Faculty Bibliography

Cocaine, an addictive psychostimulant, has a broad mechanism of action, including the induction of a wide range of alterations in brain metabolism and mitochondrial homeostasis. Our group recently identified a subpopulation of non-microvesicular, non-exosomal extracellular vesicles of mitochondrial origin (mitovesicles) and developed a method to isolate mitovesicles from brain parenchyma. We hypothesised that the generation and secretion of mitovesicles is affected by mitochondrial abnormalities induced by chronic cocaine exposure. Mitovesicles from the brain extracellular space of cocaine-administered mice were enlarged and more numerous when compared to controls, supporting a model in which mitovesicle biogenesis is enhanced in the presence of mitochondrial alterations. This interrelationship was confirmed in vitro. Moreover, cocaine affected mitovesicle protein composition, causing a functional alteration in mitovesicle ATP production capacity. These data suggest that mitovesicles are previously unidentified players in the biology of cocaine addiction and that target therapies to fine-tune brain mitovesicle functionality may be beneficial to mitigate the effects of chronic cocaine exposure.

Conventional synthetic vascular grafts are associated with significant failure rates due to their mismatched mechanical properties with the native vessel and poor regenerative potential. Though different tissue engineering approaches have been used to improve the biocompatibility of synthetic vascular grafts, it is still crucial to develop a new generation of synthetic grafts that can match the dynamics of native vessel and direct the host response to achieve robust vascular regeneration. The size of pores within implanted biomaterials has shown significant effects on macrophage polarization, which has been further confirmed as necessary for efficient vascular formation and remodeling. Here, we developed biodegradable, autoclavable synthetic vascular grafts from a new polyurethane elastomer and tailored the grafts' interconnected pore sizes to promote macrophage populations with a pro-regenerative phenotype and improve vascular regeneration and patency rate. The synthetic vascular grafts showed similar mechanical properties to native blood vessels, encouraged macrophage populations with varying M2 to M1 phenotypic expression, and maintained patency and vascular regeneration in a one-month rat carotid interposition model and in a four-month rat aortic interposition model. This innovative bioactive synthetic vascular graft holds promise to treat clinical vascular diseases.

In CM238, 76% of patients (pts) with high-risk resected melanoma (MEL) treated with adjuvant NIVO were alive at 5 years, but there is limited RW data to validate these findings in a similar patient population. This study compared the OS of pts treated with adjuvant NIVO in CM238 vs. RW pts. Pts with stage III resected MEL (AJCC 8th edition) who received adjuvant NIVO were selected from CM238 and the nationwide Flatiron Health electronic health record-derived de-identified database (the RW cohort). Pts in the RW cohort were required to meet the eligibility criteria in CM238 where possible. OS/real-world OS (rwOS) were evaluated using Kaplan-Meier methods and compared using Cox proportional hazards models after adjusting for key prognostic baseline variables (age, sex, race, disease stage, time from surgical resection to index date, ECOG, and comorbidities) between the two cohorts. In the RW cohort, 492 pts with resected stage III MEL received adjuvant NIVO and 320 pts met the eligibility criteria of CM238. Compared with pts in the CM238 cohort, pts in the RW cohort were older (median: 64 vs. 56 years), heavier (91 vs. 82 kg), had a higher proportion of diabetes (9% vs. 6%) and ECOG PS 1 (17% vs. 10%), and had slightly more stage IIIA (4% vs. 1%), less stage IIIB (29% vs. 32%), and similar stage IIIC (63% vs. 63%) and stage IIID (4% vs. 5%). Before adjustment, pts in the RW cohort had a higher risk of mortality compared with pts in the CM238 cohort (HR: 1.46; 95% CI, 1.00 to 2.13; p < 0.05). After adjusting for differences in key patient characteristics, OS was similar between the two cohorts (HR: 1.12; 95% CI, 0.68 to 1.84, p = 0.66). In conclusion, pts with stage III resected MEL treated with adjuvant NIVO in the RW setting had OS similar to those in CM238 after adjusting for key prognostic factors

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.

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.

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.

BACKGROUND/UNASSIGNED:Locking plate technology was developed approximately 25-years-ago and has been successfully used since. Newer designs and material properties have been used to modify the original design, but these changes have yet to be correlated to improved patient outcomes. The purpose of this study was to evaluate the outcomes of first-generation locking plate (FGLP) and screw systems at our institution over an 18 year period. METHODS/UNASSIGNED:Between 2001 to 2018, 76 patients with 82 proximal tibia and distal femur fractures (both acute fracture and nonunions) who were treated with a first-generation titanium, uniaxial locking plate with unicortical screws (FGLP), also known as a LISS plate (Synthes Paoli Pa), were identified and compared to 198 patients with 203 similar fracture patterns treated with 2nd and 3rd generation locking plates, or Later Generation Locking Plates (LGLP). Inclusion criteria was a minimum of 1-year follow-up. At latest follow-up, outcomes were assessed using radiographic analysis, Short Musculoskeletal Functional Assessment (SMFA), VAS pain scores, and knee ROM. All descriptive statistics were calculated using IBM SPSS (Armonk, NY). RESULTS/UNASSIGNED:A total of 76 patients with 82 fractures had a mean 4-year follow-up available for analysis. There were 76 patients with 82 fractures fixed with a First-generation locking plate. The mean age at time of injury for all patients was 59.2 and 61.0% were female. Mean time to union for fractures about the knee fixed with FGLP was by 5.3 months for acute fractures and 6.1 months for nonunions. At final follow-up, the mean standardized SMFA for all patients was 19.9, mean knee range of motion was 1.6°-111.9°, and mean VAS pain score was 2.7. When compared to a group of similar patients with similar fractures and nonunions treated with LGLPs there were no differences in outcomes assessed. CONCLUSION/UNASSIGNED:.

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.].