Search Results (89)

Background/Objectives: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder of mitochondrial fatty acid β-oxidation caused by pathogenic variants in ACADVL. The clinical spectrum is highly heterogeneous, ranging from lethal neonatal cardiomyopathy to late-onset myopathy. This study aims to characterize the rare c.215C>T (p.Ser72Phe) variant, identified in compound heterozygosity with the common pathogenic allele c.848T>C (p.Val283Ala) in a male neonate detected by newborn screening (NBS). Methods: Genetic analysis was performed using Sanger sequencing on the proband and his family members. The pathogenicity of the p.Ser72Phe variant was evaluated through multiple bioinformatic predictors and interpreted according to ACMG/AMP guidelines. To understand the functional impact on the protein, structural modeling was conducted using FoldX 4.0 for energy calculations and UCSF ChimeraX for the visualization of conformational changes and cofactor-binding site perturbations in the VLCAD homodimer. Results: At the end of the first postnatal week, liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of dried blood spots of the proband revealed a markedly abnormal acylcarnitine profile, with C14:1 levels (1.837 μmol/L) approximately five times above the reference range. Clinical reports documented hypoketotic hypoglycemia, consistent with VLCADD. Segregation analysis demonstrated transmission of both variants within the family, with additional heterozygous and homozygous carriers identified. Bioinformatic predictions uniformly classified p.Ser72Phe as deleterious. This variant has an extremely low allele frequency and affects a highly conserved residue in the FAD-binding domain. Structural modeling with FoldX yielded a mean ΔΔG of +22.63 ± 5.48 kcal/mol, indicating a significant localized thermodynamic burden. Inspection of the mutant model in ChimeraX showed perturbation of the side-chain orientation and attenuation of the local hydrogen-bonding network at the FAD-binding site, together with increased steric packing around residue 72. Taken together, the clinical, genetic, and structural evidence support reclassification of p. Ser72Phe as likely pathogenic according to ACMG criteria, specifically applying the ClinGen ACADVL VCEP specifications. Conclusions: This study expands the ACADVL mutational spectrum and underscores the value of integrating sequencing, segregation, and structural bioinformatics in interpreting rare variants detected through NBS. Full article

Fish oil (FO) has been shown to confer beneficial effects on hepatic diseases in both humans and animals. This study aimed to investigate whether dietary fish oil (FO) supplementation alleviates deoxynivalenol (DON)-induced liver injury by modulating the ferroptosis signaling pathway in weaned piglets. Twenty-four weaned piglets were allocated to a 2 × 2 factorial design, with the main factors consisting of dietary treatment (5% corn oil or 5% FO supplementation) and DON exposure (basal diet or diet contaminated with 4 mg/kg DON). After 21 days of dietary treatment, piglets were euthanized for collection of blood and liver samples. Dietary FO significantly attenuated DON-induced hepatic structural damage and inflammatory infiltration. Specifically, FO supplementation reduced the activities of aspartate transaminase (AST) and alkaline phosphatase (ALP), as well as the AST/alanine aminotransferase (ALT) ratio following DON exposure. Dietary FO also decreased malondialdehyde (MDA) concentrations in both the liver and serum, lowered hepatic 4-hydroxynonenal (4-HNE) level and Fe²⁺ content, and increased hepatic glutathione (GSH) content. Moreover, dietary FO ameliorated ultrastructural liver damage induced by DON. Furthermore, DON significantly downregulated the mRNA levels of multiple genes associated with iron metabolism and ferroptosis, including heat shock protein beta-1 (HSPB1), acyl-CoA synthetase long chain family member 4 (ACSL4), and arachidonate 15-lipoxygenase (ALOX15), and upregulated the mRNA levels of transferrin (TF), ferritin heavy chain (FTH), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor 1 (TFR1). Dietary FO counteracted these alterations by decreasing the mRNA of SLC7A11, TFR1, FTH, and TF after DON exposure. Finally, FO significantly decreased the protein expression of SLC7A11, iron-responsive element-binding protein 2 (IREB2), and FHT1 and increased the GPX4 protein expression following DON exposure. These findings suggest that FO may ameliorate DON-induced liver injury in weaned piglets, possibly through suppressing the ferroptosis signaling pathway. Full article

High ammonia nitrogen stress significantly compromises the survival of Litopenaeus vannamei under low-salinity conditions. However, existing studies predominantly focus on ammonia nitrogen responses under single stressors or normal seawater salinity. The molecular regulatory mechanisms, metabolic remodeling patterns, and key pathway interactions in shrimp subjected to high ammonia nitrogen stress under low-salinity environment remain unclear. In this study, we employed integrated transcriptomic and metabolomic analyses to unveil the underlying molecular responses and metabolic biomarkers in the gills of L. vannamei to ammonia stress under low-salinity conditions. First, L. vannamei underwent low-salinity acclimation from 30‰ to 5‰ salinity and was then reared for one week to acclimate to the experimental environment. Subsequently, shrimp were treated with 42.32 mg/L ammonia nitrogen for a consecutive 96 h period. Integrated transcriptomic and metabolomic analyses elucidated the stress response patterns in the gills of L. vannamei under low-salinity ammonia nitrogen exposure. Specifically, 352, 802, and 140 differentially expressed genes (DEGs) were identified at 12 h, 48 h, and 96 h post-exposure, respectively. GO and KEGG enrichment analyses revealed that the significant DEGs were primarily enriched in six major pathways: autophagy, immune-related pathway, ABC transporter, fatty acid degradation and metabolism, metabolic pathway, and PPAR signaling pathway. Metabolomic profiling identified numerous differentially accumulated metabolites (DAMs) in both positive and negative ion modes, with significantly altered DAMs mainly consisting of organic acids and their derivatives, phospholipids, and other related metabolites. Key DAMs included taurine, guanosine, 1-palmitoyl-sn-glycero-3-phosphocholine, pseudouridine, and betaine. Integrative multi-omics analysis revealed that L. vannamei mediates stress responses by modulating five core pathways under low-salinity/high-ammonia-nitrogen dual stress: fatty acid degradation and metabolism (e.g., acyl-CoA dehydrogenase short chain (Acads), acetyl-CoA acetyltransferase 2 (ACAT2)), autophagy (e.g., autophagy-related protein 101-like (atg101)), immune regulation pathway (e.g., V-type proton ATPase subunit H-like (VhaSFD), actin-5C-like (Act5C)), metabolic pathway (e.g., molybdopterin synthase catalytic subunit-like (Mocs2B), cytochrome P450 2U1-like (Cyp2b1)), and ABC transporter (e.g., ATP-binding cassette sub-family D member 3-like (ABCD3), ATP-binding cassette sub-family B member 10 (ABCB10)). Through characterization of these core pathways, this study reveals the fundamental mechanisms by which L. vannamei responds to high ammonia nitrogen stress following low-salinity acclimation, providing a theoretical foundation for estuarine shrimp farming. Full article

This experiment investigated the effects of dietary Artemisia ordosica Krasch. (AOK) supplementation on the n3-polyunsaturated fatty acid (n3-PUFA) profile of subcutaneous adipose tissue (SADT) in Arbas cashmere goats and explored the underlying transcriptional mechanisms. Forty healthy, weaned kids (120 ± 10 days of age; similar body weight) were randomly allocated to two groups (n = 20): a control group (CON, basal diet) and an AOK group (AOK, basal diet with 3% of the roughage replaced by AOK). The feeding trial spanned 104 days, consisting of a 14-day adaptation period and 90 days of data acquisition. Compared with the CON group, AOK significantly reduced the content of saturated fatty acids (SFAs) and n6-polyunsaturated fatty acids (n6-PUFAs)/n3-PUFAs (n6/n3). In contrast, the levels of n3-PUFAs in the SADT of cashmere goats increased markedly (p < 0.05). Compared with the CON group, AOK exhibited significantly higher activities of hormone-sensitive lipase (HSL) (p = 0.027), adenylyl cyclase 2 (ADCY2) (p = 0.010), adenylyl cyclase 5 (ADCY5) (p = 0.046), cluster of differentiation 36 (CD36) (p = 0.013), solute carrier family 27 member 4 (SLC27A4) (p = 0.021), and fatty acid binding protein 4 (FABP4) (p = 0.040), along with significantly lower activities of fatty acid synthase (FAS) (p = 0.002), lipoprotein lipase (LPL) (p = 0.048), and stearoyl-coa desaturase (SCD) (p = 0.026) in SADT. Compared with the CON group, the activities of superoxide dismutase (SOD) (p = 0.032), catalase (CAT) (p = 0.010), glutathione peroxidase (GSH-PX) (p = 0.029), and total antioxidant capacity (T-AOC) (p = 0.002) were significantly increased in the AOK group. Transcriptomic profiling revealed that AOK supplementation downregulated mRNA levels of ADCY2, ADCY5, LPL, FAS, SCD, stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), glycogen synthase 1 (GYS1), acyl-CoA oxidase 1 (ACOX1), acetyl-CoA carboxylase (ACC), diacylglycerol acyltransferase 1 (DGAT1), fatty acid desaturase 1 (FADS1), solute carrier family 27 member 2 (SLC27A2), erythroblastic leukemia viral oncogene homolog 4 (ERBB4), and carnitine palmitoyltransferase 1B (CPT1B) (p < 0.05). It also markedly induced acyl-CoA synthetase long-chain family member 4 (ACSL4) (p < 0.01) in SADT. Genes significantly enriched in the adenosine-monophosphate-activated protein kinase (AMPK) signaling pathway included LPL, SCD1, CPT1B, and GYS1 (p = 0.010). Genes significantly enriched in the phosphatidylinositol 3-kinase-akt (PI3K-Akt) signaling pathway included GYS1 and ERBB4 (p = 0.015). CPT1B, ADCY2, and GYS1 were identified as the genes significantly enriched in the insulin resistance signaling pathway (p = 0.048). LPL was the only gene significantly enriched in the cholesterol metabolism pathway (p = 0.049). Genes showing a tendency toward significant enrichment in the peroxisome-proliferator-activated receptor (PPAR) signaling pathway included ACSL4, CPT1B, SCD1, and LPL (p = 0.051). These interconnected cascades improve insulin sensitivity, stimulate triglyceride (TG) hydrolysis, and modulate n3-PUFA levels. Supplementation with AOK enhances n3-PUFA content by accelerating TG breakdown while simultaneously restraining FA oxidation in SADT. Consequently, AOK supplementation can be effectively used to enhance the nutritional value of cashmere goat meat through improved n3-PUFA deposition in SADT. Full article

Background: Glucocorticoids (GCs) are a key pathogenic factor in steroid-induced avascular necrosis of the femoral head (SANFH). GCs can directly damage bone microvascular endothelial cells (BMECs), leading to impaired intraosseous blood supply. Recent studies suggest the Hippo signaling pathway may be involved in the pathogenesis of SANFH; however, its role in vascular endothelial repair and angiogenesis remains unclear. This study aims to investigate the therapeutic effects of human umbilical cord mesenchymal stem cells (hUC-MSCs) on SANFH, with a particular focus on their protective or reparative mechanisms on BMECs. Methods: In vivo, a SANFH mouse model is established and divided into NC, MPS, and hUC-MSCs groups, followed by Micro-CT imagin, hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) (n = 8 per group). In vitro, BMECs are divided into NC, dexamethasone (Dex), hUC-MSCs, and Fer-1 groups to analyze cellular biological behaviors. Target protein expression is assessed using Western blotting and immunofluorescence microscopy. Ferroptosis-related markers are detected via biochemical assays. Mitochondrial ultrastructural changes are observed using transmission electron microscopy. Results: In vivo, the MPS group exhibited significant bone cavitation, sparse trabeculae, and disrupted trabecular architecture in the femoral head. The hUC-MSCs group showed marked improvement in bone microstructure, HE staining showed a significant decrease in the empty lacunae rate in the femoral head, and IHC results revealed markedly increased expression of cluster of differentiation 31 (CD31) and vascular endothelial growth factor (VEGF). In vitro, Dex stimulation suppressed BMECs proliferation. In Dex-treated cells, levels of intracellular reactive oxygen species (ROS), lipid peroxides, ferrous ion (Fe²⁺), malondialdehyde (MDA), acyl-CoA synthetase long chain family member 4 (ACSL4) and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) were all increased, while expression of glutathione (GSH) and glutathione Peroxidase 4 (GPX4) was reduced. Transmission electron microscopy revealed plasma membrane rupture and reduction or loss of mitochondrial cristae. Furthermore, Dex promoted Hippo-mediated phosphorylation of Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ), upregulated NOX4 expression, and suppressed CD31 and VEGF expression. Following hUC-MSCs treatment, BMECs demonstrated enhanced proliferation, migration, and tube-forming capacity. Cellular GSH and GPX4 levels increased, antioxidant capacity was restored, peroxide accumulation decreased, and cells were protected from ferroptosis-effects comparable to those in the Fer-1 group. Additionally, hUC-MSCs inhibited YAP/TAZ phosphorylation and promoted elevated expression of CD31 and VEGF. Conclusions: These findings suggest that hUC-MSCs may attenuate Dex-induced ferroptosis in BMECs, enhance BMEC migration and angiogenesis, and improve femoral head microstructure in SANFH through modulation of the Hippo-YAP/TAZ signaling pathway. This study provides novel insights into the therapeutic potential of hUC-MSCs for SANFH. Full article

Short-chain acyl-CoA dehydrogenase (SCAD) is a critical enzyme in mitochondrial fatty acid β-oxidation, catalyzing the initial dehydrogenation of short-chain acyl-CoAs. Mutations in the ACADS gene cause SCAD deficiency (SCADD), a disorder with remarkably heterogeneous clinical presentation. However, the molecular mechanisms underlying substrate specificity and the pathogenicity of most ACADS variants remain poorly understood. Here, we present high-resolution cryo-EM structures of human SCAD in complex with its physiological substrate butyryl-CoA (C₄) and the longer substrate hexanoyl-CoA (C₆). The butyryl-CoA-bound structure at 2.1 Å resolution details a pre-catalytic geometry ideal for hydride transfer, with Glu392 positioned as the catalytic base. We systematically characterized nineteen disease-associated mutations, which we classify into three functional categories: those disrupting FAD binding, those impairing substrate binding, and those compromising protein folding and stability. In addition, using the W177R mutant as a representative model, we demonstrate that folding-defective mutations provoke protein aggregation, leading to proteotoxicity, oxidative stress, and apoptosis, revealing a pathogenic mechanism beyond mere catalytic loss. In brief, our integrated findings elucidate the structural determinants of substrate specificity and catalytic mechanism in SCAD, and provide mechanistic insights into the functional impairments caused by mutations linked to SCADD. Full article

This study aimed to investigate the impacts of chestnut tannin (CT) on growth performance, immune response, and intestinal health of broilers challenged with necrotic enteritis (NE) through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-ferroptosis pathway. A total of 240 one-day-old male Cobb 500 broilers (44.54 ± 0.51 g) were randomly divided into four groups, including a Control group, NE group, 500 mg/kg CT group (L-CT), and 1000 mg/kg CT group (H-CT), with six replicates per group and ten broilers per replicate. Sporulated coccidia oocysts on day 14 and Clostridium perfringens solution from days 19 to 21 were given to all broilers except the Control group through oral administration to establish the NE infection model. The results demonstrated that dietary supplementation with CT improved (p < 0.05) growth performance, intestinal morphology, and intestinal mucosal barrier function of broilers challenged with NE. CT supplementation decreased (p < 0.05) interleukin (IL)-1β, IL-6, type I interferon, interferon-γ, and tumor necrosis factor-α concentrations and increased (p < 0.05) IL-10 concentration in the jejunal mucosa. Furthermore, CT supplementation decreased (p < 0.05) Fe²⁺ concentration, malondialdehyde concentration, mitochondrial DNA level, and mitochondrial reactive oxygen species level in the jejunal mucosa. Broilers under NE challenge had upregulated (p < 0.05) jejunal protein expression of cGAS, STING, phospho-TANK-binding kinase 1, phospho-interferon regulatory factor 7, phospho-nuclear factor kappa B, ferroptosis suppressor protein 1, prostaglandin-endoperoxide synthase 2, acyl-CoA synthetase long-chain family member 4, WD repeat domain phosphoinositide-interacting protein 2, nuclear receptor co activator factor 4 and autophagy related protein 5 and downregulated (p < 0.05) glutathione peroxidase 4, ferritin heavy chain 1, ferritin light chain and ferroportin 1 compared with the Control group, while the supplementation of CT reversed these effects. In conclusion, CT improved intestinal inflammatory damage of broilers challenged with NE by inhibiting the cGAS-STING-ferroptosis pathway, which was more effective at a dose of 1000 mg/kg in this study. Full article

Background/Objectives: Females are generally more resistant to ischemia-related ferroptosis than males, due to differences in iron metabolism, antioxidant pathways, and sex hormone-mediated regulation of ferroptosis suppressors. This has not been systematically studied in a human donor liver model. To investigate the effect of sex on ferroptosis and oxidative stress pathways in non-utilized donor livers (NDLs), we assessed patterns of gene expression in NDLs under ex vivo normothermic machine perfusion (NMP). Methods: We utilized the PROTECT dual-circuit ex vivo NMP system to assess three male and two female NDLs undergoing 6 h NMP. Perfusate and tissue samples were collected at baseline and 6 h of NMP. Malondialdehyde (MDA) levels were quantified as biochemical markers of iron overload and lipid peroxidation, respectively. Ferroptosis-related gene expression was assessed using molecular assays. Comparisons between male and female NDLs were used to determine the influence of sex on ferroptosis and oxidative injury during NMP. Results: NMP was successfully performed on NDLs (n = 5) from three male (56.3 ± 5.7 years) and two female donors (46.5 ± 0.7 years, p = 0.15). The fold-change in the oxidative stress marker MDA was comparable between female (1.2 ± 0.6) and male (1.0 ± 0.4) NDLs after 6 h NMP (p = 0.76). All livers showed upregulation of ferroptosis-related genes (Hypoxia-inducible factor 1 alpha, Iron Responsive Binding Elements 2, Ribosomal Protein L8, Ferritin Heavy Chain 1, Acyl-CoA synthetase family member 2, ATP synthase membrane subunit c locus 3, Heme-oxygenase 1, NAD(P)H Quinone Dehydrogenase 1, Tetratricopeptide Repeat Domain 35, Nuclear Factor Erythroid 2 Related Factor 2). ACSF2 expression was significantly higher in female NDLs compared with males undergoing 6 h NMP (3.6 ± 3.0 vs. 1.0 ± 0.7-fold change, p = 0.04). There were no sex-based significant differences observed in the expression of other ferroptosis-related genes (HIF-1α, IREB2, RPL8, FTH-1, ATP5G3, HO-1, NQO1, TTC35, and NRF2) between male and female NDLs. No gene reached statistical significance after false-discovery-rate (FDR) correction. Conclusions: Normothermic machine perfusion of NDLs was feasible, and no sex-related differences were observed in MDA levels or most ferroptosis-related gene expression after 6 h. Although ACSF2 showed higher expression in female livers, this was not significant after multiple testing correction, highlighting the need for larger studies to explore sex-dependent ferroptosis signaling during liver preservation. Full article

The incidence of primary liver cancer is increasing annually, with extremely high mortality and suboptimal therapeutic outcomes. The inefficient presentation of tumor antigens and low infiltration of specific cytotoxic T lymphocytes (CTLs) result in insufficient immunogenicity, which limits the efficacy of immunotherapy. Despite the popularity of immune checkpoint inhibitors (ICIs), insufficient immune activation means only a small subset of hepatocellular carcinoma (HCC) patients exhibit clinical responses to ICIs, showing significant inter-individual variability. The activation of the cyclic GMP-AMP synthase(cGAS)- stimulator of interferon genes(STING) pathway initiates the expression of type I interferons (IFNs) and inflammatory cytokines, promoting the formation of a pro-inflammatory environment at the tumor site. This pathway enhances anti-tumor immune responses by facilitating antigen processing and presentation, T cell priming and activation, and remodeling of the immunosuppressive microenvironment. Our research found that cucurbitacin B (CuB), a natural component derived from traditional Chinese medicine, had significant anti-hepatocellular carcinoma properties and exerted anti-tumor effects through the cGAS-STING pathway. Specifically, CuB regulated ferroptosis by down-regulating the expression of Solute Carrier Family 7 Member 11 (SLC7A11) and Glutathione Peroxidase 4 (GPX4) and upregulating the expression of Transferrin Receptor Protein 1 (TFR1) and Long-chain Acyl-CoA Synthetase 4 (ACSL4). These actions involved lipid substrates, iron ion homeostasis, and antioxidant defense systems. The release of mitochondrial DNA (mtDNA) triggered by ferroptosis activated the cGAS-STING immune signaling pathway, leading to the up-regulation of cGAS, phosphorylated STING (p-STING), phosphorylated TANK-binding kinase 1 (TBK1), phosphorylated Interferon regulatory factor3 (IRF3), and Interferon-β (IFN-β). This cascade activation pattern provides new insights into the drug treatment of tumors. Full article

Feline calicivirus (FCV) is a highly variable RNA virus that infects domestic cats and circulates endemically within feline populations, causing a wide spectrum of clinical manifestations, from asymptomatic infections to severe disease. Genomic analysis of 69 FCV strains revealed a high prevalence of the virus across multiple provinces in China. In vitro infection of CRFK cells with laboratory isolates FCV-BJ616 and FCV-BJDX40 resulted in significant cytotoxic effects. Serum proteomic analysis identified 221 upregulated and 123 downregulated proteins following infection with FCV-BJ616, and 233 upregulated and 165 downregulated proteins following infection with FCV-BJDX40. Among these, 215 proteins exhibited shared differential expression. Functional analyses revealed enriched pathways, including TNF signaling and ferroptosis. Notably, upregulation of Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) was correlated with lung injury, while downregulation of S100 Calcium Binding Protein A2 (S100A2) was associated with poor prognosis in FCV-associated oral disease. The differential expression of ACSL4 and S100A2 was further validated through Western blot analysis. These results suggest that ACSL4 and S100A2 are promising candidate biomarkers for monitoring FCV infection and disease progression, laying a foundation for future diagnostic and prognostic applications. Full article

Background/Objectives: We have previously demonstrated that fatty acid oxidation (FAO) enzymes physically and functionally interact with electron transfer chain supercomplexes (ETC-SC) at two contact points. The FAO trifunctional protein (TFP) and electron transfer flavoprotein dehydrogenase (ETFDH) interact with the NADH⁺-binding domain of ETC complex I (com I) and the core 2 subunit of complex III (com III), respectively. In addition, the FAO enzyme very-long-chain acyl-CoA dehydrogenase (VLCAD) interacts with TFP. These interactions define a functional FAO-ETC macromolecular complex (FAO-ETC MEC) in which FAO-generated NADH⁺ and FADH₂ can safely transfer electron equivalents to ETC in order to generate ATP. Methods: In this study, we use multiple mitochondrial functional studies to demonstrate the effect of added VLCAD protein on mutant mitochondria. Results: We demonstrate that heart mitochondria from a VLCAD knockout (KO) mouse exhibit disrupted supercomplexes, with significantly reduced levels of TFPα and TFPβ subunits, electron transfer flavoprotein a-subunit (ETFα), and NDUFV2 subunit of com I in the FAO-ETC MEC. In addition, the activities of individual oxidative phosphorylation (OXPHOS) enzymes are decreased, as is the transfer of reducing equivalents from palmitoyl-CoA to ETC (FAO-ETC flux). However, the total amount of these proteins did not decrease in VLCAD KO animals. These results suggest that loss of VLCAD affects the interactions of FAO and ETC proteins in the FAO-ETC MEC. Reconstitution of VLCAD-deficient heart mitochondria with recombinant VLCAD improved the levels of FAO-ETC MEC proteins and enzyme activities, as well as restoring FAO-ETC flux. It also reduced mitochondrial ROS levels, previously demonstrated to be elevated in VLCAD-deficient mitochondria. In contrast, incubation of VLCAD KO mitochondria with two VLCADs with mutations in the C-terminal domain of the enzyme (A450P and L462P) did not restore FAO-ETC MECs. Conclusions: These results suggest that VLCAD is a necessary component of the FAO-ETC MEC and plays a major role in assembly of the macro-supercomplex. These studies provide evidence that both the level of enzyme and its structural confirmation are necessary to stabilize the FAO-ETC MEC. Full article

Acyl-CoA-binding proteins (ACBPs) are essential lipid carrier proteins involved in plant lipid metabolism. However, the systematic identification and expression profiles of the ACBP gene family in citrus species remain poorly understood. Here, Citrus sinensis and Poncirus trifoliata were chosen as model species to examine the biological properties of citrus ACBPs. Using bioinformatics methods, five ACBP gene members were found in each species and named CsACBPs and PtrACBPs, respectively. All obtained ACBP members were divided into four subfamilies based on conserved domains and amino acid sequences. CsACBP and PtrACBP genes exhibited structural variation in motifs and exons. The predicted protein structures of CsACBPs and PtrACBPs exhibited conservation between the two species while displaying distinct variation within each species. Collinearity analysis showed one intraspecific pairing relationship in each of the two citrus species. Furthermore, there were more collinear couplings between citrus species and Arabidopsis thaliana but none between citrus species and Oryza sativa (rice). Notably, the analysis of cis-acting elements in ACBP gene promoters identified a number of motifs associated with light, abiotic stresses, and phytohormones. Expression profiling confirmed tissue-specific expression patterns of CsACBP1~5 and PtrACBP1~5. RT-qPCR analysis revealed that all CsACBP and PtrACBP genes responded to cold and salt stresses, though the magnitude of their responses varied significantly. Specially, although PtrACBP5 did not respond to low temperatures as rapidly as other members, its expression level increased significantly after 24 h of low-temperature treatment. Protein–protein interaction (PPI) network predictions indicated tight associations among four of the five CsACBPs, with CsACBP5 excluded from these interactions. Moreover, CsACBP1, CsACBP2, and CsACBP3 were predicted to be potential targets of csi-miR3952, csi-miR396a, and csi-miR477b, respectively. Overall, our research provides a solid foundation for further investigations into the biological functions and regulatory mechanisms of ACBP genes in citrus growth, development, and stress adaptation. Full article

Acyl-CoA-binding proteins (ACBPs) possess a conserved acyl-CoA-binding (ACB) domain that facilitates binding to acyl-CoA esters. In addition to their typical role in lipid metabolism, plant ACBPs have been shown to participate in various physiological processes, such as membrane biogenesis, stress response pathways and plant immunity mechanisms. Here, we identified five PutACBP members in alkaligrass (Puccinellia tenuiflora), which were divided into four distinct classes based on a phylogenetic tree constructed from 86 ACBP genes from 12 plant species. Promoter analysis identified numerous cis-acting elements linked to abiotic stresses (e.g., light, drought, heat, and cold) and hormone responses. Expression profile analyses revealed that PutACBPs exhibit broad expression patterns across many organs and respond to salinity-alkali, cold, H₂O₂, and CdCl₂ stresses. Transient expression of five PutACBP-GFPs in tobacco (Nicotiana tabacum) revealed PutACBP1 and PutACBP2 localized to the plasma membrane, cytoplasm, and cell nucleus, while PutACBP3, PutACBP4, and PutACBP5 localized around the plasma membrane and cytoplasm. Furthermore, heterologous constitutive expression of PutACBP3 in Arabidopsis (Arabidopsis thaliana) enhanced the resistance of transgenic plants to salinity stress, possibly through alterations in the levels of lipid metabolism-related and stress-responsive genes. The ACBP gene family is highly conserved across different plant species. This study provides the first comprehensive genomic and functional characterization of the PutACBP family in alkaligrass, elucidating its evolutionary conservation, phylogenetic classification, and stress-response roles. Notably, overexpression of PutACBP3 in Arabidopsis significantly enhanced salt tolerance, suggesting its critical function in salt-stress adaptation in alkaligrass. Full article

Computer-aided docking combined with AlphaFold-based protein modeling revealed strong binding affinities of the ligand DOLA ((Z)-5-(dimethylamino)-N-(octadec-9-en-1-yl)naphthalene-1-sulfonamide) to metabolic proteins in the insect species Tenebrio molitor, Tribolium castaneum, Locusta migratoria, Lucilia cuprina, and Drosophila melanogaster. Significant interactions were observed with cytochrome P450 families CYP6 and CYP4 involved in detoxification, hormone regulation, and fatty acid metabolism, with binding energies ranging from −10.4 to −8.5 kcal/mol. DOLA also showed affinity for enzymes related to fatty acid metabolism and transport, including long-chain fatty acid-CoA ligase, fatty acyl-CoA reductases, CYP18a1, lipocalins, and fatty acid desaturases. These in silico findings highlight DOLA as a promising ligand for studying insect metabolic pathways and as a potential agent for population control, warranting further experimental validation. Full article

Plasma-activated water (PAW) is an emerging disinfectant; however, its potential as a quorum sensing inhibitor (QSI) for biofilm control remains underexplored, and its action mechanisms have not been elucidated. This study investigated the effects of PAW on biofilm formation and spoilage factors secretion in Pseudomonas fluorescens under sub-inhibitory conditions. PAW generated by treating water for 60 s (PAW-60) reduced biofilm biomass by up to 1.29 log CFU/mL after 12 h incubation. It also completely inhibited protease production (100%) and decreased siderophore production by 31.87%. N-butyryl-homoserine lactone (C₄-HSL) was identified as the dominant signaling molecule, with its production decreasing by 34.34–84.07% following PAW treatments. Meanwhile, C₄-HSL activity was significantly suppressed by 42.58–65.38%. An FTIR analysis revealed the formation of a new C=O group, indicating oxidative degradation of acyl homoserine lactones (AHLs). Exogenous C₄-HSL progressively restored the biofilm biomass, spoilage factors production, and QS-related gene expression levels, with no significant difference observed compared with the control at 0.05 µg/mL (p < 0.05). The results suggest that the inhibitory effects of PAW are primarily due to the disruption of AHLs transduction in the QS pathway. Molecular docking showed that the long-lived reactive species in PAW could bind to AHLs’ synthetic protein (FadD1) and receptor protein (LuxR) via hydrogen bonding. PAW-60 reduced the spoilage activity of P. fluorescens inoculated into fish muscle juice and extended its shelf life from 8 to 10 days during storage at 4 °C. A strong positive correlation was observed between AHLs accumulation and the spoilage process. These findings demonstrate that PAW mitigates biofilm formation and food spoilage by blocking signaling transduction, which involves suppression of AHLs production, oxidative degradation of AHLs molecules, and disruption of AHLs recognition. Full article