Faculty Bibliography

BACKGROUND:Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium. METHODS:We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. RESULTS:Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and β-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. CONCLUSIONS:Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. Validating the function of these candidates in vivo is challenging, due to low efficiency and low throughput of most model systems. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked-in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. We used this system to identify both cell-autonomous and non-cell autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our high-efficiency genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Aortic stiffening is an inevitable manifestation of chronological aging, yet the mechano-molecular programs that orchestrate region- and layer-specific adaptations along the length and through the wall of the aorta are incompletely defined. Here, we show that the decline in passive cyclic distensibility is more pronounced in the ascending thoracic aorta (ATA) compared to distal segments of the aorta and that collagen content increases in both the medial and adventitial compartments of the ATA during aging. The single-cell RNA sequencing of aged ATA tissues reveals altered cellular senescence, remodeling, and inflammatory responses accompanied by enrichment of T-lymphocytes and rarefaction of vascular smooth muscle cells, compared to young samples. T lymphocyte clusters accumulate in the adventitia, while the activation of mechanosensitive Piezo-1 enhances vasoconstriction and contributes to the overall functional decline of ATA tissues. These results portray the immuno-mechanical aging of the ATA as a process that culminates in a stiffer conduit permissive to the accrual of multi-gerogenic signals priming to disease development.

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.

PURPOSE/OBJECTIVE:The primary goal of this study was to determine the anatomic relationship between the clavicle and the apical lung segment. The secondary goal was to determine the incidence of pneumothorax (PTX) in patients who underwent clavicle ORIF to analyze the utility of postoperative chest radiographs. METHODS:Six hundred thirty-one patients with a midshaft clavicle fracture who underwent superior plating at a single institution were identified. Forty-two patients had a CT scan of the chest. Three points on the uninjured clavicle were defined: 2 cm from the medial end of the clavicle, the mid-point of the clavicle, and 2 cm from the lateral end of the clavicle. At each point, the distance from both the inferior cortex and the superior cortex of the clavicle to the apical lung segment was measured. All 631 patients who underwent Open Reduction and Internal Fixation had a postoperative chest radiograph to evaluate implant placement, restoration of clavicular length, and presence of PTX. RESULTS:From the lateral end of the clavicle, the mean distance of the lung was 60.0 ± 14.9 mm (20.1 to 96.1 mm) from the inferior cortex of the clavicle. At the mid-point, the mean distance of the lung was 32.3 ± 7.2 mm (20.4 to 45.5 mm) from the inferior cortex of the clavicle. At the medial end, the mean distance of the lung was 18.0 ± 5.5 mm (8.1 to 28.9 mm) from the inferior cortex of the clavicle. A review of postoperative radiographs for all 631 patients revealed none (0%) with a postoperative iatrogenic PTX. CONCLUSION/CONCLUSIONS:The risk of injury is minimal in all three zones. Postoperative chest radiographs after clavicle fracture repair to rule out PTX are unnecessary.

Infection and chronic post-traumatic osteomyelitis of the tibia after open fracture are complex problems that cause significant morbidity and threaten the viability of a limb. Therefore, it is of utmost importance for the orthopaedic surgeon to understand both patient and treatment factors that modify the risk of developing these disastrous complications. Infection risk is largely based on severity of open injury in addition to inherent patient factors. Orthopaedic surgeons can work to mitigate this risk with prompt antibiotic administration, thorough and complete debridement, expedient fracture stabilization, and early wound closure. In the case osteomyelitis does occur, the surgeon should use a systematic multidisciplinary approach for eradication.

Although the mechanism for activation of latent TGFβ1 and TGFβ3 is understood to involve the binding of the TGFβ propeptide (LAP) to both an integrin and an insoluble substrate, the activation of latent TGFβ2 has been unclear because the TGFβ2 LAP does not have the classical integrin binding sequence found in the other two TGFβ isoform LAPs. To assess the potential requirement for covalent linkage with a matrix or cell surface protein for the activation of latent TGFβ2, we generated mice in which the TGFβ2 Cys residue predicted to be involved in binding was mutated to Ser (Tgfb2^C24S