Faculty Bibliography

Specific and effective treatments for autism spectrum disorder (ASD) are lacking due to a poor understanding of disease mechanisms. Here we test the idea that similarities between diverse ASD mouse models are caused by deficits in common molecular pathways at neuronal synapses. To do this, we leverage the availability of multiple genetic models of ASD that exhibit shared synaptic and behavioral deficits and use quantitative mass spectrometry with isobaric tandem mass tagging (TMT) to compare their hippocampal synaptic proteomes. Comparative analyses of mouse models for Fragile X syndrome (Fmr1 knockout), cortical dysplasia focal epilepsy syndrome (Cntnap2 knockout), PTEN hamartoma tumor syndrome (Pten haploinsufficiency), ANKS1B syndrome (Anks1b haploinsufficiency), and idiopathic autism (BTBR+) revealed several common altered cellular and molecular pathways at the synapse, including changes in oxidative phosphorylation, and Rho family small GTPase signaling. Functional validation of one of these aberrant pathways, Rac1 signaling, confirms that the ANKS1B model displays altered Rac1 activity counter to that observed in other models, as predicted by the bioinformatic analyses. Overall similarity analyses reveal clusters of synaptic profiles, which may form the basis for molecular subtypes that explain genetic heterogeneity in ASD despite a common clinical diagnosis. Our results suggest that ASD-linked susceptibility genes ultimately converge on common signaling pathways regulating synaptic function and propose that these points of convergence are key to understanding the pathogenesis of this disorder.

Recent advances in volume electron microscopy (vEM) allow unprecedented visualization of the electron-dense structures of cells, tissues and model organisms at nanometric resolution in three dimensions (3D). Light-based microscopy has been widely used for specific localization of proteins; however, it is restricted by the diffraction limit of light, and lacks the ability to identify underlying structures. Here, we describe a protocol for ultrastructural detection, in three dimensions, of a protein (Connexin 43) expressed in the intercalated disc region of adult murine heart. Our protocol does not rest on the expression of genetically encoded proteins and it overcomes hurdles related to pre-embedding and immunolabeling, such as the penetration of the label and the preservation of the tissue. The pre-embedding volumetric immuno-electron microscopy (pre-embedding vIEM) protocol presented here combines several practical strategies to balance sample fixation with antigen and ultrastructural preservation, and penetration of labeling with blocking of non-specific antigen binding sites. The small 1.4 nm gold along with surrounded silver used as a detection marker buried in the sample also serves as a functional conductive resin that significantly reduces the charging of samples. Our protocol also presents strategies for facilitating the successful cutting of the samples during serial block-face scanning electron microscopy (SBF-SEM) imaging. Our results suggest that the small gold-based pre-embedding vIEM is an ideal labeling method for molecular localization throughout the depth of the sample at subcellular compartments and membrane microdomains.

This study compares outcomes of patients with Lisfranc injuries treated with screw only fixation constructs to those treated with dorsal plate and screw constructs. Seventy patients who underwent surgical treatment for acute Lisfranc injury without arthrodesis and minimum 6-month (mean >1-year) follow-up were identified. Demographics, surgical information, and radiographic imaging were reviewed. Cost data were compared. The primary outcome measure was the American Orthopedic Foot and Ankle Surgery (AOFAS) midfoot score. Univariate analysis through independent sample t tests, Mann-Whitney U, and chi-squared compared the populations. Twenty-three (33%) patients were treated with plate constructs and 47 (67%) with screw only fixation. The plate group was older (49 ± 18 vs 40 ± 16 years, p = .029). More screw constructs treated isolated medial column injuries compared to plate constructs (92% vs 65%, p = .006). At latest follow-up (mean 14 ± 13 months), all tarsometatarsal joints were aligned. There was no difference in AOFAS midfoot scores. Plate patients experienced longer operations (131 ± 70 vs 75 ± 31 minutes, p < .001) and tourniquet time (101 ± 41 vs 69 ± 25 minutes, p = .001). Plate constructs were more expensive than screw ($2.3X ± $2.3X vs $X ± $0.4X, p < .001) ($X is the mean cost of screws alone). Plate patients had a higher incidence of wound complications (13% vs 0%, p = .012). Treatment of Lisfranc fracture dislocation injuries with screws only demonstrated a higher value procedure as similar outcomes were found amidst lower implant costs. Screw only fixation required a shorter operative and tourniquet time with less frequent wound complications. Screw only fixations proved mechanically sound enough to achieve goals of repair without inferior outcomes.

The purpose of this study was to determine if the use of peripheral nerve blocks in the operative management of tibial plateau fractures is associated with improved outcomes when compared with the use of spinal and general anesthesia. Over a period of 16 years, 132 patients who underwent operative repair for a low-energy tibial plateau fracture and had at least 12 months of follow-up met the inclusion criteria and formed the basis of this study. Patients were grouped into cohorts based on the anesthetic method used during surgery: peripheral nerve block in combination with conscious sedation or general anesthesia (BA), general anesthesia alone (GA), or spinal anesthesia alone (SA). Outcomes were assessed at 3 months, 6 months, and 12 months. Length of stay was greatest in the GA cohort (P<.05), and more patients in the BA cohort were discharged to home (P<.05). Patients in the GA cohort had the highest pain scores at 3 months and 6 months (P<.05). Patients in both the SA and BA cohorts had better Short Musculoskeletal Function Assessment scores at 6 and 12 months when compared with the GA cohort (P<.05). Although knee range of motion did not differ among the three cohorts at 3 months, it did differ at 6 months and 12 months postoperatively, with those who had a preoperative nerve blockade (SA and BA) having the greatest knee range of motion (P<.05). Regional anesthesia was safe and was associated with lower pain scores in the early postoperative period and greater knee range of motion and functional outcome scores in the late postoperative period. [Orthopedics. 2023;46(6):358-364.].

The development of single cell approaches has facilitated the investigation of cellular heterogeneity and cell type-specific gene expression in complex tissues. Adipose tissue depots contain lipid storing adipocytes as well as a diverse array of cell types that form the adipocyte niche and regulate adipose tissue function. Here, I describe two protocols for the isolation of single cells and nuclei from white and brown adipose tissue. Additionally, I provide a detailed workflow for isolation of cell type- or lineage-specific single nuclei using nuclear tagging and translating ribosome affinity purification (NuTRAP) mouse models.

Loss of bone is a common medical problem and, while it can be treated with available therapies, some of these therapies have critical side effects. We have previously demonstrated that CGS21680, a selective A_2A adenosine receptor agonist, prevents bone loss, but its on-target toxicities (hypotension, tachycardia) and frequent dosing requirements make it unusable in the clinic. We therefore generated a novel alendronate-CGS21680 conjugate (MRS7216), to target the agonist to bone where it remains for long periods thereby diminishing the frequency of administration and curtailing side effects. MRS7216 was synthesized from CGS21680 by sequential activation of the carboxylic acid moiety and reacting with an appropriate amino acid (PEG, alendronic acid) under basic conditions. MRS7216 was tested on C57BL/6J (WT) mice with established osteoporosis (OP) and WT or A2A KO mice with wear particle-induced inflammatory osteolysis (OL). Mice were treated weekly with MRS7216 (10mg/kg). Bone formation was studied after in vivo labeling with calcein/Alizarin Red, and μCT and histology analyses were performed. In addition, human primary osteoblasts and osteoclasts were cultured using bone marrow discarded after hip replacement. Receptor binding studies demonstrate that MRS7216 efficiently binds the A2A adenosine receptor. MRS7216-treated OP and OL mice had significant new bone formation and reduced bone loss compared to vehicle or alendronate-treated mice. Histological analysis showed that MRS7216 treatment significantly reduced osteoclast number and increased osteoblast number in murine models. Interestingly, cultured human osteoclast differentiation was inhibited, and osteoblast differentiation was stimulated by the compound indicating that MRS7216 conjugates represent a novel therapeutic approach to treat osteoporosis and osteolysis.

Abdominal aortic aneurysms (AAA) is a multifactorial complex disease with life-threatening consequences. While Genome-wide association studies (GWAS) have revealed several single nucleotide polymorphisms (SNPs) located in the genome of individuals with AAA, the link between SNPs with the associated pathological signals, the influence of risk factors on their distribution and their combined analysis is not fully understood. We integrated 86 AAA SNPs from GWAS and clinical cohorts from the literature to determine their phenotypical vulnerabilities and association with AAA risk factors. The SNPs were annotated using snpXplorer AnnotateMe tool to identify their chromosomal position, minor allele frequency, CADD (Combined Annotation Dependent Depletion), annotation-based pathogenicity score, variant consequence, and their associated gene. Gene enrichment analysis was performed using Gene Ontology and clustered using REVIGO. The plug-in GeneMANIA in Cytoscape was applied to identify network integration with associated genes and functions. 15 SNPs affecting 20 genes with a CADD score above ten were identified. AAA SNPs were predominantly located on chromosome 3 and 9. Stop-gained rs5516 SNP obtained high frequency in AAA and associated with proinflammatory and vascular remodeling phenotypes. SNPs presence positively correlated with hypertension, dyslipidemia and smoking history. GO showed that AAA SNPs and their associated genes could regulate lipid metabolism, extracellular matrix organization, smooth muscle cell proliferation, and oxidative stress, suggesting that part of these AAA traits could stem from genetic abnormalities. We show a library of inborn SNPs and associated genes that manifest in AAA. We uncover their pathological signaling trajectories that likely fuel AAA development.