Faculty Bibliography

INTRODUCTION/BACKGROUND:In city hospitals, subway-related traumatic amputations are a frequent pattern of injury, however there is a paucity of literature on this specific injury pattern. The purpose of this study was to describe the epidemiology of subway-related traumatic amputations, as well as compare them to non-subway traumatic amputations. PATIENTS AND METHODS/METHODS:Retrospective review was performed at a single Level-1 trauma center in a metropolitan area. All patients who sustained a traumatic lower-extremity amputation over a seven-year period were included. Demographics, injury, treatment-related information, and complications were collected. Subway and non-subway traumatic amputations were statistically compared. Cohorts were further subdivided into above-knee amputations (AKAs) and below-knee amputations (BKAs) for statistical comparison. RESULTS:Fifty-seven patients sustained 72 traumatic lower-extremity amputations, including 64 subway-related amputations. Fifteen patients with bilateral lower-extremity amputations all had subway-related injuries. Patients with subway-related injuries were more likely to have a history of alcohol use disorder (58.1 % vs. 0 %; P = 0.002), and experienced longer stays in the intensive care unit (ICU) (8.9 vs. 3.6 days; P = 0.006). Twenty-four amputations (33.3 %) were complicated by wound infection during the initial hospitalization, with wound cultures growing a variety of organisms, most frequently Enterococcus species and Enterobacter cloacae. When subway injuries were separated by AKAs and BKAs, patients with AKAs underwent more irrigation and debridement procedures on average (10.3 vs. 5.8; P = 0.006), had a higher rate of wound infections (58.8 % vs. 25.0 %; P = 0.018), and had longer hospital stays (50.4 vs. 32.2 days; P = 0.047). CONCLUSION/CONCLUSIONS:Subway-related amputations are associated with longer ICU stays and a history of alcohol use disorder compared to non-subway traumatic amputations. Approximately 1/3 of these patients are expected to develop a wound infection, with Enterococcus and Enterobacter species being the most commonly identified organisms. Further research into high-energy, traumatic amputations, including subway injuries, may help improve prognostication of patient outcomes, identify potential in-hospital complications, and proactively direct differences in care compared to the standard for non-subway-related amputations. LEVEL OF EVIDENCE/METHODS:Prognostic Level III.

OBJECTIVE:Genome-wide association studies implicate metabo-psychiatric origins for anorexia nervosa (AN). There are two case reports totaling six adult females who experienced complete remission of AN following a treatment comprised of ketogenic diet (targeting metabolism) with ketamine infusions (targeting psychiatric origins), but no study has determined the efficacy of ketogenic diet, alone. We addressed this gap in knowledge, with exploration of potential molecular mechanisms, using an animal model. METHOD/METHODS:Adult C57BL6 female mice underwent 2 or 3 cycles of activity-based anorexia (ABA1, ABA2, ABA3), an animal model of AN relapse, in which AN-like maladaptive behaviors of hyperactivity and voluntary food restriction are elicited when wheel access is combined with food restriction. ABA was categorized as severe, based on weight loss ≥ 20%, food restriction-evoked increase in wheel counts > 10,000/6 h, and crouching/grimace, and compared across two groups: (1) KG, fed ketogenic food continuously (N = 25); and (2) CON, fed standard diet (N = 28). RESULTS:86% of CON versus none of the KG were crouching with grimace during ABA1. 93% of CON versus 11% of KG lost weight severely during ABA2 (p < 0.001, 8% difference of group mean weights). Severe hyperactivity was prevalent among CON (86%) and rare for KG (4%) during ABA2 (p < 0.001 on all food-restricted days). ABA up-regulated BDNF (brain-derived neurotrophic factor) in the hippocampus of both groups but ketone body, β-hydroxybutyrate, in urine was increased only among KG. DISCUSSION/CONCLUSIONS:Ketogenic diet may reduce severity of AN relapse through reduction of compulsive exercise, via mechanisms that are in addition to BDNF up-regulation and involve β-hydroxybutyrate.

The outer membrane of Gram-negative bacteria provides a formidable barrier, essential for both pathogenesis and antimicrobial resistance. Biogenesis of this complex structure necessitates the transport of phospholipids across the cell envelope. Recently, YhdP was implicated as a major protagonist in the trafficking of inner membrane phospholipids to the outer membrane; however the molecular mechanism of YhdP mediated transport remains elusive. Here, utilising AlphaFold, we observe YhdP to form an elongated assembly of 60 β-strands that curve to form a continuous hydrophobic groove. This architecture is consistent with our negative stain electron microscopy data which reveals YhdP to be approximately 250 Å in length and thus sufficient to span the bacterial cell envelope. Furthermore, molecular dynamics simulations and bacterial growth assays indicate essential helical regions at the N- and C-termini of YhdP, that may embed into the inner and outer membranes respectively, reinforcing its envelope spanning nature. Our in vivo crosslinking data reveal phosphate-containing substrates captured along the length of the YhdP groove, providing direct evidence that YhdP interacts with a phosphate-containing substrate, which we propose to be phospholipids. This finding is congruent with our molecular dynamics simulations which demonstrate the propensity for inner membrane lipids to spontaneously enter the groove of YhdP. Collectively, our results support a model in which YhdP bridges the cell envelope, providing a hydrophobic environment for the transport of phospholipids to the outer membrane.

Desmosomes are adhesive cell contacts abundant in tissues exposed to mechanical strain, such as the stratified and simple epithelia of the epidermis and mucous membranes, as well as the myocardium. Besides their role in mechanical cell cohesion, desmosomes also modulate pathways important for tissue differentiation, wound healing and immune responses. Dysfunctional desmosomes, resulting from pathogenic variants in genes encoding desmosomal components, autoantibodies targeting desmosomal adhesion molecules or inflammation, cause the life-threatening diseases arrhythmogenic cardiomyopathy and pemphigus and contribute to the pathogenesis of inflammatory bowel diseases. The Alpine Desmosome Disease Meeting 2024 (ADDM 2024), held in Grainau, Germany in October 2024, connected international researchers from basic sciences with clinical experts from dermatology, cardiology, gastroenterology and surgery. The participants discussed recent advances, identified hot topics in desmosome biology and disease and provided new concepts for pathogenesis and treatment approaches.

Human endogenous retroviruses (HERVs) occupy a large portion of the human genome. Most HERVs are transcriptionally silent, but they can be reactivated during pathological states such as viral infection and certain cancers. The HERV-K HML-2 clade includes elements that recently integrated have in the human germ line and often contain intact open reading frames that possibly support peptide and protein expression. Understanding HERV-K-host interactions and their potential as biomarkers is problematic due to the high similarity among different elements. Previously, we described a long-read single molecule real-time sequencing (PacBio) strategy to analyze HERV-K RNA expression profiles in different cell types. However, identifying HERV-K HML-2 proteins accurately is difficult without robust and reliable methods and reagents. Here we present a new approach to characterize the HML-2 elements that (a) are being translated and (b) produce enough protein to be detected and identified by mass spectrometry. Our data reveal that RNA expression profiling alone cannot accurately predict which HML-2 elements are responsible for protein production, as we observe several differences between the highest expressed RNAs and the elements that are the predominant source of HERV-K HML-2 protein synthesis. These studies represent an important advance toward untangling the complexity of HERV-K-host interactions.

Advancements in cryo-EM have stimulated a revolution in structural biology. Yet, for membrane proteins near the cryo-EM size threshold of approximately 40 kDa, including transporters and G-protein coupled receptors, the absence of distinguishable structural features makes image alignment and structure determination a significant challenge. Furthermore, resolving more than one protein conformation within a sample, a major advantage of cryo-EM, represents an even greater degree of difficulty. Here, we describe a strategy for introducing a rigid fiducial marker (BRIL domain) at the C-terminus of membrane transporters from the Major Facilitator Superfamily (MFS) with AlphaFold2. This approach involves fusion of the last transmembrane domain helix of the target protein with the first helix of BRIL through a short poly-alanine linker to promote helicity. Combining this strategy with a BRIL-specific Fab, we elucidated four cryo-EM structures of the 42 kDa Staphylococcus aureus transporter NorA, three of which were derived from a single sample corresponding to inward-open, inward-occluded, and occluded conformations. Hence, this fusion construct facilitated experiments to characterize the conformational landscape of NorA and validated our design to position the BRIL/antibody pair in an orientation that avoids steric clash with the transporter. The latter was enabled through AlphaFold2 predictions, which minimized guesswork and reduced the need for screening several constructs. We further validated the suitability of the method to three additional MFS transporters (GlpT, Bmr, and Blt), results that supported a rigid linker between the transporter and BRIL. The successful application to four MFS proteins, the largest family of secondary transporters in nature, and analysis of predicted structures for the family indicates this strategy will be a valuable tool for studying other MFS members using cryo-EM.

OBJECTIVES/OBJECTIVE:To determine which in-hospital complications following the operative treatment of hip fractures are associated with increased inpatient, 30-day and 1 year mortality. METHODS:Design: Retrospective study. SETTING/METHODS:A single academic medical center and a Level 1 Trauma Center. PATIENT SELECTION CRITERIA/UNASSIGNED:All patients who were operatively treated for hip fractures (OTA/AO 31A, 31B and Vancouver A,B, and C periprosthetic fractures) at a single center between October, 2014 and June, 2023. OUTCOME MEASURES AND COMPARISONS/UNASSIGNED:Occurrence of an in-hospital complication was recorded. Cohorts were based upon mortality time points (during admission, 30-days and 1-year) and compared to patients who were alive at those time points to determine which in- hospital complications were most associated with mortality. Correlation analysis was performed between patients who died and those who were alive at each time point. RESULTS:A total of 3,134 patients (average age of 79.6 years, range 18-104 years and 66.6% female) met inclusion for this study. The overall mortality rate during admission, 30 days and 1 year were found to be 1.6%, 3.9% and 11.1%, respectively. Sepsis was the complication most associated with increased in-hospital mortality (OR: 7.79, 95% CI 3.22 - 18.82, p<0.001) compared to other in-hospital complications. Compared to other in-hospital complications, stroke was the complication most associated with 30-day mortality (OR: 7.95, 95% CI 1.82 - 34.68, p<0.001). Myocardial infarction was the complication most associated with 1-year mortality (OR: 2.86, 95% CI 1.21 - 6.77, p=0.017) compared to other in-hospital complications. CONCLUSIONS:Post-operative sepsis, stroke and myocardial infraction were the three complications most associated with mortality during admission, 30-day mortality and 1-year mortality, respectively, during the operative treatment of hip fractures. LEVEL OF EVIDENCE/METHODS:Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Estrogen deficiency is considered the most important cause of postmenopausal osteoporosis. However, the underlying mechanism is still not completely understood. In this study, progranulin (PGRN) was isolated as a key regulator of bone mineral density in postmenopausal women through high throughput proteomics screening. In addition, PGRN-deficient mice exhibited significantly lower bone mass than their littermates in an ovariectomy-induced osteoporosis model. Furthermore, estrogen-mediated inhibition of osteoclastogenesis and bone resorption as well as its protection against ovariectomy-induced bone loss largely depended on PGRN. Mechanistic studies revealed the existence of a positive feedback regulatory loop between PGRN and estrogen signaling. In addition, loss of PGRN led to the reduction of estrogen receptor α, the important estrogen receptor involved in estrogen regulation of osteoporosis, through enhancing its degradation via K48-linked ubiquitination. These findings not only provide a previously unrecognized interplay between PGRN and estrogen signaling in regulating osteoclastogenesis and osteoporosis but may also present a new therapeutic approach for the prevention and treatment of postmenopausal osteoporosis by targeting PGRN/estrogen receptor α.

Glucocorticoids (GCs) are the most prescribed anti-inflammatory and immunosuppressive drugs. However, their use is often limited by substantial side effects, such as GC-induced osteoporosis (GIO) with the underlying mechanisms still not fully understood. In this study, we identify Tau as a low-affinity binding receptor for GCs that plays a crucial role in GIO. Tau deficiency largely abolished bone loss induced by high-dose dexamethasone, a synthetic GC, in both inflammatory arthritis and GIO models. Furthermore, TRx0237, a Tau inhibitor identified from an FDA-approved drug library, effectively prevented GIO. Notably, combinatorial administration of TRx0237 and dexamethasone completely overcame the osteoporosis adverse effect of dexamethasone in treating inflammatory arthritis. These findings present Tau as a previously unrecognized GC receptor with low affinity, and provide potential strategies to mitigate a spectrum of GC-related adverse effects, particularly osteoporosis.

Hair follicle neogenesis (HFN) occurs following large skin excisions in mice, serving as a rare regenerative model in mammalian wound healing. Wound healing typically results in fibrosis in mice and humans. We previously showed small skin excisions in mice result in scarring devoid of HFN, displaying features of non-regenerative healing, and Hedgehog (Hh) activation in the dermis of such wounds can induce HFN. In this study, we sought to verify the role of dermal Wnt/β-catenin signaling in HFN, as this pathway is essential for HF development, but is also paradoxically well-characterized in fibrosis of adult wounds. By deletion of β-catenin in large wound myofibroblasts, we show Wnt/β-catenin signaling is required for endogenous mechanisms of HFN. Through utilizing a combined mouse model that simultaneously induces deletion of β-catenin and constitutive activation of Smoothened (Smo) in myofibroblasts, we also found β-catenin is required for Hh-driven DP formation. Transcriptome analysis confirms Wnt/β-catenin and Hh pathways are activated in dermal papilla (DP) cells. Our results indicate that Wnt-active fibrotic status may also create a permissive state for the regenerative function of Hh, suggesting that activation of both Wnt and Hh pathways in skin wound fibroblasts must be ensured in future strategies to promote HFN.