Faculty Bibliography

We previously found GPR133 (ADGRD1), an orphan adhesion GPCR, is De novo expressed in glioblastoma (GBM) and enriched in patient-derived glioblastoma stem cells (GSCs). Knockdown of GPR133 reduces GBM cell proliferation and tumorsphere formation, and abolishes orthotopic tumor initiation in vivo in mice. Analysis of TCGA data indicates that increased GPR133 transcription inversely correlates with patient survival in GBM. While these findings underscore the importance of GPR133 in GBM and suggest an essential role in tumor growth, its ligand and mechanism of activation remain unknown. Toward identifying GPR133 ligands, we used GPR133's N-terminal ectodomain as bait and performed affinity co-immunoprecipitation (CoIP) followed by mass spectrometry as an unbiased screening approach. We identified 490 extracellular proteins with enriched binding to GPR133 compared to control. Reverse CoIP using the 15 most abundant candidate ligands as bait to purify the receptor confirmed this interaction reproducibly in 4 candidates. Despite this binding, overexpression of these candidate ligands, or addition of purified recombinant protein, is not sufficient to increase receptor signaling as assessed by cAMP levels in HEK293 cells. This suggests that ligand binding to the GPR133 ectodomain may not be sufficient by itself to induce receptor activation. We hypothesize receptor activation requires mechanical forces in addition to ligand binding. Consistent with this hypothesis, the GPR133 binding proteins we have identified may be anchored to the extracellular matrix, mediating such mechanical force. To test whether mechanical shearing of the extracellular domain is sufficient for receptor activation, we used Dynabeads coupled to antibody against GPR133's N-terminal ectodomain, and indeed observed receptor activation leading to elevated cAMP levels. No activation was observed when Dynabeads devoid of antibody were used. This mode of GPR133 activation might indicate a role in sensing mechanical/viscoelastic properties of GBM extracellular matrix, which may be relevant to tumor cell migration and invasion

Dalbavancin is a lipoglycopeptide antibiotic with a prolonged half-life. A Phase 1 study assessed dalbavancin levels in epithelial lining fluid (ELF) in 35 healthy adults using ELF bronchial microsampling up to 168 hrs after 1500 mg dalbavancin. The penetration of dalbavancin into ELF was 36%. ELF levels of dalbavancin exceeded the MIC₉₀ of S. pneumoniae and S. aureus for ≥ 7 days.

STUDY OBJECTIVE/OBJECTIVE:To clarify the relationship of hypogastric nerves (HNs) with several pelvic anatomic landmarks and to assess any anatomic differences between the 2 sides of the pelvis, both in cadaveric and in vivo dissections. DESIGN/METHODS:Prospective observational study. SETTING/METHODS:An anatomic theater for cadaveric dissections and a university hospital for in vivo laparoscopy. PATIENTS/METHODS:Five nulliparous female cadavers underwent laparotomic dissection; 10 nulliparous patients underwent laparoscopic surgery for rectosigmoid endometriosis without posterolateral parametrial infiltration. INTERVENTIONS/METHODS:Measurements of the closest distance between HNs and ureters, the midsagittal plane, the midcervical plane, and uterosacral ligaments on both hemipelvises. A comparison of anatomic data of the 2 hemipelvises was conducted. MEASUREMENTS AND MAIN RESULTS/RESULTS:The right and left HNs were identified in all specimens, both on cadavers and in vivo dissections. A wide anatomic variability was reported. Regarding the differences between the 2 hemipelvises, we found that the right HN was significantly (p <.001) farther to the ureter (mean = 14.5 mm; range, 10-25 mm) than the left one (mean = 8.6 mm; range, 7-12 mm). The HN was closer to the midsagittal plane on the right side (mean = 14.6 mm; range, 12-17 mm) than on the left side (mean = 21.6 mm; range, 19-25 mm). The midcervical plane was found 2.7 mm (range, 2-4 mm) to the left of the midsagittal one. The right HN was found to be nonsignificantly closer to the midcervical plane and the uterosacral ligament on the right side than on the left side (p >.05). CONCLUSIONS:Despite a wide anatomic variability of position and appearance, the HNs are reproducibly identifiable using an "interfascial" technique and considering the ureters and uterosacral ligaments as anatomic landmarks.

Type 1 cardiorenal syndrome (CRS1) is characterized by acute cardiac disease (e.g., acute heart failure [AHF]), leading to acute kidney injury. Sirtuin 1 (SIRT1), an NAD⁺ -dependent deacylase, has been found to be associated with CRS1. To confirm whether a correlation exists between SIRT1 variants and the risk of CRS1, the association between the prevalence of CRS1 and single-nucleotide polymorphisms (SNPs) within the SIRT1 gene was investigated in AHF patients. A total of 316 Chinese AHF participants (158 patients with CRS1 and 158 age- and sex-matched controls) were recruited for the present observational study to investigate the association between nine common SIRT1 SNPs (i.e., rs7895833 G > A, rs10509291 T > A, rs3740051 A > G, rs932658 A > C, rs33957861 C > T, rs7069102 C > G, rs2273773 T > C, rs3818292 A > G, and rs1467568 A > G) and the susceptibility to CRS1. Significant differences in genotype distribution between the control and CRS1 groups were found for rs7895833 and rs1467568. After applying a Bonferroni adjustment, the A allele of rs7895833 was still found to be protective (p = 0.001; odds ratio [OR] = 0.77) against CRS1 in this study population. The AA genotype of rs7895833 and the GA genotype of rs1467568 were associated with a significantly reduced risk of CRS1 (OR = 0.23 and 0.49, respectively). rs7895833 and rs1467568 were further analyzed as a haplotype, and the GA haplotype (rs7895833-rs1467568) exhibited a significant association with CRS1 (p = 0.008), while the AA haplotype showed a significant protective effect (p = 0.022). Our study showed that SIRT1 rs7895833 and rs1467568 polymorphisms had a significant effect on the risk of developing CRS1 in a population in China.

Objective: Abdominal aortic aneurysm (AAA) is a fatal vascular disease on rupture with still limited mechanistic knowledge of the pathophysiologic process. We sought to determine the heterogeneous cell subtypes and to characterize the spectrum of transcriptome signatures in each cell population within the aneurysmal wall by unbiased single-cell RNA sequencing (scRNA-seq) of human AAA tissue.
Method(s): Aortic specimens were collected from AAA and control healthy organ donor. Samples were processed by enzymatic digestion and mechanical disruption to generate single-cell suspension. Single-cell RNA libraries were prepared after generation of single-cell beads in emulsion. Sequencing was performed on a NovaSeq 6000 platform (Illumina, San Diego, Calif). After alignment, barcode assignment, and sample de-multiplexing, data analysis was performed on t-distributed stochastic neighbor embedding charts of cell transcriptome. Cell clusters were identified by unsupervised proximity based on Euclidian distance and supervised identification of biologic markers within clusters. Pathway analysis algorithms were used to outline biologically relevant networks.
Result(s): Unbiased analysis of scRNA-seq data sets showed 19 different cell clusters with unique transcriptomic signatures in AAA. A total of 8826 significant differentially expressed genes were identified in AAA vs control. Notably, gene transcription-associated extracellular matrix remodeling (COL1A1, COL3A1, COL1A2, LUM), Wnt signaling modulation (SFRP2), and synthetic cellular phenotypes (RPS29, RPS27, RPL13A, RPL28) were among the top increased profiles in AAA tissue. Pathway enrichment analysis of AAA vs control libraries revealed significant modulation of cell proliferation, cell-extracellular matrix interaction, neoangiogenesis, and inflammation. Five novel cell clusters with distinct immune synthetic phenotypes were predominantly abundant in AAA wall compared with the healthy aorta. A robust enrichment in immune cell entities was identified in AAA but not in control tissues, including expansion of CD19⁺ B lymphocytes and a subset of CD3E⁺ T lymphocytes significantly expressing IL32 and CCL5. In contrast, smooth muscle cell (ACTA2⁺MYH11⁺) number declined in AAA but revealed increased transcription of the protease ADAMTS4 and inflammatory signals (CCL19, CCL21, IL6, CCL2). Intercluster pathway analysis revealed enrichment of eukaryotic initiation factor 2 and mechanistic target of rapamycin signaling in the AAA macrophage population along with an increased number of inflammatory and T-cell activation cascades.
Conclusion(s): To the best of our knowledge, this is the first report of scRNA-seq analysis on human AAA. This cutting-edge technique uncovered novel cell clusters and provided a comprehensive understanding of cellular spatiotemporal changes within the AAA wall. Here we provide novel interconnected mechanistic insights into this complex disease to enrich our understanding of AAA development.
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Dystonia is a disabling neurological syndrome characterized by abnormal movements and postures that result from intermittent or sustained involuntary muscle contractions; mutations of DYT1/TOR1A are the most common cause of childhood-onset, generalized, inherited dystonia. Patient and mouse model data strongly support dysregulation of the nigrostriatal dopamine neurotransmission circuit in the presence of the DYT1-causing mutation. To determine striatal medium spiny neuron (MSN) cell-autonomous and non-cell autonomous effects relevant to dopamine transmission, we created a transgenic mouse in which expression of mutant torsinA in forebrain is restricted to MSNs. We assayed electrically evoked and cocaine-enhanced dopamine release and locomotor activity, dopamine uptake, gene expression of dopamine-associated neuropeptides and receptors, and response to the muscarinic cholinergic antagonist, trihexyphenidyl. We found that over-expression of mutant torsinA in MSNs produces complex cell-autonomous and non-cell autonomous alterations in nigrostriatal dopaminergic and intrastriatal cholinergic function, similar to that found in pan-cellular DYT1 mouse models. These data introduce targets for future studies to identify which are causative and which are compensatory in DYT1 dystonia, and thereby aid in defining appropriate therapies.

The mitochondrial inner membrane consists of the inner boundary membrane and invaginations called cristae, which differ in protein composition and likely have distinct functions. In this issue of The EMBO Journal, Wolf et al (2019) report that the cristae carry a higher membrane potential than the intervening boundary membranes. Their data suggest electro-chemical discontinuity among segments of the inner membrane, implying that individual cristae may operate with some degree of independence.