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Aims Acyl‐CoA synthetase long chain family member 4 (ACSL4) is closely related to tumor genesis and development in certain tissues. However, the function of ACSL4 in early brain injury (EBI) caused by subarachnoid hemorrhage (SAH) is unclear. In this study, we investigated the expression patterns and role of ACSL4 in SAH and post‐SAH EBI using a rat model of SAH. Methods The rat model of SAH was induced by autologous blood injection into the prechiasmatic cistern of rats. We also used two specific inhibitors of ferroptosis (Ferrostatin‐1 and Liproxstatin‐1) to investigate the role of ferroptosis in EBI. Results We found that ACSL4 levels in brain tissue increased significantly in post‐SAH EBI. Inhibiting the expression of ACSL4 using small interfering RNAs alleviated inflammation, blood‐brain barrier (BBB) impairment, oxidative stress, brain edema, and behavioral and cognitive deficits, and increased the number of surviving neurons, after SAH. Similar effects were obtained by suppressing ferroptosis. Conclusions ACSL4 exacerbated SAH‐induced EBI by mediating ferroptosis. These findings may provide a theoretical basis for potential therapy aimed at alleviating post‐SAH EBI. ACSL4 levels in brain tissue of rats increase significantly in EBI and brain damage after SAH could be reduced by down‐regulation of ACSL4. ACSL4 could trigger ferroptosis and aggravate brain damage via catalyzing lipid metabolism. It may provide a theoretical basis for potential therapy to alleviate EBI after SAH

Background KRAS G12C-targeted therapies have transformed the treatment of KRAS G12C-mutant lung adenocarcinoma. However, acquired resistance to these therapies, whose underlying molecular mechanisms are not fully understood, presents a major obstacle to achieving long-term therapeutic success. The purpose of this study was to elucidate the mechanisms of acquired resistance to KRAS G12C inhibitors and identify potential regulators of resistance in lung adenocarcinoma. Methods Two lung adenocarcinoma cell lines (H23 and H2122) were exposed to escalating doses of two novel KRAS G12C inhibitors, fulzerasib and garsorasib, to generate resistant variants. Transcriptomic profiling was conducted to identify genes consistently upregulated in resistant cells. CRISPR/Cas9-mediated knockout (MXD4-KO) and siRNA-mediated knockdown of MXD4 were performed to assess its role in drug resistance. Mechanistic investigations employed inhibitors of ferroptosis, apoptosis, and necrosis, along with assays measuring lipid peroxidation and malondialdehyde levels. Further analysis included ferroptosis-related gene expression profiling, lipidomic profiling, ChIP-Seq, ChIP-qPCR, and dual-luciferase reporter assays. The findings were validated in patient-derived organoids (PDOs) and nude mouse models. Results Transcriptomic profiling identified 11 genes consistently upregulated in resistant cells, with MXD4 emerging as a key resistance regulator. CRISPR/Cas9-mediated knockout of MXD4 restored sensitivity to fulzerasib, garsorasib, sotorasib, and adagrasib, an effect fully reversed upon MXD4 re-overexpression. Similarly, siRNA-mediated knockdown of MXD4 in resistant cells restored drug sensitivity. Mechanistic studies revealed that MXD4 specifically suppresses ferroptosis, rather than apoptotic or necrotic pathways, by repressing ACSL4 expression and blocking its catalytic synthesis of phosphatidylethanolamine-polyunsaturated fatty acids (PE-PUFAs). ACSL4 knockout or overexpression abolished MXD4’s ability to promote ferroptosis suppression and resistance to KRAS G12C inhibitors. ChIP-Seq, ChIP-qPCR, and dual-luciferase reporter assays confirmed that MXD4 directly binds to and represses the ACSL4 promoter. These findings were validated in PDOs and nude mouse models, where MXD4 knockout restored therapeutic sensitivity, while ACSL4 knockout blocked MXD4’s resistance-promoting effects. Conclusions This study uncovers a novel resistance mechanism in which MXD4 transcriptionally silences ACSL4 to suppress ferroptosis, enabling cancer cells to evade KRAS G12C inhibitors. Targeting the MXD4-ACSL4 axis represents a promising strategy to overcome therapeutic resistance in KRAS G12C-mutant lung cancer, potentially improving long-term treatment outcomes.

Production of flavors from natural substrates by microbial transformation has become a growing and expanding field of study over the past decades. Vanillin, a major component of vanilla flavor, is a principal flavoring compound used worldwide. Streptomyces sp. strain V-1 is known to be one of the most promising microbial producers of natural vanillin from ferulic acid. Although identification of the microbial genes involved in the biotransformation of ferulic acid to vanillin has been previously reported, purification and detailed characterization of the corresponding enzymes with important functions have rarely been studied. In this study, we isolated and identified 2 critical genes, fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, which are involved in the vanillin production from ferulic acid. Both genes were heterologously expressed in Escherichia coli, and the resting cell reactions for converting ferulic acid to vanillin were performed. The corresponding crucial enzymes, Fcs and Ech, were purified for the first time and the enzymatic activity of each purified protein was studied. Furthermore, Fcs was comprehensively characterized, at an optimal pH of 7.0 and temperature of 30°C. Kinetic constants for Fcs revealed the apparent Km, kcat, and Vmax values to be 0.35 mM, 67.7 s(-1), and 78.2 U mg(-1), respectively. The catalytic efficiency (kcat/Km) value of Fcs was 193.4 mM(-1) s(-1) for ferulic acid. The characterization of Fcs and Ech may be helpful for further research in the field of enzymatic engineering and metabolic regulation.

Background This study investigates the role of fatty acid metabolism (FAM)-related genes in lymph node metastasis (LNM) and prognosis of lung adenocarcinoma (LUAD) and elucidates the underlying mechanisms. Methods Transcriptomic and single-cell RNA-seq data from TCGA and GEO were integrated to identify FAM-related genes. Non-negative matrix factorization clustering and univariate Cox regression were applied to develop a FAM-based prognostic risk model (FScore). Associations of FScore with gene mutations and tumor microenvironment features were analyzed. Immunohistochemistry, and functional assays were performed to alidate the role of ACSL3 in LUAD malignancy and lymphangiogenesis. Results A five-gene FAM-related risk signature (ACSL3, MCAT, NDUFAB1, OLAH, ACSL4) was identified. The derived FScore stratified patient prognosis across multiple independent cohorts, with high FScore linked to significantly worse overall survival. FScore increased progressively with nodal stage (N0 < N1 < N2) and correlated with an immunosuppressive “cold” tumor microenvironment and specific mutation patterns (e.g., low FLG mutation). Single-cell and spatial transcriptomics revealed cell-type–specific FAM activity, predominantly in epithelial, mast, and myeloid cells. ACSL3 was overexpressed in LUAD tissues and served as an independent poor prognostic factor. ACSL3 overexpression elevated intracellular triglyceride and phospholipid levels, upregulated key FAM enzymes (FASN, ACC, ACLY) and the c-Myc/VEGFC axis, promoted proliferation, migration, invasion, and lymphangiogenesis, while suppressing apoptosis. Conclusions The FScore serves as a robust predictor of LNM and poor prognosis in LUAD. ACSL3 drives lymphatic metastasis via the c-Myc/VEGFC axis, positioning ACSL3 as a potential therapeutic target to suppress LNM in LUAD.

Background Long-chain fatty acyl CoA synthetase 4 (ACSL4) is crucial for lipid metabolism, primarily catalyzing the formation of 12–20 carbon chain fatty acids. ACSL4 is upregulated in various cancers and linked to aggressive behavior and poor survival. A bioinformatics study showing ACSL4 upregulation in pancreatic cancer. However, utility for actual pathological diagnosis and clinical significance in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) are unexplored. This study aimed to investigate ACSL4 expression in PDAC and IPMN, and evaluate its clinical implications. Methods We examined ACSL4 expression using immunohistochemistry in 165 patients with PDAC and IPMN. Differences in ACSL4 expression between malignant and benign lesions were evaluated using the Pearson χ2 test. The association between ACSL4 expression, pathological parameters, and survival was assessed through Kaplan-Meier and Cox regression analyses in 96 patients with invasive cancer. Results Compared to normal pancreatic ducts, low-grade pancreatic intraepithelial neoplasm, and intraductal papillary mucinous adenoma (IPMA) (3.3%, 3.4%, and 2.7%, respectively), ACSL4 expression was significantly higher in invasive PDAC, noninvasive intraductal papillary mucinous carcinoma (IPMC), and invasive IPMC (77%, 86.7%, and 93.9%, respectively). In invasive cancers, low ACSL4 expression was associated with a higher frequency of lymphovascular invasion and recurrence and shorter disease-free survival ( P  = 0.006). Additionally, low ACSL4 expression was an independent prognostic factor for shorter disease-free survival in multivariable Cox regression analysis (HR = 2.409, 95% CI: 1.121–5.180, P  = 0.024). Conclusion ACSL4 expression helps differentiate cancerous from precancerous lesions in pancreatic cancer, but low expression is linked to a higher frequency of lymphovascular invasion and shorter disease-free survival in invasive cases. Due to the limited sample size and broad confidence intervals, the findings of this study should be interpreted with caution and require validation in larger, independent cohorts.

All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be relevant for other (+)RNA viruses as well.

Background 4-Hydroxycinnamaldehydes are important intermediates in several secondary metabolism pathways, including those involved in the biosynthesis of phenolic acids, flavonoids, terpenoids and monolignols. They are also involved in the biosynthesis and degradation of lignins, which are important limiting factors during the processes of papermaking and biofuel production. Access to these aromatic polymers is necessary to explore the secondary biometabolic pathways they are involved in. Coniferaldehyde, sinapaldehyde, p -coumaraldehyde and caffealdehyde are members of the 4-hydroxycinnamaldehyde family. Although coniferaldehyde and sinapaldehyde can be purchased from commercial sources, p -coumaraldehyde and caffealdehyde are not commercially available. Therefore, there is increasing interest in producing 4-hydroxycinnamaldehydes. Here, we attempted to produce 4-hydroxycinnamaldehydes using engineered Escherichia coli . Results 4-Coumaric acid: coenzyme A ligase (4CL1) and cinnamoyl coenzyme A reductase (CCR) were fused by means of genetic engineering to generate an artificial bifunctional enzyme, 4CL1–CCR, which was overexpressed in cultured E. coli supplemented with phenylpropanoic acids. Three 4-hydroxycinnamaldehydes, p -coumaraldehyde, caffealdehyde and coniferaldehyde, were thereby biosynthesized and secreted into the culture medium. The products were extracted and purified from the culture medium, and identically characterized by the HPLC–PDA–ESI–MSn. The productivity of this new metabolic system were 49 mg/L for p -coumaraldehyde, 19 mg/L for caffealdehyde and 35 mg/L for coniferaldehyde. Extracellular hydroxycinnamoyl-coenzyme A thioesters were not detected, indicating that these thioesters could not pass freely through the cellular membrane. The fusion enzyme 4CL1–CCR can catalyze sequential multistep reactions, thereby avoiding the permeability problem of intermediates, which reveals its superiority over a mixture of individual native enzymes. Moreover, we have described a highly sensitive and selective method for separation and identification of phenylpropanoic acids and their corresponding cinnamaldehydes in the present paper. The feasibility of this method has been proven in the application of the method to the analysis of the metabolites of whole-cell catalysts. Conclusions We have established a bioconversion pathway for the microbial production of valuable 4-hydroxycinnamaldehydes from phenylpropanoic acids. This biotransformation method is both convenient and environmentally friendly, and provides new insights into the biosynthesis of natural plant secondary products.

Like obese humans, Zucker diabetic fatty (ZDF) rats exhibit early β cell compensation for insulin resistance (4-fold β cell hyperplasia) followed by decompensation (>50% loss of β cells). In prediabetic and diabetic ZDF islets, apoptosis measured by DNA laddering is increased 3- and >7-fold, respectively, compared with lean ZDF controls. Ceramide, a fatty acid-containing messenger in cytokine-induced apoptosis, was significantly increased (P < 0.01) in prediabetic and diabetic islets. Free fatty acids (FFAs) in plasma are high (>1 mM) in prediabetic and diabetic ZDF rats; therefore, we cultured prediabetic islets in 1 mM FFA. DNA laddering rose to 19.6% vs. 4.6% in lean control islets, preceded by an 82% increase in ceramide. C2-Ceramide without FFA induced DNA laddering, but fumonisin B1, a ceramide synthetase inhibitor, completely blocked FFA-induced DNA laddering in cultured ZDF islets. [3H]Palmitate incorporation in [3H]ceramide in ZDF islets was twice that of controls, but [3H]palmitate oxidation was 77% less. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, both blocked DNA laddering. These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis. In ZDF obesity, β cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production.

Acyl-CoA synthetase 5 (ACSL5), a mitochondrially localized enzyme, catalyzes the synthesis of long-chain fatty acid thioesters and is physiologically involved in pro-apoptotic sensing of enterocytes. The aim of the present study is to identify an ACSL5-dependent regulation of mitochondrially expressed proteins and the characterization of related pathways in normal and diseased human intestinal mucosa. Proteomics of isolated mitochondria from ACSL5 transfectants and CaCo2 controls were performed. ACSL5-dependent protein synthesis was verified with quantitative reverse transcription plus the polymerase chain reaction, Western blotting, short-interfering-RNA-mediated gene silencing and additional cell culture experiments. Lipid changes were analyzed with tandem mass spectrometry. ACSL5-related pathways were characterized in normal mucosa and sporadic adenocarcinomas of the human intestine. In CaCo2 cells transfected with ACSL5, mortalin ( HSPA9 ) was about two-fold increased in mitochondria, whereas cytoplasmic mortalin levels were unchanged. Disturbance of acyl-CoA/sphingolipid metabolism, induced by ACSL5 over-expression, was characterized as crucial. ACSL5-related over-expression of mitochondrial mortalin was found in HEK293 and Lovo (wild-type TP53 [tumor protein p53]) and CaCo2 (p53-negative; TP53 mutated) cells but not in Colo320DM cells (mutated TP53 ). In normal human intestinal mucosa, an increasing gradient of both ACSL5 and mortalin from bottom to top was observed, whereas p53 (wild-type TP53 ) decreased. In sporadic intestinal adenocarcinomas with strong p53 immunostaining (mutated TP53 ), ACSL5-related mortalin expression was heterogeneous. ACSL5-induced mitochondrial mortalin expression is assumed to be a stress response to ACSL5-related changes in lipid metabolism and is regulated by the TP53 status. Uncoupling of ACSL5 and mitochondrial mortalin by mutated TP53 could be important in colorectal carcinogenesis.

Background Nonalcoholic fatty liver disease (NAFLD) is caused by fat metabolism disorders and thereby abnormal or excessive accumulation of fat in hepatocytes, and characterized by steatosis, inflammation, fibrosis, apoptosis or necrosis. Aim This study was carried out to explore the correlation between gene expression profiles of rat livers and the occurrence and progression of NAFLD at the transcriptional level. Methods A rat model of nonalcoholic steatohepatitis (NASH) was established by feeding male rats with high-fat emulsion via gavage, and Rat Genome 230 2.0 Array was used to detect gene expression profiles of liver tissues obtained from male rats following 0, 2, 4, and 6 weeks of high-fat emulsion feeding. Methods of bioinformatics and systems biology were applied to analyze the correlation between gene expression changes and physiological activities involved in NAFLD. Results In total, 93 function-known genes, including 36 up-regulated and 57 down-regulated, differed significantly in expression compared to those of control rats, and 18 physiological activities were closely related to NAFLD. Especially, the activity of cell differentiation was decreased during the whole process of NAFLD, and the activities of inflammation response, stimulus response, cell migration and adhesion were attenuated in the second, fourth and sixth week, respectively. In the fourth and sixth weeks, lipid metabolism and cell apoptosis were augmented, and the former might be associated with the enhanced expression of plin , acsl6 , scd2 , elovl3 , etc. Conclusion These data provide useful information on the global gene expression changes due to high-fat emulsion feeding and bring important insights into the mechanisms of NAFLD.