Faculty Bibliography

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding - thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. STATEMENT OF SIGNIFICANCE: : Direct molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. Compared with that in control mice, the protein in these mice clears from the serum faster and accumulates significantly more in fibrotic livers. This work advances the development of targeted protein probes for in vivo fibrosis imaging.

The morphology of cells in vivo can arise from a variety of mechanisms. In the Caenorhabditis elegans hermaphrodite gonad, the distal tip cell (DTC) elaborates into a complex plexus over a relatively short developmental time period, but the mechanisms underlying this change in cell morphology are not well defined. We correlated the time of DTC elaboration with the L4-to-adult molt, but ruled out a relevant heterochronic pathway as a cue for DTC elaboration. Instead, we found that the timing of gonad elongation and aspects of underlying germline flux influence DTC elaboration. We propose a 'hitch and tow' aspect of organ-level dynamics that contributes to cellular morphogenesis, whereby germline flux drags the flexible DTC cell cortex away from its stationary cell body. More broadly, we speculate that this mechanism may contribute to cell shape changes in other contexts with implications for development and disease.

BACKGROUND/UNASSIGNED:A deeper understanding of acute rejection in vascularized composite allotransplantation is paramount for expanding its utility and longevity. There remains a need to develop more precise and accurate tools for diagnosis and prognosis of these allografts, as well as alternatives to traditional immunosuppressive regimens. METHODS/UNASSIGNED:Twenty-seven skin biopsies collected from 3 vascularized composite allotransplantation recipients, consisting of face and hand transplants, were evaluated by histology, immunohistochemistry staining, and gene expression profiling. RESULTS/UNASSIGNED:significantly predicted inflammation specific to vascularized composite allografts that required therapeutic intervention. CONCLUSIONS/UNASSIGNED:The mechanism of vascularized composite allograft-specific inflammation and rejection appears to be conserved across different patients and skin on different anatomical sites. A concise gene signature can be utilized to ascertain graft status along with a continuous scale, providing valuable diagnostic and prognostic information to supplement current gold standards of graft evaluation.

BACKGROUND:The purpose of this study was to define the optimal combination of surgical technique and postoperative rehabilitation protocol for elderly patients undergoing either hemiarthroplasty (HA) or reverse total shoulder arthroplasty (rTSA) for acute proximal humerus fracture (PHF) by performing a network meta-analysis of the comparative studies in the literature. METHODS:A systematic review of the literature using Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines of MEDLINE, EMBASE, and Cochrane Library was screened from 2007 to 2023. Inclusion criteria were level I-IV studies utilizing primary HA and/or rTSA published in a peer-reviewed journal, that specified whether humeral stems were cemented or noncemented, specified postoperative rehabilitation protocol, and reported results of HA and/or rTSA performed for PHF. Early range of motion (ROM) was defined as the initiation of active ROM at ≤3 weeks after surgery. Level of evidence was evaluated based on the criteria by the Oxford Centre for Evidence-Based Medicine. Clinical outcomes were compared using a frequentist approach to network meta-analysis with a random-effects model that was performed using the netmeta package version 0.9-6 in R. RESULTS:A total of 28 studies (1119 patients) were included with an average age of 74 ± 3.7 and mean follow-up of 32 ± 11.1 months. In the early ROM cohort (Early), the mean time to active ROM was 2.4 ± 0.76 weeks compared to 5.9 ± 1.04 weeks in the delayed ROM cohort (Delayed). Overall, rTSA-Pressfit-Early resulted in statistically superior outcomes including postoperative forward elevation (126 ± 27.5), abduction (116 ± 30.6), internal rotation (5.27 ± 0.74, corresponding to L3-L1), American Shoulder and Elbow Surgeons score (71.8 ± 17), tuberosity union (89%), and lowest tuberosity nonunion rate (9.6%) in patients ≥65 year old with acute PHF undergoing shoulder arthroplasty (all P ≤ .05). In total there were 277 (14.5%) complications across the cohorts, of which 89/277 (34%) were in the HA-Cement-Delayed cohort. HA-Cement-Delayed resulted in 2-times higher odds of experiencing a complication when compared to rTSA-Cement-Delayed (P = .005). Conversely, rTSA-Cement-Early cohort followed by rTSA-Pressfit-Early resulted in a total complication rate of 4.7% and 5.4% (odds ratios, 0.30; P = .01 & odds ratios, 0.42; P = .05), respectively. The total rate of scapular notching was higher in the cemented rTSA subgroups (16.5%) vs. (8.91%) in the press fit rTSA subgroups (P = .02). CONCLUSION/CONCLUSIONS:Our study demonstrates that patients ≥65 years of age, who sustain a 3-or 4-part PHF achieve the most benefit in terms of ROM, postoperative functional outcomes, tuberosity union, and overall complication rate when undergoing rTSA with a noncemented stem and early postoperative ROM when compared to the mainstream preference-rTSA-Cement-Delayed.

The integration of artificial intelligence (AI) in healthcare, known herein as Medical AI, has seen a remarkable increase in attention over the last few years. This study aims to provide a comprehensive analysis of the trends in venture funding in the medical AI sector in comparison to venture funding in healthcare and AI as a whole over the past decade, using data from the Pitchbook financial database, and its implications for the future of healthcare quality and delivery. An extensive review of venture investments in healthcare, AI, and medical AI (the overlap between healthcare and AI) sectors was conducted for a 10-year period from October 7, 2013 to October 6, 2023. The study used Pitchbook"™s database to catalogue deals across various stages, round numbers, and series, inclusive of all ownership models and geographic locations. The analysis focused on completed transactions, extracting descriptive statistics for deal flow, capital flow, and post-funding valuations while analyzing trends. The study found that the medical AI sector experienced a higher year-over-year growth in deal volume (P=0.01 compared to healthcare, P=0.08 compared to AI) and capital flow (P=0.01 compared to healthcare and P=0.03 compared to AI) over this time period, with all sectors witnessing a sharp stimulus during the coronavirus disease 2019 (COVID-19) stimulus period, alongside marked increases at the time of introduction of seminal AI technologies. This was followed by marked drawdowns with the onset of high inflation and high interest rates. Early-stage funding was dominant in medical AI, indicating a market leaning towards emerging technologies. Despite a decrease in total deal volume in recent years, there was a steady increase in median deal sizes and valuations, highlighting the sector"™s resilience and perceived value. The findings suggest that medical AI is a rapidly growing sector with significant investor interest, particularly in early-stage ventures. The findings align with the early stages of a valuation bubble, though the sector thus far has shown resilience and value growth despite broader economic fluctuations and reduced deal volume, indicating a selective yet robust investment environment.

Myasthenia gravis (MG) is a chronic and severe disease of the skeletal neuromuscular junction (NMJ) in which the effects of neurotransmitters are attenuated, leading to muscle weakness. In the most common forms of autoimmune MG, antibodies attack components of the postsynaptic membrane, including the acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). MuSK, a master regulator of NMJ development, associates with the low-density lipoprotein-related receptor 4 (Lrp4) to form the signaling receptor for neuronal Agrin, a nerve-derived synaptic organizer. Pathogenic antibodies to MuSK interfere with binding between MuSK and Lrp4, inhibiting the differentiation and maintenance of the NMJ. MuSK MG can be debilitating and refractory to treatments that are effective for AChR MG. We show here that recombinant antibodies, derived from MuSK MG patients, cause severe neuromuscular disease in mice. The disease can be prevented by a MuSK agonist antibody, presented either prophylactically or after disease onset. These findings suggest a therapeutic alternative to generalized immunosuppression for treating MuSK MG by selectively and directly targeting the disease mechanism.

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

Phagocytosis is an intensely physical process that depends on the mechanical properties of both the phagocytic cell and its chosen target. Here, we employed differentially deformable hydrogel microparticles to examine the role of cargo rigidity in the regulation of phagocytosis by macrophages. Whereas stiff cargos elicited canonical phagocytic cup formation and rapid engulfment, soft cargos induced an architecturally distinct response, characterized by filamentous actin protrusions at the center of the contact site, slower cup advancement, and frequent phagocytic stalling. Using phosphoproteomics, we identified β2 integrins as critical mediators of this mechanically regulated phagocytic switch. Macrophages lacking β2 integrins or their downstream effectors, Talin1 and Vinculin, exhibited specific defects in phagocytic cup architecture and selective suppression of stiff cargo uptake. We conclude that integrin signaling serves as a mechanical checkpoint during phagocytosis to pair cargo rigidity to the appropriate mode of engulfment.

Muscle-specific kinase (MuSK) is essential for the formation, function, and preservation of neuromuscular synapses. Activation of MuSK by a MuSK agonist antibody may stabilize or improve the function of the neuromuscular junction (NMJ) in patients with disorders of the NMJ, such as congenital myasthenia (CM). Here, we generated and characterized ARGX-119, a first-in-class humanized agonist monoclonal antibody specific for MuSK, that is being developed for treatment of patients with neuromuscular diseases. We performed in vitro ligand-binding assays to show that ARGX-119 binds with high affinity to the Frizzled-like domain of human, nonhuman primate, rat, and mouse MuSK, without off-target binding, making it suitable for clinical development. Within the Fc region, ARGX-119 harbors L234A and L235A mutations to diminish potential immune-activating effector functions. Its mode of action is to activate MuSK, without interfering with its natural ligand neural Agrin, and cluster acetylcholine receptors in a dose-dependent manner, thereby stabilizing neuromuscular function. In a mouse model of DOK7 CM, ARGX-119 prevented early postnatal lethality and reversed disease relapse in adult Dok7 CM mice by restoring neuromuscular function and reducing muscle weakness and fatigability in a dose-dependent manner. Pharmacokinetic studies in nonhuman primates, rats, and mice revealed a nonlinear PK behavior of ARGX-119, indicative of target-mediated drug disposition and in vivo target engagement. On the basis of this proof-of-concept study, ARGX-119 has the potential to alleviate neuromuscular diseases hallmarked by impaired neuromuscular synaptic function, warranting further clinical development.

Live imaging of translation based on tag recognition by a single-chain antibody is a powerful technique to assess translation regulation in living cells. However, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system, especially in a multicellular organism. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different green fluorescent protein variants fused to the single-chain fragment variable (scFv) and uncovered photobleaching, aggregation, and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.