Utkan Demirci - US grants

[unreadable] DESCRIPTION (provided by applicant): Cryopreservation remains the only practical option for extending human fertility in modern clinical practice. We are applying nano- and micro-scale technologies to develop a novel method for automated mammalian germ cell cryopreservation. The primary problem with current cryopreservation technologies is that they either leave large mammalian cells (e.g., oocytes) susceptible to intracellular damage secondary to the formation of intracellular ice crystals, or require the use of very high levels of toxic cryoprotectant agents (CPA's). The problem of how to balance the risk of intracellular ice crystal formation with cytotoxicity from high intracellular CPA levels has proven to be one of the defining problems in the field of biopreservation. [unreadable] [unreadable] A second problem with existing germ cell freezing technologies is that all current procedures are highly labor- and time-intensive, requiring the full attention of a well-trained clinical embryologist. The reliance on highly skilled human labor is not only inefficient, but also adds an additional level of variability to the process. Third, all current cryopreservation protocols expose cells to large, step-wise changes in the CPA concentrations as the sample is manually transferred from one solution to another. This approach has two major drawbacks: (1) each abrupt change in CPA concentration causes osmotic stress to the cells; (2) manually transferring cells between different media causes shearing and other mechanical stress. [unreadable] [unreadable] The ideal cryopreservation protocol would combine the reduced chemical toxicity of conventional slow freezing with the resistance to intracellular ice crystal formation of vitrification. At the same time, the ideal protocol would load and unload CPA's in a continuous manner in order to avoid osmotic shock. Lastly, it would minimize handling of cells in order to reduce mechanical stress, diminish the labor burden on the embryology lab, and lessen (or even eliminate) human error and test-to-test variability. We believe we can develop a disposable microfluidic chip that would accomplish all of these goals. [unreadable] [unreadable] (a) sufficiently high rate of cooling to allow the use of CPA concentrations as low as those utilized in slow freezing protocols; and, (b) greatly reduced osmotic and mechanical stress on cells due to a combination of the progressive loading/unloading of CPA's and a minimum level of handling. Cryopreservation remains the only practical option for extending human fertility in modern clinical practice. The problem of how to balance the risk of intracellular ice crystal formation with cytotoxicity from high intracellular CPA levels has proven to be one of the defining problems in the field of biopreservation. We are applying nano- and micro-scale technologies to develop a novel method for automated mammalian germ cell cryopreservation that has potential to decrease intracellular damage and use lower levels of toxic cryoprotectant agents (CPA's). Further our approach has the potential to decrease laborious and time-intensive techniques and additional level of variability to the process due to the human factor. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): More than 40 million HIV-infected people live in the developing world, yet it is estimated that only one in ten persons infected with HIV has been tested and knows his/her HIV status1. The U.S National Intelligence Council (NIC) predicted that the number of HIV-infected individuals in the developing world will rise to 80 million by 20102. Effective antiretroviral therapy (ART) for HIV has been available in developed countries for more than a decade. However, worldwide, less than 10% (1.3 million) of the infected individuals currently receive treatment, since they live in developing countries. Part of the problem associated with existing ART delivery systems are the limitations of conventional methods to diagnose and monitor infected individuals living in rural poor communities. To increase access to HIV care and to improve treatment outcome requires development of low-cost diagnostic tools for developing countries3-6. We propose to develop a low-cost point-of-care HIV diagnostic tool for the developing world. At the interface between Harvard and MIT we have access to the necessary infrastructure. Moreover, the PI was recognized as one of the top 35 young innovators of the world for his work on HIV diagnostic microchips for Global Health. More than 40 million HIV-infected people live in the developing world, yet it is estimated that only one in ten persons infected with HIV has been tested and knows his/her HIV status1 and we propose to develop a low-cost point-of-care HIV diagnostic tool for the developing world.

DESCRIPTION (provided by applicant): HIV remains the most serious infectious disease challenge to public health. As a result of inadequate access to HIV prevention and treatment, everyday, more than 6800 people contract HIV and more than 5700 people die from AIDS, globally. In 2007, worldwide, 33.2 million people were living with HIV infection, 2.5 million people were newly infected and 2.1 million died from AIDS mostly due to lack of available monitoring technologies at resource limited settings. Our objective is to develop a rapid, accurate and low-cost HIV monitoring device using a novel CD4+ T lymphocyte purification and counting technology at resource limited settings. We have been working on microfluidic CD4+ T cell counting microchips and also tested them with HIV patient samples. Here, we will leverage from this expertise to create an on-chip CD4+ T cell purification technology that can eliminate CD4+ monocytes to achieve +10% clinical CD4+ T lymphocyte counting accuracy. PUBLIC HEALTH RELEVANCE: Our objective is to develop a rapid, accurate and low-cost HIV monitoring device using a novel CD4+ T lymphocyte purification and counting technology at resource limited settings. We have been working on microfluidic CD4+ T cell counting microchips and also tested them with HIV patient samples. Here, we will leverage from this expertise to create an on-chip CD4+ T cell purification technology that can eliminate CD4+ monocytes to achieve 1 10% clinical CD4+ T lymphocyte counting accuracy.

DESCRIPTION (provided by applicant): We propose to develop an automated microfluidic microchip merged with a high-throughput cell- encapsulating droplet ejection system for efficient, rapid, and inexpensive blood cryopreservation. Blood is the single most important tissue for biopreservation. In particular, red blood cells (RBCs) are required for transfusion, whenever patients suffer massive blood loss due to: (1) trauma; (2) bleeding disorders; (3) major surgery; or (4) post-partum hemorrhage. Current technology only allows frozen storage of blood and blood products, including packed RBCs. Current blood-freezing technologies employ labor and time intensive procedures that require trained clinical technicians. The complex manual handling involved in blood biopreservation results in high cost, long-processing times, and process variability. Currently, cryopreservation of blood cells is mostly done by slow-freezing. However, slow freezing leaves blood cells susceptible to intracellular damage from intracellular ice crystal formation (IIF). Therefore there is a significant need for improved technologies enabling effective cryopreservation of blood. Although it is shown in the literature that vitrification techniques could achieve better biopreservation outcomes for various cell types such as RBCs and oocytes, vitrification is not applied to blood biopreservation clinically due to throughput limitations. The current vitrification methods require microliter volumes of cells to be filled into straws that are then vitrified, which is not feasible to biopreserve liters of blood. Since these products have limited shelf lives, blood freezing must be done continually and routinely in every hospital and medical center in the US to meet the constant demand. Accordingly, there is a need for a new platform technology that will transform the operational logistics for an efficient future for the existing blood supply chain mechanisms. In this project, we will advance the clinical practice in blood cryopreservation by leveraging the advantages provided by vitrification and enabled by our novel microscale technologies. As a result we expect to achieve: ultra-rapid cooling rates (10,000 oC/sec) at low cryoprotectant agent concentrations with low levels of ice formation. These conditions will lead to improved functionality and longer shelf life of RBCs (>42 days). We are proposing to develop an enabling platform applicable to practically all cell types, especially therapeutic RBCs, peripheral blood stem cells, and primary hepatocytes. If successful, the proposed research can have a significant impact on the long-term storage of blood products for both civilian and military needs.

DESCRIPTION (provided by applicant): To reduce morbidity and improve quality of life for persons living with HIV/AIDS, the World Health Organization (WHO) is rapidly expanding access to antiretroviral therapy (ART) in developing countries. However, the expansion is significantly restricted by the lack of cost-effective point-of-care (POC) viral load assays that cn effectively reach patients living in rural, isolated settings. In developed countries, HIV-1 viral load is regularly used to closely monitor and assess the patient response to ART, to ensure drug adherence and to stage disease progression. In contrast, developing countries are using CD4+ cell count and clinical symptoms to guide ART following the WHO guidelines with the exception of infants, where viral load assays are required. This is because HIV-1 viral load assays are expensive ($50-200 per test), instrument-dependent, and technically complex. Recent studies, however, have shown that CD4 cell counting strategy cannot detect early virological failure. This failure leads to accumulation of drug-resistant strains in infected individuals and reduces the efficacy of first-line drugs. Thus, a rapid, inexpensive, and simple viral load test is urgently needed at the point of care (POC). Here, microfluidics and optical imaging technologies will be used to create a novel HIV-1 viral load microchip. It was hypothesized that the developed HIV-1 viral load microchip can: (i) selectively capture HIV from whole blood, (ii) be sensitive within the clinical cut-off (with ?10% error range), inexpensive (<$1), rapid (within 15 minutes), and automated to handle finger-prick whole blood (100 ?L) to aid in quality care and treatment at the POC. This is a technology driven proposal motivated by the urgent significant clinical need. Prior work has shown the proof-of-concept that HIV-1 can be captured and quantified from patient whole blood on-chip. The following distinct but interrelated specific aims are proposed: Aim 1: To build disposable microchips to capture HIV-1, Aim 2: To develop a portable system for on-chip HIV-1 viral load by integrating HIV-1 capturing microchips with a detection/quantification system, and Aim 3: To validate the microchip technology with 200 HIV-infected samples. This proposed technology is broadly applicable to other infectious diseases having a reasonably well-described biomarker available such as influenza, hepatitis, malaria and tuberculosis.

SUMMARY Tissue engineering holds great promise for enabling alternative therapies for diseases such as diabetes, heart and liver failure. A common approach in tissue engineering is seeding cells in biodegradable scaffolds, which brings cells together in close proximity, aiming to mimic the native tissue environment. These scaffolds are expected to degrade and replaced by cellular growth and extracellular matrix deposition to generate a natural tissue. However, challenges remain with the current approaches, such as: 1) The inability to achieve a complex three- dimensional (3D) cellular architecture and organization (i.e. cardiac tissue is made of three major type of cells; i) cardiomyocytes ii) cardiofibroblasts, and iii) endothelial cells; 2) The limited control over cell-cell proximity with microscale resolution; 3) The inability to generate micro- engineered tissue constructs at high throughput with uniform cell distribution and high cell seeding density; 4) The lack of vascularity which results in cell necrosis and loss of function limiting the biologically relevant engineered tissues (i.e. diffusion length of metabolites is, typically, smaller than 300 micron). We hypothesized that engineered nanofilms can be used to assemble microgels into 3D constructs which can be integrated in a fluidic device to increase assembly throughput. The goal of this project is to address these challenges by developing a Microscale Acoustic System of Nanocoatings (MASON). In this proposal, we will merge directional nanocoating (i.e. ratchets) for transport of microgels, acoustic micro electro- mechanical systems (MEMS), and microscale hydrogel (microgel) fabrication technologies to achieve microgel assembly with controlled cellular architecture. We propose to build complex structures of biodegradable microgels in a microfluidic device based on directed assembly. Thus, the proposed novel microgel assembly approach presents a promising direction towards synthetic tissue engineering which is applicable to multiple organ systems. We expect to show that this novel approach will be significantly more efficient in addressing the problems outlined above than existing microgel assembly technologies.

DESCRIPTION (provided by applicant): Long-term biopreservation of cells and tissues has a broad impact in multiple fields including tissue engineering, regenerative medicine, stem cells, blood banking, animal strain preservation (biodiversity protection), clinical sample storage, transplantation medicine and in vitro drug testing. Vitrification (ice/crystal-free cryopreservatio) has emerged as a novel approach over traditional slow freezing methods. Although vitrification minimizes mechanical damage due to ice crystal nucleation, it suffers from toxicity due to high concentrations of cryoprotectant agents (CPAs). The current vitrification methods require extremely high levels of CPAs of up to 8.2 M that are cytotoxic and cause osmotic shock. Also, the lengthy manual processing steps of current vitrification methods add to the technical complexity, require highly trained technicians, and result in variations between users. For instance, low CPA-level vitrification has immense potential for the stem cells compared to other methods in preserving their functionality. Recently, we demonstrated that we can achieve vitrification at ultra-rapid freezing and thawing rates with as low as 1.5M CPA concentration. We are adapting this new knowledge to the vital needs of cell cryopreservation at the clinic including discarded anonymous human oocytes. This proposal investigates a new experimental strategy to minimize the CPA concentrations and improve clinical outcomes using novel technologies (i.e., nanoliter droplet vitrification). These steps are facilitated by theoretical understanding o the underlying mechanisms governing vitrification. The expected outcome of this study is a closed-system platform technology with broad applications to human cell (e.g., hepatocytes, oocytes, sperm, stem cells), tissues (e.g., blood), micro-tissues (e.g., embryoid bodies, islets) covering areas of reproductive medicine, tissue engineering and regenerative medicine as well as to wild life preservation. These studies can also significantly impact the care of infertile couples and facilitate fertility preservation.

5.3 million American couples of reproductive age are affected by infertility. Among these cases, male factors account for up to 50%, necessitating the identification of key parameters dictating male fertility, including sperm count, morphology and motility. Assisted reproductive technologies (ARTs) have emerged as powerful tools to address male infertility problems in modern clinical practice. In vitro fertilization (IVF) with or without intra cytoplasmic sperm injection (ICSI) has become the most widely used assisted reproductive technology (ART) in modern clinical practice to overcome male infertility challenges. One of the obstacles of IVF and ICSI lies in identifying and isolating the most motile, and healthiest sperm from semen samples that have low sperm counts (oligozoospermia), low sperm motility (oligospermaesthenia).

Selection of the best performing sperm based in the selection criteria including motility is the keystone for successful outcomes of fertilization and full term pregnancy. However, it remains a clinical challenge to select the most motile normal/healthy sperm. The researchers propose to develop a GPU accelerated computational framework that will enable the multi-scale coarsegrained modeling of sperm motility in micro-channels. Towards achieving this goal, they will develop a computationally efficient model of sperm motility and interactions, and design a computer-optimized space-constrained microfluidic sorting (SCMS) system, integrated with a lensless technology, for rapid monitoring, selection and sorting of sperm. As they are utilizing such microchip technology, the proposed device can be easily transformed into a scalable device composed of multiplexed channels. In the long term, this computational platform can be used to design micro-fluidic devices for the selection and sorting of not only spermatozoa, but also other types of biological entities, such as circulating tumors cells (CTCs) or HIV from blood.

The broader impacts of this proposal include educational and scientific outcomes that will open new avenues for physical and biological research and have a considerable impact on fundamental and applied science, education, and medicine. This proposal will facilitate the participation of undergraduates in year round research activities, and directly support the training of graduate students at the interface between physics, engineering and medicine at Harvard, MIT and WPI. The PIs will actively participate in recruitment efforts to broaden the participation of underrepresented groups in the biological, physical sciences, and engineering by attending national meetings and via Harvard's underrepresented minority program. They will continue to advise undergraduates through the Undergraduate Research Opportunities Program (UROP) and the mandatory senior theses (MQPs) at WPI. The PIs will also develop graduate courses, and arrange field trips with local high schools in educating students about computational biophysics and microfluidics research. At the national and international level, the PIs will educate the students and the public at other institutions on technological and scientific challenges by giving lectures at NSF supported international summer schools, and by organizing hands-on workshops.

DESCRIPTION (provided by applicant): HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy, and a main challenge in achieving HIV-1 antiretroviral (ART)-free remission is the persistence of these latent viral reservoirs. Quantifying the replication competent viral reservoir is critical to understanding how HIV-1 persists and to measuring how this reservoir changes in response to therapeutic strategies. Quantitative co-culture assay have been developed to measure the replication competent reservoir by activating CD4+ T cells from patients in the presence of feeder cells for viral outgrowth and measurements of HIV-1 proteins or genetic material. However, traditional quantitative co-culture assays are time-consuming, labor-intensive and require parallel reactions in large-well format culture. Novel methods to miniaturize the co- culture platform and to increase sample throughput have the potential significantly to improve the study of HIV latency and could be used to streamline and standardize testing of novel reservoir eradication strategies. We propose to adapt and validate a novel, miniaturized chip-based microfluidic co-culture assay and compare its performance with the traditional co-culture assay. We hypothesize that miniaturizing these assays will allow for higher throughput and be less labor intensive than traditional platforms. The proposed method will also allow for tight control of the growth microenvironment which will help to improve sensitivity and standardize test results between laboratories. Our aims are: (1) adapt a chip-based microfluidic cell culture/expansion protocol for viral co-culture to quantify integrated, replication-competent provirus as infectious units per million cells, and, (2) validate the performance of the novel microfluidic co-culture assay compared with traditional laboratory viral outgrowth assays. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our proposed assay has the potential to be used for a variety of applications involving the isolation and growth of infected human cell subsets and tissues, and we ultimately plan to use the assay to quantify replication competent reservoirs in various cell types and from patients undergoing allogeneic stem cell transplantation and cytoreductive chemotherapy.

Routine high-sensitivity multiplexed detection of disease biomarkers in blood will have an impact on early diagnosis and therapy selection. A novel and highly effective approach for detection of broad categories of disease is multiplexed detection of antibodies using protein microarrays. As a greater number of antibody biomarkers are being identified and clinically validated for viral infection, cancer, cardiovascular disease, and autoimmune disease, there is a strong need to detect them with greater sensitivity and greater signal-to-noise ratio, while at the same time being able to perform automated biomarker analysis with low cost per test. In the proposed project, the investigators integrate photonic crystal enhanced fluorescence (PCEF) technology with a novel size-exclusion blood filtration technology to develop a multiplexed microspot fluorescent sandwich assay platform for the clinical laboratory environment. Our approach integrates several innovations into an inexpensive plastic-based sensor cartridge and desktop detection instrument: 1. A silicon-based nanostructured resonant optical photonic crystal chip with an integrated Fabry-Perot optical cavity will be used to deliver ~50x greater signal enhancement than current-generation PCEF devices, which already routinely provide <1 pg/ml limits of detection in complex media, using only 10 ?l sample volumes. 2. A laser-machined blood filter will separate plasma from a droplet of heparinized whole blood in 60 seconds, enabling the entire assay protocol to be performed automatically in <60 minutes without user intervention. 3. An innovative laser scanning approach is used to couple light from a small semiconductor laser directed to the PC surface in the optimal ?on-resonance? condition, resulting in a rugged, compact instrument with a cost of <$10K. 4. A statistical bioinformatics tool indicating the presence or absence of the biomarkers in the test sample. As an example application of the system, we will focus our effort on detection of a panel of 3 serum antibodies for human papillomavirus (HPV) as a means for identifying patients with greater susceptibility to oropharyngeal carcinoma (OPC), although the same technology can be extended to detection of broad classes of antibody biomarkers. The new instrument will be first tested upon biomarkers spiked into whole blood, with results compared against single-antibody ELISA in microplates and the Luminex bead-based system. Further validation and comparison will be performed upon clinical blood samples from patients with known HPV exposure. Our long-term goal is to demonstrate a prototype system that can be applied broadly for multiplexed serum antibody biomarker analysis.

DESCRIPTION (provided by applicant): HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy. The main challenge in achieving a cure for HIV-1 infection is the persistence of these latent viral reservoirs. Assays that allow for identification, characterization, and isolation of latently infected single cells fo downstream genomic sequencing are needed to efficiently and fully characterize HIV-1 reservoirs. However, existing assays that analyze latently-infected cells require burdensome and costly serial cell dilutions. Other proposed methods to identify and analyze HIV-infected cells require significant investment in costly equipment and reagents and have not been adapted for downstream characterization of latent reservoirs. We propose to develop and validate an innovative and novel assay using microfluidic methods with PCR for identification, enumeration, and isolation and downstream characterization of viral genomes from latently-infected human cells. The application of our approach will be particular useful in the analysis of samples from patients on combination antiretroviral therapy and/or in studies of novel modalities of reservoir eradication. Specific aims include: 1) develop and test the efficacy of the proposed method to identify and enumerate latently-infected human PBMCs, tissue derived macrophages, and other primary human cells using microfluidic methods and PCR, and, 2) validate our assay to isolate single-cell droplets with integrated HIV-1 DNA for downstream sequencing and secondary target gene quantification. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs on the single-cell level in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our prior experiences in the detection and quantification of very low-levels of HIV-1 genetic material and in the development of microfluidic devices for viral and immune characterization will be crucial to the development of novel assays to identify and characterize HIV-infection at the single-cell level. The proposed method has the potential to be adapted for a wide variety of multidisciplinary research studies, such as single-cell characterization of viral and intracellular pathogens or analysis of stem cells and/or malignant tissues.

PI: Demirci, Utkan
Proposal Number: 1547791

PI: Kaplan, David L.
Proposal Number: 1547806

The coordinated function in the brain of billions of neurons in dense and entangled networks can be seen as the epicenter of our unique higher consciousness, as well as of our vulnerability to debilitating diseases, such as schizophrenia, autism and Alzheimer's. The investigators propose a unique approach of sound waves and silk protein biomaterials, to recreate the complex three-dimensional brain network structures in a small dish, and use them to investigate their response to a laboratory model of brain concussion damage. With these studies, the investigators aspire to demonstrate how these constructs may help scientists better understand the workings of the brain in healthy and diseased states.

The complexity of the brain poses a large roadblock for scientists to examine molecular, cellular and circuit level behavior of brain physiology. Novel approaches and technologies are needed that complement and advance the existing in vivo, ex vivo and in vitro approaches. The goal of the proposed research is to develop a new flexible bioprinting platform for the in vitro fabrication of 3-dimensional (3D) neural tissue constructs that faithfully mimic the biological complexity, development, architecture and function of 3D circuits present in the brain. The key innovations include the strategy of acoustic biopatterning and silk protein scaffolds for encapsulating neurons in long-lived, 3D multilayered architectures. To prototype and validate the construct, the investigators propose in the first aim to create 6-layer cortical circuits built of primary neurons. In the second aim, they will examine the physiology of the 3D circuit tissues using a comprehensive neuro-technological tool-box. Electrophysiology, fluorescence imaging, genomics and proteomics approaches will be employed to evaluate functional and structural milestones of the developing in vitro 3-D neural circuits, including a brain damage disease model. This radically different approach for investigating brain physiology and pathophysiology has the potential to provide new tools for neuroscience, the utility of which extends to other fields because of the general applicability of the proposed advanced biomanufacturing approaches. The broader impact of this proposal includes the participation of high school, undergraduate and graduate level scientists in research at the intersection of neuroscience, tissue engineering and biomanufacturing, thus presenting a useful platform for the training of interdisciplinary scientists.

? DESCRIPTION (provided by applicant): The development of POC diagnostic technologies to detect Kaposi's sarcoma herpesvirus (KSHV or HHV8) in HIV-infected persons that utilizes oral or blood biospecimen technologies is of great clinical importance. Such technology would enable rapid POC diagnostic devices for the detection of KSHV infection, and referral for treatment for prevention and management of KSHV malignancies to prevent cancer- and AIDS-related mortality. In HIV/AIDS patients, coinfection with viruses such as KSHV, can lead to the development of Kaposi sarcoma (KS) tumors, which is the leading AIDS malignancy. KS frequently occurs in the oral cavity and KSHV is shed in saliva. KSHV also is the etiologic agent of primary effusion lymphoma (PEL) and is tightly linked with multicentric Castleman's disease, an aggessive lymphoproliferative disorder. Currently, there are no specific or highly effective treatment options for KS, PEL or multicentric Castleman's disease. KSHV transmission is currently poorly understood and most commonly occurs through saliva, where virus is shed at relatively high titers in the oral cavity. A better understanding of KSHV transmission would allow the development of strategies to disrupt KSHV transmission and prevent the risk of KSHV associated malignancies. Studies to enable better understanding of viral transmission will require frequent monitoring of KSHV shed in saliva. Second, KSHV viral load in blood has been shown to predict the onset of KS. In addition, KSHV viral load correlates with disease burden in KS patients and is lower during disease remission. Therefore, quantitative detection of KSHV is critical to optimal clinical management of those at risk for, and with, KSHV malignancy. The development of inexpensive, rapid detection of KSHV is therefore a priority. Our objective is to build a nanoplasmonic platform for the capture and detection of KSHV. Approaches will include detection of KSHV spiked in unstimulated whole saliva or blood samples and discarded, unstimulated whole saliva or blood patient specimens. Successful application of such an approach could lead to strategies for early detection of infection, cancer prevention, and monitoring of therapy. This platform technology is also widely applicable to other infectious agents including poxviruses, other herpesviruses, human papillomavirus (HPV), dengue, tuberculosis malaria, and Trichomonas vaginalis as well as early detection at the POC.

? DESCRIPTION (provided by applicant): A major obstacle to HIV-1 eradication is the existence of latently infected cells that persist despite antiretroviral therapy (ART). Current eradication strategies focus on the reactivation and clearance of infected cells with various latency reactivating agents (LRA). However, viral reactivation alone is insufficient to reduce HIV-1 DNA reservoirs, and the association between increased cell-associated HIV-1 RNA and the frequency of reactivated cells is poorly understood. It is also unknown how reactivation strategies and HIV-1 genome integrity affect the frequency or amplitude of HIV-1 transcription. As a result, we have developed and implemented a novel method that provides insights into HIV-1 persistence that are inaccessible though existing technologies. Individual cells are encapsulated into nanoliter-scale reaction droplets, followed by intra-droplet lysis and PCR amplification of unspliced (us) and multiply spliced (ms) HIV-1 RNA and downstream isolation and sequencing of genomic viral DNA. We have successfully applied this method to identify transcriptionally active CD4+ T cells from HIV-1-infected patients on suppressive ART with and without LRA stimulation. Our data suggest the frequency of infected cells may increase while total cell-associated levels decrease and vice versa. We also observed dichotomies between usRNA and msRNA responses to latent reservoir activation. These findings suggest that single-cell analysis will be crucial in providing insight into which cells and tissues are targets for eradication in shock and kill approaches. We propose to utilize our assay to perform high-throughput reservoir quantification and downstream HIV-1 genome characterization of cells isolated from latently-infected peripheral blood and organized lymphoid tissue obtained from early and late ART treated individuals. We hypothesize that effector cells will display higher frequencies and amplitudes of HIV-1 RNA reactivation than those with memory or regulatory phenotype. Early treated individuals are thought to have smaller reservoirs, observed preferentially in effector memory cells which may have the propensity to reactive HIV-1 more efficiently. Our aims are to: (1) determine HIV-1 RNA transcriptional activity in response to various HIV-1 latency reversing agents in single cells derived from peripheral blood and organized lymphoid tissues from suppressed patients, (2) determine the single-cell responses to ex vivo LRA reactivation in early and late antiretroviral treated individuals, and (3) define the relationship between single-cell HIV-1 genetic sequence integrity, and HIV-1 transcriptional activity. In future studies, we plan to apply our novel methods to characterize the in vivo responses to interventional trials of HDACi and other immune modulating therapies. We also plan to adapt our assay to directly measure viral outgrowth in individual cells.

? DESCRIPTION (provided by applicant: The HIV/AIDS pandemic has had a devastating global impact causing more than 30 million HIV-1 infections and over 25 million deaths worldwide. In addition, it is estimated that annually over 450,000 infants are infected through mother-to-child transmission (MTCT). Although antiretroviral therapy (ART) is effective to save lives and reduce MTCT, the coverage of ART in treatment-eligible patients in developing countries is only approximately 67% due to the lack of simple, inexpensive and rapid near-patient treatment monitoring tools. To address the unmet need, we propose to develop an HIV-1 viral load monitoring microfluidic platform technology development of a sensitive photonic crystal sensing technology. This technology detects and quantifies the binding of biotargets (e.g., HIV-1 virus particles) to an optical sensing surface due to the change of bulk index of refraction. The resulted shift in the peak wavelength value correlates with the concentration of biotargets in a biological sample. This technology- driven proposal addresses a significant global clinical need and aims to deliver a portable photonic crystal device that can (i) selectively capture HIV-1 from whole blood, (ii) be sensitive within the clinical cut-off (with ±10% error range), inexpensive (<$1), rapid (within 30 minutes), and (iii) handle fingerprick whole blood (up to 100 µL) to aid i HIV patient care and treatment in resource-constrained settings. The delivery of this photonic crystal-based HIV-1 viral load monitoring microchips can significantly facilitate the expansion of ART in developing countries, achieving universal access to ART to control the AIDS pandemic.

PROJECT SUMMARY/ABSTRACT: The Canary Center at Stanford for Cancer Early Detection (?Canary Center?) is a world-class facility with the mission to foster interdisciplinary research leading to the development of blood tests and molecular imaging approaches to detect and localize early cancers by integrating research in in vivo and in vitro diagnostics to deliver these tests. Embedded within this mission is the need to formally present new and innovative approaches to scientific communities at all levels. The Canary Cancer Research Education Summer Training (Canary CREST) Program fulfills an educational mission by introducing students to research education and new career paths. The overall goal of the program is to train a new generation of interdisciplinary scientists in early cancer detection by offering an integrative hands-on research experience at an early stage of their scientific education. The Canary CREST Program is a 10-week instructional summer program for 25 undergraduate students in the biological, engineering, mathematical, or physical sciences, and offers a structured research experience with a focus on early cancer detection. The program will be administered by the Canary Center and brings together a multidisciplinary group of 28 faculty whose research groups are dedicated to the field of early cancer detection using experimental and computational approaches in biochemistry, bioengineering, bioinformatics, molecular imaging, and cancer biology. Proposed Canary CREST Program activities include: (1) Mentor-directed research in one of six investigative areas, namely, development of devices for cancer diagnostics, cancer biomarker discovery and validation, cancer biology, molecular imaging of cancer, clinical imaging of cancer, and cancer bioinformatics; (2) Specially-designed classroom sessions to provide a conceptual framework of the field of early cancer detection; (3) Seminars in scientific research; (4) A comprehensive professional development component that includes career talks, student presentations, workshops on communication skills and career opportunities; and (5) Participation in an Ethics Forum. The key aspect of this program is to offer participants the opportunity to conduct mentor-directed research while developing an understanding of hypothesis-driven studies with critical interpretation of results and analysis of data. The long-term objective of this educational research program is to support the growing need of specialized researchers who will have a significant impact in the rapidly-expanding area of cancer early detection.

Abstract Multiple drug therapies have been approved for treating advanced cancer. However, the effectiveness of each is variable and the ability to monitor or predict efficacy in individual patients is underdeveloped. Our team recently demonstrated (using traditional sequencing-based methods) that expression levels of specific microRNAs (miRNAs) in blood can effectively predict treatment outcomes. The goal of this proposal is to develop innovative technologies that will allow us to measure miRNAs from a patient on a frequent basis, in a way that is convenient and rapid, to enable precise adjustment of therapy. This is currently not achievable using RT-PCR or sequencing-based detection. All cancers are associated with heterogeneous somatic genetic alterations, ushering in a new generation of nucleic-acid-based targeted treatments. The measurement of somatic genome based biomarkers to assess, monitor, and change treatments is needed. Circulating exosomal miRNAs represent one class of highly specific markers of cancer-associated genetic mutations that can be noninvasively sampled from blood, whose quantitation can provide previously-unavailable information to clinicians for generating informed decisions on selection of effective treatments among the wide array of options. In order to make effective routine use of miRNA cancer biomarkers, novel technical approaches will need to be developed that can offer a high degree of multiplexing, quantitation, ultrasensitivity, low cost, simplicity, integrated sample processing, and robust instrumentation suitable for point of care (POC) settings. We link a newly demonstrated form of microscopy, called NanoParticle Photonic Resonator Absorption Microscopy (NP-PRAM) with a simple and effective exosome isolation approach to perform sample preparation that yields exosomal miRNA for detection. Using plasmonic NPs whose resonant wavelength matches a photonic crystal surface, NP-PRAM demonstrates high contrast ?digital resolution? precision sensing of exosomal miRNAs. We plan to develop assays for simultaneous detection of 5 miRNA sequences extracted from a single droplet of blood with a rapid assay protocol that does not require fluorescent emitters or enzymatic amplification. We utilize simulation-guided miRNA probe design for ultraspecific hybridization. We will apply NP-PRAM in the context of a panel of clinically validated miRNA biomarkers for advanced prostate cancer. Our approach offers important advantages compared to existing methods for detection of circulating nucleic acid biomarkers: It requires only a ~50 µl droplet of test sample unlike 10-20 ml of blood for RT-PCR based detection methods. NP-PRAM detection produces highly quantified results because nanoparticle tags are not subject to the effects of quenching or background fluorescence that are common to fluorescent dyes. The assay is isothermal, conducted at room temperature, and highly selective, while it does not require enzyme amplification or wash steps. The approach can be applied to quantitative characterization of miRNA biomarkers for all cancer types, although here we specifically focus on a clinically validated set of miRs for prostate cancer.

Abstract Frequent, accurate, and highly sensitive HIV-1 viral load monitoring is a critical component of AIDS antiretroviral therapy, a tool for reducing the incidence of mother-to-child HIV transmission, and a required element of routine diagnostic testing to make people aware of their HIV status. Although enormous research and product development effort has been applied to point-of-care viral load testing, the current paradigm of nucleic acid tests and antigen assays continues to demonstrate fundamental limitations that derive from their inherent complexity and lack of robustness, which in turn impact their costs and practicality for adoption in resource-limited settings. We seek to address an important gap in the capabilities of existing technologies through a combination of three innovations to yield an integrated, rapid, simple, ultrasensitive, highly selective, robust, and inexpensive system for quantitative viral load measurement. First, we utilize microfluidic separation of virions from whole blood, yielding a 10-50 µl plasma sample from 20-100 µl of whole blood in <10 min, with >95% virus extraction efficiency. Second, we will achieve ultraselective recognition of intact HIV virions from the resulting serum using designer DNA nanostructures that take the form of a macromolecular ?net? whose vertices are a precise mechanical match to the spacing and positioning of the spike gp120 protein matrix displayed on the HIV outer surface. The DNA net vertices incorporate nucleic acid aptamer probes that have been selected for selectively targeting the HIV gp120, resulting in multiple sites of high affinity attachment, and thus the ?net? can be used as an effective capture probe when covalently attached to a photonic crystal biosensor surface. Finally, we will utilize a newly-invented form of biosensor microscopy called Photonic Resonator Interference Scattering Microscopy (PRISM) in which the photonic crystal surface amplifies laser light scattering from captured intact virions, enabling each one to be counted with high signal-to-noise ratio. Because PRISM does not require labels or enzymatic amplification, our approach enables dynamic, real-time counting of captured virus with digital precision and ultrasensitivity. In the proposed project, we will integrate viral separation and the photonic crystal biosensor into a plastic cartridge and develop a rapid workflow that will be simple and rapid for compatibility with point-of-care settings, with the goal of yielding a result in <30 minutes sample-to-answer. Our Aims include development of a point-of-care version of the PRISM instrument, and statistically robust characterization of detection limits, repeatability, and robustness. Our study will conclude with validation of the system using clinical specimens and direct comparison against gold-standard laboratory RT-PCR analysis.

PROJECT SUMMARY/ABSTRACT Alzheimer?s disease (AD) afflicts millions of Americans, yet no effective treatments exist. Iron has been shown to be involved in key AD pathologic processes, including amyloid and tau aggregation, inflammation, oxidative stress, and cell death mechanisms. Despite this growing evidence, it is challenging to ascertain alterations in iron metabolism in vivo, limiting potential translation to biomarkers and novel therapies. Exosomes are nanometer-sized vesicles shed by cells to transport proteins, nucleic acids, metals, lipids or metabolites. While exosomes reflect cellular processes and can reveal disease-related pathologies in human tissues and biofluids, iron abnormalities in AD exosomes have not yet been investigated. We will address this knowledge gap through state-of-the-art exosome isolation technology combined with advanced iron imaging, protein quantification and next generation sequencing methods. Our goal is to investigate iron dysregulation in exosomes from post- mortem AD brains, in order to unveil AD-specific biomarkers and facilitate the development of novel therapies. The project aims are: (1) To determine whether the quantity, oxidation state, and cellular origin of exosomal iron is altered in AD. Using MRI and synchrotron X-ray microscopy, we will quantify tissue iron content and oxidation state in human AD and control hippocampal specimens. We will then use our novel exosome isolation platform, ExoTIC, to isolate exosomes from regions of high hippocampal iron content in the same specimens. Using antibodies that target cell-surface proteins, we will enrich the isolated exosomes based on their cellular origin (e.g. neurons, microglia, etc.). We will quantify exosomal iron content from each cell type using mass spectrometry (ICP-MS), and measure exosomal iron oxidation state using electron microscopy. Taken together, we will determine whether iron content and oxidation state are altered in Alzheimer?s exosomes compared to controls, in particular in exosomes originating in microglia, the brain?s immune cells. (2) Detect dysregulation of iron-related proteins and RNAs in AD exosomes. Using Western blotting on the enriched exosomes, we will determine whether levels of proteins that play a role in iron metabolism are altered in AD compared to controls. Because exosomes are generally rich in microRNAs that are known to regulate gene expression, we will use RNA-Seq to determine whether exosomal microRNAs regulating these same iron-related proteins are also altered in AD. Machine learning algorithms will enable the creation of an atlas of microRNAs linking iron, iron-related proteins, and neuropathology, which should provide a deeper understanding of AD biology. Characterization of exosome content in the AD brain should result in cell-specific signatures of iron dysregulation associated with neurodegeneration. This approach may elucidate novel aspects of AD biology, lead to novel assays to detect early AD, and facilitate a much-needed future therapy.

The endothelium is the cellular monolayer that covers the inner lining of the entire circulatory system. Endothelial dysfunction is a feature of pulmonary arterial hypertension (PAH), a life-threatening disease associated with abnormally high pulmonary pressures and chronic right heart failure. Due to the limitations of available static cell culture and animal models, our understanding of the mechanisms that orchestrate the initiation and perseverance of endothelial dysfunction in PAH remains incomplete. Given that endothelial dysfunction is a common finding in PAH, an understanding of the mechanism behind maladaptive endothelial responses could help accelerate the discovery of novel therapies for PAH. Presently, it is believed that endothelial derived exosomes contribute to PAH by carrying signals that trigger maladaptive endothelial responses in the setting of injury. Exosomes are cell-derived small (~30-150 nm) extracellular vesicles that carry proteins, metabolites and nucleic acids involved in a variety of physiological and pathological processes. While it is known that exosomes carry molecular and genetic factors associated with angiogenesis, inflammation and vasoreactivity, a comprehensive assessment of exosome cargo of healthy and dysfunctional PMVECs has been hindered by current low-yield exosome isolation techniques. These techniques cannot perform real-time dynamic exosome isolation from pulmonary microvascular endothelial cells (PMVECs) exposed to PAH-associated stressors. To address this unmet need, we have designed the MFES (Multifunctional Exosome Sorter) that can dissect the whole exosome population into subpopulations based on size and surface markers. MFES is the first lab-on-a-chip platform that integrates: 1) a vessel-on-a-chip module for real-time characterization of PMVEC functional responses across a wide range of physiological and pathological parameters, 2) a module for high-yield exosome size-based isolation, 3) a surface marker based exosome sorting using magnetic beads, and 4) multi-omics phenotyping of exosomes of PMVECs. Here, we are proposing a technology that can enable broadly to investigate the two main defining characteristics of exosomal subtypes, i.e., size and surface markers, both separately independently, and in combination sequentially. We will characterize changes in exosome cargo in healthy and PAH PMVECs exposed to shear stress-related conditions in the MFES. We will isolate subpopulations of exosomes based on size and surface markers and characterize them for their cargo (Aim 1). Then, we will determine whether exosomes derived from stressed PMVECs can induce pathological changes in healthy PMVECs cultured in a microfluidic culture chip (Aim 2). This technological innovation enables to study endothelial exosome biology in a setting that represents the flow dynamics associated with PAH. Further, the use of cutting-edge -omics technologies, bioinformatic analysis integrated with machine learning algorithms to analyze the purified exosomes is expected to yield a comprehensive dataset of exosome cargo profiles and open exciting opportunities for investigating the biological role of exosomes in PAH pathobiology and the testing of novel therapeutic agents.