University Of Rochester

NIH Research Projects · FY 2025 · 2021-07

Factors Associated with Response to Cardiac Resynchronization Therapy in Heart Failure Patients with Non-LBBB ECG Pattern PI: Valentina Kutyifa, MD, PhD University of Rochester Medical Center, Rochester, NY Morbidity, mortality, and health care costs of the treatment of systolic heart failure (HF) are rapidly increasing. Cardiac resynchronization therapy with a defibrillator (CRT-D) is cost-effectively reducing HF events and death in HF patients with a wide QRS and low ejection fraction. However, response to CRT-D is not unequivocally present in all patients, with less benefit in those without the presence of an ECG abnormality, left bundle branch block (non-LBBB), posing a significant treatment challenge. Because of the conflicting and limited data on response to CRT-D in this cohort, it is possible that we currently treat a large proportion of HF patients with non-LBBB who have limited or no benefit from the device. Therefore, better selection of patients for this expensive therapy is warranted. One of our recent studies suggested a clinical benefit in patients with non-LBBB and marked echocardiography response, and identified predictors. However, there is a need to prospectively validate these predictors of echocardiography response to CRT-D in non-LBBB in this hard-to-treat patient population, and identify potential novel ECG and echocardiography predictors, utilizing novel statistical methods of machine learning. We propose a prospective, observational, single-arm study in a currently guideline-indicated cohort to validate and identify predictors of echocardiography response to CRT-D, including novel ECG and echocardiography markers, and to assess subsequent clinical outcomes in 270 HF patients with an implanted CRT-D and non-LBBB ECG pattern. The primary aim of the study is to prospectively validating our previously identified clinical predictors of echocardiography response to CRT-D in HF patients with non-LBBB that could enable better patient selection. Our secondary aim is to identify the incremental value of novel ECG and echocardiography variables to predict echocardiography response to CRT-D in non-LBBB patients, including ECG variables of sum absolute QRST integral and ventricular electrical activation delay, and echocardiography-derived variables of left ventricular dyssynchrony and contractility. Then we will apply the developed predictive model to prospectively identify non- LBBB patients with CRT-D at a risk of heart failure, ventricular arrhythmias, or death. Tertiary aim is to identify novel ECG and echocardiography predictors of response in non-LBBB using machine learning analysis. Study population will include 270 HF patients with non-LBBB (135 with mild HF and 135 with advanced HF) and an implanted CRT-D with 6 months echocardiography follow-up analyzed by an echocardiography core lab, and assessing clinical outcomes of heart failure, ventricular arrhythmias, or death.

NIH Research Projects · FY 2025 · 2021-07

Tendon injuries heal in a fibrotic manner via chronic deposition of excessive, disorganized extracellular matrix. Myofibroblasts are a critical driver of fibrosis in many tissues, and emerging evidence demonstrates myofibroblast heterogeneity. That is, myofibroblasts with unique molecular profiles that correspond to changes in myofibroblast function. Importantly, the fibroblast lineage from which myofibroblasts are derived also plays a major role in dictating myofibroblast function. However, very little is known about myofibroblast dynamics during fibrotic tendon healing, including their fibroblast/tenocyte-lineage, how their functions change over time, what the molecular profiles of these different myofibroblast subtypes are, and how myofibroblasts interact with other cells, such as macrophages, to mediate healing and fibrosis. We have identified Scleraxis-lineage and S100a4-lineage cells as the predominant myofibroblast precursor populations during tendon healing. Interestingly, depletion of S100a4-cells impairs early tendon healing, while Scx-cell depletion improves late tendon healing suggesting these cells may give rise to functionally distinct myofibroblast populations. In addition, our preliminary data identifies macrophages as a critical driver of the tenocyte-myofibroblast transition, and we observed prolonged macrophage presence during late healing, concomitant with NFB- mediated pro-survival signaling in myofibroblasts. These data are consistent with a pro-fibrotic feedback loop between macrophages and myofibroblasts to sustain fibrosis in many tissues. Thus, in the present study we will use a murine model of acute tendon injury and repair to rigorously define tenocyte lineage-specific contributions to myofibroblast fate and define myofibroblast heterogeneity during healing. We will test the central hypothesis that inhibiting macrophage-mediated myofibroblast differentiation and NF-B-mediated survival of lineage-specific myofibroblasts promotes regenerative tendon healing. We will test this hypothesis through the following specific aims: Aim 1: Define the temporal and tenocyte lineage-dependent immunophenotype of myofibroblasts during fibrotic tendon healing. Aim 2: Establish the requirement for extrinsic macrophages in fibrotic healing via modulation of the tenocyte-myofibroblast transition and test the translational potential of inhibiting macrophage recruitment. Aim 3: Demonstrate that disrupting myofibroblast survival or macrophage persistence inhibits sustained fibrosis and promotes regenerative tendon healing. Successful completion of these studies will establish myofibroblast lineage, molecular profile and activation mechanisms over time during fibrotic healing and define disruption of persistent myofibroblasts and macrophages as a novel means to improve tendon healing.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY The candidate: Dr. Manuel Gomez-Ramirez is an Assistant Professor in the Department of Brain and Cognitive Sciences (BCS) at the University of Rochester, whose overarching goal is to establish cutting-edge research program focused on studying neural circuits and spiking dynamics that mediate object sensing and manipulation with the hands (i.e., haptics). Dr. Gomez-Ramirez completed his Ph.D. at the City College of the City University of New York studying mechanisms of distracter suppression in humans. He transitioned to studying mechanisms of tactile perception in non-human primates using multi-electrode single-unit recordings at the Johns Hopkins University. He conducted a second post-doctoral fellowship at Brown University to learn calcium-based imaging and optogenetic methods in mice. In his new lab, Dr. Gomez-Ramirez aims to incorporate techniques learned in his post-doctoral fellowships to study haptics in a non-human primate model. Research: In this award, Dr. Gomez-Ramirez will study how selective attention controls the sensory gain of neocortical representations encoding distracter stimuli on the hand. He will use cutting-edge electrophysiological and neuromodulation methods (i.e., optogenetics) to determine the neural circuit, and its dynamics, that mediate sensory suppression of distracting tactile inputs. Aim 1 investigates the granularity of attention to modulate sensory responses via targeting of cells’ receptive field (RF) inhibitory and/or excitatory sub-regions. Aim 2 investigates the precision and flexibility of attention to deploy distracter suppression across different somatotopic representations in the somatosensory system. Aim 3 tests the role of layer 5 SST neurons in mediating sensory gain suppression in supragranular layers, the mayor cortical layer of intra- and inter-area communication. Training, Mentors, Collaborators, and Environment: The major research training goals of this award are to gain expertise in (1) Cutting-edge population decoding methods (e.g., state-space analytical methods), and (2) optogenetic stimulation in monkeys to study the how top-down signals regulate local somatosensory cortical circuits representing relevant vs. irrelevant tactile information. This award will also provide key training in grant and manuscript writing, personnel management, as well as engagement in extracurricular activities that will enhance my career and professional development. The K01 scientific mentoring team comprises world-experts in their respective field of research, and have been role models for mentoring and supporting junior faculty. The Tenure Oversight team is composed additional senior faculty that will provide another layer of monitoring to ensure progress in the activities proposed in the tenure plan. Impact and Significance: Data gathered from studies in this award will inform an R01 application focused on studying how top-down signals modulate cross-cortical somatosensory circuits mediating object sensing and manipulation. This award will help provide training on cutting-edge electrophysiological, computational, and genetic-based optical methods to personnel in the Dr. Gomez-Ramirez’s lab.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Autophagy is an important cellular antiviral defense mechanism that potentially affects HIV infection and transmission. The antiviral properties of autophagy are two-fold: (i) autophagy targets virions for lysosomal degradation and (ii) this pathway aids in antigen presentation of virus pathogens. Despite its antiviral potential, efforts to capitalize on autophagy against HIV have been limited due to a gap in our understanding of the role of autophagy in HIV infection. Our long-term goal is to manipulate the antiviral properties of autophagy to facilitate HIV clearance and improve treatments. Our overall objective is to identify the molecular interactions between HIV and the autophagy machinery. Our central hypothesis is that autophagy restricts HIV, but the virus overcomes this block using Nef, a notorious virulence factor that mediates immune evasion. This hypothesis has been formulated based on our published and novel work demonstrating that autophagy depletes the HIV proteins Gag, Vif and Vpr, but only when nef is defective 17. Gag is the driver of virion assembly; Vif counteracts the damaging effects of the restriction factor APOBEC3G; and Vpr enhances infectivity by facilitating viral transcription and arresting cell cycle at G2. Thus, a decrease in Gag, Vif and Vpr will impact virion production and infectivity. However, we found that Nef blocks autophagy, restoring in turn HIV proteins and particle release. Our studies showed that Nef sequesters the autophagosome initiator BECN1 at the endoplasmic reticulum (ER) by facilitating its association with the autophagy inhibitor BCL2. Mechanistically, we uncovered that Nef induces BCL2 mono-ubiquitination via the E3 ligase Parkin. This post-translational modification increases BCL2's inhibitory effect over BECN1. Hence, this activity of Nef impairs autophagosome formation. The significance for this research is that: (i) it will address an important knowledge gap concerning the interplay between HIV and autophagy, and will reveal how this pathway influences HIV infection and transmission; and (ii) it will provide the basis for the design of approaches aimed at enhancing autophagy to treat HIV infection and/or improve cure strategies. We will attain the overall objective by pursuing the following three specific aims: (1) Identify HIV molecules vulnerable to autophagy; (2) Dissect the mechanism by which Nef blocks autophagy; and (3) Elucidate the impact of autophagy antagonism on HIV infectivity. The proposed research is innovative because it represents a substantive departure from the status quo by revealing that HIV critically needs Nef to counteract autophagy restriction.

NIH Research Projects · FY 2025 · 2021-07

Insertable Cardiac Monitor-Guided Early Intervention to Reduce Atrial Fibrillation Burden Following Catheter Ablation (ICM-REDUCE-AF Trial) PI: Ilan Goldenberg, MD, University of Rochester Medical Center, Rochester, NY Percutaneous catheter ablation (CA) to achieve pulmonary vein electrical isolation is an effective and recommended treatment for drug-refractory paroxysmal and persistent atrial fibrillation (AF). Nevertheless, recurrence rates after a single AF ablation procedure are in the range of 30%-50%. Accordingly, more measures are needed to improve success rates following CA of AF. To date, conventional management after CA ablation has mostly been based on intervention for clinical AF recurrence. Continuous recording with insertable cardiac monitors (ICMs) can now be used to detect early recurrences of subclinical AF (SCAF) and patients-triggered mobile app transmissions post ablation. We hypothesize that early intervention following CA can prevent substrate progression that promotes the onset and maintenance of atrial arrhythmias. We therefore propose a randomized, double-blind (to SCAF data), single center, clinical trial in which 120 patients with drug-refractory paroxysmal AF or persistent AF planned to undergo CA with an ICM, will be randomized to: an intervention arm (n=60) consisting of ICM-guided early intervention based on SCAF and patient-triggered mobile app transmissions vs. a control arm (n=60) consisting of a standard intervention protocol based on clinical AF recurrence validated by the ICM. We believe that ICM-guided early intervention management provides a novel personalized approach and a paradigm shift in post-AF ablation management that will result in a significant reduction in AF burden and healthcare utilization in this expanding population, with corresponding improvements in functional capacity and quality of life, compared with conventional follow-up after AF ablation. In the proposed clinical trial, Aim 1 is to evaluate whether ICM-guided early intervention based on SCAF detection and symptom-triggered mobile app transmissions will be associated with a significant reduction in AF burden following CA for AF compared with the standard strategy of treatment upon the development of clinical AF recurrence. AF burden will be assessed from ICMs at 15 months post-AF ablation (excluding the 3-month blanking period). Aim 2 is to evaluate whether ICM-guided early intervention management will be associated with a reduction in in healthcare utilization (defined as unplanned hospitalizations, emergency department visits, cardioversions, and unplanned office visits) compared with a conventional management strategy. Aim 3 is to evaluate whether ICM-guided early intervention management will be associated with improvement in functional capacity and quality of life compared with a conventional management strategy following CA for AF.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT The use of left ventricular assist device (LVAD) for patients with advanced heart failure has continued to increase over the years. The significant improvement in survival with the current generation of LVADs is in large part due to the advances in device durability, and mechanics. However, several important factors continue to limit the benefit of LVAD support and ventricular tachyarrhythmias (VTA) following LVAD implantation has been suggested to be associated with subsequent repeat hospitalization and increased mortality. Our retrospective data from the University of Rochester have shown that patients who developed VTA following LVAD implant experienced a 7-fold increase in subsequent mortality risk. Importantly, we have also shown that the most powerful predictor of VTA post LVAD implant is a history of VTA at any time prior to the LVAD. Several ablation trials have demonstrated reduction of recurrent VTA and, in selected at-risk patient groups with ischemic heart disease, mortality. These findings suggest an effective early intervention with VTA ablation peri-LVAD implantation in high-risk patients, specifically those with a history of VTA, may reduce VTA recurrence and improve clinical outcomes. Utilization of this strategy has varied amongst centers as there are no prospective data on the efficacy and safety of this management. Accordingly, we propose a prospective, randomized, multicenter clinical trial that will evaluate prophylactic intra-operative VTA ablation in high-risk LVAD recipients on clinical outcomes. The study cohort will consist of 100 LVAD candidate patients with a prior history of VTA and ischemic cardiomyopathy randomized to either prophylactic intra-operative VTA ablation vs. conventional medical management in a 1:1 fashion. Eight high-volume LVAD implant centers with experience in VTA ablation will participate. The primary specific aim is to evaluate the effect of prophylactic intra-operative VTA ablation at the time of LVAD implantation on post-implant total recurrent VTA events after accounting for the competing risk of death. Secondary specific aims are: 1) To evaluate the effects of a management strategy that incorporates intra- operative VT ablation at the time of LVAD implantation on adverse clinical outcomes following LVAD implantation; 2) To collect prospective data on peri-procedural outcomes associated with the proposed novel approach of prophylactic intra-operative VT ablation. We will also explore the mechanisms associated with: a) recurrent VTA post LVAD implant with electrophysiology studies and b) the ramifications of recurrent VTA on right ventricular hemodynamics post LVAD implant with echocardiographic ramp studies and right heart catheterization.

NIH Research Projects · FY 2025 · 2021-07

Noise induced hearing loss (NIHL) has disabled millions of people world-wide. Individual risk for NIHL varies from person to person under similar exposure conditions, suggesting that genetic factors contribute to susceptibility. We have found that mice lacking a transcription factor called FOXO3 become severely and permanently deafened after a noise exposure that only briefly affects their wild-type littermates. FOXO3 has multiple functions in other cell types, including oxidative stress reduction, autophagy, and directly inducing apoptosis. A recent study linked human genetic variations in FOXO3 to a greater susceptibility to occupational NIHL. However, the FOXO3 alleles associated with NIHL drive increased expression of FOXO3. Thus, there is evidence to indicate that FOXO3 is important for hearing preservation, but there is also evidence that excess FOXO3 drives NIHL. In this grant, we seek to address this knowledge gap by researching the mechanisms of FOXO3 function, using translatome sequencing, cell-specific Foxo3 conditional knockouts (cKO), and CRISPR modifications to generate mouse lines that can be used to investigate the human NIHL-linked FOXO3 allele. In Foxo3-knockout (KO) mice, noise eliminates high-frequency outer hair cells (OHCs). We show that this occurs through a rapid cell death program called parthanatos, which is caspase-independent apoptosis. Parthanatos indicates that in the absence of any noise damage, Foxo3-KO OHCs are primed for death. Bulk RNA-Seq data from control Foxo3-KO and wild-type littermates show no evidence for changes in oxidative stress reducers known to be regulated by FOXO3. Instead, we see changes in actin binding genes expressed in OHCs. In Aim 1, we propose to validate this screen and identify markers of OHC distress in the Foxo3-KO through translatome sequencing. In Aim 2, we propose to make cell-specific Foxo3-cKO to identify the cells in which FOXO3 acts. Wild-type mice express FOXO3 protein in both OHCs and in the surrounding supporting cells (SCs). By using inducible DNA recombinases lines specific to either OHCs or SCs, we can ablate FOXO3 function in either cell type. We will expose such Foxo3-cKO mice to noise and determine their NIHL susceptibility. Finally, in Aim 3, we have used CRISPR genetic modification technology to create two mouse lines, one with control sequences (Foxo3-T-allele mice), as well as one homologous to the human FOXO3 allele that confers NIHL susceptibility (Foxo3-G-allele mice). We will validate that the Foxo3-G-allele mouse line has increased levels of FOXO3 in cochlear cells after noise exposure. We hypothesize that this modification promotes apoptosis from FOXO3 activation, and we will test that hypothesis by exposing Foxo3- G-allele mice to noise, measuring their hearing and analyzing potential cellular losses. In sum, through both loss-of-function and gain-of-function experiments, we will analyze FOXO3's role in hearing loss from noise.

NIH Research Projects · FY 2025 · 2021-06

Project Abstract (from original application): Virus infections are highly associated with the exacerbation of allergic diseases including allergic asthma, atopic dermatitis, and allergic rhinitis. IgE-mediated allergic sensitization has been shown to impair antiviral responses by innate immune cells such as monocytes- enhancing pro-inflammatory cytokine secretion, disrupting phagocytosis, and inhibiting virus-induced maturation, IFN production, and altering CD4 T cell priming. These findings suggest a role for IgE-mediated signals in modulating innate antiviral signaling pathways, however little is known regarding the molecular mechanisms behind these observations. Given the significant morbidity and economic impact of allergic diseases, a thorough understanding of IgE-mediated effects on antiviral responses is critical for the discovery of new therapeutics for viral and allergic diseases. The goal of this study is to determine how IgE-mediated allergic stimulation inhibits monocyte antiviral responses to regulate cellular functions. Using primary human monocytes, we will utilize established molecular and biochemical techniques in combination with advanced flow cytometry techniques (flow cytometry imaging and mass cytometry) and transcriptomics to: (Aim 1) determine how IgE-mediated signaling components regulate early antiviral recognition pathways, (Aim 2) determine the mechanisms by which IgE-induced IL-10 regulates interferon receptor signaling, and (Aim 3) translate the in vitro findings by comparing monocyte antiviral responses in individuals with high and low serum IgE. These studies will fill a large gap in our current knowledge of how IgE-mediated processes modulate antiviral responses to promote virus-induced allergic exacerbations. Dr. Regina K. Rowe, M.D. Ph.D. is currently an Assistant Professor of Pediatric Infectious Diseases at the University of Rochester Medical Center. She received her Ph.D. from Washington University in St. Louis in Molecular Microbiology and Microbial Pathogenesis where she investigated the interactions of hantaviruses with the respiratory epithelium. She then received medical training at St. Louis University followed by Pediatric Residency and Infectious Disease Fellowship training at UT Southwestern where her postdoctoral research focused on the effects of allergic stimulation on monocyte-induced T cell priming. This career development award will expand her expertise in systems immunology through training in cell signaling mechanisms, advanced flow cytometry methods, and bioinformatics. As a pediatric physician-scientist, she will utilize her skills as an infectious disease specialist trained in both human immunology and virology to establish a scientific platform to answer a breadth of questions involving host-pathogen interactions in the context of human disease. Her innovative approaches have the potential to identify new therapeutics to treat virus-induced allergic diseases, and ultimately prevent disease development, exacerbation, and progression.

NIH Research Projects · FY 2026 · 2021-05

The burden of neurological disorders disproportionately impacts resource-limited tropical settings resulting in grossly insufficient capacity for care provision or research. This global research program aims to advance our understanding of common neurological disorders in the African context to inform the local prevention and treatment of these conditions while also elucidating pathophysiological processes more broadly relevant. This will be accomplished through the continuation of Dr. Birbeck's neuro-HIV and cerebral malaria investigations plus support and mentorship for young clinician scientists engaged in a broad range of research endeavors. Research activities include-- • Cerebral malaria: In a prospective cohort study of children with CNS malaria, we will examine the role of neuroinflammation in structural injury and neurologic morbidity with laboratory assessments of acute inflammation, serial neuroimaging, and long term neurological outcomes. The novel study population will include children in a randomized controlled trial (RCT) of aggressive antipyretics as well as a more representative non-RCT population. The effects of co-infection with SARS-CoV-2 on parasite clearance, inflammatory factors associated with malarial death and neurologic sequelae and structural injuries including ischemia, bleeding, and thromboses will be ascertained. • Neuro-HIV: Given the widespread availability of HIV therapies, the next challenge in neuro-HIV care in Africa includes noncommunicable disorders associated with chronic low grade inflammation. Utilizing our network of rural and urban HIV clinics, we will study HIV-associated accelerated aging of the nervous system. Given its highly inflammatory state, SARS-CoV-2 could potentially contribute to this burden. Among children, we have recently reported high rates of subclinical cerebrovascular disease (CVD) despite long-standing, effectively treated HIV. We will pursue further imaging studies including in HIV uninfected but exposed children and community controls to identify risk factors for CVD, examine the metabolic impact of antiretroviral therapies and assess the relationship between premature CVD and infection with SARS-Co-V2. Among adults with HIV, SARS-Co-V2 infection will be evaluated for its possible role in accelerated aging in a 5-year prospective cohort study monitoring for cognitive impairment, psychiatric symptoms, strokes, neuropathies and seizures. This research will be undertaken in Zambia and Malawi where Dr. Birbeck has worked for over 25 years. This research program award will also provide infrastructure, mentorship and a vibrant environment for scholarship and trainee engagement with both US and African academics.

NIH Research Projects · FY 2026 · 2021-04

Medicare reimburses over 5 million biopsies resulting in 1.5 million surgical procedures annually for nonmelanoma skin cancer (NMSC). Despite its burden on the health care system, treatment remains time- consuming and unevenly available due to inefficient histologic methods used for diagnosis and to guide therapy. As an alternative, two photon fluorescence microscopy (TPFM) combined with rapid molecular labeling can evaluate histology dramatically faster than conventional methods. We have developed high speed TPFM imaging for dermatologic surgery, protocols for rapid molecular labeling of human tissue, and conducted preliminary studies showing that TPFM can evaluate skin cancer with similar accuracy to conventional histology. Our approach addresses the limitations of current methods, which begin with diagnosis, where the delay for histology means that patients must return for a second clinic visit if biopsies are positive. Inefficiencies are compounded during therapy, where in standard excision the delay for processing means that histology is evaluated postoperatively, and a second surgery may be required to complete therapy. Alternatively, in Mohs surgery, intraoperative frozen sections may be used, resulting in a substantial increase in procedure time and reduced clinic capacity. As a result, costs are increased and access to care is subject to regional variations, with patients in rural areas more likely to receive substandard care that puts them at risk for disfigurement or tumor recurrence. The goal of this research program is to advance two photon imaging technology for surgical applications, and then conduct clinical trials testing the use of TPFM in both diagnostic biopsy procedures and in surgical treatment of NMSC. The aims for this proposal are: Aim 1 will develop improved TPFM that incorporates recent advances in detector and laser technology to reduce cost, shrink size, and enhance contrast, providing an efficient new tool for diagnosing and treating NMSC. Concurrently, studies will examine the accuracy of TPFM for a less common type of skin cancer. Aim 2 will study patients undergoing dermatologic biopsy, testing the hypothesis that patients can be immediately diagnosed using TPFM, enabling same visit treatment for common skin cancers. Finally, Aim 3 proposes interventional surgical trials. Aim 3, Task 1 will integrate TPFM into Mohs surgery, testing the hypothesis that faster histological imaging can accelerate treatment. Aim 3, Task 2 will introduce TPFM for margin evaluation into standard (non-Mohs) excision of skin cancer, testing the hypothesis that TPFM can be used in a scenario for which no intraoperative imaging is currently practical. Collectively, these aims will test more efficient methods of diagnosing and excising skin cancer, accelerating treatment, reducing costs and morbidity while expanding access to care. Successful completion of this project would demonstrate powerful new tools for general surgical pathology and validate them for use in dermatologic surgery.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract Immune dysfunction and imbalances in synaptic pruning have been implicated as contributing factors to neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. Recent studies suggest that dysregulation of complement may be involved. Complement deposits on neuronal synapses to mediate synaptic pruning by microglia and refine neural circuits during critical windows of brain development. Complement can also aberrantly tag synapses for removal in inflammatory and neurodegenerative diseases. Self-directed complement activity is usually held in check by complement regulatory proteins expressed on cell membranes. Nevertheless, the role of complement inhibitors has been largely ignored in studies of complement-mediated synaptic pruning and is the subject of this grant. Our preliminary data shows that the Sez6 family (consisting of Sez6, Sez6L, and Sez6L2) are novel, complement inhibitors. Sez6 family members are highly expressed by neurons during development and in adulthood. Sez6 proteins have been shown to modulate synapse numbers, synaptic plasticity, and dendrite morphology. Genetic loss of Sez6 genes results in impaired cognition and motor deficits. Sez6 family members also have genetic connections to autism, schizophrenia, intellectual disability, epilepsy, and bipolar disorder. We propose that Sez6 proteins modulate synapse numbers and brain development by putting the brakes on complement-mediated synaptic pruning by microglia. Furthermore, disruptions in this process may contribute to the pathogenesis of neurodevelopmental disorders such as ASD. We will investigate mechanisms of complement regulation by Sez6 family members and whether these are disrupted by missense mutations previously identifed in ASD patients. Then will determine if Sez6 family genetic knockout phenotypes are complement-dependent and/or exacerbated by the inflammatory environment of maternal immune activation. Finally, we will investigate whether neuronal activity and the endocytic motifs within the cytoplasmic tail of Sez6 proteins differentially place Sez6 proteins and their complement inhibitory function on the cell surface of active synapses as opposed to weak and inappropriate synapses. This would couple the functional strength of specific neuronal connections to synapses that can be tagged and removed by complement-mediated pruning. This research program will provide insight into the mechanisms of how Sez6 proteins are protective factors against excessive complement-mediated pruning by microglia that may be especially relevant to the pathogenesis of various neurodevelopmental disorders such as ASD.

NIH Research Projects · FY 2025 · 2021-04

Project Summary The development of distant metastases accounts for a significant proportion of breast cancer mortality; as many as 30% of patients initially diagnosed at an early stage will eventually progress to metastatic disease. A key early step in metastatic progression is an increase in cellular plasticity that enables a subset of tumor cells to lose residual epithelial features and gain migratory and invasive behavior, molecularly reflected in the epithelial to mesenchymal transition (EMT). Considerable evidence now points to cancer-associated EMT as a highly dynamic and reversible process with disseminated cancer cells exhibiting many hybrid intermediate states (partial-EMT) proposed to possess the greatest potential for aggressive, stem-like, behavior. However, the epigenetic mechanisms that control such phenotypic plasticity and the role of this process in early invasion remain incompletely understood. Recently we discovered that the local regulation of RNA polymerase (Pol II) pause release by the histone methyltransferase SUV420H2 plays an important role in stabilizing the epithelial ‘identity’ of luminal breast cancer cells and in so doing, suppresses breast cancer cell invasion. Specifically, we find that the local conversion of H4K20me1 to me3 by SUV420H2 enforces RNA polymerase pausing by blocking recruitment of the MOF/MSL complex, which is in turn necessary for the acetylation of H4K16, recruitment of pTEFb and Pol II pause release. We further find that SUV420H2-mediated repression constrains the mesenchymal program in luminal breast epithelial cells, yet is directed to new sites upon TGF-β induced EMT. SUV420H2 is downregulated in triple negative/basal subtype of breast cancers, and its forced downregulation or inhibition promotes collective invasion in breast cell spheroids grown in 3D. These and other findings lead us to propose that the relaxation of SUV420H2-mediated Pol II pausing control is one source of the epigenetic plasticity and transcriptional heterogeneity that underlies breast cancer cell adaptation and the emergence of tumor cells with invasive properties. Using a combination of precision run-on sequencing (Pro-seq) and native chromatin analyses via CUT&Tag technology, we will determine the how the SUV420H2 mediated pause constraints enforces phenotypic stability and how loss of these constraints allows for transcriptional promiscuity. We will explore the role of the HEXIM1 / 7SK snRNP complex as a novel ‘reader’ of histone H4 modifications and its role in SUV420H2-mediated pause control. Lastly, we will determine the impact of dysregulated Pol II pausing dynamics on transcriptional diversity and the emergence of breast cells with invasive “leader” potential in a 3D spheroid model of collective invasion. Over the long term, the results of our studies will provide important insight into the mechanisms underlying epigenetic plasticity and its role in tumor cell adaptation, and a framework for the development of novel reprogramming strategies to block breast cancer metastatic progression.

NIH Research Projects · FY 2025 · 2021-03

Splicing factors are frequently altered by mutations and copy-number changes both in cancer and in germline genetic diseases resulting in multi-system developmental syndromes. Despite the fact that virtually all genes in humans undergo splicing, spliceosomal genetic alterations tend to exhibit surprisingly specific effects on subsets of splicing events, leaving most insignificantly changed. These effects can be allele-specific, cell-type specific, and dependent on the genetic background of the afflicted cell. This makes it especially challenging to determine which affected splicing events contribute to disease etiology. The fact that a limited set of introns is responsive to any specific splicing factor alteration indicates that introns and their flanking exons have evolved in structure and sequence to confer differential sensitivity to the action of different spliceosome components. This raises a fundamental question: what are the features common to sets of introns that confer this specificity? Using naturally occurring splicing gene mutations, amplifications, and deletions, these perturbations will be modelled in a genetically stable, untransformed, isogenic cell system where it is possible to isolate the effect of a single alteration on the transcriptome and on the binding patterns of the altered protein. These studies will shed light on the mechanisms of normal spliceosome function, and provide insight into which genes and biological pathways affected by splicing dysfunction likely contribute to disease states. The proposed experiments will employ three distinct methods to model spliceosome perturbations associated with human disease, with a focus on factors that physically or functionally interact with the essential spliceosome protein SF3B1. (Specific Aim 1) Introduction of an allelic series of cancer-associated SF3B1 missense-mutations into isogenic cell lines using recombinase-mediated cassette exchange (RMCE); (Specific Aim 2) CRISPRa/i-mediated activation or inhibition of transcription to up- or down- regulate splicing factors that are amplified in cancers (PUF60, SF3B4, and U2AF2) and lost in developmental syndromes (PUF60, SF3B4); and (Specific Aim 3) rapid depletion of spliceosomal RNA helicases (DDX39B, DDX46, and DHX16) and their putative co-factors (SUGP1, RBM17, and GPKOW) at the protein level using auxin-inducible degrons. Three distinct methods of RNA sequencing will be used to quantify the changes resulting from these perturbations: poly(A)-selected RNAseq, allele-specific eCLIP, and a novel intron lariat capture sequencing approach. Lastly, we will integrate these genomic data sets into models using deep learning neural networks to interrogate our central hypothesis: the sequence and structure of individual mammalian introns have evolved to confer differential dependence on specific ‘core’ components of the spliceosome, and that mutations, amplifications, and deletions in these core components causal for human disease will uncover intron-centric gene expression regulatory circuits that are controlled though modulation of the abundance or activity of the associated splicing factors in normal cells.

NIH Research Projects · FY 2026 · 2021-02

PROJECT SUMMARY Optimal flow of blood within the brain is ensured by two processes: (1) autoregulation, a collection of intrinsic mechanisms that continuously adjust the microcirculation to maintain a constant flow of blood in the face of changes in perfusion pressure, and (2) neurovascular coupling, an ensemble of cerebral vasculature physiological processes that tightly match local blood flow to the needs of metabolically active regions of the brain. These distinctive responses are necessary for brain health and function but remain incompletely understood. Further, loss of microvascular control is associated with common age-related cerebrovascular pathologies, including stroke, cerebral small vessel diseases (cSVDs), and vascular cognitive impairment and dementia (VCID). The overarching goal of this proposal is to address this critical knowledge gap by providing a better understand of how the brain’s ever-changing milieu of physical, environmental, endocrine, paracrine, metabolic, and neurochemical stimuli are sensed by the cerebral microvasculature at the cellular level, and how these signals are processed to ensure homeostasis and adaptability. The primary mechanistic focus of our research is ion channels of the transient receptor potential (TRP) family—polymodal sensors of many types of physical and chemical stimuli present in all cells. Over the past 10 years, our research team has discovered that TRPM4 (TRP melastatin 4) and TRPML1 (TRP mucolipin 1) channels in cerebral vascular smooth muscle cells are important for the development of myogenic tone, a fundamental autoregulatory mechanism, and has demonstrated critical sensory roles for TRPA1 (TRP ankyrin 1) and TRPV3 (TRP vanilloid 3) channels on the endothelium of cerebral arteries and arterioles. Continuing with this theme and using advanced biomedical imaging approaches and next-generation genetic mouse models, we will weave together the central concepts established by our independent projects to develop a comprehensive overview of TRP channels as cellular sensors in the cerebral microvasculature. Examples of proposed studies include investigations that will define the nanoscale architecture of TRP channel signaling networks in health and disease using superresolution microscopy, elucidate how TRPML1 channels are endogenously regulated in smooth muscle cells to prevent vascular hypercontractility during myogenic vasoconstriction, and test the hypothesis that TRPA1 channels on brain capillary endothelial cells act as detectors of reactive oxygen species to promote neurovascular coupling. We will layer basic science investigations intended to elucidate fundamental regulatory mechanisms with research designed to understand how processes controlled by TRP channels go wrong and contribute to the transformation of healthy small vessels in the brain to a disease state during aging. To further this goal, we are developing and characterizing new genetic models of age-related cSVDs and VCID in collaboration with investigators at UCSF, and propose to use this unique resource to explore themes that include the involvement of TRPM4, TRPML1, and TRPA1 channels in cerebral vascular dysfunction during age-related cSVDs and VCID.

NIH Research Projects · FY 2024 · 2021-01

Abstract Emerging evidence suggests that childhood cancer survivors treated without central nervous system (CNS) directed therapies are at significant risk for neurocognitive impairment that is associated with decreased social attainment and quality of life. However, the underlying biological mechanisms of neurocognitive impairment in this population are poorly understood limiting our ability to prevent or alleviate these adverse outcomes. Long- term survivors of childhood cancer have a higher frequency of frailty and chronic health conditions than sibling controls suggesting cancer therapy may accelerate the physiological and biological aging process, which may lead to neurocognitive impairment. Studies in the general population indicate systemic inflammation and oxidative stress increase with age and are associated with increased morbidity, mortality, and cognitive decline. Inflammation and oxidative stress are also important regulators of telomere length and epigenetic changes which have been associated with neurocognitive impairment in aging non-cancer populations. These biomarkers have yet to be extensively examined in childhood cancer survivors treated without CNS directed therapies. The objective of this K99R00 is to identify aging-related biological predictors of neurocognitive impairment and subsequent decline in order to inform the design of future interventions using existing data and biospecimens from 300 HL survivors and 200 community controls in the St. Jude Lifetime cohort. Specifically, the K99 phase aims to examine cross-sectional associations between markers of inflammation, oxidative stress, immunosenescence, and cellular aging (telomere length and epigenetic age acceleration) with neurocognitive impairment in long-term Hodgkin lymphoma survivors (HL). The R00 phase will expand on these findings by first describing the trajectory of neurocognitive decline in long-term HL survivors and then by examining longitudinal associations between these biomarkers and subsequent neurocognitive decline. Further, these studies will provide data on the influence of modifiable risk factors (e.g. exercise, smoking, nutrition) on these biomarkers to inform future development of interventions to mitigate neurocognitive impairment in cancer survivors. Dr. Williams is an emerging translational cancer control epidemiologist focused on underlying pathophysiologic and biologic mechanisms of neurocognitive function in cancer survivors. The K99R00 allows Dr. Williams to develop expertise in 1) neurobiology and cancer biology and treatment specific to HL, 2) aging-related biomarkers and molecular epidemiology, 3) complex statistical methods and 4) clinical and behavioral intervention trials. Dr. Williams' mentoring team has extensive expertise in neurocognitive assessments, neuropathology, molecular epidemiology, childhood cancer, and statistical methods. St. Jude Children's Research Hospital is an international leader in cancer control and survivorship and provides a resource-rich training environment for Dr. Williams. The combined training and research plan will ensure Dr. Williams' transition to independence by providing the skills and preliminary data to successfully compete for future R01-level grants.

NIH Research Projects · FY 2025 · 2021-01

The importance for vision of the tiny fovea has been established by centuries of investigation as well as observations of the devastating consequences of its damage through injury or disease. Though evidence suggests that the fovea contains the full complement of the two dozen or so classes of ganglion cells found in peripheral retina, we know little about the physiology of these foveal cells. This gap in our understanding is the result of challenges in obtaining electrophysiological recordings from this delicate and topographically-complex structure. These challenges have been overcome by a method developed in our laboratory that allows simultaneous calcium imaging of the fluorescence responses of hundreds of foveal retinal ganglion cells in response to visual stimuli. Because this technique allows recording from single cells without damage in the living eye, we can study the same cells for months or even years, offering the opportunity to characterize the performance of each cell more thoroughly than has been possible with any prior method. Since the first submission of the proposal, we have made significant improvements in the expression of calcium indicator, GCamMP6s, in ganglion cells that increases the extent of expression to greater eccentricities, the fluorescence signal from each cell, as well as reducing the loss of ganglion cells over time. Moreover, we have designed a new ophthalmoscope with two independent adaptive optics systems, one dedicated to high resolution stimulus delivery and a second dedicated to high resolution ganglion cell recording. We have also developed an extensive battery of visual stimuli to characterize the responses of each cell in space, time, and color. This battery will include a white noise stimulus capable of identifying the locations and classes of single cone inputs to the receptive fields of foveal ganglion cells. To assist in cell classification, these physiological observations will be supplemented with ex vivo and in vivo histological analysis of the morphology of ganglion cell dendritic arbors. Armed with these improvements, we will undertake a comprehensive survey of both the physiology and anatomy of the foveal ganglion cell classes that mediate primate foveal vision.

NIH Research Projects · FY 2025 · 2020-12

This proposed Mentored Patient-Oriented Research Career Development Award will support Dr. Croft's advancement as a physician-researcher studying the effects of air pollution on the immune response to respiratory viral infection (RVI). Specifically, Dr. Croft's proposed study provides an opportunity for mentored development in advanced environmental epidemiology approaches, exposure assessment, immune system transcriptomics, and research team management. RVIs are a serious cause of morbidity and mortality in adults. PM2.5 and combustion related air pollution (black carbon: a marker of traffic pollution) have been linked to an increased rate of hospitalizations for RVI. However, the immune mechanisms by which air pollution may enhance susceptibility to severe RVIs (i.e. requiring hospitalization) in adults remains unclear. Dr. Croft's overarching hypothesis is that short-term exposure to ambient air pollution increases the risk of a severe documented respiratory viral infection in adults, by disrupting key gene pathways (e.g. NF-κB and IFN-γ) within the innate immune response. To test this hypothesis, Dr. Croft will enroll patients hospitalized with rigorously adjudicated, microbiologically proven RVI from an active R01 study on improved diagnostics for respiratory infection. In this population of patients with RVI, he will then lead analyses to explore whether short-term increases in multiple air pollutant and source-specific pollutant concentrations are associated with an increased rate of hospitalization for specific RVIs (e.g. influenza or respiratory syncytial virus). In the same population of patients, he will determine whether these pollutant exposures are associated with activation or suppression of key innate immune pathways. Specifically, using a population of patients with clinically adjudicated diagnoses of RVI, his study will use a case-crossover design to estimate the rate of RVI hospitalizations associated with acute increases in the concentration of particulate and gaseous pollutants in the prior 28 days (Aim 1). Patients with confirmed infection will undergo RNA transcriptional profiling of peripheral blood, allowing Dr. Croft to also examine the association between the same short-term increases in pollutant concentrations and gene expression within the innate immune system (Aim 2). In both aims, we will determine whether this response may be sex-specific, race or ethnicity specific or unique in patients with COPD or asthma. This study may immediately benefit the clinical counseling of patients with exposure to air pollution and may also help guide future air quality policies to minimize the risk to populations vulnerable to RVI. Finally, the study results will provide valuable data upon which to base a future prospective study of the effects of air pollution on respiratory infection and innate immunity. His mentors include experts in environmental epidemiology (David Rich, ScD), virology (Ann Falsey, MD) and transcriptomics (Thomas Mariani, PhD). Dr. Croft's highly productive mentor team, including multiple content specific mentors, will help support his development into an independent investigator pursuing clinical research on the respiratory health effects of inhaled toxins.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Among neurological disorders, the fastest growing is now Parkinson's disease (PD), surpassing Alzheimer's dis- ease. PD manifests as a heterogeneous clinical syndrome and this variability in the clinical phenotype highlights the need to tailor the type and/or the dosage of treatment to the specific and changing needs of individuals living with PD. The main goal of individualized, or precision, medicine is to use patient characteristics to determine an individualized treatment strategy (ITS) to promote wellness. Due to the complex nature of PD coupled with phenotypic heterogeneity, formulating successful individualized approaches to medical care is a complex prob- lem that may benefit from a more data-driven approach. One of the challenges in developing reliable ITSs is that the analyses require studies with fairly large sample sizes and longitudinal assessment of subjects over a relatively long period of time. The data set must also include various prescribing patterns to allow the analytic method to learn the effects of different treatment sequences (strategies). These important requirements preclude investigators from using data from a single clinical study to construct data-driven ITSs. Existing guidelines for symptomatic drug therapy for PD can best be described as "permissive". The relative lack of comparative evidence for different classes of drugs has created challenges in devising recommendations to follow any specific therapeutic strategy. We fill this important gap by proposing a two phase study. The first phase (R61) focuses on creating a harmonized and curated dataset by integrating data from six clinical trials and the PPMI observational study that, in aggregate, involved 4,705 patients followed from 23.5 to 96 months. To the best of our knowledge, such comprehensive data harmonization has not been done before in PD and it can provide an excellent source of information for future studies as well. In the second phase (R33), we will leverage the harmonized data set to develop high quality ITSs for PD with respect to several clinical outcomes including UPDRS score, quality of life, and Schwab and England (SE) ADL measured at 24 and 48 months of follow-up. Specifically, the goals of the R33 phase are to (Aim 1) compare commonly used sequences of drug classes for PD; (Aim 2) identify the best individualized treatment strategies to inform optimal sequences of drug classes for PD. In pursuit of these aims, we will propose robust, rigorous and computationally efficient statistical machine learning methods for constructing data-driven optimal ITSs for PD. The proposal expands the scope of existing methods in developing ITSs by relaxing certain unrealistic assumptions and through the use of flexible modeling techniques (e.g., machine learning methods) while maintaining valid statistical inference. These new methods will be integrated into easy-to-use, publicly available software in the R language (Aim 3). This will maximize the adoption of the proposed methodology by other investigators and allow researchers to analyze other PD datasets with a goal of constructing an ITS for PD. Furthermore, because the methods are not disease-specific, our methods and software will enable similar exploration for other diseases.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT The University of University of Rochester Medical Center Post-baccalaureate Research Education Program training program (URMC-PREP) is an innovative and progressive program designed to prepare, promote, and continually assist the successful transition and retention of promising scholars from underrepresented groups (URGs) into rigorous biomedical research doctoral degree programs. URMC-PREP intends to aggressively achieve and maintain a success rate higher than 85% for transition of scholars into competitive and successful biomedical PhD degree programs via three specific aims: (1) Implement research and professional educational pathways to prepare scholars for doctoral programs; (2) Optimize scholar outcomes by improving mentoring practices; and (3) Commit to rigorous and continuous evaluation of directorship, mentorship, and scholar outcomes. Built upon a proven effective educational and research training plan directed by a complementary co- leadership that is supported by 3 years' success rate of graduate school acceptance over 87%, this URMC- PREP is reinforced by several key innovative features including: (i) an opportunity to develop independent research proficiency, reliability, and integrity with one of 70 faculty mentors and laboratories spread over the 14 scientific disciplines; (ii) a team-mentorship network approach including peer- and faculty- mentors plus a Steering Committee member that will guide, supervise, advise, and facilitate the scholar's research academic education, application, and matriculation into graduate school, as well as build autonomy, self-assessment, Individual Development Plans, and confidence in inclusion; (iii) a continued supervision and assistance by this mentoring network after leaving the program by regular bi-annual in-house or online meeting; (iv) an enhanced Deaf and Hard of Hearing (DHH) representation through collaboration with partner institutions; (v) a defined training plan to improve mentoring practices through required workshops for bench mentors, orientation, and development workshops for faculty mentors; and (v) a rigorous, comprehensive, and continual multileveled evaluation plan guided by a Logic Model to assess scholars, mentors, and the program as a whole. The co-directorship and team-based mentoring within the framework of the 3 aims will insure that URMC- PREP scholars will achieve successful professional competency and the attitudinal belief of inclusion in their pursuit of graduate education and biomedical research leadership.

NIH Research Projects · FY 2025 · 2020-09

Abstract Our studies in mice show that inhaled exposures during development to concentrated ambient ultrafine particle (UFP) air pollution produces neuropathological and behavioral features common to 3 male-biased disorders, i.e., schizophrenia (SCZ), autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD), providing biological plausibility to a growing epidemiological literature linking these disorders to air pollution. In fact, the observed features in mice are intriguingly similar to those of SCZ. Our studies were not specifically designed to test these connections. Therefore, the proposed application seeks to determine the specific contribution of developmental UFP exposures to SCZ and the mechanisms initiating these adverse effects and their sex-dependency. Aim 1 tests the hypothesis that developmental UFP exposures will produce, in a UFP concentration-dependent manner, classic as yet unexamined characteristics of SCZ (alterations in cytokine profiles, reductions in parvalbumin interneurons and synaptic density and altered pre-pulse inhibition). SCZ has been linked to increased serum copper (Cu), and markedly elevated brain Cu levels in mice were found after developmental UFP exposure. Excess brain Cu can also produce neurotoxic features consistent with SCZ. Consequently, Aim 2 tests the hypothesis that elevated Cu contamination in ambient UFP is a specific driver of the observed SCZ features. Brain microglial colonization and activation is higher in male brain during the period of our UFP exposures. Given the critical role of microglial activation and inflammation in SCZ, ASD and ADHD, and the inflammatory and redox properties of AP and of Cu, Aim 3 tests the mechanistic role of microglial activation as the initiating mechanism of neurotoxicity in males by administration of the microglial activation inhibitor, minocycline. During adolescence, female brain exhibits greater microglial number/activation state. Thus, Aim 3 also tests the hypothesis that adolescent UFP exposure will enhance vulnerability of females. Findings from these studies assist in defining mechanisms for neuropsychiatric disorders and the basis of their differential vulnerability by sex and a potential need for additional regulation of air pollution for public health protection.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract: Neurodegenerative illnesses such as Parkinson’s (PD), Lewy Body Dementia (LBD) and Alzheimer’s disease (AD) affect nearly 15% of adults over age 65 and are leading causes of death in the US. While these illnesses are traditionally defined by their neurologic symptoms, more recent research describes the high impact of other medical symptoms on patients and the immense psychosocial consequences of these illnesses on both patients and families. Unfortunately, multiple lines of evidence demonstrate that many of the needs most important to patients and caregivers (e.g. pain management, advance care planning, end-of-life care) are poorly addressed under current care models. Palliative care is an approach to caring for individuals with life-limiting illness that addresses potential causes of suffering including physical symptoms, psychosocial issues and spiritual needs. To date there have been limited attempts to apply these principles to neurodegenerative illnesses despite evidence that patients’ and caregivers’ unmet needs may be amenable to this approach. Notably, the candidate has played a central role among a growing cadre of academic centers that now offer palliative care services for neurodegenerative illnesses and presents results from a randomized trial of academic-based outpatient palliative care that convincingly demonstrate this approach improves patient and caregiver outcomes over current standards of care. While efficacy trials are critical to forwarding this field, barriers to their dissemination include a limited workforce of palliative care specialists, lack of palliative education amongst neurologists, lack of team-based resources in community settings, and patient mobility/transportation issues. The long-term goal of the candidate is to improve outcomes and raise standards of care for older adults affected by neurodegenerative illnesses through novel, efficient and effective models of delivering palliative care. The Research Aims of this award will be met through: Study 1: Determine the effectiveness and feasibility of individual palliative care training for community neurologists and team-based virtual house calls for PD/LBD patients and caregivers (funded R01); Study 2: Develop a community-based model of palliative care for AD patients and caregivers (funded NIA AD Administrative Supplement; R01 trial to stem from results); Study 3: Determine the effectiveness and feasibility of a novel online community model to support community-based palliative care for PD/LBD (R01 Under Review); and Study 4: Integrate geriatric principles and care into our neuropalliative care model to improve outcomes for patients and caregivers affected by neurodegenerative illness (future P01 grant). As this is an emerging research direction for the candidate, the Career Development Objectives will provide formal training in academic leadership, geriatric palliative care, implementation science, caregiver support, telehealth and healthcare policy. This proposal is significant because it will create a foundation for palliative care dissemination efforts relevant to neurodegenerative illness and the broader field of geriatric palliative care.

NIH Research Projects · FY 2024 · 2020-09

Abstract More than 520,000 patients with End Stage Renal Disease (ESRD) underwent routine dialysis in the US in 2017. Conventional hemodialysis (HD) uses floor-standing instruments, which contributes to the dominance of center- based dialysis for the HD delivery space. Wearable HD systems could be employed to improve clinical outcomes and quality of life for patients with ESRD by enabling continuous dialysis. Wearable HD also enables frequent dialysis on a flexible treatment schedule. While there are potential benefits of more frequent dialysis, this comes at a cost of increased burden on lifestyle, risks of access malfunction, and health care costs. Also, episodic treatments provide insufficient time to remove large toxins (small diffusion coefficients) and protein-bound toxins. The barrier is the size of the current membranes which are bulky and not easily integrated into a wearable system and require large amounts of extracorporeal blood flow to achieve appropriate toxin clearances. Achieving significant improvements will require highly efficient membranes that enable prescribed toxin removal in small device formats. Our group has developed a variety of ultrathin (< 100 nm) nanoporous, silicon-based membranes and have established their value in improving the efficiency and precision of molecular separations. Because nanomembranes are 100 to1000 times thinner than conventional hemodialysis membranes, we hypothesize their ability to reduce the format for hemodialysis by orders of magnitude. We have recently developed a lift-off technique to produce sheets of nanoporous nitride (NPN) membrane material separated from the supporting silicon wafer. We propose to develop, using COMSOL Multiphysics modeling, a two-stage hemodialyzer incorporating two NPN membrane sheets in series. The fist NPN sheet membrane (100-nm pores) will filter out the cellular material generating plasma that will then be dialyzed by the second membrane (20-nm to 30-nm pores). The two-filter system will be tested on the benchtop for its ability to separate uremic toxins from whole blood and measured for hemocompatibility (hemolysis, complement activation etc.). The devices will also be bench tested for their ability to withstand the pressures exerted by the extracorporeal blood flow and designed ultrafiltration. The two-stage hemodialyzers will be tested in a small-animal model (male and female Sprague- Dawley rats). We expect, based on previous clearance studies with chip-based NPN membranes, that NPN sheet membranes can be used to construct a mechanically reliable hemodialysis device that achieves homeostatic levels of toxins through continuous operation. By enabling effective hemodialysis is small formats, our membrane technology will hasten the adoption of not only wearable HD therapies, but of portable and implantable HD therapies. This effort supports the recently created “Advancing American Kidney Health initiative” to transform how ESRD therapy is delivered.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract MicroRNAs (miRNAs) are a class of small (18-24 nucleotide) RNAs that are essential regulators of gene expression, which act within the RNA-induced silencing complex (RISC) to bind mRNAs and suppress translation. Alterations in miRNA expression have been shown to disrupt entire cellular pathways, substantially contributing to a variety of human diseases. Despite nearly 25 years of research, miRNAs remain dicult to measure due to their short length, relatively small number, sequence similarity, and diculty to isolate from other small RNA fragments. While qPCR- and microarray-based miRNA assays are still widely used, the majority of recent studies use small RNA-seq (sRNA-seq) because it allows for the quanti cation of isomiRs (miRNA isoforms) and the possibility of identifying novel miRNAs. The processing of reads generated from sRNA-seq data globally distinguish between miRNA reads and those from other small RNAs, but do not necessarily capture the full spectrum of miRNA variation. Subsequent statistical analyses of processed sRNA-seq data are still performed using methods developed for mRNA-seq data despite the fact that sRNA-seq data violate several of the assumptions of these methods. Speci cally, methods for mRNA-seq data assume approximate independence between feature counts; however, the small total number of miRNAs and presence of a small number of very highly expressed miRNAs result in a lack of independence between miRNA counts. Additionally, normalization methods for mRNA-seq data assume either the overall level of transcription is constant across samples or an equal number of features are over- and under-expressed when comparing any two samples, neither of which hold for sRNA-seq data. The development of statistical methods that address the challenges of sRNA-seq data represents a critical need for miRNA research. Our long-term goal is to advance miRNA research by developing statistical methods that are tailored to the speci c complexities of miRNA expression data. The overall objective of this application is to improve the analysis of sRNA-seq data by developing statistical methods that account for challenges speci c to sRNA-seq data and outperform methods designed for mRNA-seq data. This addresses an urgent need for statistical methods to appropriately analyze sRNA-seq data, which are now routinely generated by large consortia such as TCGA and FANTOM. The rationale that underlies the proposed research is that methods that explicitly address the challenges inherent in measuring miRNAs are necessary to fully elucidate the role miRNAs play in many human disease processes.

NIH Research Projects · FY 2025 · 2020-09

Abstract Repair and reconstruction of bone loss due to tumor resection, trauma and infection remains a significant clinical challenge. Worldwide, autografts or allografts are used in approximately 3 million orthopaedic procedures annually, of which 6% are craniomaxillofacial in nature. Bone tissue engineering has been hailed as the ultimate solution for replacing bone autograft in repair of bone defects. However, the long-term success of bone tissue engineering is impeded by inadequate vascularization of the engineered construct. The current lack of progress in vascularization of tissue engineered scaffold is attributed to our incomplete understanding of angiogenesis and vascular beds in bone repair and regeneration. A functional blood vessel network consists of arteries, veins and a capillary interface that connects arterial and venous microvessels for proper vascular perfusion. While the specification of arterial and venous endothelium has been well studied during early embryonic development, the postnatal regulation of arterial and venous expansion and specification at capillary level during repair and regeneration is poorly understood. A series of recent studies have suggested that hypoxia affects the endothelial cell (EC) specification at the osteogenic and angiogenic interface in development and aging. Genetic manipulation of the hypoxia inducible factor 1 (HIF-1) pathway markedly affects the formation of specific subsets of capillary vessels, termed Type H (CD31highEmcnhigh) vessels that couple to OSX+ osteoblasts at the long bone metaphysis. To gain a better understanding of the critical role of hypoxia at the osteogenic and angiogenic interface in repair and regeneration, we established a series of novel imaging approaches that permit high resolution, quantitative, and functional analyses of capillary vessels that couple to Col (I) 2.3 GFP+ osteoblasts at a cranial bone defect site. Utilizing these novel imaging approaches in a layer-by-layer enabled, nanofiber-mediated cranial defect repair model, we demonstrate that osteogenesis- dependent angiogenesis consists of morphologically and functionally distinct CD31+Emcn+ and CD31+Emcn- vessels. Examination of blood vessel type distribution and bone regeneration demonstrates differential angiogenic responses and contrasting distributions of CD31+Emcn+ and CD31+Emcn- vessels associated with Col I (2.3) GFP+ osteoblasts, new bone and non-bone forming tissue, suggesting that EC specification at the capillary level is a key component of osteogenesis-dependent angiogenesis in bone repair and regeneration. Based on these findings, we propose to examine the effects of hypoxia on EC specification and the impact of dysregulation of EC specification on bone formation during cranial defect repair and regeneration. Three complementary Aims will combine imaging, genetic and engineering approaches to defining the osteogenesis- dependent EC specification and the role of hypoxia in repair and regeneration. The success of our study will provide novel insights into mechanisms of osteogenesis and angiogenesis in repair, potentially offering novel translational targets for bone regeneration.

NIH Research Projects · FY 2024 · 2020-09

Abstract Long-term cognitive impairment affects more than 70% of sepsis survivors, but the underlying mechanisms remain unknown. Though widely hypothesized, evidence of blood-brain barrier (BBB) dysfunction in septic patients is limited by practical barriers to diagnostic studies in critically ill subjects. While BBB breakdown and cognitive impairment are seen in animal models of sepsis, the complexity of sepsis in vivo and differences between animal and human responses means that animal models cannot unambiguously identify the circulating factors that cause brain injury in human sepsis. Therefore, we propose to develop the µSiM-hNVU as an `on-chip' platform featuring a human iPSC-derived neurovascular unit (NVU; brain microvascular endothelial cells, pericytes and astrocytes). The `blood side' will allow the flow-based introduction of blood- borne cells and molecules with known or hypothesized roles in sepsis related brain injury, and the `brain side' will feature iPSC-derived microglial cells serving as a reporter of the brain inflammatory status. The human NVU will be built on a device platform – the µSiM – featuring ultrathin silicon nanomembranes that provide for unhindered solute exchange between `blood' and `brain' compartments and glass-like optical quality for live cell imaging and high-resolution microscopy. In the R61 phase, the device platform will be advanced for ease-of- use including `plug-and-play' modules for flow and barrier measurements (TEER, diffusion), and compatibility with a small-volume, digital-ELISA assay for secreted proteins. The µSiM-hNVU will be validated with functional assays of blood-brain barrier (BBB) function, protein expression studies, and transcriptional analysis. We will also build a iPSC NVU in which each cellular component of the NVU carries the ApoE4 allele. The expression of the ApoE4 lipoprotein drives BBB dysfunction by a known pathway and increases the risk of cognitive impairment in humans and animals experiencing brain inflammation. We will use the ApoE4-NVU as a `diseased BBB on a chip” which we hypothesize will show enhanced vulnerabilities to candidate mechanisms of brain injury identified by our team and others. Specifically, we will test the hypotheses that 1) pre-activated monocytes invade the brain and drive microglial activation; 2) the damage associated molecular pattern (DAMP) complex S100A8/A9 drive BBB breakdown to promote leukocyte infiltration and neuroinflammation; and 3) circulating factors that degrade endothelial glycocaylx (e.g., heparinase) or contribute to systemic inflammation (cell-free hemoglobin) promote CNS infiltration of leukocytes and subsequent neuroinflammation.