Faculty Bibliography

BACKGROUND:Type 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum. METHODS:An in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (μCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells. RESULTS:The in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, μCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells. CONCLUSION/CONCLUSIONS:Rosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.

The current, rapidly diversifying pandemic has accelerated the need for efficient and effective identification of potential drug candidates for COVID-19. Knowledge on host-immune response to SARS-CoV-2 infection, however, remains limited with few drugs approved to date. Viable strategies and tools are rapidly arising to address this, especially with repurposing of existing drugs offering significant promise. Here we introduce a systems biology tool, the PHENotype SIMulator, which -by leveraging available transcriptomic and proteomic databases-allows modeling of SARS-CoV-2 infection in host cells in silico to i) determine with high sensitivity and specificity (both>96%) the viral effects on cellular host-immune response, resulting in specific cellular SARS-CoV-2 signatures and ii) utilize these cell-specific signatures to identify promising repurposable therapeutics. Powered by this tool, coupled with domain expertise, we identify several potential COVID-19 drugs including methylprednisolone and metformin, and further discern key cellular SARS-CoV-2-affected pathways as potential druggable targets in COVID-19 pathogenesis.

The field of additive manufacturing, 3D printing (3DP), has experienced an exponential growth over the past four decades, in part due to increased accessibility. Developments including computer-aided design and manufacturing, incorporation of more versatile materials, and improved printing techniques/equipment have stimulated growth of 3DP technologies within various industries, but most specifically the medical field. Alternatives to metals including ceramics and polymers have been garnering popularity due to their resorbable properties and physiologic similarity to extracellular matrix. 3DP has the capacity to utilize an assortment of materials and printing techniques for a multitude of indications, each with their own associated benefits. Within the field of medicine, advances in medical imaging have facilitated the integration of 3DP. In particular, the field of orthopedics has been one of the earliest medical specialties to implement 3DP. Current indications include education for patients, providers, and trainees, in addition to surgical planning. Moreover, further possibilities within orthopedic surgery continue to be explored, including the development of patient-specific implants. This review aims to highlight the use of current 3DP technology and materials by the orthopedic community, and includes comments on current trends and future direction(s) within the field.

BACKGROUND:The metalloprotease ADAMTS-7 (a disintegrin and metalloproteinase with thrombospondin type 1 motif 7) is a novel locus associated with human coronary atherosclerosis. ADAMTS-7 deletion protects against atherosclerosis and vascular restenosis in rodents. METHODS: RESULTS: CONCLUSIONS:ATS7vac is a novel atherosclerosis vaccine that also alleviates in-stent restenosis. The application of ATS7vac would be a complementary therapeutic avenue to the current lipid-lowering strategy for atherosclerotic disease.

UNLABELLED:The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology. ONE SENTENCE SUMMARY/UNASSIGNED:We reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.

The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogenetically conserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of the kidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, at extracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligand binding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformation of which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleterious missense variants in humans appears to impair homodimer assembly. These observations lay the foundation for further understanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRP receptor subfamily.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.

GPR133 (ADGRD1) is an adhesion G protein-coupled receptor that signals through Gαs and is required for growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca²⁺-dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca²⁺-sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca²⁺ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM impairs tumor growth in vitro, suggesting functions of ESYT1 beyond the interaction with GPR133. Our findings suggest a novel mechanism for modulation of GPR133 signaling by increased cytosolic Ca²⁺, which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3^-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.