Faculty Bibliography

Over the past decade, the field of human embryo editing has witnessed remarkable advancements and triggered significant ethical debates. The groundbreaking tool, CRISPR/Cas9, has revolutionized the landscape of genetic engineering by enabling modifications at the genomic level in germ cells. Since the first case of human embryo gene editing in 2015, the field has rapidly progressed, presenting promising avenues for therapeutic interventions. However, it still grapples with safety concerns, including off-target effects, mosaicism, and the long-term impacts of genetic alterations, as well as ongoing ethical controversies. In this review, we will systematically overview the significant research in this field and provide insights into the potential applications of basic research in early embryonic development and the treatment of genetic diseases.

Aneuploidy is common in cancer and has been implicated in promoting tumor progression, yet the underlying mechanisms remain poorly understood. By generating models of aneuploidy, we found that aneuploidy confers resistance to reactive oxygen species (ROS)-mediated cell death, independent of the specific chromosomes gained or lost. Mechanistically, poly(ADP-ribose) polymerase 1 (PARP1) is suppressed in aneuploid cells, which inhibits PARP1-mediated cell death (parthanatos). We validated aneuploidy-associated PARP1 suppression across 15 cell models and human tumors, with pronounced effects in metastatic tumors. Importantly, PARP1 downregulation promotes tumor metastasis while PARP1 upregulation suppresses it. Through a genome-wide CRISPR screen and functional validation, we identified the transcription factor CCAAT/enhancer-binding protein beta (CEBPB) as a mediator of PARP1 downregulation and ROS resistance in aneuploid cells. Lysosomal dysfunction serves as the upstream activator of CEBPB in aneuploid cells. We propose that aneuploidy-driven CEBPB activation suppresses PARP1, fostering ROS resistance and cancer progression.

LRP2 (Megalin or low-density lipoprotein-related receptor 2), together with Cubilin and Amnionless, is responsible for binding and internalizing a wide range of nutrients and toxins from the kidney's glomerular filtrate by endocytosis. Accordingly, Lrp2 deletion or mutation results in the loss of these ligands into the urine. Yet Lrp2 is essential not only for receptor-mediated but also for fluid-phase endocytosis, implicating a broader role beyond ligand binding. To identify the linkage between Lrp2 and endocytosis, we engineered Lrp2-APEX2-expressing mice and performed biotinylation in vivo to label Lrp2's cytoplasmic partners. We demonstrated the specificity and sensitivity of this technique by mass spectrometric identification of biotinylated proteins from kidney lysate and immunostaining kidney sections. We identified critical endocytic regulators interacting with Lrp2, but also many proteins functionally associated with endocytosis that are not already known to interact with Lrp2. These data suggest that Lrp2 plays a central role in organizing apical membranes through PDZ domain proteins and engages with regulators and molecular motors during endocytosis. These interactions are abolished in the absence of Lrp2.

BACKGROUND:are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy. This study tests whether plakophilin-2 (PKP2) deficiency only in cardiomyocytes is sufficient to provoke premature aging and proinflammatory senescence in nonmyocyte, cardiac resident cells. METHODS:We studied mice with cardiomyocyte-specific, tamoxifen-activated loss of PKP2 (cardiomyocyte-specific conditional knockout of plakophilin-2) using conventional and multiplex imaging, cytokine arrays, epigenetic clocks, spatial transcriptomics, expansion and structured illumination microscopy, and correlative data analysis. We examined nonmyocytes and cardiomyocytes for premature aging and senescence. RESULTS:We observed senescence-associated heterochromatin foci in nonmyocytes, predominantly in cells positive for α-smooth muscle actin staining. Cytokines in media of nonmyocyte cells were consistent with senescence-associated secretory phenotype. Epigenetic clocks identified premature aging. Multiplex immunohistochemistry showed nonmyocyte cells in niches, intermingled with cardiomyocytes. Spatial transcriptomics showed overrepresentation of senescence-associated secretory phenotype-related transcripts, predominantly in myocyte-rich areas of the left ventricle. Senescence-associated heterochromatin foci and increased epigenetic age were not found in cardiomyocytes from cardiomyocyte-specific conditional knockout of plakophilin-2 hearts, although we observed structural features associated with premature aging. Cross-reference analysis showed correlation between the cardiomyocyte-specific conditional knockout of plakophilin-2 cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. CONCLUSIONS:Loss of PKP2 expression only in adult cardiac myocytes is sufficient to induce proinflammatory senescence in nonmyocytes, and overall premature cardiac aging. This is the first study to intersect cellular senescence and premature aging with desmosomal arrhythmogenic cardiomyopathies. We speculate that cell-agnostic molecular signatures, biomarkers, and pharmacology of senescence and of neurodegenerative diseases may be relevant to diagnose or treat PKP2 arrhythmogenic right ventricular cardiomyopathy.

During development and homeostasis, tissues move and rearrange to form organs, seal wounds, or-in the case of cancer-spread in the body. To accomplish this, cells in tissues need to communicate with each other, generate force to push themselves forward, and know where to go to-all of this with little to no error. Here, we discuss how a migrating tissue-the zebrafish posterior lateral line primordium-solves these challenges. We focus on the strategies that ensure signaling within the tissue, enable the tissue to generate and transmit force to its substrate for propulsion, and allow robust directional sensing and migration by the tissue. These strategies include facilitated diffusion and ligand trapping for focal signaling, a self-generated attractant gradient for long-distance migration, clamping of the attractant concentration to the attractant receptor's K

Vascularized composite allotransplantation (VCA) is increasingly recognized as a reconstructive option that offers substantial benefits for patients with severe tissue loss, including injuries affecting the face and hand. Despite its clinical promise, VCA remains limited by surgical complexity, the need for long-term immunosuppression, and associated morbidity. Preclinical models are therefore essential for refining surgical techniques, testing novel immunomodulatory approaches, and investigating ischemia/reperfusion injury. Compared with large-animal models, rodent models offer advantages in accessibility and cost while adhering more closely to the 3Rs principles of Replacement, Reduction, and Refinement. Among them, the mouse offers unique advantages, including the availability of humanized strains, a broad panel of well-characterized antibodies, and compatibility with advanced immunological assays. These features make the mouse particularly valuable for translational VCA research. Murine VCA, however, requires demanding supermicrosurgical skills, as vascular anastomoses are performed on vessels measuring approximately 0.3-0.5 mm in diameter. To enhance reproducibility, we provide technical tips that have proven critical to the success of these procedures. These include the insertion of a nylon filament into the vessel lumen to stabilize the anastomosis, careful separation of the artery and vein over a sufficient distance along the pedicle, precise anesthetic dosing, and specific maneuvers to prevent pedicle twisting. We also highlight common pitfalls and errors, offering practical guidance to improve outcomes. Here, we describe a comprehensive, stepwise protocol for mouse heterotopic hindlimb-to-neck transplantation, accompanied by instructional video material. This non-functional model minimizes postoperative morbidity compared with orthotopic transplantation while providing robust and reproducible results. It is ideally suited for training in supermicrosurgery and for addressing key experimental questions in VCA, including immune tolerance, graft preservation, and ischemia/reperfusion mechanisms. This methodology provides investigators with a reliable murine platform to advance translational VCA research and to develop innovative strategies aimed at improving outcomes in reconstructive transplantation.

OBJECTIVES/OBJECTIVE:This study evaluated the impact of prolonged glucagon-like peptide-1 (GLP-1) receptor agonist use on postoperative outcomes, including radiographic post-traumatic osteoarthritis (PTOA), fracture nonunion, and final knee range of motion-following operative management of tibial plateau fractures across multiple BMI strata. METHODS:A retrospective cohort study was conducted at an urban academic institution, including patients who underwent surgical fixation for tibial plateau fractures between 2016-2024, with a ≥6 months follow-up. The GLP-1 cohort consisted of patients with documented long-term GLP-1 use pre- and postoperatively. GLP-1 users (Group A, n=24) were compared to three non-GLP-1 cohorts stratified by BMI: Group B (BMI 18.5-25, n=150), Group C (BMI 25-30, n=150), and Group D (BMI ≥30, n=100). Outcomes included Kellgren-Lawrence osteoarthritis grade, post-reduction fracture angulation, articular step-off, Charlson Comorbidity Index (CCI), fracture complications (infection, nonunion, PTOA, revision surgery), and final knee flexion range of motion (ROM). Statistical analyses used SPSS Statistics version 29.0 (IBM Corp., Armonk, NY) with ANOVA and Chi-square tests. RESULTS:Mean follow-up was 28.83 months. Baseline age, CCI, fracture angulation, and step-off were comparable between groups. Pre-injury osteoarthritis severity was higher in Group A (0.96±0.88) than in Groups B (0.68±0.86), C (0.54 ± 0.75), and D (0.78±0.74) (p<0.001). Radiographic PTOA incidence was highest in Group D (32%, p<0.01), while Group A rates were comparable to Groups B and C (p≈0.62). Final knee flexion ROM differed significantly (p<0.01), with Group D showing the lowest mobility (119.08±16.47°). Nonunion rates were significantly higher in Group A (p<0.01). CONCLUSIONS:Among obese patients, GLP-1 receptor agonist use was associated with a lower incidence of PTOA and preserved knee ROM compared to untreated obese individuals, with outcomes similar to non-obese patients. However, GLP-1 use was also linked to increased nonunion rates. These findings suggest that while GLP-1 therapy may mitigate obesity-related joint degeneration, it may also challenge fracture healing.

Vascularized composite allotransplantation (VCA) involves immunologically heterogeneous tissues with a high incidence of acute rejection. Reliable and timely detection of rejection onset remains a major unmet challenge in VCA management. This longitudinal exploratory case study assessed blood- and tissue-derived biomarkers for acute rejection monitoring in a full-face and bilateral hand transplant recipient over 4.6 years. Of these biomarkers, donor-derived cell-free DNA (dd-cfDNA) and short tandem repeats (STR) showed trends toward elevated recipient levels during acute rejection, though differences were not statistically significant. CD8⁺ T-cell percentages increased before acute rejection onset, highlighting a temporal association. Anti-angiotensin II type 1 receptor antibody (AT1R-Ab) levels did not differ significantly between acute rejection and non-rejection episodes, possibly due to prophylactic immune cell depletion. While dd-cfDNA and STR levels correlate with rejection episodes and reflect key graft cellular events, CD8⁺ T-cell dynamics demonstrated the strongest temporal association with rejection episodes in this patient, though no biomarker showed statistically significant differences. These exploratory findings support the need for further longitudinal, multi-patient studies to validate emerging biomarkers and refine rejection monitoring strategies in VCA.

Determining the clinical relevance of BRCA2 variants of uncertain significance is critical for informed risk management. Recently, two saturation genome editing studies assessed the functional effects of all single nucleotide variants in the BRCA2 C-terminal DNA Binding Domain. To improve the accuracy of functional data used for ACMG/AMP variant classification, we combined results from these studies in four composite models and evaluated the performance of each model using variants with known classifications. Here, we show that an "Integrated VarCall Model", which combined raw functional data for 6383 variants from the original studies, yielded 98.8% accuracy and out-performed the original studies and other combined data models. Incorporation of the "Integrated VarCall Model" functional data with other sources of evidence according to ClinGen BRCA1/2 variant curation expert panel specifications resulted in classification of 5926 (92.8%) BRCA2 variants as pathogenic (n = 735) or benign (n = 5191) and provides valuable insights for individuals with BRCA2 variants.

BACKGROUND/UNASSIGNED:Dysregulated proteolysis is implicated in thoracic (thoracic aortic aneurysm [TAA]) and abdominal aortic aneurysm (AAA) pathogenesis, but proteolytic landscapes (degradomes) of aneurysmal and normal aorta and contributions of individual proteases remain undefined. Here, a proteome-wide approach was used to define and compare TAA and AAA degradomes and uncover the specific role in aortic remodeling of 2 proteases consistently identified in the aneurysms, CMA1 (mast cell chymase) and MMP9 (matrix metalloprotease 9). METHODS/UNASSIGNED:The mass spectrometry-based N-terminomics strategy, terminal amine isotopic labeling of substrates, was applied to Marfan syndrome TAAs (n=5), AAAs (n=16), and nondiseased thoracic aorta (n=4), and abdominal aorta (n=4) in a forward degradomics application, that is, to define substrate and protease degradomes. 8-plex iTRAQ terminal amine isotopic labeling of substrates was used for quantitative comparison of the tissue cohorts. Cleavage sites of CMA1 and MMP9 were sought by reverse degradomics, that is, digestion of aortic proteins with these proteases, followed by terminal amine isotopic labeling of substrates. CMA1 and MMP9 proteolysis of biglycan was further resolved using amino-terminal oriented mass spectrometry of substrates. RESULTS/UNASSIGNED:We experimentally annotated 20 885 proteolytically derived peptides and identified 129 proteases in the aortic tissues. Quantitative substrate degradome comparisons identified specific differentially modulated pathways and networks in TAAs and AAAs. Reverse degradomics elucidated >300 CMA1 and MMP9 substrate cleavage sites, of which many, including orthogonally validated biglycan cleavages, occurred in the disease degradomes. CONCLUSIONS/UNASSIGNED:Unbiased forward degradomics of the aortic wall from TAA, AAA, and nondiseased tissue provides a systems biology view of aortic wall breakdown and a new resource for its hitherto occult proteolytic landscape, demonstrating widespread extracellular matrix remodeling with disproportionate impact on proteoglycans. The findings provided insights into aortic aneurysm pathways and disease biomarkers and suggest involvement of numerous proteases. Mapping of specific proteolytic contributions of CMA1 and MMP9 illustrates a strategy for defining the activities of all proteases involved in aortic disease.