Search Results (757)

Lipolytic and esterase-like enzymes are crucial in the food industry for flavor production and ester hydrolysis. This study presents an exploratory, baseline feasibility approach to evaluate esterase-like enzyme production by Serratia marcescens 11E via a sustainable co-valorization matrix, where vegetable wax residues serve as structural solid support and cheese whey acts as the primary lipid nutritional source. Under fixed 48-h screening conditions, the recovered cell-free extract exhibited a distinct catalytic preference for short-chain esters, showing higher specific activity toward 4-nitrophenyl acetate (0.743 U/mg) than 4-nitrophenyl palmitate (0.125 U/mg), confirming a predominant carboxylesterase-like profile. Biochemical characterization revealed an initial optimal activity at pH 9.0 and 30 °C, along with a noteworthy bimodal catalytic behavior featuring a secondary activity peak at 70 °C. Size-exclusion chromatography resolved the extract into two distinct active elution pools (17.8 and 26.2 U/mg), which corresponded to candidate protein bands of 35–40 kDa on SDS-PAGE. Although definitive molecular identification remains subject to ongoing zymographic and proteomic characterization, these foundational findings demonstrate the potential of co-valorizing lipid- and carbohydrate-rich industrial wastes to produce resilient proteins with esterase-like activity at elevated temperatures. Full article

Background: Mitochondrial dysfunction is a central event in the pathogenesis of ischemic stroke. The roles of specific mitochondrial complex subunits, such as NDUFA4 and NDUFB3, in cerebral ischemia–reperfusion injury remain poorly defined. This study aims to investigate the dynamic expressions and functional impact of NDUFA4 and NDUFB3 in ischemic stroke. Methods: A transient middle cerebral artery occlusion (MCAO) model was established in male C57BL/6J mice. Label-free quantitative proteomics and Western blotting were employed to analyze protein expression in the ischemic penumbra. Highly differentiated PC12 cells were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) or glutamate excitotoxicity to mimic ischemic injury in vitro. The functional consequences of NDUFB3 knockdown and overexpression were assessed by measuring ATP levels, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and apoptosis. The involvement of the JNK-mediated mitochondrial apoptotic pathway was also examined. Results: Proteomic analysis revealed a significant upregulation of NDUFA4 and NDUFB3 in the ischemic penumbra of MCAO mice, as verified by western blot. In highly differentiated PC12 cells, both OGD/R and glutamate exposure induced a time-dependent increase in these proteins in mitochondrial fractions. Functional studies demonstrated that NDUFB3 knockdown significantly rescued OGD/R-induced mitochondrial dysfunction, as indicated by restored ATP production, reduced ROS generation, and stabilized ΔΨm. Furthermore, NDUFB3 silencing attenuated apoptosis by inhibiting JNK phosphorylation and decreasing BAX levels. Conversely, overexpression of NDUFB3 alone was sufficient to induce mitochondrial abnormalities, including loss of ΔΨm and elevated oxidative stress in highly differentiated PC12 cells. Conclusions: Ischemic injury triggers the upregulation of mitochondrial complex subunits NDUFA4 and NDUFB3. While this may initially act as a compensatory response, our findings identify NDUFB3 as a critical mediator of ischemic stroke pathology, whose overexpression drives mitochondrial dysfunction and apoptosis. In contrast, the suppression of NDUFB3 provides protection against ischemic injury. Therefore, NDUFB3 may be a potential candidate therapeutic target for reducing mitochondrial damage in ischemic stroke, but this role requires further validation in additional experimental and translational models. Full article

The increasing prevalence of Alzheimer’s disease has created a substantial and urgent need for brain-healthy functional foods. The processing of Pacific white shrimp (Litopenaeus vannamei) generates considerable amounts of head waste, which is rich in bioactive compounds, including lipids and peptides, holding great promise for the development of nutraceuticals to support human brain health. However, traditional extraction methods are time-consuming and inefficient in fully utilizing these compounds. This study aimed to explore the functional properties of these shrimp head-derived ingredients using “one-step” three-phase partitioning (TPP) followed by successive proteolysis. The extracted polar lipid (PL-SH), protein (P-SH) and proteolytic peptidic product (Pep-SH) from shrimp heads were screened for their antioxidant, neuroprotective, and anti-neuroinflammatory activities. Antioxidant activities were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺), and hydroxyl free radical scavenging assays, all of which revealed strong antioxidant potential for all three products. Neuroprotective activities were assessed using HT-22 mouse hippocampal neuronal cells challenged with Aβ₂₅₋₃₅, and anti-neuroinflammatory activities were evaluated using BV-2 microglial cells stimulated with lipopolysaccharide (LPS). The results suggested that both PL-SH and Pep-SH exerted protective effects against Aβ₂₅₋₃₅-induced cell damage under the tested conditions, and PL-SH also reduced nitric oxide (NO) production induced by LPS, indicating potential anti-neuroinflammatory activity. However, further studies with additional biomarkers (e.g., ROS, apoptosis markers, and cytokines) are required to confirm these effects. The lipid composition of PL-SH was further characterized by thin-layer chromatography and LC-MS/MS-based lipidomics, revealing various classes of phospholipids. Furthermore, analysis of the molecular weight distribution and sequences of peptides in Pep-SH revealed peptide sizes ranging from 70 to 1700 Da and a high degree of homology to known antioxidant and neuroprotective peptide sequences. These findings suggest that lipids and peptides from Pacific white shrimp heads possess valuable functional properties, supporting their potential use in the development of functional foods for neuroprotection and anti-neuroinflammation. Full article

We explored the possibility of antioxidant and antifibrotic effects of panthenol (PL) associated with modulation of coenzyme A (CoA) biosynthesis in the liver in a rat model of chronic obstructive cholestasis induced by bile duct ligation (BDL). We found that PL increased alcohol dehydrogenase (ADH) activity in the liver of BDL rats. PL and its analog pantethine increased pantothenate kinase (PANK) activity, restored hepatic CoA levels reduced by BDL, lowered protein-bound CoA, and normalized impaired mitochondrial functions associated with induced oxidative stress after BDL. These effects were accompanied by decreased collagen deposition and improved morphological features of hepatocytes. In contrast, PANK inhibitor, hopantenic acid (HPA), reduced hepatic CoA levels, aggravated hepatocellular damage, and promoted fibrosis. In the human hepatic stellate cell line LX-2, PL exhibited no cytotoxicity over a wide concentration range, increased intracellular CoA levels, decreased reactive oxygen species (ROS) production, and attenuated collagen accumulation associated with oxidative stress in vitro. Importantly, inhibition of ADH by 4-methylpyrazole completely abolished the protective effects of panthenol, indicating that its activity depends on metabolic pathways involving CoA. Notably, PL did not directly reduce H₂O₂ or superoxide anion radical production in cell-free systems but significantly suppressed lipid peroxidation in liposomes and red blood cells in vitro. Ultimately, these findings indicate that the antioxidant and antifibrotic effects of PL are associated with modulation of CoA metabolism and enhanced resistance of biological membranes to oxidative damage. Full article

Psoriasis is a chronic, immune-mediated inflammatory skin disease for which the development of structurally novel and accessible small-molecule candidates remains of considerable interest. In this study, a series of juglone-derived naphthoquinone analogs was synthesized to explore the influence of substitution pattern on anti-inflammatory activity and cytotoxicity. Their biological profiles were first evaluated in LPS-stimulated HaCaT cells by combining cytotoxicity assessment with nitric oxide (NO) screening. Most derivatives showed reduced cytotoxicity compared with juglone, and preliminary structure–activity relationship analysis indicated that retention of a free hydroxyl group at the C-2 position was generally favorable for both reduction in NO release and cellular safety, whereas C-3 alkyl substitution tended to weaken activity and increase cytotoxicity. Among the tested compounds, compound 11 showed the most favorable balance between reduction in NO release and low cytotoxicity. Further evaluation showed that compound 11 reduced the protein levels of several inflammatory mediators in the culture supernatants of LPS-stimulated HaCaT cells, including TNF-α, IL-6, IL-1β, IL-17A, and IL-23, under the tested conditions. In an imiquimod-induced psoriasis-like mouse model, topical administration of compound 11 partially alleviated IMQ-induced psoriasis-like skin lesions, improved histopathological changes to some extent, and reduced selected inflammatory cytokine levels in serum and skin tissues under the tested conditions. Exploratory target prediction, molecular docking, and in silico ADMET analyses provided supportive computational insight into the biological profile of compound 11. Overall, these findings suggest that juglone-derived naphthoquinones may serve as useful natural-product-inspired scaffolds for further anti-inflammatory optimization, and compound 11 warrants further investigation in psoriasis-related experimental models. Full article

As a cornerstone of modern science, cell lines are the foundational platforms for key medical advances. They enable vaccinology (through viral propagation and attenuation), gene therapy (via vector development), and biopharmaceutical production (via recombinant protein expression). Designer mammalian, avian, and insect expression systems, including Vero, MDCK, HEK293, BHK21, CHO, PER.C6, EB66, and Sf21/Sf9, have become indispensable cellular platforms, delivering enhanced biologic yields, superior genetic stability, and validated end-product biosafety. Simultaneous advances in cell culture media optimization have enabled a critical shift from serum-dependent media to serum-free, chemically defined, and xeno-free alternatives, which aim to restore compositional traceability of culture media components, reduce potential residual xenogeneic proteins in serum-supplemented media, and promote reproducibility even at the molecular level. This review emphasizes the far-reaching influence of cell culture systems as the expression powerhouse that sustains modern virology, whilst focusing on recent cell-engineering methods and optimization strategies in culture media that have facilitated this shift. Full article

Background/objectives: Epithelial ovarian cancer (EOC) is the deadliest gynaecologic malignancy, largely due to late-stage diagnosis and ineffective therapy. EOC commonly spreads through the peritoneal cavity as multicellular spheroids, which are metastatic structures that enhance survival under detachment stress, promote dissemination, and contribute to therapeutic resistance. We previously showed that ULK1, a serine/threonine kinase classically linked to macroautophagy initiation, supports EOC progression, suggesting non-canonical roles in spheroid biology and pathogenesis. Methods: CRISPR/Cas9 ULK1 knockout (ULK1KO) models were generated in OVCAR8, HEYA8, and ES2 cells. Mitochondrial degradation phenotypes were assessed in spheroids by immunoblotting and fluorescence microscopy. Label-free proteomics with bioinformatic pathway analysis identified ULK1-associated programs in EOC spheroids. Bioenergetic consequences were quantified using Seahorse ATP-Rate assays. Therapeutic interactions were evaluated using multi-dose combination matrices testing the ULK1 inhibitor DCC-3116 with metformin. Results: ULK1 modulated mitochondrial degradation in a cell-line-specific manner, either promoting or protecting against mitochondrial loss through mechanisms that were uncoupled from canonical autophagy machinery. Proteomic and bioinformatic analyses revealed significant alterations in mitochondria-related processes, aligning with emerging ULK1 functions in mitochondrial homeostasis. ULK1 loss broadly reduced OXPHOS complex proteins in EOC spheroids and consistently decreased hexokinase 2 (HK2), indicating coordinated metabolic remodeling. Seahorse profiling mirrored these shifts: OVCAR8 ULK1KO spheroids showed reduced OCR and ATP production, whereas HEYA8 and ES2 ULK1KO spheroids exhibited increased mitochondrial ATP production. Combination matrices showed potential synergy between DCC-3116 and metformin. Conclusions: These data show that ULK1 differentially regulates mitochondrial degradation across EOC spheroid models through potential mechanisms alternative to canonical autophagy machinery, while reshaping spheroid metabolism and revealing potential therapeutic vulnerabilities in advanced EOC. Full article

Fatty liver hemorrhagic syndrome (FLHS) is a common metabolic disorder in laying hens, leading to reduced egg production and economic losses. Aspirin eugenol ester (AEE) has lipid-lowering, anti-inflammatory, and antioxidant properties, but its effects on FLHS are unknown. This study evaluated the protective effects of AEE using an in vivo FLHS model induced by a high-energy low-protein diet in laying hens and an in vitro steatosis model established by free fatty acid treatment in LMH cells. AEE alleviated liver histopathological damage, reduced oxidative stress (decreased ROS and MDA; increased SOD, GSH, and CAT), and suppressed inflammatory responses. The hepatoprotective effects of AEE were tentatively associated with altered molecular expression of the Nrf2 antioxidant pathway and MAPK/NF-κB inflammatory signaling; however, this correlation was speculated based on molecular detection and incomplete in vitro pharmacological interventions, lacking rigorous causal validation. These findings suggest that AEE alleviates FLHS-related liver injury in laying hens, possibly in association with altered oxidative and inflammatory status. Collectively, these preliminary findings provide a limited theoretical reference for the potential application of AEE as a preventive agent against FLHS in laying hens. Full article

Obesity is a major global health challenge characterized by chronic low-grade inflammation, oxidative stress, and an increased risk of cardiometabolic disorders. Although sex-related differences in inflammatory and redox biomarkers have been reported in obese populations, the molecular mechanisms underlying this heterogeneity remain incompletely understood. In this study, we applied a proteomics-based approach to investigate urinary extracellular vesicles from 45 obese individuals (BMI 30–40 kg/m²; age 50–70 years) in order to identify molecular signatures associated with metabolic dysregulation. Shotgun proteomics analysis performed by nanoLC–MS/MS enabled the identification of 3822 proteins. Hierarchical clustering of proteomic profiles revealed two distinct molecular groups, predominantly enriched in males (Group I) and females (Group II). Label-free quantitative analysis identified 466 differentially abundant proteins between the two clusters. Functional enrichment analysis highlighted pathways associated with immune response, metabolic regulation, and redox homeostasis, including glycolysis/gluconeogenesis, lysosome activity, leukocyte transendothelial migration, and glutathione, cysteine and methionine metabolism. Notably, proteins related to ferroptosis were enriched, suggesting the involvement of iron-dependent oxidative cell death mechanisms in the metabolic imbalance observed in a subset of subjects. Furthermore, the non-enzymatic glycosylation of urinary proteins was significantly higher in Group I compared with Group II (p = 0.0002), indicating increased formation of advanced glycation products in individuals with a more pronounced pro-oxidant state. Preliminary follow-up data suggested a higher incidence of pathological events, including cardiovascular complications, among individuals belonging to Group I. Overall, these findings demonstrate that urinary proteomic profiling can identify distinct molecular phenotypes among obese individuals and highlight oxidative stress, ferroptosis, and protein glycation as potential determinants of metabolic vulnerability, supporting the use of non-invasive proteomic approaches for improved risk stratification in obesity. Full article

The global prevalence of pulmonary infections caused by non-tuberculous Mycobacteria (NTM), particularly the Mycobacterium avium complex (MAC), is increasing. Since NTM are ubiquitous in moist environments and resistant to standard disinfectants, this study evaluated the efficacy of chlorous acid water (CAW) against them. CAW demonstrated superior sanitizing effects compared to sodium hypochlorite (NaClO), efficiently inactivating NTM at 100 mg/L free available chlorine even in the presence of organic matter, where 1000 mg/L NaClO failed. Instead, subcellular fractionation and protein analysis revealed that CAW penetrates the cell to induce extensive aggregation of internal functional proteins, leading to the rapid collapse of membrane potential and ATP production. Furthermore, CAW exhibited significantly lower cytotoxicity toward human lung-derived A549 cells than NaClO. These results indicate that CAW inactivates NTM effectively by targeting internal protein stability and the respiratory chain, offering a potent and safer disinfection strategy for clinical and domestic environments. Full article

The global tilapia industry is threatened due to the mass die-off of farmed tilapia caused by an emerging virus, Tilapia Lake Virus (TiLV). The threat of TiLV disease demands for cost-effective diagnostics and preventive measures such as protein and antibody-based detection tools and even vaccines like protein subunit vaccines. In this study, we aimed to recombinantly express the entire S4 protein of TiLV in vitro and in vivo using an extended host-range baculovirus expression vector system (EHR-BEVS) and used the purified protein to generate polyclonal anti-S4 antibodies. The parental extended host-range ABM-eIF4E baculovirus previously developed in our laboratory was used to generate the recombinant ABM-eIF4E-S4-His baculovirus expression vector using homologous recombination and was purified through multiple rounds of plaque selection and end-point titration, eliminating the need for the use of antibiotic resistance gene selection marker. Different insect cell lines including Trichoplusia ni Hübner, 1802 larvae were infected with ABM-eIF4-S4-His to produce recombinant S4 protein. Western blotting was used to detect the recombinantly expressed S4 protein. We generated an antibiotic resistance gene-free EHR-BEV and observed the permissibility of all the insect cell lines tested including T. ni larvae to infection with the recombinant ABM-eIF4E-S4-His. Secondly, immunogenic S4 protein capable of eliciting rabbit polyclonal anti-S4 antibodies was recombinantly expressed in the infected insect cells and larvae. TiLV S4 protein was recombinantly expressed in vitro and in vivo using extended host-range ABM-eIF4E-S4-His baculovirus expression vector and was shown to induce rabbit antibody production that can recognize S4 protein supporting the potential of insect larvae as alternative biofactories in the production of recombinant TiLV protein subunit for the development of diagnostics and preventive vaccines against TiLV disease. Full article

The discovery of melatonin as a multifunctional free radical scavenger and its possible synthesis in the mitochondrial matrix of peripheral eukaryotic somatic cells highlights a critical new perspective on the importance of this indole. Experimental evidence supporting these findings is substantial, but there are still lingering questions whether melatonin is a direct radical scavenger in vivo and whether it is synthesized in the mitochondrial matrix. We systematically analyze the innovative experimental approaches that support melatonin’s radical scavenging actions and assess the compelling data supporting its production in mitochondria. Melatonin concentrations are reportedly higher in this organelle than in other cellular compartments. Proteins for the enzymes required to convert serotonin to melatonin are present in the mitochondrial matrix and purified mitochondria synthesize melatonin. In the mitochondrial matrix, melatonin is likely located within the “damage radius” of highly reactive oxygen species. We also summarize novel actions of melatonin associated with its regulation of membrane fluidity, determine the molecular composition of membrane lipid rafts, and modulate liquid–liquid phase separation and biomolecular condensates intracellularly. If the findings discussed herein continue to be validated, melatonin would be in an optimal position to function as an antioxidant and may be a key driver in the context of preserving mitochondrial redox homeostasis and disease mitigation. Full article

Individuals with diabetes mellitus (DM) experience impaired wound healing, where the healing process is often compromised by a complex, hostile microenvironment characterized by persistent inflammation, high oxidative stress, and dysfunctional angiogenesis. The hyperglycemic environment damages the blood vessels and disturbs the normal hypoxia-induced upregulation of vascular endothelial growth factors, causes poor vascularization and insufficient production of new blood vessels, and leads to impaired perfusion and thickened and dysfunctional capillary basement membranes, which reduce blood flow to the wound, leading to delayed wound healing. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are the main effectors of intercellular communication and have emerged as a potent cell-free strategy for the acceleration of tissue repair. MSC-EVs can be isolated from various adult tissues, but increasing evidence suggests that placental MSC-derived EVs (PMSC-EVs) possess distinct clinical and biological advantages for enhancing diabetic wound healing. Placentas are unique cell sources for PMSCs, which can be easily acquired non-invasively from a discarded placenta and is ethically acceptable, and have superior proliferative capacity. The cargo of PMSC-derived EVs contains macromolecules such as proteins, mRNA, miRNA, and lipids, which may be tailored for fetomaternal tolerance and translates to unmatched immunomodulatory potential for resolving chronic diabetic inflammation. The PMSC-derived EVs also aid in enhancing multiple pathways, including modulation of inflammation, angiogenesis, and epithelial proliferation, that lead to increased wound healing. This article will highlight the unique advantages, specific mechanisms, and limitations of PMSC-derived EVs as an innovative non-cellular therapeutic modality in restoring vital repair processes and enhancing diabetic wound healing. Full article

Hypertension is a severe global public health issue. Food-derived angiotensin-converting enzyme (ACE)-inhibitory peptides have shown great potential as safe and effective alternatives to synthetic antihypertensive drugs. Camel milk is rich in bioactive peptides. This study aimed to screen for ACE-inhibitory peptides from hydrolyzed camel casein, explore their inhibitory mechanisms and endothelial protective effects in vitro, and reveal their potential antihypertensive pathways using network pharmacology. This study screened three peptides with angiotensin-converting enzyme (ACE) inhibitory activity from enzymatically hydrolyzed camel casein components: MVPFLQPK, VPFLQPKVM, and QKWKFL, with IC₅₀ values of 277.1, 396.9, and 486.9 μmol/L, respectively. Enzyme inhibition kinetics analysis indicated that MVPFLQPK exhibited a non-competitive inhibition pattern, VPFLQPKVM exhibited a mixed inhibition pattern, and QKWKFL exhibited a competitive inhibition pattern. Molecular docking revealed that all three peptides formed hydrogen bond interactions with ACE, and QKWKFL and VPFLQPKVM directly bound to the enzyme’s active site to inhibit substrate catalysis. Molecular dynamics simulation further confirmed the high stability of the three peptide–ACE complexes, with binding free energies from −34.24 to −51.19 kcal/mol. The primary contributing forces include hydrogen bonds, van der Waals interactions, electrostatic forces, and nonpolar solvation effects. Network pharmacology analysis suggested that these peptides may exert synergistic antihypertensive effects by regulating multiple blood pressure-related pathways, including the renin–angiotensin system, renin secretion, and calcium signaling pathways, by acting on key targets such as ACE, REN, SRC, and MMP9. Cell experiments demonstrated that all three peptides exhibited no cytotoxicity in the Ang II-induced HUVEC injury model, significantly promoted NO release, inhibited ET-1 secretion, and possessed endothelial protective potential. This study investigated the in vitro ACE-inhibitory mechanism of peptides derived from camel milk and their potential role in blood pressure regulation, providing experimental evidence for subsequent in vivo activity validation and the development of functional camel milk protein products. Full article

Extracellular vesicles (EVs) can disseminate replication-competent viral genomes complexed with selected host proteins, enabling stealth cell-to-cell transfer within lipid membrane-enclosed bubbles. In addition to complementing free-virion spread, EV-associated genomes can be protected from neutralizing antibodies and persist under conditions in which classical virion production decreases. Here, we propose a route-resolved framework in which interconnected cellular secretory pathways, including endoplasmic reticulum (ER) remodeling, multivesicular body (MVB) biogenesis, secretory autophagy, and plasma-membrane budding, jointly generate EV heterogeneity and create discrete opportunities for the capture, protection, and export of infectious cargo. We highlight reticulon-3 (RTN3), an ER-shaping protein, as an upstream regulator that can couple infection-induced ER microdomains to endosomal docking and to autophagy-linked trafficking decisions that bias intermediates toward secretion rather than degradation. Supporting this view, transmission electron microscopy of dengue virus-infected cells reveals extensive vesicular remodeling, including irregular MVBs adjacent to the plasma membrane and autophagosome-like double-membrane structures, consistent with altered vesicular routing following RTN3 perturbation. Collectively, these route-resolved, spatially organized spatio-organelle changes support a pathomechanistic model in which RTN3-mediated ER remodeling reshapes ER-endosome-autophagy trafficking interfaces, creating regulated decision points that can be leveraged to stratify infectious EV subsets (with infectivity-linked single-vesicle and quantitative proteomics approaches) and to inform host-directed strategies that curb non-lytic viral dissemination. Full article