 evolved to cope with environmental stressors that perturb the folding of proteins within cells," says Luke Whitesell, a senior author of the paper and a senior scientist at Whitehead Institute. "Yet, because HSP90's capacity is limited it can also make the mutant protein much more sensitive to environmental challenges. We observed that even mild stresses, such as fever, can provoke major effects in cells expressing HSP90-buffered mutants. This finding may help to explain why mutations can have varied clinical manifestations from person to person." Nevertheless, HSP90 cannot buffer all mutations. Some cause such problems that they cannot be rescued and these tend to be recognized by another chaperone, HSP70. Thus, the severity of the mutations' effects are reflected in the pattern of HSP90 vs. HSP70 binding to the mutant protein. "This pattern fits well with the biochemical roles of HSP90 and HSP70 during protein-folding," Karras notes. "HSP70 binds extended stretches of amino acids that are typically hidden in the core of the normally folded protein, while HSP90 binds to partially folded proteins. A severe mutation that leads to major unfolding and loss-of-function mainly drives HSP70 binding greater than HSP90 binding. Milder mutations have the opposite effect. Based on this pattern, we estimate that HSP90 can influence the consequences of up to 25 percent of missense mutations in some genes." The study was based on data mined from more than 1,500 disease-causing mutations associated with a diverse spectrum of diseases, including the cancer-predisposing syndrome Fanconi Anemia. In the long run, this new understanding of protein folding within cells could help clinicians predict who is at risk to develop a particular disease and who is most likely to respond well to specific treatment approaches. This work was supported by the Fanconi Anemia Research Fund (to S.L.), the Department of Defense (CDMRP BCRP W81XWH-14-1-0157 to S.L.), the Harold and Leila Y. Mathers Foundation, and the NIH (R37HL052725, RO1-DK43889 and PO1HL048546 to A.D.D.; P50HG004233 and R01HG001715 to M.V. and S.L.). S.L. was an investigator of the Howard Hughes Medical Institute. G.I.K. was supported by EMBO and HFSP Long-Term Fellowships. Georgios Karras is a post-doctoral researcher and Luke Whitesell is a senior scientist in the Lindquist lab at Whitehead Institute. 1 Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA 2 Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA 02215, USA 3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA 4 Center for DNA Damage and Repair and Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA 5 Department of Biology, Massachusetts Institute of Technology, and Howard Hughes Medical Institute, Cambridge MA 02139, USA 6 Correspondence to: Luke Whitesell: Georgios Karras: 7 Lead Contact 8 Deceased Whitehead Institute is a world-renowned non-profit research institution dedicated to improving human health through basic biomedical research. Wholly independent in its governance, finances, and research programs, Whitehead Institute shares a close affiliation with Massachusetts Institute of Technology through its faculty, who hold joint MIT appointments.

News Article | February 21, 2017

You can anticipate a color before you see it, based solely on the length of light waves. Music can be interpreted from notes on a page without being heard. Not so with odor. The only way to tell if something will smell like roses or turpentine, sea breeze or gasoline, is to sniff it. New research, described in Science on February 19, is making the most mysterious of our senses a little more predictable. A project initiated by Rockefeller University scientists and powered by a crowdsourcing effort has devised a mathematical model that can forecast the scent a molecule will evoke. "This is a centuries-old problem. People have attempted to work around it in many different ways, as you can see in the perfume section of a department store, when the clerks ask 'do you like something floral?' or 'do you like something musky?'" says study researcher Leslie Vosshall, the Robin Chemers Neustein Professor. "We haven't completely solved the question of how to predict an odor based on the chemical properties of the molecules that convey it, but this is the furthest anyone has pushed toward an explanation," she adds. The good, the bad, and the odorless As head of Rockefeller's Laboratory of Neurogenetics and Behavior, Vosshall studies odor perception in humans and insects. As part of this work, she and Andreas Keller, a research associate in her lab, set out to explore the link between molecules and the scent they give off. To get the data they needed, they asked volunteers to sniff a carefully curated set of molecules, each contained in a little vial. The possibilities were nearly endless--while the limits on human perception of light and sound are well known, no such boundaries have been established for odor. So in an effort to explore the full range of our sense of smell, Keller assembled a diverse cast of 476 molecules, many of which had never been tested in smell studies before. He included familiar aromas, like the sweet warmth of vanillin and the reek of methylthiobutyrate, a stinky-cheese molecule. He also selected molecules sniffers were unlikely to recognize--2-isopropylphenol, anyone?--and even those thought to be odorless, like water and glycerol. The 49 study volunteers rated each based on how strong they found it, how pleasant, and to what degree it evoked garlic, flower, urine, and 16 other attributes. Altogether, this effort generated more than 1 million data points. The researchers then sought to link this perceptual information to more than 2 million additional data points describing chemical features of the smell molecules, such as the number of sulfur atoms they contain. It took a crowd to solve this problem. Twenty-two teams of computationally savvy volunteers hailing from research institutions and companies around the world participated in the DREAM Olfaction Prediction Challenge, which was organized by study researcher Pablo Meyer, a team leader at IBM's Thomas J. Watson Research Center. Using Vosshall's and Keller's odor ratings, one of the largest sets of such data ever collected, these teams devised algorithms that could "learn" to predict an odor's attributes based on a molecule's chemical features. The best solution didn't appear in any single model. To take advantage of the wisdom of the crowd, DREAM challenges typically merge their submissions into an aggregate model, one that is often more powerful than any individual model. "A DREAM Challenge is more like hitting a piñata at a party than a normal research project: Everyone swings, and even if your algorithms don't break it open, you still contribute to the solution," Meyer says. "With this approach, a robust set of data, and a little luck, we were able to crack this particularly difficult problem." At the end of the challenge, the researchers tested the performance of the aggregate model using ratings they had held back on 69 molecules as a sort of answer key. A perfect score for matching attribute profiles to molecules would have been 1.0; the model scored 0.83, significantly better than any previous attempts to solve this problem. The smells that the models could most easily predict were garlic and fish, probably because the sniffers generally agreed on how to apply them while rating odors. Other attributes, such as cold or acid, were more challenging, probably because there is less consensus about what these terms mean for an odor, Vosshall says. While not yet perfect, the smell prediction model opens new possibilities for perfume chemists looking for more efficient ways to formulate, say, the perfect scent of dusky rose. It also shines new light on the immensely complex biology of smell perception. No one fully understands what happens when odor molecules waft into the nose and are converted into electrical signals that travel to the brain. "Once you can associate the input of the chemical structure and the output of an odor, you can start to delineate what might be happening during that translation," says Vosshall who is a Howard Hughes Medical Institute investigator. "This model is an important initial step in that direction."

News Article | February 15, 2017

LA JOLLA--(February 14, 2017) Not everyone is Fred Astaire or Michael Jackson, but even those of us who seem to have two left feet have got rhythm--in our brains. From breathing to walking to chewing, our days are filled with repetitive actions that depend on the rhythmic firing of neurons. Yet the neural circuitry underpinning such seemingly ordinary behaviors is not fully understood, even though better insights could lead to new therapies for disorders such as Parkinson's disease, ALS and autism. Recently, neuroscientists at the Salk Institute used stem cells to generate diverse networks of self-contained spinal cord systems in a dish, dubbed circuitoids, to study this rhythmic pattern in neurons. The work, which appears online in the February 14, 2017, issue of eLife, reveals that some of the circuitoids--with no external prompting--exhibited spontaneous, coordinated rhythmic activity of the kind known to drive repetitive movements. "It's still very difficult to contemplate how large groups of neurons with literally billions if not trillions of connections take information and process it," says the work's senior author, Salk Professor Samuel Pfaff, who is also a Howard Hughes Medical Institute investigator and holds the Benjamin H. Lewis Chair. "But we think that developing this kind of simple circuitry in a dish will allow us to extract some of the principles of how real brain circuits operate. With that basic information maybe we can begin to understand how things go awry in disease." Nerve cells in your brain and spinal cord connect to one another much like electronic circuits. And just as electronic circuits consist of many components, the nervous system contains a dizzying array of neurons, often resulting in networks with many hundreds of thousands of cells. To model these complex neural circuits, the Pfaff lab prompted embryonic stem cells from mice to grow into clusters of spinal cord neurons, which they named circuitoids. Each circuitoid typically contained 50,000 cells in clumps just large enough to see with the naked eye, and with different ratios of neuronal subtypes. With molecular tools, the researchers tagged four key subtypes of both excitatory (promoting an electrical signal) and inhibitory (stopping an electrical signal) neurons vital to movement, called V1, V2a, V3 and motor neurons. Observing the cells in the circuitoids in real time using high-tech microscopy, the team discovered that circuitoids composed only of V2a or V3 excitatory neurons or excitatory motor neurons (which control muscles) spontaneously fired rhythmically, but that circuitoids comprising only inhibitory neurons did not. Interestingly, adding inhibitory neurons to V3 excitatory circuitoids sped up the firing rate, while adding them to motor circuitoids caused the neurons to form sub-networks, smaller independent circuits of neural activity within a circuitoid. "These results suggest that varying the ratios of excitatory to inhibitory neurons within networks may be a way that real brains create complex but flexible circuits to govern rhythmic activity," says Pfaff. "Circuitoids can reveal the foundation for complex neural controls that lead to much more elaborate types of behaviors as we move through our world in a seamless kind of way." Because these circuitoids contain neurons that are actively functioning as an interconnected network to produce patterned firing, Pfaff believes that they will more closely model a normal aspect of the brain than other kinds of cell culture systems. Aside from more accurately studying disease processes that affect circuitry, the new technique also suggests a mechanism by which dysfunctional brain activity could be treated by altering the ratios of cell types in circuits. Other authors included: Matthew J. Sternfeld, Christopher A. Hinckley, Niall J. Moore, Matthew T. Pankratz, Kathryn L. Hilde, Shawn P. Driscoll, Marito Hayashi, Neal D. Amin, Dario Bonanomi, Wesley D. Gifford, and Martyn Goulding of Salk; and Kamal Sharma of the University of Illinois, Chicago. The work was funded by the National Cancer Institute at the National Institutes of Health; the Rose Hills Foundation; the H. A. and Mary K. Chapman Charitable Trust; the University of California, San Diego, Neurosciences Graduate Program; a U.S. National Research Service Award fellowship from the U.S. National Institutes of Health National Institute of Neurological Disorders and Stroke; the National Science Foundation; the Japanese Ministry of Education, Culture, Sports, Science, and Technology Long-Term Student Support Program; the Timken-Sturgis Foundation; the California Institute for Regenerative Medicine; the Howard Hughes Medical Institute; the Christopher and Dana Reeve Foundation; the Marshall Heritage Foundation; and the Sol Goldman Charitable Trust. Every cure has a starting point. The Salk Institute embodies Jonas Salk's mission to dare to make dreams into reality. Its internationally renowned and award-winning scientists explore the very foundations of life, seeking new understandings in neuroscience, genetics, immunology, plant biology and more. The Institute is an independent nonprofit organization and architectural landmark: small by choice, intimate by nature and fearless in the face of any challenge. Be it cancer or Alzheimer's, aging or diabetes, Salk is where cures begin. Learn more at:

Lee J.T.,Howard Hughes Medical Institute | Lee J.T.,Harvard University | Bartolomei M.S.,University of Pennsylvania
Cell | Year: 2013

X chromosome inactivation and genomic imprinting are classic epigenetic processes that cause disease when not appropriately regulated in mammals. Whereas X chromosome inactivation evolved to solve the problem of gene dosage, the purpose of genomic imprinting remains controversial. Nevertheless, the two phenomena are united by allelic control of large gene clusters, such that only one copy of a gene is expressed in every cell. Allelic regulation poses significant challenges because it requires coordinated long-range control in cis and stable propagation over time. Long noncoding RNAs have emerged as a common theme, and their contributions to diseases of imprinting and the X chromosome have become apparent. Here, we review recent advances in basic biology, the connections to disease, and preview potential therapeutic strategies for future development. © 2013 Elsevier Inc.

Ipsaro J.J.,Wm Keck Structural Biology Laboratory | Ipsaro J.J.,Howard Hughes Medical Institute | Joshua-Tor L.,Wm Keck Structural Biology Laboratory | Joshua-Tor L.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2015

Since its relatively recent discovery, RNA interference (RNAi) has emerged as a potent, specific and ubiquitous means of gene regulation. Through a number of pathways that are conserved in eukaryotes from yeast to humans, small noncoding RNAs direct molecular machinery to silence gene expression. In this Review, we focus on mechanisms and structures that govern RNA silencing in higher organisms. In addition to highlighting recent advances, we discuss parallels and differences among RNAi pathways. Together, the studies reviewed herein reveal the versatility and programmability of RNA-induced silencing complexes and emphasize the importance of both upstream biogenesis and downstream silencing factors. © 2015 Nature America, Inc. All rights reserved.

Luo W.,University of Pennsylvania | Sehgal A.,University of Pennsylvania | Sehgal A.,Howard Hughes Medical Institute
Cell | Year: 2012

Although molecular components of the circadian clock are known, mechanisms that transmit signals from the clock and produce rhythmic behavior are poorly understood. We find that the microRNA miR-279 regulates the JAK/STAT pathway to drive rest:activity rhythms in Drosophila. Overexpression of microRNA miR-279 or miR-279 deletion attenuates rest:activity rhythms. Oscillations of the clock protein PERIOD are normal in pacemaker neurons lacking miR-279, suggesting that miR-279 acts downstream of the clock. We identify the JAK/STAT ligand, Upd, as a target of miR-279 and show that knockdown of Upd rescues the behavioral phenotype of miR-279 mutants. Manipulations of the JAK/STAT pathway also disrupt circadian rhythms. In addition, central clock neurons project in the vicinity of Upd-expressing neurons, providing a possible physical connection by which the central clock could regulate JAK/STAT signaling to control rest:activity rhythms. © 2012 Elsevier Inc.

Wang H.,Oregon Health And Science University | Elferich J.,Oregon Health And Science University | Gouaux E.,Oregon Health And Science University | Gouaux E.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2012

Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT-substrate complexes in lipid bicelles and in β-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site. © 2012 Nature America, Inc. All rights reserved.

High K.A.,Children's Hospital of Philadelphia | High K.A.,Howard Hughes Medical Institute | High K.A.,University of Pennsylvania
Blood | Year: 2012

Since the isolation and characterization of the genes for FVIII and FIX some 30 years ago, a longstanding goal of the field has been development of successful gene therapy for the hemophilias. In a landmark study published in 2011, Nathwani et al demonstrated successful conversion of severe hemophilia B to mild or moderate disease in 6 adult males who underwent intravenous infusion of an adeno-associated viral (AAV) vector expressing factor IX. These 6 subjects have now exhibited expression of FIX at levels ranging from 1% to 6% of normal for periods of > 2 years. This review discusses obstacles that were overcome to reach this goal and the next steps in clinical investigation. Safety issues that will need to be addressed before more widespread use of this approach are discussed. Efforts to extend AAV-mediated gene therapy to hemophilia A, and alternate approaches that may be useful for persons with severe liver disease, who may not be candidates for gene transfer to liver, are also discussed. © 2012 by The American Society of Hematology.

Coulon A.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | Chow C.C.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | Singer R.H.,Yeshiva University | Singer R.H.,Howard Hughes Medical Institute | Larson D.R.,U.S. National Cancer Institute
Nature Reviews Genetics | Year: 2013

Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time- and energy-dependence at the molecular level. © 2013 Macmillan Publishers Limited. All rights reserved.

Mischiati M.,Howard Hughes Medical Institute | Lin H.-T.,Howard Hughes Medical Institute | Herold P.,Howard Hughes Medical Institute | Imler E.,University of Arizona | And 2 more authors.
Nature | Year: 2015

Sensorimotor control in vertebrates relies on internal models. When extending an arm to reach for an object, the brain uses predictive models of both limb dynamics and target properties. Whether invertebrates use such models remains unclear. Here we examine to what extent prey interception by dragonflies (Plathemis lydia), a behaviour analogous to targeted reaching, requires internal models. By simultaneously tracking the position and orientation of a dragonfly's head and body during flight, we provide evidence that interception steering is driven by forward and inverse models of dragonfly body dynamics and by models of prey motion. Predictive rotations of the dragonfly's head continuously track the prey's angular position. The head-body angles established by prey tracking appear to guide systematic rotations of the dragonfly's body to align it with the prey's flight path. Model-driven control thus underlies the bulk of interception steering manoeuvres, while vision is used for reactions to unexpected prey movements. These findings illuminate the computational sophistication with which insects construct behaviour. © 2015 Macmillan Publishers Limited. All rights reserved.

Shay J.E.S.,University of Pennsylvania | Celeste Simon M.,University of Pennsylvania | Celeste Simon M.,Howard Hughes Medical Institute
Seminars in Cell and Developmental Biology | Year: 2012

Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors that allow adaptation to hypoxic environments. HIFs function in the cellular response to stress: metabolic, hypoxic, or inflammatory. Metabolic changes occur during tumorigenesis that are, in part, under hypoxia and HIF regulation. Additionally, inflammatory signaling and infiltration secondary to hypoxia are clear drivers of tumor progression. HIF-1α and HIF-2α have opposing and occasionally overlapping roles in both tumor cells and inflammatory cells within the tumor microenvironment and crosstalk between these populations has clear effects on tumor metabolism, inflammation, and progression. It is becoming increasingly apparent that HIFs are one common link between hypoxia, chronic inflammation, metabolic adaptation, and tumor progression through its function in macrophages during cancer development. © 2012 Elsevier Ltd.

Majmundar A.J.,University of Pennsylvania | Wong W.J.,University of Pennsylvania | Simon M.C.,University of Pennsylvania | Simon M.C.,Howard Hughes Medical Institute
Molecular Cell | Year: 2010

Oxygen (O2) is an essential nutrient that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O2 availability, or hypoxia. In order to cope with this stress, evolutionarily conserved responses are engaged. In mammals, the primary transcriptional response to hypoxic stress is mediated by the hypoxia-inducible factors (HIFs). While canonically regulated by prolyl hydroxylase domain-containing enzymes (PHDs), the HIFα subunits are intricately responsive to numerous other factors, including factor-inhibiting HIF1α (FIH1), sirtuins, and metabolites. These transcription factors function in normal tissue homeostasis and impinge on critical aspects of disease progression and recovery. Insights from basic HIF biology are being translated into pharmaceuticals targeting the HIF pathway. © 2010 Elsevier Inc.

Ackerman D.,University of Pennsylvania | Simon M.C.,University of Pennsylvania | Simon M.C.,Howard Hughes Medical Institute
Trends in Cell Biology | Year: 2014

Solid tumors typically develop hostile microenvironments characterized by irregular vascularization and poor oxygen (O2) and nutrient supply. Whereas normal cells modulate anabolic and catabolic pathways in response to changes in nutrient availability, cancer cells exhibit unregulated growth even under nutrient scarcity. Recent studies have demonstrated that constitutive activation of growth-promoting pathways results in dependence on unsaturated fatty acids for survival under O2 deprivation. In cancer cells, this dependence represents a critical metabolic vulnerability that could be exploited therapeutically. Here we review how this dependence on unsaturated lipids is affected by the microenvironmental conditions faced by cancer cells. © 2014 Elsevier Ltd.

De Obaldia M.E.,University of Pennsylvania | De Obaldia M.E.,Howard Hughes Medical Institute | Bhandoola A.,University of Pennsylvania | Bhandoola A.,U.S. National Cancer Institute
Annual Review of Immunology | Year: 2015

The lymphocyte family has expanded significantly in recent years to include not only the adaptive lymphocytes (T cells, B cells) and NK cells, but also several additional innate lymphoid cell (ILC) types. ILCs lack clonally distributed antigen receptors characteristic of adaptive lymphocytes and instead respond exclusively to signaling via germline-encoded receptors. ILCs resemble T cells more closely than any other leukocyte lineage at the transcriptome level and express many elements of the core T cell transcriptional program, including Notch, Gata3, Tcf7, and Bcl11b. We present our current understanding of the shared and distinct transcriptional regulatory mechanisms involved in the development of adaptive T lymphocytes and closely related ILCs. We discuss the possibility that a core set of transcriptional regulators common to ILCs and T cells establish enhancers that enable implementation of closely aligned effector pathways. Studies of the transcriptional regulation of lymphopoiesis will support the development of novel therapeutic approaches to correct early lymphoid developmental defects and aberrant lymphocyte function. © 2015 by Annual Reviews. All rights reserved.

Araki Y.,Howard Hughes Medical Institute | Zeng M.,Hong Kong University of Science and Technology | Zhang M.,Hong Kong University of Science and Technology | Huganir R.L.,Howard Hughes Medical Institute
Neuron | Year: 2015

SynGAP is a Ras-GTPase activating protein highly enriched at excitatory synapses in the brain. Previous studies have shown that CaMKII and the RAS-ERK pathway are critical for several forms of synaptic plasticity including LTP. NMDA receptor-dependent calcium influx has been shown to regulate the RAS-ERK pathway and downstream events that result inAMPA receptor synaptic accumulation, spine enlargement, and synaptic strengthening during LTP. However, the cellular mechanisms whereby calcium influx and CaMKII control Ras activity remain elusive. Using live-imaging techniques, we have found that SynGAP is rapidly dispersed from spines upon LTP induction in hippocampal neurons, and this dispersion depends on phosphorylation of SynGAP by CaMKII. Moreover, the degree of acute dispersion predicts the maintenance of spine enlargement. Thus, the synaptic dispersion of SynGAP by CaMKII phosphorylation during LTP represents a key signaling component that transduces CaMKII activity to small G protein-mediated spine enlargement, AMPA receptor synaptic incorporation, and synaptic potentiation. © 2015 Elsevier Inc.

Abe M.,University of Pennsylvania | Bonini N.M.,University of Pennsylvania | Bonini N.M.,Howard Hughes Medical Institute
Trends in Cell Biology | Year: 2013

Neurodegenerative diseases are typically late-onset, progressive disorders that affect neural function and integrity. Although most attention has been focused on the genetic underpinnings of familial disease, mechanisms are likely to be shared with more predominant sporadic forms, which can be influenced by age, environment, and genetic inputs. Previous work has largely addressed the roles of select protein-coding genes; however, disease pathogenesis is complicated and can be modulated through not just protein-coding genes, but also regulatory mechanisms mediated by the exploding world of small non-coding RNAs. Here, we focus on emerging roles of miRNAs in age-associated events impacting long-term brain integrity and neurodegenerative disease. © 2012 Elsevier Ltd.

Mingozzi F.,Children's Hospital of Philadelphia | High K.A.,Children's Hospital of Philadelphia | High K.A.,Howard Hughes Medical Institute | High K.A.,University of Pennsylvania
Nature Reviews Genetics | Year: 2011

In vivo gene replacement for the treatment of inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. Successful translation to the clinic was initially slow, but long-term expression of donated genes at therapeutic levels has now been achieved in patients with inherited retinal disorders and haemophilia B. Recent exciting results have raised hopes for the treatment of many other diseases. As we discuss here, the prospects and challenges for AAV gene therapy are to a large extent dependent on the target tissue and the specific disease. © 2011 Macmillan Publishers Limited. All rights reserved.

OShaughnessy E.C.,Howard Hughes Medical Institute | Palani S.,University of Pennsylvania | Collins J.J.,Howard Hughes Medical Institute | Collins J.J.,Wyss Institute for Biologically Inspired Engineering | Sarkar C.A.,University of Pennsylvania
Cell | Year: 2011

The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems. © 2011 Elsevier Inc.

News Article | February 15, 2017

WORCESTER, MA - A father's nicotine use may have a significant impact on children's risk of some diseases. In a study published in the online biomedical sciences journal eLife, Oliver J. Rando, MD, PhD, and colleagues at UMass Medical School, demonstrate that mice born of fathers who are habitually exposed to nicotine inherit enhanced chemical tolerance and drug clearance abilities. These findings offer a powerful framework for exploring how information about a father's environmental exposure history is passed down to offspring. "Children born of fathers who have been exposed to nicotine are programmed to be not only more resistant to nicotine toxicity, but to other chemicals as well," said Dr. Rando, professor of biochemistry & molecular pharmacology. "If a similar phenomenon occurs in humans, this raises many important questions. For example, if your father smoked does that mean chemotherapy might be less effective for you? Are you more or less likely to smoke? It's important to understand what information is specifically being passed down from father to offspring and how that impacts us." Studies over the past decade in the field of epigenetics - the study of inheritable traits that are carried outside the genome - have provided unexpected support to the notion that the environmental conditions experienced by a parent can affect disease risk and other features of future generations. In mammals, many of these studies have focused on interactions between the male parent and the offspring - paternal effects - as these are in many ways easier to investigate than maternal effects. Specifically, a number of studies have linked paternal diet to metabolic changes in offspring, while others link paternal stress to anxiety-like behaviors in the next generation. Despite the growing number of these studies, only a small number of paternal exposures have been explored rigorously in the lab. In addition, it has remained unclear in these studies whether the offspring response is specific for the paternal exposure, or whether it is a more generic response to a father's overall quality of life. To address this question, Rando and colleagues set out to determine how precise the response is for the environment experienced by the male parent, by looking at a single molecular interaction. Nicotine is a commonly used drug in humans, and acts by binding to a specific molecular receptor. Providing male mice with access to nicotine, researchers sought to learn whether their offspring were more or less sensitive to nicotine, and whether the offspring response was specific to nicotine or extended to other molecules. What researchers found is that the offspring of nicotine-exposed fathers, compared to the offspring of fathers that were never exposed to nicotine, were protected from toxic levels of nicotine. Researchers then tested whether this resistance was specific for nicotine by treating both sets of offspring with cocaine, which acts via a wholly distinct molecular pathway than nicotine. Surprisingly, the children of nicotine-exposed fathers were also protected from cocaine. This multi-toxin resistance is likely a result of enhanced drug metabolism in the liver, and corresponds to an increase in expression levels of genes involved in drug metabolism. These genes were also packaged in a more open and accessible configuration in the liver cells, allowing for increased expression. "This demonstrates that 'dad' paints with very broad brush strokes. Fathers exposed to nicotine do not specifically program changes in nicotine receptors in their children, as these children are broadly resistant to multiple toxins," said Rando. To determine if multiple, distinct molecules are capable of affecting drug resistance in the next generation, Rando and colleagues treated male mice with another bioactive compound, mecamylamine, which blocks nicotine receptors and is sometimes used to help people stop smoking. Surprisingly, offspring of these mice exhibited the same chemical resistance as those exposed to nicotine. "These findings raise key questions about what drugs or molecules are sufficient to affect children of exposed fathers," said Rando. "What distinguishes nicotine and mecamylamine from the countless small molecules present in our food and environment?" The next step for Rando and colleagues is to determine how many channels of information are being passed down from parent to offspring. "We now know that this information is relatively nonspecific," he said. "But is dad only telling us, on a scale of 1 to 10, that his life was good or not, or is he telling us four or five things broadly about the amount of food, level of stress and degree of chemical exposure?" Given the prevalence of smoking in humans, Rando notes that "there are obvious reasons to be interested in whether this type of effect also happens in human beings, but given the differences between mice and humans in their metabolism of nicotine, it will need to be tested rigorously in future studies of human populations." The University of Massachusetts Medical School (UMMS), one of five campuses of the University system, is comprised of the School of Medicine, the Graduate School of Biomedical Sciences, the Graduate School of Nursing, a thriving research enterprise and an innovative public service initiative, Commonwealth Medicine. Its mission is to advance the health of the people of the Commonwealth through pioneering education, research, public service and health care delivery with its clinical partner, UMass Memorial Health Care. In doing so, it has built a reputation as a world-class research institution and as a leader in primary care education. The Medical School attracts more than $266 million annually in research funding, placing it among the top 50 medical schools in the nation. In 2006, UMMS's Craig C. Mello, PhD, Howard Hughes Medical Institute Investigator and the Blais University Chair in Molecular Medicine, was awarded the Nobel Prize in Physiology or Medicine, along with colleague Andrew Z. Fire, PhD, of Stanford University, for their discoveries related to RNA interference (RNAi). The 2013 opening of the Albert Sherman Center ushered in a new era of biomedical research and education on campus. Designed to maximize collaboration across fields, the Sherman Center is home to scientists pursuing novel research in emerging scientific fields with the goal of translating new discoveries into innovative therapies for human diseases.

News Article | February 21, 2017

GRAND RAPIDS, Mich. (Feb. 21, 2017)--An international collaboration of life scientists, including experts at Van Andel Research Institute, has described in exquisite detail the critical first steps of DNA replication, which allows cells to divide and most advanced life, including human, to propagate. Results of the study are published in the journal Nature Structural and Molecular Biology and reveal that a ring-shaped protein called origin recognition complex (ORC) possesses a special alpha-helix, which slips into a groove on DNA and initiates a cascade of microscopic interactions that copy DNA. "This is a story of one ring that lords over another ring," says Huilin Li, Ph.D., a professor in Van Andel Research Institute's Center for Epigenetics and a senior author of the paper. "Biologists have known for many years that both ORC and helicase are ring-shaped structures essential in the initiation and execution of DNA replication, but until now we never understood exactly how the ORC ring loads the helicase ring onto DNA." The work also reveals that ORC, with the help of Cdc6 and Cdt1, loads the helicase core onto DNA via paired interactions of the so-called winged helix domains. The resulting 14-protein structure completes the loading of the first helicase ring and is now prepared to load the next ring. This process represents the inception of an immensely complex and elegant system that is constantly ongoing at tens of thousands of points on the DNA in many cells of the human body, and it all starts with ORCs. "We hope that by mapping this process, others will eventually convert this knowledge into new treatments for DNA replication-related conditions, including many cancers and rare disorders," says Li. At the outset, the six-protein ORCs assemble into a crescent, which envelops the DNA duplex. The ORCs then recruit a seventh protein, called Cdc6, to encircle DNA. Next, this ring threads the second ring, called minichromosome maintenance protein (Cdt1-bound Mcm2-7 hexamer), around DNA, which completes loading of the first Mcm2-7 hexamer. "It's like threading a pearl onto a string; but unlike a short piece of string, the DNA strand is incredibly long and so the bead cannot be threaded on at one end," says Christian Speck, a professor at Imperial College of London's Institute of Clinical Sciences, leader of the DNA Replication group at MRC London Institute of Medical Sciences and a senior author of the paper. "Instead, it must somehow be opened up, slotted around the strand, and closed again." The study was conducted on the DNA of Saccharomyces cerevisiae, better known as baker's yeast, because of its biological and genomic similarity to larger organisms, including mammals, at an average resolution of 3.9 Angströms (about 40 billionths of a meter), which is roughly the diameter of a single atom of sodium. Magnification of this scale is currently possible only with cryoelectron microscopy (cryo-EM), a revolutionary technology VARI continues to invest in through its recently established Cryo-EM Core. Imaging for this study was conducted at Howard Hughes Medical Institute's Janelia Research Campus and at Scripps Research Institute. Study authors are Zuanning Yuan, Lin Bai, Jingchuan Sun and Huilin Li, of Van Andel Research Institute; Alberto Riera, Marta Barbon and Christian Speck, all of Imperial College of London and MRC London Institute of Medical Sciences; Jingchuan Sun of University of Pennsylvania; Saikat Nandi and Bruce Stillman, both of Cold Spring Harbor Laboratory; Christos Spanos, Zhuo Angel Chen and Juri Rappsilber, all of University of Edinburgh. Rappsilber is also affiliated with Technische Universität Berlin. Sun is now affiliated with University of Pennsylvania. This work was funded by the U.S. National Institutes of Health (GM111472 and OD12272 to Huilin Li and GM45436 to Bruce Stillman), the Biotechnology and Biological Sciences Research Council UK (P56061 to Christian Speck), and the Wellcome Trust (Investigator Award P56628 to Speck, Senior Research Fellowship 103139 to Juri Rappsilber, a Centre core grant 092076 to Rappsilber, and an instrument grant 108504 to Rappsilber). Van Andel Institute (VAI) is an independent nonprofit biomedical research and science education organization committed to improving the health and enhancing the lives of current and future generations. Established by Jay and Betty Van Andel in 1996 in Grand Rapids, Michigan, VAI has grown into a premier research and educational institution that supports the work of more than 360 scientists, educators and staff. Van Andel Research Institute (VARI), VAI's research division, is dedicated to determining the epigenetic, genetic, molecular and cellular origins of cancer, Parkinson's and other diseases and translating those findings into effective therapies. The Institute's scientists work in onsite laboratories and participate in collaborative partnerships that span the globe. Learn more about Van Andel Institute or donate by visiting http://www. .

News Article | February 16, 2017

The U.S. patent office has delivered a potentially lucrative victory to bioengineer Feng Zhang of the Broad Institute in Massachusetts, regarding patents for an extraordinarily useful gene-editing tool. CRISPR, a technology that's already worth billions of dollars, is shaping up to play a big role in medicine and medical research because it can edit DNA with unprecedented accuracy. But exactly who has the right to profit from the technology has been up for debate. Wednesday the U.S. Patent and Trademark Office said patents issued to the Broad Institute in 2014, and then challenged by the University of California, Berkeley, are in fact valid. "It's a pretty monumental decision here," said Jacob Sherkow, an associate professor at the New York Law School, who has been tracking the dispute closely. "It seems to reward the most valuable aspect of CRISPR to the Broad Institute," Sherkow told Shots. The proceedings aren't entirely settled, but as Sherkow sees the situation, the Broad Institute — a joint venture of Harvard University and MIT — will hold the patent for using CRISPR in human beings, other animals, and plants. Sherkow told Shots he believes Cal's patent, which has not yet been issued, could be limited to bacteria. "Obviously the patents covering the application of this technology in human cells ... are going to be much more financially valuable than using the same technology in bacteria," Sherkow says, "because one can develop drugs and other therapies from them." Investors Wednesday seemed to agree with this assessment. The value of companies that were spun off to license the Broad patents rose sharply, while the company based on the Berkeley patent lost value. Potentially, tens of billions of dollars are at stake here, both for the companies and for the universities. Biochemist Jennifer Doudna, of U.C. Berkeley and the Howard Hughes Medical Institute, discovered the biology that underlies this technology along with a European colleague, Emmanuelle Charpentier, who is now director of the Institute for Infection Biology at the Max Planck Institute in Berlin. Doudna told Shots she isn't convinced that Berkeley is the big loser here. She said the ruling paves the way for her patent application to move forward. "We're looking forward to having our patent issued," she said. "And our patent is a very broad patent that covers the composition and the use of this technology in all cell types." If the patent office rules the way Doudna hopes it will, people wanting to use CRISPR in higher organisms will have get licenses from both Berkeley and the Broad Institute. "That's the thing that I think is a bit crazy about the way the decision comes down," Doudna said. "It leaves the field — the situation — where a license would be necessary from both parties. There's not further clarity at this stage." There's yet another possibility: Berkeley could appeal Wednesday's ruling, and once again challenge the Broad Institute's patents. Doudna said the university hasn't decided what to do just yet.

Zhou H.-X.,Florida State University | McCammon J.A.,Howard Hughes Medical Institute
Trends in Biochemical Sciences | Year: 2010

Protein dynamics are essential for virtually all protein functions, certainly for gating mechanisms of ion channels and regulation of enzyme catalysis. Ion channels usually feature a gate in the channel pore that prevents ion permeation in the closed state. Some bifunctional enzymes with two distant active sites use a tunnel to transport intermediate products; a gate can help prevent premature leakage. Enzymes with a buried active site also require a tunnel for substrate entrance; a gate along the tunnel can contribute to selectivity. The gates in these different contexts show distinct characteristics in sequence, structure and dynamics, but they also have common features. In particular, aromatic residues often appear to serve as gates, probably because of their ability, through side chain rotation, to effect large changes in cross section. © 2009 Elsevier Ltd. All rights reserved.

Law J.A.,University of California at Los Angeles | Jacobsen S.E.,University of California at Los Angeles | Jacobsen S.E.,Howard Hughes Medical Institute
Nature Reviews Genetics | Year: 2010

Cytosine DNA methylation is a stable epigenetic mark that is crucial for diverse biological processes, including gene and transposon silencing, imprinting and X chromosome inactivation. Recent findings in plants and animals have greatly increased our understanding of the pathways used to accurately target, maintain and modify patterns of DNA methylation and have revealed unanticipated mechanistic similarities between these organisms. Key roles have emerged for small RNAs, proteins with domains that bind methylated DNA and DNA glycosylases in these processes. Drawing on insights from both plants and animals should deepen our understanding of the regulation and biological significance of DNA methylation. © 2010 Macmillan Publishers Limited. All rights reserved.

Poulikakos P.I.,Sloan Kettering Cancer Center | Zhang C.,Howard Hughes Medical Institute | Bollag G.,Plexxikon | Shokat K.M.,Howard Hughes Medical Institute | Rosen N.,Sloan Kettering Cancer Center
Nature | Year: 2010

Tumours with mutant BRAF are dependent on the RAFĝ€" MEKĝ€"ERK signalling pathway for their growth. We found that ATP-competitive RAF inhibitors inhibit ERK signalling in cells with mutant BRAF, but unexpectedly enhance signalling in cells with wild-type BRAF. Here we demonstrate the mechanistic basis for these findings. We used chemical genetic methods to show that drug-mediated transactivation of RAF dimers is responsible for paradoxical activation of the enzyme by inhibitors. Induction of ERK signalling requires direct binding of the drug to the ATP-binding site of one kinase of the dimer and is dependent on RAS activity. Drug binding to one member of RAF homodimers (CRAFĝ€"CRAF) or heterodimers (CRAFĝ€"BRAF) inhibits one protomer, but results in transactivation of the drug-free protomer. In BRAF(V600E) tumours, RAS is not activated, thus transactivation is minimal and ERK signalling is inhibited in cells exposed to RAF inhibitors. These results indicate that RAF inhibitors will be effective in tumours in which BRAF is mutated. Furthermore, because RAF inhibitors do not inhibit ERK signalling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumour activity than mitogen-activated protein kinase (MEK) inhibitors, but could also cause toxicity due to MEK/ERK activation. These predictions have been borne out in a recent clinical trial of the RAF inhibitor PLX4032 (refs 4, 5). The model indicates that promotion of RAF dimerization by elevation of wild-type RAF expression or RAS activity could lead to drug resistance in mutant BRAF tumours. In agreement with this prediction, RAF inhibitors do not inhibit ERK signalling in cells that coexpress BRAF(V600E) and mutant RAS. © 2010 Macmillan Publishers Limited. All rights reserved.

Feng S.,University of California at Los Angeles | Jacobsen S.E.,University of California at Los Angeles | Jacobsen S.E.,Howard Hughes Medical Institute
Current Opinion in Plant Biology | Year: 2011

Plant genomes are modified by an array of epigenetic marks that help regulate plant growth and reproduction. Although plants share many epigenetic features with animals and fungi, some epigenetic marks are unique to plants. In different organisms, the same epigenetic mark can play different roles and/or similar functions can be carried out by different epigenetic marks. Furthermore, while the enzymatic systems responsible for generating or eliminating epigenetic marks are often conserved, there are also cases where they are quite divergent between plants and other organisms. DNA methylation and methylation of histone tails on the lysine 4, 9, and 27 positions are among the best characterized epigenetic marks in both plants and animals. Recent studies have greatly enhanced our knowledge about the pattern of these marks in various genomes and provided insights into how they are established and maintained and how they function. This review focuses on the conservation and divergence of the pathways that mediate these four types of epigenetic marks. © 2010 Elsevier Ltd.

Jonkers I.,Cornell University | Kwak H.,Howard Hughes Medical Institute | Lis J.T.,Cornell University
eLife | Year: 2014

Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ~3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes. © Jonkers et al.

Pastor-Pareja J.C.,Tsinghua University | Pastor-Pareja J.C.,Howard Hughes Medical Institute | Xu T.,Howard Hughes Medical Institute | Xu T.,Fudan University
Annual Review of Genetics | Year: 2013

Cancer was seen for a long time as a strictly cell-autonomous process in which oncogenes and tumor-suppressor mutations drive clonal cell expansions. Research in the past decade, however, paints a more integrative picture of communication and interplay between neighboring cells in tissues. It is increasingly clear as well that tumors, far from being homogenous lumps of cells, consist of different cell types that function together as complex tissue-level communities. The repertoire of interactive cell behaviors and the quantity of cellular players involved call for a social cell biology that investigates these interactions. Research into this social cell biology is critical for understanding development of normal and tumoral tissues. Such complex social cell biology interactions can be parsed in Drosophila. Techniques in Drosophila for analysis of gene function and clonal behavior allow us to generate tumors and dissect their complex interactive biology with cellular resolution. Here, we review recent Drosophila research aimed at understanding tissue-level biology and social cell interactions in tumors, highlighting the principles these studies reveal. © 2013 by Annual Reviews. All rights reserved.

Bier E.,University of California at San Diego | De Robertis E.M.,Howard Hughes Medical Institute | De Robertis E.M.,University of California at Los Angeles
Science | Year: 2015

Bone morphogenetic proteins (BMPs) act in dose-dependent fashion to regulate cell fate choices in a myriad of developmental contexts. In early vertebrate and invertebrate embryos, BMPs and their antagonists establish epidermal versus central nervous system domains. In this highly conserved system, BMP antagonists mediate the neural-inductive activities proposed by Hans Spemann and Hilde Mangold nearly a century ago. BMPs distributed in gradients subsequently function as morphogens to subdivide the three germ layers into distinct territories and act to organize body axes, regulate growth, maintain stem cell niches, or signal inductively across germ layers. In this Review, we summarize the variety of mechanisms that contribute to generating reliable developmental responses to BMP gradients and other morphogen systems. © 2015, American Association for the Advancement of Science. All rights reserved.

Samuel V.T.,Yale University | Samuel V.T.,Veterans Affairs Medical Center | Shulman G.I.,Yale University | Shulman G.I.,Howard Hughes Medical Institute
Cell | Year: 2012

Insulin resistance is a complex metabolic disorder that defies explanation by a single etiological pathway. Accumulation of ectopic lipid metabolites, activation of the unfolded protein response (UPR) pathway, and innate immune pathways have all been implicated in the pathogenesis of insulin resistance. However, these pathways are also closely linked to changes in fatty acid uptake, lipogenesis, and energy expenditure that can impact ectopic lipid deposition. Ultimately, these cellular changes may converge to promote the accumulation of specific lipid metabolites (diacylglycerols and/or ceramides) in liver and skeletal muscle, a common final pathway leading to impaired insulin signaling and insulin resistance. © 2012 Elsevier Inc.

Albert F.W.,University of California at Los Angeles | Albert F.W.,Gonda Center 5309 | Kruglyak L.,University of California at Los Angeles | Kruglyak L.,Gonda Center 5309 | Kruglyak L.,Howard Hughes Medical Institute
Nature Reviews Genetics | Year: 2015

We are in a phase of unprecedented progress in identifying genetic loci that cause variation in traits ranging from growth and fitness in simple organisms to disease in humans. However, a mechanistic understanding of how these loci influence traits is lacking for the majority of loci. Studies of the genetics of gene expression have emerged as a key tool for linking DNA sequence variation to phenotypes. Here, we review recent insights into the molecular nature of regulatory variants and describe their influence on the transcriptome and the proteome. We discuss conceptual advances from studies in model organisms and present examples of complete chains of causality that link individual polymorphisms to changes in gene expression, which in turn result in physiological changes and, ultimately, disease risk. © 2015 Macmillan Publishers Limited.

Young B.C.,Beth Israel Deaconess Medical Center | Levine R.J.,Eunice Kennedy Shriver National Institute of Child Health and Human Development | Karumanchi S.A.,Beth Israel Deaconess Medical Center | Karumanchi S.A.,Howard Hughes Medical Institute
Annual Review of Pathology: Mechanisms of Disease | Year: 2010

Preeclampsia is a systemic syndrome that occurs in 3 to 5% of pregnant women and classically manifests as new-onset hypertension and proteinuria after 20 weeks of gestation. Preeclampsia is a leading cause of maternal and neonatal morbidity and mortality. The only known cure is delivery of the placenta. Recent discoveries, however, have led to important advances in understanding the pathogenesis of the condition. Placental antiangiogenic factors are upregulated and disrupt the maternal endothelium. This change in the normal angiogenic balance toward an antiangiogenic state can result in hypertension, proteinuria, glomerular endotheliosis, HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and cerebral edema' the clinical signs of preeclampsia and eclampsia. The regulation of these antiangiogenic factors in the placenta is unknown. The recent discoveries of upregulated antiangiogenic factors provide promise for future testing to predict and diagnose preeclampsia as well as therapeutic targets for amelioration of the clinical disease. Copyright © 2010 by Annual Reviews.

Stroud H.,University of California at Los Angeles | Greenberg M.V.C.,University of California at Los Angeles | Feng S.,University of California at Los Angeles | Feng S.,Howard Hughes Medical Institute | And 3 more authors.
Cell | Year: 2013

Cytosine methylation is involved in various biological processes such as silencing of transposable elements (TEs) and imprinting. Multiple pathways regulate DNA methylation in different sequence contexts, but the factors that regulate DNA methylation at a given site in the genome largely remain unknown. Here we have surveyed the methylomes of a comprehensive list of 86 Arabidopsis gene silencing mutants by generating single-nucleotide resolution maps of DNA methylation. We find that DNA methylation is site specifically regulated by different factors. Furthermore, we have identified additional regulators of DNA methylation. These data and analyses will serve as a comprehensive community resource for further understanding the control of DNA methylation patterning. © 2013 Elsevier Inc.

Li N.,Howard Hughes Medical Institute | Chen T.-W.,Howard Hughes Medical Institute | Guo Z.V.,Howard Hughes Medical Institute | Gerfen C.R.,National Institute of Mental Health | Svoboda K.,Howard Hughes Medical Institute
Nature | Year: 2015

Activity in motor cortex predicts specific movements seconds before they occur, but how this preparatory activity relates to upcoming movements is obscure. We dissected the conversion of preparatory activity to movement within a structured motor cortex circuit. An anterior lateral region of the mouse cortex (a possible homologue of premotor cortex in primates) contains equal proportions of intermingled neurons predicting ipsi- or contralateral movements, yet unilateral inactivation of this cortical region during movement planning disrupts contralateral movements. Using cell-type-specific electrophysiology, cellular imaging and optogenetic perturbation, we show that layer 5 neurons projecting within the cortex have unbiased laterality. Activity with a contralateral population bias arises specifically in layer 5 neurons projecting to the brainstem, and only late during movement planning. These results reveal the transformation of distributed preparatory activity into movement commands within hierarchically organized cortical circuits. © 2015 Macmillan Publishers Limited.

Dall S.R.X.,University of Exeter | Bell A.M.,Urbana University | Bolnick D.I.,Howard Hughes Medical Institute | Ratnieks F.L.W.,University of Sussex
Ecology Letters | Year: 2012

Individuals often differ in what they do. This has been recognised since antiquity. Nevertheless, the ecological and evolutionary significance of such variation is attracting widespread interest, which is burgeoning to an extent that is fragmenting the literature. As a first attempt at synthesis, we focus on individual differences in behaviour within populations that exceed the day-to-day variation in individual behaviour (i.e. behavioural specialisation). Indeed, the factors promoting ecologically relevant behavioural specialisation within natural populations are likely to have far-reaching ecological and evolutionary consequences. We discuss such individual differences from three distinct perspectives: individual niche specialisations, the division of labour within insect societies and animal personality variation. In the process, while recognising that each area has its own unique motivations, we identify a number of opportunities for productive 'cross-fertilisation' among the (largely independent) bodies of work. We conclude that a complete understanding of evolutionarily and ecologically relevant individual differences must specify how ecological interactions impact the basic biological process (e.g. Darwinian selection, development and information processing) that underpin the organismal features determining behavioural specialisations. Moreover, there is likely to be co-variation amongst behavioural specialisations. Thus, we sketch the key elements of a general framework for studying the evolutionary ecology of individual differences. © 2012 Blackwell Publishing Ltd/CNRS.

Schatz D.G.,Howard Hughes Medical Institute | Swanson P.C.,Creighton University
Annual Review of Genetics | Year: 2011

V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences. © 2011 by Annual Reviews. All rights reserved.

Sandoe J.,Harvard University | Sandoe J.,Harvard Stem Cell Institute | Eggan K.,Harvard Stem Cell Institute | Eggan K.,Howard Hughes Medical Institute
Nature Neuroscience | Year: 2013

Human neurodegenerative disorders are among the most difficult to study. In particular, the inability to readily obtain the faulty cell types most relevant to these diseases has impeded progress for decades. Recent advances in pluripotent stem cell technology now grant access to substantial quantities of disease-pertinent neurons both with and without predisposing mutations. While this suite of technologies has revolutionized the field of 'in vitro disease modeling', great care must be taken in their deployment if robust, durable discoveries are to be made. Here we review what we perceive to be several of the stumbling blocks in the use of stem cells for the study of neurological disease and offer strategies to overcome them. © 2013 Nature America, Inc. All rights reserved.

Schultz K.M.,Lehigh University | Kyburz K.A.,Howard Hughes Medical Institute | Anseth K.S.,Howard Hughes Medical Institute
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Biomaterials that mimic aspects of the extracellular matrix by presenting a 3D microenvironment that cells can locally degrade and remodel are finding increased applications as wound-healing matrices, tissue engineering scaffolds, and even substrates for stem cell expansion. In vivo, cells do not simply reside in a static microenvironment, but instead, they dynamically reengineer their surroundings. For example, cells secrete proteases that degrade extracellular components, attach to the matrix through adhesive sites, and can exert traction forces on the local matrix, causing its spatial reorganization. Although biomaterials scaffolds provide initially well-defined microenvironments for 3D culture of cells, less is known about the changes that occur over time, especially local matrix remodeling that can play an integral role in directing cell behavior. Here, we use microrheology as a quantitative tool to characterize dynamic cellular remodeling of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels that degrade in response to cell-secreted matrix metalloproteinases (MMPs). This technique allows measurement of spatial changes in material properties during migration of encapsulated cells and has a sensitivity that identifies regions where cells simply adhere to the matrix, as well as the extent of local cell remodeling of the material through MMP-mediated degradation. Collectively, these microrheological measurements provide insight into microscopic, cellular manipulation of the pericellular region that gives rise to macroscopic tracks created in scaffolds by migrating cells. This quantitative and predictable information should benefit the design of improved biomaterial scaffolds for medically relevant applications. © 2015, National Academy of Sciences. All rights reserved.

Wu H.,Howard Hughes Medical Institute | Wu H.,Harvard University | Wu H.,Harvard Stem Cell Institute | Zhang Y.,Howard Hughes Medical Institute | And 2 more authors.
Cell | Year: 2014

Methylation of cytosines in the mammalian genome represents a key epigenetic modification and is dynamically regulated during development. Compelling evidence now suggests that dynamic regulation of DNA methylation is mainly achieved through a cyclic enzymatic cascade comprised of cytosine methylation, iterative oxidation of methyl group by TET dioxygenases, and restoration of unmodified cytosines by either replication-dependent dilution or DNA glycosylase-initiated base excision repair. In this review, we discuss the mechanism and function of DNA demethylation in mammalian genomes, focusing particularly on how developmental modulation of the cytosine-modifying pathway is coupled to active reversal of DNA methylation in diverse biological processes. © 2014 Elsevier Inc.

Hsu Y.-C.,Howard Hughes Medical Institute | Hsu Y.-C.,Harvard Stem Cell Institute | Li L.,Howard Hughes Medical Institute | Fuchs E.,Howard Hughes Medical Institute
Nature Medicine | Year: 2014

The skin protects mammals from insults, infection and dehydration and enables thermoregulation and sensory perception. Various skin-resident cells carry out these diverse functions. Constant turnover of cells and healing upon injury necessitate multiple reservoirs of stem cells. Thus, the skin provides a model for studying interactions between stem cells and their microenvironments, or niches. Advances in genetic and imaging tools have brought new findings about the lineage relationships between skin stem cells and their progeny and about the mutual influences between skin stem cells and their niches. Such knowledge may offer novel avenues for therapeutics and regenerative medicine. © 2014 Nature America, Inc.

Enchev R.I.,ETH Zurich | Schulman B.A.,Howard Hughes Medical Institute | Peter M.,ETH Zurich
Nature Reviews Molecular Cell Biology | Year: 2015

NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin-RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here, we re-evaluate studies of non-cullin targets of NEDD8 in light of the current understanding of the neddylation pathway, and suggest criteria for identifying genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights. © 2014 Macmillan Publishers Limited.

Suzanne M.,Toulouse 1 University Capitole | Suzanne M.,French National Center for Scientific Research | Steller H.,Howard Hughes Medical Institute
Cell Death and Differentiation | Year: 2013

Programmed cell death is an important process during development that serves to remove superfluous cells and tissues, such as larval organs during metamorphosis, supernumerary cells during nervous system development, muscle patterning and cardiac morphogenesis. Different kinds of cell death have been observed and were originally classified based on distinct morphological features: (1) type I programmed cell death (PCD) or apoptosis is recognized by cell rounding, DNA fragmentation, externalization of phosphatidyl serine, caspase activation and the absence of inflammatory reaction, (2) type II PCD or autophagy is characterized by the presence of large vacuoles and the fact that cells can recover until very late in the process and (3) necrosis is associated with an uncontrolled release of the intracellular content after cell swelling and rupture of the membrane, which commonly induces an inflammatory response. In this review, we will focus exclusively on developmental cell death by apoptosis and its role in tissue remodeling. © 2013 Macmillan Publishers Limited All rights reserved.

Amat F.,Max Planck Institute of Molecular Cell Biology and Genetics | Amat F.,Howard Hughes Medical Institute
Nature methods | Year: 2014

The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min(-1) on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

Kopf M.,ETH Zurich | Schneider C.,ETH Zurich | Schneider C.,Howard Hughes Medical Institute | Nobs S.P.,ETH Zurich
Nature Immunology | Year: 2015

Gas exchange is the vital function of the lungs. It occurs in the alveoli, where oxygen and carbon dioxide diffuse across the alveolar epithelium and the capillary endothelium surrounding the alveoli, separated only by a fused basement membrane 0.2-0.5 Î 1/4m in thickness. This tenuous barrier is exposed to dangerous or innocuous particles, toxins, allergens and infectious agents inhaled with the air or carried in the blood. The lung immune system has evolved to ward off pathogens and restrain inflammation-mediated damage to maintain gas exchange. Lung-resident macrophages and dendritic cells are located in close proximity to the epithelial surface of the respiratory system and the capillaries to sample and examine the air-borne and blood-borne material. In communication with alveolar epithelial cells, they set the threshold and the quality of the immune response. © 2015 Nature America, Inc.

Dwyer D.J.,University of Maryland University College | Collins J.J.,Howard Hughes Medical Institute | Collins J.J.,Harvard University | Collins J.J.,Boston University | Walker G.C.,Massachusetts Institute of Technology
Annual Review of Pharmacology and Toxicology | Year: 2015

We face an impending crisis in our ability to treat infectious disease brought about by the emergence of antibiotic-resistant pathogens and a decline in the development of new antibiotics. Urgent action is needed. This review focuses on a less well-understood aspect of antibiotic action: the complex metabolic events that occur subsequent to the interaction of antibiotics with their molecular targets and play roles in antibiotic lethality. Independent lines of evidence from studies of the action of bactericidal antibiotics on diverse bacteria collectively suggest that the initial interactions of drugs with their targets cannot fully account for the antibiotic lethality and that these interactions elicit the production of reactive oxidants including reactive oxygen species that contribute to bacterial cell death. Recent challenges to this concept are considered in the context of the broader literature of this emerging area of research. Possible ways that this new knowledge might be exploited to improve antibiotic therapy are also considered. ©2015 by Annual Reviews. All rights reserved.

Bushdid C.,Rockefeller University | Bushdid C.,University Pierre and Marie Curie | Magnasco M.O.,Rockefeller University | Vosshall L.B.,Rockefeller University | And 2 more authors.
Science | Year: 2014

Humans can discriminate several million different colors and almost half a million different tones, but the number of discriminable olfactory stimuli remains unknown. The lay and scientific literature typically claims that humans can discriminate 10,000 odors, but this number has never been empirically validated. We determined the resolution of the human sense of smell by testing the capacity of humans to discriminate odor mixtures with varying numbers of shared components. On the basis of the results of psychophysical testing, we calculated that humans can discriminate at least 1 trillion olfactory stimuli. This is far more than previous estimates of distinguishable olfactory stimuli. It demonstrates that the human olfactory system, with its hundreds of different olfactory receptors, far outperforms the other senses in the number of physically different stimuli it can discriminate.

Plank T.-D.M.,University of Colorado at Denver | Kieft J.S.,Howard Hughes Medical Institute
Wiley Interdisciplinary Reviews: RNA | Year: 2012

Internal ribosome entry sites (IRESs) are RNA sequences that can recruit the translation machinery independent of the 5′ end of the messenger RNA. IRESs are found in both viral and cellular RNAs and are important for regulating gene expression. There is great diversity in the mechanisms used by IRESs to recruit the ribosome and this is reflected in a variety of RNA sequences that function as IRESs. The ability of an RNA sequence to function as an IRES is conferred by structures operating at multiple levels from primary sequence through higher-order three-dimensional structures within dynamic ribonucleoproteins (RNPs). When these diverse structures are compared, some trends are apparent, but overall it is not possible to find universal rules to describe IRES structure and mechanism. Clearly, many different sequences and structures have evolved to perform the function of recruiting, positioning, and activating a ribosome without using the canonical cap-dependent mechanism. However, as our understanding of the specific sequences, structures, and mechanisms behind IRES function improves, more common features may emerge to link these diverse RNAs. © 2012 John Wiley & Sons, Ltd.

Edelaar P.,CSIC - Doñana Biological Station | Edelaar P.,Pablo De Olavide University | Bolnick D.I.,Howard Hughes Medical Institute
Trends in Ecology and Evolution | Year: 2012

Dispersal is an important life-history trait involved in species persistence, evolution, and diversification, yet is one of the least understood concepts in ecology and evolutionary biology. There is a growing realization that dispersal might not involve the random sample of genotypes as is typically assumed, but instead can be enriched for certain genotypes. Here, we review and compare various sources of such non-random gene flow, and summarize its effects on local adaptation and resource use, metapopulation dynamics, adaptation to climate change, biological invasion, and speciation. Given the possible ubiquity and impacts of non-random gene flow, there is an urgent need for the fields of evolution and ecology to test for non-random gene flow and to more fully incorporate its effects into theory. © 2012 Elsevier Ltd.

Brain-computer interface advance allows fast, accurate typing by people with paralysis in Stanford-led study A clinical research publication led by Stanford University investigators has demonstrated that a brain-to-computer hookup can enable people with paralysis to type via direct brain control at the highest speeds and accuracy levels reported to date. The report involved three study participants with severe limb weakness -- two from amyotrophic lateral sclerosis, also called Lou Gehrig's disease, and one from a spinal cord injury. They each had one or two baby-aspirin-sized electrode arrays placed in their brains to record signals from the motor cortex, a region controlling muscle movement. These signals were transmitted to a computer via a cable and translated by algorithms into point-and-click commands guiding a cursor to characters on an onscreen keyboard. Each participant, after minimal training, mastered the technique sufficiently to outperform the results of any previous test of brain-computer interfaces, or BCIs, for enhancing communication by people with similarly impaired movement. Notably, the study participants achieved these typing rates without the use of automatic word-completion assistance common in electronic keyboarding applications nowadays, which likely would have boosted their performance. One participant, Dennis Degray of Menlo Park, California, was able to type 39 correct characters per minute, equivalent to about eight words per minute. This point-and-click approach could be applied to a variety of computing devices, including smartphones and tablets, without substantial modifications, the Stanford researchers said. "Our study's success marks a major milestone on the road to improving quality of life for people with paralysis," said Jaimie Henderson, MD, professor of neurosurgery, who performed two of the three device-implantation procedures. The third took place at Massachusetts General Hospital. Henderson and Krishna Shenoy, PhD, professor of electrical engineering, are co-senior authors of the study, which will be published online Feb. 21 in eLife. The lead authors are former postdoctoral scholar Chethan Pandarinath, PhD, and postdoctoral scholar Paul Nuyujukian, MD, PhD, both of whom spent well over two years working full time on the project at Stanford. "This study reports the highest speed and accuracy, by a factor of three, over what's been shown before," said Shenoy, a Howard Hughes Medical Institute investigator who's been pursuing BCI development for 15 years and working with Henderson since 2009. "We're approaching the speed at which you can type text on your cellphone." "The performance is really exciting," said Pandarinath, who now has a joint appointment at Emory University and the Georgia Institute of Technology as an assistant professor of biomedical engineering. "We're achieving communication rates that many people with arm and hand paralysis would find useful. That's a critical step for making devices that could be suitable for real-world use." Shenoy's lab pioneered the algorithms used to decode the complex volleys of electrical signals fired by nerve cells in the motor cortex, the brain's command center for movement, and convert them in real time into actions ordinarily executed by spinal cord and muscles. "These high-performing BCI algorithms' use in human clinical trials demonstrates the potential for this class of technology to restore communication to people with paralysis," said Nuyujukian. Millions of people with paralysis reside in the United States. Sometimes their paralysis comes gradually, as occurs in ALS. Sometimes it arrives suddenly, as in Degray's case. Now 64, Degray became quadriplegic on Oct. 10, 2007, when he fell and sustained a life-changing spinal-cord injury. "I was taking out the trash in the rain," he said. Holding the garbage in one hand and the recycling in the other, he slipped on the grass and landed on his chin. The impact spared his brain but severely injured his spine, cutting off all communication between his brain and musculature from the head down. "I've got nothing going on below the collarbones," he said. Degray received two device implants at Henderson's hands in August 2016. In several ensuing research sessions, he and the other two study participants, who underwent similar surgeries, were encouraged to attempt or visualize patterns of desired arm, hand and finger movements. Resulting neural signals from the motor cortex were electronically extracted by the embedded recording devices, transmitted to a computer and translated by Shenoy's algorithms into commands directing a cursor on an onscreen keyboard to participant-specified characters. The researchers gauged the speeds at which the patients were able to correctly copy phrases and sentences -- for example, "The quick brown fox jumped over the lazy dog." Average rates were 7.8 words per minute for Degray and 6.3 and 2.7 words per minute, respectively, for the other two participants. The investigational system used in the study, an intracortical brain-computer interface called the BrainGate Neural Interface System*, represents the newest generation of BCIs. Previous generations picked up signals first via electrical leads placed on the scalp, then by being surgically positioned at the brain's surface beneath the skull. An intracortical BCI uses a tiny silicon chip, just over one-sixth of an inch square, from which protrude 100 electrodes that penetrate the brain to about the thickness of a quarter and tap into the electrical activity of individual nerve cells in the motor cortex. Henderson likened the resulting improved resolution of neural sensing, compared with that of older-generation BCIs, to that of handing out applause meters to individual members of a studio audience rather than just stationing them on the ceiling, "so you can tell just how hard and how fast each person in the audience is clapping." Shenoy said the day will come -- closer to five than 10 years from now, he predicted --when a self-calibrating, fully implanted wireless system can be used without caregiver assistance, has no cosmetic impact and can be used around the clock. "I don't see any insurmountable challenges." he said. "We know the steps we have to take to get there." Degray, who continues to participate actively in the research, knew how to type before his accident but was no expert at it. He described his newly revealed prowess in the language of a video game aficionado. "This is like one of the coolest video games I've ever gotten to play with," he said. "And I don't even have to put a quarter in it." The study's results are the culmination of a long-running collaboration between Henderson and Shenoy and a multi-institutional consortium called BrainGate. Leigh Hochberg, MD, PhD, a neurologist and neuroscientist at Massachusetts General Hospital, Brown University and the VA Rehabilitation Research and Development Center for Neurorestoration and Neurotechnology in Providence, Rhode Island, directs the pilot clinical trial of the BrainGate system and is a study co-author. "This incredible collaboration continues to break new ground in developing powerful, intuitive, flexible neural interfaces that we all hope will one day restore communication, mobility and independence for people with neurologic disease or injury," said Hochberg.

News Article | February 16, 2017

In mice genetically predisposed to glaucoma, vitamin B3 added to drinking water is effective at preventing the disease, a research team led by Jackson Laboratory Professor and Howard Hughes Medical Investigator Simon W.M. John reports in the journal Science. The vitamin administration was surprisingly effective, eliminating the vast majority of age-related molecular changes and providing a remarkably robust protection against glaucoma. It offers promise for developing inexpensive and safe treatments for glaucoma patients. Glaucoma is one of the most common neurodegenerative diseases, affecting an estimated 80 million people worldwide. In most glaucoma patients, harmfully high pressure inside the eye or intraocular pressure leads to the progressive dysfunction and loss of retinal ganglion cells. Retinal ganglion cells are the neuronal cells that connect the eye to the brain via the optic nerve. Increasing age is a key risk factor for glaucoma, contributing to both harmful elevation of intraocular pressure and increased neuronal vulnerability to pressure-induced damage. "We wanted to identify key age-related susceptibility factors that change with age in the eye," John says, "and that therefore increase vulnerability to disease and in particular neuronal disease." By understanding general age-related mechanism, there is the potential to develop new interventions to generally protect from common age-related disease processes in many people. Conducting a variety of genomic, metabolic, neurobiological and other tests in mice susceptible to inherited glaucoma, compared to control mice, the researchers discovered that NAD, a molecule vital to energy metabolism in neurons and other cells, declines with age. "There's an analogy with an old motorbike," John says. "It runs just fine, but little things get less reliable with age. One day you stress it: you drive it up a steep hill or you go on really long journey and you get in trouble. It's less reliable than a new bike and it's going to fail with a higher frequency than that new bike." The decrease in NAD levels reduces the reliability of neurons' energy metabolism, especially under stress such as increased intraocular pressure. "Like taking that big hill on your old bike, some things are going to fail more often," John says. "The amount of failure will increase over time, resulting in more damage and disease progression." In essence, the treatments of vitamin B3 (nicotinamide, an amide form of vitamin B3, also called niacinamide) boosted the metabolic reliability of aging retinal ganglion cells, keeping them healthier for longer. "Because these cells are still healthy, and still metabolically robust," says JAX Postdoctoral Associate Pete Williams, first author of the study, "even when high intraocular pressure turns on, they better resist damaging processes." The researchers also found that a single gene-therapy application of Nmnat1 (the gene for an enzyme that makes NAD from nicotinamide) prevented glaucoma from developing in this mouse model. "It can be a problem for patients, especially the elderly, to take their drugs every day and in the correct dose," Williams says. "So gene therapy could be a one-shot, protective treatment." He notes that gene therapies, through injections into the eye, have been approved for a handful of very rare, human genetic eye disorders, and their demonstration of an important age-dependent factor may enable gene therapy for more common eye disease. John says that the team is pursuing clinical partnerships to begin the process of testing the effectiveness of vitamin B3 treatment in glaucoma patients. They are also exploring potential applications for the treatment in other diseases involving neurodegeneration. Collaborating with the John lab was Vittorio Porciatti of the Bascom Palmer Eye Institute at the University of Miami Miller School of Medicine, and the late Nobel Laureate Oliver Smithies of the University of North Carolina, Chapel Hill. Funding sources for the research included National Eye Institute grant EY11721, the Barbara and Joseph Cohen Foundation, and National Heart, Lung and Blood Institute grant HL49277. Simon John is an Investigator of the Howard Hughes Medical Institute, and a research assistant professor in the opthalmology department of Tufts University School of Medicine. The Jackson Laboratory is an independent, nonprofit biomedical research institution based in Bar Harbor, Maine, with a National Cancer Institute-designated Cancer Center, a facility in Sacramento, Calif., and a genomic medicine institute in Farmington, Conn. It employs 1,800 staff, and its mission is to discover precise genomic solutions for disease and empower the global biomedical community in the shared quest to improve human health.

Deal R.B.,Fred Hutchinson Cancer Research Center | Henikoff S.,Fred Hutchinson Cancer Research Center | Henikoff S.,Howard Hughes Medical Institute
Developmental Cell | Year: 2010

Understanding the production and function of specialized cells during development requires the isolation of individual cell types for analysis, but this is currently a major technical challenge. Here we describe a method for cell type-specific RNA and chromatin profiling that circumvents many of the limitations of current methods for cell isolation. We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. We identified hundreds of genes that are preferentially expressed in each cell type and show that genes with the largest expression differences between hair and non-hair cells also show differences between cell types in the trimethylation of histone H3 at lysines 4 and 27. This method should be applicable to any organism that is amenable to transformation. © 2010 Elsevier Inc.

Luger K.,Colorado State University | Luger K.,Howard Hughes Medical Institute | Dechassa M.L.,Colorado State University | Tremethick D.J.,Australian National University
Nature Reviews Molecular Cell Biology | Year: 2012

The compaction of genomic DNA into chromatin has profound implications for the regulation of key processes such as transcription, replication and DNA repair. Nucleosomes, the repeating building blocks of chromatin, vary in the composition of their histone protein components. This is the result of the incorporation of variant histones and post-translational modifications of histone amino acid side chains. The resulting changes in nucleosome structure, stability and dynamics affect the compaction of nucleosomal arrays into higher-order structures. It is becoming clear that chromatin structures are not nearly as uniform and regular as previously assumed. This implies that chromatin structure must also be viewed in the context of specific biological functions. © 2012 Macmillan Publishers Limited. All rights reserved.

Eisele E.,Johns Hopkins University | Siliciano R.,Johns Hopkins University | Siliciano R.,Howard Hughes Medical Institute
Immunity | Year: 2012

This Perspective proposes definitions for key terms in the field of HIV-1 latency and eradication. In the context of eradication, a reservoir is a cell type that allows persistence of replication-competent HIV-1 on a timescale of years in patients on optimal antiretroviral therapy. Reservoirs act as a barrier to eradication in the patient population in which cure attempts will likely be made. Halting viral replication is essential to eradication, and definitions and criteria for assessing whether this goal has been achieved are proposed. The cell types that may serve as reservoirs for HIV-1 are discussed. Currently, only latently infected resting CD4+ T cells fit the proposed definition of a reservoir, and more evidence is necessary to demonstrate that other cell types, including hematopoietic stem cells and macrophages, fit this definition. Further research is urgently required on potential reservoirs in the gut-associated lymphoid tissue and the central nervous system. © 2012 Elsevier Inc.

Zhang Y.,Howard Hughes Medical Institute | Kim T.-H.,Howard Hughes Medical Institute | Kim T.-H.,Dana-Farber Cancer Institute | Niswander L.,Howard Hughes Medical Institute
Genes and Development | Year: 2012

Hirschsprung disease (HSCR) is caused by a reduction of enteric neural crest cells (ENCCs) in the gut and gastrointestinal blockage. Knowledge of the genetics underlying HSCR is incomplete, particularly genes that control cellular behaviors of ENCC migration. Here we report a novel regulator of ENCC migration in mice. Disruption of the Phactr4 gene causes an embryonic gastrointestinal defect due to colon hypoganglionosis, which resembles human HSCR. Time-lapse imaging of ENCCs within the embryonic gut demonstrates a collective cell migration defect. Mutant ENCCs show undirected cellular protrusions and disrupted directional and chain migration. Phactr4 acts cell-autonomously in ENCCs and colocalizes with integrin and cofilin at cell protrusions. Mechanistically, we show that Phactr4 negatively regulates integrin signaling through the RHO/ROCK pathway and coordinates protein phosphatase 1 (PP1) with cofilin activity to regulate cytoskeletal dynamics. Strikingly, lamellipodia formation and in vivo ENCC chain migration defects are rescued by inhibition of ROCK or integrin function. Our results demonstrate a previously unknown pathway in ENCC collective migration in vivo and provide new candidate genes for human genetic studies of HSCR. © 2012 by Cold Spring Harbor Laboratory Press.

Haber D.A.,Harvard University | Haber D.A.,Howard Hughes Medical Institute | Velculescu V.E.,Johns Hopkins University
Cancer Discovery | Year: 2014

The ability to study nonhematologic cancers through noninvasive sampling of blood is one of the most exciting and rapidly advancing fields in cancer diagnostics. This has been driven both by major technologic advances, including the isolation of intact cancer cells and the analysis of cancer cell-derived DNA from blood samples, and by the increasing application of molecularly driven therapeutics, which rely on such accurate and timely measurements of critical biomarkers. Moreover, the dramatic efficacy of these potent cancer therapies drives the selection for additional genetic changes as tumors acquire drug resistance, necessitating repeated sampling of cancer cells to adjust therapy in response to tumor evolution. Together, these advanced noninvasive diagnostic capabilities and their applications in guiding precision cancer therapies are poised to change the ways in which we select and monitor cancer treatments. Significance: Recent advances in technologies to analyze circulating tumor cells and circulating tumor DNA are setting the stage for real-time, noninvasive monitoring of cancer and providing novel insights into cancer evolution, invasion, and metastasis. © 2014 American Association for Cancer Research.

Wang L.,California Institute of Technology | Anderson D.J.,California Institute of Technology | Anderson D.J.,Howard Hughes Medical Institute
Nature | Year: 2010

Aggression is regulated by pheromones in many animal species. However, in no system have aggression pheromones, their cognate receptors and corresponding sensory neurons been identified. Here we show that 11-cis-vaccenyl acetate (cVA), a male-specific volatile pheromone, robustly promotes male-male aggression in the vinegar fly Drosophila melanogaster. The aggression-promoting effect of synthetic cVA requires olfactory sensory neurons (OSNs) expressing the receptor Or67d, as well as the receptor itself. Activation of Or67d-expressing OSNs, either by genetic manipulation of their excitability or by exposure to male pheromones in the absence of other classes of OSNs, is sufficient to promote aggression. High densities of male flies can promote aggression by the release of volatile cVA. In turn, cVA-promoted aggression can promote male fly dispersal from a food resource, in a manner dependent on Or67d-expressing OSNs. These data indicate that cVA may mediate negative-feedback control of male population density, through its effect on aggression. Identification of a pheromone-OSN pair controlling aggression in a genetic organism opens the way to unravelling the neurobiology of this evolutionarily conserved behaviour. © 2010 Macmillan Publishers Limited. All rights reserved.

Haswell E.S.,Washington University in St. Louis | Phillips R.,California Institute of Technology | Rees D.C.,Howard Hughes Medical Institute
Structure | Year: 2011

While mechanobiological processes employ diverse mechanisms, at their heart are force-induced perturbations in the structure and dynamics of molecules capable of triggering subsequent events. Among the best characterized force-sensing systems are bacterial mechanosensitive channels. These channels reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane; the membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal, interpreted for the cell by mechanosensitive channels. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension or other functions. This review focuses on the bacterial mechanosensitive channels of large (MscL) and small (MscS) conductance and their eukaryotic homologs, with an emphasis on the outstanding issues surrounding the function and mechanism of this fascinating class of molecules. © 2011 Elsevier Ltd.

Dorrestein P.C.,University of California at San Diego | Mazmanian S.K.,California Institute of Technology | Knight R.,BioFrontiers Institute | Knight R.,Howard Hughes Medical Institute
Immunity | Year: 2014

The unexpected diversity of the human microbiome and metabolome far exceeds the complexity of the human genome. Although we now understand microbial taxonomic and genetic repertoires in some populations, we are just beginning to assemble the necessary computational and experimental tools to understand the metabolome in comparable detail. However, even with the limited current state of knowledge, individual connections between microbes and metabolites, between microbes and immune function, and between metabolites and immune function are being established. Here, we provide our perspective on these connections and outline a systematic research program that could turn these individual links into a broader network that allows us to understand how these components interact. This program will enable us to exploit connections among the microbiome, metabolome, and host immune system to maintain health and perhaps help us understand how to reverse the processes that lead to a wide range of immune and other diseases. Recent research has developed specific connections among particular microbes, metabolites, and host immune processes, but a generalized map of this interaction network is still lacking. Dorrestein etal. describe these connections and outline how we could move toward a global view. © 2014 Elsevier Inc.

Zhou Y.,Johns Hopkins University | Nathans J.,Johns Hopkins University | Nathans J.,Howard Hughes Medical Institute
Developmental Cell | Year: 2014

Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a coactivator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp coreceptor and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. Theseexperiments identify Gpr124 as a ligand-specific coactivator of canonical Wnt signaling. © 2014 Elsevier Inc.

Dillin A.,Howard Hughes Medical Institute | Cohen E.,Hebrew University of Jerusalem
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2011

Late onset is a common hallmark character of numerous disorders including human neurodegenerative maladies such as Huntington's, Parkinson's and Alzheimer's diseases. Why these diseases manifest in aged individuals and why distinct disorders share strikingly similar emergence patterns were until recently unsolved enigmas. During the past decade, invertebrate-based studies indicated that the insulin/IGF signalling pathway (IIS) mechanistically links neurodegenerative-associated toxic protein aggregation and ageing; yet, until recently it was unclear whether this link is conserved from invertebrates to mammals. Recent studies performed in Alzheimer's mouse models indicated that ageing alteration by IIS reduction slows the progression of Alzheimer's-like disease, protects the brain and mitigates the behavioural, pathological and biochemical impairments associated with the disease. Here, we review these novel studies and discuss the potential of ageing alteration as a therapeutic approach for the treatment of late onset neurodegeneration. © 2011 The Royal Society.

Dubuis J.O.,Princeton University | Samanta R.,Howard Hughes Medical Institute | Gregor T.,Princeton University
Molecular Systems Biology | Year: 2013

Quantification of gene expression has become a central tool for understanding genetic networks. In many systems, the only viable way to measure protein levels is by immunofluorescence, which is notorious for its limited accuracy. Using the early Drosophila embryo as an example, we show that careful identification and control of experimental error allows for highly accurate gene expression measurements. We generated antibodies in different host species, allowing for simultaneous staining of four Drosophila gap genes in individual embryos. Careful error analysis of hundreds of expression profiles reveals that less than ∼20% of the observed embryo-to-embryo fluctuations stem from experimental error. These measurements make it possible to extract not only very accurate mean gene expression profiles but also their naturally occurring fluctuations of biological origin and corresponding cross-correlations. We use this analysis to extract gap gene profile dynamics with ∼1 min accuracy. The combination of these new measurements and analysis techniques reveals a twofold increase in profile reproducibility owing to a collective network dynamics that relays positional accuracy from the maternal gradients to the pair-rule genes. © 2013 EMBO and Macmillan Publishers Limited.

Hong W.,Howard Hughes Medical Institute | Kim D.W.,California Institute of Technology | Anderson D.J.,Howard Hughes Medical Institute
Cell | Year: 2014

Animals display a range of innate social behaviors that play essential roles in survival and reproduction. While the medial amygdala (MeA) has been implicated in prototypic social behaviors such as aggression, the circuit-level mechanisms controlling such behaviors are not well understood. Using cell-type-specific functional manipulations, we find that distinct neuronal populations in the MeA control different social and asocial behaviors. A GABAergic subpopulation promotes aggression and two other social behaviors, while neighboring glutamatergic neurons promote repetitive self-grooming, an asocial behavior. Moreover, this glutamatergic subpopulation inhibits social interactions independently of its effect to promote self-grooming, while the GABAergic subpopulation inhibits self-grooming, even in a nonsocial context. These data suggest that social versus repetitive asocial behaviors are controlled in an antagonistic manner by inhibitory versus excitatory amygdala subpopulations, respectively. These findings provide a framework for understanding circuit-level mechanisms underlying opponency between innate behaviors, with implications for their perturbation in psychiatric disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

Losman J.-A.,Dana-Farber Cancer Institute | Kaelin Jr. W.G.,Dana-Farber Cancer Institute | Kaelin Jr. W.G.,Howard Hughes Medical Institute
Genes and Development | Year: 2013

Mutations in metabolic enzymes, including isocitrate dehydrogenase 1 (IDH1) and IDH2, in cancer strongly implicate altered metabolism in tumorigenesis. IDH1 and IDH2 catalyze the interconversion of isocitrate and 2-oxoglutarate (2OG). 2OG is a TCA cycle intermediate and an essential cofactor for many enzymes, including JmjC domain-containing histone demethylases, TET 5-methylcytosine hydroxylases, and EglN prolyl-4-hydroxylases. Cancer-associated IDHmutations alter the enzymes such that they reduce 2OG to the structurally similar metabolite (R)-2-hydroxyglutarate [(R)-2HG]. Here we review what is known about the molecular mechanisms of transformation by mutant IDH and discuss their implications for the development of targeted therapies to treat IDH mutant malignancies. © 2013 by Cold Spring Harbor Laboratory Press.

Gan L.,California Institute of Technology | Jensen G.J.,California Institute of Technology | Jensen G.J.,Howard Hughes Medical Institute
Quarterly Reviews of Biophysics | Year: 2012

The electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution three-dimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or 'tomogram'. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology. © Cambridge University Press 2011.

Cho J.Y.,California Institute of Technology | Cho J.Y.,Howard Hughes Medical Institute | Sternberg P.W.,California Institute of Technology | Sternberg P.W.,Howard Hughes Medical Institute
Cell | Year: 2014

Sleep is characterized by behavioral quiescence, homeostasis, increased arousal threshold, and rapid reversibility. Understanding how these properties are encoded by a neuronal circuit has been difficult, and no single molecular or neuronal pathway has been shown to be responsible for the regulation of sleep. Taking advantage of the well-mapped neuronal connections of Caenorhabditis elegans and the sleep-like states in this animal, we demonstrate the changed properties of both sensory neurons and downstream interneurons that mediate sleep and arousal. The ASH sensory neuron displays reduced sensitivity to stimuli in the sleep-like state, and the activity of the corresponding interneurons in ASH's motor circuit becomes asynchronous. Restoration of interneuron synchrony is sufficient for arousal. The multilevel circuit depression revealed provides an elegant strategy to promote a robust decrease in arousal while allowing for rapid reversibility of the sleep state. © 2014 Elsevier Inc.

Little S.C.,Howard Hughes Medical Institute | Tikhonov M.,Princeton University | Gregor T.,Princeton University
Cell | Year: 2013

Early embryonic patterning events are strikingly precise, a fact that appears incompatible with the stochastic gene expression observed across phyla. Using single-molecule mRNA quantification in Drosophila embryos, we determine the magnitude of fluctuations in the expression of four critical patterning genes. The accumulation of mRNAs is identical across genes and fluctuates by only ∼8% between neighboring nuclei, generating precise protein distributions. In contrast, transcribing loci exhibit an intrinsic noise of ∼45% independent of specific promoter-enhancer architecture or fluctuating inputs. Precise transcript distribution in the syncytium is recovered via straightforward spatiotemporal averaging, i.e., accumulation and diffusion of transcripts during nuclear cycles, without regulatory feedback. Common expression characteristics shared between genes suggest that fluctuations in mRNA production are context independent and are a fundamental property of transcription. The findings shed light on how the apparent paradox between stochastic transcription and developmental precision is resolved. © 2013 Elsevier Inc.

Anderson D.J.,California Institute of Technology | Anderson D.J.,Howard Hughes Medical Institute | Perona P.,California Institute of Technology
Neuron | Year: 2014

The new field of "Computational Ethology" is made possible by advances in technology, mathematics, and engineering that allow scientists to automate the measurement and the analysis of animal behavior. We explore the opportunities and long-term directions of research in this area. © 2014 Elsevier Inc.

Bruner K.M.,Johns Hopkins University | Hosmane N.N.,Johns Hopkins University | Siliciano R.F.,Johns Hopkins University | Siliciano R.F.,Howard Hughes Medical Institute
Trends in Microbiology | Year: 2015

The latent reservoir (LR) of HIV-1 in resting memory CD4+ T cells serves as a major barrier to curing HIV-1 infection. While many PCR- and culture-based assays have been used to measure the size of the LR, correlation between results of different assays is poor and recent studies indicate that no available assay provides an accurate measurement of reservoir size. The discrepancies between assays are a hurdle to clinical trials that aim to measure the efficacy of HIV-1 eradication strategies. Here we describe the advantages and disadvantages of various approaches to measuring the LR. © 2015 Elsevier Ltd.

Hu T.T.,Princeton University | Eisen M.B.,Howard Hughes Medical Institute | Thornton K.R.,University of California at Irvine | Andolfatto P.,Princeton University
Genome Research | Year: 2013

We create a new assembly of the Drosophila simulans genome using 142 million paired short-read sequences and previously published data for strain w501. Our assembly represents a higher-quality genomic sequence with greater coverage, fewer misassemblies, and, by several indexes, fewer sequence errors. Evolutionary analysis of this genome reference sequence reveals interesting patterns of lineage-specific divergence that are different from those previously reported. Specifically, we find that Drosophila melanogaster evolves faster than D. simulans at all annotated classes of sites, including putatively neutrally evolving sites found in minimal introns. While this may be partly explained by a higher mutation rate in D. melanogaster, we also find significant heterogeneity in rates of evolution across classes of sites, consistent with historical differences in the effective population size for the two species. Also contrary to previous findings, we find that the X chromosome is evolving significantly faster than autosomes for nonsynonymous and most noncoding DNA sites and significantly slower for synonymous sites. The absence of a X/A difference for putatively neutral sites and the robustness of the pattern to Gene Ontology and sex-biased expression suggest that partly recessive beneficial mutations may comprise a substantial fraction of noncoding DNA divergence observed between species. Our results have more general implications for the interpretation of evolutionary analyses of genomes of different quality.

Tagliabracci V.S.,University of California at San Diego | Pinna L.A.,University of Padua | Dixon J.E.,University of California at San Diego | Dixon J.E.,Howard Hughes Medical Institute
Trends in Biochemical Sciences | Year: 2013

Protein kinases constitute one of the largest gene families and control many aspects of cellular life. In retrospect, the first indication for their existence was reported 130 years ago when the secreted protein, casein, was shown to contain phosphate. Despite its identification as the first phosphoprotein, the responsible kinase has remained obscure. This conundrum was solved with the discovery of a novel family of atypical protein kinases that are secreted and appear to phosphorylate numerous extracellular proteins, including casein. Fam20C, the archetypical member, phosphorylates secreted proteins within Ser-x-Glu/pSer motifs. This discovery has solved a 130-year-old mystery and has shed light on several human disorders of biomineralization. © 2012 Elsevier Ltd.

Tomasetti C.,Johns Hopkins University | Vogelstein B.,Howard Hughes Medical Institute
Science | Year: 2015

Some tissue types give rise to human cancers millions of times more often than other tissue types. Although this has been recognized for more than a century, it has never been explained. Here, we show that the lifetime risk of cancers of many different types is strongly correlated (0.81) with the total number of divisions of the normal self-renewing cells maintaining that tissue's homeostasis. These results suggest that only a third of the variation in cancer risk among tissues is attributable to environmental factors or inherited predispositions. The majority is due to "bad luck," that is, random mutations arising during DNA replication in normal, noncancerous stem cells. This is important not only for understanding the disease but also for designing strategies to limit the mortality it causes.

Haber D.A.,Harvard University | Haber D.A.,Howard Hughes Medical Institute | Gray N.S.,Dana-Farber Cancer Institute | Baselga J.,Harvard University
Cell | Year: 2011

Building on years of basic scientific discovery, recent advances in the fields of cancer genetics and medicinal chemistry are now converging to revolutionize the treatment of cancer. Starting with serendipitous observations in rare subsets of cancer, a paradigm shift in clinical research is poised to ensure that new molecular insights are rapidly applied to shape emerging cancer therapies. Could this mark a turning point in the "War on Cancer"? © 2011 Elsevier Inc.

Mishra P.,California Institute of Technology | Chan D.C.,California Institute of Technology | Chan D.C.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2014

During cell division, it is critical to properly partition functional sets of organelles to each daughter cell. The partitioning of mitochondria shares some common features with that of other organelles, particularly in the use of interactions with cytoskeletal elements to facilitate delivery to the daughter cells. However, mitochondria have unique features-including their own genome and a maternal mode of germline transmission-that place additional demands on this process. Consequently, mechanisms have evolved to regulate mitochondrial segregation during cell division, oogenesis, fertilization and tissue development, as well as to ensure the integrity of these organelles and their DNA, including fusion-fission dynamics, organelle transport, mitophagy and genetic selection of functional genomes. Defects in these processes can lead to cell and tissue pathologies. © 2014 Macmillan Publishers Limited. All rights reserved.

Hoelz A.,California Institute of Technology | Debler E.W.,Laboratory of Cell Biology | Blobel G.,Laboratory of Cell Biology | Blobel G.,Howard Hughes Medical Institute
Annual Review of Biochemistry | Year: 2011

In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies. © 2011 by Annual Reviews. All rights reserved.

Berndsen C.E.,James Madison University | Wolberger C.,Johns Hopkins University | Wolberger C.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2014

E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine. Three distinct classes of E3 ligases have been identified that stimulate transfer of ubiquitin and ubiquitin-like proteins through either a direct or an indirect mechanism. Only recently have the catalytic mechanisms of E3 ligases begun to be elucidated. © 2014 Nature America, Inc.

Anderson D.J.,California Institute of Technology | Anderson D.J.,Howard Hughes Medical Institute | Adolphs R.,California Institute of Technology
Cell | Year: 2014

Since the 19th century, there has been disagreement over the fundamental question of whether "emotions" are cause or consequence of their associated behaviors. This question of causation is most directly addressable in genetically tractable model organisms, including invertebrates such as Drosophila. Yet there is ongoing debate about whether such species even have "emotions," as emotions are typically defined with reference to human behavior and neuroanatomy. Here, we argue that emotional behaviors are a class of behaviors that express internal emotion states. These emotion states exhibit certain general functional and adaptive properties that apply across any specific human emotions like fear or anger, as well as across phylogeny. These general properties, which can be thought of as "emotion primitives," can be modeled and studied in evolutionarily distant model organisms, allowing functional dissection of their mechanistic bases and tests of their causal relationships to behavior. More generally, our approach not only aims at better integration of such studies in model organisms with studies of emotion in humans, but also suggests a revision of how emotion should be operationalized within psychology and psychiatry. © 2014 Elsevier Inc.

West A.P.,California Institute of Technology | Scharf L.,California Institute of Technology | Scheid J.F.,Rockefeller University | Klein F.,Rockefeller University | And 4 more authors.
Cell | Year: 2014

Despite 30 years of effort, there is no effective vaccine for HIV-1. However, antibodies can prevent HIV-1 infection in humanized mice and macaques when passively transferred. New single-cell-based methods have uncovered many broad and potent donor-derived antibodies, and structural studies have revealed the molecular bases for their activities. The new data suggest why such antibodies are difficult to elicit and inform HIV-1 vaccine development efforts. In addition to protecting against infection, the newly identified antibodies can suppress active infections in mice and macaques, suggesting they could be valuable additions to anti-HIV-1 therapies and to strategies to eradicate HIV-1 infection. © 2014 Elsevier Inc.

Dillin A.,Howard Hughes Medical Institute | Gottschling D.E.,Fred Hutchinson Cancer Research Center | Nystrom T.,Gothenburg University
Current Opinion in Cell Biology | Year: 2014

Over 40 years ago, Francois Jacob proposed that levels of 'integrons' explain how biological systems are constructed. Today, these networks of interactions between tissues, cells, organelles, metabolic pathways, genes, and individual molecules provide key insights into biology. We suggest that the wiring and interdependency between subsystems within a network are useful to understand the aging process. The breakdown of one subsystem (e.g. an organelle) can have ramifications for other interconnected subsystems, leading to the sequential collapse of subsystem functions. But yet, the interconnected nature of homeostatic wiring can provide organisms with the means of compensating for the decline of one subsystem. This occurs at multiple levels in an organism. - for example, between organelles or between tissues. We review recent data that highlight the importance of such interconnectivity/communication in the aging process, in both progressive decline and longevity assurance. © 2013.

Hong W.,California Institute of Technology | Luo L.,Howard Hughes Medical Institute
Genetics | Year: 2014

Precise connections established between pre- and postsynaptic partners during development are essential for the proper function of the nervous system. The olfactory system detects a wide variety of odorants and processes the information in a precisely connected neural circuit. A common feature of the olfactory systems from insects to mammals is that the olfactory receptor neurons (ORNs) expressing the same odorant receptor make one-to-one connections with a single class of second-order olfactory projection neurons (PNs). This represents one of the most striking examples of targeting specificity in developmental neurobiology. Recent studies have uncovered central roles of transmembrane and secreted proteins in organizing this one-to-one connection specificity in the olfactory system. Here, we review recent advances in the understanding of how this wiring specificity is genetically controlled and focus on the mechanisms by which transmembrane and secreted proteins regulate different stages of the Drosophila olfactory circuit assembly in a coordinated manner. We also discuss how combinatorial coding, redundancy, and error-correcting ability could contribute to constructing a complex neural circuit in general. © 2014 by the Genetics Society of America.

Brunton B.W.,Princeton University | Brunton B.W.,University of Washington | Botvinick M.M.,Princeton University | Brody C.D.,Princeton University | Brody C.D.,Howard Hughes Medical Institute
Science | Year: 2013

The gradual and noisy accumulation of evidence is a fundamental component of decision-making, with noise playing a key role as the source of variability and errors. However, the origins of this noise have never been determined. We developed decision-making tasks in which sensory evidence is delivered in randomly timed pulses, and analyzed the resulting data with models that use the richly detailed information of each trial 's pulse timing to distinguish between different decision-making mechanisms. This analysis allowed measurement of the magnitude of noise in the accumulator 's memory, separately from noise associated with incoming sensory evidence. In our tasks, the accumulator's memory was noiseless, for both rats and humans. In contrast, the addition of new sensory evidence was the primary source of variability. We suggest our task and modeling approach as a powerful method for revealing internal properties of decision-making processes.

Deal R.B.,Fred Hutchinson Cancer Research Center | Henikoff S.,Fred Hutchinson Cancer Research Center | Henikoff S.,Howard Hughes Medical Institute
Nature Protocols | Year: 2011

Genomic studies of cell differentiation and function within a whole organism depend on the ability to isolate specific cell types from a tissue, but this is technically difficult. We developed a method called INTACT (isolation of nuclei tagged in specific cell types) that allows affinity-based isolation of nuclei from individual cell types of a tissue, thereby circumventing the problems associated with mechanical purification techniques. In this method nuclei are affinity-labeled through transgenic expression of a biotinylated nuclear envelope protein in the cell type of interest. Total nuclei are isolated from transgenic plants and biotin-labeled nuclei are then purified using streptavidin-coated magnetic beads, without the need for specialized equipment. INTACT gives high yield and purity of nuclei from the desired cell types, which can be used for genome-wide analysis of gene expression and chromatin features. The entire procedure, from nuclei purification through cDNA preparation or chromatin immunoprecipitation (ChIP), can be completed within 2 d. The protocol we present assumes that transgenic lines are already available, and includes procedural details for amplification of cDNA or ChIP DNA prior to microarray or deep sequencing analysis. © 2010 Nature America, Inc. All rights reserved.

Ngo H.B.,California Institute of Technology | Kaiser J.T.,California Institute of Technology | Chan D.C.,California Institute of Technology | Chan D.C.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2011

Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters. © 2011 Nature America, Inc. All rights reserved.

Blum J.S.,Indiana University | Wearsch P.A.,Case Western Reserve University | Cresswell P.,Howard Hughes Medical Institute
Annual Review of Immunology | Year: 2013

T cell recognition of antigen-presenting cells depends on their expression of a spectrum of peptides bound to major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I-and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review, we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. © Copyright 2013 by Annual Reviews. All rights reserved.

Potter C.J.,Johns Hopkins University | Luo L.,Howard Hughes Medical Institute
Nature Protocols | Year: 2011

In Drosophila, the GAL4/UAS/GAL80 repressible binary expression system is widely used to manipulate or mark tissues of interest. However, complex biological systems often require distinct transgenic manipulations of different cell populations. For this purpose, we recently developed the Q system, a second repressible binary expression system. We describe here the basic steps for performing a variety of Q system experiments in vivo. These include how to generate and use Q system reagents to express effector transgenes in tissues of interest, how to use the Q system in conjunction with the GAL4 system to generate intersectional expression patterns that precisely limit which tissues will be experimentally manipulated and how to use the Q system to perform mosaic analysis. The protocol described here can be adapted to a wide range of experimental designs. © 2011 Nature America, Inc. All rights reserved.

Bullen C.K.,Johns Hopkins University | Laird G.M.,Johns Hopkins University | Durand C.M.,Johns Hopkins University | Siliciano J.D.,Johns Hopkins University | And 2 more authors.
Nature Medicine | Year: 2014

HIV-1 persists in a latent reservoir despite antiretroviral therapy (ART). This reservoir is the major barrier to HIV-1 eradication. Current approaches to purging the latent reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects. Agents that reverse latency without activating T cells have been identified using in vitro models of latency. However, their effects on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo. Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually. © 2014 Nature America, Inc. All rights reserved.

Siliciano J.D.,Johns Hopkins University | Siliciano R.F.,Johns Hopkins University | Siliciano R.F.,Howard Hughes Medical Institute
Journal of Allergy and Clinical Immunology | Year: 2014

HIV-1 infection can now be readily controlled with combination antiretroviral therapy. However, the virus persists indefinitely in a stable latent reservoir in resting CD4+ T cells. This reservoir generally prevents cure of the infection with combination antiretroviral therapy alone. However, several recent cases of potential HIV-1 cure have generated renewed optimism. Here we review these cases and consider new developments in our understanding of the latent reservoir. In addition, we consider clinical aspects of curative strategies to provide a more realistic picture of what a generally applicable cure for HIV-1 infection is likely to entail. © 2014 American Academy of Allergy, Asthma and Immunology.

Van Berlo J.H.,University of Minnesota | Molkentin J.D.,Cincinnati Childrens Hospital Medical Center | Molkentin J.D.,Howard Hughes Medical Institute
Nature Medicine | Year: 2014

Cardiac regeneration is a rapidly evolving and controversial field of research. The identification some 12 years ago of progenitor cells that reside within the heart spurred enthusiasm for cell-based regenerative therapies. However, recent evidence has called into question both the presence of a biologically important stem cell population in the heart and the ability of exogenously derived cells to promote regeneration through direct formation of new cardiomyocytes. Here, we discuss recent developments that suggest an emerging consensus on the ability of different cell types to regenerate the adult mammalian heart. © 2014 Nature America, Inc. All rights reserved.

Howard Hughes Medical Institute and Johns Hopkins University | Date: 2012-07-16

The present invention is directed to nucleic acid and amino acid sequences of a novel piggyBac transposase enzymes created by modifying the transposase of Trichoplusia ni. The piggyBac transposases of the present invention are functionally active or hyperactive for excision and have decreased integration activity compared to wild type Trichoplusia ni piggyBac transposase enzyme. These transposases are ideal for use in methods of transforming cells and organisms. In particular embodiments, the present invention provides methods of transient integration and expression of transgenes.

Griffin E.E.,Howard Hughes Medical Institute | Odde D.J.,University of Minnesota | Seydoux G.,Howard Hughes Medical Institute
Cell | Year: 2011

Protein concentration gradients encode spatial information across cells and tissues and often depend on spatially localized protein synthesis. Here, we report that a different mechanism underlies the MEX-5 gradient. MEX-5 is an RNA-binding protein that becomes distributed in a cytoplasmic gradient along the anterior-to-posterior axis of the one-cell C. elegans embryo. We demonstrate that the MEX-5 gradient is a direct consequence of an underlying gradient in MEX-5 diffusivity. The MEX-5 diffusion gradient arises when the PAR-1 kinase stimulates the release of MEX-5 from slow-diffusive, RNA-containing complexes in the posterior cytoplasm. PAR-1 directly phosphorylates MEX-5 and is antagonized by the spatially uniform phosphatase PP2A. Mathematical modeling and in vivo observations demonstrate that spatially segregated phosphorylation and dephosphorylation reactions are sufficient to generate stable protein concentration gradients in the cytoplasm. The principles demonstrated here apply to any spatially segregated modification cycle that affects protein diffusion and do not require protein synthesis or degradation. © 2011 Elsevier Inc.

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