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The Scripps Research Institute is a nonprofit American medical research facility that focuses on research and education in the biomedical science. Headquartered in San Diego, California with a sister facility in Jupiter, Florida, the institute is home to 3,000 scientists, technicians, graduate students, and administrative and other staff, making it among the largest private, non-profit biomedical research organizations in the world. Wikipedia.

Kasirer-Friede A.,University of California at San Diego | Ruggeri Z.M.,Scripps Research Institute | Shattil S.J.,University of California at San Diego
Blood | Year: 2010

Binding of platelets to fibrinogen via integrin αIIbβ3 stimulates cytoskeletal reorganization and spreading. These responses depend on tyrosine phosphorylation of multiple proteins by Src family members and Syk. Among Src substrates in platelets is adhesion- and degranulationpromoting adapter protein (ADAP), an adapter with potential binding partners: SLP-76, VASP, and SKAP-HOM. During studies of platelet function under shear flow, we discovered that ADAP-/- mouse platelets, unlike ADAP+/+ platelets, formed unstable thrombi in response to carotid artery injury. Moreover, fibrinogen-adherent ADAP-/- platelets in shear flow ex vivo showed reduced spreading and smaller zones of contact with the matrix. These abnormalities were not observed under static conditions, and they could not be rescued by stimulating platelets with a PAR4 receptor agonist or by direct αIIbβ3 activation with MnCl2, consistent with a defect in outside-in αIIbβ3 signaling. ADAP+/+ platelets subjected to shear flow assembled F-actin-rich structures that colocalized with SLP-76 and the Rac1 exchange factor, phospho-Vav1. In contrast, platelets deficient in ADAP, but not those deficient in VASP or SKAP-HOM, failed to form these structures. These results establish that ADAP is an essential component of αIIbβ3-mediated platelet mechanotransduction that promotes F-actin assembly and enables platelet spreading and thrombus stabilization under fluid shear stress. © 2010 by The American Society of Hematology.

Kelso J.M.,Scripps Research Institute
Pediatric, Allergy, Immunology, and Pulmonology | Year: 2016

With appropriate management, children with asthma should expect few symptoms, no limits on activity, rare exacerbations, and normal lung function. Appropriate education of parents and other caregivers of children with asthma has clearly been shown to help achieve these goals. Although recommended in asthma guidelines, providing written asthma action plans does not improve outcomes beyond asthma education alone. © Copyright 2016, Mary Ann Liebert, Inc. 2016.

Goodsell D.S.,Scripps Research Institute
Biochemistry and Molecular Biology Education | Year: 2011

Diverse biological data may be used to create illustrations of molecules in their cellular context. This report describes the scientific results that support an illustration of a eukaryotic cell, enlarged by one million times to show the distribution and arrangement of macromolecules. The panoramic cross section includes eight panels that extend from the nucleus to the cell surface, showing the process of protein synthesis and export. Results from biochemistry, electron microscopy, NMR spectroscopy and x-ray crystallography were used to create the image. Copyright © 2011 Wiley Periodicals, Inc.

Rhind N.,University of Massachusetts Medical School | Russell P.,Scripps Research Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

Cell division requires careful orchestration of three major events: entry into mitosis, chromosomal segregation, and cytokinesis. Signaling within and between the molecules that control these events allows for their coordination via checkpoints, a specific class of signaling pathways that ensure the dependency of cell-cycle events on the successful completion of preceding events. Multiple positive- and negative-feedback loops ensure that a cell is fully committed to division and that the events occur in the proper order. Unlike other signaling pathways, which integrate external inputs to decide whether to execute a given process, signaling at cell division is largely dedicated to completing a decision made in G1 phase-to initiate and complete a round of mitotic cell division. Instead of deciding if the events of cell division will take place, these signaling pathways entrain these events to the activation of the cell-cycle kinase cyclin-dependent kinase 1 (CDK1) and provide the opportunity for checkpoint proteins to arrest cell division if things go wrong. © 2012 Cold Spring Harbor Laboratory Press.

Marin O.,University Miguel Hernandez | Muller U.,Scripps Research Institute
Current Opinion in Neurobiology | Year: 2014

Neocortical circuits are assembled from subtypes of glutamatergic excitatory and GABAergic inhibitory neurons with divergent anatomical and molecular signatures and unique physiological properties. Excitatory neurons derive from progenitors in the pallium, whereas inhibitory neurons originate from progenitors in the subpallium. Both classes of neurons subsequently migrate along well-defined routes to their final target area, where they integrate into common neuronal circuits. Recent findings show that neuronal diversity within the lineages of excitatory and inhibitory neurons is in part already established at the level of progenitor cells before migration. This poses challenges for our understanding of how radial units of interconnected excitatory and inhibitory neurons are assembled from progenitors that are spatially segregated and diverse in nature. © 2014.

Fujioka K.,Scripps Research Institute
Diabetes, Obesity and Metabolism | Year: 2015

Recently, the recognition of obesity as a complex disease that requires chronic management has become more widespread. There has also been a movement away from a focus on body mass index alone, and toward the management of obesity-related comorbidities as well as excess weight. This article examines the current and emerging pharmacological options for weight management in people with overweight or obesity who have, or are at a high risk of, weight-related comorbidities. In the USA, the current options for pharmacological weight management are phentermine (indicated for short-term use only), orlistat, combined phentermine/topiramate extended release, lorcaserin, naltrexone/bupropion and liraglutide 3.0mg. Currently, orlistat, naltrexone/bupropion and liraglutide 3.0mg are approved in Europe. All of the above-mentioned medications have shown weight-loss efficacy versus placebo. Those approved for long-term weight management have also been associated with improvements in weight-related comorbidities, such as hypertension, prediabetes, diabetes or dyslipidaemia, or related biomarkers. As with all drugs, the safety and tolerability profiles of medications for weight management should be considered alongside their efficacy to ensure correct use. Additional medications for weight management that are in clinical development include bupropion/zonisamide and beloranib. The field of obesity treatment is advancing with a number of medications being recently approved, and with other pharmacological options emerging. © 2015 John Wiley & Sons Ltd.

Mechanical injury induces cell death in cartilage and triggers a remodeling process that ultimately can manifest as osteoarthritis. Autophagy is a process for turnover of intracellular organelles and macromolecules that protects cells during stress responses. This study was undertaken to determine changes in and functions of autophagy following mechanical injury to cartilage. Bovine and human cartilage explants were subjected to mechanical impact (40% strain for 500 msec). Cell viability, sulfated glycosaminoglycan (sGAG) release, and changes in the levels of the autophagy markers ULK1, beclin 1, and microtubule-associated protein 1 light chain 3 (LC3) were evaluated. Cartilage explants were treated with the mammalian target of rapamycin complex 1 (mTORC-1) inhibitor and the autophagy inducer rapamycin and tested for protective effects against mechanical injury. Explants were also treated with the cell death inducers nitric oxide and tumor necrosis factor α (TNFα) plus actinomycin D, and the proinflammatory cytokine interleukin-1α (IL-1α). Mechanical injury induced cell death and loss of sGAG in a time-dependent manner. This was associated with significantly decreased ULK1, beclin 1, and LC3 expression in the cartilage superficial zone (P < 0.05) 48 hours after injury. The levels of LC3-II were increased 24 hours after injury but decreased at 48 and 96 hours. Rapamycin enhanced expression of autophagy regulators and prevented cell death and sGAG loss in mechanically injured explants. Rapamycin also protected against cell death induced by sodium nitroprusside and TNFα plus actinomycin D and prevented sGAG loss induced by IL-1α. Our findings indicate that mechanical injury leads to suppression of autophagy, predominantly in the superficial zone where most of the cell death occurs. Pharmacologic inhibition of mTORC-1, at least in part by enhancement of autophagy, prevents cell and matrix damage, suggesting a novel approach for chondroprotection. Copyright © 2012 by the American College of Rheumatology.

Naji S.,Scripps Research Institute
Molecular & cellular proteomics : MCP | Year: 2012

The HIV-1 Rev protein plays a key role in the late phase of virus replication. It binds to the Rev Response Element found in underspliced HIV mRNAs, and drives their nuclear export by the CRM1 receptor pathway. Moreover, mounting evidence suggests that Rev has additional functions in viral replication. Here we employed proteomics and statistical analysis to identify candidate host cell factors that interact with Rev. For this we studied Rev complexes assembled in vitro with nuclear or cytosolic extracts under conditions emulating various intracellular environments of Rev. We ranked the protein-protein interactions by combining several statistical features derived from pairwise comparison of conditions in which the abundance of the binding partners changed. As a validation set, we selected the eight DEAD/H box proteins of the RNA helicase family from the top-ranking 5% of the proteins. These proteins all associate with ectopically expressed Rev in immunoprecipitates of cultured cells. From gene knockdown approaches, our work in combination with previous studies indicates that six of the eight DEAD/H proteins are linked to HIV production in our cell model. In a more detailed analysis of infected cells where either DDX3X, DDX5, DDX17, or DDX21 was silenced, we observed distinctive phenotypes for multiple replication features, variously involving virus particle release, the levels of unspliced and spliced HIV mRNAs, and the nuclear and cytoplasmic concentrations of these transcripts. Altogether the work indicates that our top-scoring data set is enriched in Rev-interacting proteins relevant to HIV replication. Our more detailed analysis of several Rev-interacting DEAD proteins suggests a complex set of functions for the helicases in regulation of HIV mRNAs. The strategy used here for identifying Rev interaction partners should prove effective for analyzing other viral and cellular proteins.

Blackmond D.G.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2015

The use of modern kinetic tools to obtain virtually continuous reaction progress data over the course of a catalytic reaction opens up a vista that provides mechanistic insights into both simple and complex catalytic networks. Reaction profiles offer a rate/concentration scan that tells the story of a batch reaction time course in a qualitative "fingerprinting" manner as well as in quantitative detail. Reaction progress experiments may be mathematically designed to elucidate catalytic rate laws from only a fraction of the number of experiments required in classical kinetic measurements. The information gained from kinetic profiles provides clues to direct further mechanistic analysis by other approaches. Examples from a variety of catalytic reactions spanning two decades of the author's work help to delineate nuances on a central mechanistic theme. © 2015 American Chemical Society.

Bouchecareilh M.,Scripps Research Institute
Proceedings of the American Thoracic Society | Year: 2010

The function of the human proteome is defined by the proteostasis network (PN) (Science 2008;319:916; Science 2010;329:766), a biological system that generates, protects, and, where necessary, degrades a protein to optimize the cell, tissue, and organismal response to diet, stress, and aging. Numerous human diseases result from the failure of proteins to fold properly in response to mutation, disrupting the proteome. In the case of the exocytic pathway, this includes proteostasis components that direct folding, and export of proteins from the endoplasmic reticulum (ER). Included here are serpin deficiencies, a class of related diseases that result in a significant reduction of secretion of serine proteinase inhibitors from the liver into serum. In response to misfolding, variants of the serine protease α(1)-antitrypsin (α1AT) fail to exit the ER and are targeted for either ER-associated degradation or autophagic pathways. The challenge for developing α1AT deficiency therapeutics is to understand the PN pathways involved in folding and export. Herein, we review the role of the PN in managing the protein fold and function during synthesis in the ER and trafficking to the cell surface or extracellular space. We highlight the role of the proteostasis boundary to define the operation of the proteome (Annu Rev Biochem 2009;78:959). We discuss how manipulation of folding energetics or the PN by pharmacological intervention could provide multiple routes for restoration of variant α1AT function to the benefit of human health.

Zhang A.P.P.,University of Chicago | Pigli Y.Z.,University of Chicago | Pigli Y.Z.,Scripps Research Institute | Rice P.A.,University of Chicago
Nature | Year: 2010

The eubacterial SOS system is a paradigm of cellular DNA damage and repair, and its activation can contribute to antibiotic resistance. Under normal conditions, LexA represses the transcription of many DNA repair proteins by binding to SOS 'boxes'-in their operators. Under genotoxic stress, accumulating complexes of RecA, ATP and single-stranded DNA (ssDNA) activate LexA for autocleavage. To address how LexA recognizes its binding sites, we determined three crystal structures of Escherichia coli LexA in complex with SOS boxes. Here we report the structure of these LexA-DNA complexes. The DNA-binding domains of the LexA dimer interact with the DNA in the classical fashion of a winged helix-turn-helix motif. However, the wings of these two DNA-binding domains bind to the same minor groove of the DNA. These wing-wing contacts may explain why the spacing between the two half-sites of E. coli SOS boxes is invariant. © 2010 Macmillan Publishers Limited. All rights reserved.

Picciotto M.R.,Yale University | Kenny P.J.,Scripps Research Institute
Cold Spring Harbor Perspectives in Medicine | Year: 2013

Tobacco smoking results in more than 5 million deaths each year and accounts for almost 90% of all deaths from lung cancer. Nicotine, the major reinforcing component of tobacco smoke, acts in the brain through the neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are allosterically regulated, ligand-gated ion channels consisting of five membrane-spanning subunits. Twelve mammalian α subunits (α2-α10) and β subunits (β2-β4) have been cloned. The predominant nAChR subtypes in mammalian brain are those containing α4 and β2 subunits (denoted as α4β2* nAChRs). The α4β2* nAChRs mediate many behaviors related to nicotine addiction and are the primary targets for currently approved smoking cessation agents. Considering the large number of nAChR subunits in the brain, it is likely that nAChRs containing subunits in addition to α4 and β2 also play a role in tobacco smoking. Indeed, genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and β4 nAChR subunits, respectively, has been shown to increase vulnerability to tobacco dependence and smoking-associated diseases including lung cancer. Moreover, mice in which expression of α5 or β4 subunits has been genetically modified have profoundly altered patterns of nicotine consumption. In addition to the reinforcing properties of nicotine, the effects of nicotine on appetite, attention, and mood are also thought to contribute to establishment and maintenance of the tobacco smoking habit. Here we review recent insights into the behavioral actions of nicotine and the nAChRs subtypes involved, which likely contribute to the development of tobacco dependence in smokers. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

Taffe M.A.,Scripps Research Institute
Journal of Psychopharmacology | Year: 2012

Cannabis remains the most commonly abused illicit drug and is rapidly expanding in quasi-licit use in some jurisdictions under medical marijuana laws. Effects of the psychoactive constituent δ9tetrahydrocannabinol (δ9THC) on cognitive function remain of pressing concern. Prior studies in monkeys have not shown consistent evidence of memory-specific effects of δ9THC on recognition tasks, and it remains unclear to what extent δ9THC causes sedative versus specific cognitive effects. In this study, adult male rhesus monkeys were trained on tasks which assess spatial working memory, visuo-spatial associative memory and learning as well as motivation for food reward. Subjects were subsequently challenged with 0.1-0.3 mg/kg δ9THC, i.m., in randomized order and evaluated on the behavioral measures. The performance of both vsPAL and SOSS tasks was impaired by δ9THC in a dose and task-difficulty dependent manner. It is concluded that δ9THC disrupts cognition in a way that is consistent with a direct effect on memory. There was evidence for interference with spatial working memory, visuo-spatial associative memory and incremental learning in the latter task. These results and the lack of specific effect of δ9THC in prior visual recognition studies imply a sensitivity of spatial memory processing and/or working memory to endocannabinoid perturbation. © The Author(s) 2012.

Kelso J.M.,Scripps Research Institute
Journal of Allergy and Clinical Immunology | Year: 2014

Excipients are substances in pharmaceuticals other than the active ingredients. Some excipients are foods or substances derived from foods, raising the possibility that these substances would pose a hazard to patients with food allergy. This review describes which food-derived substances are used as pharmaceutical excipients in which medications and reviews published data regarding the safety of the administration of these medications to recipients with food allergy. Such reactions are rare, usually because the amount of food protein is not present in a large enough quantity to elicit a reaction. When a food protein appears as an unintentional contaminant, the amount, if any, that is present might be variable and might elicit reactions only from some lots of medication or only in some patients. In most circumstances these medications should not be routinely withheld from patients who have particular food allergies because most will tolerate the medications uneventfully. However, if a particular patient has had an apparent allergic reaction to the medication, potential allergy to the food component should be investigated. © 2014 American Academy of Allergy, Asthma & Immunology.

Mayford M.,Scripps Research Institute | Siegelbaum S.A.,Howard Hughes Medical Institute | Kandel E.R.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

The synapse is the functional unit of the brain. During the last several decades we have acquired a great deal of information on its structure, molecular components, and physiological function. It is clear that synapses are morphologically and molecularly diverse and that this diversity is recruited to different functions. One of the most intriguing findings is that the size of the synaptic response in not invariant, but can be altered by a variety of homoand heterosynaptic factors such as past patterns of use or modulatory neurotransmitters. Perhaps the most difficult challenge in neuroscience is to design experiments that reveal how these basic building blocks of the brain are put together and how they are regulated to mediate the information flow through neural circuits that is necessary to produce complex behaviors and store memories. In this reviewwe will focus on studies that attempt to uncover the role of synaptic plasticity in the regulation of whole-animal behavior by learning and memory. © 2012 Cold Spring Harbor Laboratory Press; all rights reserved.

Schnermann M.J.,U.S. National Cancer Institute | Shenvi R.A.,Scripps Research Institute
Natural Product Reports | Year: 2015

Covering up to 2014 Isocyanoterpenes (ICTs) are marine natural products biosynthesized through an unusual pathway that adorns terpene scaffolds with nitrogenous functionality derived from cyanide. The appendage of nitrogen functional groups-isonitriles in particular-onto stereochemically-rich carbocyclic ring systems provides enigmatic, bioactive molecules that have required innovative chemical syntheses. This review discusses the challenges inherent to the synthesis of this diverse family and details the development of the field. We also present recent progress in isolation and discuss key aspects of the remarkable biological activity of these compounds. © 2015 The Royal Society of Chemistry.

Koob G.F.,Scripps Research Institute | Volkow N.D.,U.S. National Institute on Drug Abuse
Neuropsychopharmacology | Year: 2010

Drug addiction is a chronically relapsing disorder that has been characterized by (1) compulsion to seek and take the drug, (2) loss of control in limiting intake, and (3) emergence of a negative emotional state (eg, dysphoria, anxiety, irritability) reflecting a motivational withdrawal syndrome when access to the drug is prevented. Drug addiction has been conceptualized as a disorder that involves elements of both impulsivity and compulsivity that yield a composite addiction cycle composed of three stages: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation (craving). Animal and human imaging studies have revealed discrete circuits that mediate the three stages of the addiction cycle with key elements of the ventral tegmental area and ventral striatum as a focal point for the binge/intoxication stage, a key role for the extended amygdala in the withdrawal/negative affect stage, and a key role in the preoccupation/anticipation stage for a widely distributed network involving the orbitofrontal cortex-dorsal striatum, prefrontal cortex, basolateral amygdala, hippocampus, and insula involved in craving and the cingulate gyrus, dorsolateral prefrontal, and inferior frontal cortices in disrupted inhibitory control. The transition to addiction involves neuroplasticity in all of these structures that may begin with changes in the mesolimbic dopamine system and a cascade of neuroadaptations from the ventral striatum to dorsal striatum and orbitofrontal cortex and eventually dysregulation of the prefrontal cortex, cingulate gyrus, and extended amygdala. The delineation of the neurocircuitry of the evolving stages of the addiction syndrome forms a heuristic basis for the search for the molecular, genetic, and neuropharmacological neuroadaptations that are key to vulnerability for developing and maintaining addiction. © 2010 Nature Publishing Group All rights reserved.

Steinhoff A.K.,Kaiser Permanente | Bugbee W.D.,Scripps Research Institute
Knee Surgery, Sports Traumatology, Arthroscopy | Year: 2014

Purpose: To compare the following subjective scoring instruments: the Knee Society Function (KS-F) Score and Knee Injury and Osteoarthritis Outcome Score (KOOS) in patients who underwent total knee arthroplasty (TKA) by assessing standard metrics related to the utility of each instrument, including responsiveness, ceiling effect, and distribution of scores.Methods: Patients undergoing TKA for osteoarthritis (n = 82) were evaluated pre-operatively and post-operatively at a median time of 1 year (range 0.7–3.6 years) using the KS-F score and KOOS. Responsiveness, the ability of a tool to measure change over time, was evaluated by computing the standardized response mean. Ceiling effects were determined by calculating the percentage of patients who attained the best-possible score.Results: The KOOS scale had higher standardized response mean than KS-F scale in the majority of subsections (except sports and recreation). The KOOS had a lower ceiling effect in the majority of subsections (except pain) and was significantly lower on the symptoms and quality of life subscales (both p < 0.05). Scores were not normally distributed for either KS-F or KOOS scales.Conclusions: Since the KOOS scale had a higher responsiveness and a lower ceiling effect, it appears to be a superior outcome tool versus the KS-F scale when evaluating the outcomes of TKA patients.Level of evidence: Diagnostic Study, Level I. © 2014 European Society of Sports Traumatology, Knee Surgery, Arthroscopy (ESSKA)

Microfluidic technology - the manipulation of fluids at micrometer scales - has revolutionized many areas of synthetic biology. The bottom-up synthesis of "minimal" cell models has traditionally suffered from poor control of assembly conditions. Giant unilamellar vesicles (GUVs) are good models of living cells on account of their size and unilamellar membrane structure. In recent years, a number of microfluidic approaches for constructing GUVs has emerged. These provide control over traditionally elusive parameters of vesicular structure, such as size, lamellarity, membrane composition, and internal contents. They also address sophisticated cellular functions such as division and protein synthesis. Microfluidic techniques for GUV synthesis can broadly be categorized as continuous-flow based approaches and droplet-based approaches. This review presents the state-of-the-art of microfluidic technology, a robust platform for recapitulating complex cellular structure and function in synthetic models of biological cells. © 2012 WILEY Periodicals, Inc.

Childs-Disney J.L.,Scripps Research Institute
Nature communications | Year: 2013

The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)(exp)). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. A thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)(exp) and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)(exp). This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)(exp) and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of myotonic dystrophy type 1.

The 21st Annual Antibody Engineering and 8th Annual Antibody Therapeutics international conferences, and the 2010 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 5-9, 2010 in San Diego, CA. The conferences were organized with a focus on antibody engineering only on the first day and a joint engineering/therapeutics session on the last day. Delegates could select from presentations that occurred in two simultaneous sessions on days 2 and 3. Day 1 included presentations on neutralizing antibodies and the identification of vaccine targets, as well as a historical overview of 20 years of phage display utilization. Topics presented in the Antibody Engineering sessions on day 2 and 3 included antibody biosynthesis, structure and stability; antibodies in a complex environment; antibody half-life; and targeted nanoparticle therapeutics. In the Antibody Therapeutics sessions on days 2 and 3, preclinical and early stage development and clinical updates of antibody therapeutics, including TRX518, SYM004, MM111, PRO140, CVX-241, ASG-5ME, U3-1287 (AMG888), R1507 and trastuzumab emtansine, were discussed, and perspectives were provided on the development of biosimilar and biobetter antibodies, including coverage of regulatory and intellectual property issues. The joint engineering/therapeutics session on the last day focused on bispecific and next-generation antibodies.

Dyson H.J.,Scripps Research Institute
Quarterly Reviews of Biophysics | Year: 2011

Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional (3D) structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question 'why would a particular domain need to be unstructured?' are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but also of partially structured and highly dynamic members of the disorder-order continuum. © 2011 Cambridge University Press.

Lazzerini-Denchi E.,Scripps Research Institute | Sfeir A.,New York University
Nature Reviews Molecular Cell Biology | Year: 2016

Mammalian cells have evolved specialized mechanisms to sense and repair double-strand breaks (DSBs) to maintain genomic stability. However, in certain cases, the activity of these pathways can lead to aberrant DNA repair, genomic instability and tumorigenesis. One such case is DNA repair at the natural ends of linear chromosomes, known as telomeres, which can lead to chromosome-end fusions. Here, we review data obtained over the past decade and discuss the mechanisms that protect mammalian chromosome ends from the DNA damage response. We also discuss how telomere research has helped to uncover key steps in DSB repair. Last, we summarize how dysfunctional telomeres and the ensuing genomic instability drive the progression of cancer. © 2016 Macmillan Publishers Limited. All rights reserved.

Guo M.,Scripps Research Institute
Topics in current chemistry | Year: 2014

When compared to other conserved housekeeping protein families, such as ribosomal proteins, during the evolution of higher eukaryotes, aminoacyl-tRNA synthetases (aaRSs) show an apparent high propensity to add new sequences, and especially new domains. The stepwise emergence of those new domains is consistent with their involvement in a broad range of biological functions beyond protein synthesis, and correlates with the increasing biological complexity of higher organisms. These new domains have been extensively characterized based on their evolutionary origins and their sequence, structural, and functional features. While some of the domains are uniquely found in aaRSs and may have originated from nucleic acid binding motifs, others are common domain modules mediating protein-protein interactions that play a critical role in the assembly of the multi-synthetase complex (MSC). Interestingly, the MSC has emerged from a miniature complex in yeast to a large stable complex in humans. The human MSC consists of nine aaRSs (LysRS, ArgRS, GlnRS, AspRS, MetRS, IleRS, LeuRS, GluProRS, and bifunctional aaRs) and three scaffold proteins (AIMP1/p43, AIMP2/p38, and AIMP3/p18), and has a molecular weight of 1.5 million Dalton. The MSC has been proposed to have a functional dualism: facilitating protein synthesis and serving as a reservoir of non-canonical functions associated with its synthetase and non-synthetase components. Importantly, domain additions and functional expansions are not limited to the components of the MSC and are found in almost all aaRS proteins. From a structural perspective, multi-functionalities are represented by multiple conformational states. In fact, alternative conformations of aaRSs have been generated by various mechanisms from proteolysis to alternative splicing and posttranslational modifications, as well as by disease-causing mutations. Therefore, the metamorphosis between different conformational states is connected to the activation and regulation of the novel functions of aaRSs in higher eukaryotes.

Payne R.J.,University of Sydney | Wong C.-H.,Academia Sinica, Taiwan | Wong C.-H.,Scripps Research Institute
Chemical Communications | Year: 2010

A number of recent advances in the chemical synthesis of glycopeptides and glycoproteins are described, with particular focus on the development of peptide ligation strategies and their implementation in the convergent assembly of complex glycopeptides. Recent applications in the synthesis of full length homogeneous glycoproteins are also highlighted. © 2010 The Royal Society of Chemistry.

Levesque B.G.,Scripps Research Institute | Loftus E.V.,Mayo Medical School
Clinical Gastroenterology and Hepatology | Year: 2012

Azathioprine (AZA) and 6-mercaptopurine are therapeutic options for patients with moderate to severe inflammatory Crohn's disease. AZA has both a complex metabolism and potential for adverse events that can be clinically challenging. AZA has been shown to maintain remission and reduce corticosteroid use in patients with Crohn's disease. There is heterogeneous thiopurine methyltransferase metabolism among patients, which has implications for clinical dosing and risk for adverse events. Routine thiopurine methyltransferase testing before the initiation of AZA will reduce early leukopenia and is mandatory to avoid potentially life-threatening myelotoxicity. Thiopurine metabolite assays may aid in the assessment of adherence and adverse events. Patients who do not respond to AZA therapy may benefit from the addition of biologic therapy or methotrexate. © 2012 AGA Institute.

Plate L.,University of California at Berkeley | Marletta M.A.,University of California at Berkeley | Marletta M.A.,Scripps Research Institute
Molecular Cell | Year: 2012

Nitric oxide (NO) signaling in vertebrates is well characterized and involves the heme- nitric oxide/oxygen-binding (H-NOX) domain of soluble guanylate cyclase as a selective NO sensor. In contrast, little is known about the biological role or signaling output of bacterial H-NOX proteins. Here, we describe a molecular pathway for H-NOX signaling in Shewanella oneidensis. NO stimulates biofilm formation by controlling the levels of the bacterial secondary messenger cyclic diguanosine monophosphate (c-di-GMP). Phosphotransfer profiling was used to map the connectivity of a multicomponent signaling network that involves integration from two histidine kinases and branching to three response regulators. A feed-forward loop between response regulators with phosphodiesterase domains and phosphorylation-mediated activation intricately regulated c-di-GMP levels. Phenotypic characterization established a link between NO signaling and biofilm formation. Cellular adhesion may provide a protection mechanism for bacteria against reactive and damaging NO. These results are broadly applicable to H-NOX-mediated NO signaling in bacteria. © 2012 Elsevier Inc.

Gottesfeld J.M.,Scripps Research Institute | Rusche J.R.,Repligen | Pandolfo M.,Free University of Colombia
Journal of Neurochemistry | Year: 2013

The genetic defect in Friedreich's ataxia (FRDA) is the expansion of a GAA·TCC triplet in the first intron of the FXN gene, which encodes the mitochondrial protein frataxin. Previous studies have established that the repeats reduce transcription of this essential gene, with a concomitant decrease in frataxin protein in affected individuals. As the repeats do not alter the FXN protein coding sequence, one therapeutic approach would be to increase transcription of pathogenic FXN genes. Histone posttranslational modifications near the expanded repeats are consistent with heterochromatin formation and FXN gene silencing. In an effort to find small molecules that would reactivate this silent gene, histone deacetylase inhibitors were screened for their ability to up-regulate FXN gene expression in patient cells and members of the pimelic 2-aminobenzamide family of class I histone deacetylase inhibitors were identified as potent inducers of FXN gene expression and frataxin protein. Importantly, these molecules up-regulate FXN expression in human neuronal cells derived from patient-induced pluripotent stem cells and in two mouse models for the disease. Preclinical studies of safety and toxicity have been completed for one such compound and a phase I clinical trial in FRDA patients has been initiated. Furthermore, medicinal chemistry efforts have identified improved compounds with superior pharmacological properties. © 2013 International Society for Neurochemistry.

Williamson J.R.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2014

Post-transcriptional RNA modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. The current lack of a comprehensive method for a fast quantitative analysis of rRNA modifications significantly limits our understanding of how individual modification steps are coordinated during biogenesis inside the cell. Here, an LC-MS approach has been developed and successfully applied for quantitative monitoring of 29 out of 36 modified residues in the 16S and 23S rRNA from Escherichia coli. An isotope labeling strategy is described for efficient identification of ribose and base methylations, and a novel metabolic labeling approach is presented to allow identification of MS-silent pseudouridine modifications. The method was used to measure relative abundances of modified residues in incomplete ribosomal subunits compared to a mature 15N-labeled rRNA standard, and a number of modifications in both 16S and 23S rRNA were present in substoichiometric amounts in the preribosomal particles. The RNA modification levels correlate well with previously obtained profiles for the ribosomal proteins, suggesting that RNA is modified in a schedule comparable to the association of the ribosomal proteins. Importantly, this study establishes an efficient workflow for a global monitoring of ribosomal modifications that will contribute to a better understanding of mechanisms of RNA modifications and their impact on intracellular processes in the future. © 2014 American Chemical Society.

Morris K.V.,University of New South Wales | Morris K.V.,Scripps Research Institute | Mattick J.S.,Garvan Institute of Medical Research | Mattick J.S.,University of New South Wales
Nature Reviews Genetics | Year: 2014

Discoveries over the past decade portend a paradigm shift in molecular biology. Evidence suggests that RNA is not only functional as a messenger between DNA and protein but also involved in the regulation of genome organization and gene expression, which is increasingly elaborate in complex organisms. Regulatory RNA seems to operate at many levels; in particular, it plays an important part in the epigenetic processes that control differentiation and development. These discoveries suggest a central role for RNA in human evolution and ontogeny. Here, we review the emergence of the previously unsuspected world of regulatory RNA from a historical perspective. © 2014 Macmillan Publishers Limited. All rights reserved.

Johnson J.E.,Scripps Research Institute
Current Opinion in Structural Biology | Year: 2010

Similar modes of virus maturation have been observed in dsDNA bacteriophages and the structurally related herpes viruses and some type of maturation occur in most animal viruses. Recently a variety of biophysical studies of maturation intermediates of bacteriophages P22, λ, and HK97 have suggested an energy landscape that drives the transitions and structure-based mechanisms for its formation. Near-atomic resolution models of subunit tertiary structures in an early intermediate of bacteriophage HK97 maturation revealed a remarkable distortion of the secondary structures when compared to the mature particle. Scaffolding proteins may induce the distortion that is maintained by quaternary structure interactions following scaffold release, making the intermediate particle metastable. © 2010 Elsevier Ltd.

Shang M.,CAS Shanghai Institute of Organic Chemistry | Wang H.-L.,CAS Shanghai Institute of Organic Chemistry | Sun S.-Z.,Shanghai University | Dai H.-X.,CAS Shanghai Institute of Organic Chemistry | And 2 more authors.
Journal of the American Chemical Society | Year: 2014

Cu(II)-promoted ortho alkynylation of arenes and heteroarenes with terminal alkynes has been developed to prepare aryl alkynes. A variety of arenes and terminal alkynes bearing different substituents are compatible with this reaction, thus providing an alternative disconnection to Sonogashira coupling. © 2014 American Chemical Society.

Mousseau G.,Scripps Research Institute
Cell host & microbe | Year: 2012

The human immunodeficiency virus type 1 (HIV) Tat protein, a potent activator of HIV gene expression, is essential for integrated viral genome expression and represents a potential antiviral target. Tat binds the 5'-terminal region of HIV mRNA's stem-bulge-loop structure, the transactivation-responsive (TAR) element, to activate transcription. We find that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid from a marine sponge, inhibits Tat-mediated transactivation of the integrated provirus by binding specifically to the TAR-binding domain of Tat. Working at subnanomolar concentrations, dCA reduces Tat-mediated transcriptional initiation/elongation from the viral promoter to inhibit HIV-1 and HIV-2 replication in acutely and chronically infected cells. Importantly, dCA abrogates spontaneous viral particle release from CD4(+)T cells from virally suppressed subjects on highly active antiretroviral therapy (HAART). Thus, dCA defines a unique class of anti-HIV drugs that may inhibit viral production from stable reservoirs and reduce residual viremia during HAART. Copyright © 2012 Elsevier Inc. All rights reserved.

Roberts T.C.,Scripps Research Institute | Roberts T.C.,University of Oxford
Molecular Therapy - Nucleic Acids | Year: 2014

MicroRNAs (miRNAs) are a class of genome-encoded small RNAs that are primarily considered to be post-transcriptional negative regulators of gene expression acting in the cytoplasm. Over a decade of research has focused on this canonical paradigm of miRNA function, with many success stories. Indeed, miRNAs have been identified that act as master regulators of a myriad of cellular processes, and many miRNAs are promising therapeutic targets or disease biomarkers. However, it is becoming increasingly apparent that the canonical view of miRNA function is incomplete. Several lines of evidence now point to additional functions for miRNAs in the nucleus of the mammalian cell. The majority of cellular miRNAs are present in both the nucleus and the cytoplasm, and certain miRNAs show specific nuclear enrichment. Additionally, some miRNAs colocalize with sub-nuclear structures such as the nucleolus and chromatin. Multiple components of the miRNA processing machinery are present in the nuclear compartment and are shuttled back and forth across the nuclear envelope. In the nucleus, miRNAs act to regulate the stability of nuclear transcripts, induce epigenetic alterations that either silence or activate transcription at specific gene promoters, and modulate cotranscriptional alternative splicing events. Nuclear miRNA-directed gene regulation constitutes a departure from the prevailing view of miRNA function and as such, warrants detailed further investigation. © 2014 The American Society of Gene & Cell Therapy All rights reserved.

Rambo R.P.,Lawrence Berkeley National Laboratory | Tainer J.A.,Lawrence Berkeley National Laboratory | Tainer J.A.,Scripps Research Institute
Current Opinion in Structural Biology | Year: 2010

Small-angle X-ray scattering (SAXS) is changing how we perceive biological structures, because it reveals dynamic macromolecular conformations and assemblies in solution. SAXS information captures thermodynamic ensembles, enhances static structures detailed by high-resolution methods, uncovers commonalities among diverse macromolecules, and helps define biological mechanisms. SAXS-based experiments on RNA riboswitches and ribozymes and on DNA-protein complexes including DNA-PK and p53 discover flexibilities that better define structure-function relationships. Furthermore, SAXS results suggest conformational variation is a general functional feature of macromolecules. Thus, accurate structural analyses will require a comprehensive approach that assesses both flexibility, as seen by SAXS, and detail, as determined by X-ray crystallography and NMR. Here, we review recent SAXS computational tools, technologies, and applications to nucleic acids and related structures.

Shang M.,CAS Shanghai Institute of Organic Chemistry | Sun S.-Z.,CAS Shanghai Institute of Organic Chemistry | Dai H.-X.,CAS Shanghai Institute of Organic Chemistry | Yu J.-Q.,CAS Shanghai Institute of Organic Chemistry | Yu J.-Q.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2014

A Cu(OAc)2-mediated C-H amidation and amination of arenes and heteroarenes has been developed using a readily removable directing group. A wide range of sulfonamides, amides, and anilines function as amine donors in this reaction. Heterocycles present in both reactants are tolerated, making this a broadly applicable method for the synthesis of a family of inhibitors including 2-benzamidobenzoic acids and N-phenylaminobenzoates. © 2014 American Chemical Society.

Nicolaou K.C.,Scripps Research Institute
Angewandte Chemie (International ed. in English) | Year: 2012

Thiostrepton, a powerful antibiotic belonging to the thiopeptide class, was synthesized in the laboratory for the first time in 2004 through an arduous campaign involving novel strategies and tactics, scenic detours, and unexpected roadblocks. In this Review the author narrates the long journey to success, not so dissimilar to Odysseus' return voyage to Ithaca, full of adventure, knowledge, and wisdom. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Blackmond D.G.,Scripps Research Institute
Tetrahedron Asymmetry | Year: 2010

The Kagan ML n models developed for rationalizing non-linear effects of catalyst enantiopurity have become a valuable mechanistic tool for probing complex asymmetric catalytic reactions. This work demonstrates how these models also provide clues about reactivity that may be used for further evidence to test a mechanistic hypothesis. Special considerations for probing non-linear effects in asymmetric synthesis using stoichiometric chiral auxiliaries and in asymmetric autocatalysis are highlighted in comparison with asymmetric catalysis. © 2010 Elsevier Ltd. All rights reserved.

Rudolf J.D.,University of Utah | Rudolf J.D.,Scripps Research Institute | Poulter C.D.,University of Utah
ACS Chemical Biology | Year: 2013

The tyrosine O-prenyltransferase SirD in Leptosphaeria maculans catalyzes normal prenylation of the hydroxyl group in tyrosine as the first committed step in the biosynthesis of the phytotoxin sirodesmin PL. SirD also catalyzes normal N-prenylation of 4-aminophenylalanine and normal C-prenylation at C7 of tryptophan. In this study, we found that 4-mercaptophenylalanine and several derivatives of tryptophan are also substrates for prenylation by dimethylallyl diphosphate. Incubation of SirD with 4-mercaptophenylalanine gave normal S-prenylated mercaptophenylalanine. We found that incubation of the enzyme with tryptophan gave reverse prenylation at N1 in addition to the previously reported normal prenylation at C7. 4-Methyltryptophan also gave normal prenylation at C7 and reverse prenylation at N1, whereas 4-methoxytryptophan gave normal and reverse prenylation at C7, and 7-methyltryptophan gave normal prenylation at C6 and reverse prenylation at N1. The ability of SirD to prenylate at three different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is similar to behavior seen for dimethylallyltryptophan synthase. The multiple products produced by SirD suggests it and dimethylallyltryptophan synthase use a dissociative electrophilic mechanism for alkylation of amino acid substrates. © 2013 American Chemical Society.

Matyskiela M.E.,University of California at Berkeley | Lander G.C.,Lawrence Berkeley National Laboratory | Lander G.C.,Scripps Research Institute | Martin A.,University of California at Berkeley
Nature Structural and Molecular Biology | Year: 2013

The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains and translocates them into the associated peptidase after unfolding, but detailed mechanisms remain unknown. Here we present the 26S proteasome structure from Saccharomyces cerevisiae during substrate degradation, showing that the regulatory particle switches from a preengaged to a translocation-competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase and a spiral orientation of pore loops that suggests a rapid progression of ATP-hydrolysis events around the ring. Notably, Rpn11 moves from an occluded position to directly above the central pore, thus facilitating substrate deubiquitination concomitant with translocation.

Benjamin D.I.,University of California at Berkeley | Cravatt B.F.,Scripps Research Institute | Nomura D.K.,University of California at Berkeley
Cell Metabolism | Year: 2012

Cancer cells possess fundamentally altered metabolism that provides a foundation to support tumorigenicity and malignancy. Our understanding of the biochemical underpinnings of cancer has benefited from the integrated utilization of large-scale profiling platforms (e.g., genomics, proteomics, and metabolomics), which, together, can provide a global assessment of how enzymes and their parent metabolic networks become altered in cancer to fuel tumor growth. This review presents several examples of how these integrated platforms have yielded fundamental insights into dysregulated metabolism in cancer. We will also discuss questions and challenges that must be addressed to more completely describe, and eventually control, the diverse metabolic pathways that support tumorigenesis. © 2012 Elsevier Inc.

Amini-Bavil-Olyaee S.,University of Southern California | Choi Y.J.,University of Southern California | Lee J.H.,University of Southern California | Shi M.,University of Southern California | And 3 more authors.
Cell Host and Microbe | Year: 2013

Vesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) regulate intracellular cholesterol homeostasis, which is required for many virus infections. During entry, viruses or virus-containing vesicles can fuse with endosomal membranes to mediate the cytosolic release of virions, and alterations in endosomal cholesterol can inhibit this invasion step. We show that the antiviral effector protein interferon-inducible transmembrane protein 3 (IFITM3) interacts with VAPA and prevents its association with OSBP, thereby disrupting intracellular cholesterol homeostasis and inhibiting viral entry. By altering VAPA-OSBP function, IFITM3 induces a marked accumulation of cholesterol in multivesicular bodies and late endosomes, which inhibits the fusion of intraluminal virion-containing vesicles with endosomal membranes and thereby blocks virus release into the cytosol. Consequently, ectopic expression or depletion of the VAPA gene profoundly affects IFITM3-mediated inhibition of viral entry. Thus, IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry, further underscoring the importance of cholesterol in virus infection. © 2013 Elsevier Inc.

Ezzet K.A.,Scripps Research Institute | McCauley J.C.,Shiley Center for Orthopaedic Research and Education at Scripps Clinic
Journal of Arthroplasty | Year: 2014

Proper femoral and acetabular component position and leg length equality are important intraoperative considerations during total hip arthroplasty. Unfortunately, traditional surgical techniques often lead to suboptimal component position, and such deviations have been associated with increased rates of prosthetic wear, dislocation, component loosening, and patient dissatisfaction. Although surgical navigation has been shown to improve reproducibility of component alignment, such technology is not universally available and is associated with significant costs and additional surgical/anesthetic time. In the current study, we found that a routine intraoperative pelvic radiograph could successfully identify malpositioned components and leg length inequalities and could allow for successful correction of identified problems. Unexpected component malposition and leg length inequality occurred in only 1.5% of cases where an intraoperative pelvic radiograph was utilized. © 2014 Elsevier Inc.

Pollard K.M.,Scripps Research Institute
Journal of Autoimmunity | Year: 2012

Autoimmunity is thought to result from a combination of genetics, environmental triggers, and stochastic events. Gender is also a significant risk factor with many diseases exhibiting a female bias. Although the role of environmental triggers, especially medications, in eliciting autoimmunity is well established less is known about the interplay between gender, the environment and autoimmunity. This review examines the contribution of gender in autoimmunity induced by selected chemical, physical and biological agents in humans and animal models. Epidemiological studies reveal that environmental factors can be associated with a gender bias in human autoimmunity. However many studies show that the increased risk of autoimmunity is often influenced by occupational exposure or other gender biased activities. Animal studies, although often prejudiced by the exclusive use of female animals, reveal that gender bias can be strain specific suggesting an interaction between sex chromosome complement and background genes. This observation has important implications because it argues that within a gender biased disease there may be individuals in which gender does not contribute to autoimmunity. Exposure to environmental factors, which encompasses everything around us, adds an additional layer of complexity. Understanding how the environment influences the relationship between sex chromosome complement and innate and adaptive immune responses will be essential in determining the role of gender in environmentally-induced autoimmunity. © 2011 Elsevier Ltd.

Gonzalez B.,Scripps Research Institute
Nature protocols | Year: 2010

Engineered zinc-finger transcription factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this protocol, we describe a user-friendly ZF technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The new technology described here simplifies the building of ZF libraries, avoids PCR-introduced bias and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9-11 d.

Guidotti L.G.,San Raffaele Scientific Institute | Isogawa M.,Nagoya City University | Chisari F.V.,Scripps Research Institute
Current Opinion in Immunology | Year: 2015

Hepatitis B virus (HBV) is a noncytopathic, hepatotropic, double-stranded DNA virus that causes acute and chronic hepatitis. Although HBV does not induce a measurable innate immune response in the infected liver, the outcome of infection is determined by the kinetics, breadth, vigor, trafficking, and effector functions of HBV-specific adaptive T cell responses, and the development of neutralizing antibodies. Dysregulation of one or more of these events leads to persistent HBV infection and a variably severe chronic necroinflammatory liver disease that fosters the development of hepatocellular carcinoma. Deeper understanding of the mechanisms responsible for immunological tolerance to HBV is needed in order to devise immunotherapeutic strategies to cure chronic HBV infection and prevent its life-threatening sequelae. © 2015 .

Kelso J.M.,Scripps Research Institute
Current Opinion in Allergy and Clinical Immunology | Year: 2012

PURPOSE OF REVIEW: To review recent publications on the safety of influenza vaccines [both the injectable, inactivated trivalent influenza vaccine (TIV) and the intranasal, live attenuated influenza vaccine (LAIV)] and new recommendations regarding their use. RECENT FINDINGS: Numerous studies have demonstrated that TIV can be safely administered to patients with egg allergy. Influenza vaccines are very unlikely to cause or exacerbate Guillain- Barré syndrome (GBS). TIV cannot cause asthma exacerbations, whereas there may be some slight risk that LAIV could do so. TIV is well tolerated by patients with immunocompromise. Some brands of influenza vaccine are not indicated for certain age groups due to lack of effectiveness or possible side effects. SUMMARY: TIV should be administered to patients with egg allergy with appropriate precautions. Influenza vaccines should be withheld from patients with a history of GBS only if the GBS began within 6 weeks of prior influenza immunization. TIV should be given to patients with asthma, but they should not receive LAIV. TIV should be given to immunocompromised patients, but they should not receive LAIV. Contacts of most immunocompromised patients can receive either TIV or LAIV. Age appropriate brands of influenza vaccine should be used. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Karbstein K.,Scripps Research Institute
Current Opinion in Chemical Biology | Year: 2011

Ribosome assembly involves rRNA transcription, modification, folding and cleavage from precursor transcripts, and association of ribosomal proteins (Rps). In bacteria, this complex process requires only a handful of proteins in addition to those needed for rRNA transcription, modification and cleavage, while in eukaryotes a large machinery comprising ∼200 proteins in the yeast S. cerevisiae has been identified. Furthermore, while the bacterial assembly factors generally produce only cold-sensitive phenotypes upon deletion, most of the eukaryotic assembly factors are essential, comprising ∼20% of essential yeast proteins. This review explores recent rapid progress in the structural and functional dissection of the 40S assembly machinery. © 2011 Elsevier Ltd.

Leonard S.E.,University of Michigan | Carroll K.S.,Scripps Research Institute
Current Opinion in Chemical Biology | Year: 2011

Oxidative cysteine modifications have emerged as a central mechanism for dynamic post-translational regulation of all major protein classes and correlate with many disease states. Elucidating the precise roles of cysteine oxidation in physiology and pathology presents a major challenge. This article reviews the current, targeted proteomic strategies that are available to detect and quantify cysteine oxidation. A number of indirect methods have been developed to monitor changes in the redox state of cysteines, with the majority relying on the loss of reactivity with thiol-modifying reagents or restoration of labeling by reducing agents. Recent advances in chemical biology allow for the direct detection of specific cysteine oxoforms based on their distinct chemical attributes. In addition, new chemical reporters of cysteine oxidation have enabled in situ detection of labile modifications and improved proteomic analysis of redox-regulated proteins. Progress in the field of redox proteomics should advance our knowledge of regulatory mechanisms that involve oxidation of cysteine residues and lead to a better understanding of oxidative biochemistry in health and disease. © 2010 Elsevier Ltd.

Ronald P.C.,University of California at Davis | Ronald P.C.,Joint BioEnergy Institute | Ronald P.C.,Kyung Hee University | Beutler B.,Scripps Research Institute
Science | Year: 2010

The last common ancestor of plants and animals may have lived 1 billion years ago. Plants and animals have occasionally exchanged genes but, for the most part, have countered selective pressures independently. Microbes (bacteria, eukaryotes, and viruses) were omnipresent threats, influencing the direction of multicellular evolution. Receptors that detect molecular signatures of infectious organisms mediate awareness of nonself and are integral to host defense in plants and animals alike. The discoveries leading to elucidation of these receptors and their ligands followed a similar logical and methodological pathway in both plant and animal research.

Rangaraju S.,Scripps Research Institute
Molecular Psychiatry | Year: 2016

Antidepressants have been shown to improve longevity in C. elegans. It is plausible that orthologs of genes involved in mood regulation and stress response are involved in such an effect. We sought to understand the underlying biology. First, we analyzed the transcriptome from worms treated with the antidepressant mianserin, previously identified in a large-scale unbiased drug screen as promoting increased lifespan in worms. We identified the most robust treatment-related changes in gene expression, and identified the corresponding human orthologs. Our analysis uncovered a series of genes and biological pathways that may be at the interface between antidepressant effects and longevity, notably pathways involved in drug metabolism/degradation (nicotine and melatonin). Second, we examined which of these genes overlap with genes which may be involved in depressive symptoms in an aging non-psychiatric human population (n=3577), discovered using a genome-wide association study (GWAS) approach in a design with extremes of distribution of phenotype. Third, we used a convergent functional genomics (CFG) approach to prioritize these genes for relevance to mood disorders and stress. The top gene identified was ANK3. To validate our findings, we conducted genetic and gene-expression studies, in C. elegans and in humans. We studied C. elegans inactivating mutants for ANK3/unc-44, and show that they survive longer than wild-type, particularly in older worms, independently of mianserin treatment. We also show that some ANK3/unc-44 expression is necessary for the effects of mianserin on prolonging lifespan and survival in the face of oxidative stress, particularly in younger worms. Wild-type ANK3/unc-44 increases in expression with age in C. elegans, and is maintained at lower youthful levels by mianserin treatment. These lower levels may be optimal in terms of longevity, offering a favorable balance between sufficient oxidative stress resistance in younger worms and survival effects in older worms. Thus, ANK3/unc-44 may represent an example of antagonistic pleiotropy, in which low-expression level in young animals are beneficial, but the age-associated increase becomes detrimental. Inactivating mutations in ANK3/unc-44 reverse this effect and cause detrimental effects in young animals (sensitivity to oxidative stress) and beneficial effect in old animals (increased survival). In humans, we studied if the most significant single nucleotide polymorphism (SNP) for depressive symptoms in ANK3 from our GWAS has a relationship to lifespan, and show a trend towards longer lifespan in individuals with the risk allele for depressive symptoms in men (odds ratio (OR) 1.41, P=0.031) but not in women (OR 1.08, P=0.33). We also examined whether ANK3, by itself or in a panel with other top CFG-prioritized genes, acts as a blood gene-expression biomarker for biological age, in two independent cohorts, one of live psychiatric patients (n=737), and one of suicide completers from the coroner’s office (n=45). We show significantly lower levels of ANK3 expression in chronologically younger individuals than in middle age individuals, with a diminution of that effect in suicide completers, who presumably have been exposed to more severe and acute negative mood and stress. Of note, ANK3 was previously reported to be overexpressed in fibroblasts from patients with Hutchinson–Gilford progeria syndrome, a form of accelerated aging. Taken together, these studies uncover ANK3 and other genes in our dataset as biological links between mood, stress and longevity/aging, that may be biomarkers as well as targets for preventive or therapeutic interventions. Drug repurposing bioinformatics analyses identified the relatively innocuous omega-3 fatty acid DHA (docosahexaenoic acid), piracetam, quercetin, vitamin D and resveratrol as potential longevity promoting compounds, along with a series of existing drugs, such as estrogen-like compounds, antidiabetics and sirolimus/rapamycin. Intriguingly, some of our top candidate genes for mood and stress-modulated longevity were changed in expression in opposite direction in previous studies in the Alzheimer disease. Additionally, a whole series of others were changed in expression in opposite direction in our previous studies on suicide, suggesting the possibility of a “life switch” actively controlled by mood and stress.Molecular Psychiatry advance online publication, 24 May 2016; doi:10.1038/mp.2016.65. © 2016 Macmillan Publishers Limited

Saphire E.O.,Scripps Research Institute
Cell Host and Microbe | Year: 2015

The unprecedented scale of the recent Ebola virus outbreak caused many to wonder whether this virus is different, and raised concerns about how to contain the outbreak. Two recent studies published in Science (Hoenen et al., 2015; Marzi et al., 2015) shed light on the subject and offer a new solution. © 2015 Elsevier Inc.

Griffin J.H.,Scripps Research Institute | Zlokovic B.V.,University of California at San Diego | Mosnier L.O.,University of Southern California
Blood | Year: 2015

The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a cytoprotective on the basis of endothelial barrier stabilization and anti-inflammatory and antiapoptotic actions; and (3) a regenerative on the basis of stimulation of neurogenesis, angiogenesis, and wound healing. Pharmacologic therapies using recombinant human and murine APCs indicate that APC provides effective acute or chronic therapies for a strikingly diverse range of preclinical injury models. APC reduces the damage caused by the following: ischemia/reperfusion in brain, heart, and kidney; pulmonary, kidney, and gastrointestinal inflammation; sepsis; Ebola virus; diabetes; and total lethal body radiation. For these beneficial effects, APC alters cell signaling networks and gene expression profiles by activating protease-activated receptors 1 and 3. APC's activation of these G protein-coupled receptors differs completely from thrombin's activation mechanism due to biased signaling via either G proteins or β-arrestin-2. To reduce APC-associated bleeding risk, APC variants were engineered to lack >90% anticoagulant activity but retain normal cell signaling. Such a neuroprotective variant, 3K3A-APC (Lys191-193Ala), has advanced to clinical trials for ischemic stroke. A rich data set of preclinical knowledge provides a solid foundation for potential translation of APC variants to future novel therapies. © 2015 by The American Society of Hematology.

Soliven B.,University of Chicago | Miron V.,Queens Medical Research Institute | Chun J.,Scripps Research Institute
Neurology | Year: 2011

Sphingosine 1-phosphate receptors (S1PRs) are G protein-coupled receptors expressed by many cell types, including immune and neural cells. These receptors are promising targets for immunomodulatory and possibly neuromodulatory therapies. Fingolimod (FTY720) is a sphingosine analog that, when phosphorylated, becomes a prototypical S1PR modulator. It has recently been approved as the first oral treatment for relapsing forms of multiple sclerosis in some countries. Fingolimod has documented effects on lymphocyte egress, selectively retaining lymphocytes within the lymph nodes. In addition, fingolimod can enter the CNS and can act on S1PRs expressed by most neural lineages. In this article, we discuss recent results supporting the concept that S1PR modulators may exert neuroprotective and regenerative actions in the CNS as well as having anti-inflammatory effects. ©2011 American Academy of Neurology.

Kenny P.J.,Scripps Research Institute
Pharmacology Biochemistry and Behavior | Year: 2011

Tobacco use is a major cause of disease and premature death in the United States. Nicotine is considered the key component of tobacco responsible for addiction in human smokers. Accumulating evidence supports an important role for the hypocretin (orexin) neuropeptide system in regulating the reinforcing properties of most major drugs of abuse, including nicotine. Here, data showing that nicotine activates hypocretin-producing neurons in the lateral hypothalamus, and that disruption of hypocretin transmission decreases nicotine self-administration behavior in rats will be reviewed. Recent findings suggesting that plasma hypocretin levels may be related to the magnitude of cigarette craving in abstinent smokers will be discussed. Finally, the data suggesting that hypocretin transmission in the insular cortex may play an important role in regulating nicotine self-administration behavior in rats will be reviewed. This latter finding may provide mechanistic insight into the apparent disruption of tobacco addiction reported in human smokers with stroke-associated damage to the insular cortex. © 2010 Elsevier Inc.

Yates J.R.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2013

Mass spectrometry has evolved at an exponential rate over the last 100 years. Innovations in the development of mass spectrometers have created powerful instruments capable of analyzing a wide range of targets, from rare atoms and molecules to very large molecules, such as a proteins, protein complexes, and DNA. These performance gains have been driven by sustaining innovations, punctuated by the occasional disruptive innovation. The use of mass spectrometry for proteome analysis was driven by disruptive innovations that created a capability for large-scale analysis of proteins and modifications. © 2013 American Chemical Society.

Truong T.H.,University of Michigan | Carroll K.S.,Scripps Research Institute
Biochemistry | Year: 2012

Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes, including growth, proliferation, and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a number of human malignancies, making this receptor an important target for the development of anticancer drugs. In addition to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H 2O2 by NADPH-dependent oxidases. In turn, H 2O2 functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for the development of new therapeutic strategies for targeting this and other H2O 2-modulated pathways. © 2012 American Chemical Society.

Mayford M.,Scripps Research Institute
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2014

Understanding the molecular and cellular changes that underlie memory, the engram, requires the identification, isolation and manipulation of the neurons involved. This presents a major difficulty for complex forms of memory, for example hippocampus-dependent declarative memory, where the participating neurons are likely to be sparse, anatomically distributed and unique to each individual brain and learning event. In this paper, I discuss several new approaches to this problem. In vivo calcium imaging techniques provide a means of assessing the activity patterns of large numbers of neurons over long periods of time with precise anatomical identification. This provides important insight into how the brain represents complex information and how this is altered with learning. The development of techniques for the genetic modification of neural ensembles based on their natural, sensory-evoked, activity along with optogenetics allows direct tests of the coding function of these ensembles. These approaches provide a new methodological framework in which to examine the mechanisms of complex forms of learning at the level of the neurons involved in a specific memory. © 2013 The Authors.

Eisele Y.S.,Scripps Research Institute | Duyckaerts C.,Laboratoire Of Neuropathologie Raymond Escourolle
Acta Neuropathologica | Year: 2016

In brains of patients with Alzheimer’s disease (AD), Aβ peptides accumulate in parenchyma and, almost invariably, also in the vascular walls. Although Aβ aggregation is, by definition, present in AD, its impact is only incompletely understood. It occurs in a stereotypical spatiotemporal distribution within neuronal networks in the course of the disease. This suggests a role for synaptic connections in propagating Aβ pathology, and possibly of axonal transport in an antero- or retrograde way—although, there is also evidence for passive, extracellular diffusion. Striking, in AD, is the conjunction of tau and Aβ pathology. Tau pathology in the cell body of neurons precedes Aβ deposition in their synaptic endings in several circuits such as the entorhino-dentate, cortico-striatal or subiculo-mammillary connections. However, genetic evidence suggests that Aβ accumulation is the first step in AD pathogenesis. To model the complexity and consequences of Aβ aggregation in vivo, various transgenic (tg) rodents have been generated. In rodents tg for the human Aβ precursor protein, focal injections of preformed Aβ aggregates can induce Aβ deposits in the vicinity of the injection site, and over time in more distant regions of the brain. This suggests that Aβ shares with α-synuclein, tau and other proteins the property to misfold and aggregate homotypic molecules. We propose to group those proteins under the term “propagons”. Propagons may lack the infectivity of prions. We review findings from neuropathological examinations of human brains in different stages of AD and from studies in rodent models of Aβ aggregation and discuss putative mechanisms underlying the initiation and spread of Aβ pathology. © 2015, Springer-Verlag Berlin Heidelberg.

Oldstone M.B.A.,Scripps Research Institute
Annual Review of Pathology: Mechanisms of Disease | Year: 2016

This odyssey is mine from early junior high school, where my dreams for adventure were shaped by Arthur Conan Doyle's Sherlock Holmes, Percival Christopher Wren's Beau Geste, and best of all the remarkable explorers in Paul de Kruif's Microbe Hunters. My birth site was in Manhattan (my mother was a Vogue model and my father worked in retail), and I traveled to college at the University of Alabama, Tuscaloosa, where my love of history and English literature was shaped along with a sufficient exposure to biology, chemistry, and genetics to meet requirements for entering medical school. By the second year at the University of Maryland School of Medicine, through expert teachers such as Theodore (Ted) Woodward and Sheldon (Shelly) Greisman in medicine and Charles Weissmann in virology and microbiology, I found that understanding why and how people became ill was more my cup of tea than identifying and treating their illnesses. Although I was becoming competent in diagnosis and treatment, I left medical school at the end of my sophomore year to seek a more basic understanding of biology and chemistry. I achieved this by working toward a PhD in biochemistry at Johns Hopkins McCollum-Pratt Institute combined with study of rickettsial toxin at Maryland. This was a very important time in my life, because it convinced me that addressing biologic and medical questions in a disciplined scientific manner was what my life voyage should be. That voyage led me initially, through Woodward's contact, to work a summer in Joe Smadel's unit at Walter Reed (Smadel being one of the deans of American virology) and to meet several times with Carleton Gajdusek and then John Enders at Harvard, who pointed me to Frank Dixon at Scripps in La Jolla, California, for postdoctoral training. Dixon was among the founders of modern immunology and a pathfinder for immunopathology. Training by and association with Dixon and his other postdoctoral fellows, my independent position at Scripps, early polishing by Karl Habel (a superb senior virologist who left the National Institutes of Health and came to Scripps), and the gifted postdoctoral fellows who joined my laboratory over four decades form the log of my scientific voyage. The strong friendships and collaborations developed with other young but growing experimentalists like Bernie Fields and Abner Notkins are the fabric of the tale I will weave and were pivotal in the establishment of viral pathogenesis as a discipline. © 2016 by Annual Reviews. All rights reserved.

Catz S.D.,Scripps Research Institute
Journal of Leukocyte Biology | Year: 2013

The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. © Society for Leukocyte Biology.

Muller U.,Scripps Research Institute | Barr-Gillespie P.G.,Oregon Health And Science University
Nature Reviews Drug Discovery | Year: 2015

Hearing loss is the most common form of sensory impairment in humans and affects more than 40 million people in the United States alone. No drug-based therapy has been approved by the Food and Drug Administration, and treatment mostly relies on devices such as hearing aids and cochlear implants. Over recent years, more than 100 genetic loci have been linked to hearing loss and many of the affected genes have been identified. This understanding of the genetic pathways that regulate auditory function has revealed new targets for pharmacological treatment of the disease. Moreover, approaches that are based on stem cells and gene therapy, which may have the potential to restore or maintain auditory function, are beginning to emerge. © 2015 Macmillan Publishers Limited.

Vogt P.K.,Scripps Research Institute
Nature Reviews Cancer | Year: 2012

Retroviruses are the original source of oncogenes. The discovery and characterization of these genes was made possible by the introduction of quantitative cell biological and molecular techniques for the study of tumour viruses. Key features of all retroviral oncogenes were first identified in src, the oncogene of Rous sarcoma virus. These include non-involvement in viral replication, coding for a single protein and cellular origin. The MYC, RAS and ERBB oncogenes quickly followed SRC, and these together with PI3K are now recognized as crucial driving forces in human cancer. © 2012 Macmillan Publishers Limited. All rights reserved.

Background: Until recently, the presence of a permanent pacemaker or an implantable cardioverter-defibrillator has been a relative contraindication for the performance of magnetic resonance imaging (MRI). A number of small studies have shown that MRI can be performed with minimal risk when patients are properly monitored and device programming is modified appropriately for the procedure. However, the risk of performing MRI for patients with implanted cardiac devices has not been sufficiently evaluated to advocate routine clinical use. The aim of the present protocol is to prospectively determine the rate of adverse clinical events and device parameter changes in patients with implanted non-MRI-conditional cardiac devices undergoing clinically indicated nonthoracic MRI at 1.5 T. Methods: The MagnaSafe Registry is a multicenter, prospective cohort study of up to 1500 MRI examinations in patients with pacemakers or implantable cardioverter-defibrillators implanted after 2001 who undergo clinically indicated nonthoracic MRI following a specific protocol to ensure that preventable potential adverse events are mitigated. Adverse events and changes in device parameter measurements that may be associated with the imaging procedure will be documented. Results: Through August 2012, 701 MRI studies have been performed, representing 47% of the total target enrollment. Conclusions: The results of this registry will provide additional documentation of the risk of MRI and will further validate a clinical protocol for screening and the performance of clinically indicated MRI for patients with implanted cardiac devices. © 2013 Mosby, Inc. All rights reserved.

Pockros P.J.,Scripps Research Institute
Expert Opinion on Biological Therapy | Year: 2011

The approval of the first direct-acting antiviral (DAA) drugs for treatment of HCV in 2011 has lead to improved sustained viral response rates of up to 79% in treatment-nave or relapse genotype 1 patients. Areas covered: Clinical trial data, the clinical skills required for the use of DAA drugs, the use of genetic tests and HCV RNA assays, new small molecules, resistance-associated variants, combinations of two or more DAAs, treatment of special populations, and future directions. The results of the pivotal Phase III trials with telaprevir and boceprevir, including the efficacy, safety and tolerability, drugdrug interactions and management of the most common side-effects. Resistance-associated variant data and treatment strategies implemented to minimize the development of resistance with these first-generation protease inhibitors. Expert opinion: Combination therapies of protease inhibitors with nucleoside or non-nucleoside polymerase inhibitors, non-structural protein 5A (NS5A) inhibitors and cyclophylin inhibitors are currently underway in regimens that use pegylated interferon and ribavirin or are interferon-free. The explosion of new drug development will probably move the field forward and offer both improved efficacy and tolerability to patients with hepatitis C infections. The use of these drugs ushers in a new era for the treatment of HCV but must be done with care and caution. © 2011 Informa UK, Ltd.

Carvalho P.C.,Carlos Chagas Institute | Yates J.R.,Scripps Research Institute | Barbosa V.C.,Federal University of Rio de Janeiro
Bioinformatics | Year: 2012

We present an updated version of the TFold software for pinpointing differentially expressed proteins in shotgun proteomics experiments. Given an FDR bound, the updated approach uses a theoretical FDR estimator to maximize the number of identifications that satisfy both a fold-change cutoff that varies with the t-test P-value as a power law and a stringency criterion that aims to detect lowly abundant proteins. The new version has yielded significant improvements in sensitivity over the previous one. © The Author 2012. Published by Oxford University Press. All rights reserved.

Cohen J.A.,Cleveland Clinic | Chun J.,Scripps Research Institute
Annals of Neurology | Year: 2011

Until recently, all approved multiple sclerosis (MS) disease treatments were administered parenterally. Oral fingolimod was approved in September 2010 by the US Food and Drug Administration to reduce relapses and disability progression in relapsing forms of MS. In the clinical trials that led to approval, fingolimod reduced not only acute relapses and magnetic resonance imaging lesion activity but also disability progression and brain volume loss, suggesting preservation of tissue. Fingolimod's mechanism of action in MS is not known with certainty. Its active form, fingolimod-phosphate (fingolimod-P), is a sphingosine 1-phosphate receptor (S1PR) modulator that inhibits egress of lymphocytes from lymph nodes and their recirculation, potentially reducing trafficking of pathogenic cells into the central nervous system (CNS). Fingolimod also readily penetrates the CNS, and fingolimod-P formed in situ may have direct effects on neural cells. Fingolimod potently inhibits the MS animal model, experimental autoimmune encephalomyelitis, but is ineffective in mice with selective deficiency of the S1P 1 S1PR subtype on astrocytes despite normal expression in the immune compartment. These findings suggest that S1PR modulation by fingolimod in both the immune system and CNS, producing a combination of beneficial anti-inflammatory and possibly neuroprotective/ reparative effects, may contribute to its efficacy in MS. In clinical trials, fingolimod was generally safe and well tolerated. Its interaction with S1PRs in a variety of tissues largely accounts for the reported adverse effects, which were seen more frequently with doses 2.5 to 10× the approved 0.5mg dose. Fingolimod's unique mechanism of action distinguishes it from all other currently approved MS therapies. © 2011 American Neurological Association.

Puthanveettil S.V.,Scripps Research Institute
RNA Biology | Year: 2013

Several studies have shown that synthesis of new proteins at the synapse is a prerequisite for the storage of long-term memories. Relatively little is known about the availability of distinct mRNA populations for translation at specific synapses, the process that determines mRNA localization, and the temporal designations of localized mRNA translation during memory storage. Techniques such as synaptosome preparation and microdissection of distal neuronal processes of cultured neurons and dendritic layers in brain slices are general approaches used to identify localized RNAs. Exploration of the association of RNA-binding proteins to the axonal transport machinery has led to the development of a strategy to identify RNAs that are transported from the cell body to synapses by molecular motor kinesin. In this article, RNA localization at the synapse, as well as its mechanisms and significance in understanding longterm memory storage, are discussed. © 2013 Landes Bioscience.

Koob G.F.,Scripps Research Institute
Handbook of Clinical Neurology | Year: 2014

Alcoholism, more generically drug addiction, can be defined as a chronically relapsing disorder characterized by: (1) compulsion to seek and take the drug (alcohol); (2) loss of control in limiting (alcohol) intake; and (3) emergence of a negative emotional state (e.g., dysphoria, anxiety, irritability), reflecting a motivational withdrawal syndrome, when access to the drug (alcohol) is prevented (defined here as dependence). The compulsive drug seeking associated with alcoholism can be derived from multiple neuroadaptations, but the thesis argued here, derived largely from animal models, is that a key component involves decreased brain reward function, increased brain stress function, and compromised executive function, all of which contribute to the construct of negative reinforcement. Negative reinforcement is defined as drug taking that alleviates a negative emotional state. The negative emotional state that drives such negative reinforcement is hypothesized to derive from decreases in reward neurotransmission in the ventral striatum, such as decreased dopamine and opioid peptide function in the nucleus accumbens (ventral striatum), but also recruitment of brain stress systems, such as corticotropin-releasing factor (CRF), in the extended amygdala. Data from animal models that support this thesis show that acute withdrawal from chronic alcohol, sufficient to produce dependence, increases reward thresholds, increases anxiety-like responses, decreases dopamine system function, and increases extracellular levels of CRF in the central nucleus of the amygdala. CRF receptor antagonists also block excessive drug intake produced by dependence. Alcoholism also involves substantial neuroadaptations that persist beyond acute withdrawal and trigger relapse and deficits in cognitive function that can also fuel compulsive drinking. A brain stress response system is hypothesized to be activated by acute excessive drug intake, to be sensitized during repeated withdrawal, to persist into protracted abstinence, and to contribute to the compulsivity of alcoholism. Other components of brain stress systems in the extended amygdala that interact with CRF and may contribute to the negative motivational state of withdrawal include increases in norepinephrine function, increases in dynorphin activity, and decreases in neuropeptide Y. The combination of impairment of function in reward circuitry and recruitment of brain stress system circuitry provides a powerful neurochemical basis for the negative emotional states that are responsible for the negative reinforcement that drives the compulsivity of alcoholism. © 2014 Elsevier B.V.

Pockros P.J.,Scripps Research Institute
Therapeutic Advances in Gastroenterology | Year: 2010

A large number of new therapies are in development for chronic hepatitis C including direct-acting antiviral drugs (DAA), which target specific hepatitis C virus enzymes. Two of these compounds have already advanced into phase 3 development in the USA and EU, and many more are in phase 2 trials and likely to advance. In this review, the results of recent studies on ribavirin analogues, nonstructural (NS) 3/4 serine protease inhibitors, NS5B polymerase inhibitors, cyclophilin inhibitors, silimarin components, and thiazolides have been updated. Each compound includes a brief summary of its proposed mechanism of action, results of early clinical trials, and more advanced trial data where available. These compounds are likely to be the first approved in the USA and EU and will initially be used in combination with the current standard of care. It is possible that future treatment paradigms with these agents will offer the potential of interferon-free regimens. It is most likely that patients for these new therapies will be selected carefully by identifying and treating first those who have excellent sustained virologic response rates with 24 weeks of pegylated interferon and ribavirin, the current standard of care. It is also likely that there will be a need to identify those patients who are not likely to have a sustained virologic response with the addition of a protease inhibitor to the current standard of care and delaying their therapy until combination viral suppression therapy becomes an option. The cost and side effects of the DAA will be important considerations for treating physicians. This review is current through 2009; however, data are rapidly changing. © The Author(s), 2010.

Multiple sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved by the FDA as the first oral treatment for relapsing forms of MS. Fingolimod is a novel compound produced by chemical modification of a fungal precursor. Its active metabolite, formed by in vivo phosphorylation, modulates sphingosine 1-phosphate (S1P) receptors that are a subset of a larger family of cell-surface, G protein-coupled receptors (GPCRs) mediating the effects of bioactive lipids known as lysophospholipids. Fingolimod's mechanism of action in MS is not completely understood; however, its relevant biology indicates a fundamentally different mechanism compared to all previously approved MS therapies, with evolving research supporting both immunological and nervous system activities. This duality may herald a paradigm shift in the treatment of MS and other neurological disorders.

Members of the nuclear receptor superfamily differentiate in terms of specificity for DNA recognition and binding, oligomeric state, and ligand binding. The wide range of specificities are impressive given the high degree of sequence conservation in the DNA binding domain (DBD) and moderate sequence conservation with high structural similarity within the ligand binding domains (LBDs). Determining sequence positions that are conserved within nuclear receptor subfamilies can provide important indicators into the structural dynamics that translate to oligomeric state of the active receptor, DNA binding specificity and ligand affinity and selectivity. Here we present a method to analyze sequence data from all nuclear receptors that facilitates detection of co-evolving pairs using Mutual Information (MI). Using this method we demonstrate that MI can reveal functionally important sequence positions within the superfamily and the approach identified three sequence positions that have conserved sequence patterns across all nuclear receptors and subfamilies. Interestingly, two of the sequence positions identified are located within the DBD CII and the third was within Helix c of the DBD. These sequences are located within the heterodimer interface of PPARγ (CII) and RXRα (Helix c) based on PDB:3DZU. Helix c of PPARγ, which is not involved in the DBD dimer interface, binds the minor groove in the 5' flanking region in a consensus PPARγ response element (PPRE) and the corresponding RXRα (CII) is found in the 3' flanking region of RXRE (3DZU). As these three sequence positions represent unique identifiers for all nuclear receptors and they are located within the dimer interface of PPARγ-RXRα DBD (3DZU) interfacing with the flanking regions of the NRRE, we conclude they are critical sequence positions perhaps dictating nuclear receptor (NR) DNA binding specificity.

Robertson M.P.,Scripps Research Institute
Cold Spring Harbor perspectives in biology | Year: 2012

The general notion of an "RNA World" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool, and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this "RNA-first" view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA.

Derbyshire E.R.,Harvard University | Marletta M.A.,Scripps Research Institute
Annual Review of Biochemistry | Year: 2012

Nitric oxide (NO) is an essential signaling molecule in biological systems. In mammals, the diatomic gas is critical to the cyclic guanosine monophosphate (cGMP) pathway as it functions as the primary activator of soluble guanylate cyclase (sGC). NO is synthesized from l-arginine and oxygen (O2) by the enzyme nitric oxide synthase (NOS). Once produced, NO rapidly diffuses across cell membranes and binds to the heme cofactor of sGC. sGC forms a stable complex with NO and carbon monoxide (CO), but not with O2. The binding of NO to sGC leads to significant increases in cGMP levels. The second messenger then directly modulates phosphodiesterases (PDEs), ion-gated channels, or cGMP-dependent protein kinases to regulate physiological functions, including vasodilation, platelet aggregation, and neurotransmission. Many studies are focused on elucidating the molecular mechanism of sGC activation and deactivation with a goal of therapeutic intervention in diseases involving the NOcGMP-signaling pathway. This review summarizes the current understanding of sGC structure and regulation as well as recent developments in NO signaling. © 2012 by Annual Reviews. All rights reserved.

Olia A.S.,Purdue University | Prevelige Jr P.E.,University of Alabama at Birmingham | Johnson J.E.,Scripps Research Institute | Cingolani G.,Thomas Jefferson University
Nature Structural and Molecular Biology | Year: 2011

DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25-Å-resolution structure of the portal-protein core bound to 12 copies of gene product 4 (gp4) reveals a ~1.1-MDa assembly formed by 24 proteins. Unexpectedly, a lower-resolution structure of the full-length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200-Å-long α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell.

Rao S.V.,The Duke Clinical Research Institute | Cohen M.G.,University of Miami | Kandzari D.E.,Scripps Research Institute | Bertrand O.F.,Quebec Heart Lung Institute | Gilchrist I.C.,Penn State Hershey Medical Center
Journal of the American College of Cardiology | Year: 2010

Periprocedural bleeding complications after percutaneous coronary intervention (PCI) are associated with increased short- and long-term morbidity and mortality. Although clinical trials have primarily assessed pharmacological strategies for reducing bleeding risk, there is a mounting body of evidence suggesting that adoption of a transradial rather than a transfemoral approach to PCI may permit greater reductions in bleeding risk than have been achieved with pharmacological strategies alone. However, despite a long history of use, a lack of widespread uptake by physicians coupled with the technological limitations of available devices has in the past confined transradial PCI to the status of a niche procedure, and many operators lack experience in this technique. In this review, we examine the history of the transradial approach to PCI and discuss some of the circumstances that have hitherto limited its appeal. We then review the current state of the peer-reviewed literature supporting its use and summarize the unresolved issues affecting broader application of this technique, including lack of operator familiarity and an insufficient evidence base for guiding practice. Finally, we describe potential directions for future investigation in the transradial realm. © 2010 American College of Cardiology Foundation.

Bansal V.,Scripps Research Institute
Bioinformatics | Year: 2010

Motivation: Next-generation sequencing technologies have enabled the sequencing of several human genomes in their entirety. However, the routine resequencing of complete genomes remains infeasible. The massive capacity of next-generation sequencers can be harnessed for sequencing specific genomic regions in hundreds to thousands of individuals. Sequencing-based association studies are currently limited by the low level of multiplexing offered by sequencing platforms. Pooled sequencing represents a costeffective approach for studying rare variants in large populations. To utilize the power of DNA pooling, it is important to accurately identify sequence variants from pooled sequencing data. Detection of rare variants from pooled sequencing represents a different challenge than detection of variants from individual sequencing. Results: We describe a novel statistical approach, CRISP [Comprehensive Read analysis for Identification of Single Nucleotide Polymorphisms (SNPs) from Pooled sequencing] that is able to identify both rare and common variants by using two approaches: (i) comparing the distribution of allele counts across multiple pools using contingency tables and (ii) evaluating the probability of observing multiple non-reference base calls due to sequencing errors alone. Information about the distribution of reads between the forward and reverse strands and the size of the pools is also incorporated within this framework to filter out false variants. Validation of CRISP on two separate pooled sequencing datasets generated using the Illumina Genome Analyzer demonstrates that it can detect 80-85% of SNPs identified using individual sequencing while achieving a low false discovery rate (3-5%). Comparison with previous methods for pooled SNP detection demonstrates the significantly lower false positive and false negative rates for CRISP. Availability: Implementation of this method is available at http://polymorphism.scripps.edu/~vbansal/software/CRISP/. Contact: vbansal@scripps.edu. © The Author(s) 2010. Published by Oxford University Press.

Avila J.L.,Wistar Institute | Kissil J.L.,Scripps Research Institute
Trends in Molecular Medicine | Year: 2013

The Notch signaling pathways are known to play critical roles during pancreatic development, but it remains unclear what functions are important in the adult organ. One area of debate is the role of Notch signaling in the development of pancreatic ductal adenocarcinoma (PDAC) and proposed precursor lesions, pancreatic intraepithelial neoplasia (PanIN). Initial studies revealed that Notch signaling is reactivated during PDAC initiation and development, suggesting that Notch promotes PDAC and may therefore represent a target for drug development. However, more recent work reveals a tumor suppressive role for Notch receptors in the context of PanIN development. Here, we summarize the current literature describing Notch signaling in the development of PDAC, and discuss the potential of the Notch pathway as a therapeutic target. © 2013 Elsevier Ltd.

Haase S.B.,Duke University | Wittenberg C.,Scripps Research Institute
Genetics | Year: 2014

Nearly 20% of the budding yeast genome is transcribed periodically during the cell division cycle. The precise temporal execution of this large transcriptional program is controlled by a large interacting network of transcriptional regulators, kinases, and ubiquitin ligases. Historically, this network has been viewed as a collection of four coregulated gene clusters that are associated with each phase of the cell cycle. Although the broad outlines of these gene clusters were described nearly 20 years ago, new technologies have enabled major advances in our understanding of the genes comprising those clusters, their regulation, and the complex regulatory interplay between clusters. More recently, advances are being made in understanding the roles of chromatin in the control of the transcriptional program. We are also beginning to discover important regulatory interactions between the cell-cycle transcriptional program and other cell-cycle regulatory mechanisms such as checkpoints and metabolic networks. Here we review recent advances and contemporary models of the transcriptional network and consider these models in the context of eukaryotic cellcycle controls. © 2014 by the Genetics Society of America.

Koob G.F.,Scripps Research Institute
Brain Research | Year: 2010

Drug addiction is a chronically relapsing disorder characterized by a compulsion to seek and take drugs, the development of dependence, and the manifestation of a negative emotional state when the drug is removed. Activation of brain stress systems is hypothesized to be a key element of the negative emotional state produced by dependence that drives drug-seeking through negative reinforcement mechanisms, defined as the "dark side" of addiction. The focus of the present review is on the role of corticotropin-releasing factor (CRF) and CRF-related peptides in the dark side of addiction. CRF is a key mediator of the hormonal, autonomic, and behavior responses to stressors. Emphasis is placed on the role of CRF in extrahypothalamic systems in the extended amygdala, including the central nucleus of the amygdala, bed nucleus of the stria terminalis, and a transition area in the shell of the nucleus accumbens, in the dark side of addiction. The urocortin/CRF2 systems have been less explored, but results suggest their role in the neuroadaptation associated with chronic drug use, sometimes in opposition to the effects produced by the CRF1 receptor. Compelling evidence argues that the CRF stress system, including its activation of the hypothalamic-pituitary-adrenal axis, plays a key role in engaging the transition to dependence and maintaining dependence once it is initiated. Understanding the role of the CRF systems in addiction not only provides insight into the neurobiology of the dark side of addiction, but also provides novel targets for identifying vulnerability to addiction and the treatment of addiction. © 2009 Elsevier B.V. All rights reserved.

Johnsson P.,Karolinska Institutet | Morris K.V.,University of New South Wales | Morris K.V.,Scripps Research Institute
Cell Research | Year: 2014

New findings bring to light a previously unappreciated mechanism involved in the regulation of the oncoprotein MYC. Interesting observations find that the long noncoding RNA (lncRNA) PVT1 is active in controlling levels of MYC through regulation of MYC protein stability. © 2014 IBCB, SIBS, CAS.

Fonslow B.R.,Scripps Research Institute
WormBook : the online review of C. elegans biology | Year: 2014

Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.

Karbstein K.,Scripps Research Institute
Trends in Cell Biology | Year: 2013

Protein synthesis on ribosomes is carefully quality-controlled to ensure the faithful transmission of genetic information from mRNA to protein. Many of these mechanisms rely on communication between distant sites on the ribosomes, and thus on the integrity of the ribosome structure. Furthermore, haploinsufficiency of ribosomal proteins, which increases the chances of forming incompletely assembled ribosomes, can predispose to cancer. Finally, release of inactive ribosomes into the translating pool will lead to their degradation together with the degradation of the bound mRNA. Together, these findings suggest that quality control mechanisms must be in place to survey nascent ribosomes and ensure their functionality. This review gives an account of these mechanisms as currently known. © 2013 Elsevier Ltd.

Gruol D.L.,Scripps Research Institute
Cerebellum | Year: 2013

Recent studies have established the existence of an innate immune system in the central nervous system (CNS) and implicated a critical role for this system in both normal and pathological processes. Astrocytes and microglia, normal components of the CNS, are the primary cell types that comprise the innate immune system of the CNS. Basic to their role during normal and adverse conditions is the production of neuroimmune factors such as cytokines and chemokines, which are signaling molecules that initiate or coordinate downstream cellular actions. During adverse conditions, cytokines and chemokines function in defensive and repair. However, if expression of these factors becomes dysregulated, abnormal CNS function can result. Both neurons and glial cells of the CNS express receptors for cytokines and chemokines, but the biological consequence of receptor activation has yet to be fully resolved. Our studies show that neuroadaptive changes are produced in primary cultures of rat cerebellar cells chronically treated with the cytokine interleukin-6 (IL-6) and in the cerebellum of transgenic mice that chronically express elevated levels of IL-6 in the CNS. In the cerebellum in culture and in vivo, the neuroadaptive changes included alterations in the level of expression of proteins involved in gene expression, signal transduction, and synaptic transmission. Associated with these changes were alterations in neuronal function. A comparison of results from the cultured cerebellar cells and cerebellum of the transgenic mice indicated that the effects of IL-6 can vary across neuronal types. However, alterations in mechanisms involved in Ca2+ homeostasis were observed in all cell types studied. These results indicate that modifications in cerebellar function are likely to occur in disorders associated with elevated levels of IL-6 in the cerebellum. © 2013 Springer Science+Business Media New York.

Blackmond D.G.,Scripps Research Institute
Cold Spring Harbor perspectives in biology | Year: 2010

The single-handedness of biological molecules has fascinated scientists and laymen alike since Pasteur's first painstaking separation of the enantiomorphic crystals of a tartrate salt more than 150 yr ago. More recently, a number of theoretical and experimental investigations have helped to delineate models for how one enantiomer might have come to dominate over the other from what presumably was a racemic prebiotic world. This article highlights mechanisms for enantioenrichment that include either chemical or physical processes, or a combination of both. The scientific driving force for this work arises from an interest in understanding the origin of life, because the homochirality of biological molecules is a signature of life.

The goal of this study was to assess the offset of the antiplatelet effects of prasugrel and clopidogrel. Guidelines recommend discontinuing clopidogrel at least 5 days and prasugrel at least 7 days before surgery. The pharmacodynamic basis for these recommendations is limited. Aspirin-treated patients with coronary artery disease were randomly assigned to either prasugrel 10 mg or clopidogrel 75 mg daily for 7 days. Platelet reactivity was measured before study drug administration and for up to 12 days during washout. The primary endpoint was the cumulative proportion of patients returning to baseline reactivity after study drug discontinuation. A total of 56 patients were randomized; 54 were eligible for analysis. Platelet reactivity was lower 24 h after the last dose of prasugrel compared with clopidogrel. After prasugrel, ≥75% of patients returned to baseline reactivity by washout day 7 compared with day 5 after clopidogrel. Recovery time was dependent on the level of platelet reactivity before study drug exposure and the initial degree of platelet inhibition after study drug discontinuation but not on treatment assignment. Recovery time after thienopyridine discontinuation depends on the magnitude of on-treatment platelet inhibition, resulting, on average, in a more delayed recovery with prasugrel compared with clopidogrel. The offset of prasugrel was consistent with current guidelines regarding the recommended waiting period for surgery after discontinuation. (Prasugrel/Clopidogrel Maintenance Dose Washout Study; NCT01014624). Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

In this article, I discuss the hallmarks of hypoxia in vitro and in vivo and review work showing that many types of stem cell proliferate more robustly in lowered oxygen. I then discuss recent studies showing that alterations in the levels and the types of cell and substrate adhesion molecules are a notable response to reduced O2 levels in both cultured primary neural stem cells and brain tissues in response to hypoxia in vivo. The ability of O2 levels to regulate adhesion molecule expression is linked to the Wnt signaling pathway, which can control and be controlled by adhesion events. The ability of O2 levels to influence cell adhesion also has far-reaching implications for development, ischemic trauma and neural regeneration, as well as for cancer and other diseases. Finally I discuss the possibility that the fluctuations in O2 levels known to have occurred over evolutionary time could, by influencing adhesion systems, have contributed to early symbiotic events in unicellular organisms and to the emergence of multicellularity. It is not my intention to be exhaustive in these domains, which are far from my own field of study. Rather this article is meant to provoke and stimulate thinking about molecular evolution involving O2 sensing and signaling during eras of geologic and atmospheric change that might inform modern studies on development and disease. © 2012 Landes Bioscience.

Eberwine J.,University of Pennsylvania | Bartfai T.,Scripps Research Institute
Pharmacology and Therapeutics | Year: 2011

We report on an 'unbiased' molecular characterization of individual, adult neurons, active in a central, anterior hypothalamic neuronal circuit, by establishing cDNA libraries from each individual, electrophysiologically identified warm sensitive neuron (WSN). The cDNA libraries were analyzed by Affymetrix microarray. The presence and frequency of cDNAs were confirmed and enhanced with Illumina sequencing of each single cell cDNA library. cDNAs encoding the GABA biosynthetic enzyme Gad1 and of adrenomedullin, galanin, prodynorphin, somatostatin, and tachykinin were found in the WSNs. The functional cellular and in vivo studies on dozens of the more than 500 neurotransmitters, hormone receptors and ion channels, whose cDNA was identified and sequence confirmed, suggest little or no discrepancy between the transcriptional and functional data in WSNs; whenever agonists were available for a receptor whose cDNA was identified, a functional response was found. Sequencing single neuron libraries permitted identification of rarely expressed receptors like the insulin receptor, adiponectin receptor 2 and of receptor heterodimers; information that is lost when pooling cells leads to dilution of signals and mixing signals. Despite the common electrophysiological phenotype and uniform Gad1 expression, WSN transcriptomes show heterogeneity, suggesting strong epigenetic influence on the transcriptome. Our study suggests that it is well-worth interrogating the cDNA libraries of single neurons by sequencing and chipping. © 2010 Elsevier Inc. All rights reserved.

Encalada S.E.,Scripps Research Institute | Goldstein L.S.B.,University of California at San Diego
Annual Review of Biophysics | Year: 2014

Axonal transport is indispensable for the distribution of vesicles, organelles, messenger RNAs (mRNAs), and signaling molecules along the axon. This process is mediated by kinesins and dyneins, molecular motors that bind to cargoes and translocate on microtubule tracks. Tight modulation of motor protein activity is necessary, but little is known about the molecules and mechanisms that regulate transport. Moreover, evidence suggests that transport impairments contribute to the initiation or progression of neurodegenerative diseases, or both, but the mechanisms by which motor activity is affected in disease are unclear. In this review, we discuss some of the physical and biophysical properties that influence motor regulation in healthy neurons. We further discuss the evidence for the role of transport in neurodegeneration, highlighting two pathways that may contribute to transport impairment-dependent disease: genetic mutations or variation, and protein aggregation. Understanding how and when transport parameters change in disease will help delineate molecular mechanisms of neurodegeneration. Copyright © 2014 by Annual Reviews. All rights reserved.

Jackson S.P.,Monash University | Jackson S.P.,Scripps Research Institute
Nature Medicine | Year: 2011

The formation of blood clots-thrombosis-at sites of atherosclerotic plaque rupture is a major clinical problem despite ongoing improvements in antithrombotic therapy. Progress in identifying the pathogenic mechanisms regulating arterial thrombosis has led to the development of newer therapeutics, and there is general anticipation that these treatments will have greater efficacy and improved safety. However, major advances in this field require the identification of specific risk factors for arterial thrombosis in affected individuals and a rethink of the 'one size fits all' approach to antithrombotic therapy. © 2011 Nature America, Inc. All rights reserved.

Truong T.H.,University of Michigan | Carroll K.S.,Scripps Research Institute
Critical Reviews in Biochemistry and Molecular Biology | Year: 2013

Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous hydrogen peroxide (H2O2) by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H 2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiological and pathological H2O2 responses. © 2013 Informa Healthcare USA, Inc.

Schimmel P.,Scripps Research Institute
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2011

Aminoacyl tRNA synthetases are ancient proteins that interpret the genetic material in all life forms.They are thought to have appeared during the transition from the RNA world to the theatre of proteins.During translation, they establish the rules of the genetic code, whereby each amino acid isattached to a tRNA that is cognate to the amino acid. Mistranslation occurs when an amino acidis attached to the wrong tRNA and subsequently is misplaced in a nascent protein. Mistranslationcan be toxic to bacteria and mammalian cells, and can lead to heritable mutations. The great challengefor nature appears to be serine-for-alanine mistranslation, where even small amounts of thismistranslation cause severe neuropathologies in the mouse. To minimize serine-for-alanine mistranslation,powerful selective pressures developed to prevent mistranslation through a special editingactivity imbedded within alanyl-tRNA synthetases (AlaRSs). However, serine-for-alanine mistranslationis so challenging that a separate, genome-encoded fragment of the editing domain ofAlaRS is distributed throughout the Tree of Life to redundantly prevent serine-to-alanine mistranslation.Detailed X-ray structural and functional analysis shed light on why serine-for-alaninemistranslation is a universal problem, and on the selective pressures that engendered the appearanceof AlaXps at the base of the Tree of Life. © 2011 The Royal Society.

Kelso J.M.,Scripps Research Institute
Immunology and Allergy Clinics of North America | Year: 2015

Most children with a history of penicillin allergy are labeled allergic and denied treatment with penicillin and sometimes other beta-lactam antibiotics. Most of these children never were or are no longer allergic to penicillin. Penicillin skin testing and oral challenge can identify patients who are not currently allergic, allowing them to be treated with penicillin. Children with egg allergy are often denied influenza vaccination, because the vaccine contains a small amount of egg protein. However, recent studies have demonstrated that children with even severe egg allergy can safely receive the vaccine, reducing their risk of the morbidity and mortality associated with influenza. © 2015 Elsevier Inc.

Shenvi R.A.,Scripps Research Institute
Natural Product Reports | Year: 2016

Covering: 1860-2016 A mechanistic link may exist between convulsant plant substances typified by picrotoxinin, and 'neurotrophic' sesquiterpenes like jiadifenolide. Picrotoxinin elicits convulsion by anion blockade of the Cys-loop family of neurotransmitter-gated ion channels. These same receptors mediate neuronal development and neurite outgrowth prior to synapse formation. Due to its structural homology with picrotoxin and anisatin, it is possible that jiadifenolide enhances NGF-stimulated neurite outgrowth by modulation of the Cys-loop family of receptors. © The Royal Society of Chemistry 2016.

Saphire E.O.,Scripps Research Institute
Immunotherapy | Year: 2013

Multiple recent, independent studies have confirmed that passively administered antibodies can provide effective postexposure therapy in nonhuman primates after exposure to an otherwise lethal dose of Ebola virus or Marburg virus. In this article, we review composition and performance of the antibody cocktails tested thus far, what is known about antibody epitopes on the viral glycoprotein target and ongoing research questions in further development of such cocktails for pre-exposure or emergency postexposure use. © 2013 Future Medicine Ltd.

Gibson G.,Ford Motor Company | Asahara H.,Scripps Research Institute
Journal of Orthopaedic Research | Year: 2013

microRNAs are small non-coding RNAs that in the last decade have emerged as overarching regulators of gene expression. Their abundance, ability to repress a large number of target genes and overlapping target specificity indicate a complex network of interactions that is still being defined. A number of studies focused on the role of microRNAs in cartilage have identified a small number, including miR-140 and -675 as playing important roles in regulation of cartilage homeostasis and together with the broader description of the activity of microRNAs in other tissues are beginning to define the function of microRNAs in cartilage development and homeostasis. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1333-1344, 2013. Copyright © 2013 Orthopaedic Research Society.

Ferreon A.C.,Scripps Research Institute
Methods in enzymology | Year: 2010

Intrinsically disordered proteins (IDPs) (also referred to as natively unfolded proteins) play critical roles in a variety of cellular processes such as transcription and translation and also are linked to several human diseases. Biophysical studies of IDPs present unusual experimental challenges due in part to their broad conformational heterogeneity and potentially complex binding-induced folding behavior. By minimizing the averaging over an ensemble (which is typical of most conventional experiments), single-molecule fluorescence (SMF) techniques have recently begun to add advanced capabilities for structural studies to the experimental arsenal of IDP investigators. Here, we briefly discuss a few common SMF methods that are particularly useful for IDP studies, including SMF resonance energy transfer and fluorescence correlation spectroscopy, along with site-specific protein-labeling methods that are essential for application of these methods to IDPs. We then present an overview of a few studies in this area, highlighting how SMF methods are being used to gain valuable information about two amyloidogenic IDPs, the Parkinson's disease-linked alpha-synuclein and the NM domain of the yeast prion protein Sup 35. SMF experiments provided new information about the proteins' rapidly fluctuating IDP forms, and the complex alpha-synuclein folding behavior upon its binding to lipid and membrane mimics. We anticipate that SMF and single-molecule methods, in general, will find broad application for structural and mechanistic studies of a wide variety of IDPs, both of their disordered conformations, and their ordered ensembles relevant for function and disease. Copyright 2010 Elsevier Inc. All rights reserved.

Smith R.G.,Scripps Research Institute
Science Signaling | Year: 2016

The gut-derived hormone ghrelin regulates growth hormone release, appetite, metabolism, and immune function through its receptor, the growth hormone secretagogue receptor 1a (GHSR1a). In this issue of Science Signaling, Chebani et al. decode GHSR1a signaling by using transgenic rats with a mutation in GHSR1a that prevents its interaction with β-arrestin. These mutant rats are more responsive to endogenous ghrelin, resulting in increased fat deposition and insulin resistance without affecting food intake.

Miles L.,Scripps Research Institute | Parmer R.,University of California at San Diego
Seminars in Thrombosis and Hemostasis | Year: 2013

The interaction of plasminogen with cell surfaces results in promotion of plasmin formation and retention on the cell surface. This results in arming cell surfaces with the broad-spectrum proteolytic activity of plasmin. Over the past quarter century, key functional consequences of the association of plasmin with the cell surface have been elucidated. Physiologic and pathophysiologic processes with plasmin-dependent cell migration as a central feature include inflammation, wound healing, oncogenesis, metastasis, myogenesis, and muscle regeneration. Cell surface plasmin also participates in neurite outgrowth and prohormone processing. Furthermore, plasmin-induced cell signaling also affects the functions of inflammatory cells, via production of cytokines, reactive oxygen species, and other mediators. Finally, plasminogen receptors regulate fibrinolysis. In this review, we highlight emerging data that shed light on longstanding controversies and raise new issues in the field. We focus on (1) the impact of the recent X-ray crystal structures of plasminogen and the development of antibodies that recognize cell-induced conformational changes in plasminogen on our understanding of the interaction of plasminogen with cells; (2) the relationship between apoptosis and plasminogen binding to cells; (3) the current status of our understanding of the molecular identity of plasminogen receptors and the discovery of a structurally unique novel plasminogen receptor, Plg-RKT; (4) the determinants of the interplay between distinct plasminogen receptors and cellular functions; and (5) new insights into the role of colocalization of plasminogen and plasminogen activator receptors on the cell surface. © 2013 by Thieme Medical Publishers, Inc.

Posokhova E.,Scripps Research Institute
PloS one | Year: 2013

Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-I(KACh) intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-I(KACh) signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-I(KACh) signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.

Montenegro J.,University of Santiago de Compostela | Ghadiri M.R.,Scripps Research Institute | Granja J.R.,University of Santiago de Compostela
Accounts of Chemical Research | Year: 2013

The lipid bilayer membranes are Nature's dynamic structural motifs that individualize cells and keep ions, proteins, biopolymers and metabolites confined in the appropriate location. The compartmentalization and isolation of these molecules from the external media facilitate the sophisticated functions and connections between the different biological processes accomplished by living organisms. However, cells require assistance from minimal energy shortcuts for the transport of molecules across membranes so that they can interact with the exterior and regulate their internal environments.Ion channels and pores stand out from all other possible transport mechanisms due to their high selectivity and efficiency in discriminating and transporting ions or molecules across membrane barriers. Nevertheless, the complexity of these smart "membrane holes" has driven researchers to develop simpler artificial structures with comparable performance to the natural systems. As a broad range of supramolecular interactions have emerged as efficient tools for the rational design and preparation of stable 3D superstructures, these results have stimulated the creativity of chemists to design synthetic mimics of natural active macromolecules and even to develop artificial structures with functions and properties.In this Account, we highlight results from our laboratories on the construction of artificial ion channel models that exploit the self-assembly of conformationally flat cyclic peptides (CPs) into supramolecular nanotubes. Because of the straightforward synthesis of the cyclic peptide monomers and the complete control over the internal diameter and external surface properties of the resulting hollow tubular suprastructure, CPs are the optimal candidates for the fabrication of ion channels. The ion channel activity and selective transport of small molecules by these structures are examples of the great potential that cyclic peptide nanotubes show for the construction of functional artificial transmembrane transporters. Our experience to date suggests that the next steps for achieving conceptual devices with better performance and selectivity will derive from the topological control over cyclic peptide assembly and the functionalization of the lumen. © 2013 American Chemical Society.

Hopkins S.,Autoimmune Technologies, Llc | Gallay P.,Scripps Research Institute
Viruses | Year: 2012

The advent of the replicon system together with advances in cell culture have contributed significantly to our understanding of the function of virally-encoded structural and nonstructural proteins in the replication cycle of the hepatitis C virus. In addition, in vitro systems have been used to identify several host proteins whose expression is critical for supporting such diverse activities as viral entry, RNA replication, particle assembly, and the release of infectious virions. Among all known host proteins that participate in the HCV replication cycle, cyclophilins are unique because they constitute the only host target that has formed the basis of pharmaceutical drug discovery and drug development programs. The introduction of the nonimmunosuppressive cyclophilin inhibitors into clinical testing has confirmed the clinical utility of CsA-based inhibitors for the treatment of individuals with chronic hepatitis C infection and has yielded new insights into their mechanism(s) of action. This review describes the biochemical evidence for the potential roles played by cyclophilins in supporting HCV RNA replication and summarizes clinical trial results obtained with the first generation of nonimmunosuppressive cyclophilin inhibitors. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

Candeias N.R.,University of Lisbon | Candeias N.R.,Scripps Research Institute | Montalbano F.,University of Lisbon | Cal P.M.S.D.,University of Lisbon | Gois P.M.P.,University of Lisbon
Chemical Reviews | Year: 2010

Researchers conducted studies to demonstrate the use of boronic acids and esters in the Petasis-borono Mannich multicomponent reaction. Investigations revealed that the reaction consisted in the formation of an imine or an iminium after condensation of an aldehyde with a primary or secondary amine. The C-N double bond reacted with the boronic acid to yield the secondary or tertiary amine. The reaction between the boronic acid and the iminium or the imine was extremely facilitated by the presence of an adjacent OH functional group that activated the boronic acid via the formation of a tetrahedron boronate salt despite its inertness towards the aldehyde functional group. The Petasis-borono Mannich multicomponent reaction had also been evaluated in the preparation of several small organic molecules with distinct functional groups such as functionalized α-amino acids, α-hydroxyl amines, 2-hydroxyl morpholines, alkylaminophenols, and 2H-chromenes.

Balch W.E.,Scripps Research Institute
Cold Spring Harbor perspectives in biology | Year: 2011

Cystic fibrosis (CF) is a consequence of defective recognition of the multimembrane spanning protein cystic fibrosis conductance transmembrane regulator (CFTR) by the protein homeostasis or proteostasis network (PN) (Hutt and Balch (2010). Like many variant proteins triggering misfolding diseases, mutant CFTR has a complex folding and membrane trafficking itinerary that is managed by the PN to maintain proteome balance and this balance is disrupted in human disease. The biological pathways dictating the folding and function of CFTR in health and disease are being studied by numerous investigators, providing a unique opportunity to begin to understand and therapeutically address the role of the PN in disease onset, and its progression during aging. We discuss the general concept that therapeutic management of the emergent properties of the PN to control the energetics of CFTR folding biology may provide significant clinical benefit.

Evans C.H.,Harvard University | Ghivizzani S.C.,Florida College | Robbins P.D.,Scripps Research Institute
Translational Research | Year: 2013

Arthritis is a disease of joints. The biology of joints makes them very difficult targets for drug delivery in a manner that is specific and selective. This is especially true for proteinaceous drugs ("biologics"). Gene transfer is the only technology that can solve the delivery problem in a clinically reasonable fashion. There is an abundance of preclinical data confirming that genes can be efficiently transferred to tissues within joints by intra-articular injection using a variety of different vectors in conjunction with ex vivo and in vivo strategies. Using the appropriate gene transfer technologies, long-term, intra-articular expression of anti-arthritic transgenes at therapeutic concentrations can be achieved. Numerous studies confirm that gene therapy is effective in treating experimental models of rheumatoid arthritis (RA) and osteoarthritis (OA) in the laboratory. A limited number of clinical trials have been completed, which confirm safety and feasibility but only 3 protocols have reached phase II; as yet, there is no unambiguous evidence of efficacy in human disease. Only 2 clinical trials are presently underway, both phase II studies using allogeneic chondrocytes expressing transforming growth factor-β1 for the treatment of OA. Phase I studies using adeno-associated virus to deliver interleukin-1Ra in OA and interferon-β in RA are going through the regulatory process. It is to be hoped that the recent successes in treating rare, Mendelian diseases by gene therapy will lead to accelerated development of genetic treatments for common, non-Mendelian diseases, such as arthritis. © 2013 Mosby, Inc. All rights reserved.

Fuss J.O.,Lawrence Berkeley National Laboratory | Tainer J.A.,Lawrence Berkeley National Laboratory | Tainer J.A.,Scripps Research Institute
DNA Repair | Year: 2011

Helicases must unwind DNA at the right place and time to maintain genomic integrity or gene expression. Biologically critical XPB and XPD helicases are key members of the human TFIIH complex; they anchor CAK kinase (cyclinH, MAT1, CDK7) to TFIIH and open DNA for transcription and for repair of duplex distorting damage by nucleotide excision repair (NER). NER is initiated by arrested RNA polymerase or damage recognition by XPC-RAD23B with or without DDB1/DDB2. XP helicases, named for their role in the extreme sun-mediated skin cancer predisposition xeroderma pigmentosum (XP), are then recruited to asymmetrically unwind dsDNA flanking the damage. XPB and XPD genetic defects can also cause premature aging with profound neurological defects without increased cancers: Cockayne syndrome (CS) and trichothiodystrophy (TTD). XP helicase patient phenotypes cannot be predicted from the mutation position along the linear gene sequence and adjacent mutations can cause different diseases. Here we consider the structural biology of DNA damage recognition by XPC-RAD23B, DDB1/DDB2, RNAPII, and ATL, and of helix unwinding by the XPB and XPD helicases plus the bacterial repair helicases UvrB and UvrD in complex with DNA. We then propose unified models for TFIIH assembly and roles in NER. Collective crystal structures with NMR and electron microscopy results reveal functional motifs, domains, and architectural elements that contribute to biological activities: damaged DNA binding, translocation, unwinding, and ATP driven changes plus TFIIH assembly and signaling. Coupled with mapping of patient mutations, these combined structural analyses provide a framework for integrating and unifying the rich biochemical and cellular information that has accumulated over forty years of study. This integration resolves puzzles regarding XP helicase functions and suggests that XP helicase positions and activities within TFIIH detect and verify damage, select the damaged strand for incision, and coordinate repair with transcription and cell cycle through CAK signaling. © 2011 Elsevier B.V.

Disney M.D.,Scripps Research Institute
Drug Discovery Today | Year: 2013

RNA is an important yet vastly underexploited target for small molecule chemical probes or lead therapeutics. Small molecules have been used successfully to modulate the function of the bacterial ribosome, viral RNAs and riboswitches. These RNAs are either highly expressed or can be targeted using substrate mimicry, a mainstay in the design of enzyme inhibitors. However, most cellular RNAs are neither highly expressed nor have a lead small molecule inhibitor, a significant challenge for drug discovery efforts. Herein, I describe the design of small molecules targeting expanded repeating transcripts that cause myotonic muscular dystrophy (DM). These test cases illustrate the challenges of designing small molecules that target RNA and the advantages of targeting repeating transcripts. Lastly, I discuss how small molecules might be more advantageous than oligonucleotides for targeting RNA. © 2013 Elsevier Ltd. All rights reserved.

Lazar D.C.,Scripps Research Institute
Molecular Therapy | Year: 2015

HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The “shock and kill” strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a “hotspot” for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a “functional cure” of HIV/AIDS.Molecular Therapy (2016); doi:10.1038/mt.2015.202. © 2015 Official journal of the American Society of Gene & Cell Therapy

Cherqui S.,Scripps Research Institute
Kidney International | Year: 2012

Cystinosis as a clinical entity is a progressive dysfunction of multiple organs caused by the accumulation of cystine in the tissues, leading, for example, to end-stage renal failure, diabetes, hypothyroidism, myopathy, and central nervous system deterioration. Brodin-Sartorius and colleagues present a long-term study on the impact of cysteamine therapy on these complications. The data show that cysteamine improves the outcome and complications of cystinosis but does not prevent them. © 2012 International Society of Nephrology.

Crabbe J.C.,Oregon Health And Science University | Harris R.A.,University of Texas at Austin | Koob G.F.,Scripps Research Institute
Annals of the New York Academy of Sciences | Year: 2011

Binge drinking is prevalent and has serious biomedical consequences. In children, adolescents, and young adults, it is a prominent risk factor for later development of alcohol-use disorders. Many preclinical models have been employed to study the genetic risks for and biomedical consequences of alcohol drinking. However, these models historically did not result in blood-alcohol concentrations (BACs) exceeding 80 mg%; this relatively modest level is the threshold that currently defines a binge session, according to the NIAAA and CDC. Nevertheless, in alcohol-dependent rodents, binge drinking has been well documented. Key neurobiological substrates localized to brain reward and stress systems have been identified. Studies of newer models of binge drinking without dependence are reviewed here. In these models, rodents, non-human primates, and flies will drink enough to reach high BACs. They often display observable signs of intoxication. The neurobiological consequences of these episodes of binge drinking without dependence are reviewed, and preliminary evidence for roles for GABA, glutamate, opioid peptides, and corticotropin releasing factor are discussed, as is the need for more work to identify the antecedents and consequences of binge drinking in both animal models and humans. © 2011 New York Academy of Sciences.

Kenny P.,Scripps Research Institute
Neuron | Year: 2011

Food is consumed in order to maintain energy balance at homeostatic levels. In addition, palatable food is also consumed for its hedonic properties independent of energy status. Such reward-related consumption can result in caloric intake exceeding requirements and is considered a major culprit in the rapidly increasing rates of obesity in developed countries. Compared with homeostatic mechanisms of feeding, much less is known about how hedonic systems in brain influence food intake. Intriguingly, excessive consumption of palatable food can trigger neuroadaptive responses in brain reward circuitries similar to drugs of abuse. Furthermore, similar genetic vulnerabilities in brain reward systems can increase predisposition to drug addiction and obesity. Here, recent advances in our understanding of the brain circuitries that regulate hedonic aspects of feeding behavior will be reviewed. Also, emerging evidence suggesting that obesity and drug addiction may share common hedonic mechanisms will also be considered. © 2011 Elsevier Inc.

He B.,Cornell University | Du J.,Cornell University | Du J.,Scripps Research Institute | Lin H.,Cornell University
Journal of the American Chemical Society | Year: 2012

Sirtuins, a class of enzymes known as nicotinamide adenine dinucleotide-dependent deacetylases, have been shown to regulate a variety of biological processes, including aging, transcription, and metabolism. Sirtuins are considered promising targets for treating several human diseases. There are seven sirtuins in humans (Sirt1-7). Small molecules that can target a particular human sirtuin are important for drug development and fundamental studies of sirtuin biology. Here we demonstrate that thiosuccinyl peptides are potent and selective Sirt5 inhibitors. The design of these inhibitors is based on our recent discovery that Sirt5 prefers to catalyze the hydrolysis of malonyl and succinyl groups, rather than an acetyl group, from lysine residues. Furthermore, among the seven human sirtuins, Sirt5 is the only one that has this unique acyl group preference. This study demonstrates that the different acyl group preferences of different sirtuins can be conveniently utilized to develop small molecules that selectively target different sirtuins. © 2012 American Chemical Society.

Topol E.J.,Scripps Research Institute
Nature Reviews Genetics | Year: 2015

I call for an international open medical resource to provide a database for every individual's genomic, metabolomic, microbiomic, epigenomic and clinical information. This resource is needed in order to facilitate genetic diagnoses and transform medical care. © 2015 Macmillan Publishers Limited.

Fuhrmann J.,Scripps Research Institute | Clancy K.W.,University of Massachusetts Medical School | Thompson P.R.,University of Massachusetts Medical School
Chemical Reviews | Year: 2015

Chemical biology of protein arginine modifications in epigenetic regulation is investigated. Prevailing knowledge surrounding the post-translational modification (PTM) of histone arginine residues, focusing on enzyme classes that catalyze the citrullination and methylation of arginine residues along with non-canonical arginine modifications such as phosphorylation, ADP-ribosylation and arginylation are investigated. The major focus of the investigation is on the protein arginine deiminases (PADs) and protein arginine methyltransferases (PRMTs).

Forli S.,Scripps Research Institute
Current Topics in Medicinal Chemistry | Year: 2014

Epothilones are natural compounds isolated from a myxobacterium at the beginning of the 1990s, and showed a remarkable anti-neoplastic activity. They act through the same mechanism of action of paclitaxel, by stabilizing microtubules and inducing apoptosis. Although, their chemical structure, simpler than taxanes, makes them more suitable for derivatization. Their interesting pharmacokinetic and bioavailabilty profiles, and the activity against paclitaxel-resistant cell lines make them interesting therapeutic agents. Here a brief historical perspective of epothilones is presented, since their isolation, the identification of their mechanism of action and activity, to the recent clinical trials. © 2014 Bentham Science Publishers.

Fowler V.M.,Scripps Research Institute
Current Topics in Membranes | Year: 2013

The mammalian erythrocyte, or red blood cell (RBC), is a unique experiment of nature: a cell with no intracellular organelles, nucleus or transcellular cytoskeleton, and a plasma membrane with uniform structure across its entire surface. By virtue of these specialized properties, the RBC membrane has provided a template for discovery of the fundamental actin filament network machine of the membrane skeleton, now known to confer mechanical resilience, anchor membrane proteins, and organize membrane domains in all cells. This chapter provides a historical perspective and critical analysis of the biochemistry, structure, and physiological functions of this actin filament network in RBCs. The core units of this network are nodes of ~35-37nm-long actin filaments, interconnected by long strands of (α1β1)2-spectrin tetramers, forming a 2D isotropic lattice with quasi-hexagonal symmetry. Actin filament length and stability is critical for network formation, relying upon filament capping at both ends: tropomodulin-1 at pointed ends and αβ-adducin at barbed ends. Tropomodulin-1 capping is essential for precise filament lengths, and is enhanced by tropomyosin, which binds along the short actin filaments. αβ-adducin capping recruits spectrins to sites near barbed ends, promoting network formation. Accessory proteins, 4.1R and dematin, also promote spectrin binding to actin and, with αβ-adducin, link to membrane proteins, targeting actin nodes to the membrane. Dissection of the molecular organization within the RBC membrane skeleton is one of the paramount achievements of cell biological research in the past century. Future studies will reveal the structure and dynamics of actin filament capping, mechanisms of precise length regulation, and spectrin-actin lattice symmetry. © 2013 Elsevier Inc.

Hud N.V.,Georgia Institute of Technology | Cafferty B.J.,Georgia Institute of Technology | Krishnamurthy R.,Scripps Research Institute | Williams L.D.,Georgia Institute of Technology
Chemistry and Biology | Year: 2013

The origin of RNA is one of the most formidable problems facing prebiotic chemists. We consider RNA as a product of evolution, as opposed to the more conventional view of RNA as originally being the product of abiotic processes. We have come to accept that life's informational polymers have changed in chemical structure since their emergence, which presents a quandary similar to the paradox of "My Grandfather's Axe". Here, we discuss reasons why all contemporary components of RNA - the nucleobases, ribose, and phosphate - are not likely the original components of the first informational polymer(s) of life. We also evaluate three distinct models put forth as pathways for how the earliest informational polymers might have assembled. We see the quest to uncover the ancestors of RNA as an exciting scientific journey, one that is already providing additional chemical constraints on the origin of life and one that has the potential to produce self-assembling materials, novel catalysis, and bioactive compounds. © 2013 Elsevier Ltd.

Niu W.,University of Nebraska - Lincoln | Schultz P.G.,Scripps Research Institute | Guo J.,University of Nebraska - Lincoln
ACS Chemical Biology | Year: 2013

We have utilized in vitro evolution to identify tRNA variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to a quadruplet codon in both bacterial and mammalian cells. This approach will facilitate the creation of an optimized and standardized system for the genetic incorporation of unnatural amino acids using quadruplet codons, which will allow the biosynthesis of biopolymers that contain multiple unnatural building blocks. © 2013 American Chemical Society.

Fujioka K.,Scripps Research Institute
Obesity | Year: 2015

In 2014 we have 4 new weight loss medications and one older medication with very different mechanisms of action all approved for chronic weight management. Each medication has its own unique risk profile that makes patient selection important. Knowledge of the contraindications and safety issues can guide physicians to the most appropriate choice for a particular patient. Obesity medicine is entering a new era where our available options for prescribing have been very well studied. There should be no surprises, because bupropion, naltrexone, phentermine, topiramate\ and liraglutide have been prescribed for many years in millions of patients and lorcaserin has high specificity for a single receptor subtype. The FDA demanded very detailed risk-oriented studies to have these medications approved. In addition, the FDA has established REMS programs or risk management strategies to help ensure that the patients do not receive inapppropriate medications. These medications were approved by the US FDA after very thorough testing. The decision to approve these medications was based on the benefits out-weighing the risks. Thus, if following the appropriate guidelines according to package labels, the practitioner can feel safe in prescribing these medications. © 2015 The Obesity Society.

Saayman S.M.,Scripps Research Institute
Molecular Therapy | Year: 2016

Cystic fibrosis (CF) is a life-shortening genetic disease. The root cause of CF is heritable recessive mutations that affect the cystic fibrosis transmembrance conductance regulator (CFTR) gene and the subsequent expression and activity of encoded ion channels at the cell surface. We show that CFTR is regulated transcriptionally by the actions of a novel long noncoding RNA (lncRNA), designated as BGas, that emanates from intron 11 of the CFTR gene and is expressed in the antisense orientation relative to the protein coding sense strand. We find that BGas functions in concert with several proteins including HMGA1, HMGB1, and WIBG to modulate the local chromatin and DNA architecture of intron 11 of the CFTR gene and thereby affects transcription. Suppression of BGas or its associated proteins results in a gain of both CFTR expression and chloride ion function. The observations described here highlight a previously underappreciated mechanism of transcriptional control and suggest that BGas may serve as a therapeutic target for specifically activating expression of CFTR.Molecular Therapy (2016); doi:10.1038/mt.2016.112. © 2016 Official journal of the American Society of Gene & Cell Therapy

Lauer J.,Scripps Research Institute
Nature Structural and Molecular Biology | Year: 2016

Neddylation is a post-translational modification that controls the cell cycle and proliferation by conjugating the ubiquitin-like protein NEDD8 to specific targets. Here we report that glycyl-tRNA synthetase (GlyRS), an essential enzyme in protein synthesis, also plays a critical role in neddylation. In human cells, knockdown of GlyRS, but not knockdown of a different tRNA synthetase, decreased the global level of neddylation and caused cell-cycle abnormality. This function of GlyRS is achieved through direct interactions with multiple components of the neddylation pathway, including NEDD8, E1, and E2 (Ubc12). Using various structural and functional approaches, we show that GlyRS binds the APPBP1 subunit of E1 and captures and protects activated E2 (NEDD8-conjugated Ubc12) before the activated E2 reaches a downstream target. Therefore, GlyRS functions as a chaperone that critically supports neddylation. This function is probably conserved in all eukaryotic GlyRS enzymes and may contribute to the strong association of GlyRS with cancer progression. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

Reisfeld R.A.,Scripps Research Institute
Critical Reviews in Oncogenesis | Year: 2013

Cancer immunotherapy is in the midst of a major paradigm shift from an approach primarily focused on attacking tumor cells to a strategy also targeting the tumor microenvironment (TME). This strategy is designed for the use of combination therapies, several of which are reviewed here. Particular emphasis is placed on targeting such components of the TME as tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) overexpressing specific therapy targets such as legumain, an asparaginyl endopeptidase, proto-oncogene Fra-1, transcription factor Stat3 and fibroblast activation protein (FAP) as well as HER-2, respectively. The use of DNA vaccines directed against some of these targets overexpressed on breast tumor cells as well as TAMs and CAFs in the TME results in the elimination of tumor growth, progression, metastasis, and recurrence in mouse tumor models. This type of cancer therapy is significantly more effective; the strategy is specifically designed to combine immunotherapies, including DNA vaccines, with a novel chemotherapy featuring highly effective nanoparticle-mediated drug delivery, specifically targeted to tumor cells as well as key components of the TME, leading to its modulation and subsequent elimination of tumor growth, metastasis, and, most importantly, suppression of tumor recurrence. © 2013 by Begell House, Inc.

Zanetti G.,Howard Hughes Medical Institute | Pahuja K.B.,Howard Hughes Medical Institute | Studer S.,Scripps Research Institute | Shim S.,Hanwha Chemical Bio Business Unit | Schekman R.,Howard Hughes Medical Institute
Nature Cell Biology | Year: 2012

Secretory proteins are transported to the Golgi complex in vesicles that bud from the endoplasmic reticulum. The cytoplasmic coat protein complex II (COPII) is responsible for cargo sorting and vesicle morphogenesis. COPII was first described in Saccharomyces cerevisiae, but its basic function is conserved throughout all eukaryotes. Nevertheless, the COPII coat has adapted to the higher complexity of mammalian physiology, achieving more sophisticated levels of secretory regulation. In this review we cover aspects of mammalian COPII-mediated regulation of secretion, in particular related to the function of COPII paralogues, the spatial organization of cargo export and the role of accessory proteins.

Dyson H.J.,Scripps Research Institute | Komives E.A.,University of California at San Diego
IUBMB Life | Year: 2012

The paradigmatic transcription factors of the NFκB family provide an increasingly complex view of the mechanism of signal-mediated transcriptional activation. Although the primary event, phosphorylation and subsequent ubiquitin-dependent degradation of IκBα, the inhibitor of the canonical NFκB (p50/p65), is reasonably well understood, the means whereby the activation is turned off by postinduction repression are less well understood. Recent work highlighted in this review suggests that the inhibitor IκBα participates in the "stripping" of NFκB from the DNA, and that this process relies heavily on the disordered and weakly ordered segments of IκBα. Kinetic and equilibrium measurements in vitro as well as genetic screens in vivo convincingly demonstrate not only that IκBα greatly increases the dissociation rate of NFκB from DNA but also that further control of the process is mediated by the extremely short half-life of free IκBα, doubtless a result of the overall weakly folded nature of the free protein. These studies illustrate the versatility of protein systems that use not only well-structured proteins and protein complexes but also the full range of available weakly structured and disordered states to maximize functional efficiency and metabolic control. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Chen M.Z.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2012

A de novo synthesis of substituted pyridines is described that proceeds through nucleophilic addition of a dithiane anion to an α,β- unsaturated carbonyl followed by metallacycle-mediated union of the resulting allylic alcohol with preformed trimethylsilane-imines (generated in situ by the low-temperature reaction of lithium hexamethyldisilazide with an aldehyde) and Ag(I)- or Hg(II)-mediated ring closure. The process is useful for the convergent assembly of di- through penta-substituted pyridines with complete regiochemical control. © 2011 American Chemical Society.

Kenny P.J.,Molecular Therapeutics | Kenny P.J.,Scripps Research Institute
Nature Reviews Neuroscience | Year: 2011

The hedonic properties of food can stimulate feeding behaviour even when energy requirements have been met, contributing to weight gain and obesity. Similarly, the hedonic effects of drugs of abuse can motivate their excessive intake, culminating in addiction. Common brain substrates regulate the hedonic properties of palatable food and addictive drugs, and recent reports suggest that excessive consumption of food or drugs of abuse induces similar neuroadaptive responses in brain reward circuitries. Here, we review evidence suggesting that obesity and drug addiction may share common molecular, cellular and systems-level mechanisms. © 2011 Macmillan Publishers Limited. All rights reserved.

Waalen J.,Scripps Research Institute
Translational Research | Year: 2014

The heritability of obesity has long been appreciated and the genetics of obesity has been the focus of intensive study for decades. Early studies elucidating genetic factors involved in rare monogenic and syndromic forms of extreme obesity focused attention on dysfunction of hypothalamic leptin-related pathways in the control of food intake as a major contributor. Subsequent genome-wide association studies of common genetic variants identified novel loci that are involved in more common forms of obesity across populations of diverse ethnicities and ages. The subsequent search for factors contributing to the heritability of obesity not explained by these 2 approaches ("missing heritability") has revealed additional rare variants, copy number variants, and epigenetic changes that contribute. Although clinical applications of these findings have been limited to date, the increasing understanding of the interplay of these genetic factors with environmental conditions, such as the increased availability of high calorie foods and decreased energy expenditure of sedentary lifestyles, promises to accelerate the translation of genetic findings into more successful preventive and therapeutic interventions. © 2014 Elsevier Inc.

Prapainop K.,University of Oxford | Witter D.P.,University of Oxford | Wentworth Jr. P.,University of Oxford | Wentworth Jr. P.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2012

A major challenge in nanomaterial science is to develop approaches that ensure that when administered in vivo, nanoparticles can be targeted to their requisite site of action. Herein we report the first approach that allows for cell-specific uptake of nanomaterials by a process involving reprogramming of the behavior of the ubiquitous protein corona of nanomaterials. Specifically, judicious surface modification of quantum dots with a small molecule that induces a protein-misfolding event in a component of the nanoparticle-associated protein corona renders the associated nanomaterials susceptible to cell-specific, receptor-mediated endocytosis. We see this chemical approach as a new and general method for exploiting the inescapable protein corona to target nanomaterials to specific cells. © 2012 American Chemical Society.

Hastie K.M.,Scripps Research Institute
Nature Structural and Molecular Biology | Year: 2016

Arenaviruses exist worldwide and can cause hemorrhagic fever and neurologic disease. A single glycoprotein expressed on the viral surface mediates entry into target cells. This glycoprotein, termed GPC, contains a membrane-associated signal peptide, a receptor-binding subunit termed GP1 and a fusion-mediating subunit termed GP2. Although GPC is a critical target of antibodies and vaccines, the structure of the metastable GP1–GP2 prefusion complex has remained elusive for all arenaviruses. Here we describe the crystal structure of the fully glycosylated prefusion GP1–GP2 complex of the prototypic arenavirus LCMV at 3.5 Å. This structure reveals the conformational changes that the arenavirus glycoprotein must undergo to cause fusion and illustrates the fusion regions and potential oligomeric states. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

Pockros P.J.,Scripps Research Institute
Clinics in Liver Disease | Year: 2013

Non-nucleoside polymerase inhibitors have several limitations including low to moderate potency, a low barrier to resistance, unlikely to cross genotype activity, genotype potency 1b > 1a, and hyperbilirubinemia for 2 of the drugs (tegobuvir and BI-207127). These drugs will have no role in monotherapy and may have only a limited role in triple therapy. They could be part of a quadruple therapy regimen or a triple or quadruple interferon-free regimen. Several issues remain unclear at the time of this review; the role of these compounds including minimal dosing required, safety, and cost remains to be clarified. © 2013 Elsevier Inc.

Wright N.E.,Columbia University | Snyder S.A.,Scripps Research Institute
Angewandte Chemie - International Edition | Year: 2014

Explorations into a series of different approaches for 9-membered carbocycle formation have afforded the first reported example of a 9-exo-dig ring closure via a AuIII-promoted reaction between an alkyne and an aryl ring as well as several additional, unique Friedel-Crafts-type cyclizations. Analyses of the factors leading to the success of these transformations are provided, with the application of one of the developed 9-membered ring closures affording an efficient and scalable synthesis of the bioactive resveratrol trimer caraphenol A. That synthesis proceeded with an average yield of 89 % per step (7.8 % overall yield) and has provided access to more than 600 mg of the target molecule. Fun with Friedel-Crafts: Explorations into 9-membered carbocycle formation have afforded the first 9-exo-dig ring closure via a AuIII-promoted reaction between an alkyne and an aryl ring along with several additional, unique Friedel-Crafts-type cyclizations. Application of one of the 9-membered ring closures enabled an efficient and scalable (600 mg) synthesis of the resveratrol trimer caraphenol A in an average yield of 89 % per step. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mosnier L.O.,Scripps Research Institute
Journal of Biological Chemistry | Year: 2011

Thrombomodulin (TM) is a cofactor for thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) and thereby helps coordinate coagulation, anticoagulation, fibrinolysis, and inflammation. Platelet factor 4 (PF4), a platelet α-granule protein and a soluble cofactor for TM-dependent protein C activation, stimulates protein C activation in vitro and in vivo. In contrast to stimulation of protein C activation, PF4 is shown here to inhibit activation of TAFI by thrombin-TM. Consequences of inhibition of TAFI activation by PF4 included loss of TM-dependent prolongation of clot lysis times in hemophilia A plasma and loss of TM-stimulated conversion of bradykinin (BK) to des-Arg9-BK by TAFIa in normal plasma. Thus, PF4 modulates the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, thereby preventing the generation of the antifibrinolytic and anti-inflammatory activities of TAFIa. To block the inhibitory effects of PF4 on TAFI activation, heparin derivatives were tested for their ability to retain high affinity binding to PF4 despite having greatly diminished anticoagulant activity. N-acetylated heparin (NAc-Hep) lacked detectable anticoagulant activity in activated partial thromboplastin time clotting assays but retained high affinity binding to PF4 and effectively reversed PF4 binding to immobilized TM. NAc-Hep permitted BK conversion to des-Arg9-BK by TAFIa in the presence of PF4. In a clot lysis assay on TM-expressing cells using hemophilia A plasma, NAc-Hep prevented PF4-mediated inhibition of TAFI activation and the antifibrinolytic functions of TAFIa. Accordingly, NAc-Hep or similar heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Barros C.S.,Scripps Research Institute
Cold Spring Harbor perspectives in biology | Year: 2011

An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system.

Conti B.,Scripps Research Institute | Hansen M.,Sanford Burnham Institute for Medical Research
Cell | Year: 2013

In this issue, Xiao et al. challenge the notion that cold temperatures promote longevity solely through thermodynamic effects. They show that low temperatures activate a cold-sensitive cation channel, TRPA-1, which triggers a complex signaling pathway in both neurons and nonneuronal cells to extend the lifespan of Caenorhabditis elegans. © 2013 Elsevier Inc.

Koziol J.A.,Scripps Research Institute
FEBS Letters | Year: 2010

Breitling et al. (2004) [1] introduced a statistical technique, the rank product method, for detecting differentially regulated genes in replicated microarray experiments. The technique has achieved widespread acceptance and is now used more broadly, in such diverse fields as RNAi analysis, proteomics, and machine learning. In this note, we extend the rank product method to the two sample setting, provide distribution theory attending the rank product method in this setting, and give numerical details for implementing the method. © 2010 Federation of European Biochemical Societies.

Aird W.C.,Beth Israel Deaconess Medical Center | Mosnier L.O.,Scripps Research Institute | Fairhurst R.M.,National Institute of Allergy and Infectious Diseases
Blood | Year: 2014

Of all the outcomes of Plasmodium falcipa-rum infection, the coma of cerebral malaria (CM) is particularly deadly. Malariologists have long wondered how some patients develop this organ-specific syndrome. Data from two recent publications support a novel mechanism of CM pathogenesis in which infected erythrocytes (IEs) express specific virulence proteins that mediate IE binding to the endothelial protein C receptor (EPCR). Malaria-associated depletion of EPCR, with subsequent impairment of the protein C system promotes a proin-flammatory, procoagulant state in brain microvessels.

Kodadek T.,Scripps Research Institute
Chemistry and Biology | Year: 2014

The ability to monitor and manipulate antigen-specific immune responses would have a major impact on several areas of biology and medicine. In this perspective, I consider pharmacological methods to do this, with a focus on the development of abiological "antigen surrogates" capable of binding to the antigen-binding sites of antibodies and B cell receptors with high affinity and selectivity. I describe the application of combinatorial library screening to identify antigen surrogates for monoclonal antibodies of therapeutic interest using chronic lymphocytic leukemia as an example. Furthermore, I discuss the use of multiplexed assays for the quantification of antigen surrogate-antibody complexes as diagnostic tools and antigen surrogate discovery via serum screening. Although antigen surrogates are a fairly new concept, I argue that they will open new avenues for both basic and clinical research and that major advances can be expected over the next few years. © 2014 Elsevier Ltd All rights reserved.

Breaker R.R.,Howard Hughes Medical Institute | Joyce G.F.,Scripps Research Institute
Chemistry and Biology | Year: 2014

RNA and DNA are simple linear polymers consisting of only four major types of subunits, and yet these molecules carry out a remarkable diversity of functions in cells and in the laboratory. Each newly discovered function of natural or engineered nucleic acids enforces the view that prior assessments of nucleic acid function were far too narrow and that many more exciting findings are yet to come. This Perspective highlights just a few of the numerous discoveries over the past 20 years pertaining to nucleic acid function, focusing on those that have been of particular interest to chemical biologists. History suggests that there will continue to be many opportunities to engage chemical biologists in the discovery, creation, and manipulation of nucleic acid function in the years to come. ©2014 Elsevier Ltd All rights reserved.

Dyson H.J.,Scripps Research Institute
Molecular BioSystems | Year: 2012

Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with them. The interactions of disordered proteins with DNA most often manifest as molding of the protein onto the B-form DNA structure, although some well-known instances involve remodeling of the DNA structure that seems to require that the interacting proteins be disordered to various extents in the free state. By contrast, induced fit in RNA-protein interactions has been recognized for many years - the existence and prevalence of this phenomenon provides the clearest possible evidence that RNA and its interactions with proteins must be considered as highly dynamic, and the dynamic nature of RNA and its multiplicity of folded and unfolded states is an integral part of its nature and function.

Tabarean I.V.,Scripps Research Institute
PLoS ONE | Year: 2012

Thermoregulatory neurons of the median preoptic nucleus (MnPO) represent a target at which histamine modulates body temperature. The mechanism by which histamine excites a population of MnPO neurons is not known. In this study it was found that histamine activated a cationic inward current and increased the intracellular Ca2+ concentration, actions that had a transient component as well as a sustained one that lasted for tens of minutes after removal of the agonist. The sustained component was blocked by TRPC channel blockers. Single-cell reverse transcription-PCR analysis revealed expression of TRPC1, TRPC5 and TRPC7 subunits in neurons excited by histamine. These studies also established the presence of transcripts for the glutamatergic marker Vglut2 and for the H1 histamine receptor in neurons excited by histamine. Intracellular application of antibodies directed against cytoplasmic sites of the TRPC1 or TRPC5 channel subunits decreased the histamine-induced inward current. The persistent inward current and elevation in intracellular Ca2+ concentration could be reversed by activating the PKA pathway. This data reveal a novel mechanism by which histamine induces persistent excitation and sustained intracellular Ca2+ elevation in glutamatergic MnPO neurons. © 2012 Iustin V.

Pi-Sunyer X.,Columbia University | Astrup A.,Copenhagen University | Fujioka K.,Scripps Research Institute | Greenway F.,Louisiana State University | And 6 more authors.
New England Journal of Medicine | Year: 2015

BACKGROUND Obesity is a chronic disease with serious health consequences, but weight loss is difficult to maintain through lifestyle intervention alone. Liraglutide, a glucagonlike peptide-1 analogue, has been shown to have potential benefit for weight management at a once-daily dose of 3.0 mg, injected subcutaneously. METHODS We conducted a 56-week, double-blind trial involving 3731 patients who did not have type 2 diabetes and who had a body-mass index (BMI; the weight in kilograms divided by the square of the height in meters) of at least 30 or a BMI of at least 27 if they had treated or untreated dyslipidemia or hypertension. We randomly assigned patients in a 2:1 ratio to receive once-daily subcutaneous injections of liraglutide at a dose of 3.0 mg (2487 patients) or placebo (1244 patients); both groups received counseling on lifestyle modification. The coprimary end points were the change in body weight and the proportions of patients losing at least 5% and more than 10% of their initial body weight. RESULTS At baseline, the mean (±SD) age of the patients was 45.1±12.0 years, the mean weight was 106.2±21.4 kg, and the mean BMI was 38.3±6.4; a total of 78.5% of the patients were women and 61.2% had prediabetes. At week 56, patients in the liraglutide group had lost a mean of 8.4±7.3 kg of body weight, and those in the placebo group had lost a mean of 2.8±6.5 kg (a difference of -5.6 kg; 95% confidence interval, -6.0 to -5.1; P<0.001, with last-observation-carried-forward imputation). A total of 63.2% of the patients in the liraglutide group as compared with 27.1% in the placebo group lost at least 5% of their body weight (P<0.001), and 33.1% and 10.6%, respectively, lost more than 10% of their body weight (P<0.001). The most frequently reported adverse events with liraglutide were mild or moderate nausea and diarrhea. Serious events occurred in 6.2% of the patients in the liraglutide group and in 5.0% of the patients in the placebo group. CONCLUSIONS In this study, 3.0 mg of liraglutide, as an adjunct to diet and exercise, was associated with reduced body weight and improved metabolic control. (Funded by Novo Nordisk; SCALE Obesity and Prediabetes NN8022-1839 ClinicalTrials.gov number, NCT01272219.). Copyright © 2015 Massachusetts Medical Society.

Amara N.,Ben - Gurion University of the Negev | Krom B.P.,University of Groningen | Kaufmann G.F.,Scripps Research Institute | Meijler M.M.,Ben - Gurion University of the Negev
Chemical Reviews | Year: 2011

Different signaling systems have been discovered, with still more proteins and small molecules involved in QS likely to be uncovered in future. Interactions between plants and microorganisms are ubiquitous. The ascent of plants on land and its subsequent first interactions with soil microbes has been estimated to have occurred around 500 million years ago, and the ability of plants to defend themselves against continuous attacks of pathogens has been paramount to their evolutionary success. The first QSM-inactivating enzyme was identified by Dong by screening treated soil samples and laboratory bacterial collections. The use of AHL-degrading enzymes has been proposed as a strategy to attenuate bacterial virulence. Reimmann and co-workers reported that over-expression of aiiA homologues of two Bacillus spp in P. aeruginosa PAO1 resulted in decreased production of elastase (LasB).

Phinney D.G.,Scripps Research Institute
Journal of Cellular Biochemistry | Year: 2012

The term mesenchymal stem cell (MSCs) was adopted in the 1990s to describe a population of bone-marrow-derived cells that demonstrated the capacity for tri-lineage differentiation at a clonal level. Research conducted during the ensuing decades has demonstrated that MSCs fulfill many functions in addition to connective tissue progenitors including contributing to the HSC niche and regulating the function of immune effector cells of both the innate and adaptive immune system. Despite these advances, fundamental aspects of MSC biology remain indeterminate. For example, the embryonic origin of MSCs and their niche in vivo remains a highly debated topic. More importantly, the mechanisms that regulate self-renewal and lineage specification have also been largely unexplored. The later is significant in that MSC population's exhibit considerable donor-to-donor and intra-population heterogeneity but knowledge regarding how different functional attributes of MSCs are specified at the population level is unknown. This poses significant obstacles in research and in efforts to develop clinical manufacturing protocols that reproducibly generate functionally equivalent MSC populations. Herein, I discuss data demonstrating that MSC populations are intrinsically heterogeneous, elaborate on the molecular basis for this heterogeneity, and discuss how heterogeneity impacts clinical manufacturing and the therapeutic potency of MSCs. © 2012 Wiley Periodicals, Inc.

Schork N.J.,Scripps Research Institute
Genome Medicine | Year: 2013

Integration of clinical evaluations and whole-genome sequence data from eight individuals in a recent study demonstrates that genetic and clinical information can be combined and applied to preventive medicine. Statistical and graphical tools were developed to assess and visualize the genetic risk of common chronic conditions and to show the changes in disease risk that result from monitoring clinical symptoms over time. This approach provides a direction to consider in the adoption of genetic information in health care, but, like all provocative scientific articles, it raises as many questions as it answers.Please see related Research: http://genomemedicine.com/content/5/6/58. © 2013 BioMed Central Ltd.

Clauson C.,University of Pittsburgh | Scharer O.D.,State University of New York at Stony Brook | Niedernhofer L.,University of Pittsburgh | Niedernhofer L.,Scripps Research Institute
Cold Spring Harbor Perspectives in Medicine | Year: 2013

DNA inter strand cross-links (ICLs) are lesions caused by a variety of endogenous metabolites, environmental exposures, and cancer chemotherapeutic agents that have two reactive groups. The common feature of these diverse lesions is that two nucleotides on opposite strands are covalently joined. ICLs prevent the separation of two DNA strands and therefore essential cellular processes including DNA replication and transcription. ICLs are mainly detected in S phase when a replication fork stalls at an ICL. Damage signaling and repair of ICLs are promoted by the Fanconi anemia pathway and numerous posttranslational modifications of DNA repair and chromatin structural proteins. ICLs are also detected and repaired in non replicating cells, although the mechanism is less clear. Aunique feature of ICL repair is that both strands of DNA must be incised to completely remove the lesion. This is accomplished in sequential steps to prevent creating multiple double-strand breaks. Unhooking of an ICL from one strand is followed by translesion synthesis to fill the gap and create an intact duplex DNA, harboring a remnant of the ICL. Removal of the lesion from the second strand is likely accomplished by nucleotide excision repair. Inadequate repair of ICLs is particularly detrimental to rapidly dividing cells, explaining the bone marrow failure characteristic of Fanconi anemia and why cross-linking agents are efficacious in cancer therapy. Herein, recent advances in our understanding of ICLs and the biological responses they trigger are discussed. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

Raehal K.M.,Scripps Research Institute
Handbook of experimental pharmacology | Year: 2014

Pain is a complex disorder with neurochemical and psychological components contributing to the severity, the persistence, and the difficulty in adequately treating the condition. Opioid and cannabinoids are two classes of analgesics that have been used to treat pain for centuries and are arguably the oldest of "pharmacological" interventions used by man. Unfortunately, they also produce several adverse side effects that can complicate pain management. Opioids and cannabinoids act at G protein-coupled receptors (GPCRs), and much of their effects are mediated by the mu-opioid receptor (MOR) and cannabinoid CB1 receptor (CB1R), respectively. These receptors couple to intracellular second messengers and regulatory proteins to impart their biological effects. In this chapter, we review the role of the intracellular regulatory proteins, β-arrestins, in modulating MOR and CB1R and how they influence the analgesic and side-effect profiles of opioid and cannabinoid drugs in vivo. This review of the literature suggests that the development of opioid and cannabinoid agonists that bias MOR and CB1R toward G protein signaling cascades and away from β-arrestin interactions may provide a novel mechanism by which to produce analgesia with less severe adverse effects.

Cahalan S.M.,Scripps Research Institute
Current Topics in Microbiology and Immunology | Year: 2014

The zwitterionic lysophospholipid Sphingosine 1-Phosphate (S1P) is a pleiotropic mediator of physiology and pathology. The synthesis, transport, and degradation of S1P are tightly regulated to ensure that S1P is present in the proper concentrations in the proper location. The binding of S1P to five G protein-coupled S1P receptors regulates many physiological systems, particularly the immune and vascular systems. Our understanding of the functions of S1P has been aided by the tractability of the system to both chemical and genetic manipulation. Chemical modulators have been generated to affect most of the known components of S1P biology, including agonists of S1P receptors and inhibitors of enzymes regulating S1P production and degradation. Genetic knockouts and manipulations have been similarly engineered to disrupt the functions of individual S1P receptors or enzymes involved in S1P metabolism. This chapter will focus on the development and utilization of these chemical and genetic tools to explore the complex biology surrounding S1P and its receptors, with particular attention paid to the in vivo findings that these tools have allowed for. © Springer International Publishing Switzerland 2014.

Kelso J.M.,Scripps Research Institute
Expert Review of Vaccines | Year: 2014

The most serious form of type I or IgE-mediated hypersensitivity reaction is anaphylaxis. A standardized case definition of anaphylaxis as an adverse event after immunization has been developed. Such reactions to vaccines, including influenza vaccine, are rare but potentially life-threatening. Until recently, all influenza vaccines were manufactured in eggs. Residual egg protein in the vaccines was thought to pose a risk to egg-allergic vaccine recipients. However, a large number of recent studies have demonstrated that egg-allergic recipients are no more likely than those without egg allergy to suffer such reactions. Published guidelines have been updated to recommend that patients with egg allergy receive annual influenza vaccination. Any patient who has an anaphylactic reaction to influenza vaccine should be carefully evaluated by an allergist for guidance on subsequent immunization. © Informa UK, Ltd.

Histone deacetylases (HDACs) enzymes, which affect the acetylation status of histones and other important cellular proteins, have been recognized as potentially useful therapeutic targets for a broad range of human disorders. Emerging studies have demonstrated that different types of HDAC inhibitors show beneficial effects in various experimental models of neurological disorders. HDAC enzymes comprise a large family of proteins, with18 HDAC enzymes currently identified in humans. Hence, an important question for HDAC inhibitor therapeutics is which HDAC enzyme(s) is/are important for the amelioration of disease phenotypes, as it has become clear that individual HDAC enzymes play different biological roles in the brain. This review will discuss evidence supporting the involvement of HDAC1 and HDAC3 in polyglutamine disorders, including Huntington's disease, and the use of HDAC1- and HDAC3-selective HDAC inhibitors as therapeutic intervention for these disorders. Further, while HDAC inhibitors are known alter chromatin structure resulting in changes in gene transcription, understanding the exact mechanisms responsible for the preclinical efficacy of these compounds remains a challenge. The potential chromatin-related and non-chromatin-related mechanisms of action of selective HDAC inhibitors will also be discussed. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Shen K.,California Institute of Technology | Zhang X.,Scripps Research Institute | Shan S.-O.,California Institute of Technology
RNA | Year: 2011

During cotranslational protein targeting by the Signal Recognition Particle (SRP), the correct cargo accelerates stable complex assembly between the SRP and SRP receptor (FtsY) by several orders of magnitude, thus enabling rapid and faithful cargo delivery to the target membrane. The molecular mechanism underlying this cargo-induced rate acceleration has been unclear. Here we show that the SRP RNA allows assembly of the SRP-FtsY complex to be specifically stimulated by a correct cargo, and, reciprocally, a correct cargo enables the SRP RNA to optimize its electrostatic interactions with FtsY. These results combined with recent structural work led us to suggest a "conformational selection" model that explains the synergistic action of the SRP RNA with the cargo in accelerating complex assembly. In addition to its previously proposed role in preventing the premature dissociation of SRP and FtsY, we found that the SRP RNA also plays an active role in ensuring the formation of productive assembly intermediates, thus guiding the SRP and FtsY through the most efficient pathway of assembly. Copyright © 2011 RNA Society.

Barouch-Bentov R.,Stanford University | Sauer K.,Scripps Research Institute
Expert Opinion on Investigational Drugs | Year: 2011

Introduction: Because of their important roles in disease and excellent druggability, kinases have become the second largest drug target family. The great success of the BCR-ABL inhibitor imatinib in treating chronic myelogenous leukemia illustrates the high potential of kinase inhibitor (KI) therapeutics, but also unveils a major limitation: the development of drug resistance. This is a significant concern as KIs reach large patient populations for an expanding array of indications. Areas covered: We provide an up-to-date understanding of the mechanisms through which KIs function and through which cells can become KI-resistant. We review current and future approaches to overcome KI resistance, focusing on currently approved KIs and KIs in clinical trials. We then discuss approaches to improve KI efficacy and overcome drug resistance and novel approaches to develop less drug resistance-prone KI therapeutics. Expert opinion: Although drug resistance is a concern for current KI therapeutics, recent progress in our understanding of the underlying mechanisms and promising technological advances may overcome this limitation and provide powerful new therapeutics. © 2011 Informa UK, Ltd.

Nicolaou K.C.,Scripps Research Institute | Nicolaou K.C.,University of California at San Diego
Angewandte Chemie - International Edition | Year: 2012

Thiostrepton, a powerful antibiotic belonging to the thiopeptide class, was synthesized in the laboratory for the first time in 2004 through an arduous campaign involving novel strategies and tactics, scenic detours, and unexpected roadblocks. In this Review the author narrates the long journey to success, not so dissimilar to Odysseus' return voyage to Ithaca, full of adventure, knowledge, and wisdom. Inspired by the writing form of Mahaffy,1 the author has taken liberties with the style in which this Review is written. An adventurous undertaking: The synthetic conquest of thiostrepton was achieved in 2004. In this vivid account the author describes the laboratory odyssey with its many intriguing twists and turns that led to this memorable total synthesis. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plummer E.M.,Scripps Research Institute | Plummer E.M.,University of California at San Diego | Manchester M.,University of California at San Diego
Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology | Year: 2011

Current vaccines that provide protection against infectious diseases have primarily relied on attenuated or inactivated pathogens. Virus-like particles (VLPs), comprised of capsid proteins that can initiate an immune response but do not include the genetic material required for replication, promote immunogenicity and have been developed and approved as vaccines in some cases. In addition, many of these VLPs can be used as molecular platforms for genetic fusion or chemical attachment of heterologous antigenic epitopes. This approach has been shown to provide protective immunity against the foreign epitopes in many cases. A variety of VLPs and virus-based nanoparticles are being developed for use as vaccines and epitope platforms. These particles have the potential to increase efficacy of current vaccines as well as treat diseases for which no effective vaccines are available. Copyright © 2010 John Wiley & Sons, Inc.

The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC's cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC's cytoprotective versus thrombin's proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects.

Interleukin-7 (IL-7) is essential to T-cell survival as well as homeostatic proliferation, and clinical trials that exploit the mitogenic effects of IL-7 have achieved success in treating human diseases. In mice, the in vivo potency of IL-7 improves dramatically when it is administered as a complex with the anti-IL-7 neutralizing monoclonal antibody clone M25. However, the mechanism whereby M25 augments IL-7 potency is unknown. We have analyzed the discrete contributions of the antibody constant (Fc) and IL-7-binding (Fab) domains to the mechanism. By engaging the neonatal Fc receptor the Fc domain extends the in vivo lifespan of IL-7/M25 complexes and accounts for the majority of their activity. Unexpectedly, the IL-7-neutralizing Fab domain provides an additional, albeit smaller, contribution, possibly by serving as a cytokine depot. This study is the first to demonstrate that the neutralizing aspect of the monoclonal antibody is directly involved in enhancing the potency of a cytokine with a single form of receptor. Lessons from the mechanism of IL-7/M25 complexes inform the design of next-generation cytokine therapeutics.

Gulder T.,RWTH Aachen | Baran P.S.,Scripps Research Institute
Natural Product Reports | Year: 2012

Covering: up to 2011 Cyclophane natural products comprise an intriguing class of structurally diverse compounds. As inherent for all cyclic compounds regardless of their origin, macrocyclization is naturally the most decisive step, which defines the overall efficiency of the synthetic pathway. Especially in small cyclophane molecules, this key step constitutes an even greater challenge. Due to the strain imparted by the macrocyclic system, free rotation of the benzene ring(s) is often restricted depending on both the constitution of the tether and the aromatic portions. Not surprisingly, the synthesis of natural cyclophanes with their often outstanding pharmaceutical activities and the inherent issues associated with their preparation has attracted much attention among the synthetic community. In particular, it stimulated the development of new strategies for the ring-closing step, as often otherwise well established and robust reactions fail to perform effectively. In this review, we describe the challenges synthetic chemists are facing during the synthesis of this small, but structurally and biologically fascinating class of natural products, concentrating on the representatives exhibiting configurational stability. The main focus will be on the different concepts for the installation of the macrocyclic system, in most cases the central problem in assembling these extremely rigid molecules. This journal is © The Royal Society of Chemistry 2012.

Leow D.,Scripps Research Institute | Tan C.-H.,National University of Singapore
Synlett | Year: 2010

This account chronicles our discovery and development of catalytic reactions using chiral guanidines and guanidinium salts. 1 Introduction 2 Chiral Guanidines as Brnsted Bases 2.1 Synthetic Route for the Catalyst 2.2 Michael Reactions 2.3 Henry Reactions 2.4 Phospha-Michael Reactions 2.5 Diels-Alder Reactions 2.6 Protonation Reactions 2.7 Isomerization Reactions 2.8 Mannich Reactions 3 Catalytic Reactions of Chiral Guanidinium Salts 3.1 Michael Reactions 3.2 Phospha-Mannich Reactions 4 Chiral Guanidines as Ligands 5 Conclusions and Outlook.

Rambo R.P.,Physical Biosciences Division | Tainer J.A.,Lawrence Berkeley National Laboratory | Tainer J.A.,Scripps Research Institute
Annual Review of Biophysics | Year: 2013

Posttranslational modification is an evolutionarily conserved mechanism for regulating protein activity, binding affinity, and stability. Compared with established posttranslational modifications such as phosphorylation or ubiquitination, posttranslational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, posttranslational modification by protons can drive dynamic changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and, in contrast to most other posttranslational modifications, does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton posttranslational modification of pH-sensing proteins regulating different cellular processes. Copyright © 2013 by Annual Reviews.

Signal transduction systems are influenced by positive and negative forces resulting in an output reflecting the sum of the opposing forces. The Rap family of regulatory protein modules control the output of two-component signal transduction systems through protein:protein and protein:peptide interactions. These modules and their peptide regulators are found in complex signaling pathways, including the bacterial developmental pathway to sporulation, competence, and protease secretion. Two articles published in the current issue of PLOS Biology reveal by means of crystallographic analyses how the Rap proteins of bacilli are regulated by their inhibitor Phr peptide and provide a mechanistic explanation for a genetic phenotype isolated decades earlier. The Rap-Phr module of bacterial regulators was the prototype of a family that now extends to other bacterial signaling proteins that involve the use of the tetratricopeptide repeat structural fold. The results invite speculation regarding the potential exploitation of this module as a molecular tool for applications in therapeutic design and biotechnology. © 2013 Marta Perego.

Paegel B.M.,Scripps Research Institute
Current Opinion in Chemical Biology | Year: 2010

Evolution at its heart is an iterative algorithm composed of three steps: selection, amplification and mutagenesis. This algorithm can be applied to complex inputs such as populations of whole organisms and viruses, or mixtures of bare nucleic acids and proteins. The output is the same: evolutionary adaptation of new and improved function subject to selection. Recent breakthroughs in microfluidic technology have introduced automation and process monitoring to in vitro evolution, and reproducible preparation of emulsions and other multi-phase reaction landscapes. It is at this intersection of compartmentalization and in vitro evolution where miniaturization is again redefining experimental design in contemporary chemistry and biology. © 2010 Elsevier Ltd.

Ruiz-Castillo P.,Massachusetts Institute of Technology | Blackmond D.G.,Scripps Research Institute | Buchwald S.L.,Massachusetts Institute of Technology
Journal of the American Chemical Society | Year: 2015

We report the Pd-catalyzed arylation of very hindered α,α,α-trisubstituted primary amines. Kinetics-based mechanistic analysis and rational design have led to the development of two biarylphosphine ligands that allow the transformation to proceed with excellent efficiency. The process was effective in coupling a wide range of functionalized aryl and heteroaryl halides under mild conditions. © 2015 American Chemical Society.

Rumbaugh G.,Scripps Research Institute
Methods in molecular biology (Clifton, N.J.) | Year: 2011

Recent years have witnessed an explosion of research on the role of epigenetic modifications, such as DNA methylation and histone protein acetylation and phosphorylation, in neuroscience. These changes exert control over gene expression and have been shown to play important roles in a variety of neural processes, including learning and memory. We and others have also recently shown that epigenetic changes may contribute to neurodegenerative disorders, such as Alzheimer's disease. Western blot analysis with antibodies raised against specific histone modifications is a relatively simple technique able to reveal the type, location, and degree of histone posttranslational modifications produced by an experimental manipulation. Here we provide a step-by-step protocol for isolating histone proteins from tissue and measuring these posttranslational modifications.

Ruf W.,Scripps Research Institute
Thrombosis Research | Year: 2012

The hemostatic system is involved in multiple interactions with transformed cells that progress from a dormant, non-vascularized tumor to highly metastatic phenotypes. Oncogenic transformations up regulate not only the initiator of the coagulation cascade, tissue factor (TF), but also induce other molecules that are required for TF's direct cell signaling activity, including the protease activated receptor (PAR) 2 and factor VIIa. TF-dependent signaling is a major driver for primary tumor progression, whereas TF-initiated coagulation and other components of the hemostatic system support metastasis. Basic research continues to identify pivotal molecular interactions in these processes and provides potential leads for targeting specific tumor promoting pathways associated with hemostasis and thrombosis. © 2012 Elsevier Ltd.

Miyaki S.,Hiroshima University | Asahara H.,Scripps Research Institute
Nature Reviews Rheumatology | Year: 2012

Osteoarthritis (OA), the most common musculoskeletal disorder, is complex, multifaceted, and characterized by degradation of articular cartilage and alterations in other joint tissues. Although some pathogenic pathways have been characterized, current knowledge is incomplete and effective approaches to the prevention or treatment of OA are lacking. Understanding novel molecular mechanisms that are involved in the maintenance and destruction of articular cartilage, including extracellular regulators and intracellular signalling mechanisms in joint cells that control cartilage homeostasis, has the potential to identify new therapeutic targets in OA. MicroRNAs control tissue development and homeostasis by fine-tuning gene expression, with expression patterns specific to tissues and developmental stages, and are increasingly implicated in the pathogenesis of complex diseases such as cancer and cardiovascular disorders. The emergent roles of microRNAs in cartilage homeostasis and OA pathogenesis are summarized in this Review, alongside potential clinical applications. © 2012 Macmillan Publishers Limited. All rights reserved.

Tan E.M.,Scripps Research Institute
Journal of Clinical Investigation | Year: 2012

The appearance of autoantibody to DNA followed sequentially by the disappearance of anti-DNA and appearance of DNA antigen in a patient with systemic lupus erythematosus demonstrated that autoantibodies participate in immune complex-mediated pathogenesis. Continuing studies showed that autoantibodies are also useful biomarkers in clinical diagnosis and important reagents for elucidating the structure and function of intracellular proteins in cell biology. Recently, autoantibodies to tumor-associated antigens have been identified in cancer, and these findings have expanded the field of cancer immunodiagnostics.

Fujioka K.,Scripps Research Institute
Diabetes, Obesity and Metabolism | Year: 2010

Weight loss is a primary goal of therapy in overweight patients with type 2 diabetes. This review examines whether positive patient outcomes are observed even after relatively small amounts of weight loss, that is, weight loss being more easily attainable in practice. Clinical studies demonstrate that therapeutic benefit rises with increasing weight loss, but that losses as low as 0.45-4 kg (1-9 lb) have positive effects on metabolic control, cardiovascular risk factors and mortality rates. Even the intention to lose weight, without significant success, can improve outcomes in patients with diabetes, presumably because of the healthy behaviours associated with the attempt. The current data support a continued focus on weight loss, including moderate weight loss, as a key component of good care for overweight patients with type 2 diabetes. © 2010 Blackwell Publishing Ltd.

Topol E.J.,Scripps Research Institute
Science Translational Medicine | Year: 2012

Contrary to the impression given by Roberts et al. and its coverage in the media, the notion that genetic information cannot with certainty predict disease in an individual is well known, but its precision will improve as more kinds of genetic variability can be ascertained.

The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O 2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by n O→σ* S-OH orbital interactions, which force the -OH group to adopt a position trans to the S· · ·O interaction, leading to an almost linear arrangement of the O· · ·S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S· · ·N or S· · ·O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide. © 2013 Elsevier B.V. All rights reserved.

Desponts C.,Scripps Research Institute
Methods in molecular biology (Clifton, N.J.) | Year: 2010

Induction of pluripotent stem cells from somatic cells by defined factors was shown to be possible only recently, but already several laboratories have made tremendous strive toward improving and understanding the process. Originally, Oct4, Sox2, Klf4, and cMyc were identified as being the combination of genes necessary to induce reprogramming. It was later shown that cMyc was dispensable; however, in its absence the process was less efficient and took a considerably longer period of time to occur. Furthermore, others have shown that the combination of Oct4, Sox2, Nanog, and Lin28 could also induce reprogramming. One major caveat associated with these techniques remains the need for overexpression of several genes using viral systems. Until very recently, most studies were done using integrating viruses such as retroviruses and lentiviruses. This method ensured that the protein of interested would be expressed at a high concentration and for an adequate period of time necessary to induce reprogramming. Up to date, others have now been able to use different nonintegrative method such as adenovirus and plasmid transfection to induce reprogramming. Furthermore, piggyBac transposition was successfully used to induce reprogramming of murine cells. Most importantly, it was recently published that reprogramming can be induced in the absence of virus, with proteins and small molecules. All of the later methods are appealing since they do not require the integration of the virus or plasmid to exert its effect. However, one avenue that would be all the more therapeutically appealing would be to induce reprogramming in the absence of gene overexpression systems, using small molecules to modulate signaling pathways in the somatic cells. A few molecules have already been identified with the ability to either improve the process or replace one or two of the genes deemed necessary for reprogramming. We have screened successfully for compounds that can replace some of these factors, and share the methods developed following these screens.

Yang J.,A GSK Company | Sundrud M.S.,Scripps Research Institute | Skepner J.,A GSK Company | Yamagata T.,A GSK Company
Trends in Pharmacological Sciences | Year: 2014

T helper 17 (Th17) cells have been implicated in the pathogenesis of most common autoimmune diseases, including psoriasis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and multiple sclerosis (MS). Although anti-interleukin-17 (IL-17) antibodies show marked clinical efficacy in psoriasis, targeting IL-17 alone is not sufficient to improve clinical end points in other autoimmune conditions, namely RA and Crohn's disease. Given that Th17 cells express IL-17 together with many other proinflammatory cytokines [IL-17F, IL-22, IL-26, and granulocyte-macrophage colony-stimulating factor (GM-CSF)], targeting the Th17 cell lineage may be superior to blocking a single effector cytokine. Here, we discuss the rationale for targeting two checkpoints in the development and inflammatory function of Th17 cells, retinoid-related orphan receptor-γt (RORγt) and IL-23, and we review recent progress in the development of both RORγt small molecule inhibitors and IL-23 neutralizing antibodies. © 2014 Elsevier Ltd.

Oldstone M.B.A.,Scripps Research Institute
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy | Year: 2014

On a clonal level, certain antibodies and T cells can interact with dissimilar antigens found in microbes and in host cells. More than 5% of over 800 monoclonal antibodies derived from multiple RNA and DNA viruses, as well as from a large number of T cell clones, engage in such interactions. Several of these cross-reactions, which we termed molecular mimicry, are against unique host proteins involved in autoimmune responses and diseases. Thus, molecular mimicry initiated as a host response to a virus or a microbial infection, but alternatively cross-reacting with an appropriate host-antigen, can be a mechanism for instigating an autoimmune disease. Molecular mimicry provides an explanation for the genetic observation that identical twins rarely manifest the same autoimmune disease and the documented epidemiologic evidence that microbial and/or viral infections often precede autoimmune disorders. © 2014, Mary Ann Liebert, Inc. 2014.

Miyamoto T.,Scripps Research Institute
Nature communications | Year: 2011

Nitric oxide (NO) is an unstable signalling molecule synthesized de novo mainly from L-arginine by NO synthase (NOS) enzymes. Nitrite reduction can also produce NO, predominantly within body fluids (for example, saliva, sweat and blood plasma) and under extreme hypoxic and acidic conditions. It remains unknown if intracellular canonical signalling pathways regulate nitrite-dependent NO production. Here we examine NO production in the skin, a hypoxic tissue enriched in nitrites wherein NO has important roles in wound healing and other biological processes. We show that activation of TRPV3, a heat-activated transient receptor potential ion channel expressed in keratinocytes, induces NO production via a nitrite-dependent pathway. TRPV3 and nitrite are involved in keratinocyte migration in vitro and in wound healing and thermosensory behaviours in vivo. Our study demonstrates that activation of an ion channel can induce NOS-independent NO production in keratinocytes.

Feeney A.J.,Scripps Research Institute
Current Opinion in Immunology | Year: 2011

Recent studies of the regulation of antigen receptor rearrangement have revealed several completely new levels of control. Not only do antigen receptor loci undergo changes in histone modifications as they become accessible for recombination, but also the number of different histone modifications and the variation at different parts of each receptor locus reveal great complexity. RAG2 is now known to bind to one of these histone modifications, H3K4me3, and this targets the initial RAG binding events to the J genes. The large megabase receptor loci undergo 3D changes in their structure during rearrangement, and receptor loci move throughout the nucleus, transiently binding to heterochromatin, and transiently pairing with each other. RAG-mediated DNA breaks promote some of these movements, and also result in widespread changes in the transcriptional profile promoting differentiation. © 2010 Elsevier Ltd.

Rambo R.P.,Lawrence Berkeley National Laboratory | Tainer J.A.,Lawrence Berkeley National Laboratory | Tainer J.A.,Scripps Research Institute
Biopolymers | Year: 2011

Unstructured proteins, RNA or DNA components provide functionally important flexibility that is key to many macromolecular assemblies throughout cell biology. As objective, quantitative experimental measures of flexibility and disorder in solution are limited, small angle scattering (SAS), and in particular small angle X-ray scattering (SAXS), provides a critical technology to assess macromolecular flexibility as well as shape and assembly. Here, we consider the Porod-Debye law as a powerful tool for detecting biopolymer flexibility in SAS experiments. We show that the Porod-Debye region fundamentally describes the nature of the scattering intensity decay by capturing the information needed for distinguishing between folded and flexible particles. Particularly for comparative SAS experiments, application of the law, as described here, can distinguish between discrete conformational changes and localized flexibility relevant to molecular recognition and interaction networks. This approach aids insightful analyses of fully and partly flexible macromolecules that is more robust and conclusive than traditional Kratky analyses. Furthermore, we demonstrate for prototypic SAXS data that the ability to calculate particle density by the Porod-Debye criteria, as shown here, provides an objective quality assurance parameter that may prove of general use for SAXS modeling and validation. © 2011 Wiley Periodicals, Inc.

Sutherland M.R.,University of British Columbia | Ruf W.,Scripps Research Institute | Pryzdial E.L.G.,University of British Columbia
Blood | Year: 2012

The coagulation system provides physiologic host defense, but it can also be exploited by pathogens for infection. On the HSV1 surface, host-cell-derived tissue factor (TF) and virus-encoded glyco-protein C (gC) can stimulate protease activated receptor 1 (PAR1)-enhanced infection by triggering thrombin production. Using novel engineered HSV1 variants deficient in either TF and/or gC, in the present study, we show that activated coagulation factors X (FXa) or VII (FVIIa) directly affect HSV1 infection of human umbilical vein endothelial cells in a manner that is dependent on viral TF and gC. The combination of FXa and FVIIa maximally enhanced infection for TF +/gC +HSV1 and receptor desensitization and Ab inhibition demonstrated that both proteases act on PAR2. Inhibitory TF Abs showed that the required TF source was viral. Individually, TF or gC partly enhanced the effect of FXa, but not FVIIa, revealing gC as a novel PAR2 cofactor for FVIIa. In sharp contrast, thrombin enhanced infection via PAR1 independently of viral TF and gC. Thrombin combined with FXa/FVIIa enhanced infection, suggesting that PAR1 and PAR2 are independently involved in virus propagation. These results show that HSV1 surface cofactors promote cellular PAR2-mediated infection, indicating a novel mode by which pathogens exploit the initiation phase of the host hemostatic system. © 2012 by The American Society of Hematology.

Kodadek T.,Scripps Research Institute
Chemical Communications | Year: 2011

Combinatorial chemistry provides a powerful tool for the rapid creation of large numbers of synthetic compounds. Ideally, these libraries should be a rich source of bioactive molecules, but there is the general feeling that the initial promise of combinatorial chemistry has not yet been realized. In particular, enthusiasm for conducting unbiased (non-structure-guided) screens of large libraries for protein or RNA ligands has waned. A central challenge in this area is to devise methods for the synthesis of chemically diverse, high-quality libraries of molecules with many of the desirable features of natural products. These include diverse functionality, a significant representation of chiral sp 3 centers that provide conformational bias to the molecule, significant skeletal diversity, and good pharmacokinetic properties. However, these libraries must be easy to make from cheap, readily available building blocks, ideally those that would support convenient hit optimization/structure reactivity relationship studies. Meeting these challenges will not be easy. Here I review some recent advances in this area and provide some thoughts on likely important developments in the next few years. © 2011 The Royal Society of Chemistry.

Bruce K.D.,Scripps Research Institute
Current Diabetes Reports | Year: 2014

The global prevalence of diabetes mellitus has reached epidemic proportions. In 2010, it was estimated that 6.4 % of the adult population (285 million) have diabetes. In recent years, the incidence of type 2 diabetes (T2D), a condition traditionally associated with aging, has been steadily increasing among younger individuals. It is now a well-established notion that the early-life period is a critical window of development and that influences during this period can "developmentally prime" the metabolic status of the adult. This review discusses the role of maternal and in utero influences on the developmental priming of T2D risk. Both human epidemiological studies and experimental animal models are beginning to demonstrate that early dietary challenges can accelerate the onset of age-associated metabolic disturbances, including insulin resistance, T2D, obesity, hypertension, and cardiovascular disease. These findings show that poor maternal nutrition can prime a prediabetes phenotype, often manifest as insulin resistance, by very early stages of life. Thus, the maternal diet is a critical determinant of premature T2D risk. While the mechanisms that link early nutrition to age-associated metabolic decline are currently unclear, preliminary findings suggest perturbations in a number of processes involved in cellular aging, such as changes in longevity-associated Sirtuin activity, epigenetic regulation of key metabolic genes, and mitochondrial dysfunction. Preliminary studies show that pharmacological interventions in utero and dietary supplementation in early postnatal life may alleviate insulin resistance and reduce T2D risk. However, further studies are warranted to fully understand the relationship between the early environment and long-term effects on metabolism. Such mechanistic insights will facilitate strategic interventions that prevent accelerated metabolic decline and the premature onset of T2D in the current and future generations. © 2013 Springer Science+Business Media New York.

Morris K.V.,University of New South Wales | Morris K.V.,Scripps Research Institute
Epigenetics | Year: 2015

Observations over the last decade suggest that some RNA transcripts, such as non-coding RNAs, function in regulating the transcriptional and epigenetic state of gene expression. DNA methylation appears to be operative in non-coding RNA regulation of gene expression. Interestingly, methylated cytosines undergo deamination to remove the methylation, which if not properly repaired results in the methylated cytosine being recognized by the cell as a thymine. This observation suggests that the process of non-coding RNA-directed epigenetic targeting also has the potential to alter the genomic landscape of the cell by changing cytosines to thymines and ultimately influence the evolution of the cell. This proposed theory of “RNA-mediated gene evolution” might be one possible mechanism of action whereby RNA participates in the natural selective process to drive cellular and possibly organismal evolution. © 2015 Taylor & Francis Group, LLC.

Chun J.,Scripps Research Institute | Hla T.,Cornell University | Lynch K.R.,University of Virginia | Spiegel S.,Virginia Commonwealth University | Moolenaar W.H.,Netherlands Cancer Institute
Pharmacological Reviews | Year: 2010

Lysophospholipids are cell membrane-derived lipids that include both glycerophospholipids such as lysophosphatidic acid (LPA) and sphingoid lipids such as sphingosine 1-phosphate (S1P). These and related molecules can function in vertebrates as extracellular signals by binding and activating G protein-coupled receptors. There are currently five LPA receptors, along with a proposed sixth (LPA 1-LPA 6), and five S1P receptors (S1P 1-S1P 5). A remarkably diverse biology and pathophysiology has emerged since the last review, driven by cloned receptors and targeted gene deletion ("knockout") studies in mice, which implicate receptor-mediated lysophospholipid signaling in most organ systems and multiple disease processes. The entry of various lysophospholipid receptor modulatory compounds into humans through clinical trials is ongoing and may lead to new medicines that are based on this signaling system. This review incorporates IUPHAR Nomenclature Committee guidelines in updating the nomenclature for lysophospholipid receptors (http://www.iuphar-db.org/DATABASE/ FamilyMenuForward?familyId=36). Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.

Kunicki T.J.,Scripps Research Institute | Nugent D.J.,Childrens Hospital of Orange County
Blood | Year: 2010

Genetic and environmental factors contribute to a substantial variation in platelet function seen among normal persons. Candidate gene association studies represent a valiant effort to define the genetic component in an era where genetic tools were limited, but the single nucleotide polymorphisms identified in those studies need to be validated by more objective, comprehensive approaches, such as genome-wide association studies (GWASs) of quantitative functional traits in much larger cohorts of more carefully selected normal subjects. During the past year, platelet count and mean platelet volume, which indirectly affect platelet function, were the subjects of GWAS. The majority of the GWAS signals were located to noncoding regions, a consistent outcome of all GWAS to date, suggesting a major role for mechanisms that alter phenotype at the level of transcription or posttranscriptional modifications. Of 15 quantitative trait loci associated with mean platelet volume and platelet count, one located at 12q24 is also a risk locus for coronary artery disease. In most cases, the effect sizes of individual quantitative trait loci are admittedly small, but the results of these studies have led to new insight into regulators of hematopoiesis and megakaryopoiesis that would otherwise be unapparent and difficult to define. © 2010 by The American Society of Hematology.

Chun J.,Scripps Research Institute
Trends in Endocrinology and Metabolism | Year: 2010

Lysophosphatidic acid (LPA) is a cell membrane phospholipid metabolite that can act as an extracellular signal. Its effects are mediated through at least five G protein-coupled receptors, LPA 1-5, and probably others as well. Studies in multiple species including LPAR-deficient mice and humans have identified or implicated important roles for receptor-mediated LPA signaling in multiple aspects of vertebrate reproduction. These include ovarian function, spermatogenesis, fertilization, early embryo development, embryo implantation, embryo spacing, decidualization, pregnancy maintenance and parturition. LPA signaling can also have pathological consequences, influencing aspects of endometriosis and ovarian cancer. Here we review recent progress in LPA signaling research relevant to female and male reproduction. © 2009 Elsevier Ltd. All rights reserved.

Kodadek T.,Scripps Research Institute
Current Opinion in Chemical Biology | Year: 2010

Antibodies are critical reagents in biological research and are increasingly being developed as therapeutic agents. They typically exhibit very high affinity and selectivity for their ligands. Synthetic protein-binding agents rarely achieve the combination of high affinity and selectivity for their target protein that is typical of a good antibody. However, significant efforts are underway to develop a new generation of protein ligands with improved properties. This article reviews progress towards this goal and suggests fruitful strategies for future research. © 2010 Elsevier Ltd.

Hayes D.F.,University of Michigan | Markus H.S.,University of Cambridge | Leslie R.D.,St. Bartholomews Hospital | Topol E.J.,Scripps Research Institute
BMC Medicine | Year: 2014

Personalized medicine is increasingly being employed across many areas of clinical practice, as genes associated with specific diseases are discovered and targeted therapies are developed. Mobile apps are also beginning to be used in medicine with the aim of providing a personalized approach to disease management. In some areas of medicine, patient-tailored risk prediction and treatment are applied routinely in the clinic, whereas in other fields, more work is required to translate scientific advances into individualized treatment. In this forum article, we asked specialists in oncology, neurology, endocrinology and mobile health technology to discuss where we are in terms of personalized medicine, and address their visions for the future and the challenges that remain in their respective fields. © 2014 Hayes et al.; licensee BioMed Central Ltd.

Nicolaou K.C.,Scripps Research Institute | Adsool V.A.,University of California at San Diego | Hale C.R.H.,Gilman Drive
Organic Letters | Year: 2010

(Figure Presented) PhI(OAc)2 In the presence of OsO4 (cat.) and 2,6-lutidine cleaves oleflnlc bonds to yield the corresponding carbonyl compounds, albeit, In some cases, with a-hydroxy ketones as byproduct. A more practical and clean protocol to effect oxidative cleavage of olefinic bonds involves NMO, OsO4 (cat.), 2,6-lutidine, and PhI(OAc) 2. © 2010 American Chemical Society.

Durieux J.,Salk Institute for Biological Studies | Wolff S.,Scripps Research Institute | Dillin A.,Salk Institute for Biological Studies
Cell | Year: 2011

The life span of C. elegans can be increased via reduced function of the mitochondria; however, the extent to which mitochondrial alteration in a single, distinct tissue may influence aging in the whole organism remains unknown. We addressed this question by asking whether manipulations to ETC function can modulate aging in a cell-non-autonomous fashion. We report that the alteration of mitochondrial function in key tissues is essential for establishing and maintaining a prolongevity cue. We find that regulators of mitochondrial stress responses are essential and specific genetic requirements for the electron transport chain (ETC) longevity pathway. Strikingly, we find that mitochondrial perturbation in one tissue is perceived and acted upon by the mitochondrial stress response pathway in a distal tissue. These results suggest that mitochondria may establish and perpetuate the rate of aging for the whole organism independent of cell-autonomous functions. © 2011 Elsevier Inc.

Webb D.R.,Scripps Research Institute
Biochemical Pharmacology | Year: 2014

Animals have been used as models to study inflammation and autoimmunity for more than 80 years. During that time it has been understood that although the use of such models is an important and necessary part of understanding human disease, they inevitably display significant differences from the human disease state. Since our understanding of human inflammation and autoimmunity is necessarily incomplete, it may be concluded that the animal models will also be reflective of the state of knowledge regarding such diseases. Nevertheless, animal models of rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis have been successfully used to enhance the understanding of the human disease and have made significant contributions to the development of powerful new therapies. However, there are exceptions. One of the most persistent has been the study of sepsis where the animal models have been woefully inadequate in uncovering targets for drug discovery and have led to repeated clinical failures. As will be explained, only by using newly developed genomics tools has it been possible to uncover the differences between sepsis in mice and sepsis in man. It is concluded that approaches using the newer genomic and proteomic data derived from human tissues, will make possible the development of animal models with more predictive power as aids to drug discovery. © 2013 Elsevier Inc.

Mosnier L.O.,Scripps Research Institute
Blood | Year: 2014

In this issue of Blood, Dinarvand and colleagues identify polyphosphate (polyP) as a potent mediator of proinflammatory effects induced by nuclear proteins such as histone H4 and high mobility group box 1 (HMGB1). Coagulation, platelet activation, and inflammation are intricately linked and regulatory mechanisms ensure a balanced response to infection and inflammation. Not only do these observations promote polyP to the ranks of an all-round proinflammatory and procoagulant agent, but also protection by activated protein C (APC) against these proinflammatory effects reveals an intricate battle between polyP and APC that is fought on multiple fronts. © 2014 by The American Society of Hematology.

Morris K.V.,Scripps Research Institute
Seminars in Cell and Developmental Biology | Year: 2011

Recent evidence suggests that particular species of non-coding RNAs can modulate gene transcription in human cells. While such observations were in the past relegated to imprinted genes, it is now becoming apparent that several different genes in differentiated cells may be under some form of RNA based regulatory control. Studies carried out to date have begun to discern the mechanism of action whereby non-coding RNAs modulate gene transcription by the targeted recruitment of epigenetic silencing complexes to homology containing loci in the genome. The results of these studies will be considered in detail as well as the implications that a vast array of non-coding RNA based regulatory networks may be operative in human cells. © 2011 Elsevier Ltd.

Lerner R.A.,Scripps Research Institute
Molecular BioSystems | Year: 2011

Convergence of observations from different sources is the norm in science. However, when convergence occurs in man for antibodies it is remarkable because the repertoire of possible immunoglobulin products is very large and diverse. Thus, one would not expect to see the same antibody twice from divergent populations unless there is special significance as to why the immune response is constrained. Now, broadly neutralizing antibodies isolated from combinatorial libraries from three separate populations have been shown to all use the same (V H 1-69) germ line gene and interact with the influenza virus in very similar ways. Here we discuss the reasons for this convergence in terms of how the immunological repertoire responds to emergency situations where time is short as occurs, for example, in potentially lethal infections. It is suggested that there is a first responder or S.O.S. component of the antibody repertoire that evolved to initiate rapid defense against infectious agents. The discovery of the homologies between these commonly produced antibodies may have significance for the design of novel vaccines. Finally, these convergent results may give much insight into why antibodies encoded by the V H 1-69 germ line gene are highly over represented in B-cell lymphomas. © The Royal Society of Chemistry.

Avram L.,Tel Aviv University | Cohen Y.,Tel Aviv University | Rebek Jr. J.,Scripps Research Institute
Chemical Communications | Year: 2011

Basic research in the chemistry of hexameric resorcin[4]arenes and pyrogallol[4]arenes during the last decade is reviewed. Applications of NMR methods to determine solution structures, host guest properties and exchange dynamics are discussed. The scientific issue is the behavior of molecules in small spaces; the challenge is to translate this information to practical applications in, say, catalysis or transport. © 2011 The Royal Society of Chemistry.

Bar-Peled L.,Scripps Research Institute | Sabatini D.M.,Whitehead Institute For Biomedical Research | Sabatini D.M.,Massachusetts Institute of Technology | Sabatini D.M.,Howard Hughes Medical Institute | Sabatini D.M.,The Broad Institute of MIT and Harvard
Trends in Cell Biology | Year: 2014

The mechanistic target of rapamycin complex I (mTORC1) is a central regulator of cellular and organismal growth, and hyperactivation of this pathway is implicated in the pathogenesis of many human diseases including cancer and diabetes. mTORC1 promotes growth in response to the availability of nutrients, such as amino acids, which drive mTORC1 to the lysosomal surface, its site of activation. How amino acid levels are communicated to mTORC1 is only recently coming to light by the discovery of a lysosome-based signaling system composed of Rags (Ras-related GTPases) and Ragulator v-ATPase, GATOR (GAP activity towards Rags), and folliculin (FLCN) complexes. Increased understanding of this pathway will not only provide insight into growth control but also into the human pathologies triggered by its deregulation. © 2014 Elsevier Ltd.

Colby D.A.,Gilead Sciences | Tsai A.S.,Scripps Research Institute | Bergman R.G.,University of California at Berkeley | Ellman J.A.,Yale University
Accounts of Chemical Research | Year: 2012

Over the last several decades, researchers have achieved remarkable progress in the field of organometallic chemistry. The development of metalcatalyzed cross-coupling reactions represents a paradigm shift in chemical synthesis, and today synthetic chemists can readily access carbon-carbon and carbon-heteroatom bonds from a vast array of starting compounds. Although we cannot understate the importance of these methods, the required prefunctionalization to carry out these reactions adds cost and reduces the availability of the starting reagents. The use of C-H bond activation in lieu of prefunctionalization has presented a tantalizing alternative to classical cross-coupling reactions. Researchers have met the challenges of selectivity and reactivity associated with the development of C-H bond functionalization reactions with an explosion of creative advances in substrate and catalyst design. Literature reports on selectivity based on steric effects, acidity, and electronic and directing group effects are now numerous. Our group has developed an array of C-H bond functionalization reactions that take advantage of a chelating directing group, and this Account surveys our progress in this area. The use of chelation control in C-H bond functionalization offers several advantages with respect to substrate scope and application to total synthesis. The predictability and decreased dependence on the inherent stereoelectronics of the substrate generally result in selective and high yielding transformations with broad applicability. The nature of the chelating moiety can be chosen to serve as a functional handle in subsequent elaborations. Our work began with the use of Rh(I) catalysts in intramolecular aromatic C-H annulations, which we further developed to include enantioselective transformations. The application of this chemistry to the simple olefinic C-H bonds found in r, β-unsaturated imines allowed access to highly substituted olefins, pyridines, and piperidines. We observed complementary reactivity with Rh(III) catalysts and developed an oxidative coupling with unactivated alkenes. Further studies on the Rh(III) catalysts led us to develop methods for the coupling of C-H bonds to polarized p bonds such as those in imines and isocyanates. In several cases the methods that we have developed for chelation-controlled C-H bond functionalization have been applied to the total synthesis of complex molecules such as natural products, highlighting the utility of these methods in organic synthesis. © 2011 American Chemical Society.

Cherezov V.,Scripps Research Institute
Current Opinion in Structural Biology | Year: 2011

Lipidic cubic phase (LCP) is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment. LCP technologies, however, have not been fully embraced by the membrane protein structural biology community, primarily because of the difficulties associated with handling viscous materials. Recent developments of pre-crystallization assays and improvements in crystal imaging, successes in obtaining high resolution structures of G protein-coupled receptors (GPCRs), and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research. This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein structures, shedding light on their functional mechanisms and on structural details of lipid-protein interactions. © 2011 Elsevier Ltd.

Gale A.J.,Scripps Research Institute
Toxicologic pathology | Year: 2011

The goal of this review is to briefly summarize the two primary pathways of hemostasis, primary hemostasis and secondary hemostasis, as well as to summarize anticoagulant mechanisms and fibrinolysis. In addition, this review will discuss pathologies of hemostasis and the mechanisms of the various drugs that are available to impact these pathways to prevent either thrombosis or bleeding. While many of the main drugs that are used to treat disorders of hemostasis have been used for decades, greater understanding of hemostasis has led to development of various new drugs that have come onto the market recently or are close to coming onto the market. Thus, improved understanding of hemostasis continues to lead to benefits for patients.

De La Cruz J.,University of Seville | Karbstein K.,Scripps Research Institute | Woolford J.L.,Carnegie Mellon University
Annual Review of Biochemistry | Year: 2015

The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth. Copyright © 2015 by Annual Reviews. All rights reserved.

Of the many approaches proposed to generalize the native chemical ligation approach for protein synthesis, the simple procedure of global desulfurization of peptide thiols has become the most widely adopted. In this review, the development of the native ligation-desulfurization strategy is described, focusing on the conversion of Cys to Ala following ligation at N-terminal Cys residues. Subsequent variations on this theme have broadened the scope to other natural amino acids including Phe, Leu, Val, and Lys, and even non-native peptide linkages such as isopeptide bonds on lysine side chains. Using insights from both selenocysteine-peptide side reactions and radical initiated desulfurization procedures, a new method for the selective deselenization of peptides containing both selenocysteine and cysteine residues has been developed. Together, these approaches represent a robust and flexible methodology for the synthesis of complex polypeptides without the use of protecting groups. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Au J.S.,Scripps Research Institute | Frenette C.T.,Center for Organ and Cell Transplantation
Gut and Liver | Year: 2015

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide. This cancer commonly arises against a background of chronic liver disease. As a result, a patient with HCC requires multidisciplinary care. Treatment options vary widely based on tumor burden and metastases. The most widely utilized staging system is the Barcelona Clinic Liver Cancer staging system, which recommends treatments based on tumor size and the underlying liver disease and functional status of the patient. Treatment options range from surgical resection or transplantation to locoregional therapies with modalities such as radiofrequency ablation and transarterial chemoembolization to systemic chemotherapies. Future care involves the development of combination therapies that afford the best tumor response, further clarification of the patients best suited for therapies and the development of new oral chemotherapeutic agents. © This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Tsutakawa S.E.,Cyclotron Road | Lafrance-Vanasse J.,Cyclotron Road | Tainer J.A.,Cyclotron Road | Tainer J.A.,Scripps Research Institute
DNA Repair | Year: 2014

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency. © 2014 Elsevier B.V.

Holmes D.R.,Mayo Medical School | Doshi S.K.,St Johns Health Center | Kar S.,Cedars Sinai Medical Center | Price M.J.,Scripps Research Institute | And 4 more authors.
Journal of the American College of Cardiology | Year: 2015

Abstract Background The risk-benefit ratio of left atrial appendage closure (LAAC) versus systemic therapy (warfarin) for prevention of stroke, systemic embolism, and cardiovascular death in nonvalvular atrial fibrillation (NVAF) requires continued evaluation. Objectives This study sought to assess composite data regarding left atrial appendage closure (LAAC) in 2 randomized trials compared to warfarin for prevention of stroke, systemic embolism, and cardiovascular death in patients with nonvalvular AF. Methods Our meta-analysis included 2,406 patients with 5,931 patient-years (PY) of follow-up from the PROTECT AF (Watchman Left Atrial Appendage System for Embolic Protection in Patients with Atrial Fibrillation) and PREVAIL (Prospective Randomized Evaluation of the Watchman LAA Closure Device In Patients With Atrial Fibrillation Versus Long Term Warfarin Therapy) trials, and their respective registries (Continued Access to PROTECT AF registry and Continued Access to PREVAIL registry). Results With mean follow-up of 2.69 years, patients receiving LAAC with the Watchman device had significantly fewer hemorrhagic strokes (0.15 vs. 0.96 events/100 patient-years [PY]; hazard ratio [HR]: 0.22; p = 0.004), cardiovascular/unexplained death (1.1 vs. 2.3 events/100 PY; HR: 0.48; p = 0.006), and nonprocedural bleeding (6.0% vs. 11.3%; HR: 0.51; p = 0.006) compared with warfarin. All-cause stroke or systemic embolism was similar between both strategies (1.75 vs. 1.87 events/100 PY; HR: 1.02; 95% CI: 0.62 to 1.7; p = 0.94). There were more ischemic strokes in the device group (1.6 vs. 0.9 and 0.2 vs. 1.0 events/100 PY; HR: 1.95 and 0.22, respectively; p = 0.05 and 0.004, respectively). Both trials and registries identified similar event rates and consistent device effect in multiple subsets. Conclusions In patients with NVAF at increased risk for stroke or bleeding who are candidates for chronic anticoagulation, LAAC resulted in improved rates of hemorrhagic stroke, cardiovascular/unexplained death, and nonprocedural bleeding compared to warfarin. © 2015 American College of Cardiology Foundation.

A proangiogenic function of tissue-infiltrating monocytes/macrophages has long been attributed to their matrix metalloproteinase-9 zymogen (proMMP-9). Herein, we evaluated the capacity of human monocytes, mature M0 macrophages, and M1- and M2-polarized macrophages to induce proMMP-9-mediated angiogenesis. Only M2 macrophages induced angiogenesis at levels comparable with highly angiogenic neutrophils previously shown to release their proMMP-9 in a unique form, free of tissue inhibitor of metalloproteinases-1 (TIMP-1). Macrophage differentiation was accompanied by induction of low-angiogenic, TIMP-1-encumbered proMMP-9. However, polarization toward the M2, but not the M1 phenotype, caused a substantial downregulation of TIMP-1 expression, resulting in production of angiogenic, TIMP-deficient proMMP-9. Correspondingly, the angiogenic potency of M2 proMMP-9 was lost after its complexing with TIMP-1, whereas TIMP-1 silencing in M0/M1 macrophages rendered them both angiogenic. Similar to human cells, murine bone marrow-derived M2 macrophages also shut down their TIMP-1 expression and produced proMMP-9 unencumbered by TIMP-1. Providing proof that angiogenic capacity of murine M2 macrophages depended on their TIMP-free proMMP-9, Mmp9-null M2 macrophages were nonangiogenic, although their TIMP-1 was severely downregulated. Our study provides a unifying molecular mechanism for high angiogenic capacity of TIMP-free proMMP-9 that would be uniquely produced in a pathophysiological microenvironment by influxing neutrophils and/or M2 polarized macrophages.

Vaccination with SIVmac239Δnef provides robust protection against subsequent challenge with wild type SIV, but safety issues have precluded designing an HIV-1 vaccine based on a live attenuated virus concept. Safe immunogens and adjuvants that could reproduce identified immune correlates of SIVmac239Δnef protection therefore offer an alternative path for development of an HIV vaccine. Here we describe SIV envelope trimeric gp41 (gp41t) immunogens based on a protective correlate of antibodies to gp41t concentrated on the path of virus entry by the neonatal Fc receptor (FcRn) in cervical vaginal epithelium. We developed a gp41t immunogen-MPLA adjuvant liposomal nanoparticle for intra-muscular immunization and a gp41t-Fc immunogen for intranasal immunization for pilot studies in mice, rabbits, and rhesus macaques. Repeated immunizations to mimic persistent antigen exposure in infection elicited gp41t antibodies in rhesus macaques that were detectable in FcRn+ cervical vaginal epithelium, thus recapitulating one key feature of SIVmac239Δnef vaccinated and protected animals. While this strategy did not reproduce the system of local production of antibody in SIVmac239Δnef-vaccinated animals, passive immunization experiments supported the concept that sufficiently high levels of antibody can be concentrated by the FcRn at mucosal frontlines, thus setting the stage for assessing protection against vaginal challenge by gp41t immunization. Copyright © 2016 Wolters Kluwer Health, Inc.

Wieland S.F.,Scripps Research Institute
Cold Spring Harbor Perspectives in Medicine | Year: 2015

Even before the discovery of hepatitis B virus (HBV), it was known that chimpanzees (Pan troglodytes) are susceptible to human hepatitis viruses. The chimpanzee is the only primate animal model for HBV infections. Much like HBV-infected human patients, chimpanzees can develop acute and chronic HBV infections and consequent hepatitis. Chimpanzees also develop a cellular immune response similar to that observed in humans. For these reasons, the chimpanzee has proven to be an invaluable model for investigations on HBV-driven disease pathogenesis and also the testing of novel antiviral therapies and prophylactic approaches. © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

Koob G.F.,Scripps Research Institute
Frontiers in Psychiatry | Year: 2013

Drug addiction can be defined by a three-stage cycle - binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation - that involves allostatic changes in the brain reward and stress systems. Two primary sources of reinforcement, positive and negative reinforcement, have been hypothesized to play a role in this allostatic process. The negative emotional state that drives negative reinforcement is hypothesized to derive from dysregulation of key neurochemical elements involved in the brain reward and stress systems. Specific neurochemical elements in these structures include not only decreases in reward system function (within-system opponent processes) but also recruitment of the brain stress systems mediated by corticotropin-releasing factor (CRF) and dynorphin-κ opioid systems in the ventral striatum, extended amygdala, and frontal cortex (both between-system opponent processes). CRF antagonists block anxiety-like responses associated with withdrawal, block increases in reward thresholds produced by withdrawal from drugs of abuse, and block compulsive-like drug taking during extended access. Excessive drug taking also engages the activation of CRF in the medial prefrontal cortex, paralleled by deficits in executive function that may facilitate the transition to compulsive-like responding. Neuropeptide Y, a powerful anti-stress neurotransmitter, has a profile of action on compulsive-like responding for ethanol similar to a CRF1 antagonist. Blockade of the ? opioid system can also block dysphoric-like effects associated with withdrawal from drugs of abuse and block the development of compulsive-like responding during extended access to drugs of abuse, suggesting another powerful brain stress system that contributes to compulsive drug seeking. The loss of reward function and recruitment of brain systems provide a powerful neurochemical basis that drives the compulsivity of addiction. © 2013 Koob.

Roberts T.C.,Scripps Research Institute | Morris K.V.,University of New South Wales | Wood M.J.,Oxford Genetics
Philosophical transactions of the Royal Society of London. Series B, Biological sciences | Year: 2014

Long non-coding RNAs (lncRNAs) are transcripts with low protein-coding potential that represent a large proportion of the transcriptional output of the cell. Many lncRNAs exhibit features indicative of functionality including tissue-restricted expression, localization to distinct subcellular structures, regulated expression and evolutionary conservation. Some lncRNAs have been shown to associate with chromatin-modifying activities and transcription factors, suggesting that a common mode of action may be to guide protein complexes to target genomic loci. However, the functions (if any) of the vast majority of lncRNA transcripts are currently unknown, and the subject of investigation. Here, we consider the putative role(s) of lncRNAs in neurodevelopment and brain function with an emphasis on the epigenetic regulation of gene expression. Associations of lncRNAs with neurodevelopmental/neuropsychiatric disorders, neurodegeneration and brain cancers are also discussed. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Seo Y.H.,University of Michigan | Carroll K.S.,Scripps Research Institute
Angewandte Chemie - International Edition | Year: 2011

Quantitative proteomics: The new technique mentioned in the title-in short, ICDID-enables quantification of sulfenic acid modifications in proteins (see picture). The approach permits S-hydroxylation site occupancy to be monitored at individual cysteines within a single protein and is compatible with peptide-based proteomic strategies. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Yung Y.C.,Scripps Research Institute
Science translational medicine | Year: 2011

Fetal hydrocephalus (FH), characterized by the accumulation of cerebrospinal fluid, an enlarged head, and neurological dysfunction, is one of the most common neurological disorders of newborns. Although the etiology of FH remains unclear, it is associated with intracranial hemorrhage. Here, we report that lysophosphatidic acid (LPA), a blood-borne lipid that activates signaling through heterotrimeric guanosine 5'-triphosphate-binding protein (G protein)-coupled receptors, provides a molecular explanation for FH associated with hemorrhage. A mouse model of intracranial hemorrhage in which the brains of mouse embryos were exposed to blood or LPA resulted in development of FH. FH development was dependent on the expression of the LPA(1) receptor by neural progenitor cells. Administration of an LPA(1) receptor antagonist blocked development of FH. These findings implicate the LPA signaling pathway in the etiology of FH and suggest new potential targets for developing new treatments for FH.

Ruf W.,Scripps Research Institute
Thrombosis Research | Year: 2012

The generation of procoagulant Tissue Factor (TF) is crucial for thrombosis. TF contains a surface exposed allosteric disulfide bond that stabilizes the carboxyl-terminal domain involved in ligand interactions with coagulation factors VIIa and X. TF procoagulant activation typically occurs following cellular perturbations that also cause the appearance of procoagulant phosphatidylserine in the outer leaflet of cell membranes. However, thiol modifying agents, without suppressing phosphatidylserine exposure, can prevent TF activation, implicating thiol-disulfide exchange reactions in the regulation of TF procoagulant activity of primary cells. Protein disulfide isomerase (PDI), a regulator of extracellular thiol exchange, is associated with cell surface TF and required for TF-dependent thrombosis in vivo. PDI regulates the thiol-dependent biogenesis of procoagulant microparticles that are released from myeloid cells and smooth muscle cells following activation of the purinergic P2X7 receptor. Genetic deletion of P2X7 signaling attenuates FeCl 3-induced carotid artery thrombosis in mice, indicating that TF prothrombotic activity is regulated by specific cell signaling pathways in vivo. © 2012 Elsevier Ltd. All rights reserved.

Pockros P.J.,Scripps Research Institute
Drugs | Year: 2012

Two oral direct-acting antivirals (DAA) are now available for the treatment of chronic hepatitis C infection and several generations of DAA are in development. Expectations are that, at some time in the near future, hepatitis C will be 'curable' with an all-oral DAA regimen. This article reviews the current problems associated with interferon-based hepatitis C treatments that are combined with DAAs, including adverse events and complications of therapy, contraindications, drug-drug interactions and cost. The article further discusses difficulties with new drug development and provides an opinion on the research issues still to be dealt with and the requirements for the successful implementation of such a strategy. These include lack of efficacy in certain populations, unexpected side effects, antiviral resistance, late relapse, lack of cooperation between drug developers and cost. © 2012 Springer International Publishing AG. All rights reserved.

Murphy R.T.,University of California at Irvine | Pennock A.T.,Rady Childrens Hospital | Bugbee W.D.,Scripps Research Institute
American Journal of Sports Medicine | Year: 2014

Background: Multiple studies in adults have shown that osteochondral allograft transplantation is an effective treatment option for large chondral and osteochondral defects of the knee. Limited outcome data are available on osteochondral allografts in the pediatric and adolescent patient populations. Purpose: To describe a 28-year experience with osteochondral allograft transplantation in patients younger than 18 years with a focus on subjective outcome measures, return to activities, and allograft survivorship. Study Design: Case series; Level of evidence, 4. Methods: A total of 39 patients (43 knees) underwent fresh osteochondral allograft transplantation for treatment of chondral and osteochondral lesions. Twenty-six male and 17 female knees with a mean age of 16.4 years (range, 11.0-17.9 years) at index surgery were followed-up at a mean of 8.4 years (range, 1.7-27.1 years). Thirty-four knees (79%) had at least 1 previous surgery. The most common underlying causes of the lesions were osteochondritis dissecans (61%), avascular necrosis (16%), and traumatic chondral injury (14%). Mean allograft size was 8.4 cm2. The most common allograft location was the medial femoral condyle (41.9%), followed by the lateral femoral condyle (35%). Each patient was evaluated with the International Knee Documentation Committee pain, function, and total scores; a modified Merle d'Aubigné -Postel (18-point) scale; and Knee Society function score. Failure was defined as revision osteochondral allograft or conversion to arthroplasty. Results: Five knees experienced clinical failure at a median of 2.7 years (range, 1.0-14.7 years). Four failures were salvaged successfully with another osteochondral allograft transplant. One patient underwent prosthetic arthroplasty 8.6 years after revision allograft. Graft survivorship was 90% at 10 years. Of the knees whose grafts were in situ at latest follow-up, 88% were rated good/excellent (18-point scale). The mean International Knee Documentation Committee scores improved from 42 preoperatively to 75 postoperatively, and the Knee Society function score improved from 69 to 89 (both P<.05). Eighty-nine percent of patients reported "extremely satisfied" or "satisfied." Conclusion: With 88% good/excellent results and 80% salvage rate of clinical failures with an additional allograft, osteochondral allograft transplantation is a useful treatment option in pediatric and adolescent patients. © 2013 The Author(s).

Srinivasan S.,Scripps Research Institute
Annual Review of Physiology | Year: 2015

Over the past decade, studies conducted in Caenorhabditis elegans have helped to uncover the ancient and complex origins of body fat regulation. This review highlights the powerful combination of genetics, pharmacology, and biochemistry used to study energy balance and the regulation of cellular fat metabolism in C. elegans. The complete wiring diagram of the C. elegans nervous system has been exploited to understand how the sensory nervous system regulates body fat and how food perception is coupled with the production of energy via fat metabolism. As a model organism, C. elegans also offers a unique opportunity to discover neuroendocrine factors that mediate direct communication between the nervous system and the metabolic tissues. The coming years are expected to reveal a wealth of information on the neuroendocrine control of body fat in C. elegans. Copyright © 2015 by Annual Reviews. All rights reserved.

Kamat S.S.,Texas A&M University | Kamat S.S.,Scripps Research Institute | Raushel F.M.,Texas A&M University
Current Opinion in Chemical Biology | Year: 2013

Phosphonates are ubiquitous organophosphorus compounds that contain a characteristic C. P bond which is chemically inert and hydrolytically stable. Bacteria have evolved pathways to metabolize these phosphonate compounds and utilize the products of these pathways as nutrient sources. This review aims to present all of the known bacterial enzymes capable of transforming phosphonates to phosphates. There are three major classes of enzymes known to date performing such transformations: phosphonatases, the C-P lyase complex and an oxidative pathway for C. P bond cleavage. A brief description of each class is presented. © 2013 Elsevier Ltd.

Versteeg H.H.,Leiden University | Ruf W.,Scripps Research Institute
Current Opinion in Hematology | Year: 2011

Purpose of Review: The transmembrane protein tissue factor (TF), the initiator of the extrinsic coagulation cascade and a risk factor for thrombotic disease, largely resides on the cell surface in an inactive, encrypted conformation. Whereas TF procoagulant activation, or decryption, is closely associated with the exposure of procoagulant phosphatidylserine in the outer leaflet of cell membranes, thiol pathways and protein disulfide isomerase (PDI) play increasingly recognized roles in regulating TF prothrombotic pathways. Recent Findings: TF contains a solvent-exposed allosteric disulfide bond that stabilizes the carboxyl-terminal domain involved in ligand interactions with coagulation factors VIIa and X. PDI is a prime candidate to modify the allosteric disulfide by reduction, S-nitrosylation and glutathionation, implicated as regulators of TF procoagulant activity. In-vivo studies localize PDI to sites of thrombus formation and inhibition of PDI attenuates arterial thrombosis. PDI also contributes to the biogenesis of procoagulant microparticles that are released in the context of TF decryption following ATP-mediated activation of the purinergic P2X7 receptor. Genetic deletion of P2X7 signaling attenuates TF and PDI-dependent thrombosis, identifying a new connection between prothrombotic and proinflammatory pathways. Summary: Although the precise biochemical events of TF encryption and decryption require further study, PDI and thiol pathways emerge as important regulators of vascular thrombotic diseases. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Kishi S.,Scripps Research Institute
Translational Research | Year: 2014

Can we reset, reprogram, rejuvenate, or reverse the organismal aging process? Certain genetic manipulations could at least reset and reprogram epigenetic dynamics beyond phenotypic plasticity and elasticity in cells, which can be manipulated further into organisms. However, in a whole complex aging organism, how can we rejuvenate intrinsic resources and infrastructures in an intact and noninvasive manner? The incidence of diseases increases exponentially with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but essentially inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these 2 phenomena to rejuvenate over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states based on diverse epigenotypes in response to intrinsic or extrinsic environmental cues and genetic perturbations. We hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds and windows of plasticity and its robustness by molecular genetic and chemical epigenetic approaches, we have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during their embryonic and/or larval stages ("embryonic/larval senescence"). Subsequently, at least some of these mutant animals were found to show a shortened life span, whereas others would be expected to live longer into adulthood. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and its regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes, genotypes, and epigenotypes that can be linked to the senescence phenotype, which facilitates searching for the evolutionary and developmental origins of aging in vertebrates. © 2014 Mosby, Inc. All rights reserved.

Mcheyzer-Williams M.G.,Scripps Research Institute
Cell | Year: 2014

B cells expressing potent and broadly neutralizing antibodies to HIV-1 have been found in many infected individuals, suggesting that adaptive immunity has the capacity to defeat viral escape. Gao et al. uncover an intermediate stage in this process when viral escape from one "helper" B cell lineage selects variants that drive a separate B cell lineage toward broad neutralization. © 2014 Elsevier Inc.

Dube H.,Allegheny College | Ams M.R.,Scripps Research Institute | Rebek Jr. J.,Allegheny College
Journal of the American Chemical Society | Year: 2010

The trans to cis isomerization of 4,4'-dimethylazobenzene by the nonchemical stimuli light and heat alters its guest binding properties, allowing control over encapsulation of a second guest. We show here how this remote control for reversible encapsulation can be used as a supramolecular fluorescence on/off switch. If trans-4-ethyl-4'-methylstilbene is used as the second guest its fluorescence is altered depending on whether it is free in solution or encapsulated. We demonstrate that the change in fluorescence is indeed a consequence of the controlled encapsulation state of the stilbene by correlation of fluorescence and 1H NMR data. © 2010 American Chemical Society.

Tainer J.A.,Scripps Research Institute | McCammon J.A.,Howard Hughes Medical Institute | Ivanov I.,Georgia State University
Journal of the American Chemical Society | Year: 2010

Proliferating cell nuclear antigen (PCNA, sliding clamp) is a toroidal-shaped protein that encircles DNA and plays a pivotal role in DNA replication, modification and repair. To perform its vital functions, the clamp has to be opened and resealed at primer-template junctions by a clamp loader molecular machine, replication factor C (RFC). The mechanism of this process constitutes a significant piece in the puzzle of processive DNA replication. We show that upon clamp opening the RFC/PCNA complex undergoes a large conformational rearrangement, leading to the formation of an extended interface between the clamp and RFC. Binding of ring-open PCNA to all five RFC subunits transforms the free-energy landscape underlying the closed- to open state transition, trapping PCNA in an open conformation. Careful comparison of free-energy profiles for clamp opening in the presence and absence of RFC allowed us to substantiate the role of RFC in the initial stage of the clamp-loading cycle. RFC does not appreciably destabilize the closed state of PCNA. Instead, the function of the clamp loader is dependent on the selective stabilization of the open conformation of the clamp. © 2010 American Chemical Society.

Strack R.L.,Cornell University | Disney M.D.,Scripps Research Institute | Jaffrey S.R.,Cornell University
Nature Methods | Year: 2013

Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells. © 2013 Nature America, Inc.

Johnson J.E.,Scripps Research Institute
Journal of Electron Microscopy | Year: 2013

This is a personal history of my structural studies of icosahedral viruses that evolved from crystallographic studies, to hybrid methods with electron cryo-microscopy and image reconstruction (cryoEM) and then developed further by incorporating a variety of physical methods to augment the high resolution crystallographic studies. It is not meant to be comprehensive, even for my own work, but hopefully provides some perspective on the growth of our understanding of these remarkable biologic assemblies. The goal is to provide a historical perspective for those new to the field and to emphasize the limitations of any one method, even those that provide atomic resolution information about viruses. © The Author 2013. Published by Oxford University Press [on behalf of The Japanese Society of Microscopy]. All rights reserved.

Hutt D.M.,Scripps Research Institute
Cold Spring Harbor perspectives in medicine | Year: 2013

The folding biology common to all three kingdoms of life (Archaea, Bacteria, and Eukarya) is proteostasis. The proteostasis network (PN) functions as a " cloud" to generate, protect, and degrade the proteome. Whereas microbes (Bacteria, Archaea) have a single compartment, Eukarya have numerous subcellular compartments.We examine evidence that Eukarya compartments use coat, tether, and fusion (CTF) membrane trafficking components to form an evolutionarily advanced arm of the PN that we refer to as the " trafficking PN" (TPN). We suggest that the TPN builds compartments by generating a mosaic of integrated cargo-specific trafficking signatures (TRaCKS). TRaCKS control the temporal and spatial features of protein-folding biology based on the Anfinsen principle that the local environment plays a critical role in managing protein structure. TPN-generated endomembrane compartments apply a " quinary" level of structural control to modify the secondary, tertiary, and quaternary structures defined by the primary polypeptide-chain sequence. The development of Anfinsen compartments provides a unifying foundation for understanding the purpose of endomembrane biology and its capacity to drive extant Eukarya function and diversity.

Gascoigne N.R.J.,Scripps Research Institute | Palmer E.,University of Basel
Current Opinion in Immunology | Year: 2011

T cell receptor signaling allows the developing thymocyte to undergo positive or negative selection, which is required for the formation of a useful mature T cell repertoire. Recent developments include the finding that much of the Lck kinase (required to initiate T cell signaling) is already in an active configuration before signaling. The analog strength of antigen binding to the T cell receptor binding may be translated into a digital signal by the amount of time the TCR is paired with a co-receptor carrying Lck. Downstream, the cellular localization of MAP kinase signaling is determined by the strength of the signal and in turn predicts positive or negative selection. A novel protein, Themis, is important in crossing the positive selection developmental checkpoint, but its mode of action is still uncertain. Commitment to the CD4 or CD8 lineage is influenced by the amount of ZAP-70 signaling and also by closely regulated responsiveness to intrathymic cytokines such as IL7. © 2011 Elsevier Ltd.

McKay D.B.,Scripps Research Institute
Seminars in Immunopathology | Year: 2011

Solid organ transplantation is a life saving procedure for patients with end-stage organ disease, and great care is taken to ensure that healthy organs are procured from deceased or live donors. Despite rigorous efforts to avoid injury, all organs experience some degree of damage from a process called ischemia reperfusion injury (IRI). The first part of the injury (ischemia) occurs when the donor organ's blood supply is compromised, and the second part (reperfusion) occurs when the blood supply is reestablished. The pathophysiology of the IRI is complex, but data from many laboratories have demonstrated that the inciting events of ischemia/reperfusion injury are triggered through a phylogenetically conserved system called the innate immune system. The innate immune system is a complex array of molecules, receptors and cellular elements present in species as diverse as plants to humans. This review discusses the role of the innate immune system in renal IRI and focuses on mechanisms of injury during organ procurement and transplantation. Although there are overlapping complex mechanisms, blockade of the innate immune system will likely provide a novel approach to preventing the earliest events associated with renal ischemia. Potentially, blockade of innate immune activation will provide the opportunity to increase the use marginal donors, especially those from patients deceased after cardiac death. © 2011 Springer-Verlag.

Poceta J.S.,Scripps Research Institute
Journal of Clinical Sleep Medicine | Year: 2011

Study Objectives: To describe zolpidem-associated complex behaviors, including both daytime automatisms and sleep-related parasomnias. Methods: A case series of eight clinical patients and six legal defendants is presented. Patients presented to the author after an episode of confusion, amnesia, or somnambulism. Legal defendants were being prosecuted for driving under the influence, and the author reviewed the cases as expert witness for the defense. Potential predisposing factors including comorbidities, social situation, physician instruction, concomitant medications, and patterns of medication management were considered. Results: Patients and defendants exhibited abnormal behavior characterized by poor motor control and confusion. Although remaining apparently interactive with the environment, all reported amnesia for 3 to 5 hours. In some cases, the episodes began during daytime wakefulness because of accidental or purposeful ingestion of the zolpidem and are considered automatisms. Other cases began after ingestion of zolpidem at the time of going to bed and are considered parasomnias. Risk factors for both wake and sleep-related automatic complex behaviors include the concomitant ingestion of other sedating drugs, a higher dose of zolpidem, a history of parasomnia, ingestion at times other than bedtime or when sleep is unlikely, poor management of pill bottles, and living alone. In addition, similar size and shape of two medications contributed to accidental ingestion in at least one case. Conclusions: Sleep driving and other complex behaviors can occur after zolpidem ingestion. Physicians should assess patients for potential risk factors and inquire about parasomnias. Serious legal and medical complications can occur as a result of these forms of automatic complex behaviors.

Blackmond D.G.,Scripps Research Institute
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2011

The single handedness of biological molecules has fascinated scientists and laymen alike since Pasteur's first painstaking separation of the enantiomorphic crystals of a tartrate salt over 150 years ago. More recently, a number of theoretical and experimental investigations have helped to delineate models for how one enantiomer might have come to dominate over the other from what presumably was a racemic prebiotic world. Mechanisms for enantioenrichment that include either chemical or physical processes, or a combination of both, are discussed in the context of experimental studies in autocatalysis and in the phase behaviour of chiral molecules. © 2011 The Royal Society.

Kandel E.R.,Columbia University | Kandel E.R.,Howard Hughes Medical Institute | Dudai Y.,Weizmann Institute of Science | Mayford M.R.,Scripps Research Institute
Cell | Year: 2014

Learning and memory are two of the most magical capabilities of our mind. Learning is the biological process of acquiring new knowledge about the world, and memory is the process of retaining and reconstructing that knowledge over time. Most of our knowledge of the world and most of our skills are not innate but learned. Thus, we are who we are in large part because of what we have learned and what we remember and forget. In this Review, we examine the molecular, cellular, and circuit mechanisms that underlie how memories are made, stored, retrieved, and lost. © 2014 Elsevier Inc.

Nomura D.K.,Scripps Research Institute | Casida J.E.,University of California at Berkeley
Journal of Agricultural and Food Chemistry | Year: 2011

Organophosphorus (OP) and thiocarbamate (TC) agrochemicals are used worldwide as insecticides, herbicides, and fungicides, but their safety assessment in terms of potential off-targets remains incomplete. In this study, a chemoproteomic platform, termed activity-based protein profiling, was used to broadly define serine hydrolase targets in mouse brain of a panel of 29 OP and TC pesticides. Among the secondary targets identified, enzymes involved in the degradation of endocannabinoid signaling lipids, monoacylglycerol lipase, and fatty acid amide hydrolase were inhibited by several OP and TC pesticides. Blockade of these two enzymes led to elevations in brain endocannabinoid levels and dysregulated brain arachidonate metabolism. Other secondary targets include enzymes thought to also play important roles in the nervous system and unannotated proteins. This study reveals a multitude of secondary targets for OP and TC pesticides and underscores the utility of chemoproteomic platforms in gaining insights into biochemical pathways that are perturbed by these toxicants. © 2010 American Chemical Society.

Lotz M.K.,Scripps Research Institute
Arthritis Research and Therapy | Year: 2010

Joint trauma can lead to a spectrum of acute lesions, including osteochondral fractures, ligament or meniscus tears and damage to the articular cartilage. This is often associated with intraarticular bleeding and causes posttraumatic joint infl ammation. Although the acute symptoms resolve and some of the lesions can be surgically repaired, joint injury triggers a chronic remodeling process in cartilage and other joint tissues that ultimately manifests as osteoarthritis in a majority of cases. The objective of the present review is to summarize information on pathogenetic mechanisms involved in the acute and chronic consequences of joint trauma and discuss potential pharmacological interventions. The focus of the review is on the early events that follow joint trauma since therapies for posttraumatic joint infl ammation are not available and this represents a unique window of opportunity to limit chronic consequences © 2010 BioMed Central Ltd.

Nguyen Q.H.,Scripps Research Institute
Patient Preference and Adherence | Year: 2014

Glaucoma is one of the leading causes of blindness and is characterized by optic nerve damage that results in visual field loss. Elevated intraocular pressure (IOP) has been associated with glaucoma progression; thus, IOP-lowering medications are the standard of care for glaucoma. Guidelines suggest monotherapy with IOP-lowering agents such as β-blockers (eg, timolol), prostaglandin analogs, carbonic anhydrase inhibitors (eg, brinzolamide), and α2-receptor agonists (eg, brimonidine). However, monotherapy may provide insufficient IOP reduction in some patients, thereby necessitating the use of multiple IOP-lowering medications. Multidrug regimens may be complex, may increase the risk of preservative-related ocular symptoms, and may potentially reduce overall drug exposure as a consequence of drug washout during closely timed sequential administrations; these difficulties may reduce overall drug efficacy and decrease patient persistence and adherence with multidrug treatment regimens. Fixed-combination medications that provide two IOP-lowering therapies within a single solution are available and may overcome some of these challenges. However, all currently available fixed combinations combine timolol with another IOP-lowering agent, indicating that additional fixed-combination alternatives would be beneficial. To meet this demand, a novel fixed combination of brinzolamide 1% and brimonidine 0.2% (BBFC) has recently been developed. In two randomized, double-masked, multinational clinical trials, BBFC had greater IOP-lowering efficacy than brinzolamide or brimonidine monotherapy after 3 months of treatment in patients with open-angle glaucoma or ocular hypertension. In both studies, the overall safety profile of BBFC was consistent with that of brinzolamide and brimonidine. Comparative studies with BBFC versus other IOP-lowering monotherapy and fixed-combination medications are not available, but the IOP reductions observed with BBFC are similar to or greater than those reported in the literature for other glaucoma treatments; thus, BBFC provides an additional fixed-combination therapeutic option for patients who require further efficacious IOP reduction and improved convenience and tolerability versus concomitant administration of two separate medications. © 2014 Nguyen.

Gruber C.W.,Medical University of Vienna | Muttenthaler M.,Scripps Research Institute
PLoS ONE | Year: 2012

Natural peptides of great number and diversity occur in all organisms, but analyzing their peptidome is often difficult. With natural product drug discovery in mind, we devised a genome-mining approach to identify defense- and neuropeptides in the genomes of social ants from Atta cephalotes (leaf-cutter ant), Camponotus floridanus (carpenter ant) and Harpegnathos saltator (basal genus). Numerous peptide-encoding genes of defense peptides, in particular defensins, and neuropeptides or regulatory peptide hormones, such as allatostatins and tachykinins, were identified and analyzed. Most interestingly we annotated genes that encode oxytocin/vasopressin-related peptides (inotocins) and their putative receptors. This is the first piece of evidence for the existence of this nonapeptide hormone system in ants (Formicidae) and supports recent findings in Tribolium castaneum (red flour beetle) and Nasonia vitripennis (parasitoid wasp), and therefore its confinement to some basal holometabolous insects. By contrast, the absence of the inotocin hormone system in Apis mellifera (honeybee), another closely-related member of the eusocial Hymenoptera clade, establishes the basis for future studies on the molecular evolution and physiological function of oxytocin/vasopressin-related peptides (vasotocin nonapeptide family) and their receptors in social insects. Particularly the identification of ant inotocin and defensin peptide sequences will provide a basis for future pharmacological characterization in the quest for potent and selective lead compounds of therapeutic value. © 2012 Gruber, Muttenthaler.

Cherezov V.,Scripps Research Institute
Methods in molecular biology (Clifton, N.J.) | Year: 2010

G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using an LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment, extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.

Chun J.,Scripps Research Institute | Hartung H.-P.,Heinrich Heine University Dusseldorf
Clinical Neuropharmacology | Year: 2010

Fingolimod (FTY720) is a first-in-class orally bioavailable compound that has shown efficacy in advanced clinical trials for the treatment of multiple sclerosis (MS). In vivo, fingolimod is phosphorylated to form fingolimod-phosphate, which resembles naturally occurring sphingosine 1-phosphate (S1P), an extracellular lipid mediator whose major effects are mediated by cognate G protein-coupled receptors. There are at least 5 S1P receptor subtypes, known as S1P subtypes 1-5 (S1P1-5), 4 of which bind fingolimod-phosphate. These receptors are expressed on a wide range of cells that are involved in many biological processes relevant to MS. S1P1 plays a key role in the immune system, regulating lymphocyte egress from lymphoid tissues into the circulation. Fingolimod-phosphate initially activates lymphocyte S1P1 via high-affinity receptor binding yet subsequently induces S1P1 down-regulation that prevents lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into the central nervous system (CNS). S1P receptors are also expressed by many CNS cell types and have been shown to influence cell proliferation, morphology, and migration. Fingolimod crosses the blood-brain barrier and may therefore have direct CNS effects, distinguishing it from immunologically targeted MS therapies. Prophylactic administration of fingolimod to animals with experimental autoimmune encephalitis (EAE), a model of MS, completely prevents development of EAE features, whereas therapeutic administration significantly reduces clinical severity of EAE. Therapeutic efficacy observed in animal studies has been substantiated in phase 2 and 3 trials involving patients with relapsing or relapsing-remitting MS. Copyright © 2010 by Lippincott Williams & Wilkins.

Gaglani S.M.,Johns Hopkins University | Topol E.J.,Scripps Research Institute
Academic Medicine | Year: 2014

Mobile health (mHealth) technologies have experienced a recent surge in attention because of their potential to transform the delivery of health care. This enthusiasm is partly due to the near ubiquity of smartphones and tablets among clinicians, as well as to the stream of mobile medical apps and devices being created. While much discussion has been devoted to how these tools will impact the practice of medicine, surprisingly little has been written on the role these technologies will play in medical education. In this commentary the authors describe the opportunities, applications, and challenges of mHealth apps and devices in medical education and argue that medical schools should make efforts to integrate these technologies into their curricula. By not doing so, medical educators risk producing a generation of clinicians underprepared for the changing realities of medical practice brought on by mHealth technologies.

Pockros P.J.,Scripps Research Institute
Frontiers of Medicine in China | Year: 2014

Chronic hepatitis C virus (HCV) infection afflicts a reported 170 million people worldwide and is often complicated by cirrhosis and hepatocellular carcinoma. Morbidity and mortality are decreased with the successful treatment of chronic HCV infection. Increased understanding of the HCV has allowed further development of new direct-acting antiviral (DAA) agents against the HCV and has also allowed the development of IFN-free oral treatment regimens. In late 2013 the first nucleotide polymerase inhibitor regimen with RBV alone for genotypes 2/3 and in combination with a 12-week regimen of PEG-IFN + RBV for genotypes 1, 4 was approved for use in the US. A number of promising new DAA regimens which are IFN-free are in phase 3 development and the first will likely be approved for use in the US in 2014. The currently approved regimens are discussed in detail and currently available data on future regimens are reviewed herein. © 2014 Higher Education Press and Springer-Verlag Berlin Heidelberg.