Wen-Ji Dong - US grants

DESCRIPTION (provided by applicant): Force development during striated muscle contraction is initiated by the binding of Ca2+ to the specific sites in troponin C (TnC), triggering a series of functional structural changes within the thin filament, including opening of the N-domain of TnC, conformational change of the inhibitory region of troponin I (Tnl), and switching interaction between Tnl and actin to Tnl and TnC, which ultimately lead to a cyclic interaction between actin and myosin to form strong force-generating cross-bridges. Full muscle activation requires both Ca2+ binding and feed back modulation of cross-bridge cycling. In cardiac muscle it is also modulated by protein phosphorylation which plays important roles in heart failing/hypertrophic process. To fully understand muscle regulatory mechanism requires structural, thermodynamic and kinetic information on each of these structural transitions during force development. My long-term research goal is to elucidate the kinetics of movements of the thin finlament betweem extremes of contraction/relaxation, and understand how they are modified by cross-bridge cycling and phosphorylation. To achieve the goal, this proposal addresses the following three issues: (1) What is the kinetics of each individual activation/deactivation process of the thin filament? (2) How does the cross-bridge cycling affect these kinetic processes? And (3) what is the role of phsophorylation in modulating these transitions? Newly designed conformational markers based on Forster resonance energy transfer to monitor these structural transitions will be used for stopped-flow kinetic and Ca2+ titration measurements at different activation conditions to acquire the desired information. These markers will be incorporated into reconstituted thin filament, myofibrils and skinned fibers along with/without phosphotylated proteins to specify the time-dependent changes of specific domain movements of the thin filament in response to Ca2+. Results of this study will enhance our understanding of molecular mechanisms of thin filament activation in response of Ca2+ and the role of protein phosphorylation in heart failure.

DESCRIPTION (provided by applicant): Heart failure results from impaired activation or deactivation of the heart at the level of the myofilament. Current dogma suggests that cardiac muscle contracts upon Ca2+ binding to cTnC, which regulates an on process in the thin filament (TF) leading to crossbridge (XB) attachment to generate force. Cardiac relaxation is regulated by a reverse off process in the TF triggered by rapid dissociation of Ca2+ from cTnC. It is thus believed that the kinetics of these structural changes modulate the kinetics of the XB cycle, such that pathology may arise from alterations in the relationship between the structural kinetics of the TF and XB cycling kinetics. However, previous ensemble studies failed to define the kinetic linkage between the TF processes and XB cycling. A main feature associated with TF regulation is Ca2+-induced dynamic interactions among the TF proteins, including multiple reversible structural changes at the TF protein interfaces. These forward and backward structural transitions represent the discreet signaling steps of the TF switching process that regulates XB cycling. Based on the findings from our recent in vitro dynamics study, we hypothesize that the microscopic kinetics of these forward and backward transitions in conformational state dictate equilibrium relationships between conformational populations and are tunable, and may thus provide the linkage between the rapid kinetics of Ca2+ exchange with cTnC and slow kinetics of XB cycling. However, the microscopic rate constants of individual steps cannot be easily determined by our current strategies that rely on ensemble-averaged measurements which obscure the spatial and temporal inhomogeneity of the protein dynamics present in the ensemble. Single-molecule spectroscopy has the unique advantage of unraveling this spatial and temporal heterogeneity inherent in ensemble samples. Accordingly, the overall objective of this project is to explore the use of single-molecule Forster Resonance Energy Transfer (smFRET) approaches to define the kinetic linkage between Ca2+-signaling and XB cycling by further characterizing the equilibrium relationships governing transitions between TF conformational populations. Importantly, microscopic forward or backward transition rate constants for each Ca2+-induced TF structural transition will be acquired. Two Specific Aims will be pursued using smFRET techniques to test our hypothesis: (1) examine the equilibrium relationships between conformational populations of cTnC at the level of single reconstituted regulatory units and (2) at the single-molecule level, determine microscopic rate constants associated with each Ca2+-induced reversible structural transitions of the C-domain of cTnI within single reconstituted regulatory units. Outcomes of this project will be of critical importane in addressing the current issue of the regulatory role of the TF in controlling XB cycling kinetics We expect that the information obtained from our proposed single-molecule studies will help to vertically advance the knowledge gained from our ensemble studies and muscle fiber research.

There are about 250 million episodes of acute infectious disease annually in the U.S., leading to 40 million patients being admitted to emergency rooms. A major challenge to treating infected patients effectively is the clinical difficulty in distinguishing between diseases caused by bacterial infection and those caused by viral infection, because they often present with similar clinical symptoms. This uncertainty directly leads to antibiotic misuse which, in turn, contributes to the development of antibiotic resistant microbes. Therefore, it is important to help physicians make evidence-based antibiotic treatment decisions during the time of a patient visit by providing rapid, accurate diagnostic tools that can distinguish between the bacterial and viral infections. Currently, there is no such technology that can meet the need. This research project aims to develop a novel technology that can quickly and accurately detect biomarkers useful for distinguishing between bacterial and viral infections. The method involves a paper-based device for separating and concentrating protein components and a mobile phone-based detection unit. The outcomes of this research could lead to a cost-effective diagnostic tool that will help to improve patient outcomes, lower healthcare costs, and reduce the over administration of broad-spectrum antibiotics and, therefore, antibiotic resistance. The research project serves as a training ground for graduate and undergraduate students to perform cutting edge research. The results of this research will be disseminated widely by making the technology accessible to other institutions across the nation, publishing outcomes in peer-reviewed journals, and presenting results to the science and engineering community at national meetings and conferences.

Routine diagnostic tests in central laboratories for pathogen detection can assist physicians in the etiological determination of an underlying infectious process. However, such tests can take days to return results, can be expensive, and usually require special instruments and well-trained personnel. Recently, a host-based diagnostic protein panel, including both the bacteria-induced proteins procalcitonin and C-reactive protein (CRP) and the virus-induced tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and interferon-induced protein-10 (IP-10), has been established to discriminate between bacterial and viral infection with remarkable sensitivity and specificity. However, detecting this protein panel is difficult and slow because of the ultra-low concentrations of the proteins in circulation and the interference caused by abundant plasma proteins. Therefore, the panel test must be run by a diagnostic laboratory and not at the point-of-care. This research project aims to develop an integrated paper-based cascade isotachophoresis (ITP) platform on which the panel proteins in blood will be 1) separated from the abundant plasma contaminant proteins by cationic ITP in the presence of specific target immune-bindings, 2) continuously enriched through cascade ITP focusing, 3) immune-captured at an elevated concentration by a unique single test line with graded target binding, and 4) multiplex-detected with a miniaturized smartphone-based point-of-care optosensing module. By combining the unique on-board purification and enrichment power of ITP stacking, paper-based lateral flow, and smartphone-based detection, this approach can create an integrated, point-of-care technology capable of the multiplex detection of circulating disease markers that are present either at an ultralow level or within a broad dynamic concentration range.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Abstract Exosomes are small-sized (30?120 nm) extracellular vesicles. They are secreted by most cell types and play important roles in extracellular communication in normal and pathological processes. The exosomes derived from cancers shuttle signaling molecules (e.g. proteins and miRNAs) from parental cancer cells and tissue to distal recipient cells to reprogram the recipient cells and promote tumor growth and metastasis. Therefore, circulating tumor-derived exosomes carrying signature protein markers of the tumor hold great potential as invaluable liquid biopsy tools for the noninvasive diagnosis of early-stage cancers. Despite their potential clinical significance, translating disease-derived exosomes into point-of-care (POC) applications for the early diagnosis of cancers is hampered by a critical technical barrier: lack of a cost-effective POC approach capable of the simultaneous analysis of specific exosomes and their content markers in clinical samples. Although a number of methods for exosome isolation and characterization have been developed, either they are singly functionalized, nonspecific, laborious, or time-consuming, or they lack the robustness to be adopted as a cost-effective POC technique. Therefore, there is an urgent need for an effective, precise, easy to use, low-cost approach to multiplex POC sample analysis to detect trace levels of specific populations of exosomes released by specific cancer cells at an early stage and comprehensively profile the cancer markers carried by the exosomes. This application aims to fill the gap by taking a multidisciplinary approach to developing a novel, disposable two- dimensional paper-based multistage isotachophoresis (ITP) technology platform capable of the simultaneous analysis of specific target exosomes and exosomal proteins in a cost-effective way. Our objective is to develop an integrated paper-based isotachophoretic platform on which: 1) anionic cascade ITP is used to deplete high- abundance plasma proteins and enrich target exosomes before their capture and analysis; 2) a second ITP process simultaneously analyzes multiplex exosomal proteins released by lysing the exosomes captured in the first ITP process; and 3) a miniaturized smartphone-based detection module quantifies the target exosomes and protein markers captured by novel graded-binding test lines. We expect that integrating effective isolation/identification of specific exosomes with multiplex analysis of their contents in a cost-effective modular platform will provide a robust POC approach to advancing basic and clinical translational research on disease- derived exosomes. Exosomes derived from breast cancer will be used as model targets to validate our technology. The success of this project will not only provide a clinically compatible POC tool for tracking specific exosomes and markers for early screening for cancers, but also prompt research on the profile analysis of exosomal markers for the precision diagnosis of other diseases. Therefore, the technique may have broad translational potential in managing patients with malicious diseases and improving their quality of life.

Regenerating healthy cartilage from stem cells is challenging. In this Reproducible Cells and Organoids via Directed-Differentiation Encoding (RECODE) project a team of researchers from Washington State University and Cornell University aim to provide a new tissue engineering strategy to make cartilage. Adult stem cells will be stimulated to become mature chondrocytes, the cells that make cartilage, to produce the type of tissue needed for repairing damaged cartilage. The project's approaches seek to prevent cells from transitioning to osteocytes, the cells that make bone. This will be done by using small molecules that interfere with the genes that control the production of bone. Real time imaging of fluorescing molecules will monitor whether the cells are likely to make cartilage or bone. Feedback is used to change the cell growth conditions to keep cells within an ideal range. The properties of the tissues created will be tested for their flexibility and composition. Both Washington State and Cornell will work with minority participation programs to include underrepresented students in the research.<br/><br/>This RECODE project aims to enhance tissue engineering by driving mesenchymal stem cells (MSCs) toward a stable chondrogenic lineage. The goal is to move tissue constructs in the direction of a functionally organized articular cartilage extracellular matrix (ECM) and prevent osteogenesis. It is hypothesized that cell proliferation and differentiation can be controlled by feedback bioprocess shear stresses, growth factor concentration, oscillating hydrostatic pressure, and gene silencing. The team will interrogate MSC maturation with a fiber optic bundle to sense, in real-time, co-upregulation of Sox9 mRNA, critically involved in orchestrating chondrogenesis, and Runx2 mRNA, which if overexpressed will promote unwanted progression toward hypertrophic calcification and ossification. Process variables will be tied to the cellular mRNA architecture and ECM manufacture through an experimental design model that will further refine feedback control rules. Spatial and temporal validation will be performed through bulk and single cell mRNA transcriptomics, and confocal strain and Fourier-transform infrared spectroscopy tissue mapping. The team will leverage Research Experience for Undergraduates (REU) support and partnerships with Louis Stokes Alliance for Minority Participation programs at Washington State University and Cornell University to recruit underrepresented students with the goal to see these students transition into tissue engineering related PhD programs.<br/><br/>This RECODE project is jointly funded by the Engineering Biology and Health Cluster in the Division of Chemical, Bioengineering, Environmental, and Transport Systems and the Biomechanics and Mechanobiology Program in the Division of Civil, Mechanical, and Manufacturing Innovation.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.