Search Results (4,434)

This study aimed to identify and characterize a high-efficiency chitinase gene from Brevibacillus laterosporus (B. laterosporus), characterize its enzymatic traits, and assess its degrading activity against Caenorhabditis elegans (C. elegans) and Parascaris equorum (P. equorum) eggs. A nematicidal B. laterosporus isolate was subjected to whole-genome sequencing for chitinase gene screening, cloning, and molecular identification. The target gene was heterologously expressed in Escherichia coli BL21 (DE3), and biochemical properties including temperature, pH, metal ions, and substrate conditions were investigated, its kinetic parameters were determined, and its biological effects on C. elegans and P. equorum eggs were analyzed. The protein BLChi79 (79.3 kDa) belongs to GH18 chitinases with a typical carbohydrate-binding module. Its optimum activity occurred at 60 °C and pH 6.0, with colloidal chitin as the optimal substrate. Mg²⁺, Fe²⁺, and Mn²⁺ boosted its activity, while K⁺, Cu²⁺, Zn²⁺, and Ca²⁺ suppressed catalysis. Its K_m was 6.14 mg·mL⁻¹, V_max 7.78 μmol·min⁻¹·mg⁻¹, and kcat 10.16 min⁻¹. The purified recombinant enzyme could degradethe C. elegans chitin layer and the P. equorum egg vitelline envelope. In summary, the BLChi79 derived from B. laterosporus targets chitin-enriched eggshells, acting as a green biocatalyst for controlling livestock gastrointestinal nematodes. Full article

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger downstream of G protein-coupled receptors (GPCRs) and a central regulator of cellular signaling. Genetically encoded exchange proteins directly activated by cAMP (Epac)-based Förster resonance energy transfer (FRET) biosensors enable real-time monitoring of cAMP dynamics in living cells, but commonly used cyan/yellow FRET pairs require short-wavelength excitation, limiting compatibility with multiplex imaging and blue-light optogenetic tools such as bacterial photoactivated adenylyl cyclases (bPACs). Here, we engineered and systematically characterized four red-shifted Epac-based single-chain FRET cAMP sensors combining yellow or orange FRET donors with red fluorescent FRET acceptors. Using ratiometric live-cell imaging, we quantified stimulus-evoked FRET responses and identified Epac_red4 as the best-performing variant, showing an approximately 55% decrease in normalized FRET after forskolin stimulation. Epac_red4 also reliably detected G_i/o-mediated decreases in cAMP following μ-opioid receptor activation. Brief 405 nm light pulses induced graded and reversible cAMP elevations using the low dark-activity variant bPAC-F198Y. Furthermore, Epac_red4 enabled analysis of cAMP recovery kinetics during phosphodiesterase inhibition and multiplex imaging of cAMP and intracellular Ca²⁺ using Fura-2 with minimal spectral and pH-related interference under physiological imaging conditions. Together, Epac_red4 represents a robust red-shifted cAMP sensor for optogenetic and multiplex signaling studies. Full article

Accessory proteins of SARS-CoV-2 play crucial roles in viral pathogenesis, yet their structural properties remain elusive. ORF7b, a small accessory protein comprising only 43 amino acids, is widely assumed to parallel the structure–function relationships of its SARS-CoV ortholog based solely on sequence homology. In this study, we challenge this paradigm through direct physicochemical and structural characterization. Sequence analysis and electrostatic profiling reveal that the SARS-CoV-2 protein is a macromolecular polyanion with a net charge of −4 at neutral pH, featuring a diffuse negative surface that is highly responsive to pH changes. Complete 3D structures generated via ab initio modeling display a helical core flanked by two highly fluctuating, disordered termini. Residue Interaction Network (RIN) topology and Normal Mode Analysis (NMA) identified specific hinges governing these flexible extremities. Furthermore, the calculated dipole moment vector is tilted outward by 24°, misaligning with the central axis. Molecular dynamics simulations suggest that while the soluble structure is highly stable in water, it undergoes severe distortions and insufficient solvation within a membrane-mimetic environment. Thermodynamic association profiles and verified interactomic data from BioGRID reveal a strong propensity for ORF7b to participate in liquid–liquid phase transitions alongside human and viral partners. Taken together, these unique properties suggest that ORF7b operates as a dynamic peripheral membrane protein rather than a sedentary transmembrane component, providing a fresh framework for future therapeutic targeting. Overall, these in silico findings shift the current paradigm on ORF7b2 topology and provide a robust, physically grounded framework that identifies specific molecular priorities for future in vitro and in vivo validation. Full article

This work reports the development of innovative enzyme–titanium nanobiohybrids synthesized via a protein-assisted approach to obtain efficient and sustainable photocatalysts for environmental remediation. By addressing the limitations of conventional TiO₂ nanoparticle synthesis, this strategy enables controlled material properties under milder, potentially scalable conditions for enhanced ROS-driven degradation of persistent dye pollutants. This work employs a bio-assisted synthesis approach using β-glucosidase as a protein scaffold, TiCl₄ as the titanium precursor, and H₂O₂ in bicarbonate buffer at room temperature, eliminating the need for harsh conditions and high temperatures. The biological moiety guides the nanoparticle formation, controlling size and morphology while preventing aggregation, all performed under mild conditions. X-ray diffraction determined that the Ti hybrid was composed of TiO₂ brookite species. TEM analyses demonstrated the formation of well-dispersed nanostructures of around 700 nm. The resulting nanobiohybrids showed excellent photocatalytic activity, achieving >99% Rhodamine B degradation under UV light in only 1 h compared to visible light. The catalyst was capable of degrading Rhodamine B at a concentration approximately 36 times above the recommended threshold for water. Furthermore, a preactivation of the catalyst by direct exposition of it to UV-395 nm light greatly enhanced the efficiency in the photocatalytic process, being inactive in visible light. The Ti–enzyme hybrid showed excellent recyclability over five consecutive cycles and retained good activity after storage, demonstrating its stability. This study introduces a sustainable and efficient route for synthesizing Ti-based nanobiohybrids, providing a promising strategy for advanced photocatalytic applications in water treatment and environmental remediation. Full article

Background/Objectives: Malignant melanoma is characterized by constitutive PI3K/Akt/mTOR and MAPK activation, driving aggressive behavior and therapeutic resistance. Alpha-lipoic acid (αLA), a naturally occurring dithiol compound with an established clinical safety profile, has shown anticancer potential; however, its integrated molecular mechanisms in melanoma remain poorly defined. This study aimed to comprehensively evaluate the cytotoxic and mechanistic effects of αLA in B16F10 murine melanoma cells. Methods: Antiproliferative effects were assessed by MTT assay at four concentrations (250, 500, 750, 1000 µM) over 48 h. Protein levels of apoptotic markers (Bax, Bcl-2, Caspase-3, AIF), kinase signaling components (p-Akt, p-mTOR, p-ERK, p-JNK), ER stress markers (GRP78, GADD153/CHOP), and cell cycle regulator Wee1 were quantified by ELISA at a specifically selected sub-lethal concentration of 750 µM (inducing ~38% growth inhibition). Results: αLA dose-dependently inhibited B16F10 proliferation. At 750 µM, it triggered robust intrinsic apoptotic signaling, evidenced by a nearly 10-fold shift in the Bax/Bcl-2 ratio and greater than 9-fold Caspase-3 activation. Elevated AIF suggested profound mitochondrial stress and the potential priming of concurrent caspase-independent cell death mechanisms. αLA suppressed survival signaling by reducing p-Akt (44%), p-mTOR, p-ERK, and p-JNK. Treatment triggered lethal ER stress via GRP78 and GADD153/CHOP upregulation and upregulated Wee1, suggesting the induction of stress-responsive checkpoint signaling. The simultaneous CHOP upregulation and p-Akt suppression highlight a concurrent dysregulation of stress and survival pathways, suggesting a potential pro-apoptotic interplay. Conclusions: αLA exerts potent multi-target anticancer effects by inducing a broad spectrum of associated molecular changes, including the suppression of PI3K/Akt/mTOR and MAPK networks, induction of ER stress, engagement of cell cycle checkpoints, and activation of the mitochondrial Bax/Bcl-2/Caspase-3 axis. Importantly, these correlative findings do not establish proven pathway dependencies. Nevertheless, this concurrent dysregulation positions αLA as a potential disruptor of inter-pathway resilience underlying drug resistance. Full article

Asthma is a heterogeneous chronic airway disease in which oxidative stress (OS) plays a central mechanistic role beyond classical immune-mediated inflammation. Reactive oxygen and nitrogen species (ROS/RNS), generated by recruited inflammatory cells and activated airway structural cells, drive epithelial injury, mucus hypersecretion, airway remodeling, and modulate key transcription factors including nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. This review synthesizes current evidence on the multilevel redox-based determinants of asthma severity, spanning from genetic polymorphisms to circulating biomarkers. We examine serum antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), peroxiredoxins (PRDXs), and the thioredoxin (Trx) system as dynamic indicators of systemic redox status and disease severity, alongside oxidative enzymes including NADPH oxidases and dual oxidases (NOX/DUOX), xanthine oxidase (XO), and myeloperoxidase (MPO) that serve as upstream sources of airway oxidant burden. Functional genetic polymorphisms in antioxidant genes (SOD2, CAT, glutathione S-transferase mu 1/glutathione S-transferase theta 1 (GSTM1/GSTT1), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/KEAP1)) and oxidative enzyme genes including nitric oxide synthase 1/2/3 (NOS1/2/3), MPO, cytochrome b-245 alpha chain (CYBA), and xanthine dehydrogenase (XDH) are reviewed as modulators of individual redox capacity and asthma susceptibility, with particular attention to gene–environment interactions. We further discuss oxidative damage biomarkers, including malondialdehyde (MDA), 8-isoprostanes, 4-hydroxynonenal, 8-oxo-7, 8-dihydro-2′-deoxyguanosine, protein carbonyls, 3-nitrotyrosine, and advanced oxidation protein products as indicators of lipid, DNA, and protein oxidation that correlate with disease activity and control. The roles of micronutrient cofactors in modulating antioxidant enzyme function and their potential as contextual biomarkers are also addressed. Additionally, emerging evidence on microRNAs (miRNAs) linked to OS biology in asthma is presented. Finally, we critically evaluate the challenges limiting clinical translation, including biomarker non-specificity, analytical variability, gene–environment complexity, and the absence of standardized reference ranges. This integrated framework supports the development of multilevel redox prognostic panels combining genetic, enzymatic, and oxidative damage readouts for improved asthma phenotyping, severity stratification, and personalized therapeutic approaches. Full article

Ginseng Alternaria leaf and stem blight (GALSB), caused by Alternaria alternata, poses a severe threat to ginseng cultivation. Although azoxystrobin is a cornerstone fungicide for GALSB management, the emergence of widespread adaptive resistance has severely curtailed its field efficacy. This study integrated molecular, transcriptomic, and genetic approaches to unravel the underlying resistance mechanisms. Targeted gene sequencing and molecular docking revealed that resistant strains harbor a conserved G143A point mutation in the AaCyt b protein. This mutation weakens the azoxystrobin–AaCyt b protein binding affinity by elevating the binding energy from −8.31 to −7.08 kcal/mol. Additionally, comparative transcriptomics and RT-qPCR demonstrated pronounced upregulation of the alternative oxidase gene (AaAOX) and core energy metabolism pathways in resistant strain TYC8-2, with AaAOX expression increasing 4.45–6.91-fold. Fungicidal inhibition of AOX via salicylhydroxamic acid (SHAM) restored fungal sensitivity, increasing azoxystrobin sensitivity by 11.66-fold. Crucially, genetic knockout of AaAOX enhanced sensitivity by approximately 2.7 × 10⁴-fold. Phenotypic assays further established AaAOX as a multifunctional regulator; the AaAOX mutant exhibited attenuated virulence on ginseng leaves and increased sensitivity to oxidative and osmotic stresses (NaCl, H₂O₂, NaAc). The G143A mutation in AaCyt b and the transcriptional overexpression of AaAOX contribute independently to drive azoxystrobin resistance in A. alternata. These findings provide comprehensive mechanistic insights to guide resistance surveillance, rational fungicide application, and precision prevention of GALSB in ginseng cultivation. We conclude that the G143A mutation in AaCyt b and the transcriptional overexpression of AaAOX act independently to drive azoxystrobin resistance in A. alternata. These findings provide comprehensive mechanistic insights to guide resistance monitoring, optimize fungicide applications, and develop precision strategies for GALSB management. Full article

Global warming has increasingly positioned heat stress (HS) as a major threat to public health, as it can inflict damage on multiple organs including the kidneys, liver, and heart. However, effective targeted therapeutic strategies remain limited. This investigation employed an integrated approach combining Network pharmacology, in silico binding simulations, and cell-based assays to elucidate the cytoprotective properties and molecular basis of oxymatrine action under heat-stressed conditions. Network analysis identified 36 overlapping targets common to oxymatrine and the pathological processes of HS-related acute kidney injury (AKI), acute liver injury (ALI), and acute myocardial injury (AMI). These targets were strongly enriched in the PI3K-AKT signaling cascade. Molecular docking showed that oxymatrine binds tightly to key pathway proteins such as PIK3CA and GSK3B, with Vina scores below −8 kcal/mol. In 293T cells, the half-maximal cytotoxic concentration (CC₅₀) of oxymatrine exceeded 2000 μM. Under heat stress, oxymatrine (31.25–1000 μM) dose-dependently increased cell viability by about 30% and significantly lowered HSP90 and HSP70 expression. Similar protective effects were observed in H9C2 cardiomyocytes under heat stress. RT-qPCR further confirmed that oxymatrine reduced the transcript levels of PI3K-AKT pathway-related genes, including CASP3, EGFR, RXRα, and MMP9 in 293T cells. We also found 18 overlapping targets between oxymatrine and ferroptosis, most of which matched the core targets above. Molecular docking analysis predicted binding of oxymatrine to the ferroptosis regulator GPX4. Together, these results suggested that oxymatrine potentially alleviates HS injury by modulating the PI3K-AKT signaling pathway andregulating potential ferroptotic targets such as GPX4. Full article

The role of gluconeogenesis in kidney disease has increasingly drawn attention. Fructose-1,6-bisphosphatase 1 (FBP1) is a key rate-limiting enzyme in gluconeogenesis that suppresses glycolysis and reduces lactate production. In this study, we first analyzed public transcriptomic datasets of diabetic kidney disease (DKD) and validated the findings in 24-week-old BKS-db mice and in high-glucose-induced human renal tubular epithelial (HK-2) cells. We further constructed tubular-specific FBP1 overexpression/knockdown mouse models via adeno-associated virus serotype 9 (AAV-9) and combined pharmacological inhibition of lactate dehydrogenase B (LDHB) to dissect the underlying mechanism. Analysis of public clinical transcriptomic datasets showed that renal tubular FBP1 expression was positively correlated with estimated glomerular filtration rate (eGFR). In vivo, tubular-specific FBP1 overexpression in BKS-db mice reduced 24-h urinary protein and decreased renal lactate accumulation (p < 0.05) compared with diabetic controls. In vitro, high glucose-induced lactate elevation in HK-2 cells was reversed by FBP1 overexpression, while co-treatment with an LDHB inhibitor abolished this protective effect. Our findings suggest that FBP1 represents a potential experimental therapeutic target associated with alleviation of renal lactic acid accumulation and mitochondrial injury in preclinical DKD models. Full article

Camelliasaponins, bioactive constituents abundant in the by-products of Camellia oleifera oil production, exhibit diverse biological activities. However, their potential in regulating neuroprotective mitophagy remains largely unexplored. This study identifies camelliasaponin B1 (CSB1) as an abundant component in C. oleifera seeds and investigates its cytoprotective mechanisms against oxidative stress. Using an in vitro model of H₂O₂-induced oxidative damage in PC12 cells, we found that CSB1 pretreatment significantly alleviated oxidative stress, as evidenced by reduced reactive oxygen species (ROS) accumulation and enhanced antioxidant enzyme activities (SOD, CAT, GSH-Px). CSB1 also preserved mitochondrial function, restoring membrane potential (ΔΨm), ultrastructure, and respiratory capacity. Mechanistically, CSB1 reduces the expression of BNIP3/NIX-LC3B pathway-related proteins, suggesting a modulatory effect on mitophagy, as supported by transcriptomic analysis, Western blotting, and immunofluorescence. Molecular docking computationally predicted potential interactions between CSB1 and BNIP3/NIX proteins, which require experimental validation. Collectively, these findings suggest that CSB1 acts as a cytoprotective agent that enhances antioxidant defenses, safeguards mitochondrial integrity, and is associated with reduced BNIP3/NIX-LC3B expression and co-localization, offering a potential molecular basis for its development as a neuroprotective agent targeting oxidative stress-related mitochondrial dysfunction. Full article

As a traditional element of Chinese medicine, Hippocampus erectus is well known for promoting adolescent growth, yet its active fractions and underlying molecular mechanisms remain unclear. In this study, the aqueous extract of H. erectus was subjected to in vitro simulated gastrointestinal digestion and ultrafiltration to separate three molecular weight fractions (<10 kDa, 10–30 kDa, >30 kDa). Their chemical profiles were characterized, and osteogenic activities were systematically evaluated using cell assays, a juvenile rat model, and integrated transcriptomics and data-independent acquisition (DIA) proteomics. Results revealed that chemical profiling showed the >30 kDa fraction was mainly composed of hemocyanin subunits, and the 10–30 kDa fraction was enriched in growth-related amino acids and steroid derivatives; functionally, the 10–30 kDa fraction promoted preosteoblast proliferation and early differentiation via enhanced alkaline phosphatase (ALP) activity, while the >30 kDa fraction dominated late osteoblast maturation and mineralization. Both fractions significantly increased rat body and bone length by expanding growth plate proliferative zones and elevating serum insulin-like growth factor-1 (IGF-1)/bone morphogenetic protein-2 (BMP-2) levels. Transcriptomic and proteomic analyses identified vascular endothelial growth factor (VEGF), Wingless-related integration site (Wnt), phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt), and extracellular matrix (ECM)–receptor interaction as potential core regulatory pathways. Integrated multi-omics analysis further confirmed Frizzled-related protein B (Frzb) and AKT1 substrate 1 (Akt1s1) as candidate key regulatory targets enriched in the Wnt and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways. These findings elucidate the multi-fraction, multi-pathway mechanism of H. erectus in promoting skeletal development, providing scientific evidence for its traditional use and a theoretical basis for growth-promoting functional food development. Full article

For over 3000 years, the perennial herb mugwort (Artemisia argyi) has served as a cornerstone of traditional Asian medicine. Its clinical efficacy is driven by a diverse array of specialized metabolites, most notably flavonoids and volatile oils. While B-box (BBX) transcription factors are known to dictate photomorphogenic development and secondary metabolic pathways in plants, this specific gene family has not yet been systematically analyzed in A. argyi. Leveraging a chromosome-level genomic assembly, we comprehensively identified and analyzed the complete repertoire of AarBBX genes, profiling their structural organization, physicochemical attributes, conserved motifs, promoter architecture, and spatial expression dynamics. The AarBBX family segregates into five distinct evolutionary clades and comprises 114 members, exceeding the gene counts in the diploid relatives Artemisia annua (27) and Arabidopsis thaliana (32), a numerical increase potentially attributable to the tetraploid genome architecture of A. argyi. Promoter scanning revealed a high density of cis-acting elements linked to light perception and environmental stress responses. Integrating RNA-seq transcriptomics with tissue-specific expression profiling, we identified prominent candidate light-responsive AarBBX genes that are highly active in green, photosynthetic tissues and acutely responsive to shifts in light conditions, providing a foundation for future exploration of their potential relationship with secondary metabolic pathways, including flavonoid and terpenoid biosynthesis. Furthermore, we validated the potential operational compartments and structural interactions of these proteins utilizing green fluorescent protein (GFP) subcellular localization and yeast two-hybrid (Y2H) screenings. Collectively, these findings provide new insights into the evolutionary trajectory and regulatory potential of the B-box (BBX) proteins in A. argyi, offering a prioritized candidate gene set for subsequent investigations into their potential roles in light-regulated secondary metabolism, including flavonoid and terpenoid pathways. Full article

Host-adapted probiotics offer a promising strategy for improving stingless bee nutrition and colony sustainability. In this study, gut-derived lactic acid bacteria (LAB) isolated from Heterotrigona itama were evaluated for probiotic potential and used to develop fermented bee pollen. Of 37 presumptive LAB isolates, three strains (BP-2, BP-3, and BPW-B1) exhibited strong tolerance to simulated gastrointestinal conditions, favorable adhesion-related properties, and acceptable safety profiles. Phylogenetic and biochemical analyses identified the selected isolates as Apilactobacillus kunkeei. The LAB strains were co-cultured with the osmophilic yeasts Zygosaccharomyces bailii TSU_YK2 and Starmerella meliponinorum TSU_YP10 to establish a host-associated LAB–yeast co-fermentation model that mimics stingless bee pollen fermentation. Co-fermentation significantly improved protein digestibility, organic acid production, antioxidant activity, and microbial viability relative to spontaneous fermentation controls (p < 0.05). Feeding experiments demonstrated that probiotic-fermented pollen increased feed intake, body weight, abdominal lipid reserves, hypopharyngeal gland development, and survival among H. itama workers. In addition, probiotic supplementation was associated with shifts in the dominant gut-associated bacterial taxa, including Lactobacillus, Bifidobacterium, and Snodgrassella. This study demonstrates the potential of combining gut-derived A. kunkeei with osmophilic yeasts as a functional fermentation starter culture to develop biologically relevant probiotic feed supplements for stingless bees. Full article

A three-factor completely randomized design was used to evaluate the effects of Lactococcus lactis, Bacillus subtilis, and Saccharomyces cerevisiae on the fermentation quality and nutritional composition of mixed silage prepared from navel orange pomace and rice straw. The addition of L. lactis increased lactic acid content and Flieg’s score of the mixed silage. The addition of B. subtilis decreased the pH value and increased the lactic acid content of the mixed silage. The addition of S. cerevisiae decreased the lactic acid content and Flieg’s score of the mixed silage. However, the interaction between the three strains significantly affected many parameters of the mixed silage. With respect to fermentation quality (Flieg’s score), adding 0.3 g kg⁻¹ L. lactis and not adding S. cerevisiae achieved the best performance of the mixed silage. Under these conditions, increasing the level of B. subtilis resulted in a higher crude protein content of the mixed silage, whereas neutral detergent fiber and acid detergent fiber contents first decreased and then increased, and water-soluble carbohydrate content first increased and then decreased. Therefore, within the selected combination of adding 0.3 g kg⁻¹ L. lactis and not adding S. cerevisiae, the addition of 0.8 g kg⁻¹ B. subtilis gave the best nutritional quality. Full article

Background/Objectives: Exposure to high and sustained levels of non-esterified fatty acids (NEFA) in the peripartal period is the main cause of fatty liver disease in dairy cows. Rumen-protected choline is often fed as part of the nutritional management of peripartal cows, with in vivo and in vitro data indicating positive effects of this nutrient on alleviating liver lipid accumulation. Although hepatic molecular mechanisms associated with choline supply have been studied using a target gene, protein, or metabolite approach, application of high-throughput technologies could vastly enhance fundamental knowledge on the functional role of choline. The main objective was to challenge isolated hepatocytes with a mixture of NEFA and determine proteome- and metabolome-wide effects in response to choline supply. Methods: Three healthy female calves (1 d old, 30–45 kg) were sacrificed to harvest hepatocytes. During a 12 h incubation, isolated hepatocytes were challenged without NEFA (control), 1.2 mM NEFA (c9-18:1, 18:2, 16:0, 18:0, and c9-16:1 at 43.5%, 4.9%, 31.9%, 14.4%, and 5.3% of total NEFA, respectively), or NEFA for 6 h followed by 10 μM choline chloride for another 6 h (NEFA + Chol). iTRAQ labeling-based protein profiling and GC/MS-based metabolomics profiling were used to determine changes in proteins and metabolites. Differentially abundant proteins for each group comparison were determined at a threshold of 1.4-fold change. Differences in metabolite profiles were assessed via pairwise comparisons. A subset of differentially abundant proteins was validated via qRT-PCR and Western blotting. Results: Compared with the control, there were 90 proteins and 22 metabolites in the NEFA group, and 83 proteins and 29 metabolites in the NEFA + Chol. Compared with NEFA, there were 49 proteins and 17 metabolites in the NEFA + Chol group. Greater abundance of hexokinase-1 (HK1), fructose-bisphosphate aldolase (ALDOA), mitochondrial pyruvate carrier 1 (MPC1), and increased concentrations of lactate with high NEFA treatment alone suggested greater glycolytic and TCA cycle activity. Accumulation of triacylglycerol in the NEFA group was associated with lipotoxicity and markers of inflammation, such as greater abundance of prostaglandin reductase 1 (PTGR1), serious cell autophagy processes, such as greater abundance of cell division cycle 42 (CDC42), and NFκB-related proteins. Choline supplementation reduced TAG partly due to greater VLDL secretion driven by greater abundance of diacylglycerol acyltransferase (DGAT1), perilipin 3 (PLIN3), and apolipoprotein C-III (APOC3). In addition, a greater abundance of carnitine O-palmitoyltransferase 1b (CPT1B) with choline suggested enhanced mitochondrial β-oxidation. Activation of the CDC42/JNK pathway and ROS/NFκB axis-related proteins, along with depressed PI3K/AKT/RAC-related proteins, indicated enhanced mitochondrial autophagy in response to NEFA. Conclusions: Overall, data confirmed published effects of choline on TAG accumulation, VLDL secretion, and fatty acid oxidation, while highlighting negative effects of NEFA on the respiratory electron transport chain, autophagy, and inflammatory processes. Full article