Faculty Bibliography

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.

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:.

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.

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.

Objectives: Smoking is the commonest cause of oral cavity squamous cell carcinoma (OC-SCC), but the etiology of OC-SCC in nonsmokers is unknown. Our primary goal was to use metagenomic shotgun sequencing (MSS) to define the taxonomic composition and functional potential of oral metagenome in nonsmokers with OC-SCC. Methods: We conducted a case"“control study with 42 OC-SCC case and 45 control nonsmokers. MSS was performed on DNA extracted from mouthwash samples. Taxonomic analysis and pathway analysis were done using MetaPhlAn2 and HUMAnN2, respectively. Statistical difference was determined using the Mann"“Whitney test controlling false discovery rate. Results: There was no significant difference in age, sex, race, or alcohol consumption between OC-SCC and control patients. There was a significant difference in beta diversity between OC-SCC and controls. At the phylum level, Bacteroidetes and Synergistetes were overly represented in OC-SCC while Actinobacteria and Firmicutes were overly represented in controls. At the genus level, Fusobacterium was overly represented in OC-SCC compared with controls, while Corynebacterium, Streptococcus, Actinomyces, Cryptobacterium, and Selenomonas were overly represented in controls. Bacterial pathway analysis identified overrepresentation in OC-SCC of pathways related to metabolism of flavin, biotin, thiamin, heme, sugars, fatty acids, peptidoglycans, and tRNA and overrepresentation of nucleotides and essential amino acids in controls. Conclusions: The oral microbiome in nonsmoker patients with OC-SCC is significantly different from that of nonsmoker control patients in taxonomic compositions and functional potentials. Our study"™s MSS findings matched with previous 16S-based methods in taxonomic differentiation but varied greatly in functional differentiation of microbiomes in OC-SCC and controls.

INTRODUCTION/BACKGROUND:The purpose of this study was to assess how quality and volume of common orthopaedic care varies across private, municipal, and federal healthcare delivery systems (HDSs). METHODS:Hip and knee arthroplasty, knee and shoulder arthroscopy, and hip fracture repair were audited over a two-year period. Electronic medical records were reviewed for demographics, diagnosis, lengths of stay (LoSs), surgical wait times, inpatient complication, readmission, and revision surgery rates. Multivariate regression controlled for differences in age, sex, diagnosis, and Charlson Comorbidity Index to determine how HDS correlated with surgical wait time, length of stay, complication rates, readmission, and revision surgery. RESULTS:The 5,696 included patients comprise 87.4% private, 8.6% municipal, and 4.0% federal HDSs. Compared with private HDS for arthroplasty, federal surgical wait times were 18 days shorter (95% CI = 9 to 26 days, P < 0.001); federal LoS was 4 days longer (95% CI = 3.6 to 4.3 days, P < 0.001); municipal LoS was 1 day longer (95% CI = 0.8 to 1.4, P < 0.001); municipal 1-year revision surgery odds were increased (odds ratio [OR] = 2.8, 95% CI = 1.3 to 5.4, P = 0.045); and complication odds increased for municipal (OR = 12.2, 95% CI = 5.2 to 27.4, P < 0.001) and federal (OR = 12.0, 95% CI = 4.5 to 30.8, P < 0.001) HDSs. Compared with private HDS for arthroscopy, municipal wait times were 57 days longer (95% CI = 48 to 66 days, P < 0.001) and federal wait times were 34 days longer (95% CI = 21 to 47 days, P < 0.001). Compared with private HDS for fracture repair, municipal wait times were 0.6 days longer (95% CI = 0.2 to 1.0, P = 0.02); federal LoS was 7 days longer (95% CI = 3.6 to 9.4 days, P < 0.001); and municipal LoS was 4 days longer (95% CI = 2.4 to 4.8, P < 0.001). Only private HDS fracture repair patients received bone health consultations. DISCUSSION/CONCLUSIONS:The private HDS provided care for a markedly larger volume of patients seeking orthopaedic care. In addition, private HDS patients experienced reduced surgical wait times, LoSs, and complication odds for inpatient elective cases, with better referral patterns for nonsurgical orthopaedic care after hip fractures within the private HDS. These results may guide improvements for federal and municipal HDSs.

BACKGROUND:The quality of evidence of the orthopedic literature has been often called into question. The fragility index (FI) has emerged as a means to evaluate the robustness of a significant result. Similarly, reverse fragility index (RFI) can be used for nonsignificant results to evaluate whether one can confidently conclude that there is no difference between groups. The analysis of FI and RFI in proximal humerus fracture (PHF) management is of particular interest, given ongoing controversy regarding optimal management and patient selection. The aim of this study was to report the FI, RFI and quality of the evidence in the proximal humerus fracture literature. METHODS:A systematic review was conducted based on the PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines, which utilized EMBASE, MEDLINE and Cochrane Library databases. Inclusion criteria included randomized controlled clinical trials related to the management of proximal humerus fractures, published from 2000 to 2020 with dichotomous outcome measures and 1:1 allocation. The FI and RFI were calculated by successively changing one nonevent to an event for each outcome measure until the result was made nonsignificant or significant, respectively. The fragility quotient, (FQ), calculated by dividing the FI by the total sample size, was calculated as well. RESULTS:There were 25 studies that met our criteria with 48 outcome measures recorded. A total of 21 studies had at least one fragile result, with ten studies including a fragile result in the conclusion of the abstract. A total of 31 outcome measures had nonsignificant results and the median RFI was found to be 4, with 71% greater than number of patients lost to follow up. Seventeen outcomes had significant results, with a median FI of 1, with 65% greater than or equal to the number patients lost to follow up. A total of 18 of 25 studies (72%) included a power analysis. In particular, ten studies reported a statistical analysis of complication rates, 90% of which were fragile. The median FQ was found to be 0.037. CONCLUSIONS:The literature on PHF management is frequently fragile. Outcome measures are often fragile, particularly with regards to comparing complication rates and reoperation rates in treatment arms. Comparing to the studies in other subspecialties PHF RCTs are relatively more fragile and underpowered. Standardized reporting of FI, FQ and RFI can help the reader to reliably draw conclusions based on the fragility of outcome measures.

BACKGROUND AND AIMS/OBJECTIVE:Atherosclerosis is widely accepted to be an inflammatory disease driven by lipid accumulation and leukocyte recruitment. More recently, galectins, a family of β-galactoside binding proteins, have been shown to play a role in leukocyte recruitment among other immunomodulatory functions. Galectin (Gal) -9, a tandem repeat type galectin expressed by the endothelium in inflammatory environments, has been proposed to promote leukocyte recruitment. However, the role of Gal-9 in the context of monocyte recruitment remains elusive. METHODS AND RESULTS/RESULTS:monocytes in a β2-integrin and glycan dependent manner, while adhesion of monocytes to stimulated endothelium is reduced when Gal-9 is knocked down. Gal-9 also facilitates enhanced recruitment of leukocytes from peripheral arterial disease (PAD) patients compared to healthy young and aged controls. We further characterise the endothelium as source of circulating Gal-9, which is increased in plasma of PAD patients compared to healthy controls. CONCLUSIONS:These results highlight a pathological role for Gal-9 as promoter of monocyte recruitment and atherosclerotic plaque progression, making it a novel target in the prevention of plaque formation and progression.