# data[].attributes.abstract
Project Summary: We propose the acquisition of a BD FACSAria Fusion cell sorter from BD Biosciences to serve as a crucial instrument for the Ross Flow Cytometry Core (RFCC) in Johns Hopkins University (JHU) School of Medicine (SOM). This 4-laser (Violet 405nm, Blue 488nm, Yellow-Green 561nm, and Red 640nm) model with 16 fluorescent detectors and a fully integrated Class II biosafety cabinet will significantly enhance the research capabilities of the users with NIH funded projects. The current cell sorter, a BD FACSAria IIu with 3 lasers and biosafety cabinet at the RFCC, has limited technical capabilities and declining reliability. It is failing to meet the sorting requirements for advanced research projects. Having served for over two decades, it has reached its End-of-Life stage without manufactory upgrade and coverages. The proposed instrument addresses the pressing and unmet needs of 22 user groups within JHU SOM, spanning diverse research areas with mostly NIH-funded projects or some from other federal funding agencies. These users rely on cell sorter as a critical tool in their research endeavors. The benefits of the Fusion sorter over the current Aria IIu include: 1) the inclusion of a biosafety cabinet enables biosafety level-2 cell sorting with enhanced precautions during sorting operations; 2) the 4 lasers and ample fluorescent detectors reduce spectral overlap, facilitating multi-color panel design; 3) the Yellow-Green laser can optimally excite some unique fluorescent proteins and fluorochromes; 4) improved index sorting into multi-well plates; 5) temperature controls both input and output chambers to improve cell viability and gene stability; 6) easy aseptic setup and cleaning procedures ensure sterile sorting; 7) the well- established cell sorter offers increased reproducibility and expanded capabilities; 8) Conventional sorter easily and simply handle panels with fluorescent protein. The RFCC, under the administration and management of Department of Medicine in SOM, is oversighted by an advisory committee that provides recommendations for successful installation and management of the Fusion cell sorter. For the past 5 years, including COVID pandemic period, the RFCC has provided over 4,750 hours of cell sorting services to about 270 users from JHU SOM and local research community. However, the current BD FACSAria IIu, upgraded from Aria I in 2012 and addition of violet laser in 2015, no longer has a service contract from BD since the end of 2023. With its inability to have further upgrades of lasers, detectors and other parts, as well as timely repair, it is incapable of fulfilling advanced sorting projects. Although expensive high-end cell sorter, especially spectral sorters, are available, the proposed Fusion cell sorter is vital and cost-effective to maintaining a fully functional flow core facility. It should be sufficient to enable innovative researches across a wide range of sorting experiments, from sorting cells expressing fluorescent proteins to deep immunophenotyping, cellular research, genomic research, and other high-performance, high-throughput applications.

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United States National Institutes of Health

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MECHANISMS AND MACROMOLECULAR INTERACTIONS UNDERLYING CELLULAR RESPONSES TO STRESS SIGNALS

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National Institute of General Medical Sciences (NIGMS)

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Proteins carrying out DNA repair, replication and transcription, processes essential for the stable transmission and expression of genomic information, share the same track – the DNA – and require delicate coordination to achieve their function. Often, regulation of these processes occurs at the level of targeting or well-timed recruitment, but also catalysis and even turnover of the factor in question. We will address these three principal modalities of regulation in the context of bacterial responses to stress signals. Our questions and hypotheses are grouped into two large areas, (1) regulation of the general stress response mediated by the promoter specificity subunit RpoS; this is exemplary of a general stress response mechanism that affect transcription initiation, and (2) specific responses to DNA damage such as subpathways of nucleotide excision repair; these are exemplary of how the DNA damage response interfaces with transcription elongation, termination as well as replication. The principal questions that will be addressed by my research plan are: A. How are activating and inhibitory regulatory inputs integrated to tune the RpoS core pathway and  globally reprogram transcription in response to an adverse environment? How does the ClpXP  adaptor RssB and stress-specific ClpXP anti-adaptors tune the proteolysis of RpoS in a stress-specific manner? B. What are the mechanisms for preferential recruitment of the NER machinery to specific lesions,  or to the template strand, directly read by RNA polymerase? How is the availability of early NER  factors regulated by proteolysis in the context of DNA damage and transcription-coupled DNA repair?  How and when do transcription-repair coupling factors collaborate with key players in the SOS response  to promote mutagenesis? Can we leverage these mechanisms towards developing novel anti-evolution  drugs for combination therapies against infection with diverse pathogens? Previous work has already allowed us to build a critical collection of reagents and expertise in the structural biology and biochemistry of transcription-coupled repair and RpoS biology. This gives us now an excellent entry point for a mechanistic dissection of the processes listed above and the development of novel antimicrobial strategies. The significance of our work is thus not only fundamental and conceptual in nature, but has immediate applications in controlling the worldwide public health crisis of antimicrobial resistance, particularly in the age of the COVID-19 pandemic.

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Engineered Lipid Nanoparticles and Microgel Matrix to Program Th1/Th2 Immune Response

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The overall objectives of this study are to (1) develop mRNA lipid nanoparticle (LNP) formulations capable of eliciting dual or biased Type 1 T helper (Th1) and/or Type 2 T helper (Th2) immune responses via a machine learning-aided screening platform, (2) engineer mRNA LNP- loaded microgels as an immunostimulatory niche in vivo to recruit and transfect host immune cells and potentiate antigen-specific immune responses, and (3) demonstrate the efficacy and safety of this new LNP-based vaccine platform in murine cancer models. The success of mRNA COVID-19 vaccines utilizing lipid nanoparticle (LNP) delivery has underscored the potential of mRNA LNP-based cancer vaccines in advancing immunotherapy by training the immune system to respond effectively to tumor antigens. To maximize their potency in eliciting robust immune responses for cancer immunotherapy, it is important to engineer effective approaches for systematic programming of immune activation profile via optimization of LNP compositions and improving delivery for mRNA LNP vaccines. Our preliminary data have demonstrated that LNP compositions and helper lipid structure influence the polarization of immune activation, and LNPs with dual Th1-plus-Th2 activation profile yielded the most potent antitumor efficacy in mouse tumor models. In addition, a candidate mRNA LNP vaccine loaded into a nanofiber-based microgel matrix, which facilitated recruitment and retention of host immune cells, enhanced antigen presentation, and elicited equally strong anti-tumor response with a single dose compared to a standard three-dose immunization regimen. In this proposed study, we will first optimize LNP composition for efficient transfection of antigen-encoding mRNA into antigen-presenting cells (APCs) and non-APCs via an machine learning-guided, iterative design-build-screen/test-learn process, and evaluate antigen presentation, and immune activation profiles in vivo; then develop an LNP-incorporated nanofiber microgel matrix termed LiNx as an immunostimulatory niche for recruitment of host immune cells and assess gene delivery efficiency and immune response profiles; and finally demonstrate the therapeutic efficacy and biosafety of the optimized LNPs and LiNx in suppressing tumor growth in therapeutic models of melanoma, lymphoma, and colon carcinoma in mice, as well as an orthotopic, immunoquiescent, pancreatic cancer mouse model. The innovation in this study lies in programming the immune activation profile generated by mRNA LNP vaccine by tunning cell-preferential transfection activity of LNPs and engineering an immunostimulatory niche using a nanofiber microgel matrix to recruit and retain host immune cells and deliver LNP vaccines, thus potentiating the therapeutic efficacy against cancer. Findings from this study can inspire rational design of new mRNA-based immunotherapies for the treatment of cancer and other diseases.

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Adaptive Neurostimulation to Restore Sleep in Parkinson's Disease: An Investigation of STN LFP Biomarkers in Sleep Dysregulation and Repair

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National Institute of Neurological Disorders and Stroke (NINDS)

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Parkinson’s disease (PD) results from progressive loss of substantia nigra dopaminergic cells and manifests clinically as impaired motor control. Non-motor symptoms are far more disabling for patients, precede the onset of motor symptoms by a decade or more, are more insidious in onset and less effectively treated. Sleep dysfunction is often the most burdensome of non-motor symptoms, is pervasive in PD patients, and includes sleep fragmentation, insomnia, excessive daytime sleepiness, and rapid eye movement (REM) behavior disorder. Changes in sleep macro-architecture are also seen in PD, with less total sleep time, increased wake after sleep onset (WASO), increased non-REM stage 1 (NREM1) sleep, and decreased NREM2, NREM3, and REM. Building on our earlier observations of spectral patterns in subthalamic nucleus local field potentials (STN LFP) that correspond to specific sleep stages, we have used a novel investigational DBS programmable generator (RC+S Summit System; Medtronic) to enable exploration of sleep biomarkers and prototyping of therapeutic closed-loop, stimulation (DBS) algorithms. Specifically, in PD patients undergoing STN DBS, we examined whether STN oscillations correlated with sleep-stage transitions, then constructed and evaluated sensing and adaptive stimulation (aDBS) paradigms that allow ongoing sleep-stage identification, and attempted to induce through aDBS an increase in sleep-stage duration associated with restorative sleep. This work addresses an unmet clinical need—i.e., the significant sleep dysfunction of PD—and enabled evaluation of STN aDBS in PD patients, specifically for the treatment of sleep dysfunction. Our primary hypothesis for UH3NS113768A1 was that STN—a highly interconnected basal ganglia node—affects the regulation and disruption of human sleep behavior and may be modulated for therapeutic advantage. We tested whether dysfunctional PD STN activity correlates with sleep fragmentation, and whether STN aDBS algorithms could be developed that improve sleep-stage maintenance and sleep quality. Results demonstrate that PD sleep disturbance is marked by pronounced fragmentation and under-expression of NREM3 and REM, reinforcing prior reports of disrupted macroarchitecture. Sub-clinical STN DBS did not alter overall sleep-stage expression, though effects on sleep spindle density support microarchitectural benefits. Efforts to implement closed-loop aDBS for sleep in the home environment revealed practical challenges, including wearable limitations and subject compliance, but also highlighted how next-generation DBS devices with enhanced recording and user interface capabilities can empower novel aDBS strategies. While these findings do not yet confirm a definitive approach to restoring sleep in PD, they underscore that STN-LFP signals hold promise as biomarkers for identifying and modulating specific sleep states in real-world settings and set the stage for more targeted, long-term interventions to improve sleep quality and potentially slow disease progression. Progress on study Aims 1 and 2 described above was impeded by the COVID Pandemic, and has gathered steam with navigation past obstacles to patient recruitment and surgeries. Enrollment for Aim 1 and 2 and investigation of Aim 1 subjects are complete, and the 3-week in-home sleep trial for Aim 2 subjects will conclude prior to Year-5 end. We recently developed novel methods for analyzing study data and are requesting additional funding to accomplish this.

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Introducing a novel computational framework for B-cell epitope prediction based on immune-induced selection signatures

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National Institute of Allergy and Infectious Diseases (NIAID)

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The accurate prediction of B-cell epitopes is essential for disease analysis, diagnostic tests, and vaccine design, yet it lags behind T-cell epitope prediction in precision. This is due to the complex nature of B-cell epitopes and the undersampling of mapped antibody-antigen structural data. To address this, we propose calculating the intensity of immune selection as an indicator of immunodominance because immune response-driven selection leaves detectable genetic signatures around common regions targeted by antibodies. Our preliminary data suggests that surface-adapted immune selection statistics recover common epitope sites in Sars-Cov-2 Spike, Influenza HA1, and malaria antigens. We have set out two primary aims to enhance B-cell epitope prediction. Aim 1: we plan to build a novel and comprehensive antigen database from 68 human pathogens, which maps 3D immune selection profiles onto the surfaces of antigens from common pathogens. Antigens will be selected from IEDB and their underlying population- level variation will be extracted from public genomic resources. Population genetics scores, such as Tajima's D and BetaScan, will be calculated on antigen surfaces. The resulting database will be deployed online for easy public access. Aim 2: we will develop a B-cell epitope predictor with two innovations. First, the training output comes from the normalized selection statistics of antigen surface instead of relying on antigen-antibody structures. Second, structural features for training inputs will be encoded through the Holographic-CNN model. The predictor's efficacy will then be compared against the state-of-the-art models using a distinct test set of experimentally resolved antigen-antibody structures. Upon completion, our predictor is expected to substantially elevate the predictive power of B-cell epitopes. Our antigen selection database will provide unparalleled new information for various research purposes of antigen evolution. These tools will be instrumental for reverse vaccinology, especially the design of epitope-based vaccines and the evaluation of the potential effectiveness of immunological interventions.

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PET to Assess Post-TB Lung Disease in TB Patients with / without HIV coinfection

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National Institute of Allergy and Infectious Diseases (NIAID)

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Mycobacterium tuberculosis remains the second leading cause of death from a single infectious agent globally, after COVID-19. The risk of developing tuberculosis (TB) is substantially higher in human immunodeficiency virus (HIV) coinfected individuals, with TB and HIV potentiating each other, causing substantial declines in immune function. Globally in 2022, there were an estimated 1.3 million deaths and 10.6 million new cases due to TB. Importantly, it is estimated that more than 58 million patients have survived TB in this century alone. However, unlike other respiratory infections, many patients with TB have permanently damaged tissues and successful TB treatments only transition these patients from harboring a communicable infectious disease, to a syndrome of chronic pulmonary morbidity. This syndrome, commonly referred as post- TB lung disease, affects ~50% of patients with pulmonary TB, who develop chronic adverse outcomes beyond the TB treatments, including bronchiectasis, poor lung function and respiratory symptoms. Although post-TB lung disease remains poorly characterized, it is primarily mediated by M. tuberculosis-induced tissue destruction (necrosis) and subsequent adverse lung tissue remodeling and fibrosis, which in addition to causing pulmonary disease, also impairs vascular supply and reduces antibiotic access to the infected TB lesions. Currently, there are no approved treatments to prevent post-TB lung disease.  We have characterized lung disease in animal models of TB to develop adjunctive treatments that prevent post-TB lung disease. Importantly, we have also developed and utilized novel, clinically translatable positron emission tomography (PET)-based imaging to longitudinally profile lesional characteristics in animal models and TB patients: 11C-rifampin (chemically identical to rifampin) PET to measure tissue antibiotic area under the curve exposures (Ordonez . . . Jain. Nat Med. 2020) and 18F-FAPI-74 PET for monitoring fibrosis. Our central hypothesis is that 18F-FAPI-74 PET could be used as a noninvasive biomarker to assess post-TB lung disease and fibrosis in TB patients. Additionally, we hypothesize that lesions with significantly reduced 11C-rifampin exposures in TB patients within 6-weeks of treatment initiation (early time-point) will develop post-TB lung disease sequelae at those sites after 6-months of TB treatments.  Here, we will leverage our expertise in TB pathogenesis and molecular imaging to conduct longitudinal, observational studies to visualize lesional rifampin exposures and lung fibrosis in patients with recently diagnosed pulmonary TB, with or without HIV coinfection, within 6-weeks and after 6-months of initiating TB treatments. Our goals are to better characterize post-TB lung disease sequelae and identify key factors, such as lesional antibiotic exposures in TB patients that could subsequently lead to post-TB lung disease. In the future, we anticipate that these imaging approaches could be used to noninvasively characterize post TB-lung disease and evaluate novel treatments that prevent or mitigate post TB-lung disease.

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2025 Nucleosides, Nucleotides and Oligonucleotides Gordon Research Conference and Gordon Research Seminar

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National Center for Advancing Translational Sciences (NCATS)

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This proposal seeks funding to support the 2025 Nucleosides, Nucleotides and Oligonucleotides (NNO) Gordon Research Conference (GRC) and the associated Gordon Research Seminar (GRS). This will be the 22nd edition of this conference, initiated in 1976. Throughout its history, NNO GRC has addressed topics directly related to the fundamental science of human biology and the treatment of disease. Modified nucleosides have been extremely effective drugs, most notably antivirals such as AZT and Harvoni, for the treatment of HIV/AIDS and hepatitis C, respectively. Meanwhile, the field has undergone transformative changes to include oligonucleotides as therapeutics for metabolic and genetic diseases, for example Spinraza, which treats spinal muscular atrophy. The recent invention of mRNA vaccines to prevent COVID is another compelling example of the transformative impact of modified nucleosides. The 2025 NNO GRC, entitled “Challenges at the Interface of Structure, Function and Chemistry of Nucleosides and Nucleic Acids” will cover the fundamental science that enabled the recent success of nucleic acid therapeutics while focusing on cutting-edge, current research in novel and emerging technologies. The specific aims for the conference are to: (1) facilitate discussions of the latest research in the field; (2) introduce early-career and inexperienced scientists to the field; and (3) provide opportunities for new collaborations and networking. These specific aims will contribute to the long-term goal of the conference to have a broad and sustainable impact on both basic science and clinical translation of nucleosides, nucleotides and oligonucleotides into therapeutics to improve human health. The 2025 NNO GRC will include sessions on (1) RNA epigenetics, modification and translation, (2) synthesis and chemistry of modified nucleotides, (3) genome and RNA editing, (4) nucleic acids chemical biology, and (5) oligonucleotide therapeutics. Co-Chairs have solicited nominations from experts in the field, emphasizing the desire to identify young scientists as potential speakers. The affiliated Gordon Research Seminar (GRS) will feature oral presentations by students and postdocs, a panel discussion with established scientists in the field, and the opportunity to create a sense of community prior to the start of the GRC. Thus, while the cutting-edge science described during the oral sessions will undoubtedly inspire others to move their own research forward in new and creative ways, the combined GRS and GRC efforts to support young and early-career scientists in general will strengthen the field and its impact on human health for decades to come.

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National Institute of General Medical Sciences (NIGMS)

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It is well understood how lipids are synthesized and metabolized in cells and that many lipids exhibit signalling functions to regulate cellular processes in a spatially and temporally defined way. The latter requires the build- up and turnover of lipid species in membranes either in a site-specific fashion or, alternatively, a directed form of lipid transport. This work aims to investigate the intracellular transfer of lipids from one membrane to another by several proteins that we discovered to be involved in lipid transport. In the previous funding period, we synthesized multifunctional lipid derivatives of five phosphoinositides and four common glycerophospholipids. These feature a photo-activatable protecting group (”cage”) to release the lipid derivative by light and a photo-crosslinking diazirine to covalently attach the lipid derivative to binding proteins. An alkyne group for click chemistry is useful for isolating lipid-protein conjugates or for determining the lipid location in cells by fluorescent tagging and microscopy. In published work, we identified specific lipid binding proteins for phosphatidylinositol 3,4,5-trisphosphate (PIP3), phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol (PI) via proteomic analysis. We then used siRNAs to block lipid transport and validated two hits that were required for transporting PIP3 and PI(3,4)P2: cytosolic MPP6 and transmembrane ATP11A. This R35 application proposes the continuation of work described in the application of R01 GM127631, namely the characterization of the lipid transport by the two above mentioned proteins (Project 1). This includes the purification and characterization of recombinant proteins and their functional mutants. We will use purified proteins to determine the 3D structure of MPP6 and its mutants by cryo-electron microscopy with and without crosslinked lipid derivatives. In Project 2, we will synthesize lipid derivatives featuring the photo-crosslinking diazirine closer to the membrane interphase to reach more transiently binding proteins such as those with a PH domain. Comparative proteomic analysis of the lipid interactomes will then be used to identify proteins involved in signalling with and without receptor stimulation. In Project 3, we will use multifunctional lipid derivatives to investigate the lipid interactomes of healthy and virus-infected cells. We recently discovered that an RNA virus infection leads to massive changes in the host cell lipidome. One exciting aspect is that one group of cellular phosphoinositides featuring a particular fatty acid composition is strongly up regulated. We will measure the lipid interactomes of flavivirus- and COVID-infected cells and identify targets crucial for viral infection and replication. Hits will be validated by protein knock-down and the effect on virus infection will be studied. Our unique lipid tools will help to better understand the lipid and lipid binding components of a viral infection.

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3STEP: A swab-seal-read assay for tuberculosis and RIF resistance enabled by autonomous extraction technology

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National Institute of Allergy and Infectious Diseases (NIAID)

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In 2021 there were 10.6 million new cases of Mycobacterium tuberculosis (TB) and 1.6 million TB-related deaths, making TB the second leading infectious cause of death worldwide behind only COVID-19. Standard TB diagnostic methods are insufficient in that they either 1) utilize challenging sample types and are often invasive (e.g., sputum, gastric aspiration, lymph node biopsy), 2) lack sufficient sensitivity and/or specificity (e.g., culture, reliance on clinical observation alone), or 3) cannot be widely implemented in community point-of-care (POC) settings. As a result, there remains a significant unmet need for affordable POC technologies that can be used at local or temporary clinics, urgent care facilities, pharmacies, and school health clinics with easier to acquire samples (e.g., urine or oral swabs). In addition, TB is notoriously difficult to lyse adding another challenge to the creation of a POC solution. Salus Discovery has developed 3STEP, a new LAMP-based POC NAT for TB that directly overcomes the above mentioned challenges. 3STEP provides lab-quality, PCR sensitivity (<10 CFU/swab analytical sensitivity) with the speed and simplicity of a lateral flow assay (LFA), (i.e., a simple tongue swab, 3 user steps, no liquid transfers, and results within ~30 minutes). 3STEP also addresses challenges of spectral multiplexing in LAMP reactions by enabling spatial multiplexing for integrated detection of drug-resistant infections. Importantly, our novel stabilized interface technology (SIFT) used to create 3STEP enables both an ultra-cheap disposable and an affordable isothermal instrument design. In this SBIR Fast Track Phase I/Phase II proposal we will continue developing 3STEP for the detection of TB and rifampin resistant (RR) TB from tongue swabs by: In Aim 1 (Phase I), optimizing 3STEP for TB oral swabs using contrived samples, including designing and implementing RR testing into the spatial multiplexing capabilities of 3STEP; In Aim 2 (Phase I), prototyping and testing a POC lysis module; In Aim 3 (Phase I), assessing clinical sensitivity in a small pilot study using bio-banked samples collected through FIND’s FEND-TB program; In Aim 4 (Phase II), finalizing the 3STEP assay and consumable for an expanded clinical evaluation; And in Aim 5 (Phase II), performing a larger scale clinical evaluation of the multiplexed 3STEP TB/RR assay. Results will be important to subsequent funding efforts for 3STEP commercial development (assay and reader) and to demonstrate field-readiness for participation in TB diagnostic evaluation studies similar to R2D2, DriveDx4TB, SMART4TB, and FEND-TB.

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Small Business Innovation Research Grant

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Use of HIF-1alpha mRNA to Promote Pedicle Flap Healing.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)

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Summary/Abstract. Surgical debridement is often used when treating major wounds, and pedicle flaps are a critical component in subsequent surgical reconstructions. Distal portions of the flap require vascular perfusion for overall success of wound repair and reconstruction. Hypoxia-inducible factor-1α (HIF-1α) is an important inducible transcription factor that orchestrates and controls cellular responses to hypoxia when paired with constitutively expressed HIF-1. HIF-1α enhances cell survival in wounds by regulating the expression of over 200 genes, including many angiogenic growth factors responsible for restoration of vascular beds. By combining the concept of increasing HIF-1α in wounds to promote cell survival and revascularization, with contemporary approaches for RNA transduction pioneered in COVID vaccines, our goal is to create a novel therapeutic strategy for enhancing pedicle flap survival. Experiments that support progress toward this goal are proposed in two specific aims: Specific Aim 1A: Quantitation of HIF-1α mRNA and response genes following intradermal injection in Sprague Dawley rats. Based on encouraging preliminary results both in vitro and in vivo, we are developing new versions of our HIF-1 mRNA reagents that are designed to improve activity when delivered in vivo using a proprietary lipid nanoparticle carrier. Rat and porcine versions of our HIF-1 mRNA reagents are being developed for use in pedicle flap assays described in Aims 1B and 2. Specific Aim 1B: In vivo assessment of HIF-1α mRNA therapeutics in a Sprague Dawley rat model of pedicle flap surgery. Findings in Aim 1A will provide preliminary data on best performing reagent structures, dose, and delivery formulations to inform testing in pedicle flap assays in male and female rats. In this aim we will measure HIF-1 expression and downstream gene induction as in Aim1A and add macroscopic monitoring of wound resolution and molecular phenotyping of the wound site using quantitative PCR and immunohistochemistry. Specific Aim 2: In vivo assessment of HIF-1α mRNA therapeutics in a porcine model of pedicle flap surgery. Pigs are valuable preclinical models for testing novel wound healing strategies. The mRNA reagents and formulations that show promise in the rat pedicle flap model in Aim 1B will subsequently be tested in a porcine model of pedicle flap survival and wound healing. Surgical outcomes will be assayed as in Aim 1B to monitor potential translatability of our studies for human clinical applications.

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Small Business Innovation Research Grant