Citations

Abstract CCAAT/enhancer-binding protein alpha (C/EBPα) is a critical lineage-defining transcription factor and tumor suppressor. Despite its clinical significance in conditions like acute myeloid leukemia and hepatocellular carcinoma (HCC), C/EBPα has remained undruggable due to its intrinsically disordered regions and lack of small-molecule binding pockets. In this study, we describe the development of a novel Oligonucleotide-based PROteolysis-TArgeting Chimera (Oligo-PROTAC or O'PROTAC) strategy designed to achieve targeted degradation of endogenous C/EBPα. We designed and synthesized a 10-nucleotide double-stranded DNA decoy derived from a C/EBPα-binding motif and conjugated it via variable linkers to the E3 ligase ligands pomalidomide or VH032. A total of six candidates were synthesized and evaluated in 3T3-J2 and Huh7 cell lines. We demonstrate that these O'PROTACs successfully localize to the nucleus and achieve dose-dependent degradation of C/EBPα, with the most potent candidates exhibiting sub-micromolar DC50 via lipofection delivery. Notably, this degradation occurred without inducing significant cytotoxicity. We observe a transcriptional shift consistent with loss of C/EBPα, including downregulation of cooperative factors involved in liver lineage specification and their downstream target genes. This work establishes O'PROTACs as a viable modality for targeting C/EBPα, offering a novel strategy for mechanistic investigation and future therapeutic targeting.

Low protein (LP) diets improve metabolic health in rodents and humans. In rodents, LP diets are typically implemented by replacing protein with carbohydrates like sucrose or cornstarch, keeping diets isocaloric. However, humans can choose from many different types of carbohydrate, and how dietary carbohydrate quality - the precise composition of the dietary sugars - impacts the response to dietary protein remains largely unexplored. Here, mice were fed control (21% protein) or LP (7% protein) diets with four different carbohydrate sources: sucrose, a 1:1 glucose/fructose mixture, glucose, or fructose. While LP diets improved metabolic health across all groups in male mice, carbohydrate quality also significantly altered specific health outcomes, with fructose-fed mice having the lowest body weight and adiposity of all control diets. In female mice, responses to LP diets were influenced by carbohydrate quality, with certain sugars inducing a stronger metabolic response to LP diets than previously seen. Finally, in female APP/PS1 mice, a model of Alzheimer's disease, we find that although LP diets reduce A-beta; plaque burden irrespective of carbohydrate type, dietary sugar type does influence spatial memory. Together, these results demonstrate that while dietary protein is a critical determinant of metabolic and neurological health, carbohydrate quality influences these outcomes in a sex-specific manner.

Background: Peripheral arterial disease (PAD) is a manifestation of systemic atherosclerosis characterized by chronic inflammation and immune dysregulation. Single-cell RNA sequencing (scRNA-seq) studies of human atherosclerotic plaques have identified distinct transcriptional programs within plaque-associated immune cells. We sought to determine whether transcriptional features described within these plaque-resident cells can be identified in peripheral blood mononuclear cells (PBMCs). Methods: PBMCs from patients with PAD (n = 4) and healthy controls (HC) (n = 5) were profiled using scRNA-seq using the Parse Biosciences platform, yielding approximately 135,000 high-quality cells. Data analysis was conducted in R, using Seurat for quality control, dimensionality reduction and clustering. Cell-type annotation was performed using Azimuth reference-based mapping. Plaque-associated inflammatory and metabolic gene programs were curated from published human plaque single-cell atlases. Results: Gene expression differences between HC and PAD cells were visualized using UMAP plots. CD14+ mononuclear phagocytes (MNPs) exhibited the most prominent and consistent transcriptional differences between HC and PAD samples (Figure 1). Within CD14+ MNPs, genes involved in lipid handling and macrophage metabolic function demonstrated a higher median percentage of expressing cells in PAD compared with HC (Figure 2). Further analysis of AQP9 and CD36, genes implicated in macrophage metabolic activation and lipid uptake in atherosclerotic plaques, with z-score normalization and subject-level averaging revealed modest but consistent increases in relative expression in PAD across subjects. These findings support the relevance of these circulating transcriptional shifts to PAD pathophysiology. Conclusions: Circulating CD14+ MNPs in PAD have detectable transcriptional features consistent with potential engagement of plaque-associated myeloid activation programs. In PAD, increased expression of CD36, which encodes the immune cell scavenger receptor for oxidized LDL, and AQP9, associated with unstable plaques and the endothelial-to-mesenchymal transition, suggest altered immunometabolism states in circulating MNPs. Although effect sizes are modest, they are consistent across subjects and statistically robust. These findings highlight PBMC scRNA-seq as a minimally invasive approach to interrogate systemic immune programs relevant to PAD pathobiology.

Background: Integrating machine learning (ML) with population pharmacokinetic (PPK) modeling may improve therapeutic drug monitoring predictions. Methods: Tacrolimus trough concentrations from lung transplant patients were split into a training data set (1151 concentrations in 80 patients) and a testing data set (224 concentrations in 20 patients). A PPK model was developed using NONMEM, followed by the development of 10 ML models to fit individual pharmacokinetic parameters from the PPK model with Bayesian forecasting. The best performing ML model was selected as the final model. Both the final PPK and ML models were compared for prediction performance. A web-based dashboard was established with R-shiny to recommend dosing regimens based on patient data. Results: In the PPK model, postoperative days, hematocrit, aspartate aminotransferase, tacrolimus daily dose, coadministered voriconazole or posaconazole, and the CYP3A5*3 genotype were identified significant covariates on the clearance. The Cubist ML model outperformed the PPK model, showing lower root mean squared error for tacrolimus concentrations in the testing data set. An online web-based precision dosing dashboard was created, accessible at (https://tac-dose-ml.shinyapps.io/shiny/). Conclusions: Integrating ML with PPK modeling could yield superior tacrolimus concentration predictions for lung transplant patients, offering an efficient alternative to Bayesian forecasting. The online dashboard provides rapid, individualized dosing recommendations.

ABSTRACT Cholangiocarcinoma (CCA) is a lethal biliary cancer in which chemoresistance is nearly universal, and its tumor immune microenvironment is dominated by immunosuppressive tumor-associated macrophages (TAMs) that exclude cytotoxic CD8 + T cells. How tumor cells sustain this immunosuppressive state during chemotherapy is undefined. Here we demonstrate that therapy-induced senescent-like (Sen-L) cancer cells accumulate after gemcitabine/cisplatin in human and murine CCA, are predominantly cancer cells, and predict shorter survival. Genetic elimination of Sen-L cancer cells reduces tumor burden, lowers TAM abundance, and restores intratumoral CD8 + T cells, establishing them as causal drivers. Growth differentiation factor 15 (GDF-15) is the dominant Sen-L-secreted factor and reprograms macrophages to suppress CD8 + T cells through the non-canonical receptor TGFBR2 and STAT6, and p16-restricted Gdf15 silencing phenocopies Sen-L elimination. Combined with chemotherapy, Sen-L elimination improves survival beyond chemotherapy alone. These findings establish Sen-L cancer cells and their GDF-15 output as causal, targetable drivers of macrophage-mediated immune evasion in CCA. SIGNIFICANCE STATEMENT Therapy-induced senescent-like cancer cells, not stromal cells, are the dominant senescent-like and immunosuppressive population in cholangiocarcinoma, and their elimination restores antitumor immunity. GDF-15 is their dominant secreted effector and engages a non-canonical macrophage receptor, TGFBR2, identifying a cancer-cell-to-macrophage axis and a Sen-L-elimination strategy to restore chemosensitivity. Abstract Figure Graphical abstract. Senescent-like CCA cells promote tumor immunosuppression through TAMs polarization by GDF-15

Ex vivo liver perfusion (EVLP) sustains human or large animal livers outside the body under near-physiological conditions, enabling functional monitoring for lactate clearance, bile production, and oxygen consumption and allowing targeted therapeutic interventions. Originally developed to optimize donor grafts for transplantation, EVLP has evolved into a powerful translational research platform bridging preclinical discovery and early clinical translation. This review discusses EVLP as a platform for gene therapy, immunotherapy, pharmacology, and personalized medicine, with particular emphasis on gene- and immune-based interventions as mechanistically mature exemplars. We consolidate advances in pharmacological testing and toxicity modeling, viral and non-viral gene delivery, genome engineering, and immunomodulation using perfused livers. We further describe emerging applications, including autologous EVLP pathways for organ-confined therapy, ex vivo liver surgery, and bioengineering strategies such as biliary organoid repair, RNA interference, and mitochondrial transfer. We highlight how these applications align with a paradigm shift in biomedical research, including the NIH’s recent initiative to prioritize human-based experimental models over animal-only studies. By leveraging transplant-declined or surgically resected organs that would otherwise be unused, ex vivo perfusion bridges the gap between pre-clinical testing and clinical practice, enabling real-time evaluation of interventions in functional human tissue. We discuss both the scientific opportunities afforded by EVLP and the technical, biosafety, and ethical challenges that must be addressed to enable responsible clinical translation.

Abstract Background & Objectives Patients with cholangiocarcinoma (CCA) commonly progress within months on first-line gemcitabine/cisplatin–based therapy with immune checkpoint inhibition (ICI), consistent with the emergence of treatment-induced adaptive resistance. Cellular senescence and its secretory program (senescence-associated secretory phenotype, SASP) can remodel the tumor immune microenvironment. We hypothesized that gemcitabine/cisplatin induces tumor-cell senescence that promotes immunosuppression and progression and prioritized SASP mediators that are mechanistically linked to this phenotype. Method We quantified senescence and lineage markers in chemotherapy-naïve (n = 18) and gemcitabine/cisplatin treated (n = 23) human cholangiocarcinoma specimens and analyzed single-nucleus RNA sequencing in 9 human CCA tumors. In murine orthotopic CCA models, senescent tumor cells were ablated using p16 or p21 promoter restricted inducible apoptosis (ATTAC), followed by immune profiling by flow cytometry in endpoint cohorts, or tested alone and in combination with gemcitabine/cisplatin-based therapy and immune checkpoint inhibition (ICI) for survival cohorts. Conditioned media from proliferating versus senescent tumor cells were applied to bone marrow-derived macrophages (BMDMs) to assess polarization and suppression on CD8+ T-cell proliferation. SASP candidates were screened by cytokine arrays and Growth/Differentiation Factor 15 (GDF-15) was validated by expression and genetic perturbation. Results Senescent cells were enriched in the tumor compartment in human cholangiocarcinoma and increased after chemotherapy, as assessed by p16, p21and telomere-associated DNA foci (TAF). In mice, ablation of p16+ or p21+ senescent tumor cells reduced tumor burden, prolonged survival, and improved response to gemcitabine/cisplatin-based regimens. Chemotherapy-induced senescence increased immunosuppressive tumor-associated macrophages (TAMs), an effect reversed by senescent tumor cell ablation. Mechanically, GDF-15 was the top enriched secreted factor from senescent human CCA cells, tracked with senescence associated transcriptional programs, and decreased after senescent tumor cell ablation. GDF-15 promoted immunosuppressive macrophage phenotype switching and macrophage-mediated suppression on CD8+ T cell proliferation. In comparison, senescence-restricted GDF-15 knockdown reduced tumor growth and macrophage-mediated immunosuppression. Conclusion Chemotherapy-induced tumor cell senescence promotes an immunosuppressive microenvironment through GDF-15 linked macrophage remodeling. Targeting senescent tumor cells or intercepting senescence associated GDF-15 signaling may improve response to first-line therapy in CCA.

Abstract Objective Invasive presurgical evaluation plays a key role in pediatric epilepsy surgery, particularly in magnetic resonance imaging (MRI)‐negative cases, by guiding resective, disconnective, or ablative procedures. This International League Against Epilepsy (ILAE) Pediatric Epilepsy Surgery Taskforce study provides an updated global overview of current invasive evaluation practices. Methods Group‐level data were collected from 61 epilepsy surgery programs (49 pediatric‐only) in 29 countries across six continents. Included were children and adolescents who underwent presurgical evaluation and epilepsy surgery in 2023. The study was designed to enable comparison with the similar ILAE survey conducted in 2004. Results A total of 2427 patients were included. Invasive evaluations were performed in 21.1% of cases, most frequently in North America (33.7%, higher than Europe: 18.0%, p = .003). Among invasive cases, 32.3% had no detectable MRI abnormalities. The main indication for invasive evaluation was seizure onset localization (88.1%), followed by motor or sensory mapping (17.2%) and language mapping (14.2%). Stereoelectroencephalography (SEEG) was the predominant technique (19.8% overall, 93.6% of invasive cases), more common in North America (30.0%, p = .021) and less common in South America (7.4%, p < .001). Subdural electrodes were used in only 3.2% of invasive cases, and combined depth and subdural approaches in 3.2%. SEEG‐guided radiofrequency thermocoagulation (RF‐TC) was performed in 40.9% of SEEG cases, most commonly in Asia (63.8%). In 16.2% of invasive evaluations, patients did not proceed to resection, disconnection, or ablation, with the highest rate in Europe (28.5%). Significance This global survey provides the first broad overview of invasive evaluation practices in pediatric epilepsy surgery across participating centers worldwide. It highlights the widespread adoption of SEEG, declining use of subdural electrodes, and increasing application of SEEG‐guided RF‐TC. The high proportion of MRI‐negative cases and the considerable proportion of patients not proceeding to resection, disconnection, or ablation underscore the complexity of contemporary surgical candidates and the need for further refinement of selection strategies.

Abstract Objective Pediatric epilepsy surgery is well established, but contemporary global data on referral and presurgical evaluation practices are lacking. This International League Against Epilepsy (ILAE) Pediatric Epilepsy Surgery Task Force study provides an updated overview of current trends and regional differences. Methods Group‐level data were collected from 61 epilepsy surgery programs (49 pediatric‐only) across 29 countries and six continents, identified through ILAE networks, and included all children and adolescents treated in 2023 who underwent presurgical evaluation/epilepsy surgery. Results Group‐level data were available for 2427 patients. Mean age at surgery was 9.1 ± 4.9 years; mean epilepsy duration was 5.3. At surgery, 3.2% were <1 year old (highest in Oceania: 5.1%), and 6.1% were nonpharmacoresistant (highest in Europe: 15.0%). Prior neurosurgery was reported in 14.2% (highest in North America: 28.8%), including 8.0% resections (6.1% for epilepsy, 1.5% for tumors; highest in Oceania: 16.5%), 2.3% disconnections (1.3% corpus callosotomy; highest in South America: 4.7%), and 4.2% neuromodulation (3.7% vagal nerve stimulation, .2% responsive neurostimulation, one deep brain stimulation; highest in North America: 12.2%). Developmental and epileptic encephalopathies (DEEs) at surgery included Lennox–Gastaut syndrome (7.4%), infantile epileptic spasms syndrome (5.1%), and DEE with spike–wave activation in sleep (1.5%). Presurgical investigations included fluorodeoxyglucose positron emission tomography (52.6%; highest in Oceania: 79.7%), genetic testing (46.8%; highest in Asia: 54.3%), magnetic resonance imaging (MRI) postprocessing (32.4%; highest in South America: 53.0%), functional MRI (fMRI; 15.2%; highest in North America: 40.3%), magnetoencephalography (11.9%; highest in North America: 39.3%), single photon emission computed tomography (9.6%; highest in North America: 22.2%), high‐density electroencephalography (EEG; 1.9%; highest in Europe: 4.7%), source localization (1.6%; highest in Oceania: 7.6%), Wada test (1.2%; highest in North America: 3.5%), and EEG‐fMRI (.5%; highest in Europe: 1.1%). Significance Despite some early surgeries, including in infancy and before pharmacoresistance, mean epilepsy duration before surgery remains >5 years. Reoperations are common, with resection more frequent than neuromodulation. Genetic testing in nearly half of patients reflects its growing relevance, and the high rate of DEEs underscores the complexity of surgical candidates.