Nair, Asha

Abstract Glioblastoma (GBM) remains the most lethal primary brain cancer due to its remarkable metabolic plasticity and therapeutic resistance. Here, we identify cholesterol dependency as a therapeutically exploitable vulnerability in GBM using two FDA-approved drugs: the H1 histamine antagonist clemastine and the retinoid X receptor agonist bexarotene. Combined treatment induces potent synergistic anti-tumor activity across patient-derived glioma models, suppressing proliferation, stemness, and survival at sub-IC50 concentrations. Mechanistically, this therapy disrupts cholesterol biosynthesis, transport, and homeostasis, triggering endoplasmic reticulum stress and activation of the unfolded protein response, ultimately leading to autophagy and apoptotic cell death. Orthotopic patient-derived glioma models recapitulate these mechanisms in vivo, where local intracranial administration significantly reduces tumor progression and prolongs survival using fourfold lower doses than systemic intraperitoneal delivery. Single-cell RNA sequencing revealed activation of regeneration and plasticity programs, accompanied by immune microenvironment remodeling and enhanced inflammatory signaling. Importantly, syngeneic models preserved immune cell composition, supporting future integration with immunotherapeutic strategies. Together, these findings establish cholesterol dysregulation–induced metabolic collapse as a promising therapeutic approach for GBM.

Abstract Background Vitamin D supplementation by human individuals has significantly increased in the world. In US the total stored 25-OH-Vitamin D3 and 25-OH-Vitamin D2 has significantly increased for both in summers and winters. Under sunlight exposure, 7 dehydrocholesterol is converted to Vitamin D3 and is endogenous source in mammals. However, most of the exogenous source for Vitamin D used to be Vitamin D2 which used to be extracted only from mushrooms but is now manufactured by supplement industry as well. In last decade most of the supplementation has been due to chemically synthesized Vitamin D3. Food is now supplemented by both D2 and D3. It is not clear what is the prevalence of D2 supplementation/consumption in American population. Competitive immunoassays do not consistently recover 25-OH-D2 compared to 25-OH-D3 and some antibodies used have variable or no cross reactivity with 25-OH-D2. LC-MS/MS method can easily distinguish the two and can help in providing the precise and accurate results for 25-OH-D2 and 25-OH-D3. Here we present what is the prevalence of Vitamin D2 consumption and toxicity in American population based on our LC-MS/MS methods. Methods The study measured vitamin 25-OH-D levels (total, D2, and D3) in 148,654 patients from Mayo Clinic as well as from other medical centers within the U.S. Patients age ranging from new-born babies to over 100 years, and represents Male, Female and Neutral genders. Samples were analyzed for 25-OH-Vitamin D by LC-MS/MS as published earlier. Unlike immunoassays, LC-MS/MS gave us separate results for 25-OH-D3 and 25-OH-D2 with 100 percent specificity. Results 25-OH-Vitamin D2 levels of greater than 10 was present in at least 10,358 and ranged from 10 to 205 ng/mL. Of 10,358, 51 percent had 20-50 ng/mL, 10 percent had 51-80 ng/mL, and 1.3 percent had >80 ng/mL, ranging from 81-205 ng/mL with a median of 92 ng/mL. Of the patients found at Total Vitamin D >80 ng/mL, toxic levels of both D2 and D3 were found more in females than males. There was no significant gender difference for Vitamin D3 consumption but for D2 consumption, mean and median levels were different in males and females. Conclusion Clinical lab testing should accurately measure both 25-OH-D3 and 25-OH-D2, otherwise the patient total Vitamin D values in circulation will be underestimated.

Abstract Background The development of Crohn’s disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated. Materials and Methods Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells. Results CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites. Conclusions The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells.

Background: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy. Currently, hypertrophy pathways responsible for HCM have not been fully elucidated. Their identification could serve as a nidus for the generation of novel therapeutics aimed at halting disease development or progression. Herein, we performed a comprehensive multi-omic characterization of hypertrophy pathways in HCM. Methods: Flash-frozen cardiac tissues were collected from genotyped HCM patients (n=97) undergoing surgical myectomy and tissue from 23 controls. RNA sequencing and mass spectrometry–enabled deep proteome and phosphoproteomic assessment were performed. Rigorous differential expression, gene set enrichment, and pathway analyses were performed to characterize HCM-mediated alterations with emphasis on hypertrophy pathways. Results: We identified transcriptional dysregulation with 1246 (8%) differentially expressed genes and elucidated downregulation of 10 hypertrophy pathways. Deep proteomic analysis identified 411 proteins (9%) that differed between HCM and controls with strong dysregulation of metabolic pathways. Seven hypertrophy pathways were upregulated with antagonistic upregulation of 5 of 10 hypertrophy pathways shown to be downregulated in the transcriptome. Most upregulated hypertrophy pathways encompassed the rat sarcoma-mitogen-activated protein kinase signaling cascade. Phosphoproteomic analysis demonstrated hyperphosphorylation of the rat sarcoma-mitogen-activated protein kinase system suggesting activation of this signaling cascade. There was a common transcriptomic and proteomic profile regardless of genotype. Conclusions: At time of surgical myectomy, the ventricular proteome, independent of genotype, reveals widespread upregulation and activation of hypertrophy pathways, mainly involving the rat sarcoma-mitogen-activated protein kinase signaling cascade. In addition, there is a counterregulatory transcriptional downregulation of the same pathways. Rat sarcoma-mitogen-activated protein kinase activation may serve a crucial role in hypertrophy observed in HCM.

Abstract Glioblastoma (GBM) patients have a median survival of 15 months despite aggressive treatment. Treatment-related pseudo-progression confounds outcome assessment by MRI, particularly in patients receiving immunotherapy. Extracellular vesicles (EVs) contain tumor-specific miRNA that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. Small plasma EVs were isolated by serial density gradient ultracentrifugation from 20 newly-diagnosed GBM patients enrolled in a clinical trial of allogeneic tumor lysate-pulsed autologous dendritic cell (DC) vaccination. Short non-coding RNA sequencing was performed for each patient at three time points (TP): pre-vaccine (TP1), post-initial vaccine (TP2), and at end of treatment (TP3). Ingenuity Pathway Analysis from 31 differentially expressed miRNAs across time points (p-value <0.05, |logFC|>1) revealed that glioma-related signaling pathways previously seen in small EVs from glioma patients compared to normal donors were replicated when comparing patients at the beginning of the treatment (TP1-vs-TP2) and between early to late time points (TP1-vs-TP3). In addition, pathways corresponding to immunological responses typically seen in GBM patients undergoing treatment, such as IL-10, IL-6, and Toll-like Receptor (TLR) signaling, were exclusively found after the initial vaccine towards the end of treatment (TP2-vs-TP3). In conclusion, signaling pathways derived from differentially expressed miRNAs across time points suggest that as patients progress through treatment, consistent differences in plasma small EVs miRNA expression profile and immunological responses could be utilized as predictors of treatment response.