Grants

Volunteers Improving Neighborhood Environments Inc.

For repair of urban farms that were damaged by the flooding of Tropical Storms Irene and Lee, and expansion of the farms to benefit community residents.

Regional Council Capital Fund - RC5

BioBAT Capital

NYC Department of Cultural Affairs

Biodesign Challenge

National Institutes of Health

Bioengineering Partnerships with Industry (U01 Clinical Trial Optional)

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Cardiac IKr is a critical repolarizing potassium current shaping the human ventricular action potential. It is conducted by heteromeric assemblies of the human ether-à-go-go-related gene (hERG1) 1a and 1b subunits. These subunits are encoded by alternate transcripts of the hERG/KCNH2 gene and differ only in their amino- terminal regions. hERG1a/1b heteromerization is vital for normal CM function, as the imbalance of subunit expression and/or function results in cellular pro-arrhythmic behaviors. hERG1a/1b assembly is mediated by the co-translational association of the encoding mRNAs in HEK293 cells, cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), and human myocardium. Evidence suggests that interaction between the nascent proteins is not required for the co-translational complex assembly. This grant's preliminary findings indicate that this complex assembly occurs post-transcriptionally and is promoted by direct interactions between hERG1a and 1b mRNAs governed by their secondary structures. In preliminary studies, RNA binding proteins DDX3X and DDX5 were identified as part of the complex, and purified DDX3X promoted hERG1a/1b mRNAs' association in vitro. In the K99 phase, I will define the mRNA structural features promoting the co-translational association and determine the affinity and energies of the RNA/RNA interaction using in vitro systems, isothermal calorimetry (ITC), mutagenesis, hybrid protein-RNA immunoprecipitation (RIP), and live-cell imaging. I will also determine whether DDX3X and DDX5 affect hERG1a and 1b mRNAs stability, translation, and association in hiPSC-CMs using qPCR, electrophysiology, Western Blot, ribosome profiling, RIP, and single molecule fluorescent in situ hybridization (smFISH). I will use quantitative ITC and in vitro reconstitution approaches to determine the specificity, affinity, and energies of the interaction between purified DDX3X and DDX5 with hERG1a and 1b mRNAs. I will also evaluate if DDX3X and DDX5 promote the association of the mRNAs in in vitro systems. In the R00 phase, I will determine whether the stability, translation, and association of hERG1a and 1b mRNAs are impaired in arrhythmias associated with type 2 long QT syndrome (LQT2). I will use hiPSC-CM disease models to evaluate half-life, translation rate, and association of the mRNAs with qPCR, ribosome profiling, RIP, and smFISH. These experiments will contribute to understanding ion channel biogenesis and elucidate molecular mechanisms underlying LQT2 related arrhythmias. This proposal is designed to fulfill my short-term goals of expanding my skills in cardiovascular research and biophysics and transitioning into the independent phase of my career. This will ultimately allow me to obtain my long-term purpose of linking RNA and ion channel biophysics to translational cardiovascular research.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Crohn's disease and ulcerative colitis, known collectively as inflammatory bowel disease (IBD), exhibit distinct and progressive patterns of inflammation in the gastrointestinal (GI) tract, sharing overlapping symptoms with other illnesses and complicating diagnosis. Although endoscopy provides visualization useful for diagnostics, it has limited reach within the GI tract and cannot provide explicit physiological measurements. Bioimpedance, a surrogate for epithelial barrier integrity, is a promising technique for quantifying dielectric tissue properties relevant to the state of inflammation in the gastrointestinal tract. However, research studying the physiological implications of bioimpedance is limited beyond the esophagus due to variations in inflammatory response and inconsistent sensor contact in the GI tract, obfuscating the real-time correlation between tissue impedance and inflammatory state. To address this, we propose to adapt a previously developed ingestible capsule platform to collect real-time measurements of pH, pressure, and bioimpedance of epithelial tissues. In prior work, we have successfully demonstrated capsule-embedded bioimpedance sensors for wireless monitoring of tissue permeability of excised colonic tissue from mice and in a cecum using a rat colitis model to evaluate the potential of bioimpedance for disease assessment. We hypothesize that a multimodal sensing approach utilizing ingestible capsule technology will enable access to barrier integrity information in local tissues throughout the GI tract, expanding the toolbox of approaches for early detection of inflammatory GI diseases. Through two distinct aims, the proposed capsule device will correlate changes in bioimpedance and intestinal permeability in the GI tract—phenomena not easily detected by traditional capsule endoscopy—and demonstrate safe GI transit using animal models. Aim 1 will focus on benchtop evaluation. Bioimpedance data will be collected from freshly excised ex vivo tissues of adult pigs with altered intestinal barrier integrity. Additionally, we will enhance measurement reliability while reducing the capsule size by integrating an array of bioimpedance and pressure sensors around the capsule to confirm tissue contact and microfabricating a pH sensor for capsule localization. The capsule will be evaluated in simulated intestinal fluids, and by ex vivo peristaltic simulators (i.e., modified organ chamber) to characterize the device under interferent GI conditions such as motion and acidic pH levels. In Aim 2, we will collect bioimpedance, contact pressure, and pH data in vivo during two animal studies in healthy adult pigs. One study will feature surgically implanted capsules fixed at stationary locations during measurement to ensure sensor contact and separate measurements by tissue type. Additionally, a small-scale pig study will be performed to understand capsule motility and ensure the safe passage of the capsule throughout GI transit while validating the reliability of the measurement. This aim will unveil measurement variability in vivo, generate datasets for statistical interpretation, and assist with determining feasibility of our technology toward future studies of permeability as an early-stage biomarker for inflammation.

U.S. National Science Foundation

Biological Anthropology Program Senior Research Awards

DARPA - Biological Technologies Office

Biological Technologies

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

SUMMARY/ABSTRACT Growing evidence suggests that whole grain (WG) intake may play an important role in cardiometabolic disease prevention. However, numerous individual human studies have failed to support clear-cut conclusions on the topic. This inconsistency may be because the prevailing nutrition research relies heavily on self-reported measures of diet that are often prone to measurement error. In addition, these instruments cannot reflect the complexity of the chemical compositions of each WG, the impact of varieties, environment, cooking, and food processing on WG chemical compositions, and the interindividual variations on the absorption and metabolisms of WG phytochemicals. The molecular signatures of WG intake remain largely undefined. The major dietary cereals - wheat, rye, oat, barley, rice and corn- contain different unique phytochemicals, which can be used to reflect the intake of each individual WG. We recently found that the combination of targeted and non-targeted metabolomics approaches can effectively identify biomarkers of WG wheat and oat intake. However, the biomarkers of WG barley, rice, and corn have not been identified and these biomarkers have not been validated and associated with coronary heart disease (CHD) in an epidemiological setting. This application is aimed to test the hypothesis that bioactive WG phytochemicals, and their metabolites, are objective biomarkers of WG intake, integrate inter-individual differences, affect endogenous metabolome, and are likely more intimately associated with cardiometabolic diseases than traditional dietary assessments. This hypothesis will be tested in two aims. Aim 1 is to identify biomarkers of WG barley, rice, and corn intake following an acute exposure in a pharmacokinetic feeding study. In this aim, we will conduct a randomized crossover acute pharmacokinetic study of WG barley, rice, and corn, respectively. We will then use a targeted metabolomics approach to identify the exposure markers of WG barley, rice, and corn intake, further develop analytical methods to study their pharmacokinetics, and build our in-house WG metabolite database, and an untargeted metabolomics approach to investigate post-prandial biomarkers of WG barley, rice, and corn intake. The goals of Aim 2 are to determine 1) whether biomarkers of WG intake identified can represent habitual intake from two well phenotyped observational studies – the Men’s Lifestyle Validation Study (MLVS) and the Women’s Lifestyle Validation Study (WLVS), which have detailed and repeated measurements of diet using multiple 7-day diet records (7DDRs); and 2) whether biomarkers of WG intake are prospectively associated with risk of CHD in a cohort of female nurses. At the completion of these studies, our expectation is that we will identify WG phytochemicals and their metabolites as objective biomarkers of total and individual WGs that are commonly consumed by Americans. We also expect that the markers are robustly associated with WG intake and a lower CHD risk in free-living individuals.

U.S. National Science Foundation

Biomechanics and Mechanobiology

National Institutes of Health

Biomedical Research Environment and Sponsored Programs Administration Development (BRE-SPAD) Program (UC2- Clinical Trial Not Allowed)

U.S. National Science Foundation

Biophotonics

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT The objective of this proposal is to develop a quantitative understanding of how the biophysical properties of antibodies impact their capacity to evolve affinity to divergent SARS-CoV-2 spike variants. Though there is substantial evidence that mutations acquired during affinity maturation impact antibody expression, affinity for distinct viral variants, and self-reactivity, we lack a quantitative understanding of (1) how mutations impact these biophysical properties and (2) how these properties, and trade-offs between them, collectively determine the fate of the corresponding B-cell lineage. Here, we propose three Aims to test our hypothesis that mutations differentially impact antibody expression, affinity, and self-reactivity, resulting in biophysical trade-offs that constrain the evolution of antibodies that bind divergent SARS-CoV-2 spike variants. In Aim 1, we quantitate the biophysical effects of mutations in anti-SARS-CoV-2 spike antibodies, using high-throughput mammalian cell- display methods we recently developed. By measuring the expression, affinity, and self-reactivity for millions of anti-spike antibodies, including broadly neutralizing antibodies (bnAbs) that bind divergent spike variants, their evolutionary predecessors, and systematically mutagenized antibody sequences, we will unveil biophysical constraints that shape affinity maturation to rapidly evolving viral antigens. In Aim 2, we evaluate the contributions of antibody biophysical properties to B-cell fitness, or proliferation, using longitudinally-sampled patient B-cells following exposure to divergent strains of SARS-CoV-2. This approach will reveal the relative importance of distinct antibody biophysical properties in driving B-cell evolutionary dynamics in human repertoires and enable development of quantitative models for predicting the outcomes of affinity maturation. In Aim 3, we define the impact of selection pressure during affinity maturation on the biophysical properties of the resulting antibodies, focusing on selection regimes known to favor the maturation of bnAbs that bind distinct spike variants. To this end, we leverage a B-cell directed evolution platform that mimics the mutagenic load of somatic hypermutation, enables fine-tuning of the antibody selection conditions, and supports longitudinal B-cell sampling to profile the evolutionary dynamics of the B-cell response and the biophysical properties of the corresponding antibody lineages. The resulting data will be used to define the impact of the selection regime on the biophysical determinants of B-cell fitness. Successful completion of these Aims will yield quantitative insight into (1) how antibody biophysical properties change during affinity maturation, (2) how they collectively determine B-cell fate in human repertoires, and (3) how their relative importance varies across distinct selection regimes. Thus, this work will advance our fundamental understanding of the biophysical mechanisms that shape antibody affinity maturation to rapidly evolving pathogens like SARS-CoV-2, supporting efforts to design and elicit antibodies that bind existing and novel viral variants.

U.S. National Science Foundation

Biosensing

Middlebury

Bioski Road Top

NIAID - National Institute of Allergy and Infectious Diseases

Natural products (aka specialized metabolites) are small molecules produced by bacteria, fungi, plants, and animals that have found many uses in agricultural, veterinary, and human health. More than half of all currently FDA approved drugs are natural products or derived from natural products. In addition, to their far- ranging biological activities (anti-cancer to anti-bacterial to anti-viral) the pathways that organisms utilize to construct natural products from simple, readily available building blocks, particularly those used by microbes, have triggered much interest. Both as a way to understand the mechanisms and enzymes to allow manipulation of the proteins and pathways to produce new-to-nature natural products with altered biological activity and from an intellectual standpoint. In this proposal, we dissect the mechanism of ureido bond formation in the biosynthesis of three families of bioactive bacterial natural products, the anabaenopeptins, the syringolins, and the muramycins, which derive from diverse bacterial genera. These biologically active molecules contain the proteolytically stable ureido bond, which is a key contributor to their biological activity. While the mechanism of ureido formation in biotin biosynthesis has been studied, we hypothesize that the biosynthesis of the anabaenopeptins, syringolins, and muramycins, which are all assembled through the action of a non-ribosomal peptide synthetase (NRPS), are distinct due to the fact that the intermediates are tethered to the NRPS in contrast to the untethered biotin intermediate, 7,8-diaminononanoate. Our preliminary data suggests that the mechanism utilized in the biosynthesis of the anabaenopeptins is distinct from the mechanism previously proposed for the biosynthesis of the syringolins. This proposal describes a multidisciplinary approach consisting of protein overexpression, isotope labeling, and chemical trapping in Aim 1, which is complemented by cryo-EM, and site directed mutagenesis in Aim 2. These specific aims will allow the elucidation of the mechanism utilized during ureido bond formation while identifying the protein residues and motifs involved in substrate binding and catalysis in proteins found in different bacterial genera and with different protein architectures. The identification of the motifs will allow the proper annotation of ureido bond forming condensation domains found in deposited sequenced genomes, which is an issue as they are currently annotated as having amide bond forming activity by popular annotation software like AntiSMASH. This prevents the accurate prediction of compound structure from genomic data and represents a current hurdle in the field of natural products. The knowledge gained in this proposal will set the stage for the future engineering of new natural products with altered biological activity by introducing ureido bonds to increase proteolytic stability and alter physiochemical properties.

City of Richmond

Bioxytech Retina, Inc

Tolland Town Treasurer

Priority

Tolland Town Treasurer

Non-Priority

Lower East Side Girls Club of New York

To support the establishment of a research lab focused on pollinator habitat around the organization's recently installed rooftop meadow.

Lower East Side Girls Club of New York

To support the establishment of a research lab focused on pollinator habitat around the organization's recently installed rooftop meadow.

Gallagher Asphalt Corporation

Land and Buildings and Structures and Thoroughfares

Builder's Asphalt LLC

Bituminous material distributors

GALLAGHER MATERIALS CORP

Land and Buildings and Structures and Thoroughfares