Faculty Bibliography

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 advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. Validating the function of these candidates in vivo is challenging, due to low efficiency and low throughput of most model systems. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked-in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. We used this system to identify both cell-autonomous and non-cell autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our high-efficiency genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

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.

As efforts to study the mechanisms of melanoma metastasis and novel therapeutic approaches multiply, researchers need accurate, high-throughput methods to evaluate the effects on tumor burden resulting from specific interventions. We show that automated quantification of tumor content from whole slide images is a compelling solution to assess in vivo experiments. In order to increase the outflow of data collection from preclinical studies, we assembled a large dataset with annotations and trained a deep neural network for the quantitative analysis of melanoma tumor content on histopathological sections of murine models. After assessing its performance in segmenting these images, the tool obtained consistent results with an orthogonal method (bioluminescence) of measuring metastasis in an experimental setting. This AI-based algorithm, made freely available to academic laboratories through a web-interface called MetFinder, promises to become an asset for melanoma researchers and pathologists interested in accurate, quantitative assessment of metastasis burden.

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.

BACKGROUND:Dickkopf-related protein 1 (DKK1) is a Wingless-related integrate site (Wnt) signaling modulator that is upregulated in prostate cancers (PCa) with low androgen receptor expression. DKN-01, an IgG4 that neutralizes DKK1, delays PCa growth in pre-clinical DKK1-expressing models. These data provided the rationale for a clinical trial testing DKN-01 in patients with metastatic castration-resistant PCa (mCRPC). METHODS:(combination) for men with mCRPC who progressed on ≥1 AR signaling inhibitors. DKK1 status was determined by RNA in-situ expression. The primary endpoint of the phase 1 dose escalation cohorts was the determination of the recommended phase 2 dose (RP2D). The primary endpoint of the phase 2 expansion cohorts was objective response rate by iRECIST criteria in patients treated with the combination. RESULTS:18 pts were enrolled into the study-10 patients in the monotherapy cohorts and 8 patients in the combination cohorts. No DLTs were observed and DKN-01 600 mg was determined as the RP2D. A best overall response of stable disease occurred in two out of seven (29%) evaluable patients in the monotherapy cohort. In the combination cohort, five out of seven (71%) evaluable patients had a partial response (PR). A median rPFS of 5.7 months was observed in the combination cohort. In the combination cohort, the median tumoral DKK1 expression H-score was 0.75 and the rPFS observed was similar between patients with DKK1 H-score ≥1 versus H-score = 0. CONCLUSION/CONCLUSIONS:DKN-01 600 mg was well tolerated. DKK1 blockade has modest anti-tumor activity as a monotherapy for mCRPC. Anti-tumor activity was observed in the combination cohorts, but the response duration was limited. DKK1 expression in the majority of mCRPC is low and did not clearly correlate with anti-tumor activity of DKN-01 plus docetaxel.

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of Kras^G12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

The interaction between tumors and their microenvironment is complex and heterogeneous. Recent developments in high-dimensional multiplexed imaging have revealed the spatial organization of tumor tissues at the molecular level. However, the discovery and thorough characterization of the tumor microenvironment (TME) remains challenging due to the scale and complexity of the images. Here, we propose a self-supervised representation learning framework, CANVAS, that enables discovery of novel types of TMEs. CANVAS is a vision transformer that directly takes high-dimensional multiplexed images and is trained using self-supervised masked image modeling. In contrast to traditional spatial analysis approaches which rely on cell segmentations, CANVAS is segmentation-free, utilizes pixel-level information, and retains local morphology and biomarker distribution information. This approach allows the model to distinguish subtle morphological differences, leading to precise separation and characterization of distinct TME signatures. We applied CANVAS to a lung tumor dataset and identified and validated a monocytic signature that is associated with poor prognosis.

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.

Spleen marginal zone (MZ) B cells are important for antibody responses against blood-borne antigens. The signals they use to detect exposure to blood are not well defined. Here, using intravital two-photon microscopy in mice, we observe transient contacts between MZ B cells and red blood cells that are in flow. We show that MZ B cells use adhesion G-protein-coupled receptor ADGRE5 (CD97) for retention in the spleen. CD97 function in MZ B cells depends on its ability to undergo autoproteolytic cleavage and signaling via Gα₁₃ and ARHGEF1. Red blood cell expression of the CD97 ligand CD55 is required for MZ B cell homeostasis. Applying a pulling force on CD97-transfected cells using an optical C-trap and CD55⁺ beads leads to accumulation of active RhoA and membrane retraction. Finally, we show that CD97 deficiency leads to a reduced T cell-independent IgM response. Thus, our studies provide evidence that MZ B cells use mechanosensing to position in a manner that enhances antibody responses against blood-borne antigens.