Faculty Bibliography

BACKGROUND:Plakophilin-2 (PKP2) is a component of the desmosome. Pathogenic variants can lead to arrhythmogenic cardiomyopathy (PKP2-ACM). In PKP2-ACM patients, exercise and catecholamine surges negatively impact arrhythmia incidence and severity. OBJECTIVE:To characterize remodeling of the sympathetic input and adrenergic response in hearts of PKP2-deficient mice (PKP2cKO) subjected to endurance exercise. METHODS:transient dynamics. Separately, we evaluated distribution of sympathetic terminals in PKP2cKO trained hearts vs controls. RESULTS:Exercise led to increased abundance of sarcolemma β1-ARs in control, and decreased abundance in PKP2cKO-myocytes. OCT3 knockdown drastically reduced the response of trained PKP2cKO-myocytes to norepinephrine but not isoproterenol, indicating preserved response to native catecholamines by intracellular (dyad-associated) receptors in the setting of a reduced sarcolemma pool. In tissue, we observed reduced abundance of sympathetic terminals, and heterogeneous distribution across the myocardium. CONCLUSION/CONCLUSIONS:Endurance exercise in PKP2-deficient myocytes leads to reduced pool of functional β1-ARs in the sarcolemma and yet availability of intracellular receptors, which can activate selected (and heterogeneous) routes of intracellular signaling cascades. We speculate that remodeling of nerve terminals affects sympathetic input distribution and hence, regional modulation of excitability and conduction. These changes can facilitate cell-generated triggered activity and heterogeneity of the underlying substrate, setting the stage for life-threatening arrhythmias.

Directional interactions that generate regular coordination geometries are a powerful means of guiding molecular and colloidal self-assembly, but implementing such high-level interactions with proteins remains challenging due to their complex shapes and intricate interface properties. Here we describe a modular approach to protein nanomaterial design inspired by the rich chemical diversity that can be generated from the small number of atomic valencies. We design protein building blocks using deep learning-based generative tools, incorporating regular coordination geometries and tailorable bonding interactions that enable the assembly of diverse closed and open architectures guided by simple geometric principles. Experimental characterization confirms the successful formation of more than 20 multicomponent polyhedral protein cages, two-dimensional arrays and three-dimensional protein lattices, with a high (10%-50%) success rate and electron microscopy data closely matching the corresponding design models. Due to modularity, individual building blocks can assemble with different partners to generate distinct regular assemblies, resulting in an economy of parts and enabling the construction of reconfigurable networks for designer nanomaterials.

INTRODUCTION/BACKGROUND:Several types of fifth metatarsal (MT) fractures exist and are treated with various methods of immobilization, weight bearing restrictions, and occasionally operative procedures. This study evaluated the differences in clinical and radiographic outcomes among pseudo-Jones fractures (Zones 1 and 2), true Jones fractures (Zone 3), and fifth metatarsal shaft and neck fractures. METHODS:A retrospective review of a consecutive series of patients presenting to a single academic medical center with a fifth metatarsal fracture between 2012 and 2022 was conducted. Radiographs obtained at the initial presentation were reviewed, and fracture patterns were categorized as either Zone 1, Zone 2, Zone 3, shaft, neck, or head fractures. RESULTS:In total, 1314 patients with isolated fifth metatarsal fractures were treated (mean age = 49.6 ± 18.0 years). In total, 1217 fractures (92.5%) were initially treated nonoperatively, and 97 fractures (7.5%) were treated operatively. The overall time to clinical and radiographic healing for all fifth metatarsal fractures treated nonoperatively was 9.9 ± 8.7 weeks and 17.9 ± 15.6 weeks, respectively (P = .245, P = .088). Immediate weightbearing led to a faster time to clinical healing by (P = .035). There was no statistically significant difference in time to clinical or radiographic union among the different fracture types (P = .496, P = .400). Likewise, there was no evidence of any difference in time to clinical or radiographic union for patients treated operatively versus nonoperatively (P > .05). CONCLUSION/CONCLUSIONS:.

While weight loss is highly recommended for those with obesity, >60% will regain their lost weight. This weight cycling is associated with elevated risk of cardiovascular disease, relative to never having lost weight. How weight loss/regain directly influence atherosclerotic inflammation is unknown. Thus, we studied short-term caloric restriction (stCR) in obese hypercholesterolemic mice, without confounding effects from changes in diet composition. Weight loss was found to promote atherosclerosis resolution independent of plasma cholesterol. From single-cell RNA-sequencing and subsequent mechanistic studies, this can be partly attributed to a unique subset of macrophages accumulating with stCR in epididymal white adipose tissue (eWAT) and atherosclerotic plaques. These macrophages, distinguished by high expression of Fcgr4, help to clear necrotic cores in atherosclerotic plaques. Conversely, weight regain (WR) following stCR accelerated atherosclerosis progression with disappearance of Fcgr4+ macrophages from eWAT and plaques. Furthermore, WR caused reprogramming of immune progenitors, sustaining hyper-inflammatory responsiveness. In summary, we have developed a model to investigate the inflammatory effects of weight cycling on atherosclerosis and the interplay between adipose tissue, bone marrow, and plaques. The findings suggest potential approaches to promote atherosclerosis resolution in obesity and weight cycling through induction of Fcgr4+ macrophages and inhibition of immune progenitor reprogramming.

The Drosophila cell-adhesion molecule Sidekick is a key component of tricellular adherens junctions in epithelia and localizes to specific synaptic layers in the optic lobes. Using mutagenesis of endogenous Sidekick, we showed that its enrichment at apical tricellular junctions and its function in cell rearrangement require its fifth and sixth immunoglobulin domains, but not the first four, although these mediate homophilic adhesion of mammalian Sidekick homologues. The C-terminal PDZ-binding motif of Sidekick contributes to localizing both Sidekick and its intracellular binding partner Canoe to tricellular adherens junctions. We found that the PAK4 homologue Mushroom bodies tiny also binds to the cytoplasmic domain of Sidekick. Its kinase activity is necessary for Sidekick accumulation at tricellular junctions, and over-activity mislocalizes Sidekick along bicellular junctions. However, mutating predicted Mushroom bodies tiny phosphorylation sites in Sidekick itself did not affect its localization. Sidekick localizes to the dendrites of T4 and T5 neurons independently of its extracellular and PDZ-binding domains, and of Mushroom bodies tiny. Our findings reveal an important role for a PAK4 family member in establishing specialized structures at tricellular adherens junctions.

Adipose tissue has emerged as a central regulator of human physiology, with its dysfunction driving the global rise in obesity-associated diseases, such as type 2 diabetes, cardiovascular and liver diseases, and several cancers. Once thought to be inert, adipocytes are now recognized as dynamic, responsive cells essential for energy homeostasis and interorgan communication, including the brain. Distinct adipose depots support specialized functions across development, sex, and aging. Technologies like single-cell RNA sequencing are unraveling depot-specific mechanisms, with the potential of identifying new therapeutic targets. This review highlights major scientific advancements leading to our current appreciation of the pivotal role of adipose tissue in health and disease. Many key discoveries in this field have been catalyzed by National Institutes of Health funding, particularly through the National Institute of Diabetes, Digestive and Kidney Diseases, now celebrating its 75th anniversary.

Targeted therapies and immunotherapy have improved treatment outcomes for many melanoma patients. However, patients whose melanomas harbor driver mutations in the neurofibromin 1 (NF1) tumor suppressor gene often lack effective targeted treatment options when their tumors do not respond to immunotherapy. In this study, we utilized patient-derived short-term cultures (STCs) and multiomics approaches to identify molecular features that could inform the development of therapies for patients with NF1 mutant melanoma. Differential gene expression analysis revealed that the epidermal growth factor receptor (EGFR) is highly expressed and active in NF1 mutant melanoma cells, where it hyper-activates ERK and AKT, leading to increased tumor cell proliferation, survival, and growth. In contrast, genetic or pharmacological inhibition of EGFR hindered cell proliferation and survival and suppressed tumor growth in patient-derived NF1 mutant melanoma models but not in NF1 wild-type models. These results reveal a connection between NF1 loss and increased EGFR expression that is critical for the survival and growth of NF1 mutant melanoma cells in patient-derived culture and xenograft models, irrespective of their BRAF and NRAS mutation status.

PURPOSE/OBJECTIVE:The purpose of this study is to examine the effect of age on outcomes following repair of acute displaced patella fractures Methods: 248 patients who sustained a displaced patella fracture and underwent open reduction and internal fixation were identified. Patients included underwent a similar operative protocol, were prescribed a standard post-operative protocol of therapy, and were seen at standard follow-up intervals. Patients were divided into groups of < 65 years old (young) and ≥ 65 years old (older). Statistical analysis was run to determine if there was a significant difference in range of knee motion and rate of major complications. RESULTS:Of the 248 patients, 149 were young and 99 were older. The mean age of the older group was 74.5 ± 6.7 and the mean age of the young group was 50 ± 12. Fracture pattern and BMI were similar the groups, however the older group had a higher average CCI (p<0.001). Additionally, the groups had similar length of follow up (p=0.693) and similar mean time to radiographic healing (p=0.533). Older patients had limited knee extension at 6 months (compared young patients (p=0.031). Finally, older patients had a higher rate of all complications compared to young patients. Two percent of older patients developed a fracture related infection (FRI), 4% developed a symptomatic nonunion and 11% were underwent re-operation including removal of hardware, total knee replacement, irrigation and debridement and manipulation under anesthesia. CONCLUSION/CONCLUSIONS:Complication rates following patella fracture fixation in older patients were higher than young patients, despite having similar injury patterns, surgical treatment and follow up. These findings can better inform treating physicians during surgical intervention of older patients with patella fractures.

Decreased brain levels of coenzyme Q₁₀ (CoQ₁₀), an endogenously synthesized lipophilic antioxidant^1,2, underpin encephalopathy in primary CoQ₁₀ deficiencies^3,4 and are associated with common neurodegenerative diseases and the ageing process^5,6. CoQ₁₀ supplementation does not increase CoQ₁₀ pools in the brain or in other tissues. The recent discovery of the mammalian CoQ₁₀ headgroup synthesis pathway, in which 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) makes 4-hydroxymandelate (4-HMA) to synthesize the CoQ₁₀ headgroup precursor 4-hydroxybenzoate (4-HB)⁷, offers an opportunity to pharmacologically restore CoQ₁₀ synthesis and mechanistically treat CoQ₁₀ deficiencies. To test whether 4-HMA or 4-HB supplementation promotes CoQ₁₀ headgroup synthesis in vivo, here we administered 4-HMA and 4-HB to Hpdl^-/- mice, which model an ultra-rare, lethal mitochondrial encephalopathy in humans. Both 4-HMA and 4-HB were incorporated into CoQ₉ and CoQ₁₀ in the brains of Hpdl^-/- mice. Oral treatment of Hpdl^-/- pups with 4-HMA or 4-HB enabled 90-100% of Hpdl^-/- mice to live to adulthood. Furthermore, 4-HB treatment stabilized and improved the neurological symptoms of a patient with progressive spasticity due to biallelic HPDL variants. Our work shows that 4-HMA and 4-HB can modify the course of mitochondrial encephalopathy driven by HPDL variants and demonstrates that CoQ₁₀ headgroup intermediates can restore CoQ₁₀ synthesis in vivo.