Grant List
Represents Grant table in the DB
GET /v1/grants?sort=start_date
{ "links": { "first": "https://cic-apps.datascience.columbia.edu/v1/grants?page%5Bnumber%5D=1&sort=start_date", "last": "https://cic-apps.datascience.columbia.edu/v1/grants?page%5Bnumber%5D=1392&sort=start_date", "next": "https://cic-apps.datascience.columbia.edu/v1/grants?page%5Bnumber%5D=2&sort=start_date", "prev": null }, "data": [ { "type": "Grant", "id": "7694", "attributes": { "award_id": "1ZIAEY000306-26", "title": "Role of Protein Interactions in Retina Development and Function", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Eye Institute (NEI)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 728296, "principal_investigator": { "id": 23489, "first_name": "SOFIA P", "last_name": "BECERRA", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1611, "ror": "https://ror.org/03wkg3b53", "name": "National Eye Institute", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1611, "ror": "https://ror.org/03wkg3b53", "name": "National Eye Institute", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true }, "abstract": "A study on the role of the PNPLA2 gene in the regulation of oxidative stress damage of RPE was completed. Oxidative stress-mediated injury of the retinal pigment epithelium (RPE) can precede progressive retinal degeneration and ultimately lead to blindness (e.g., age-related macular degeneration). The RPE expresses the PNPLA2 gene and produces its protein product PEDF-R. We showed that transient PNPLA2 overexpression decreases dead-cell proteolytic activity and that synthetic peptides derived from a central region of PEDF-R efficiently protect ARPE-19 and pig primary RPE cells from oxidative stress. We evaluated the effect of loss of PNPLA2 in RPE cells undergoing oxidative stress and found that loss of PNPLA2 conferred increased resistance to cells when subjected to oxidative stress. The molecular pathways triggered by PEDF that are involved in retinal survival activity was reviewed. PEDF is involved in signal transduction cascades necessary for protection of the retina. The interaction between PEDF and retinal cells elicits neuroprotective effects in vitro and in vivo. The direct substrates and signaling mechanisms involved in the survival response derived from such interaction were summarized. Three semi-automated cell-based protocols to identify retinoprotective factors with two retinal cell lines, rat R28 cells and mouse 661W cells were described in detail. In these protocols, cells are induced to undergo death by photo-oxidation stress, growth factor depletion or cytotoxicity with sodium iodate. PEDF, an established neurotrophic factor for retinal cells, was used as a positive control. These protocols will prove useful in high-throughput quantitative screening of potential retinoprotective factors. The polarized RPE releases PEDF in an apicolateral fashion. We determined the levels of PEDF released by polarized iPSC-derived RPE attached to porous membranes in transwells (in collaboration with the laboratory of V. Canto-Soler). The media from the basal and apical compartments were subjected to western blotting and ELISA using specific antibodies to human PEDF. Photoreceptor outer segments were isolated from bovine retina. Cells exposed to photoreceptor outer segments were assayed for phagocytosis, by following ingestion and degradation of the outer segments. Western blots of cell lysates using specific antibodies to rhodopsin were performed to determine ingestion of outer segments by the cells. The levels of a byproduct of POS degradation, beta-hydroxybutyrate, were measured in the media of the cells. The role of PEDF-R in the phagocytosis of photoreceptor outer segments continued to be investigated. The PNPLA2 gene was transiently silenced in the ARPE-19 cell line using siScramble and siPNPLA2 oligonucleotide duplexes. mRNA and protein levels of fatty acid metabolism related genes ACAD9 (acyl-CoA dehydrogenase family member 9), CPT1C (carnitine palmitoyltransferase 1C), and FASN (fatty acid synthase) were measured in PEDF-R-deficient cells challenged with photoreceptor outer segments. Cell viability of ARPE-19 cells incubated with bromoenol lactone, a phospholipase inhibitor was measured. The effects of bromoenol lactone on the degradation of photoreceptor outer segments, as well as the secretion of the ketone body beta-hydroxybutyrate were determined. PEDF-R protein levels following photoreceptor outer segments addition were measured. Total phospholipids and fatty acids were determined from cell lysates of transiently transfected cells challenged with photoreceptor outer segments (collaboration with the laboratory of MP Agdaba). An in vitro model of cultured primary retinal neurons was used to examine intracellular signaling stimulated by exogenous additions of PEDF peptide fragments. The newborn rat retinal cells in culture die spontaneously in the absence of trophic factors. CRX immunostaining was performed to identify photoreceptor cells. We performed mitochondrial assays using Mitotracker, and quantification of the changes in mitochondrial membrane potential in live cells by flow cytometry, microplate spectrophotometry and fluorescent microscopy using tetramethylrhodamine, ethyl ester (TMRE). Results show that PEDF peptides protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an enzymatic inhibitor of the PEDF-R, or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The mRNA levels of Bcl2 gene were determined in cells treated with and without effectors. Protein extracts from cells treated with and without PEDF were obtained for large-scale study of proteins. The effects of PEDF and derived peptides on regulation of Bcl2, Bax and RAC1 proteins were examined by western blotting of the rat primary cultures treated with and without the effectors. We continued to purify large amounts of recombinant human PEDF protein versions from conditioned media of stably transfected Hek.Ebna 293 cells with PEDF expression plasmids. Cells expressing for full length human PEDF and versions with single point alterations at H105A were cultured at large scale and secreted proteins in their serum free media were collected, concentrated and dialyzed (collaboration with Y. Shiloachs laboratory). The proteins were purified using a two-step ion-exchange chromatography. The purified PEDF protein combined with a peptide fragment of the ectodomain of PEDF-R was used in high-throughput crystallization screening (collaboration with V Sagar in G. Wistows laboratory). Recombinant PEDF-R versions were produced in E. coli cells induced with isopropyl - d-1-thiogalactopyranoside. The recombinant PEDF-R versions overproduced in bacteria were insoluble and solubilization studies were performed using urea. Conditions for overproduction, solubilization with urea and purification by ion exchange column chromatography were optimized. PEDF-R variants retained the ability to bind PEDF. Mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for use in structural and biological studies. A proposal to investigate the effects of PEDF and its derived peptides to inhibit the hallmark of COVID-19 cytokine storm, in particular regulation of the IL-6 gene and the IL-6 protein levels and its consequences in the context of COVID-19, was prepared and submitted to ITAC. Another proposal in collaboration with R Star, NIDDK, to study PEDF as biomarker for intervention in COVID-16 during cytokine storm was also prepared and submitted to ITAC.", "keywords": [ "Age related macular degeneration", "Amino Acids", "Angiogenesis Inhibitors", "Animal Model", "Antibodies", "Apical", "Apoptosis", "BCL-2 Protein", "BCL2 gene", "Bacteria", "Bax protein", "Binding", "Biological", "Biological Assay", "Biological Markers", "Blindness", "COVID-19", "Carnitine Palmitoyltransferase I", "Cattle", "Cell Extracts", "Cell Line", "Cell Survival", "Cells", "Cessation of life", "Choroidal Neovascularization", "Collaborations", "Column Chromatography", "Corneal Neovascularization", "Crystallization", "Development", "Developmental Process", "Enzyme-Linked Immunosorbent Assay", "Escherichia coli", "Esters", "Exposure to", "Eye", "FASN gene", "Family", "Family member", "Family suidae", "Fatty Acids", "Fatty-acid synthase", "Flow Cytometry", "Genes", "Growth Factor", "Human", "Hydrolysis", "IL6 gene", "In Vitro", "Incubated", "Ingestion", "Inherited", "Injury", "Interest Group", "Interleukin-6", "Intervention", "Ion Exchange", "Ion-Exchange Chromatography Procedure", "Ketone Bodies", "Laboratories", "Lactones", "Lead", "Length", "Ligands", "Light", "Link", "Lipase", "Longevity", "Maintenance", "Mammals", "Measures", "Mediating", "Membrane", "Membrane Potentials", "Messenger RNA", "Microscopy", "Mitochondria", "Molecular", "Mus", "National Institute of Diabetes and Digestive and Kidney Diseases", "Neoplasm Metastasis", "Neurites", "Neuronal Differentiation", "Neurons", "Newborn Infant", "Oligonucleotides", "Opsin", "Oxidative Regulation", "Oxidative Stress", "Pathway interactions", "Peptide Fragments", "Peptides", "Phagocytosis", "Phospholipase", "Phospholipases A", "Phospholipids", "Photoreceptors", "Plasmids", "Proteins", "Protocols documentation", "Rattus", "Recombinants", "Regulation", "Research", "Resistance", "Retina", "Retinal Degeneration", "Retinal Neovascularization", "Rhodopsin", "Role", "Serpin Superfamily", "Serum-Free Culture Media", "Signal Transduction", "Spectrophotometry", "Stress", "Structure", "Structure of retinal pigment epithelium", "Triglycerides", "Urea", "Variant", "Western Blotting", "Work", "acyl-CoA dehydrogenase", "base", "beta-Hydroxybutyrate", "coronavirus disease", "cytokine release syndrome", "cytotoxicity", "fatty acid metabolism", "in vitro Model", "in vivo", "induced pluripotent stem cell", "inhibitor/antagonist", "member", "mitochondrial membrane", "neuronal survival", "neurotrophic factor", "overexpression", "oxidatio" ], "approved": true } }, { "type": "Grant", "id": "7707", "attributes": { "award_id": "1ZIADK075104-08", "title": "Understanding protein folding, evolution and function via molecular simulation", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 866673, "principal_investigator": { "id": 23501, "first_name": "Robert", "last_name": "Best", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true }, "abstract": "The project has addressed the following areas in the past year: 1. Studies of SARS-Cov2 Main Protease. In collaboration with John Louis (NIDDK), we have investigated the autoprocessing mechanism of the SARS-Cov2 Main Protease. A reaction coordinate was developed that can describe the conformational change between the native protease structure and a putative transient state required for protease autoprocessing, a key step for viral replication. Simulations show that this state is only slightly higher in free energy than the native state and should be populated on a microsecond to millisecond time scale, making the proposed autoprocessing mechanism extremely plausible. Future work will investigate this mechanism further and whether it can be exploited to design novel inhibitors for SARS proteases (R. Best). 2. Association of highly charged intrinsically disordered proteins. Recent work in collaboration with Ben Schuler's single molecule FRET group in Zurich has shown that high affinity disordered complexes of proteins or proteins and nucleic acids may be ubiquitous in cell nuclei. We are seeking to develop a predictive model for the affinity and structure of these complexes, initially via molecular simulation, but then via a semi-empirical theory fitted to experimental data collected in the Schuler lab (M. Ivanovic). 3. Development of coarse-grained models for complex coacervation of intrinsically disordered proteins with single- and double-stranded nucleic acids. Going beyond the 1:1 complexes studied in project 1, it is also possible for oppositely charged macromolecules such as proteins and DNA or RNA to undergo complex coacervation, forming a separate phase with high macromolecular density, under the correct conditions. Such a phenomenon may provide a physical basis for the formation of some of the membraneless organelles observed in the cell nucleus. We have developed a coarse-grained simulation model of protein-nucleic acid interactions, and used it to study the ordering induced on formation of the condensed phase. Future work will include improving the transferability of the model to different sequences and making it more sensitive to sequence-specific effects (K. Lebold). 4. Development of transferable sequence-specific models for liquid-liquid phase separation (LLPS) of intrinsically disordered proteins. We had previously shown that a simple coarse-grained model could be useful for modelling qualitative effects on protein liquid-liquid phase separation, the basis for formation of many membraneless organelles within cells. However, this model was not very predictive of which proteins would undergo phase separation. We have therefore undertaken a comprehensive refitting of the energy function in order to describe both the properties of isolated disordered chains and also those of proteins which are known to phase separate. The inclusion of the latter data set results in a great improvement of the overall accuracy of the model, owing to some of the effects which are important for driving LLPS not being well represented in the database of isolated (non phase-separating) proteins. This work is currently in preparation for publication (T. Dannenhoffer-Lafage). 5. All-atom simulations of protein phase separation and complex coacervation. Using time obtained on the Anton supercomputer, together with novel multiscale simulation methodology, we have performed the first all-atom simulations of a protein-rich phase representative of those obtained in protein LLPS. We have characterized the interactions driving formation of this phase, the partitioning of ions into the dense phase, and the dynamics of proteins within the phase - paper currently under review. We now intend to apply similar methodology to the more challenging problem of coacervation of oppositely charged proteins, in order to elucidate the interactions responsible for stabilizing these phases (M. Ivanovic). 6. Co-translational protein folding. In collaboration with Gunnar von Heijne, we have used our previously developed model for co-translational folding on the ribosome to investigate more directly the relationship between the forces arising from the folding nascent chain and the yield of full length protein obtained in arrest peptide experiments. We also devised a method for obtaining these forces directly from experiment by using a series of different arrest peptides with the same protein constructs (5). In a second collaboration with the group of Sander Tans, we are using our coarse-grained co-translational folding model to understand the effects of the ribosome on the folding and unfolding rates of ADR1a in the ribosome exit tunnel, as probed by single molecule force and fluorescence spectroscopy. We have recently started a new collaboration with Alexey Amunts in Stockholm to interpret their cryo-EM results on mitochondrial ribosomes (R. Best, P. Tian). 7. Using sequence-based energy functions to describe protein fitness landscapes and for protein design. Building on our success in describing the fitness landscape of a single fold using coevolutionary models, we are seeking to design sequences which can fold into two different structures as envisaged in our recent theoretical work (7). We are collaborating with Susan Marqusee's group to test some of these ideas (P. Tian). We are also looking to develop similar ideas to identify proteins which naturally switch folds (such as RfaH), using sequence information (L. Frechette). 8. Modelling sensitivity of single molecule experiments to protein folding transition paths using molecular simulations. Recent single molecule fluorescence experiments have been able to detect transition paths between folded and unfolded states of proteins by combining photon by photon detection with sophisticated maximum likelihood analysis algorithms. However, it is not clear how the inferred transition path durations relate to the actual folding transition path lengths, since they cannot be independently measured. We have used simulations as a model to generate coarse-grained folding trajectories for two proteins (alpha3D, protein G), in which we can unambiguously assign transition paths. We then generated synthetic photon trajectories from these simulations and analyzed them in the same way as the experimental data. We found that the experimentally inferred transition path durations are of the right magnitude, but systematically shorter than the true durations. Beyond current analysis methods, we are also testing the feasibility of obtaining information besides just the length of the transition path, i.e. transition path \"shape\", from this type of experiment, using synthetic data generated from our simulations (G. Taumoefolau). 9. Using transition-path sampling to study the mechanism and rate of assembly of transmembrane protein dimers, as represented by Glycophorin A. We have used our force field developed to best reproduce the stability of Glycophorin A in POPC membranes to study the dynamics of protein association using enhanced sampling methods (transition-path sampling) (4). We find that association occurs via an intermediate in which non-native interactions are initially formed between the helices, followed by a second step driven by native interactions. The same approach should be applicable to study oligomerization of other transmembrane domains. (J. Domanski). Group members or jointly supervised external collaborators involved in each project are listed at the end of each section.", "keywords": [ "2019-nCoV", "Address", "Affinity", "Algorithmic Analysis", "Area", "Automobile Driving", "Base Sequence", "Cell Nucleus", "Cells", "Charge", "Collaborations", "Complex", "Cryoelectron Microscopy", "Cytoplasmic Granules", "DNA", "Data", "Data Set", "Databases", "Development", "Disease", "Evolution", "Fluorescence", "Fluorescence Spectroscopy", "Free Energy", "Future", "G-substrate", "GYPA gene", "Goals", "Grain", "Integral Membrane Protein", "Ions", "Length", "Liquid substance", "Measures", "Membrane", "Membrane Proteins", "Methodology", "Methods", "Mitochondria", "Modeling", "Molecular", "Molecular Conformation", "National Institute of Diabetes and Digestive and Kidney Diseases", "Nucleic Acids", "Organelles", "Paper", "Peptide Hydrolases", "Peptides", "Phase", "Photons", "Preparation", "Property", "Protein Dynamics", "Protein Engineering", "Proteins", "Publications", "RNA", "Reaction", "Refit", "Resolution", "Ribosomes", "Sampling", "Series", "Severe Acute Respiratory Syndrome", "Shapes", "Structure", "Supervision", "Testing", "Time", "Transmembrane Domain", "Virus Replication", "Work", "amyloid fibril formation", "density", "design", "dimer", "experimental study", "fitness", "improved", "inhibitor/antagonist", "interest", "macromolecule", "member", "millisecond", "models and simulation", "non-Native", "novel", "photon-counting detector", "predictive modeling", "protein aggregation", "protein complex", "protein folding", "protein function", "protein misfolding", "simulation", "single molecule", "single-molecule FRET", "success", "supercomputer", "theories", "time use" ], "approved": true } }, { "type": "Grant", "id": "7690", "attributes": { "award_id": "1ZIADK075141-04", "title": "Study of protein folding and misfolding by NMR spectroscopy", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 995227, "principal_investigator": { "id": 23487, "first_name": "Adriaan", "last_name": "Bax", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true }, "abstract": "In close collaboration with Philip Anfinrud, novel hardware was designed and developed that demonstrates, for the first time, that it is readily possible to monitor the folding of the protein chain in a residue-specific manner upon jumping the applied pressure. Pressure changes of up to 2.5 kbar, requiring 1-2 ms, are feasible and compatible with the recording of high quality NMR data. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin (VA2-ubiquitin), we have demonstrated that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record two- and three-dimensional NMR spectra of the unfolded protein at atmospheric pressure, providing new, residue specific information on the folding process. Protein folding, as commonly portrayed, is an exploration of a rough, high-dimensional landscape ending with a final descent into a low-energy folded state. During that journey, the protein may visit shallow basins corresponding to metastable structures, potentially of biological significance. Structural characterization of metastable states has remained challenging because of their low populations, which limit traditional NMR, and short lifetimes that make crystallization for X-ray diffraction difficult without stabilizing mutations, covalent modifications, or the addition of antibodies. We carried out the structural characterization of a pressure-sensitized ubiquitin mutant during folding and identified an on-pathway folding intermediate with non-native beta sheet registry, previously observed to be necessary in the PINK1 mitophagy pathway. Specifically, we used fast pressure jumps, synchronized with advanced NMR measurements on the evolving ensemble of protein conformations during folding, including NOE and residual dipolar coupling interactions. We found that that non-native beta sheet hydrogen bond registry can act as a metastable trap during protein folding. This work provides a template for future investigation of metastable conformations and protein folding with rich structural detail. Brain tissue of Alzheimers disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Abeta40 and Abeta42. It remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (< 1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Oligomeric states of Abeta40 were shown to exhibit a high degree of order, reflected by slow longitudinal 15N relaxation (T1 >5 s) for residues 18-21 and 31-34, whereas the N-terminal 10 residues relax much faster (T1 1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca 1000 s-1 after the oligomerization is initiated, indicating that the oligomers then have accumulated a size on the order of 1 MDa, or ca 250 peptides. Pressure-jump experiments reveal detailed information on the kinetics of the initiation of oligomer formation, as well as on the details of the structural arrangements of the peptides in the oligomer. Such experiments indicate a 3d order power dependence of the oligomerization rate on monomer concentration, indicative that a tetrameric state is the critical species forming the barrier to formation of the much larger oligomers. Experiments were interrupted by the COVID-19 pandemic but are to be resumed shortly.", "keywords": [ "3-Dimensional", "Address", "Alzheimer&apos", "s Disease", "Alzheimer&apos", "s disease patient", "Amyloid beta-42", "Amyloid beta-Protein Precursor", "Antibodies", "Atmospheric Pressure", "Back", "Biological", "COVID-19 pandemic", "Cells", "Collaborations", "Coupling", "Crystallization", "Data", "Dependence", "Deposition", "Disease", "Equilibrium", "Exhibits", "Future", "Hydrogen Bonding", "Hydrostatic Pressure", "Interruption", "Investigation", "Kinetics", "Length", "Measurement", "Modification", "Molecular Conformation", "Monitor", "Motion", "Mutation", "N-terminal", "NMR Spectroscopy", "Neurodegenerative Disorders", "PINK1 gene", "Parkinson Disease", "Pathway interactions", "Peptide Fragments", "Peptides", "Physiological", "Population", "Process", "Protein Conformation", "Proteins", "Registries", "Relaxation", "Residual state", "Resolution", "Sampling", "Signal Transduction", "Solvents", "Structure", "Temperature", "Theoretical Studies", "Thermodynamics", "Time", "Ubiquitin", "Visit", "Work", "X ray diffraction analysis", "beta pleated sheet", "brain tissue", "computer studies", "design", "experimental study", "high dimensionality", "insight", "monomer", "mutant", "non-Native", "novel", "polypeptide", "pressure", "protein folding", "protein misfolding", "two-dimensional" ], "approved": true } }, { "type": "Grant", "id": "7697", "attributes": { "award_id": "1ZIAAI001306-01", "title": "tissue tropism of coronavirus", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Institute of Allergy and Infectious Diseases (NIAID)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 41824, "principal_investigator": { "id": 23492, "first_name": "Yasmine", "last_name": "Belkaid", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1540, "ror": "https://ror.org/043z4tv69", "name": "National Institute of Allergy and Infectious Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1540, "ror": "https://ror.org/043z4tv69", "name": "National Institute of Allergy and Infectious Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true }, "abstract": "The COVID-19 condition caused by the novel coronavirus SARS-CoV-2 is an active, existential global health threat. A substantial fraction of those infected exhibit no symptoms, while others progress to severe illness resulting in multi-organ failure and death. The factors dictating whether an infected subject will progress to develop symptoms or will experience asymptomatic infection remains poorly understood. Using experimental models, work that we and others have conducted over the past few years revealed that the ability of a given microbe to cause disease can be highly contextual. Indeed, numerous factors can contribute to pathogen-induced morbidity or mortality including age, microbiota, nutritional status as well as co-infection or previous infections. Our combined laboratories have pioneered the development of murine models of infectious diseases and developed novel tools to track microbial tropism, tissue specific pathologies and immune responses to pathogens. Using models of murine coronavirus infection, this proposal aims at addressing the circumstances controlling anti-viral immunity and pathogenicity.", "keywords": [ "Acute", "Address", "Age", "COVID-19", "Cessation of life", "Coronavirus", "Coronavirus Infections", "Development", "Disease", "Exhibits", "Experimental Models", "Failure", "Family", "Goals", "Immune response", "Immunity", "Infection", "Intestines", "Intranasal Administration", "Laboratories", "Lung", "Microbe", "Modeling", "Morbidity - disease rate", "Murine hepatitis virus", "Mus", "Nutritional status", "Organ", "Pathogenicity", "Pathology", "Pulmonary Inflammation", "RNA Viruses", "Research Personnel", "Respiratory Signs and Symptoms", "Symptoms", "Tissues", "Tropism", "Virus", "Work", "antiviral immunity", "co-infection", "experience", "global health", "human disease", "infectious disease model", "member", "microbial", "microbiota", "mortality", "mouse model", "novel", "novel coronavirus", "nutrition", "pathogen", "tissue tropism", "tool" ], "approved": true } }, { "type": "Grant", "id": "7699", "attributes": { "award_id": "1ZIAES103345-01", "title": "SARS-CoV-2 Studies", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Institute of Environmental Health Sciences (NIEHS)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 438205, "principal_investigator": { "id": 23493, "first_name": "DOUGLAS", "last_name": "BELL", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1605, "ror": "https://ror.org/00j4k1h63", "name": "National Institute of Environmental Health Sciences", "address": "", "city": "", "state": "NC", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1605, "ror": "https://ror.org/00j4k1h63", "name": "National Institute of Environmental Health Sciences", "address": "", "city": "", "state": "NC", "zip": "", "country": "United States", "approved": true }, "abstract": "Smoking, Immune Senescence and COVID-19 morbidity. The general approach is to prospectively establish a bank of cryopreserved PBMCs from smokers and nonsmokers before any COVID-19 exposure and use CyTOF to analyze detailed immune profiles. Subjects will return for second post-COVID-19 pandemic visit to provide a second blood sample for analysis. The focus is primarily on immune profiles compared with COVID-19 incidence (or levels of morbidity) in smokers. As seen in Figure 2, there is a large variance in CD16+CD8+ T cell frequency among smokers. We hypothesize that senescent CD16+CD8+ T cells will be higher in smokers who develop COVID-19 or who have more severe morbidity. Specific Aims and Experimental Plans Specific Aim 1. Establish CyTOF immune profiles in 60 smokers and 60 matched nonsmokers who are COVID-19 negative and will be followed up on and recalled after the COVID-19 pandemic has subsided. Specific Aim 2. Compare immune profiles determined from a post-COVID-19 visit to the pre-COVID-19 negative sample. COVID-19 Diagnostics. Aim 1. Develop cDNA library methods for SARS-CoV-2 and host cells suitable for Next Generation sequencing.", "keywords": [ "2019-nCoV", "Bar Codes", "Blood specimen", "CD8-Positive T-Lymphocytes", "COVID-19", "COVID-19 pandemic", "Cells", "Communicable Diseases", "Cryopreservation", "Diagnostic", "Disease Outbreaks", "FCGR3B gene", "Frequencies", "Genetic Transcription", "Goals", "Guide RNA", "Healthcare Systems", "Immune", "Immune System Diseases", "Incidence", "Infection", "Methods", "Morbidity - disease rate", "Peripheral Blood Mononuclear Cell", "Predisposition", "Premature aging syndrome", "Regulatory T-Lymphocyte", "Sampling", "Screening procedure", "Severe Acute Respiratory Syndrome", "Smoker", "Smoking", "T-Lymphocyte Subsets", "Viral", "Visit", "cDNA Library", "follow-up", "high risk", "indexing", "mortality", "next generation sequencing", "non-smoker", "prospective", "response", "screening", "senescence" ], "approved": true } }, { "type": "Grant", "id": "7691", "attributes": { "award_id": "1ZIADK029046-14", "title": "Methods to accelerate protein structure determination by solution NMR", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 995227, "principal_investigator": { "id": 23487, "first_name": "Adriaan", "last_name": "Bax", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true }, "abstract": "For unfolded proteins, the Rh provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (Rh) of biomolecules in solution. However, while the impact of pressure on the viscosity of water is well known, we find that elevated pressures increase the Rh of dioxane, a commonly used reference standard, and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction observed by 13C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurements of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure and as a function of urea concentration show that Rh values of both the folded and the unfolded states remain nearly invariant. At ca 23 angstrom, the Rh of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) alpha-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of both unfolded ubiquitin and alpha-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions. Small heat-shock proteins (sHSPs) are molecular chaperones that respond to cellular stresses to combat protein aggregation. HSP27 is a critical human sHSP that forms large, dynamic oligomers whose quaternary structures and chaperone activities depend on environmental factors. Upon exposure to cellular stresses, such as heat shock or acidosis, HSP27 oligomers can dissociate into dimers and monomers, which leads to significantly enhanced chaperone activity. The structured core of the protein, the alpha-crystallin domain (ACD), forms dimers and can prevent the aggregation of substrate proteins to a similar degree as does the full-length protein. We have shown that when the ACD dimer dissociates into monomers, it partially unfolds and exhibits enhanced activity. Using solution-state NMR spectroscopy to characterize the structure and dynamics of the HSP27 ACD monomer, we have shown that the monomer is stabilized at low pH and that its backbone chemical shifts, N-15 relaxation rates, and 1H-15N residual dipolar couplings suggest structural changes and rapid motions in the region responsible for dimerization. By analyzing the solvent accessible and buried surface areas of sHSP structures in the context of a database of dimers that are known to dissociate into disordered monomers, we predict that ACD dimers from sHSPs across all kingdoms of life may partially unfold upon dissociation. This resulted in a general model in which conditional disorder-the partial unfolding of ACDs upon monomerization-is a common mechanism for sHSP activity. A series of novel three- and four-dimensional NMR experiments has been developed that provides access to the structure and dynamics of the 608-residue homodimeric Main protease of the SARS-CoV-2 virus. This protein is larger than what is readily accessible to the standard NMR approaches used, but the novel methods combined with 900 MHz high-field measurements provided sufficient data for a detailed structural analysis which reveals subtle but statistically very significant differences relative to all X-ray structures available so far. A characterization of the backbone dynamics shows a strong effect of ligands on the dynamics of the protein backbone in the active site region.", "keywords": [ "2019-nCoV", "3-Dimensional", "Acidosis", "Active Sites", "Algorithms", "Area", "Biological", "Cellular Stress", "Chemicals", "Collection", "Core Protein", "Coupling", "Data", "Data Analyses", "Databases", "Diffusion", "Dimerization", "Dioxanes", "Disease", "Dissociation", "Environmental Risk Factor", "Exhibits", "Exposure to", "Four-dimensional", "Friction", "Goals", "HSPB1 gene", "Heat shock proteins", "Heat-Shock Response", "Human", "Hydrophobic Surfaces", "Hydrophobicity", "Hydrostatic Pressure", "Length", "Life", "Ligands", "Measurement", "Measures", "Methods", "Modeling", "Molecular Chaperones", "Molecular Conformation", "Molecular Structure", "Motion", "NMR Spectroscopy", "Pattern", "Peptide Hydrolases", "Physiologic pulse", "Protein Dynamics", "Proteins", "Radial", "Reference Standards", "Relaxation", "Reporter", "Residual state", "Roentgen Rays", "Rotation", "Sampling", "Series", "Solvents", "Structure", "Surface", "Time", "Ubiquitin", "Urea", "Vertebral column", "Virus", "Viscosity", "Water", "alpha synuclein", "alpha-Crystallins", "combat", "data acquisition", "dimer", "experimental study", "monomer", "mutant", "novel", "polypeptide", "pressure", "prevent", "protein aggregation", "protein folding", "protein structure", "restraint", "small molecule" ], "approved": true } }, { "type": "Grant", "id": "7682", "attributes": { "award_id": "1ZIEBC011384-10", "title": "Anatomic Pathology Residency Program", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Cancer Institute (NCI)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 852774, "principal_investigator": { "id": 23485, "first_name": "Frederic", "last_name": "Barr", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1601, "ror": "", "name": "DIVISION OF BASIC SCIENCES - NCI", "address": "", "city": "", "state": "", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1601, "ror": "", "name": "DIVISION OF BASIC SCIENCES - NCI", "address": "", "city": "", "state": "", "zip": "", "country": "United States", "approved": true }, "abstract": "Residents in the Anatomic Pathology Residency Program contribute to the clinical research program of the NCI and the NIH. Through their efforts as anatomic pathology residents in training, they help illuminate the pathological changes associated initial presentation and therapy of both neoplastic and non-neoplastic diseases and explore new techniques to improve diagnosis of these diseases. These residents are critical to the patient care activities of the NCI and the NIH, and contribute to the diagnosis and management of disease, especially COVID-19 this year. Residents have also contributed to publications dealing with characterization, diagnosis, and pathogenesis of a number of disease entities.", "keywords": [ "Accreditation", "Anatomy", "Area", "Autopsy", "Biological Assay", "COVID-19", "Case Study", "Clinical", "Clinical Research", "Clinical Trials", "Clonality", "Communicable Diseases", "Consult", "Consultations", "Cytopathology", "DNA Methylation", "DNA analysis", "Diagnosis", "Diagnostic", "Digestive System Disorders", "Disease", "Disease Management", "Educational process of instructing", "Enrollment", "Etiology", "Exposure to", "Extramural Activities", "Flow Cytometry", "Fluorescent in Situ Hybridization", "Functional disorder", "Funding", "Gene Mutation", "Genes", "Genetic study", "Hematopathology", "Institutes", "Investigation", "Kidney Diseases", "Laboratories", "Learning", "Lymphoid", "Malignant Neoplasms", "Medicine", "Mission", "Molecular Genetics", "Mutation", "Oncology", "Outcome", "Pathogenesis", "Pathologic", "Pathology", "Patient Care", "Patients", "Philosophy", "Play", "Protocols documentation", "Publications", "Research", "Research Activity", "Research Training", "Residencies", "Role", "Services", "Surgical Pathology", "Techniques", "Tissues", "Training", "Training Programs", "United States National Institutes of Health", "base", "cancer therapy", "clinical center", "disease diagnosis", "epidemiology study", "experience", "improved", "insight", "molecular diagnostics", "neoplastic", "nervous system disorder", "neuropathology", "next generation sequencing", "novel", "programs", "recruit", "research clinical testing", "soft tissue", "urologic" ], "approved": true } }, { "type": "Grant", "id": "7698", "attributes": { "award_id": "1ZICAI001226-03", "title": "Center for Human Immunology", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Institute of Allergy and Infectious Diseases (NIAID)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 3460631, "principal_investigator": { "id": 23492, "first_name": "Yasmine", "last_name": "Belkaid", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1540, "ror": "https://ror.org/043z4tv69", "name": "National Institute of Allergy and Infectious Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1540, "ror": "https://ror.org/043z4tv69", "name": "National Institute of Allergy and Infectious Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true }, "abstract": "In response to the SARS-CoV-2 pandemic we have made the technologies and platforms at CHI available for characterization of COVID-19 patient samples, as a priority ahead of our ongoing collaborative studies. By early April we had completed adaptation of our transcriptomic and flow cytometry platforms, and operating procedures, to begin analysing peripheral blood from Covid-19 patients. CHI began receiving samples from Northern Italy and CITE-Seq was performed for more than 100 samples in close collaboration with the Tsang lab. BCR sequencing and scATAC seq was performed for a subset of these, and proteomic analysis was obtained for all of the samples by outsourcing this work at SomaLogic. 27-color flow cytometry phenotyping was also performed by CHI to validate the novel CITE-seq platform. Further, CHIs automated pipeline was used to isolate high quality RNA from PAXgene samples of 200 Covid-19 samples, to be used for both RNA-Seq at CHI as well as distribution to the Goldbach-Mansky lab. Together these efforts have contributed to initial immune characterization of acutely infected Covid-19 patients. We hope these platforms can now be applied to new cohorts becoming available which are able to address questions such as variation in the outcomes of infection. In addition to this COVID work CHI has continued to advance its previous studies that comprise 25 active collaborations across 8 Institutes and the Clinical Center. These are divided into 3 stages: initiation/sample collection (12 studies), assay (8 studies), and analysis/write up (5 studies). These all comprise multi-modal high-dimensional immune phenotyping of primary human samples. The current studies include characterizing immune changes in vaccine trials (malaria, HSV), clinical intervention trials (lupus, CHAPLE), and cohort studies of rare diseases (mitochondrial dysfunction). One of the new studies added is an exciting collaboration with Kevin Hall at NIDDK to characterize immune differences after periods of highly controlled diet changes. The monoclonal antibody therapy study of CHAPLE disease patients, with Mike Lenardo, is an example of how high dimensional phenotyping can lead to better understanding of patient pathology, and may inform use of a nascent therapy. The proteomic changes we observed give basic biology insight into, in this case, CD55 function and the mechanisms of complement inhibition, and this work is under review at Nature Immunology. Studies published this year included an integrated proteomic and cellular phenotyping analysis of maternal peripheral blood in longitudinally sampled human pregnancies, and characterization of changes in peripheral blood during treatment of healthy subjects with statins, and obese individuals undergoing a clinical trial of colchicine treatment. To support CHIs mission we continue to develop wet-lab and computational infrastructure. For single cell sequencing technologies using the 10x platform, we have expanded the number of protein antigens measured in CITE-Seq, and developed scATAC-seq approaches with greater throughput and lower cost. CITE-Seq, in particular, led to a high-profile finding characterizing variation in baseline immune states that predicts immune responses (see section Scientific Advances). For flow cytometry, on a new Cytek Aurora we have validated a 27 color cytometry for broad immune phenotyping, as well as a 33 color T cell focused panel run after PMA stimulation to detect cells primed for production of characteristic cytokines or transcription factors. For flow cytometry analysis we are applying methods to enable conventionally gated populations to be recognized and interrogated using high dimensional parameters, to help overcome the difficulties of interpreting unbiased clustering of high dimensional data. For mass cytometry analysis we are finalising a package that uses high dimensional observations to discriminate only the subset of cells that respond in ex vivo stimulation assays, such as our mass cytometry assay that quantifies phosphorylation of 10 intracellular proteins, in addition to 20 cell phenotype markers, in response to 12 in vitro stimulation conditions. CHI also provides fee-for-service access to assays not otherwise accessible to NIH researchers. The SomaLogic proteomic assay has been run for over 1000 samples, for 8 investigators, in the last year. This included human and murine samples from serum, plasma, cerebrospinal fluid, bronchioalveolar lavage fluid, and tissue homogenates. We continue to monitor alternative platforms and have run tests on O-Link that is offering increasingly high parameter antibody based analysis, although currently plan to transition our SomaLogic platform from the current 1.3k antigen panel to an analysis of 4.5k antigens.", "keywords": [ "2019-nCoV", "Acute", "Address", "Antibodies", "Antigens", "Behavior", "Biological", "Biological Assay", "Biology", "Bronchoalveolar Lavage", "COVID-19", "Cells", "Cerebrospinal Fluid", "Characteristics", "Clinical", "Clinical Trials", "Cohort Studies", "Colchicine", "Collaborations", "Color", "Complement", "Computer Analysis", "Computer Models", "Coupled", "Cytometry", "Data", "Development", "Diet", "Disease", "Fee-for-Service Plans", "Flow Cytometry", "Goals", "Health", "Health Services Accessibility", "Human", "Immune", "Immune response", "Immunity", "Immunologic Monitoring", "Immunology", "Immunotherapy", "In Vitro", "Individual", "Infection", "Institutes", "Intervention", "Intervention Trial", "Italy", "Laboratories", "Lead", "Leadership", "Link", "Liquid substance", "Lupus", "Malaria", "Measures", "Methods", "Mission", "Modeling", "Monitor", "Monoclonal Antibody Therapy", "Mus", "National Institute of Allergy and Infectious Disease", "National Institute of Diabetes and Digestive and Kidney Diseases", "Nature", "Obesity", "Outcome", "Outsourcing", "Pathology", "Patients", "Phenotype", "Phosphorylation", "Plasma", "Population", "Pregnancy", "Procedures", "Production", "Proteins", "Proteomics", "Publishing", "RNA", "Rare Diseases", "Research Personnel", "Resources", "Review Committee", "Running", "Sampling", "Scientific Advances and Accomplishments", "Serum", "Services", "Simplexvirus", "System", "T-Lymphocyte", "Technology", "Testing", "Tissues", "Training", "United States National Institutes of Health", "Vaccination", "Variant", "Work", "Writing", "base", "clinical application", "clinical center", "cohort", "collaborative approach", "computer infrastructure", "coronavirus disease", "cost", "cytokine", "data mining", "data modeling", "design", "high dimensionality", "insight", "microbiome", "microbiome research", "microbiota", "mitochondrial dysfunction", "multidimensional data", "multimodality", "novel", "pandemic disease", "peripheral blood", "phenotypic biomarker", "programs", "recruit", "response", "sample collection", "single cell sequencing", "tool", "transcription factor", "transcriptome sequencing", "transcriptomics", "vaccine trial" ], "approved": true } }, { "type": "Grant", "id": "7689", "attributes": { "award_id": "1ZIADK075023-12", "title": "Structural study of the HIV1 gp41 coat protein", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 995227, "principal_investigator": { "id": 23487, "first_name": "Adriaan", "last_name": "Bax", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1600, "ror": "https://ror.org/00adh9b73", "name": "National Institute of Diabetes and Digestive and Kidney Diseases", "address": "", "city": "", "state": "MD", "zip": "", "country": "United States", "approved": true }, "abstract": "The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine and bicelles consisting of DMPC and dihexanoyl phosphatidylcholine suggested the importance of trimerization of gp41s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion. The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We have found that in aqueous solution the MPER fragment (gp160 residues 660-674) exists in a monomer-trimer equilibrium with an association constant in the micro-molar range. Thermodynamic analysis revealed that the association is exothermic, more favorable in D2O than H2O, and increased with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13C chemical shifts, NOE, and hydrogen exchange rates revealed that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an alpha-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state. Preliminary experiments applied to the ecto-domain of the Spike protein of SARS-CoV-2 indicate this protein contains a three-helical N-terminal heptad repeat (NHR) that is considerably more stable than the corresponding region of gp41, and the propensity for the C-terminal heptad (CHR) repeat albeit lower than that of NHR, is also substantially higher than for the CHR of gp41, whereas the propensity to trimerize for its membrane proximal region is substantially lower. Work has been submitted for publication but remains under review.", "keywords": [ "2019-nCoV", "Affinity", "Antibodies", "Binding", "C-terminal", "Capsid Proteins", "Cell membrane", "Cells", "Chemicals", "Circular Dichroism", "Detergents", "Dissociation", "Ebola", "Epitopes", "Equilibrium", "Free Energy", "Glycoproteins", "HIV Envelope Protein gp120", "HIV-1", "Hemagglutinin", "Hydrogen", "Hydrophobicity", "Influenza", "Ionic Strengths", "Lecithin", "Lipid Bilayers", "Lipids", "Mediating", "Membrane", "Membrane Fusion", "Micelles", "Modeling", "Molecular Conformation", "N-terminal", "Phosphorylcholine", "Play", "Process", "Property", "Proteins", "Publications", "Reporting", "Role", "Structure", "Surface", "Tertiary Protein Structure", "Thermodynamics", "Transmembrane Domain", "Viral", "Viral Fusion Proteins", "Virus", "Work", "aqueous", "biophysical techniques", "design and construction", "driving force", "experimental study", "gp160", "influenzavirus", "intermolecular interaction", "melting", "monomer", "neutralizing antibody", "prevent" ], "approved": true } }, { "type": "Grant", "id": "7706", "attributes": { "award_id": "1ZIABC011941-01", "title": "Studies of the SARS-CoV-2 Spike Protein", "funder": { "id": 4, "ror": "https://ror.org/01cwqze88", "name": "National Institutes of Health", "approved": true }, "funder_divisions": [ "National Cancer Institute (NCI)" ], "program_reference_codes": [], "program_officials": [], "start_date": null, "end_date": null, "award_amount": 512699, "principal_investigator": { "id": 23500, "first_name": "JAY A", "last_name": "BERZOFSKY", "orcid": null, "emails": "", "private_emails": "", "keywords": null, "approved": true, "websites": null, "desired_collaboration": null, "comments": null, "affiliations": [ { "id": 1601, "ror": "", "name": "DIVISION OF BASIC SCIENCES - NCI", "address": "", "city": "", "state": "", "zip": "", "country": "United States", "approved": true } ] }, "other_investigators": [], "awardee_organization": { "id": 1601, "ror": "", "name": "DIVISION OF BASIC SCIENCES - NCI", "address": "", "city": "", "state": "", "zip": "", "country": "United States", "approved": true }, "abstract": "This project just began in April, 2020, during the COVID-19-induced closure of NIH labs, with very few people allowed to work, so it is very early ( 3 months) and progress is limited on each of the above sub-studies. In vitro studies in macaque bronchioalveolar lavage cells: So far, we have seen some effects in vitro on these cells of stimulating them with recombinant spike protein to affect expression of ACE2 and of interferons, that may play a role in infection. Little effect was seen on other cytokine or chemokine production. This work is in progress. In vivo vaccine studies in macaques: the animals have been primed IM with spike in different adjuvants and boosted systemically with spike in nanoparticles. Some T cell responses have been induced by the different regimens, and serum and BAL fluid for antibody responses have been collected and are being assayed. It is too soon to determine which regimen is more immunogenic. If we can arrange a challenge at a BSL3 facility that can handle the virus (being negotiated), we hope to challenge the animals with SARS-CoV-2 to measure vaccine efficacy and correlate that with different immune responses. In vivo studies in mice: ACE2-transgenic mice have been ordered but their shipment was delayed until the end of July. In wild type B6 mice, we have immunized with recombinant spike protein in several different adjuvants to determine the best formulation. That work is in progress. The DNA vaccine with spike protein coupled to a chemokine is being constructed and that work is in progress. Human cell lines: We have received the immortalized human lung epithelial cell lines, that express ACE2, from John Minna at UTSW, as well as some of his non-small-cell lung cancer cell lines that also express ACE2. We have just obtained (mid-July) an antibody to ACE2 that we will use to verify expression. These lines have now been thawed and are being grown up, but no results of planned studies to stimulate with spike protein have been obtained yet. We intend to measure production of chemokines, cytokines, and other factors that may affect the COVID-19 disease course, as noted above.", "keywords": [ "2019-nCoV", "Adjuvant", "Affect", "Animal Model", "Animals", "Antibodies", "Antibody Response", "Binding", "Bioinformatics", "Biological Assay", "Bronchoalveolar Lavage", "COVID-19", "Cancer cell line", "Cell Line", "Cells", "Clinical Trials", "Coupled", "DNA Vaccines", "Dendritic Cells", "Disease", "Electroporation", "Engineering", "Epithelial Cells", "Epitopes", "Formulation", "Human", "Human Cell Line", "Immune", "Immune response", "Immunize", "In Vitro", "Industrialization", "Infection", "Interferons", "Liquid substance", "Lung", "Macaca", "Malignant neoplasm of lung", "Measures", "Mediation", "Medical center", "Methods", "Modeling", "Morbidity - disease rate", "Mucosal Immunity", "Mucous Membrane", "Mus", "Natural Immunity", "Non-Small-Cell Lung Carcinoma", "Phase", "Play", "Production", "Proteins", "Recombinants", "Regimen", "Respiratory Mucosa", "Role", "Route", "SIV Vaccines", "Serum", "Ships", "Site", "Structure of respiratory epithelium", "T cell response", "Texas", "Transgenic Mice", "United States National Institutes of Health", "Universities", "Vaccines", "Vascular Endothelial Cell", "Viral", "Viral Vaccines", "Virus", "Work", "biosafety level 3 facility", "chemokine", "cytokine", "immunogenic", "immunogenicity", "immunopathology", "in vivo", "mucosal vaccine", "nanoparticle", "neutralizing antibody", "nonhuman primate", "preclinical study", "receptor", "receptor binding", "response", "vaccine candidate", "vaccine efficacy", "vaccine trial", "viral transmission" ], "approved": true } } ], "meta": { "pagination": { "page": 1, "pages": 1392, "count": 13920 } } }