Search Results (1,922)

Strawberry fruit is highly perishable and susceptible to oxidative stress and rapid quality deterioration during postharvest storage. This study evaluated the effectiveness of an edible coating based on xanthan gum (XG), enriched with citric acid (CA) and/or ascorbic acid (AA), in preserving the quality of ‘Rossetta’ strawberries stored at 4 ± 1 °C for 9 days. Coated fruits showed higher values of firmness, titratable acidity, and color parameters compared to the control, along with a more gradual increase in total soluble solids, indicating reduced dehydration and delayed ripening. In addition, treated samples retained higher levels of bioactive compounds, including total phenolics, flavonoids, and anthocyanins, as well as antioxidant activity, confirming improved nutraceutical quality during storage. By the end of storage, the combined XG+CA+AA coating modulated the antioxidant enzymatic system, enhancing the activity of superoxide dismutase (44.6%), catalase (31.6%) and ascorbate peroxidase (44.6%) in counteracting oxidative stress, accompanied by a 32.8, 45.9 and 29.8% reduction in polyphenol oxidase and lipoxygenase activities, as well as malondialdehyde content, compared to the control, respectively. Overall, the combined XG+CA+AA coating was the most effective, highlighting a synergistic action of the acids and confirming its potential to extend shelf life and preserve strawberry quality. Full article

Bacillus thuringiensis Cry toxins are well known for their high insecticidal activity against Lepidoptera, Diptera, and Coleoptera and have been widely used in Bt transgenic crops. However, their activity against Hemipteran aphids remains relatively low. Identifying novel Cry proteins and elucidating their action mechanisms can facilitate the development of effective aphid control strategies. In this study, we found that ingestion of Cry2Ab12 did not kill Myzus persicae adults but significantly reduced their offspring number and exerted a lethal effect on M. persicae nymphs. After identifying Cry2Ab12 toxin-binding proteins in M. persicae, we further characterized the interaction with Obg-like ATPase 1 (OLA1), a conserved protein involved in growth regulation. Bio-layer interferometry (BLI), ELISA, and enzyme activity assays revealed that Cry2Ab12 and OLA1 do not interact directly. Interestingly, heat shock protein 60 (HSP60) was shown to mediate the interaction among Cry2Ab12, HSP60, and OLA1, leading to inhibition of OLA1 enzymatic activity. Based on these findings and bioinformatics simulations, we proposed a mechanistic model for Cry2Ab12 toxicity against M. persicae: upon ingestion of a sufficient amount of Cry2Ab12, the formation of the Cry2Ab12–HSP60–OLA1 complex impairs the cellular stress response, disrupts normal OLA1 expression, and ultimately restricts larval growth and development, resulting in lethality. This study provides new insights into the action of Cry toxins in aphids and offers a basis for developing enhanced aphid biocontrol strategies. Full article

Vibrio tubiashii infection has led to several Babylonia areolata pandemics on the southeast coast of China, yet the immune response of the ivory shell against V. tubiashii and the specific pathogen–host interaction remain unclear. This dynamic study aimed to characterize the response of B. areolata to V. tubiashii infection with the use of pathology, transcriptomics, an enzymatic assay, and inflammatory cytokines. Hepatopancreatic cells showed marked vacuolar degeneration with intact cell membrane and extensive cytoplasmic vacuolization after infection. The dynamic transcriptome of the hepatopancreatic tissue was analyzed by RNA-seq after V. tubiashii infection, and a total of 2733 (3 h), 5610 (24 h), 3323 (48 h), and 418 (72 h) differentially expressed genes (DEGs) were identified during infection. The GO and KEGG analyses showed that the DEGs were enriched in metabolic regulation, lysosome, and multiple immune-related pathways such as the MAPK signaling pathway. The immune response of B. areolata was distinct, where the early stage of immune response (3 h) showed binding, focal adhesion, and apoptosis, as well as an activated antioxidant system. Here, expression of TNF-α, IL-1, and IL-8 was significantly increased in the hepatopancreas, whereas expression of IL-6 and IL-17 increased afterward. During the middle stage (24 h and 48 h), a large number of DEGs were suppressed, especially those associated with metabolism and lysosomes, although their expression returned to normal during prolonged infection (72 h). The PPI network showed that ppp2, atp6, and sos1 were the top immune-related DEGs during infection. Key infection-related and time-course-related genes were analyzed by WGCNA. This study illustrates that oxidative stress, inflammation, and apoptosis are strategies of the hepatopancreatic immune response in B. areolata against V. tubiashii infection and enlightens conservation and production by furthering our understanding of gastropod immunity. Full article

Background/Objective: Bisphenol AF (BPAF), a fluorinated analogue of bisphenol A, is an environmental contaminant associated with hepatotoxicity and metabolic disruption. However, the systematic molecular mechanisms linking early transcriptional events to metabolic dysfunction in the liver remain poorly defined. The aim of this study is to elucidate the association between BPAF exposure and hepatic lipid accumulation by integrating transcriptomics, cellular metabolomics, and targeted phenotypic assays. Methods: We performed RNA-sequencing on livers from mice exposed to BPAF (0.1–10 mg/kg/day, 28 days), and performed non-targeted metabolomics on AML12 murine hepatocytes co-cultured with RAW264.7 macrophages in a Transwell system (0–2500 nM BPAF, 48 h). Key metabolic pathways were identified through integrated bioinformatics and validated using enzymatic assays, qRT-PCR, Western blotting, and phenotypic staining (lipid droplets, ROS). Results: Multi-omics integration revealed significant disruption of PPAR signaling and the tricarboxylic acid (TCA) cycle. A striking dose-dependent accumulation of succinate was observed in exposed cells, concomitant with a significant inhibition of succinate dehydrogenase (SDH) activity (52% reduction at 2500 nM, p < 0.001). Transcriptomic data confirmed the downregulation of mitochondrial fatty acid β-oxidation genes. Phenotypic validation indicated that BPAF exposure is associated with oxidative stress, pro-inflammatory cytokine release (TNF-α, IL-6), and pronounced intracellular lipid droplet accumulation in hepatocytes. Conclusions: This study suggests that BPAF exposure is associated with SDH dysfunction, TCA cycle arrest, and lipid dysregulation. Whether BPAF directly inhibits SDH or acts through upstream mitochondrial targets warrants further structural and kinetic investigation. Full article

Introduction: The aglomerular kidney, characterised by the absence of functional glomeruli and reliance on tubular secretion alone, has evolved independently across multiple teleost lineages occupying diverse environments, including notothenioids of the Southern Ocean, gadids of cold North Atlantic and Arctic waters, and syngnathids distributed across temperate and tropical seas. Despite sharing this independently derived renal architecture, these groups face markedly distinct osmotic, thermal and chemical challenges in their natural habitats. How aglomerular kidneys cope with environmental stressors, including fluctuations in temperature and salinity, and how they handle the excretion of xenobiotics and other exogenous compounds through exclusively tubular mechanisms, remains poorly understood. The physiological and molecular responses underlying renal function in these lineages have received comparatively little attention relative to their glomerular counterparts. Objective: This study investigates how aglomerular kidneys across phylogenetically distinct teleost lineages respond, at molecular and physiological levels, to contrasting environmental conditions, namely temperature and salinity challenges predicted to alter the functional demands on renal osmoregulation. Methodology: Adult and juvenile specimens from target lineages were subjected to controlled exposure experiments combining different temperature and salinity regimes. Blood and urine samples were collected to assess osmolality and ionic composition. Renal tissues were processed for gene expression analysis of key transport and structural genes, histology, immunohistochemistry and enzymatic activity assays, providing an integrated picture of renal function under each condition. Results: Molecular analyses are currently underway. Preliminary work has established protocols for RNA extraction and quantitative PCR across target species, enabling comparative gene expression profiling to proceed across the full dataset. Conclusions: A comparative physiological and molecular approach across aglomerular teleost lineages will clarify whether shared renal architecture translates into shared functional responses to environmental challenge and identify lineage-specific mechanisms of renal adaptation, with broader relevance for predicting how these fishes may respond to ongoing environmental change. Full article

Crimean–Congo hemorrhagic fever virus (CCHFV) remains a major public health threat due to its high mortality rates and the absence of approved antiviral therapies. The viral ovarian tumor (OTU) protease is a critical virulence factor that suppresses host innate immunity through its deubiquitinase activity, making it an attractive therapeutic target. In this study, we employed a structure-guided approach to identify and validate novel small-molecule inhibitors targeting the non-catalytic Y89-W99 pocket of the OTU protease. Recombinant OTU protease was successfully expressed, purified, and refolded, yielding a soluble and enzymatically active protein. Cellular assays confirmed that the enzyme retains robust deubiquitinase activity, significantly reducing global ubiquitin conjugates in mammalian cells. In silico analysis of a putative DUB inhibitor library identified several candidate inhibitors with favorable binding interactions within the Y89-W99 pocket. Biochemical validation using a fluorometric Ub-AMC assay revealed that multiple small molecules strongly inhibit OTU activity, including OTUi-10 (~93% inhibition), OTUi-13 (~87%), OTUi-1 (~85%), OTUi-4 and OTUi-11 (~81%), and OTUi-9 (~76%). Additional moderate inhibitors included OTUi-12 (~67%), OTUi-19 and OTUi-21 (~66%), and OTUi-5 (~57%). In silico drug-likeness and toxicity profiling filtered the library to four fully compliant candidates, OTUi-4, OTUi-10, OTUi-11, and OTUi-12, all free of predicted toxicity alerts. These findings suggest that the Y89–W99 pocket may be a pharmacologically relevant site worthy of further investigation and identify OTUi-10, OTUi-4, and OTUi-11 as promising preliminary hit compounds. The results also provide initial insights that may guide future optimization and mechanistic studies of OTU protease inhibitors targeting CCHFV. Full article

Heparanase 1 (HPSE1) is the only mammalian endoglycosidase that cleaves heparan sulfate (HS), a glycosaminoglycan (GAG), and is frequently upregulated in cancers, thereby promoting tumor progression. Despite extensive efforts to develop inhibitors of its HS-degrading activity, its non-enzymatic functions limit therapeutic efficacy and pose a major challenge for therapeutic development. Thus, inhibiting HPSE1 expression is critical for controlling its enzymatic and non-enzymatic functions; however, no FDA-approved inhibitors are currently available. Here, we identify auranofin (AUF), an oral gold-containing drug used to treat rheumatoid arthritis, as a potent inhibitor of HPSE1 promoter activity. High-throughput screening revealed that an atypical protein kinase C (aPKC)–NF-κB signaling axis is a key regulator of HPSE1 expression. Notably, AUF treatment reduced HPSE1 expression and significantly suppressed the invasive capacity of MDA-MB-231 cells in a Transwell migration assay. We then investigated the role of HPSE1 in the invasive activity of MDA-MB-231 cells, which produce higher levels of hyaluronan (HA) and HS than non-invasive cells. Neither HS degradation, HA supplementation in Matrigel during Transwell migration, nor HPSE1 overexpression alone was sufficient to drive invasion, suggesting that invasive capacity depends on mesenchymal features and coordinated induction of HPSE1 and GAGs rather than HS degradation. Collectively, our findings demonstrate that AUF-mediated inhibition of aPKC suppresses HPSE1 expression, thereby inhibiting both its enzymatic and non-enzymatic functions and limiting cancer progression, metastasis, and angiogenesis. These results highlight the therapeutic potential of AUF for targeting HPSE1-driven tumor progression and support its repurposing for cancer treatment. Full article

The presence of organic micropollutants such as pharmaceuticals and pesticides in aquatic systems poses risks to environmental and public health, as conventional wastewater treatment plants are often ineffective at removing them, highlighting the need for alternative solutions. This study evaluates the combined use of biochar and laccase to remove trimethoprim, clindamycin, and fipronil, selected for their ubiquity, persistence, and physicochemical properties. Commercial wood-derived biochar was used, and removal performance was assessed through adsorption isotherms, time-dependent evaluation of removal efficiency, and quantification by UPLC-MS/MS. Toxicity after treatment was evaluated using bacterial growth assays with Escherichia coli and Rhodococcus erythropolis. Adsorption of trimethoprim and clindamycin followed the Langmuir model (Qmax 2.27 and 1.49 mg/g), while that of fipronil followed the Temkin model (Qmax 0.98 mg/g). The combined biochar–laccase system enabled up to 99% removal of trimethoprim and clindamycin within one hour, demonstrating synergy between adsorption and enzymatic removal. Enhanced removal was also observed for clindamycin and fipronil in mixtures. Bacterial assays showed partial restoration of growth after treatment, suggesting reduced antibacterial activity of transformation products, although effects remained species-dependent. Overall, the biochar–laccase system shows promise for micropollutant removal, supporting green remediation strategies, but further work is required to characterize transformation products and assess ecological impacts. Full article

Machupo virus (MACV) is the causal agent of Bolivian Hemorrhagic fever. It is highly pathogenic, has a high mortality rate, and currently lacks specific treatments or vaccines. MACV belongs to the Arenaviridae family, which uses a cap-snatching mechanism during the transcription process. Its viral polymerase, the L protein, harbors the endonuclease activity required for cap snatching, making it a suitable target for the development of antiviral therapeutics. We combined experimental and computational methods to characterize MACV endonuclease activity and evaluate inhibitors. A fluorescence resonance energy transfer (FRET) assay was used to measure the enzymatic activity of endonuclease and identify potent inhibitors via high-throughput screening. FRET assays identified BW-148, an inhibitor with a 48.4 µM (95% CI: 37.3–59.3 µM; R² = 0.98) IC₅₀, and a K_D of 13.7 µM (95% CI: 8.2–19.2 µM, n = 3). Docking studies reveal that BW-148 may bind near the MACV endonuclease catalytic site, inhibiting enzymatic activities by metal chelating. BW-148 is a useful lead compound for further optimization of Machupo endonuclease inhibitors. Full article

Ubiquitin-specific protease 22 (USP22) regulates epigenetic gene expression by deubiquitinating histone H2B (H2Bub1) and upregulating oncogenic proteins and pathways, while antagonizing p53-mediated tumor suppression. USP22 is frequently overexpressed in cancers and associated with therapy resistance and poor prognosis yet remains largely untargeted pharmacologically. Here, using a fluorescence-based USP22 deubiquitinase assay to screen the LOPAC^®1280 library, we identified β-Lapachone, a natural ortho-naphthoquinone with strong anticancer activities, as a potent USP22 inhibitor. β-Lapachone potently inhibited USP22 enzymatic activity, with a half-maximal inhibitory concentration (IC₅₀) of ~0.75 μM, and molecular docking revealed its occupation of the catalytic pocket adjacent to the USP22 active-site triad, supporting a potential binding mode. Functionally, β-Lapachone suppressed proliferation and induced apoptosis in A549 and H1299 RAS-mutant lung adenocarcinoma (LUAD) cells, while USP22 knockout conferred marked resistance, indicating partial USP22 dependence. In patient-derived LUAD models, β-Lapachone inhibited sphere formation and reduced CD133⁺ cancer stem cell populations. Notably, it synergized with cisplatin to enhance DNA damage and apoptosis. In vivo, β-Lapachone significantly suppressed tumor growth in a syngeneic KRAS-mutant/p53-Null mouse lung cancer model and further potentiated cisplatin-induced antitumor effects. Collectively, these findings identify β-Lapachone as a potent inhibitor of USP22 and validate USP22 inhibition as a key mechanism underlying its anticancer activity in LUAD cells, both in vitro and in vivo. Full article

Monieziosis presents a considerable challenge to livestock farming, mainly due to the parasite’s resistance to common anthelmintics, prompting the need for alternative control strategies. This study examined the in vitro effect of two enzymatic sources (plant and fungal) on the structural integrity of Moniezia expansa eggs, aiming to find new environmental management solutions. The plant enzyme papain was tested at various concentrations and time points, while the fungal enzyme was produced through solid-state fermentation using Duddingtonia flagrans (AC001), resulting in an active crude enzymatic extract (ACEE). Papain at 10% w/v showed nonlinear degradation (R² = 0.998), achieving 95% egg reduction after 48 h. The fungal extract ACEE (71.2 U mL⁻¹) caused a 60% reduction after 72 h. Morphological studies indicated significant eggshell damage following both treatments. A compatibility assay showed an antagonistic interaction, with enzyme activity decreasing by 83.83% within 48 h, likely due to cross-proteolysis. Although each agent is effective individually, combining them is not feasible. This is the first study documenting the activity of ACEE and papain against M. expansa eggs, and it recommends using them separately or sequentially for effective parasite control. Full article

The lysosomal enzyme α-galactosidase A (AGAL) degrades globotriaosylceramide (Gb₃). While this enzymatic function in lysosomal metabolism is well characterized, interaction partners and alternative functions are unknown. This study aims to identify new potential AGAL-interacting proteins. AGAL was fused to the mutated biotin ligase BirA from E. coli (TurboID). Expression of the fusion protein was confirmed by Western blot and immunofluorescence, while enzymatic activity was verified by functional assays. In three experimental settings (AGAL wild-type (WT), AGAL missense variant (p.N215S), and the control cell line), TurboID-biotinylated proximal proteins were enriched by streptavidin pull-down and analyzed by mass spectrometry. Gene Ontology (GO) terms were subsequently evaluated to characterize biological functions and localizations of the identified proteins. Selected candidates were co-immunoprecipitated with AGAL to confirm direct interactions. The AGAL-TurboID fusion protein was successfully expressed in AB8/13 podocytes. Immunofluorescence and enzyme activity assays confirmed the presence and functionality of the fusion protein. Subsequent functional analysis (GO term analysis) showed enrichment of driver terms, including extracellular matrix organization (ECM), multicellular organism development, and protein metabolic process, in the biological process category. The identified top-hit proteins were predominantly involved in the organization of ECM, cell proliferation and cytokinesis, unfolded protein response during endoplasmic reticulum stress, and protein ubiquitination. Co-immunoprecipitation confirmed the interaction between AGAL and the candidate Galectin-3-binding protein (Gal-3BP). Our results suggest that AGAL may play a role in other pathways and/or the ECM organization beyond its lysosomal function. The confirmed interaction with Gal-3BP can now be functionally investigated in further studies. Full article

Background: Ganoderma lucidum is a medicinal mushroom widely recognized for its high content of bioactive polysaccharides, particularly β-glucans with immunomodulatory properties. This study aimed to optimize polysaccharide extraction conditions to maximize yield and glucan content, and to evaluate the biological activity of the obtained fractions in an experimental model of intestinal mucositis. Methods: Polysaccharides were extracted using a combination of hot-water extraction and ethanol precipitation, optimized by response surface methodology. Optimal conditions (121 °C for 120 min followed by 90% ethanol precipitation) yielded a crude polysaccharide fraction (Poli-GL). A subsequent freeze–thaw process generated a soluble fraction (S-Poli-GL). Structural and compositional characterization was performed using enzymatic assays, monosaccharide profiling, and NMR spectroscopy. The biological effects of Poli-GL and S-Poli-GL were evaluated in a 5-fluorouracil-induced intestinal mucositis model following oral administration at doses of 30, 100, and 300 mg/kg. Results: The optimized extraction protocol enabled efficient recovery of polysaccharides enriched in glucans. S-Poli-GL exhibited a high total glucan content, including 43.3% β-glucans and 3.45% α-glucans, along with minor amounts of galactose and mannose. Structural analysis confirmed the predominance of branched β-(1→3),(1→6)-D-glucans. While Poli-GL did not prevent mucositis development, S-Poli-GL significantly reduced the disease activity index and attenuated intestinal inflammation, indicating enhanced biological activity associated with the soluble glucan-rich fraction. Conclusions: Optimization of extraction and fractionation improves the functional properties of G. lucidum polysaccharides. The soluble glucan-enriched fraction (S-Poli-GL) demonstrated significant protective effects in intestinal mucositis, supporting its potential as a therapeutic candidate and warranting further investigation for clinical application. Full article

Accurate detection of Campylobacter in chicken liver is hindered by strong matrix inhibition. This study evaluated sample-processing strategies to improve droplet digital PCR (ddPCR) quantification of Campylobacter coli and Campylobacter jejuni in chicken liver. Mechanical homogenization (Stomacher) and enzymatic/mechanical dissociation (gentleMACS), with and without 8 μm filtration, were compared. Particle-size analysis showed that filtration, especially following gentleMACS treatment, produced smaller, more uniform particles and reduced variability. Percent-degradation assays confirmed that gentleMACS achieved substantially greater tissue disruption than Stomacher homogenization. The multiplex ddPCR assay, which simultaneously targets C. coli and C. jejuni, produced droplet counts comparable to single-target reactions, indicating minimal interference between targets under the conditions tested. In inoculated liver samples, gentleMACS processing yielded droplet counts similar to those obtained from pure cultures, whereas unprocessed liver caused severe matrix interference and inconsistent quantification. Furthermore, gentleMACS-treated samples exhibited strong log-to-log linearity for quantifying C. coli and C. jejuni, enabling detection near 1 genome copy equivalent per reaction. Overall, the results indicate that enzymatic/mechanical dissociation combined with fine-pore filtration improves ddPCR detection of Campylobacter species in chicken liver. Full article

The valorization of salmon farming by-products is an essential strategy within the circular economy. This study optimized the enzymatic hydrolysis of salmon frame proteins using Alcalase 2.5L and response surface methodology (RSM). The effects of temperature (50–60 °C), substrate concentration (50–100% w/w), and protease dose (1–13 mAU/g salmon frames) were evaluated on two key responses: degree of hydrolysis (DH) and nitrogen recovery (NR). The 20 experimental assays showed that substrate concentration and enzyme dosage strongly influenced both responses, whereas temperature had a moderate effect. The fitted models exhibited R²_adjusted values above 70% and met statistical assumptions, confirming their predictive reliability. Optimal conditions for maximizing DH were 55 °C, 50% w/w substrate, and 13 mAU/g protease, yielding a predicted DH of 6.65%. In contrast, the highest NR (48.35%) was observed at 50 °C, 50% w/w substrate, and 13 mAU/g protease, indicating that solubilization does not depend solely on hydrolysis intensity. Validation experiments showed no significant differences between predicted and experimental values (p > 0.05), supporting the robustness of the models. These results demonstrate the usefulness of RSM for optimizing enzymatic hydrolysis and advancing sustainable valorization of salmon by-products. Full article