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Winners Sorted by Institute-Center FARE2020WINNERS Sorted By Institute Ji Chen Postdoctoral Fellow CC Neuroscience - General Toward a Wearable Pediatric Robotic Knee Exoskeleton for Real World Overground Gait Rehabilitation in Ambulatory Individuals Crouch gait, or excessive knee flexion, is a debilitating gait pathology in children with cerebral palsy (CP). Surgery, bracing and therapy provide only short term correction of crouch and more sustainable solutions remain a significant challenge in children with CP. One major hurdle is achieving the required dosage and intensity of gait training necessary to produce meaningful long term improvements in walking ability. Rather than replace lost or absent function, gait training in CP population aims to improve the participant’s baseline walking pattern by encouraging longer bouts of training and exercise, which is different than in those with paralysis. Wearable robotic exoskeletons, as a potential strategy, can assist individuals with CP to gradually regain knee extension over time and help maintain it for longer periods through intense task-specific gait training. We previously tested our initial prototype which produced significant improvement in knee extension comparable in magnitude to reported results from orthopedic surgery. Children continued to exert voluntary knee extensor muscle when walking with the exoskeleton which indicated the device was assisting but not controlling their gait. These positive initial results motivated us to design second prototype to expand the user population, and to enable its effective use outside of the laboratory environment. The current version has individualized control capability and device portability for home use as it implemented a multi-layered closed loop control system and a microcontroller based data acquisition system. The benchtop evaluation shows that the exoskeleton has very small torque assistance activation latency (<33ms). The torque bandwidth was 12 Hz with a 10 Nm amplitude chirp in knee flexion and extension. The actuator had low output impedance (<1.5 Nm) at low frequencies expected during use. Three different control strategies were developed for use with the exoskeleton: stated-based assistance, impedance based assist-as-needed control, and a real-time adaptive control. Exoskeleton performance was validated in a healthy subject. These three control strategies had different effects on knee angle, and spatiotemporal parameters (cadence, step length and step width). Future work will investigate the effects of providing children with CP assistance during walking on gait biomechanics and identify the optimal individualized control strategies for exoskeleton prescription in this population. __________________________________________________________________________________ Samuel Kotler Postdoctoral Fellow NCATS Chemistry Determination of E/Z-Conformation for 5-Methylene Substituted Hydantoins by EXSIDE NMR The determination of stereochemistry within a compound is critical for the development of small molecule therapeutics as structure is highly involved in a drug’s activity. NMR spectroscopy is a primary method for the determination of molecular structure and configuration, especially when acquiring an x-ray crystal structure is hindered by the inability obtain to well-diffracting crystals. However, NMR can often provide data that are difficult to interpret or inaccessible due to molecular flexibility or complexities in 3D configuration. Moreover, NMR experiments to correctly identify stereochemistry (i.e., E/Z-conformation) of small molecules are often very time consuming. Thus, it is imperative to develop an NMR platform which can rapidly and easily identify stereoisomers of small molecules through easy-to-interpret NMR parameters; e.g., the chemical shift and long-range 1H, 13C coupling constants (3JCH), the latter of which can distinctly identify E/Z conformation. Here, we developed and demonstrated the viability of this approach for nearly 40 5-methylene substituted hydantoins utilizing a variation of the standard HSQC experiment called the Excitation-Sculptured Indirect-Detection Experiment (EXSIDE). After structural assignment for each compound by the straightforward measurement of chemical shifts (1H and 13C) and homonuclear coupling constants (3JHH) with a standard suite of 1D and 2D NMR experiments, EXSIDE was used to measure long-range heteronuclear coupling constants (3JCH) to accurately identify the E/Z-conformation. Additionally, we implemented a rapid acquisition technique, non-uniform sampling (NUS), which reduced data acquisition time of the EXSIDE NMR experiment for a single compound from 12 hours to 2 hours. The development of analytical techniques to expeditiously acquire quantitative markers for structural deconvolution of small molecules is essential, especially in the design of therapeutic treatments. As such, our application of the EXSIDE NMR coupled with NUS provided an efficient method for the structural identification of 5-methylene substituted hydantoins where biological activity was imparted by the stereochemistry of the exocyclic double bond. The stereospecific assignment of these compounds enabled us to positively identify an active Z-stereoisomer that inhibited the Six1-Eya2 transcriptional protein-protein complex whose overexpression is implicated in diverse tumor types such as Wilms’ tumor, ovarian cancer, and breast cancer. __________________________________________________________________________________ Xue Liu Postdoctoral Fellow NCATS Vascular Disease and Biology 3D bio-printed Vascularized Human Skin as a Novel Drug Screening Assay Platform for Atopic Dermatitis In vitro cell assays and animal models are extensively used in early stage drug development for efficacy, risk and toxicity tests. However, these in vitro and in vivo models lack human physiological relevance, and therefore, they have limited predictive value of drug responses in humans. Three-dimensional (3D) tissue models are currently being developed as alternative, more predictive assays for high throughput screening (HTS) for drug discovery. In this study, we have developed a novel bio-printed vascularized full-thickness skin equivalent (VFTS) and a reconstructed human epidermis (RhE) in a multi-well plate platform for the purpose of drug testing. The morphology and functionality of the skin tissue equivalents were confirmed with H&E staining, immunohistological characterization, and transepithelial electrical resistance (TEER) measurements. In the dermis of the VFTS, collagen IV and CD31 showed the formation of a 3D microvascular network and basement membrane of microvessels. Late epidermal differentiation markers illustrated that the epidermal layer was fully differentiated in both VFTS and RhE tissues. Interleukin (IL)-4 treatment was used to induce atopic dermatitis (AD)-like phenotype in the skin equivalents constructed. These AD models were able to reproduce several clinical hallmarks of AD including (i) spongiosis and hyperplasia; (ii) early and terminal expression of differentiation proteins; and (iii) increases in levels of pro-inflammatory cytokines. The assay system for the AD VFTS model includes measurements of tight junction barrier values, quantification of inflammatory cytokines, pro-angiogenic factors measurements and structural morphology changes in the microvasculature. Janus Kinase (JAK) inhibitors have been reported to be efficacious for the treatment of AD. Three JAK inhibitors were shown to correct the disease phenotypes. In our study, we first provide an innovative and reproducible use of the bio-printing technology to create a vascularized human skin equivalent. Second, this construct combined with RhE is used in screening and developing pre-clinical drugs for skin disease. Third, endpoint readouts are quantifiable, robust, AD relevant, and can be scaled up to allow HTS for compound discovery. Thus, the skin equivalents developed in this study offers an in vitro approach for understanding pathological mechanism, efficacy of action and toxicity of dermal drugs. __________________________________________________________________________________ David Morse Doctoral Candidate NCATS Biophysics Spatially-segmented single-cell transcriptomics by diffusional accessibility to a small-molecule dye Single-cell RNA-sequencing (scRNA-seq) is useful for describing cell states, but identifying the spatial arrangement of these states in tissues remains challenging. We describe SEgmentation by Exogenous Perfusion (SEEP), a rapid, integrated, method for providing spatial-segmentation to scRNA-seq data. Tissues are divided into layers based on accessibility of a fluorescent dye allowing sorted cells to be characterized by transcriptomic and regional identity. We use SEEP to explore the transcriptional states of cells in direct contact with peritoneal fluid in high grade serous ovarian cancer. High throughput scRNA-seq is used to describe complex tissues by characterizing transcriptional states of individual cells. Defining a cell’s position, both in regard to tissue margins and its social context, is essential for understanding the intrinsic and extrinsic variables that effect the transcriptional identity of individual cells. Mainstream high-throughput scRNA-seq assays, however, decouple cells from their original locations within tissues. In situ hybridization readouts of gene expression and in situ sequencing preserve spatial information in tissues, but currently have a lower total read threshold than NextGen sequencing – imposing
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