Faculty Bibliography

OBJECTIVES/OBJECTIVE:Triglycerides (TGs) associate with apolipoprotein B100 (apoB100) to form very low density lipoproteins (VLDLs) in the liver. The repertoire of factors that facilitate this association is incompletely understood. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytosolic lipid droplet (LD) biogenesis in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. METHODS:Experiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, stimulated Raman scattering (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. MAIN FINDINGS/RESULTS:1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to low density lipoprotein (LDL) density; 3) Both in vitro and in vivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. PRINCIPAL CONCLUSIONS/CONCLUSIONS:The results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be an important factor in the partitioning of TG between cytosolic LDs and VLDL particles.

The intricate network of protein-chaperone interactions is crucial for maintaining cellular function. Recent discoveries have unveiled the existence of specialized chaperone assemblies, known as epichaperomes, which serve as scaffolding platforms that orchestrate the reconfiguration of protein-protein interaction networks, thereby enhancing cellular adaptability and proliferation. This study explores the structural and regulatory aspects of epichaperomes, with a particular focus on the role of post-translational modifications (PTMs) in their formation and function. A key finding is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 within an intrinsically disordered region, as critical determinants of epichaperome assembly. Our data demonstrate that phosphorylation of these serine residues enhances HSP90's interactions with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Moreover, we establish a direct link between epichaperome function and cellular physiology, particularly in contexts where robust proliferation and adaptive behavior are essential, such as in cancer and pluripotent stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone assemblies in diseases characterized by epichaperome dysregulation, thereby bridging the gap between fundamental research and precision medicine.

We describe a modular bond-centric approach to protein nanomaterial design inspired by the rich diversity of chemical structures that can be generated from the small number of atomic valencies and bonding interactions. We design protein building blocks with regular coordination geometries and bonding interactions that enable the assembly of a wide variety of closed and opened nanomaterials using simple geometrical principles. Experimental characterization confirms successful formation of more than twenty multi-component polyhedral protein cages, 2D arrays, and 3D protein lattices, with a high (10-50 %) success rate and electron microscopy data closely matching the corresponding design models. Because of the 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 systems.

Gaucher disease (GD) is caused by biallelic GBA1/Gba1 mutations that encode defective glucocerebrosidase (GCase). Progranulin (PGRN, encoded by GRN/Grn) is a modifier of GCase, but the interplay between PGRN and GCase, specifically GBA1/Gba1 mutations, contributing to GD severity is unclear. Mouse models were developed with various dosages of Gba1 D409V mutation against the PGRN deficiency (Grn-/-) [Grn-/-;Gba1D409V/WT (PG9Vwt), Grn-/-;Gba1D409V/D409V (PG9V), Grn-/-;Gba1D409V/Null (PG9VN)]. Disease progression in those mouse models was characterized by biochemical, pathological, transcriptomic, and neurobehavioral analyses. Compared to PG9Vwt, Grn-/-;Gba1WT/Null and Grn-/- mice that had a higher level of GCase activity and undetectable pathologies, homozygous or hemizygous D409V in PG9V or PG9VN, respectively, resulted in profound inflammation and neurodegeneration. PG9VN mice exhibited much earlier onset, shorter life span, tissue fibrosis, and more severe phenotypes than PG9V mice. Glycosphingolipid accumulation, inflammatory responses, lysosomal-autophagy dysfunction, microgliosis, retinal gliosis, as well as α-Synuclein increases were much more pronounced in PG9VN mice. Neurodegeneration in PG9VN was characterized by activated microglial phagocytosis of impaired neurons and programmed cell death due to necrosis and, possibly, pyroptosis. Brain transcriptomic analyses revealed the intrinsic relationship between D409V dosage, and the degree of altered gene expression related to lysosome dysfunction, microgliosis, and neurodegeneration in GD, suggesting the disease severity is dependent on a GCase activity threshold related to Gba1 D409V dosage and loss of PGRN. These findings contribute to a deeper understanding of GD pathogenesis by elucidating additional underlying mechanisms of interplay between PGRN and Gba1 mutation dosage in modulating GCase function and disease severity in GD and GBA1-associated neurodegenerative diseases.

PURPOSE OF REVIEW/OBJECTIVE:This review examines the evolving role of the fat-inducing transcript 2 (FIT2) protein in lipid droplet (LD) biology and its broader implications in cellular physiology and disease. With recent advancements in understanding FIT2 function across various model systems, this review provides a timely synthesis of its mechanisms and physiological significance. RECENT FINDINGS/RESULTS:FIT2, an endoplasmic reticulum (ER)-resident protein, has been established as a critical regulator of LD formation in diverse organisms, from yeast to mammals. It facilitates LD biogenesis by sequestering diacylglycerol (DAG) and potentially influencing ER membrane dynamics. Beyond its role in lipid metabolism, FIT2 intersects with the ER-associated degradation (ERAD), is critical for protein homeostasis, and is linked to the unfolded protein response (UPR). Dysregulation of FIT2 has also been linked to metabolic disorders such as insulin resistance and lipodystrophy, highlighting its clinical relevance. SUMMARY/CONCLUSIONS:Insights into FIT2 function underscore its pivotal role in LD formation and lipid homeostasis. Understanding its involvement in ER proteostasis and very low density lipoprotein biogenesis has broad implications for metabolic diseases and cancer. Therapeutic strategies targeting FIT2 may offer novel approaches to modulate lipid metabolism and mitigate associated pathologies. Further research is needed to elucidate the full spectrum of FIT2's interactions within cellular lipid and protein networks, potentially uncovering new therapeutic avenues for metabolic and ER stress-related disorders.

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: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.

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.

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.