BACKGROUND:Oral lichen planus (OLP) is a chronic, immune-mediated inflammatory disorder of the oral mucosa, primarily involving T-lymphocyte activity. It is recognized by the World Health Organization as an oral potentially malignant disorder due to its established risk for malignant transformation. Although OLP has been the subject of extensive research, its pathogenesis remains partially understood. Vitamin D, a fat-soluble hormone essential for calcium-phosphorus homeostasis and immunological regulation, has emerged as a significant factor in the onset, progression, and clinical course of OLP. SUMMARY:This review systematically examines the association between vitamin D-related local metabolic pathways and the oral mucosal microenvironment, synthesizing epidemiological evidence from diverse geographic regions that support an association between vitamin D deficiency and the occurrence of OLP. The molecular mechanisms by which vitamin D modulates OLP are discussed within a comprehensive framework including cellular protection, immune modulation, and regulation via non-coding RNAs. In addition, recent advances in the clinical application of vitamin D are summarized, particularly in relation to its potential role in non-invasive diagnostics and as an adjunctive therapy for OLP. Unresolved questions and areas of debate in current research are critically evaluated. Based on contemporary high-quality evidence, future research directions are proposed, emphasizing the need for individualized therapeutic strategies through multicenter, large-scale randomized controlled trials. KEY MESSAGES:Additional exploration of the interactions among vitamin D/vitamin D receptor signaling pathways, non-coding RNA networks, and the gut microbiota is recommended to enhance mechanistic insights and optimize clinical management strategies for individuals with OLP.

Preclinical studies have established that common pediatric anesthetics such as sevoflurane and propofol can induce neurotoxicity, yet their molecular mechanisms remain incompletely elucidated. This study combined LC-MS-based metabolomics with caspase-3 and cleaved caspase-3 immunoblotting, Y-maze, and Morris water maze testing to investigate metabolic alterations linked to anesthesia-induced neurotoxicity in neonatal Sprague-Dawley rats. Animals at postnatal day 7 (P7) were exposed to a single 2-hour session of sevoflurane or propofol anesthesia and assessed at 6 h (P7) and 35 days (P42) post-exposure. Both anesthetics significantly upregulated hippocampal caspase-3 and cleaved caspase-3 expression at both time points and resulted in long-term decline of working memory in the Y-maze test at P42. However, no significant cognitive alteration was detected in the Morris water maze test at P42. Metabolomic analysis revealed anesthetic-specific metabolic disturbances in the hippocampus. In propofol-treated rats, six metabolites—indole, isoleucine, lysine, phenylalanine, D-ribose 5-phosphate, and biotin—were consistently and significantly elevated at 6 h and 35 days post-anesthesia. All six metabolites showed a strong positive correlation with caspase-3 levels. In contrast, sevoflurane anesthesia led to a marked and sustained downregulation of phosphatidylcholine PC (16:0/16:0), which correlated negatively with caspase-3 expression. This study identifies distinct hippocampal metabolite profiles associated with prolonged neurotoxicity induced by propofol and sevoflurane. The propofol-related metabolites—indole, isoleucine, lysine, phenylalanine, D-ribose 5-phosphate, and biotin—and the sevoflurane-associated metabolite PC (16:0/16:0) represent potential intervention targets for mitigating the adverse neurodevelopmental effects of general anesthesia in neonates.

SUMMARY Cellular heterogeneity and plasticity are hallmarks of cancer that contribute to tumor growth and therapy resistance. Here we investigated metabolic heterogeneity in small cell lung cancer (SCLC), an aggressive neuroendocrine (NE) cancer type. Through integrated transcriptomic and metabolomic analyses, we identified a universal dependency on exogenous cysteine/cystine (Cys) across all NE/non-NE SCLC cell states. Notably, NE and non-NE cells with low levels of the ASCL1 transcription factor die from ferroptosis upon Cys depletion. In contrast, ASCL1-high cells die from apoptosis but are ferroptosis resistant. This resistance to ferroptosis is driven by the direct upregulation of the gene coding for the GCH1 enzyme by ASCL1, which results in higher levels of the BH4/BH2 antioxidants. Accordingly, combining cysteine depletion with BH4/BH2 synthesis inhibition effectively reduces tumor growth in patient-derived xenografts. This work elucidates distinct metabolic states in SCLC and suggests new approaches to induce cell death in this lethal form of cancer.

S1. Mitochondrial uncoupler NEN increases cellular NAD+/NADH ratio and inhibits reductive carboxylation.

S2. Mitochondrial uncoupler NEN accelerates forward TCA cycle and inhibits reductive carboxylation.

S7. Mitochondrial uncoupling inhibits reductive carboxylation in spheroid culture.

Regional counting of differential methylated probes in SK-N-BE(2) cells treated with NEN for 24hrs under nomoxia. Go enrichment pathways of the differential methylated probes of the CpG Island in the promoter (B) (C) and gene body (D) (E)

Reduced mitochondrial quality and quantity in tumors is associated with dedifferentiation and increased malignancy. However, it remains unclear how to restore mitochondrial quantity and quality in tumors and whether mitochondrial restoration can drive tumor differentiation. Our study shows that restoring mitochondrial function using retinoic acid (RA) to boost mitochondrial biogenesis and a mitochondrial uncoupler to enhance respiration synergistically drives neuroblastoma differentiation and inhibits proliferation. U- 13 C-glucose/glutamine isotope tracing revealed a metabolic shift from the pentose phosphate pathway to oxidative phosphorylation, accelerating the tricarboxylic acid cycle and switching substrate preference from glutamine to glucose. These effects were abolished by electron transport chain (ETC) inhibitors or in ρ 0 cells lacking mitochondrial DNA, emphasizing the necessity of mitochondrial function for differentiation. Dietary RA and uncoupler treatment promoted tumor differentiation in an orthotopic neuroblastoma xenograft model, evidenced by neuropil production and Schwann cell recruitment. Single-cell RNA sequencing of xenografts revealed that this strategy effectively eliminated the stem cell population, promoted differentiation, and increased mitochondrial gene signatures along the differentiation trajectory, potentially improving patient outcomes. Collectively, our findings establish a mitochondria-centric therapeutic strategy for inducing tumor differentiation, suggesting that maintaining/driving differentiation in tumor requires not only ATP production but also continuous ATP consumption and sustained ETC activity.

S6. Mitochondrial uncoupling inhibits reductive carboxylation in ccRCC cells.

S5. Mitochondrial uncoupler NEN, FCCP and BAM15 inhibit reductive carboxylation under hypoxia.

Dendrobium huoshanense (DH) belongs to the Dendrobium genus of the Orchidaceae family and is a herbaceous plant that protects the liver and nourishes the Yin according to traditional Chinese Medicine (TCM) theory. This research aimed to determine the therapeutic effect and mechanisms of DH on a nonalcoholic fatty liver disease (NAFLD) mouse model and its chemical composition. For pharmacological research, the pathological damage and lipid accumulation in liver tissues were evaluated using HE and oil red staining, respectively. The differential proteins between the model and DHH groups were screened using 4D label-free quantitative proteomics, and the proteomic results were verified using Western blot. The potential mechanism was validated by metabolomic analysis. The main active ingredients in a DH aqueous extract were identified using UHPLC-Q Exactive HF HRMS. Pathological staining results showed that DH can reverse liver pathological damage and lipid accumulation in the NAFLD model. Quantitative proteomics revealed that the differential proteins were mainly associated with liver lipid deposition (LAL, AMPK, TM7SF2, SBCAD, and SIRT1), insulin resistance (GYS1, GYS2, PYGL, FoxO1, and PPAR-γ), and inflammation (TLR2 and MAPKAPK). Western blot verified the above-mentioned results. Metabolomic analysis also indicated that the DH aqueous extract ameliorated NAFLD in mice by affecting cholesterol metabolism and AMPK signaling pathway, proving its significant therapeutic effects on the NAFLD model. Sixty-five compounds were identified from DH aqueous extract by analyzing the precise molecular weight and MS/MS fragmentation pathway. The pharmacological mechanism of DH in treating NAFLD mainly involved the TLR2-NF-κB and AMPK-SREBP1-SIRT1 signaling pathways.

SK-N-BE(2) cells were treated with NEN for 16hrs. (A) Upregulated and (B) downregulated genes from RNA-seq data (n=3) (sleuth q-value < 0.05 and fold change estimate b > abs(ln(2))). (C) The top 10 gene ontology (GO) pathways enriched from upregulated genes by using DAVID analysis. (D) The top 10 gene ontology (GO) pathways enriched from downregulated genes by using DAVID analysis.

S9. ETC inhibition only inhibits proliferation, but not spheroid formation, while Mitochondrial uncoupling inhibits both.

Favorable and (B) unfavorable gene list (p-value<0.05) from 11 available neuroblastoma databases from R2 (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi) (C) Overlapped the favorable prognosis gene sets (p-value<0.05, gene number >1000) from 7 available neuroblastoma databases from R2 (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). (D) (E) Go analysis of overlapped favorable and unfavorable prognosis gene sets. (F) (G) GSEA analysis of Favorable and unfavorable gene list genes from RNA-seq experiments in Figure (1D).

S8. Mitochondrial uncoupling inhibits proliferation upon anchorage-independent growth.

S3. Mitochondrial uncoupler FCCP and BAM5 inhibit reductive carboxylation.

One-Sentence SummaryRaf1 is present within the mitochondrial matrix, where it binds GLS to regulate glutamine catabolism and tumorigenesis.In cancer, Raf1 activation occurs via mechanisms that include mutation of upstream regulators, such as receptor tyrosine kinases and Ras GTPases, as well as by mutations that affectRAF1itself, including via gene amplification (1–4). Once recruited to the plasma membrane (PM) Raf1 can engage downstream mitogen-activated protein kinase (MAPK) pathway signaling through phosphorylation of the MEK kinases (5). In addition to Raf1, A-Raf and B-Raf can also activate MEK and these other two Raf isoforms can compensate for MAPK activation in the event of Raf1 loss (6,7). Despite this, Raf1 remains essential for the development and maintenance of some tumors through mechanisms independent of MAPK activity (7,8). In this regard, Raf1 has well-described interactions outside the canonical MAPK pathway, including several with outer mitochondrial membrane (OMM) proteins (9,10), although Raf1 has not been previously identified inside mitochondria. Mitochondria comprise a hub for various metabolic processes modulated in cancer cells to accommodate rapid proliferation. One such process is glutaminolysis, which involves the catabolism of glutamine to generate both ATP as well as precursors for the synthesis of fatty acids, nucleotides, and nonessential amino acids (11–13). Glutaminase (GLS) proteins, which catalyze the first and rate-limiting step of this process by converting glutamine to glutamate, are often upregulated in cancer (14–16). GLS activation has been previously associated with tumors driven by Ras, upstream regulators of Raf kinases (13,17). Here we identify Raf1 protein inside mitochondria where Raf1 associates with GLS in the mitochondrial matrix to enable glutamine catabolism and tumorigenic growth.Raf kinases play vital roles in normal mitogenic signaling and cancer, however, the identities of functionally important Raf-proximal proteins throughout the cell are not fully known. Raf1 proximity proteomics/BioID in Raf1-dependent cancer cells unexpectedly identified Raf1-adjacent proteins known to reside in the mitochondrial matrix. Inner-mitochondrial localization of Raf1 was confirmed by mitochondrial purification and super-resolution microscopy. Inside mitochondria, Raf1 associated with glutaminase (GLS) in diverse human cancers and enabled glutaminolysis, an important source of biosynthetic precursors in cancer. These impacts required Raf1 kinase activity and were independent of canonical MAP kinase pathway signaling. Kinase-dead mitochondrial matrix-localized Raf1 impaired glutaminolysis and tumorigenesis in vivo. These data indicate that Raf1 localizes inside mitochondria where it interacts with GLS to engage glutamine catabolism and support tumorigenesis.