In Vivo Genetic Screen Identifies Chromatin Remodeling Gene SET As a Collaborating Oncogenic Driver In Initiation and Maintenance of Acute Lymphoblastic Leukemia

Innovative treatment options have greatly improved outcomes for acute lymphoblastic leukemia (ALL) patients, yet relapse and refractory disease remain a significant clinical challenge. The complex genomic heterogeneity of molecular subtypes contributes to poor prognosis, high morbidity, and recurrent drug resistance in relapse and refractory ALL patients. Identifying novel genetic interactions and molecular pathways capable of initiating ALL will help to develop new diagnostic criteria and to identify actionable drug targets. Here, we developed a novel in vivo screening method using transgenic zebrafish to unbiasedly identify collaborating oncogenes that when co-expressed lead to T- and B-ALL. Our high-throughput approach allows for rapid screening of collaborating oncogenic drivers across hundreds of individual animals in a cost-effective manner. Specifically, we screened a transgene pool of 68 oncogenes identified from relapsed patients for leukemia induction in zebrafish. Using our approach, complex gene networks involved in cancer initiation in human patients can be rapidly interrogated and unraveled with in vivo resolution. We found that SET, a marker of a rare T-ALL subtype, collaborates with NOTCH1 and IL7R to initiate T-ALL in vivo, validating their synergistic effects in leukemia initiation and suggesting a critical role for epigenetic dysregulation during oncogenic transformation. We have generated a robust transcriptome atlas of zebrafish lymphoid malignancies, representing at least 7 novel models of leukemia and lymphoma molecular subtypes. In addition, this screening approach can discover additional genetic factors capable of initiating pediatric leukemias, creating a molecular “roadmap” to leukemogenesis, and identify vulnerable pathways for clinical therapies.

Investigating SLC25A46 in Breast Cancer Patients with Admixed Ancestry

Triple-negative breast cancer (TNBC) is an aggressive, highly malignant breast cancer (BC) subtype characterized by poor prognosis and chemoresistance. TNBC disproportionately affects African American and Latina women, where a better understanding of treatment outcomes requires a deep analysis of the ancestral-related underpinnings. Metabolic reprogramming within TNBC cells significantly affects treatment response, underscoring the importance of mitochondrial function. In this study, we focused on the mitochondrial protein, SLC25A46, which is essential for maintaining mitochondrial dynamics, cristae integrity, and respiratory functions. Although SLC25A46 is poorly characterized, it may have new importance in TNBC. Accounting for the role of ancestry in BC prognosis and progression, we hypothesize that ancestry significantly influences BC etiology. To test this hypothesis, we employed computational genetic approaches, utilizing PrediXcan, a gene-based statistical algorithm, to calculate genetically predicted expression levels of SLC25A46 in large cohorts (70,349 patients of European descent and 14,462 patients of African descent) from Vanderbilt's BioVU database. We observed that patients with high predicted expression were more likely to receive diagnosis of ‘abnormal findings in mammograms’ (P=2.76E-06, OR=2.26) or ‘lump or mass in breast’ (P=8.9E-06, OR=2.61) in the European descent group, which is well powered; however, we are not powered to replicate this finding in the African descent group. Therefore, we investigated SLC25A46 expression in an international RNAseq cohort enriched with women of African ancestry with TNBC (n=26). Interestingly, SLC25A46 shows significantly higher expression among self-reported AA than Ghanaian or Ethiopian women with TNBC (P=0.003), where these data are explicitly observed in patients with admixed ancestry. To complement these data further, we utilize various imaging modalities to identify unique mitochondrial structures within the tumor and spatial biological techniques that show novel complex phenotypes. Our work aims to shed light on the critical role of SLC25A46 in the context of ancestral differences and treatment outcomes in BC.

MAPK Alteration is a Targetable Vulnerability in Bladder Cancer

Bladder cancer (BC) is the fourth most common cancer in males, accounting for about 80,000 new cases annually in the US. Approximately 25% of BC patients present with either muscle-invasive bladder cancer (MIBC) or later develop metastatic BC. The five-year survival rate for metastatic BC is only 15%, resulting in about 17,000 deaths annually. Recently, multiple immunotherapeutic agents and Antibody-Drug Conjugates have been approved, but only a small subset of patients respond to these treatments, highlighting the need for novel therapeutic strategies. Large-scale genomic studies of MIBC have identified numerous recurrent genomic alterations, including mutations, copy number changes, and chromosomal translocations. In our studies, we found that approximately 20% of MIBC patients harbor alterations in the mitogen-activated protein kinase (MAPK) pathway and its upstream activators. MAPK is a three-tiered signaling cascade consisting of the proteins RAF, MEK, and ERK, which control critical cellular processes such as differentiation, proliferation, and survival. Using our institutional cohorts of bladder cancer specimens, we confirmed the presence and frequency of these alterations, particularly RAF1 amplification. To investigate whether MAPK alteration is a driver in BC, we used BC cell lines with RAF1 amplification and performed pharmacological inhibition of RAF and MEK, along with gene depletion approaches. Our preclinical studies in mouse models of BC using patient-derived xenografts demonstrated the feasibility of targeting MAPK alterations. In summary, MAPK pathway alterations represent a novel therapeutic target in BC, potentially improving outcomes for patients with emerging RAF inhibitors.

An In-Vitro Investigation to Understand the Phototoxic Properties of Porphyrin Derivates on Tetrahymena Thermophila and Basal Cell Carcinomas

Porphyrins are vital macrocyclic molecules that exist naturally and are significant to the metabolic processes of living organisms. Some of the best-known examples include iron-containing porphyrins in heme and magnesium-containing porphyrins in chlorophyll. These compounds are substantial for life on Earth. Therefore, gaining a deep understanding of porphyrins and their properties is essential for understanding many biological processes. This research investigates the synthesis, photochemical characterization, and applications of porphyrin derivatives, focusing on their potential in photodynamic therapy. Initially, the synthesis of non-water-soluble Dimethylamino-phenylporphyrin (DMAPP) and its transformation into water-soluble derivatives TAPPI4 (Tetra-p-trimethyl-ammonium-phenyl-porphyrin-tetraiodide) and its metallo derivatives ZnTAPPI4 (Tetra-(p-trimethyl-ammonium-phenyl) zinc (II)-porphyrin- tetraiodide) and CuTAPPI4 (Tetra-(p-trimethyl-ammonium-phenyl) copper (II)-porphyrin- tetraiodide) were produced. Comprehensive photochemical analysis revealed unique absorption and emission abilities, aggregation behaviors, and fluorescence lifetimes suggesting different photostability and reactivity under light exposure with lifetimes measured at 3.26 x 10-5 seconds for TAPPI4, 1.49 x 10-11 seconds for ZnTAPPI4, and 3.66 x 10-9 seconds for CuTAPPI4. These compounds were then tested in-vitro on tetrahymena thermophila, a unicellular eukaryote, and basal cell carcinoma cells (BCC) from the salivary gland and the lung with the following concentrations: 100 ppm, 50 ppm, 30 ppm, 15 ppm, three ppm, and 0 ppm. Results indicate no dark toxicity except for 100 ppm CuTAPPI4, and studies showed 90% tetrahymena thermophila cell death within 12h of continuous red-light radiation in 50 ppm TAPPI4 solutions. Furthermore, studies demonstrated 75% effectiveness on benign salivary gland BCC cells and 94% on malignant lung BCC cells at a concentration of 100ppm TAPPI4 within 12h of continuous red-light radiation. Finally, Photochemical and photophysical studies included fluorescence quenching of TAPPI4 with the following quinones: 2,6-dichloro-1,4-benzoquinone, Methyl-p-benzoquinone and Benzoquinone. The quenching constant was established for each quinone and is at kq = 1.36 x 107 L mol−1 s−1 for Benzoquinone, at kq 3.37 x 106 L mol−1 s−1, 2,6-dichloro-1,4-benzoquinone and at kq = 7.41 x 105 L mol−1 s−1 for Methyl-p-benzoquinone. This research involves several analytical methods, including UV-Vis absorption spectroscopy, emission spectroscopy, and GC-MS analysis to characterize reaction products and intermediates.

From Organoids to Autopsies: Unraveling the Mysteries of Frontotemporal Dementia (FTD) and Progressive Supranuclear Palsy (PSP)

Microtubule-associated protein tau (MAPT) dysfunction is implicated in over 20 neurodegenerative diseases. In healthy neurons, tau is axon-enriched. However, in tauopathies such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), and progressive supranuclear palsy (PSP), tau abnormally accumulates in the somatodendritic compartment, leading to neuronal death. My published work in a mouse cell culture AD model showed that tau accumulation, caused by extracellular tau oligomers, decreases the axon initial segment (AIS) length and intensity while leaving the somatodendritic compartment intact. This selective damage has significant consequences for neuronal function since the AIS is crucial for action potential initiation and neuronal polarity maintenance. My postdoctoral research aims to elucidate the genetic and neuropathological mechanisms underlying tauopathy in FTD and PSP. Specifically, I investigate how FTD-associated MAPT mutations, such as V337M, influence neuronal structure and activity. Using three isogenic iPSC lines (wild type, heterozygous, and homozygous), I employ biochemical, confocal, calcium imaging, and mass spectrometry techniques to uncover early neurodegeneration contributors. Building on my AD study, I also examine whether neurofibrillary tangle pathology correlates with AIS damage in PSP. Using PSP hippocampus tissue from the Michigan Brain Bank, I use biochemical, confocal, and machine-learning techniques to assess phosphorylated tau and AIS. My goal is for my independent lab to investigate AD-associated genes using hiPSC-derived cortical organoids and post-mortem human brain tissue, focusing on Black Americans. Overall, I aim to bridge basic and clinical neuroscience, develop early neurodegeneration interventions, and promote equity in academic research.

Continuous Production of Cu-based Catalysts Precursor Materials for Methanol Synthesis

Copper-based mixed metal oxide catalysts are used ubiquitously in the chemical industry for the conversion of syngas (CO & H2) into methanol. The precursor materials such as carbonate hydroxides and oxalates are generally prepared in semi-batch reactors. Some of the challenges faced with preparing the catalyst precursor materials in semi-batch reactors include inhomogeneous heating & mixing, long residence time, and the generation of nonuniform and agglomerated particles. In order to circumvent these challenges, I utilized a patented continuous manufacturing technique to prepare the catalyst precursor materials which can subsequently be employed for the efficient conversion of syngas to methanol. For my presentation, I will share on utilizing the three-phase slug-flow reactor which consists of liquid-liquid-gas slugs that is used for the precipitation of metal ions into metal oxalates. This technique provides a more continuous and reproducible production of uniform nano and microparticles, facilitate better control of the particle size, and shorter residence time for product generation. My presentation will highlight the preparation of copper zinc aluminum (CZA) oxalate precursors and its subsequent transformation into Cu/ZnO/Al2O3 catalysts.

Deletion of SGK1 in Smooth Muscle Cells Induces Sex-specific Differences in Obesitymediated Metabolic Dysfunction and Adipose Tissue Remodeling

Background/Significance: Obesity, characterized by an excess of adipose tissue, is an important risk factor for the development of metabolic diseases such as type 2 diabetes. In obesity, poor metabolic health is partly due to glucose intolerance, a condition in which whole-body glucose regulation is impaired. Adipose tissue is an endocrine organ central for optimal metabolic health and its expansion by excessive fat intake induces metabolic dysfunction. Obesity is caused in part by expansion of adipose tissue due to excess fat intake. The molecular mechanisms that promote metabolic dysfunction in obesity are not fully understood. Adipose tissue function is dependent on the presence of new vascular networks capable of supplying nutrients and oxygen during adipose tissue expansion. Thus, pathological vascular remodeling may promote adipose tissue expansion and dysfunction in obesity. In this regard, our previous studies have shown that serum and glucocorticoid-inducible kinase 1 (SGK1) in vascular smooth muscle cells (vSMCs) mediates pathological vascular remodeling in obesity. SGK1 is a serine/threonine kinase that becomes elevated in the vasculature during obesity and is implicated in the development of metabolic syndrome. Therefore, we hypothesize that smooth muscle cell derived-SGK1 promotes pathological adipose tissue remodeling during obesity by perturbing adipose tissue vascularization. Methods: To test this hypothesis, these studies utilized our novel SMC-specific SGK1 knockout (smSGK1KO) mouse model to study the influence of SMC-derived SGK1 on adipose tissue remodeling and overall metabolic health in vivo. Male and female 8-week-old smSGK1WT and smSGK1KO mice were subjected to either a 10% low-fat diet (LF) or a high-fat diet (HF) for 8 weeks with weekly body weight measurements. Upon completion of respective diets, mice were subjected to glucose tolerance tests (GTT) to assess glucose tolerance and metabolic health. Adipose tissues from the subcutaneous (inguinal) and intra-abdominal (epididymal/gonadal) white adipose tissue (WAT) depots were harvested to assess overall adiposity and vascularization. Results: Our data show sexual dimorphism exists in the way smSGK1KO mice respond to a chronic high-fat diet. When fed a HF diet, female smSGK1KO mice gained approximately 60% more weight than their female smSGK1WT counterparts. In contrast, weight gain was about 50% lower in male smSGK1KO compared to male smSGK1WT mice on the same HF diet. Despite the differences in weight gain, inguinal and epididymal/gonadal WAT were reduced in male and female smSGK1KO mice fed a HF diet. Surprisingly, GTT was similar between female smSGK1KO and smSGK1WT mice, despite the increased weight gain. Moreover, male smSGK1KO mice appeared to have enhanced vascularization in WAT depots as evidenced by increased vascular branching in fat pads. Conclusions and Implications: These studies suggest that SGK1 in smooth muscle cells contributes to adipose tissue development and possibly remodeling under obesogenic conditions. Additionally, these studies suggests crosstalk between the vasculature and adipose tissue modulates obesity and overall metabolic health.

Addressing Racial Disparities in Pulse Oximetry Using Polarized Light

Measuring blood oxygen saturation with pulse oximeters shows biases against highly pigmented skin tones. Retrospective and prospective studies reveal that these devices often fail to detect hypoxemia, a medical condition where there is a low level of oxygen in the blood, in Black and Brown patients compared to White patients. This discrepancy stems from inadequate testing standards, limited access to testing data, and misleading manufacturer claims. Additionally, factors like low perfusion, severe anemia and variations in person-to-person skin anatomy, which affect performance, are not fully addressed in current regulatory standards. This talk will discuss these challenges and present ongoing progress on our innovative solution that uses a polarized light photoplethysmography sensing to mitigate biases related to skin pigmentation and motion artifacts, ensuring more accurate and equitable pulse oximeter wearables for diverse populations.

Investigating The Association Between Physical Activity and Quality of Life Among Breast Cancer Survivors in The Black Women’s Health Study

Purpose: Black breast cancer survivors (BCS) experience suboptimal quality of life (QoL) compared to White BCS. Physical activity (PA) has been shown to improve QoL in cancer survivors. However, there is limited evidence demonstrating associations between PA and multiple QoL domains among Black BCS. This study examined the association between PA intensity (e.g., moderate activity) and QoL (e.g., physical health) among BCS in the Black Women’s Health Study (BWHS). Methods: A total of 1,085 BWHS participants were diagnosed with invasive breast cancer after study enrollment and completed the 2019 BWHS follow-up questionnaire, which assessed multiple QoL domains. Adjusted multinomial logistic regression models were fit to estimate the odds ratios and 95% confidence intervals for the associations with each explanatory variable. Multiple imputation was used to address missing data. Results: The average participant age was 67, and the average body mass index was 29 kg/m² at the time of QoL assessment. There were significant and positive associations between PA intensity and multiple domains of QoL (p<.05). BCS who engaged in higher durations of moderate and vigorous PA (e.g., ≥60 minutes/week) had increased odds of optimal QoL than those who engaged in lower durations of moderate and vigorous PA (e.g., <60 minutes/week). Conclusions: Black BCS who engage in higher durations of moderate and vigorous PA tend to have better QoL. These findings may help to build upon the relevance of PA in the context of supportive care for Black BCS. Future longitudinal studies are needed to establish clear temporal associations between PA and QoL in this group.

mRNA Translation Control in Pancreatic Beta Cells

Pancreatic beta-cells are specialized for high capacity mRNA translation that is tightly regulated by the nutrient environment. Acute glucose exposure robustly increases translation of insulin and other secretory granule proteins, but chronic high glucose decreases insulin translation and secretion. I used ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on beta-cell mRNA translation. Sustained high glucose downregulated translation of insulin and proteins, such as SCGN, IDH2, VPS41, SLC2A2, IGF2, SLC30A8 and PFKFB3, which are involved in insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with chronic high glucose, and in an in vivo partial pancreatectomy model of chronic hyperglycemia. Analysis of translating ribosomes and RNA surveillance pathways showed that sustained high glucose remodels ribosomes and suppresses nonsense mediated RNA decay (NMD). Altered ribosome composition and activity of RNA decay pathways could result in changes in the abundance and fidelity of the proteins produced. I hypothesize that hyperglycemia-induced remodeling of ribosomes and suppression of NMD alter expression of key beta-cell genes and increase neo-antigen production. My studies are focused on investigating how ribosome remodeling on production of beta cell proteins and the impact of reduced RNA surveillance and altered translation on neoantigen production and autoimmunity in T1D. These studies could uncover novel therapeutic targets for prevention of progressivebeta-cell failure in T1D and for optimizing functionality of ex vivo generated beta-cells for cell replacement therapy.

Targeting ROS-TAp63 Signaling Restores Skin Integrity in Fanconi Anemia

Individuals with Fanconi anemia (FA), an inherited disorder of DNA interstrand crosslink (ICL) repair, exhibit epithelial-to-mesenchymal transition phenotypes and reduced intercellular desmosome junctions within cutaneous and mucosal stratified squamous epithelia (SE). These cellular alterations align with accelerated induced blistering, indicative of compromised SE fragility which may promote early-onset, aggressive squamous cancers that are a hallmark of FA. While the FA pathway maintains the structure and function of SE, underlying cellular and molecular mechanisms have remained elusive. We demonstrate that loss of the FA pathway in human keratinocytes, engineered organotypic epithelial rafts, and FA patient skin greatly stimulates reactive oxygen species (ROS) accumulation and production of the derivative aldehyde and DNA crosslinker 4-hydroxy-nonenal (4-HNE). ROS was both necessary and sufficient to block expression of the tumor suppressor TAp63 and downstream desmosomal complex components. The antioxidant Quercetin blocked 4-HNE, and rescued TAp63 and desmosomal protein accumulation. Clinically, in the context of a phase II cancer prevention trial, long-term Quercetin supplementation ameliorated skin fragility in a majority of FA patients exhibiting baseline cutaneous vulnerability. Collectively, these findings implicate excessive ROS, induced by germline or somatic FA gene mutations, as a central cause of SE fragility, and support Quercetin supplementation as a safe, sustainable therapeutic intervention. Given that 4-HNE is known to actively generate ICLs, we propose a model wherein aberrant ROS/TAp63 signaling within the ICL repair deficient SE establishes a positive feedback loop capable of sustaining and amplifying genomic instability and epithelial fragility.

Broadening the Scope: Cross-tissue Gene Expression of Polycystic Kidney Disease Associated Genes

Polycystic kidney diseases, autosomal dominant (ADPKD) & autosomal recessive (ARPKD), cause enlargement and dysfunction of patient kidneys from fluid-filled cysts. Although other organ systems are also affected in PKD, most investigations primarily focus on the kidney or its contribution to extrarenal dysfunctions. While the field has identified PKD associated genes, there is still ambiguity around which tissues these genes are expressed in, relative levels of expression across tissues, and their role(s) in organ development and function. Leveraging single-cell RNA-sequencing data from multiple public datasets empowers us to investigate molecular signatures using more cells than previously possible from a single experiment. We can apply scalable, probabilistic tools that accurately model data, accounting for technical and biological variation. Here, I use the CZ CellxGene Discover Census to access multiple datasets across the available adult (minimum age 20) human tissues. I integrated these datasets within their respective tissues and visualized the distribution of gene expression for ADPKD (PKD1, PKD2) and ARPKD (PKHD1, DZIP1L) associated genes. Gene expression profiles for PKD genes are the first steps toward modeling their regulatory interactions (network inference) or their involvement in cellular interactions (cell-cell communication inference). Combined, these molecular signatures can provide insight on gene functions within and across cell types and tissues.

Uncovering the Roles of the Immune System in Spinal Column Tissue Repair and Homeostasis

The spinal column is an invaluable structure of the musculoskeletal system and the defining characteristic of vertebrate animals. An essential component of the spinal column is the intervertebral disc (IVD), a connective tissue that provides the shock absorption and weight distribution properties of the spine. IVDs are prone to cumulative damage as we age due to a limited regenerative capacity, which leads to debilitating back pain. Successful healing of connective tissue injuries relies on immune cells that rapidly infiltrate damaged tissues and initiate repair. However, the identity, temporal regulation, and effect of immune cells during IVD repair remains understudied. The objective of this study is to prevent IVD degeneration by defining the role of immune cells after injury to improve tissue repair. Cd3+ T lymphocytes have been shown to be critical mediators of repair, but their role in IVD healing is unknown. To determine the role of immune cells after IVD damage, we induced the robust infiltration of Cd45+ immune cells by causing a severe injury to IVD tissue. Our findings show a sex divergent response of anti-inflammatory Cd3+ γδ T cells during the injury response and a dysregulation in IVD tissue mechanics in mice lacking lymphocytes. We hypothesize that Cd3+ γδ T cells are essential for IVD tissue repair. By utilizing methodologies such as qPCR, bulk RNA sequencing, flow cytometry, and drug therapy, this study offers a targeted approach to modulate IVD repair by elucidating the types and temporal regulation of key immune cells important during IVD repair.overing the Roles of the Immune System in Spinal Column Tissue Repair and Homeostasis

ArcHIVed Gag-like Communication in the Brain

The immediate early gene Arc is predominantly expressed in excitatory glutamatergic neurons and is critical for long-term synaptic plasticity and memory. Arc is a repurposed retrotransposon that can form capsid-like particles, which encapsulate and transfer RNA between neurons via extracellular vesicles (EVs). Here, we characterize Arc particles, the pathway, and the cellular factors essential for their release, as well as their RNA content. We find that the release of Arc particles varies greatly between cell lines, with neuronal cell lines being the most efficient. We report that Arc has retained conserved motifs found in retroviral Gag proteins that are important for assembly and release, such as an MHR and a late domain. In line with these observations, intracellular Arc, like HIV-1 Gag, exists in various higher-order assemblies. Interestingly, cellular restriction factors that inhibit HIV Gag release do not affect Arc release. However, siRNA-mediated knockdown of Alix (the ESCRT adaptor protein that interacts with viral Gags) prevents the release of Arc particles. RNA sequencing analysis of Arc-enriched EVs and RNA immunoprecipitation of Arc from these fractions (but not from control fractions) reveals an overrepresentation of long noncoding and LINC RNAs. Notably, several of these RNAs are associated with neurodegenerative and neuroatypical disorders, such as ASD and cognitive decline. These results highlight the importance of examining the functional contributions of these viral fossils in cellular functions.

Endothelial to Mesenchymal Transition in Atherosclerosis in Response to Chronic Air Pollution Exposure

Background: Air pollution is the world’s leading environmental risk factor for atherosclerotic cardiovascular disease (ASCVD). Pro-atherogenic pathways include inflammation, innate and adaptive immune mechanisms and broad transcriptional reprogramming that results in transition of cells to primordial states. Endothelial to mesenchymal transition (EndMT) whereby an endothelial cell transforms to a mesenchymal-like cellular phenotype (e.g. myofibroblast) has been implicated in the pathogenesis of atherosclerosis. Methods: Male ApoE-/- mice were inhalationally exposed to concentrated particulate matter <2.5μ component (PM2.5)or filtered air (FA, controls) using a Versatile Aerosol Concentration Enrichment System (VACES, 5–6 h/day, 5 d/week for 36 weeks, n = 7/group. PM2.5 concentrations were 70-80 μg/m3 or ~10X ambient levels. Following euthanasia, vascular function was assessed followed by single-cell RNA-sequencing of aortic cells. Unsupervised clustering, UMAP projections, Gene-ontology and KEGG pathway analysis followed by pseudotime distribution assessed along specific lineages and exposure groups and statistically tested using GLM and GAM Models. Results. Endothelium-dependent relaxation was impaired in the aortas of PM-exposed mice with exaggeration of vasoconstriction. Aortic scRNA-seq data revealed 7 major cell types and 1 unknown cell type. Cell lineage trajectory inference depicted 4 lineages from endothelial cells. Comparison of the 4 pseudotime lineages revealed almost perfect interposition of endothelial to vascular smooth muscle cell transition (EndoMT) with the transition noted in response to PM2.5 versus FA. Differential gene expression profiling in response to PM2.5 revealed augmented GO terms in EC upon PM2.5 exposure include cell differentiation, cell migration, and regulation of cell population proliferation Conclusion: Chronic exposure to PM2.5 may result in EndoMT which may help explain endothelial dysfunction and acceleration of ASCVD. Analysis of cell-specific pathways including transcriptional networks may provide a renewed understanding of the mechanisms underlying environmental exposures and the progression of atherosclerosis.

Food is a Weak Zeitgeber for Food-Entrainable Oscillator

Animals use the food-entrainable oscillator (FEO) to anticipate the time of food availability. Recently, we used the open-source operant feeding device (FED3) and recorded food-seeking nose-poking behavior. Unexpectedly, we found that food anticipation is encoded to the light-entrainable circadian oscillator located outside of the suprachiasmatic nucleus (Ehichioya et al., 2023, Current Biology, Vol 33, p5233-5239). In our current study, we asked the question: can the FEO actually entrain to the feeding cycle without a light cue? To test the food entrainability of the FEO, we used Period 1/2/3 triple knockout (KO) mice, which lack canonical circadian oscillators. The mice were placed in constant darkness and exposed to a timed-restricted feeding paradigm. As our lab had previously shown, Period 1/2/3 KO mice cannot entrain to a 24 h feeding cycle due to their short endogenous period (~21 h), so we used T21 and T20 feeding cycles because the cycles closely matched their estimated autonomous period. Our results show that under the T21 feeding cycle, all 6 mice showed consolidated food anticipatory poking. Still, only 2 of these demonstrated consolidated food anticipatory poking that was phase locked to feeding time. Upon changing from T21 to T20 feeding cycle, 5 mice showed consolidated food anticipatory poking aligned with the scheduled feeding time. When mice were subjected to a 6 h delay in the feeding cycle under the T20 cycle, 2 mice showed consolidated anticipatory poking that shifted parallel with the delayed feeding time. The results indicate that food can act as a weak zeitgeber for the FEO in the absence of light-dark cycle.

Bridging the Role of Mitochondria Function in Developmental and Age-related Neurological Diseases

Disorders affecting neurological function vary on a large scale. These conditions include neurodevelopmental disorders such as autism, and neurodegenerative diseases such as Alzheimer’s. According to the World Health Organization, over 1 in 3 people are affected by neurological conditions, and it is now emerging as the leading cause of illness and disability world-wide. Mitochondria dysfunction is a central player in many neurological diseases. During my pre-doctoral work, I studied aspects of mitochondrial function as implicated in Tusc2 activity. Tusc2, a mitochondria Ca2+ sensor, aids in regulation of mitochondrial/cytosolic Ca2+ levels. Previous work found that deficiency of Tusc2 leads to increased oxidative stress, altered mitochondrial membrane potential and energy production in immune and cancer cells. However, the role of Tusc2 in neurobehavioral activity and its connection to cognitive and neuroimmune function has not been studied. Here, I hypothesize that mitochondrial Tusc2 deficiency generates a pro-inflammatory environment in the brain that disrupts homeostatic processes, thereby producing early events of cognitive dysfunction and memory impairments. Using a Tusc2 knockout mouse model, mice underwent behavioral testing including Y-maze and Open field. Immunophenotyping and molecular pathway analyses (via immunoblotting) were performed. Tusc2 KO mice showed impaired short-term spatial memory as assessed with Y- maze test (p<0.05). Immune analysis of Tusc2 KO brain immune subsets showed changes associated with neurodegeneration, with increased number of activated microglia. Western Blot analysis of hippocampal tissue revealed prominent activation of molecular pathways associated with neurodegeneration, mTOR, and synaptic plasticity, calbindin in KO mice. Overall, Tusc2 deficiency plays a pivotal role in the development of molecular processes leading to cognitive impairment. In my postdoctoral work, I aim to leverage my knowledge and skills to further my work in mitochondria dysfunction and its role in neurological disease, by studying X-linked intellectual disability type Nascimento syndrome (NS). NS is caused by deficiency in the UBE2A gene, which is involved in the ubiquitin proteosome pathway and in the clearance of dysfunctional mitochondria. Further defining NS at the molecular level will give us insight into why UBE2A deficiency leads to the clinical phenotype of the disease.

Single-Cell Transcriptomic Comparison of Patient-Derived Ovarian Cancer Organoids and Primary Tumor Epithelial Signatures

High-grade serous ovarian cancer (HGSOC) is a rare but deadly disease. Platinum-based therapy along with PARPi maintenance therapy, is recommended to 80% of patients. However, acquired resistance to platinum and PARPi remains an ongoing clinical challenge, highlighting the need for the development of targeted therapies. To address this, there is a need for better in vitro models that mimic the tumor microenvironment (TME). Unlike traditional 2D culturing methods, patient-derived ovarian cancer organoids (PDOs) form 3D structures that more accurately recapitulate multilayered, multicellular tissue. The overall objective of this study is to examine how HGSOC PDOs can be used to identify gene pathways and networks that drive drug resistance, as well as to identify novel drug therapies that exploit these pathways. Primary and metastatic tumor samples were collected from diagnosed HGSOC patients for organoid generation. In short, the collected samples underwent mechanical and enzymatic digestion, were plated in solubilized basement membrane matrix, and grown in a published HGSOC media recipe. Organoids were cultured for 1-5 passages before being submitted for sc-RNAseq. In addition to organoid samples, matched tumor FFPE samples were submitted for comparison. Preliminary results from our pilot study reveal that PDOs contain epithelial, stromal, and immune cells. We next performed cell trajectory analysis using Monocle3, which assesses cell development and differentiation over time. We identified distinct cell trajectory projections across all samples. Interestingly, our organoid sample contained many branch points, representing different cell fate decisions. This result suggests the existence of numerous intermediate cell states that are not present or are masked in whole tumor FFPE samples. In conclusion, patient-derived ovarian cancer organoids recapitulate some cell populations of the TME and can be used to identify cell fate decisions of epithelial tumor cells.

Truncating mutations in PPM1D cooperate with PI3K alterations to drive pediatric Diffuse Midline Gliomas

Mutations in PPM1D – coding for the phosphatase WIP1 - are present in 8 -12% of all Diffuse Midline Glioma (DMG), H3 K27-altered – a universally fatal subtype of pediatric high-grade tumors. PPM1D mutations are mutually exclusive with TP53 mutations and mainly lead to a truncation of its C-terminal regulatory domain. Truncated PPM1D (PPM1Dtr) has increased protein stability and impairs the p53-dependent DNA Damage Response (DDR) by dephosphorylating its numerous targets (e.g p53, H2A.X, CHK1) which contributes to its oncogenicity. However, because wildtype p53 is present in PPM1Dtr tumors, its activation is suppressed but not completely ablated suggesting that PPM1D mutant tumors may evolve differently from p53 mutant ones. Our analysis of whole genome sequencing datasets indeed found that PPM1D mutations uniquely co-occur with alterations in the PI3K/mTOR pathway suggesting potential cooperativity in enhancing tumorigenesis. Using mouse neural stem cells (mNSCs) as an isogenic model system, we have engineered specific combinations of oncogenic alterations to study their impact on cell proliferation, PI3K/mTOR pathway activation and response to DNA damage and replication stress. We find that PPM1Dtr cooperates with PIK3CA alterations to increase proliferation and signaling through the PI3K/mTOR pathway. Notably, in an in utero electroporation model, mutant PIK3CA accelerates the development of high-grade tumors that infiltrate deep into the brainstem. Our study also indicates a vulnerability to inhibition of the PI3K/mTOR pathway in PPM1Dtr-PI3K co-altered tumors. Future work will aim at 1) exploring the mechanism of cooperation and impact on replicative stress response and 2) developing and testing a rational therapeutic strategy for PPM1D-mutant tumors.

Role of B Cell in Influenza Vaccine Response Single Cell Data Analysis Pipeline

Influenza vaccines have been shown to elicit a more robust immune response in younger individuals compared to the aged. We conducted a study to understand the role of B cells in this age-related vaccine response. We collected PBMCs from 12 donors-- with seven aged (65+ years old) individuals and five younger individuals (18-40 years old). We then performed single-cell RNA sequencing on B cells isolated from PBMCs from three timepoints: baseline (before vaccination), seven days, and 14 days post-vaccination. Our poster presents the computational analysis methodology employed in our study, including quality control measures, data transformation techniques, and a discussion on the potential of transformer models as the future of analysis in this domain.

Genetic Mechanisms Controlling Development: Insights from Metabolomics

Many phenotypes are multifactorial, polygenic in nature, and also influenced by environmental factors. Candidate genes and genetic pathways that specify phenotypes of interest can be identified using metabolomics, and then functional genetic assays can validate the roles they play in phenotypic specification. Here, we used untargeted metabolomics to discover the molecular mechanisms underlying increased fecundity and fertility responses of Drosophila sechellia to a nutritional input. We identified pathways with potential contributions to nutritionally responsive reproductive and developmental traits, which are highly heritable and plastic phenotypes, including tryptophan metabolism via the kynurenine pathway, methionine metabolism and urate biosynthesis.

Examining the Relationship Between Community-based Support Services Use and Mental Health in Black Family Caregivers of Persons Living with Dementia

The mental health of family caregivers, especially those caring for persons with dementia, is a critical concern. This study explores the relationship between community-based support services (CSS) use and mental health outcomes in Black family caregivers. Using Pearlin’s caregiver stress model, the study examines how CSS can alleviate stress and improve mental health. A mixed-methods approach was employed, secondary data analysis of the 2015 NHATS and NSOC datasets and focus group interviews. Findings indicate that higher CSS utilization is significantly associated with better mental health outcomes, such as reduced depression and anxiety. Qualitative data highlight the importance of dementia training, financial constraints, and service accessibility. These results emphasize the need for tailored support services to reduce caregiver burden and enhance mental health. This study contributes to the literature by focusing on the specific needs and experiences of Black family caregivers, advocating for culturally sensitive interventions. The implications for policy and practice include developing targeted programs that address both psychological and practical needs, ultimately improving caregivers’ quality of life and caregiving capacity.

Tcf15 Negatively Regulates T and B Lymphopoiesis

Expression of genes associated with lymphopoiesis has been shown to be uniquely antagonistic to hematopoietic stem cell (HSC) maintenance and function. Additionally, reduced expression of lymphoid associated genes in HSCs has been shown to promote HSC expansion and self-renewal. These findings demonstrate that maintaining lymphoid developmental potential while preserving HSC function and identity requires a carefully calibrated molecular and cellular program. The transcription factor Tcf15 was recently shown to be indispensable to HSC maintenance and function. Tcf15 expression in HSCs conferred long term regenerative potential while absence of Tcf15 was shown to inhibit this. Because HSC maintenance and regenerative programs are so closely linked to regulation of lymphoid potential, we hypothesized that Tcf15 could function as a negative regulator of lymphopoiesis. To examine whether Tcf15 OE could selectively inhibit T and B lymphopoiesis, we first generated a Tcf15 overexpression (OE) mouse model, Col1a1TetOTcf15 /TetOTcf15 R26M2rtTA /M2rtTA (TetO-Tcf15). Tcf15 OE mice showed a dramatic reduction in bone marrow B220+ cells and thymic CD4+CD8+ cells, as early as 10 days. In contrast, no significant difference was observed in the frequency of myeloid Gr-1+ cells. We next sought to identify the cellular mechanisms by which Tcf15 OE inhibits T and B lymphopoiesis. While TetO-Tcf15 MPP4s differentiated normally, dox treated TetO-Tcf15 MPP4s failed to generate T or B cells in vitro. Additionally, initial scRNA sequence analysis has revealed that Tcf15 OE attenuates DNTT expression in the bone marrow. Our data suggests that Tcf15 expression may repress regulatory networks that promote T and B lymphopoiesis.

Targeting the Sphingolipid-1-phosphate Signaling Pathway in Preterm Labor

Objective: To explore the effects of the bioactive lipid S1P as a novel target in preterm labor management. Methods: For our preterm labor mouse model, C57BL/6L female mice were administered 15ug/mouse LPS intravaginally at 16.5 days post coitus (dpc). S1P pathway inhibitors - SKII, an S1P kinase 1 and 2 (SPHK1/2) inhibitor, Ex26, an S1P receptor 1 (S1PR1) inhibitor, or vehicle control – were administered 1 hour before LPS administration. Time of delivery was defined as birth of the first pup. At 24h post-LPS injection, all dams were sacrificed, and necropsy was performed to assess total pup delivered vs retained and pup viability. In a separate group of gravid mice, uterine tissue was collected at 4h post LPS or vehicle injection and flash frozen. Quantitative PCR and western blot analysis were used to quantify gene expression. Results: LPS induced preterm birth within 24h of administration, with a mean delivery time of 8h. Inhibition of SPHK or S1PR1 significantly decreased the number of dams delivering within 24h of LPS administration (100% for LPS vs. 33% for Ex26; p<0.05). Of the litters delivered, the proportion of pups delivered was also significantly lower with inhibition of S1PR1 (100% for LPS vs 22.2%; P<0.05). Further, S1PR1 and SPHK1 mRNA and protein expression increased in mice uterine tissue with advancing gestation. Mice treated with LPS also had increased S1PR1 protein expression compared to control (p<0.05). Conclusion: Inhibition of SPHK1/2 or S1PR1 inhibits LPS-induced preterm birth. The S1P signaling pathway may be a potential target for preventing preterm birth.

The Intersection of Endocrine Signaling and Neuroimmune Communication Regulates Muscle Inflammation-Induced Nociception in Neonatal Mice

Neonatal pain is a significant clinical issue but the mechanisms by which pain is produced early in life are poorly understood. Our recent work has linked the transcription factor (TF) serum response factor downstream of local growth hormone (GH) signaling to incision-related hypersensitivity in neonates. However, it remains unclear if similar mechanisms contribute to inflammatory pain in neonates. Therefore, we investigated if distinct TFs or GH modulated nociceptive behaviors in neonates following hindpaw muscle inflammation using 1% carrageenan. We found that local delivery of GH to the muscles of neonates with carrageenan induced inflammation, blocked spontaneous paw guarding and inhibited mechanical hypersensitivity to muscle squeezing. Results also correlated to reduced calcium responses in neurons from GH+Carrageenan treated mice compared to mice with muscle inflammation only using a novel ex vivo muscle/nerve/DRG/spinal cord GCaMP6 imaging preparation. Interestingly, we also detected a reduction in paw edema of GH treated mice that corresponded to an inhibition of the inflammation induced increase in macrophages and mast cells in the muscles. GH also suppressed neuronal STAT1 expression induced by muscle inflammation. Nerve specific siRNA mediated knockdown of STAT1 not only inhibited pain related behaviors but also reduced the upregulation of macrophages in the muscles. Neuronal STAT1 appeared to regulate DRG expression of monocyte chemoattractant protein 1 (MCP1). Knockdown of neuronal MCP1 also modulated pain-like behaviors and reversed the increase in tissue concentrations of multiple cytokines/chemokines detected after inflammation. Data suggests that local GH treatment may be a novel treatment for pain and inflammation in children.

Growth Hormone Regulates Neonatal Pain and Peripheral Inflammation Through STAT1 Dependent Transcription in Sensory Neurons

Neonatal pain is a significant clinical issue but the mechanisms by which pain is produced early in life are poorly understood. Our recent work has linked the transcription factor (TF) serum response factor downstream of local growth hormone (GH) signaling to incision-related hypersensitivity in neonates. However, it remains unclear if similar mechanisms contribute to inflammatory pain in neonates. Therefore, we investigated if distinct TFs or GH modulated nociceptive behaviors in neonates following hindpaw muscle inflammation using 1% carrageenan. We found that local delivery of GH to the muscles of neonates with carrageenan induced inflammation, blocked spontaneous paw guarding and inhibited mechanical hypersensitivity to muscle squeezing. Results also correlated to reduced calcium responses in neurons from GH+Carrageenan treated mice compared to mice with muscle inflammation only using a novel ex vivo muscle/nerve/DRG/spinal cord GCaMP6 imaging preparation. Interestingly, we also detected a reduction in paw edema of GH treated mice that corresponded to an inhibition of the inflammation induced increase in macrophages and mast cells in the muscles. GH also suppressed neuronal STAT1 expression induced by muscle inflammation. Nerve specific siRNA mediated knockdown of STAT1 not only inhibited pain related behaviors but also reduced the upregulation of macrophages in the muscles. Neuronal STAT1 appeared to regulate DRG expression of monocyte chemoattractant protein 1 (MCP1). Knockdown of neuronal MCP1 also modulated pain-like behaviors and reversed the increase in tissue concentrations of multiple cytokines/chemokines detected after inflammation. Data suggests that local GH treatment may be a novel treatment for pain and inflammation in children.

The Role of Canonical Glycolytic Endpoints in the Development and Function of Hematopoietic Stem and Progenitor Cell Types

Glycolysis is an essential metabolic pathway which converts glucose to pyruvate. It terminates via either the reduction of pyruvate to lactate by lactate dehydrogenase (LDH) or oxidative decarboxylation to acetyl-CoA by pyruvate dehydrogenase (PDH), followed by entry into the tricarboxylic acid cycle. The two endpoint arms of glycolysis through LDH and PDH have never been simultaneously deleted in any tissue to test if canonical glucose catabolism is required to maintain cell survival in vivo. In addition, the cell type specific dependency of hematopoietic stem and progenitor cells for LDH and PDH has not been systemically mapped in vivo. To test this and to understand how glycolysis operates in vivo in the absence of LDH and PDH, we performed individual or combined conditional deletions of Ldha, Ldhb, and Pdha1 in the hematopoietic system using Mx1Cre. Ldha deletion alone or in combination with Ldhb and/or PDH caused anemia. PDH and LDHA deletion exacerbated the anemia compared to LDHA deletion alone due to a block in accumulation of CD71+Ter119+ erythroid progenitors, suggesting that LDH and PDH redundantly support erythropoiesis. Analysis of the thymus revealed a persistent decrease in thymocytes across different stages of T cell development in LdhaΔ/Δ;Pdha1Δ or LdhaΔ/Δ;LdhbΔ/Δ;Pdha1Δ mice but not in LdhaΔ/Δ or LdhaΔ/Δ;LdhbΔ/Δ mice. Our results suggest that LDH and PDH are required either alone or redundantly at different stages of T cell development. Metabolomics analysis revealed multiple hematopoietic cell-type specific metabolic changes of blocking LDH or PDH. Altogether, these results suggest cell-type specific usage of glucose catabolic pathways in different differentiation stages.

Targeted Delivery of AAV9 based therapy in Huntington’s Disease Mice Using Focused Ultrasound

Huntington’s disease (HD) is a fatal, hereditary neurodegenerative disorder caused by an expanded CAG repeat sequence in exon 1 of the huntingtin gene (HTT). Despite great progress since the initial discovery of the causative HTT gene, there are still no cures for HD. Advancements have been made in HTT lowering strategies using different approaches such as ribonucleic acid (RNA) interference, however, delivery is a key challenge for HTT-lowering therapies as these agents cannot cross the blood-brain barrier (BBB). Focused Ultrasound (FUS) with microbubbles (MB) is a promising emerging technology that can be used to temporarily disrupt the BBB in a non-invasive manner to deliver therapeutic genes to the CNS. FUS BBB opening Phase I clinical trials have demonstrated that the BBB can be opened safely and reversibly in humans. The breakdown of the BBB has been described in Huntington’s disease and other neurodegenerative diseases. Here, we sought to determine the impact of HD neuropathology on FUS-mediated BBB opening and gene delivery. We compared FUS-mediated delivery of ssAAV9 containing a miRNA (miR1050) targeting HTT mRNA in zQ175 2-month, 6-month, and 12-month-old mice (N=6 per group). FUS-mediated AAV delivery led to strong GFP expression in the FUS-treated hemisphere compared to the untreated hemisphere. Both neurons and astrocytes were infected following delivery, however, neuronal transduction efficiency was significantly lower in the 12-month cohort compared to the younger cohorts. These findings show that FUS+MB can be used to deliver an AAV-miRNA based gene therapy to specific brain regions in the zQ175 HD mouse model.

Elucidating the Role of Prion Proteins in Malaria Pathogenesis and Treatment

Malaria constitutes a heavy global disease burden. Plasmodium falciparum is the species most responsible for transmission and causes the most severe form of malaria, cerebral malaria (CM).The World Health Organization (WHO) broadly defines CM as P. falciparum asexual stage parasites in peripheral blood and an unexplainable loss of consciousness. The pathogenesis of cerebral malaria is not yet fully elucidated. However, it is known that endothelial cell and blood-brain barrier (BBB) dysfunction and neuronal damage can occur due to the cytoadherence and sequestration of infected red blood cells in the brain microvasculature. Even after antimalarial treatment, CM is fatal in 15-20% of cases in children under age 5, with studies documenting long-term behavioral, neurological, and cognitive impairments in survivors of pediatric CM. Studies have linked similar disruptions in the BBB and neuronal damage as seen in CM in the neurodegenerative Alzheimer's disease (AD), which is characterized by Amyloid-Beta (Aβ) protein aggregation. Interestingly, the P. falciparum proteome is notably rich (10%) in proteins containing prion-like domains. Although these prion-like domains are prevalent, suggesting an evolutionary advantage, their amyloidogenic potential has yet to be identified. These prion-like proteins may aid the parasites’ evasion of host immune response and overall survival. Our preliminary results, using an established in vivo yeast reporter system and high-resolution microscopy in live P. falciparum cultures, identify amyloids formed by several Plasmodium proteins. Through additional genetic, biochemical, and proteomic analyses, we plan to elucidate prion domains' amyloidogenic and therapeutic utility in P. falciparum proteins.

HIV-Induced Immunomodulatory Effects and the Development of Cardiometabolic Disease in High-Fat Diet-Conditioned Mice

The development of therapies to treat people living with HIV (PLWH) has significantly increased their life expectancy, nearing that of people without HIV (PWoH). While ART has reduced viremia, chronic inflammation persists, increasing the risk of comorbidities such as cardiometabolic diseases. Our understanding of how HIV-related inflammation leads to cardiometabolic disease remains limited. However, we hypothesize that subsets of immune cells in peripheral blood mononuclear cells (PBMCs) are involved in the pathways leading to HIV-associated cardiovascular disease. To interrogate this hypothesis, we reconstituted the immune system of NSG mice with PBMCs from patients with or without HIV. Recipient mice were placed on either a standard-chow diet (SCD) or a high-fat diet (HFD) and then assessed for weight, metabolism, and immune cell composition changes. By day 30, mice with HIV on a high-fat diet (HFD-PLWH) experienced an 11.5-fold increase in weight compared to a 5.7-fold increase in non-HIV mice on the same diet (HFD-PWoH). Glucose tolerance tests revealed that mean plasma insulin levels after 10 minutes were notably higher in HFD mice compared to those on a SCD. Interestingly, we observed a strong correlation between the proportion of CGC+ CD4+ T cells in PBMCs and weight gain in HFD-PLWH mice but not in other groups. Overall, HFD-PLWH had the worst metabolic phenotypes, implicating a causal role for immune cells in cardiometabolic outcomes. These data suggest that HIV interacts with a HFD and mediates immunomodulatory effects that promote the development of phenotypes indicative of cardiometabolic disease in mice.

The Metabolic Landscape of Fontan-associated Liver Disease

Background: Single ventricle congenital heart disease (SVCHD) is a severe form of cardiac malformation in which there is only one functional ventricle. The Fontan operation is the current standard of care for SVCHD. Almost all patients who have undergone the Fontan operation develop liver fibrosis at a young age, resulting in a condition known as Fontanassociated liver disease (FALD). The pathogenesis and mechanisms underlying FALD remain little understood, hindering development of effective therapies. Objectives: We aimed to present a comprehensive multiomic analysis of human FALD, thereby revealing the fundamental biology and pathogenesis of FALD. Methods: We recently generated a single-cell transcriptomic and epigenomic atlas of human FALD using snRNA-ATAC-seq, which revealed profound metabolic reprogramming in FALD. Here we applied liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics to unveil the metabolomic landscape of human FALD, using liver samples/biopsies from age and gender-matched healthy donors and FALD patients (n=12 per group). Extracted liver metabolites were analyzed by C18 high performance liquid chromatography (HPLC) and hydrophilic interaction chromatography (HILIC) followed by high resolution MS on Orbitrap. Statistical and bioinformatics analyses were performed to identify altered metabolites and metabolic pathways in FALD. These results were integrated with recently published snRNA-ATAC-seq and serum metabolomics datasets to present a comprehensive multiomic atlas of FALD. Results: We discovered profound metabolic abnormalities in livers of patients with early-stage FALD, particularly amino acid metabolism, peroxisomal fatty acid oxidation, cytochrome P450 system, glycolysis, TCA cycle, ketone body metabolism, and bile acids metabolism. Integrated analyses with liver snRNA-ATAC-seq and serum metabolomics data unveiled the transcriptional mechanisms driving this metabolic reprogramming and the crosstalk between liver and the rest of the body. Comparison with human metabolic dysfunction associated fatty liver disease (MAFLD) and metabolic dysfunction-associated steatohepatitis (MASH) revealed dysregulated amino acid metabolism as a common metabolic abnormality. Conclusions Our comprehensive multiomic atlas of human FALD reveals the fundamental biology and pathogenesis mechanisms of FALD.

A Genome-wide Analysis of Pleiotropy Between Morning Circadian Preference and BMI Reveals the Tissue-specific Rhythmicity of ADCY3 in Adipose Tissue

Background: Chronotype is the behavioral manifestation of our internal circadian clock, influencing whether individuals are early birds (morning people) or night owls (evening people). Chronotype significantly impacts complex diseases such as obesity. This study dissects the relationship between circadian preference and body mass index (BMI) using a genome-wide pleiotropy approach. Methods: We conducted a genome-wide pleiotropy analysis to explore the associations between morning circadian preference and BMI, focusing on the tissue-specific expression and rhythmicity of key genes. Results: We identified a lead locus, rs11676272 in ADCY3, associated with increased morning preference and decreased BMI. The rs11676272 (S107P) is a missense variant that destabilizes the ADCY3 protein structure, highlighting the gene's role in both circadian behavior and metabolic processes. In mouse models, Adcy3 expression was rhythmic in white and brown adipose tissues during constant darkness, and it oscillated anti-phase to the canonical circadian genes Clock and Bmal1. The presence of the Bmal1-specific E-box motif CACGTG on the Adcy3 gene suggests Bmal1 regulation of this rhythmicity, indicating a novel mechanism of circadian control within these tissues. Conclusion: Findings from our genome-wide pleiotropy approach underscore a novel role of ADCY3 at the intersection of circadian regulation and metabolic health. This study offers new insights into the genetic architecture of these complex traits, highlighting the significance of tissue-specific rhythmicity in adipose tissue. Keywords: Chronotype, BMI, genome-wide pleiotropy, ADCY3, circadian regulation, metabolic health, adipose tissue.

Lessons Learned: Challenges in Implementing an Evidence-based Workplace Wellness Program Among Early Care Educators

Early care and education (ECE) workers are low-resourced and encounter significant health challenges. This study aims to understand barriers and facilitators in adopting a 12-month healthy lifestyle e-intervention tailored for ECE workers. In an ongoing clinical trial, preliminary data from centers (n=8) randomized to the intervention group, two educators from two 2-5-year-old classrooms per center received a Fitbit, a digital scale, and a web-based program (primary users, PU; n=16). Other staff (independent users, IU; n=138) were offered program access but no devices. Data from website registration, PU interviews at 6-months (high-engaged n=3, low-engaged n=2), and PU 12-months (n=9) and IU (n=18) surveys were analyzed. We met enrollment goals for PU (n=16), but not IU (10, 6.5%). High-engaged PU reported multiple benefits, increased awareness and improved diet through food logging, more physical activity, and practical classroom applications. Low-engaged PU expressed a preference for in-person education and more social support. Approval and encouragement from social networks were important; participants highlighted the need for workplace support to increase involvement. Few reported difficulties, which included health issues, time, and workload due to staffing shortages. IU reported no prior experience with worksite health programming. Although 78% of IU were aware of the intervention, they reported not participating because they were not a PU or did not understand the program requirements. Less than half (44%) indicated interest in participating in a similar program in future. Clearer communication and increased workplace support at individual and organizational levels are needed for future initiatives to foster a healthier workforce.

Opposing Effects of the Aryl Hydrocarbon Receptor and Type I Interferon Control Generation of CXCL13+ T Cells in Systemic Lupus Erythematosus

Expansion of B cell helper-T cells, including T follicular helper (Tfh) and T peripheral helper cells (Tph) is a prominent feature of systemic lupus erythematosus (SLE), an autoimmune disease with broad autoantibody production. Human Tfh and Tph cells highly express the B cell chemoattractant CXCL13; however, regulation of CXCL13 production by T cells remains largely unknown. Here, mass cytometry immunophenotyping revealed a marked imbalance in CD4 T cell phenotypes in SLE patients, with expansion of CXCL13+ Tfh/Tph cells and specific reduction of CD96hi IL-22+ T cells. We hypothesized that a common regulator might drive this skewed balance. Using CRISPR screens of human CD4 T cells, we identified the transcription factor aryl hydrocarbon receptor (AHR) as a central regulator of an axis of T cell polarization with CXCL13+ and IL-22+ states at opposing ends. Transcriptomic, epigenetic, CUT&RUN, and functional studies demonstrated that 1) AHR activation drives T cells away from a CXCL13+ phenotype and towards an IL-22+ phenotype, and 2) AHR engages the AP-1 family member JUN to regulate CXCL13- and IL-22-associated phenotypes. Treatment of T cells from SLE patients with an AHR agonist reduced the frequency of PD-1+ Tph cells. In contrast, type I interferon (IFN-I), a central mediator in SLE, represses AHR activation in T cells and synergizes with AHR inhibition to boost CXCL13 production and promote a Tph cell phenotype. These results reveal AHR, JUN, and IFN-I as regulators of a previously unrecognized CXCL13 <—> IL-22 polarization axis and highlight an imbalance of this axis in SLE.

Reduction in Hepatic PC Biosynthesis Augments Copper Metabolism and Drives MASH Pathogenesis

PCYT1A encodes for CTP: phosphocholine cytidyltransferase, alpha (CCT-α), rate-limiting enzyme required for ~70% of Phosphatidylcholine (PC) production via the Kennedy pathway. Hepatic PC production is also significantly decreased in patients with metabolic dysfunction-associated steatohepatitis (MASH), an advanced disorder which affects a subset of patients with MAFLD and T2D. To date however, It remains unknown how the reduction of hepatic PC biosynthesis, promotes the fibrosis and inflammation associated with MASH. Thus, in this study, we used a diet-focused model, the low methionine, choline deficient, 42% kcal/fat high-fat diet (LMCD-HFD), and a genetic model lacking PCYT1A expression in the liver. Given the complexity of MASH, as well as the heterogeneity of the liver, we used 10X single-nuclei RNA-Seq for this study. Interestingly, transcriptomics analysis of DE of genes found in hepatocytes in our MASH models associated with Metal Ion Binding and Signaling. Additional cluster analysis showed decreases in several Cu-dependent proteins. Notably, ICP-MS analysis of MASH serum and livers showed significant decrease in copper content. Pro-fibrotic or activated stellate cell (aHSCs) clusters were also enriched for genes associated with Cu-associated ECM organization, TGFB-mediated signaling, and fibrosis (ex., SMAD3; COL3A1; CD44). From these data we hypothesize that PC-dependent impairments in hepatocyte-dependent copper import, caused by PC-dependent changes in membrane plasticity copper transporter function/activity, leads to increases in circulating copper, that acts on aHSCs, promoting inflammation and fibrosis in MASH. Notably, in patients with Menkes disease, a genetic loss-of-function disorder that affects the function of ATP7A, copper exporter from the gut, leading to decreases in hepatic copper, reduction in copper-dependent enzymes, and tissue-specific copper accumulation, phenotypes similar to what we observed in our MASH mouse models. Preliminary results from in vivo studies using cell impermeable copper chelator BCS, demonstrates partial protection against diet-induced MASH through reductions in fibrosis as observed by reductions in Sirius red and decreases in pro-fibrogenic gene expression of PDGFRB and SAA1. In vitro studies using HUH-7 cells, CuSO4 can be seen inducing the expression of several cu-dependent genes in a dose-dependent manner, a finding not observed in HUH-7 cells deprived of copper using serum-free media or treated with BCS. Interestingly, in HUH-7 cells treated with PCYT1A siRNA, CuSO4 was unable to promote significant induction in copper-dependent genes similar to our observations in our in vivo MASH models. Given the FDA-approval of copper chelators such as D-Penicillamine (DPA), and Tetrathiomolybdate (TTM), for diseases such as cancer, and Wilson’s disease, our work may highlight an alternative use for these therapeutics against MASH in the future.

Impacts of State Empathy on Observationally Induced Placebo Effects

Observing another person experiencing pain reduction from a treatment influences one’s placebo effects in response to the same treatment. It has been posited that empathy traits may play a role in mediating these effects. Current studies have primarily used trait empathy measurements as potential mediators of placebo effects, however, the role of state empathy measurements are unknown. Forty-seven healthy participants completed a social learning study while we measured state empathy changes towards a human or avatar demonstrator in VR and real-world settings. Subsequently, participants underwent the same treatments seen during observation but with identical thermal intensities to test for observationally induced placebo effects, using a visual analogue scale to report both pain intensity and pain unpleasantness levels. Independent of setting, observation increased state empathy for pain intensity and unpleasantness. Moreover, observation of the human demonstrator resulted in greater state empathy levels as compared to the avatar. Observation induced significant placebo effects for pain intensity and unpleasantness, and these effects were mediated by state empathy changes. At the EEG level, faster PAF in the somatosensory and frontal regions predicted larger individual placebo effects.

Racial Disparities in ICU Delirium is Linked to Dysregulated NAD+ Metabolism

Background: Emerging evidence supports an inter-relationship between delirium, an acute neuropsychiatric condition, and dementia: dementia is a predisposing risk factor for delirium and vice versa. Yet, despite evidence supporting shared pathophysiology, similarities in non-modifiable risk factors remain inconclusive. Specifically, although Blacks experiencing a two-fold greater risk for dementia, there is conflicting data as to if racial disparities exist in delirium. Whereas Boltz et al showed that Black patients with dementia had more delirium at admission than White patients, Khan et al., observed no racial differences in the prevalence of incident and prevalent delirium and others have reported White race as a predisposing risk factor for delirium. These inconsistencies are likely attributable to inadequate sample size racial diversity to draw conclusion on racial differences. Objective: The primary goal of this study was to determine if Blacks experienced more severe delirium than Non-Black patients. As an exploratory aim, we further examined the intersectionality of race and sex on delirium severity. Methods: We conducted a secondary analysis of a randomized control trial focused on adult Intensive Care Unit (ICU) patients with delirium. Inclusion criteria consisted of: >24hr ICU stay, age >18yrs, screened positive for delirium, English speaking, no history of severe mental illness, cognitive impairment, or dementia per electronic medical record, not pregnant or nursing, no alcohol related delirium or aphasic stroke diagnosis. Primary outcome was delirium intensity as assessed by Delirium/Coma free days (duration) and delirium severity (0-7; 0-2 = no delirium, 3-5 = mild to moderate delirium, 6-7 = severe delirium). Wilcoxson signed rank test was used to test racial differences in delirium intensity. One-way ANOVA was used to compared race by sex differences. Results: We enrolled 545 patients total (Black = 248 and Non-Black = 297) with comparable critical illness exposure, baseline cognition, and activities of daily living (p > 0.05). At admission (p = 0.16), within 1 week (p = 0.041), and at discharge (p = 0.012) Black patients experienced greater delirium severity but not duration compared to non-Black patients. At discharge, Black females experienced greater delirium severity (p = 0.025), compared to Black Males, Non-Black Males, and Non-Black Females. Conclusions: Despite similar delirium duration, Black patients experience more severe delirium throughout an ICU stay compared to Non-Black patients. Although Black females experienced the most severe delirium at discharge, Non-Black males and females had similar delirium severity scores which suggest that there is not a sex difference. Our finding is likely to do under recruitment of Black male patients. Nonetheless, these results are consistent with disparities reported in the extant dementia literature.