Faculty Bibliography

Ulcerative colitis is an idiopathic, chronic inflammatory bowel disease. Its pathogenesis is multifactorial involving inflammation and immune dysregulation. Proinflammatory TNFα/NFκB signaling is believed to play a cardinal role in ulcerative colitis. Growing evidence indicates the molecular interactions between the cellular metabolites and different phases of inflammation. This study aims to identify the metabolites that can inhibit TNFα/NFκB signaling and are potentially therapeutic against various TNFα-associated inflammatory diseases, particularly inflammatory bowel diseases. We performed in vitro and in vivo screening of cellular metabolites to inhibit TNFα/NFκB signaling. Multiple confirmation assays, including NFκB translocation, quantitative real-time PCR, ELISA, immunofluorescence staining, and RNA sequencing analysis were executed. Drug affinity-responsive target stability assay with proteomics was utilized for target identification. cPLA2 ablated mice with dextran sodium sulfate-induced colitis were employed to assess pyruvate's dependence on its molecular target in attenuating ulcerative colitis pathogenesis. Metabolite screening and subsequent validation with multiple approaches led to the isolation of pyruvate, a glycolytic metabolite, and a critical node in several metabolic pathways, as a novel inhibitor of TNFα/NFκB signaling. Importantly, pyruvate suppressed inflammation, preserved colonic histology, maintained tight junction proteins, and regulated permeability in the ulcerative colitis model. Additionally, cPLA2 was identified as a previously unknown target of pyruvate and pyruvate largely lost its therapeutic effects against ulcerative colitis in cPLA2-deficient mice. Conclusively, this study not only unveils pyruvate as an antagonist of TNFα/NFκB signaling and therapeutic intervention against colitis but also provides mechanistic insight into the mode of action of pyruvate.

The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. The main advantage of this system compared to existing techniques is maintenance of endogenous regulatory elements. We used this system to identify both cell-autonomous and non-cell-autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

Pathology in large vessels frequently develops at specific locations, implying that local stressors and spatially restricted gene expression are likely contributors to disease susceptibility. Here we perform single-cell transcriptomics in the carotids, the aortic arch and the thoracic and abdominal aorta to identify site- and sex-specific differences that could inform about vulnerability. Our findings revealed (1) regionally defined transcriptional profiles, (2) signatures associated with embryonic origins and (3) differential contributions of sex-specific effectors. Furthermore, cross-referencing regional-specific signatures with available genome-wide association study and expression quantitative trait loci databases identified 339 disease candidates associated with aorta distensibility, stiffness index and blood pressure. CPNE8 and SORBS2 were further evaluated and highlighted as strong causal candidates. Sex differences were predominantly observed in the thoracic and abdominal aorta. MCAM (CD146), a transcript with sex-skewed expression and lower in male mice and men, had significantly reduced expression in human aortic aneurysms. The findings reveal underlying diversity within vascular smooth muscle cell populations relevant to understanding site-specific and sex-specific variation of vascular pathologies.

The current generation of highly successful atherosclerosis treatments, such as low-density lipoprotein (LDL)-cholesterol reduction, blood pressure management, and smoking cessation, has largely focused on ameliorating factors perceived to drive incident disease and its complications. The adverse contributions of these factors have typically been identified through epidemiological studies. The therapeutic strategies that arose in response focused on risk factors for disease development and tended to overlook the fact that patients already have established disease, by the time of presentation. However, by capitalizing on contemporary biological knowledge and technologies, it is becoming increasingly possible to shift from a model based on population-derived risk factor management to next-generation treatments (including monoclonal antibodies, small interfering RNA [siRNA], mRNA, epigenetic reprogramming, and gene editing) for atherosclerosis that are tailored to patient-level disease processes, informed by mechanistic characterization, offer potential to reverse or regress disease, and incorporate systems-level interventions that extend beyond the atherosclerotic plaque.

Auxins are plant hormones that direct the growth and development of organisms on the basis of environmental cues. Indole-3-acetic acid (IAA) is the most abundant auxin in most plants. A variety of membrane transport proteins work together to distribute auxins. These include the AUX/LAX protein family that mediate auxin import from the apoplast to the cytosol. Here we use structural and biophysical approaches combined with molecular dynamics to study transport by Arabidopsis thaliana LAX3, which is essential for plant root formation. Transport assays document high-affinity transport of IAA, as well as competitive behaviour of the synthetic phenoxyacetic acid auxin herbicide 2,4-dichlorophenoxyacetic acid and the auxin transport inhibitors 1-naphthoxyacetic acid and 2-naphthoxyacetic acid. Four cryo-EM structures were solved with resolutions of 2.9-3.4 Å: an inward open apo structure, two inward semi-occluded structures in complex with IAA and 2,4-dichlorophenoxyacetic acid, and a fully occluded structure in complex with 2-naphthoxyacetic acid. Structurally, LAX3 consists of a bundle and a scaffold domain. The ligand-binding site is sandwiched between these domains with two histidines occupying positions analogous to the sodium-binding sites in distantly related sodium:neurotransmitter transporters. This architecture suggests that these histidines couple transport to the proton motive force. Molecular dynamics simulations are used to explore substrate binding and release, including their dependence on specific protonation states. This study advances our understanding of auxin recognition and transport by AUX/LAX, providing insights into a fundamental aspect of plant physiology and development.

BACKGROUND:Motor stereotypies (MS) represent one of the transdiagnostic symptom dimensions identified by the NIMH Research Domain Criteria work group as relevant to psychopathology. MS are common in neurodevelopmental conditions, but they remain poorly understood, particularly in early childhood. The present study examined MS in 648 toddlers with autism spectrum disorder (autism, n = 455) and other neurodevelopmental conditions (non-autism, n = 193) and their concurrent and prospective links with other phenotypic characteristics. METHODS:Toddlers were recruited between February 2000 and October 2018 and evaluated at 24 +/- 5 months (Time 1, N = 648) and 41 +/- 6 months (Time 2, N = 455). The presence of MS was determined based on the Autism Diagnostic Observation Schedule assessment. The phenotypic measures included adaptive socialization skills, severity of social symptoms of autism, and verbal, nonverbal, and motor skills. The analysis was conducted using the general linear models while controlling for age, sex, visit year, group, and other relevant covariates. RESULTS: CONCLUSIONS:Motor stereotypies are present in toddlers with and without autism and may represent a distinct transdiagnostic dimension expressed early in development, associated with core developmental skills and, putatively, characterized by shared pathophysiology across neurodevelopmental conditions.

Ribosomes are central to protein synthesis in all organisms. Among mammals, the ribosome functional core is highly conserved. Remarkably, two rodent species, the naked mole-rat (NMR) and tuco-tuco display fragmented 28S rRNA, coupled with high translational fidelity and long lifespan. The unusual ribosomal architecture in the NMR and tuco-tuco has been speculated to be linked to high translational fidelity. Here we show, by single-particle cryo-electron microscopy (cryo-EM), that despite the fragmentation of their rRNA, NMR and tuco-tuco ribosomes retain their core functional architecture. Compared to ribosomes of the guinea pig, a phylogenetically related rodent without 28S rRNA fragmentation, ribosomes of NMR and tuco-tuco exhibit poorly resolved, certain expansion segments. In contrast, the structure of the guinea pig ribosome shows high similarity to human ribosome. Enhanced translational fidelity in the NMR and tuco-tuco may stem from subtle, allosteric effects in dynamics, linked to rRNA fragmentation.

BACKGROUND:Patients with peripheral artery disease have an increased risk of cancer development. Aging-associated changes in hematopoietic stem and progenitor cells (HSPCs), including inflammation and increased myelopoiesis, are implicated in both cardiovascular disease and cancer, but their contributions to cardiovascular disease-driven tumor progression are unclear. OBJECTIVES/OBJECTIVE:This study sought to study tumor growth after peripheral ischemia and consequent changes within the HSPC bone marrow compartment to uncover mechanisms through which altered hematopoiesis promotes cancer. METHODS:Mammary cancer (E0771) growth was monitored in C57BL/6J mice after hind limb ischemia (HLI) or sham surgery. The tumor immune microenvironment, circulatory immune cells, and HSPC compartment were assessed by flow cytometry. Next-generation single-cell RNA and assay for transposase-accessible chromatin sequencing of bone marrow progenitors was performed to assess the distinct and synergistic transcriptomic and epigenetic changes of cancer and peripheral ischemia. The functional impact on tumor progression and persistence of ischemia-induced epigenetic reprogramming of HSPCs and their myeloid progeny was examined by bone marrow transplantation. RESULTS:myeloid-biased hematopoietic stem cells. This was associated with accelerated cancer growth and enrichment of tumors with myeloid cells (monocytes, macrophages, neutrophils) and regulatory T cells. Increased myelopoiesis was also supported by sequencing analyses showing HLI and tumor-induced transcriptional and epigenetic enrichment for inflammatory (NLRP3 inflammasome) and aging-associated neogenin-1, thrombospondin-1) signatures in subsets of monocyte/dendritic progenitors. HLI-accelerated tumor growth and myeloid-skewing was transmissible via bone marrow transplantation, indicating long-term reprogramming of innate immune responses. CONCLUSIONS:Peripheral ischemia enhances inflammaging of hematopoietic stem cells and long-lasting alterations to antitumoral immunity, accelerating breast tumor growth.