Search Results (1,417)

Herbal melanin (HM), previously reported for its antiproliferative and pro-apoptotic properties, has garnered interest as a promising anti-colorectal cancer drug. However, HM’s biological effects and underlying molecular mechanisms and the related signaling pathways in colorectal cancer (CRC) cell motility are poorly investigated. To evaluate the impact of various concentrations (50, 100, and 200 μg/mL) of HM on cell migration, invasion, and tumorigenicity on human HT29 and SW620 CRC cell lines, a real-time cell analyzer instrument and colony formation assays were employed, respectively. An angiogenesis-related protein array was also used, and the levels of protein expression contributing to colony formation and extracellular proteolysis-driven cell migration and invasion, such as E-cadherin, N-cadherin and urokinase-type plasminogen activator receptor (uPAR), were monitored using Western blotting and RT-qPCR technologies. HM significantly decreased CRC cell motility, invasiveness, and formation of colonies, associated with E-cadherin upregulation and N-cadherin downregulation. In addition, HM specifically inhibited uPAR expression levels, which were also decreased by the pharmacological mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor UO126 and Jun N-terminal kinase (JNK) inhibitor SP600125, in both CRC cell lines, including metastatic CRC (mCRC) SW620 cell line. Addition of HM to cells pretreated with JNK and MEK inhibitors attenuated the blockade of JNK and ERK phosphorylation and alleviated HM-downregulated uPAR expression and HM-inhibited mCRC cell migration. In conclusion, our in vitro studies demonstrate that HM exhibits an inhibitory effect on CRC migration and invasiveness, associated with uPAR downregulation through JNK and ERK pathways. Full article

Background: Lipoprotein(a) [Lp(a)] is a genetically determined risk factor for atherosclerotic cardiovascular disease (ASCVD). Proteomic studies suggest that Lp(a)-associated proteins mediate inflammation, thrombosis, and vascular calcification, but methodological variability may influence proteome definition. Methods: Lp(a) was immunoprecipitated from human plasma using an apo(a)-specific monoclonal antibody and analyzed by mass spectrometry following either in-gel digestion or automated in-solution proteolysis. Proteins identified by ≥3 unique peptides and consistently detected across all samples by both methods were considered high confidence. Functional enrichment and interaction networks were assessed using STRING. Results: In-solution proteolysis identified 92 proteins and in-gel digestion identified 55 proteins, with 34 proteins shared between methods. These high-confidence proteins were enriched for pathways involved in lipoprotein remodeling, coagulation regulation, vesicle-mediated transport, lipid binding, and extracellular matrix organization, providing biological insight into mechanisms linking Lp(a) to inflammation, thrombosis, and calcification. Conclusions: Proteome composition of Lp(a) is method-dependent; however, a rigorously defined core proteome of 34 proteins was consistently identified across analytical approaches, highlighting biologically relevant pathways that may underlie Lp(a)-mediated ASCVD risk. Full article

Glyphosate is a widely used herbicide with increasing concern regarding its non-target impacts in coastal ecosystems and mariculture species. Here, we profiled acute physiological stress signatures of waterborne glyphosate exposure in the sea cucumber Apostichopus japonicus, integrating measured exposure concentrations, tissue residues, digestive and oxidative/innate immune biomarkers, and gut microbiota. After 24 h exposure, measured waterborne glyphosate confirmed the intended gradient (0.09 ± 0.02, 1.26 ± 0.09, and 4.49 ± 1.12 mg/L for low-, medium-, and high-dose treatments, respectively), and overt stress phenotypes with mortality occurred only at the high dose (36.67%), enabling separation of high-dose survivors (HS) and high-dose dead (HD) for downstream analyses. Tissue measurements showed low/background levels in controls, with compartment-specific distribution: the respiratory tree exhibited higher burdens at the medium dose, whereas coelomic fluid showed the highest burdens in HS at the 24 h endpoint. Functionally, most intestinal digestive enzymes were unchanged, but trypsin activity was consistently suppressed across exposed groups (p < 0.05). In coelomic fluid, oxidative stress responses were evident, with elevated MDA (L and M), reduced CAT (L, M, and HS), and reduced GSH-PX in HS (all p < 0.05), while SOD, GR, and lysozyme showed no significant changes. Gene sequencing of 16S rRNA (n = 3 per group) revealed significant shifts in community diversity/evenness (Shannon p = 0.0497; Simpson p = 0.0484) and beta diversity (PCo1 = 30.08%, PCo2 = 26.30%; PERMANOVA F = 1.816, p = 0.008), with LEfSe indicating discriminative taxa associated with exposure/outcomes. Collectively, these multi-level endpoints define an acute glyphosate stress signature in A. japonicus, linking internal dose distribution to oxidative disruption, impaired intestinal proteolysis, and microbiome restructuring. Full article

Cells rely heavily on DNA repair networks to survive genomic damage. For repairing double-strand breaks, Non-Homologous End Joining (NHEJ) remains the primary pathway, which is largely controlled by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Researchers have long studied how phosphorylation drives this kinase. However, recent data point to an important additional layer of control. Drawing on evidence accumulated over the past two decades, we propose a “Spatiotemporal Logic Circuit” model for DNA-PKcs regulation. In this model, SUMO-associated interactions may help stabilize synaptic assembly, HUWE1-mediated neddylation may facilitate kinase activation at Lys4007, and K48-linked ubiquitination—potentially involving RNF144A—may contribute to the turnover of persistent repair complexes. Importantly, we frame these UBL-mediated events within the broader autophosphorylation-driven conformational cycle of DNA-PKcs, which remains central to NHEJ progression. Additionally, we highlight the structural interface where activation and degradation signals may converge and the extraction barrier posed by the massive DNA-PKcs scaffold. From a translational perspective, we argue that the exceptional size of DNA-PKcs (~470 kDa) and its topological entrapment on DNA render it an unusually challenging PROTAC target—one that may require p97/VCP-assisted extraction before proteolysis can proceed. We also highlight the underappreciated risk that E3 ligase loss-of-function, already documented in BET-PROTAC resistance, may similarly undermine DNA-PKcs degrader strategies. Full article

Underutilised, nutrient-dense legumes in their sprouted form provide promising substrates for developing functional fermented foods capable of influencing gut microbial activity and metabolite production. This study evaluated the effects of probiotic lactic acid bacteria-fermented beverages derived from sprouted Bambara groundnut (Vigna subterranea) and cowpea (Vigna unguiculata) on gut microbiota composition and short-chain fatty acid (SCFA) production using an in vitro colonic fermentation model. The beverages were fermented with either Bifidobacterium animalis BB-12 (BCBF24) or Lactiplantibacillus plantarum 75 (BCL7524). During colonic fermentation, at 0, 12, 24, and 38 h, faecal slurries were collected for SCFA analysis using gas chromatography–mass spectrometry (GC-MS) and deoxyribonucleic acid (DNA) sequencing (Oxford Nanopore Technologies). Microbial diversity decreased, indicating selective enrichment of taxa. BCL7524 induced a major shift, significantly (p < 0.05) enriching Bacillota and driving Megasphaera to ~42% dominance within 24 h. This reflected cross-feeding from L. plantarum to lactate-utilising Megasphaera spp. Spearman correlation linked Megasphaera to a broad SCFA profile, including isobutyric, isovaleric, valeric, and hexanoic acids, with a significant (p < 0.05) positive correlation observed for hexanoic acid. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated proteolysis and mapped hexanoic acid to fatty acid biosynthesis pathways, suggesting chain-elongation activity contributing to hexanoate formation. In line with this, BCL7524 produced significantly (p < 0.05) higher levels of hexanoate (3–14 mM) and valerate (10–15 mM), supporting chain-elongation activity within the community. In contrast, BCBF24 enriched Actinomycetota and Bifidobacterium, correlating with acetate production (18–23 mM). This study demonstrates that specific synbiotic beverages can modulate gut microbial ecology and metabolic output under in vitro conditions. Full article

Background: Staphylococcus aureus bacteremia (SAB) is a common and life-threatening bloodstream infection often caused by methicillin-resistant SA (MRSA) isolates. Up to 35% of SAB patients fail to clear infection with gold-standard anti-MRSA antibiotics, even if the isolate meets susceptibility breakpoints in conventional assays in vitro. Such outcomes are termed persistent and may involve small colony variant (SCV) adaptation of SA in vivo. Methods: In this study, we assessed virulence phenotypes and mechanisms in persistent (PB) vs. resolving (RB) MRSA isolates from SAB. Results: Overall, PB isolates caused less hemolysis or biofilm formation than RB isolates, but proteolysis was equivalent. Attenuation of these virulence phenotypes increased longitudinally during the course of SAB. Although PB vs. RB isolates had similar human endothelial cell invasion rates, PB isolates more frequently formed SCVs intracellularly and inversely correlated with pH. Study PB and RB isolates exhibited distinct susceptibilities to prototypic human host defense peptides (HDPs), which were influenced by antibiotics and pH. Furthermore, mechanistic signatures of HDPs differed between PB and RB isolates. Conclusions: Together, these results reveal that MRSA isolates from PB vs. RB outcomes of SAB have differential virulence profiles that suggest coordinated immune subversion in PB. Understanding MRSA adaptations that promote persistence in SAB may enable innovative agents and strategies to address these challenging infections. Full article

This study evaluated the effects of essential oils, such as limonene (LIM) and a cinnamaldehyde–carvacrol blend (CCB), on the fermentation, chemical composition, in vitro digestibility, and aerobic stability of corn silage stored for 150 and 200 days. Treatments included a control, CCB (100 and 200 mg/kg DM), and LIM (100 and 200 mg/kg DM). Essential oils improved fermentation by increasing lactic acid (up to 7.46% of DM) and reducing proteolysis (NH₃-N: 0.46–0.59% of total N). Limonene, particularly at 100 mg/kg DM, enhanced the lactic:acetic acid ratio (up to 3.07), better preserved non-fiber carbohydrates (≈32.7%), and increased in vitro DM digestibility (up to 81.5%) compared to the control (≈76.0%). The CCB treatment raised acetic acid concentrations (up to 3.04% of DM). Extending storage to 200 days reduced DM recovery (≈84.0%) versus 150 days (92.5%). Treated silages showed greater aerobic stability, with a lower pH increase after 72 h of air exposure, most notably in the LIM 200 treatment (pH 4.10) compared to the control (pH 5.40). Essential oils, particularly limonene, effectively improve the fermentative quality, nutritional value, and aerobic stability of corn silage under prolonged storage. Full article

The growing demand for functional foods reflects greater consumer awareness of diet–health links, with bioactive peptides receiving increasing attention for their health-promoting effects. In this study, bioactive peptides exhibiting antioxidant, dipeptidyl peptidase-IV (DPP-IV) inhibitory, and angiotensin-converting enzyme (ACE) inhibitory activities were produced from a jack bean (Canavalia ensiformis) protein isolate using a continuous proteolysis system with two enzymes. This study encompassed two major phases: isolating protein from jack beans and implementing a continuous enzymatic hydrolysis process. Key variables examined included the enzyme-to-substrate ratio ([E]/[S]), pH level, and residence time (τ). Optimal performance was achieved at [E]/[S] = 5%, pH = 7.5, and τ = 12 h, yielding a permeate with peptide content of 0.6143 mg SE/mL, along with notable antioxidant capacity and ACE inhibition of 0.0454 mg TEAC/mL and 92.18%, respectively. These results confirm that the jack bean protein isolate is a viable substrate for generating multifunctional bioactive peptides. This study provides a foundation for scalable and sustainable production of functional food ingredients from underutilized legumes using continuous bioprocessing technology. Industrial relevance: Integrating a stirred tank reactor with membrane separation provides a promising approach for continuous bioactive peptide production using a free-enzyme system, helping to streamline processing, reduces the demand for enzyme immobilization, and minimizes batch-to-batch variability. This study shows that continuous hydrolysis of jack bean protein isolate in EMR can enhance antioxidant activity and ACE inhibition of the hydrolysates. This approach offers a safer and more efficient route to support the commercialization of jack bean-based functional products. Full article

Chlorogenic acid (CGA), which is ubiquitous in diverse botanical sources, demonstrates considerable anticancer potential through modulation of multiple targets or signaling pathways, thereby posing substantial challenges for mechanistic elucidation and target identification. Based on the proteolysis targeting chimera (PROTAC) technology’s ability to induce targeted protein degradation via ubiquitin-proteasome pathway recruitment, we synthesized a panel of CGA-PROTACs. These compounds incorporated the natural product CGA as the target-binding ligand, conjugated to pomalidomide (an E3 ligase-recruiting moiety) via various synthetic linkers. The findings indicated that compound A7, linked with an alkane linker, exhibited a notable anti-proliferative effect on 4T1 and A375 cells in vitro. The IC₅₀ value of A7 on A375 cells reached 69.70 μM, which is 2.2 times better than the effect of the precursor compound CGA (IC₅₀ = 148.80 μM). Mouse double minute 4 (MDM4) was confirmed as a potential target of compound A7 through a combination of proteomics, Western blot analysis and molecular dynamics simulation. CGA-PROTAC A7 treatment led to a dose-dependent reduction in MDM4 protein levels while significantly upregulating p53 and p21 protein expression, and thus inhibited proliferation, induced G2/M phase cell cycle arrest, and markedly enhanced apoptosis in melanoma A375 cells. This study successfully applied an effective strategy for target identification and medication discovery of natural compounds. In addition, CGA-PROTAC A7 was synthesized in one step with an overall yield of 45.96%, providing a feasible route for synthesis and establishing a basis for the combination of natural product polyphenols with PROTAC technology. Full article

Soybean meal (SBM) is a foundational protein source, but its industrial application is constrained by a complex matrix of anti-nutritional factors (ANFs). This review provides a critical synthesis of the biochemical transition from raw SBM to fermented SBM (FSBM), focusing on the synergistic mechanisms of fungal and bacterial co-fermentation. We identify that the efficacy of FSBM is primarily driven by the microbial proteolysis of glycinin into low-molecular-weight bioactive peptides (<1000 Da). These peptides serve as the primary drivers for improved intestinal morphology (increased villus height) and the modulation of the gut microbiota, providing a mechanistic basis for reported probiotic effects. Furthermore, we establish that the 5–10% improvement in the feed conversion ratio (FCR) documented for swines mathematically offsets the processing premium of fermentation. However, critical gaps remain in the standardization of solid-state fermentation (SSF) protocols, specifically regarding the selection of fungal (Aspergillus) and bacterial (Bacillus or Lactobacillus) strains, whose distinct metabolic pathways significantly diversify the functional profile of the resulting FSBM. Full article

Pathological cardiac hypertrophy, a major contributor to heart failure, is characterized by an abnormal increase in the size of atria and ventricles. In the context of ventricular hypertrophy, the right ventricle (RV) exhibits less resistance to hypertrophy than the left one (LV). Insulin-like growth factors (IGF-1 and IGF-2) are critical for cell growth and provide cardioprotective effects. Pregnancy-associated plasma protein-A (PAPP-A) is a protease that cleaves insulin-like growth factor-binding protein-4 (IGFBP-4) and enhances IGF bioavailability. This study investigated PAPP-A-mediated IGFBP-4 proteolysis—one possible mechanism of IGF release regulation in rat models of right ventricular (RVH) and left ventricular (LVH) hypertrophy. RVH was induced with monocrotaline, and LVH via renovascular hypertension (1 Kidney 1 Clip (1K1C) model). Systolic blood pressure was measured using tail-cuff plethysmography. Heart morphometry was used to assess the mass of cardiac chambers. Cardiomyocyte purity was confirmed via troponin I immunocytochemistry. Plasma natriuretic type-B peptide (BNP) and C-terminal IGFBP-4 (CT-IGFBP-4) concentrations were quantified by fluoroimmunoassay. RVH and LVH were successfully modelled, with 1.6-fold and 1.3-fold increases in RV (p < 0.0001) and LV masses (p < 0.05), respectively. Plasma BNP was 2–3 times higher in LVH versus control rats. Hypertrophied cardiomyocytes secreted significantly more BNP than controls, showing 3.3-fold and 4.1-fold increases in LVH and RVH, respectively. PAPP-A-mediated IGFBP-4 proteolysis was 4-fold higher in RVH compared to control, but unaffected in LVH. These findings suggest that PAPP-A-specific elevation of IGFBP-4 proteolysis occurs predominantly in RVH, suggesting a differential IGF bioavailability in both ventricles and highlighting PAPP-A as a potential target to increase RVH resistance to hypertrophy. Full article

The rapid expansion of the poultry industry has led to the large-scale generation of feather waste, creating serious environmental and public health concerns due to the recalcitrant nature of keratin. Poultry feathers are composed mainly of highly cross-linked keratin proteins stabilized by numerous disulfide bonds, which confer resistance to conventional proteolytic enzymes and natural degradation processes. This review examines the potential of keratinolytic fungi and their enzymes as sustainable, eco-friendly, and value-added strategies for poultry feather waste management and resource recovery. It discusses the environmental and health risks associated with improper feather disposal, such as pathogen proliferation, odor generation, and ecosystem contamination. Conventional management approaches, steam pressure hydrolysis, mechanical grinding, thermal treatment, acid–alkali hydrolysis, and oxidation, are critically evaluated in terms of efficiency and environmental impact. The review further highlights biological degradation pathways mediated by keratinolytic fungi and enzymes, with emphasis on fungal genera such as Aspergillus and Chrysosporium. Key mechanisms of fungal keratin degradation, including sulfitolysis, proteolysis, deamination, hyphal penetration, enzyme secretion, and biofilm formation, are discussed. Finally, industrial, agricultural, and feed applications of keratinases, along with advances in strain improvement, omics technologies, synthetic biology, and associated biosafety and regulatory considerations, are addressed. Full article

The oncogenic transcription factor MYC is a key driver of the development and progression of various types of cancer, but its intrinsically disordered structure and dependence on protein–protein interactions make it a difficult therapeutic target. Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that can induce the selective degradation of disease-relevant proteins. In this study, we report the synthesis and biological testing of a series of novel MYC-targeted PROTACs derived from the MYC inhibitor EN4. These ligands were conjugated to cereblon (CRBN) or von Hippel–Lindau (VHL) E3 ligase recruiters using different linker architectures and connection sites. The resulting PROTACs were synthesized in high purity and characterized analytically. Cellular evaluation in HEK293T, Panc-1 and HCT-116 cancer cells revealed only moderate reductions in cell viability. Unfortunately, none of the synthesized PROTACs showed detectable MYC degradation at biologically relevant concentrations. Testing the stability of the PROTACs in microsomes showed rapid degradation, which may be a reason for the observed inactivity in cells. These results underscore the significant challenges associated with the targeted protein degradation of intrinsically disordered transcription factors such as MYC. Further studies are necessary to identify additional causes for the lack of MYC degradation and to optimize the chemical structures accordingly. Full article

Over the past two decades, numerous studies have examined the etiological significance of DNA fragmentation in human sperm using methods such as the comet assay (CA), the sperm chromatin structure assay, the sperm chromatin dispersion assay, and the TUNEL assay. We developed single-cell pulsed-field gel electrophoresis techniques, including one-dimensional (1D-SCPFGE) and angle-modulated two-dimensional (2D-SCPFGE), to detect early signs of naturally occurring DNA fragmentation. Comparative studies using purified human sperm with and without DNA fragmentation revealed some technical limitations in the conventional methods. This technical review outlines the procedures to ensure the quantitative performance of SCPFGE: (1) The mass of naked DNA was prepared through simultaneous in-gel swelling and proteolysis, which are highly sensitive to chemical and physical factors. Notably, these processes are vulnerable to reactive oxygen species (ROS). We developed the anti-ROS SCPFGE system to prevent artifactual cleavages. (2) 1D-SCPFGE discharges long-chain fibers from the origin, separating fibrous and granular segments beyond the tips of the fibers. (3) During continuous electrophoresis after 150° rotation (2D-SCPFGE-0-150), long-chain fibers unexpectedly extended diagonally backward from the origin, with long fibrous segments pulled out from a bundle that extended during the first electrophoresis, indicating some fibrous segments were embedded within the long-chain fibers. Even when SCPFGE was employed, one-directional current led to false negatives. (4) 2D-SCPFGE with angle rotation is currently the most sensitive imaging method for single-nuclear DNA fibers. However, without knowing the size of DNA fragments, it remains a semi-quantitative analysis. (5) To prevent artifactual DNA cleavage caused by ice crystals, low-temperature liquid storage is recommended. (6) The in-gel proteolyzed naked DNA is suitable as a substrate for chemical and enzymatic DNA cleavage analyses. Full article

Microbial fermentation is attracting attention as a key process in reconstructing the profile of functional components in foods. This review summarizes the main mechanisms by which microbial fermentation generates antioxidants and functional compounds in fermented dairy products. In particular, we focus on (i) the production of bioactive peptides driven by fermentation-induced proteolysis, (ii) modulation of reactive oxygen species (ROS) homeostasis associated with shifts in metabolite composition, and (iii) the remodeling of organic acids, fatty acids, and other low-molecular-weight metabolites. We also discussed an analytical framework for evaluating antioxidant function in various analytical methods such as the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and ferric reducing antioxidant power (FRAP) assay, as well as cell-based measurements of reactive oxygen species (ROS)/nitric oxide (NO) and determining oxidative damage in animal models. Overall, the antioxidant functionality of fermented dairy products should be understood not merely as an increase in radical-scavenging capacity but as the outcome of fermentation-driven molecular remodeling and physiological regulatory effects. This review defines fermented dairy products as functional foods, highlighting the need for an omics-based approach in future fermented food studies. Full article