Mannan-oligosaccharide (MOS) produced from the hydrolysis of mannan by β-mannanase, demonstrate several beneficial properties, such as prebiotic and modulation of tight junction activity. In this study, two novel GH26 β-mannanases, MAN26HM7/A and MAN26HM7/B, from Bacillus subtilis HM7 were cloned and expressed in Escherichia coli BL21 (DE3). The optimal pH and temperature of MAN26HM7/A and MAN26HM7/B were pH 5.0 and 40 °C, and pH 6.0 and 60 °C, respectively. The β-mannanases from Bacillus subtilis HM7 exhibited different MOS product patterns from DP2-DP6 oligo-mannose. MAN26HM7/A and MAN26HM7/B preferentially hydrolyzed substrates with more than five mannose residues. The amino acid residues, A146, F65, and H331 surrounding the active sites of MAN26HM7/A and MAN26HM7/B were compared with those of other β-mannanases from Bacillus species. These amino acid differences may be responsible for altered substrate binding, substrate specificity, and hydrolytic product pattern of MAN26HM7/A and MAN26HM7/B. MOS produced from konjac, copra meal and green coffee bean by MAN26HM7, MAN26HM7/A, and MAN26HM7/B were tested for prebiotic activity. MOS produced from coffee bean hydrolyzed by MAN26HM7/B exhibited the highest prebiotic activity score on Lacticaseibacillus rhamnosus GG (ATCC 53103). This study provides an understanding of the characteristics, hydrolysis pattern of the novel GH26 β-mannanases, and the application of these novel GH26 β-mannanase for prebiotic production from agricultural products.

The natural structural barriers and resistance of different biomass types are major obstacles to broadly applicable high-efficiency biorefining. This study employed a CTAB/LA/Cu[2+] ternary pretreatment mixture composed of cetyltrimethylammonium bromide (CTAB), lactic acid (LA), and copper chloride (CuCl2, as the Cu[2+] source) at a molar ratio of 1:4:0.012 to pretreat five lignocellulosic feedstocks: agricultural residues (corn stover, CS; sorghum stover, SS), processing by-products (soybean hulls, SH), and forestry hardwoods (maple wood, MW; poplar debris, PD). Under the selected common pretreatment condition of 140 °C for 60 min, the ternary mixture exhibited feedstock-dependent fractionation, with the highest xylan removal of 64.8% observed for MW and the highest lignin removal of 74.2% observed for CS, while increasing the subsequent enzymatic hydrolysis efficiency by 3.0-7.4-fold across the five feedstocks. In addition, total oligomeric xylose and furfural were detected in the crude pretreatment liquors, with maximum concentrations of 5.6 g/L XOSs and 2.8 g/L furfural were detected in the crude pretreatment liquors. These measurements indicate the formation of hemicellulose-derived soluble products but do not represent isolated product yields or purities. Multi-scale characterization using SEM, XRD, and FT-IR clearly indicated the evolution from surface delamination to internal structural collapse of the substrates. Furthermore, based on the hemicellulose models of the five biomass types, qualitative non-covalent interaction patterns between representative hemicellulose oligomers and the pretreatment-mixture components were examined using molecular modeling and IGMH visualization. This study provides a key theoretical paradigm and potentially foundational technical demonstration for the integrated biorefining of multi-source biomass.

Cutaneous squamous cell carcinoma (cSCC) is a prevalent skin malignancy characterized by aggressive biological behaviors. Lipocalin 2 (LCN2) has been implicated in the progression of various cancers; however, the specific mechanisms by which LCN2 modulates malignant phenotypes of cSCC remain insufficiently understood. The expression levels of LCN2, ubiquitin specific peptidase 48 (USP48), and methyltransferase like 1 (METTL1) were analyzed in cSCC tissues, paracancerous tissues, and/or cell lines using western blotting and quantitative real-time PCR. Functional assays, including cell counting kit-8 (CCK-8), flow cytometry, wound healing, Transwell invasion, and colorimetric assays, were conducted to evaluate cell proliferation, apoptosis, migration, invasion, and ferroptosis indicators. The regulatory mechanisms were investigated via methylated RNA immunoprecipitation (MeRIP), dual-luciferase reporter, co-immunoprecipitation (Co-IP), and ubiquitination assays. Furthermore, in vivo tumorigenicity was assessed using a xenograft mouse model. LCN2 was significantly upregulated in cSCC tissues and cell lines. LCN2 expression was associated with clinical stage of cSCC patients. Knockdown of LCN2 suppressed cell proliferation, migration, and invasion, while promoting apoptosis and ferroptosis. Mechanistically, USP48 was found to interact with LCN2 and enhance its stability by removing K48-linked ubiquitin chains. Functionally, USP48 silencing inhibited the malignant behaviors of cSCC cells, whereas these effects were rescued by LCN2 overexpression. Upstream analysis revealed that METTL1 stabilized USP48 mRNA expression via m7G methylation modification. Knockdown of METTL1 inhibited the key malignant phenotypes of cSCC cells, however, these effects were effectively reversed by USP48 overexpression. Consistent with in vitro findings, USP48 silencing reduced tumor growth and LCN2 and Ki67 expression in vivo, but these inhibitory effects were attenuated by the restoration of LCN2. This study elucidated a novel oncogenic axis in cSCC, wherein METTL1 stabilized USP48 mRNA through m7G methylation modification, and USP48 in turn deubiquitinated and stabilized LCN2 protein. This axis promoted cSCC progression. Clinically, these findings identify the METTL1/USP48/LCN2 pathway as a potential therapeutic target for cSCC.

Hypoxia is a major determinant of tumor progression, yet most chemical hypoxia models rely on HIF stabilization rather than true oxygen depletion. Here, we evaluated sodium sulfite (SS), a direct oxygen scavenger, as an oxygen-dependent in vitro hypoxia model. Cancer cell lines were treated with optimized SS concentrations, and dissolved oxygen levels were monitored for up to 96 h. Cell viability, cell death, cell cycle distribution, morphological alterations, and hypoxia-associated metabolic and redox responses were assessed and compared with conventional hypoxia mimetics. SS reduced dissolved oxygen levels below 1 mg/L in a dose- and time-dependent manner. Based on their oxygen-consumption profiles under SS exposure, cell lines were operationally grouped into sustained and limited oxygen-consuming phenotypes. SS induced moderate cytotoxicity without reducing viability below 50%, caused S-phase accumulation in the sustained oxygen-consuming group and G0/G1 accumulation in the limited oxygen-consuming group, and altered cell morphology. Importantly, SS promoted HIF-1α and HIF-2α nuclear translocation, increased VEGF-A, LDHA, GLUT1, and HK-2 expression, decreased extracellular pH, suppressed eNOS, and enhanced antioxidant capacity more effectively than CoCl2 or DFO. Together, these findings indicate that SS induces direct oxygen depletion and captures selected signaling, metabolic, and redox-related responses associated with hypoxia. SS therefore represents a simple and accessible oxygen-depletion-based chemical model that may complement conventional hypoxia mimetics for studying selected features of hypoxic adaptation in vitro, although further studies are needed to distinguish oxygen depletion-dependent effects from sulfite-specific contributions.

Reynoutrin, a bioactive flavonoid, exhibits various biological effects. Despite its toxic side effects, cisplatin is widely used to treat malignancies. This study aimed to assess the in vitro antiproliferative activity of reynoutrin in MCF-7 and PC-3 cell lines by evaluating its effects on inflammation, oxidative stress, and apoptosis-related pathways. The cytotoxic effects of reynoutrin on MCF-7 and PC-3 cells were evaluated using an MTT assay after 24 h. Total antioxidant status (TAS) and total oxidant status (TOS) were measured using ELISA kits. The mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, caspase-3, caspase-9, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were analyzed by RT-qPCR. Apoptotic morphological changes were assessed by Hoechst staining. Reynoutrin inhibited MCF-7 and PC-3 cell viability in a dose-dependent manner, with IC50 values of 220 and 412 µg/mL, respectively. Reynoutrin increased TOS and decreased TAS (p < 0.05), indicating an increase in oxidative stress. RT-qPCR showed that TNF-α, IL-1β, and IL-6 mRNA expression levels decreased in a dose-dependent manner following reynoutrin treatment in MCF-7 and PC-3 cells (p < 0.05), suggesting anti-inflammatory effects. In both cancer cell lines, reynoutrin increased caspase-3 and caspase-9 mRNA expression levels and decreased the Bcl-2/Bax expression ratio in a dose-dependent manner (p < 0.05). These findings suggest that reynoutrin exerts antiproliferative effects that are associated with modulation of inflammatory, oxidative stress, and apoptosis-related pathways in cancer cells, highlighting its potential as a natural candidate for further investigation as an anticancer agent.

The present study aimed to synthesize Ag2CO3 and Ag/Ag2CO3 nanostructures and to systematically investigate their physicochemical characteristics, biocompatibility, metabolic effects, wound healing potential, antioxidant activity, and antibacterial efficacy. Both nanostructures were synthesized using a controlled chemical reduction method. X-ray diffraction analysis confirmed the crystalline Ag2CO3 phase and the successful incorporation of metallic silver in the Ag/Ag2CO3 composite, while zeta potential analysis revealed enhanced colloidal stability for Ag/Ag2CO3 (approximately - 38 mV) compared with Ag2CO3 (approximately - 28 mV). Biocompatibility and safety evaluation in non-diabetic mice, including complete blood count, liver and renal function tests, and lipid profiling, demonstrated that both nanostructures were well tolerated at the administered doses without evidence of severe toxicity. In alloxan-induced diabetic mice, treatment with Ag2CO3 and Ag/Ag2CO3 resulted in significant glucose-lowering effects from Day 7 onward. Serum insulin, triiodothyronine (T3), and thyroxine (T4) levels were significantly improved at Days 14 and 28 compared with diabetic controls. Antioxidant assessment after 28 days showed reduced malondialdehyde levels and enhanced activities of superoxide dismutase, catalase, and reduced glutathione. In a diabetic excision wound model with an initial wound area of 1.2 cm[2], complete wound healing occurred by Day 19 in diabetic controls, Day 17 in Ag2CO3-treated mice, and Day 14 in Ag/Ag2CO3-treated mice. Additionally, both nanostructures exhibited concentration-dependent antibacterial activity against Staphylococcus aureus and Escherichia coli, with Ag/Ag2CO3 producing inhibition zones of up to approximately 17 mm at 100 µg/mL. Overall, Ag/Ag2CO3 nanostructures demonstrated superior multifunctional biomedical performance.

The global swine industry suffers substantial economic losses from Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which causes reproductive and respiratory disorders. With NADC30- and NADC34-like strains now predominant, differentiation is complicated by their similar biology, despite characteristic Nsp2 gene deletions of 131 aa and 100 aa, respectively, highlighting a need for improved diagnostics. Moving fundamentally beyond conventional broad-spectrum detection, this assay innovatively targets the characteristic Nsp2 deletion signatures. A diagnostic platform integrating CRISPR-Cas13a with recombinase-aided amplification (RT-RAA) was constructed. Utilising aligned Nsp2 sequences, conserved crRNAs, and RT-RAA primers, NADC30- and NADC34-like strains were evaluated for their assay time, specificity, sensitivity, and consistency with RT-qPCR. The assay achieved rapid, specific detection with no cross-reactivity to common swine pathogens, and highly sensitive detection limits of 1.7 × 10[1] and 2.3 × 10[1] copies/µL for NADC30- and NADC34-like strains, respectively. Clinical validation using 30 samples demonstrated complete concordance with RT-qPCR, and the integration of T7 transcription enabled the single-tube differentiation of both strains. In summary, this one-tube, two-step fluorescent system presents a robust alternative for PRRSV diagnostics, combining specificity, sensitivity, and ease of use. It is well-suited for rapid field identification, molecular typing, and surveillance, thereby aiding in the implementation of targeted control measures.

As a nuclear transport receptor, KPNB1 transport proteins from cytoplasm into nucleus, performing a vital function in intracellular traffic. The aberrant upregulation of KPNB1 has been associated with various malignancies and transformed cells, where it is indispensable for cellular proliferation and survival. Transcription factors like E2F1 possess a nuclear localization motif that triggers the expression of a range of crucial factors during tumorigenesis. This investigation explores the influence of KPNB1 in cervical cancer (CC) cell metastasis and proliferation through the modulation of E2F1 nuclear import. Overexpression of KPNB1 was consistently detected in cervical carcinoma tissues and associated cell lines, with notably higher levels observed relative to normal cervical mucosa. Functional studies demonstrated that KPNB1 silencing induced a marked attenuation in the invasive, migratory, and proliferative capacities of HeLa and SiHa cells. Furthermore, inhibition of KPNB1 hindered the nuclear translocation of E2F1. Importantly, the application of ARQ171, a small-molecule compound that functionally elevates E2F1 expression, negated partially the suppressive effects of KPNB1 on migration, invasion, and growth of CC cells via elevating the total amount of nuclear E2F1, suggesting that E2F1 mediates the oncogenic role of KPNB1 in CC progression. The research findings highlight the potential utility of targeting KPNB1-E2F1 axis as an innovative treatment modality, underscoring its promise as a novel approach in cervical cancer treatment.

Brown seaweeds are the primary industrial source of alginates and are increasingly valued as marine-based feedstocks in integrated biorefineries. However, the substantial solid residue generated in the alginate extraction industry remains underexplored as a secondary source of carbon and energy. This study investigated the potential of the alginate-extracted residue (AER) from Sargassum siliquosum biomass collected from the southwestern coast of Taiwan as a substrate for bioethanol production. The alkaline extraction method yielded 39.00% alginate and 33.80% residue on a dry weight basis. Comprehensive characterization of the AER revealed that the solid fraction is enriched in carbohydrates (33.00%), especially the cellulose fraction, with a monosaccharide composition of glucose, fucose, and xylose in addition to guluronic acid. Elemental analysis revealed that the residue is enriched in carbon (35.29%) and shows notable reductions in ash, sulfur, and phenolic compounds. Fourier-transform infrared spectroscopy (FT-IR) analysis confirmed the selective removal of mannuronic and guluronic acid residues in AER compared to biomass, while cellulose-associated functional groups have been preserved. Enzymatic hydrolysis with commercial fungal cellulase released 2.90 g/L glucose from biomass and 5.10 g/L glucose from AER, respectively. Fermentation of the hydrolysates with Saccharomyces cerevisiae BCRC 22458 yielded ethanol titers of 0.89 and 1.20 g/L from biomass and AER hydrolysate, respectively. Concentration of the hydrolysates to increase the sugar concentration increased the ethanol titer to 2.47 g/L (yield 0.098 g/g biomass; productivity 0.33 g/L/h) for AER and 1.18 g/L for biomass. These results demonstrated that S. siliquosum AER is a promising secondary feedstock for bioethanol fermentation within a cascading biorefinery approach for brown seaweeds.

Colorectal cancer (CRC), particularly the microsatellite-stable (MSS) subtype accounting for ~ 85% of cases, remains refractory to immune checkpoint inhibitors due to persistent immunosuppression. Shaoyao Decoction (SYD), a traditional Chinese herbal formula, is known for its anti-inflammatory and anticancer effects. However, the mechanisms by which it modulates immune responses in colorectal cancer (CRC) remain poorly understood. Here, we evaluated SYD's efficacy in AOM/DSS-induced colitis-associated CRC and PD-L1-overexpressing MC38 syngeneic models, using the PD-1/PD-L1 inhibitor BMS-202 as a positive control. In vivo, SYD significantly suppressed tumor growth, normalized colon structure, and reduced tumor burden, with efficacy comparable to BMS-202 but without overt toxicity. Mechanistically, SYD downregulated PD-L1 expression in tumor and spleen tissues, shifted serum cytokines toward a pro-inflammatory profile characterized by increased IL-2 and IFN-γ as well as decreased IL-10 and TGF-β, and restored CD4[+]/CD8[+] T cell balance. In vitro, SYD inhibited the viability of PD-L1-overexpressing MC38 cells and reduced PD-L1 mRNA expression. Collectively, SYD exerts potent anti-CRC effects by targeting PD-L1 to remodel the immunosuppressive tumor microenvironment. As a multi-target agent, it holds great promise for MSS-CRC, bridging TCM with modern immuno-oncology.

Ergothioneine (EGT) is a natural sulfur-containing amino acid possessing potent antioxidant activity. It demonstrates a range of beneficial biological functions, including anti-inflammatory, neuroprotective, and cardioprotective effects. Microbial fermentation presents a promising alternative to traditional extraction from edible fungi for EGT production, offering greater scalability, though its feasibility hinges critically on production titer and cost. In this study, to achieve food-grade EGT synthesis, the probiotic Escherichia coli Nissle 1917 was employed as the host, and the heterologous enzymes methyltransferase EgtD and C-S lyase EgtE from Mycobacterium, as well as the bifunctional enzyme Egt1 from Neurospora crassa, were introduced. As a result, shake flask fermentations with the designed strain E1 achieved an EGT titer of 109.2 mg/L. Subsequently, overexpression of the EgtE mutant EgtE[Y106F] along with the incorporation of the protein fusion tag NT11 at the N-terminal via a dual-plasmid system increased the yield to 159.9 mg/L. Furtherly, the cellular metabolic homeostasis regulator RpoS was introduced and placed under the control of an arabinose-inducible promoter, thus achieving an EGT titer of 429.2 mg/L for the engineered strain E14 within 24 h of shake flask fermentation. Finally, fed-batch fermentation conducted in a 5-L bioreactor resulted in an EGT titer of 12.3 g/L at 120 h, representing the highest level reported to date. This study developed a scalable and cost-effective platform for food-grade EGT production, paving the way for its industrial application.

Antimicrobial peptides (AMPs) are a diverse class of bioactive molecules that exert their effects through multiple mechanisms, including membrane disruption, inhibition of cell wall synthesis, interference with intracellular targets, and binding to nucleic acids or proteins. They exhibit broad-spectrum antimicrobial activity and a low propensity to induce drug resistance, positioning them as promising candidates for combating multidrug-resistant infections. However, their clinical translation faces significant challenges, including protease susceptibility, potential cytotoxicity, high production costs, and suboptimal pharmacokinetic properties. In this study, a strain of Bacillus licheniformis SAM-D318 isolated from Hu sheep intestine was used as the peptide-producing strain; genomic analysis confirmed the absence of classical virulence toxin genes despite intrinsic multidrug resistance. A previously unreported cationic α-helical antimicrobial peptide, SAM-KB40 (4176.92 Da, pI 10.886), was obtained from the fermentation supernatant of B. licheniformis SAM-D318 and purified using cation-exchange chromatography and C18 reversed-phase chromatography, achieving a 70.45-fold increase in specific activity. Structural prediction by PEP-FOLD4 suggested an α-helical propensity in the C-terminal region, while circular dichroism spectroscopy indicated that SAM-KB40 adopts a predominantly random coil conformation in aqueous solution. The peptide exhibited potent antibacterial activity against Clostridium perfringens (MIC = 0.5 µg/mL, MBC = 4 µg/mL) and showed selective inhibition against Staphylococcus aureus and Listeria monocytogenes. SAM-KB40 demonstrated exceptional thermal stability (> 97% activity retained at 100 °C for 60 min), good tolerance to acidic conditions (pH 3-4) and bile salts (up to 0.50%), and remarkably low hemolytic activity (1.96% at 1000 µg/mL). Notably, the peptide neutralized recombinant C. perfringens α-toxin at 16 µg/mL in a Vero cell-based assay and exhibited no cytotoxicity at concentrations up to 128 µg/mL, demonstrating a favorable in vitro safety profile.Sequence homology analysis against UniProt, NCBI, APD, and BACTIBASE databases confirmed that SAM-KB40 has not been previously reported. While the peptide shows promising in vitro dual antibacterial and toxin-neutralizing capacity, its high sensitivity to proteinase K (39.78% residual activity) limits oral application. These findings position SAM-KB40 as a promising lead candidate for further development as a feed additive or therapeutic agent against C. perfringens-associated diseases, pending subsequent in vivo validation.

This study aimed to evaluate the effects of antioxidant dipeptides (AH, TY, and FC) on yeast stress tolerance, beer physicochemical properties, and sensory quality during high-gravity brewing. The results demonstrated that antioxidant dipeptides maintained yeast cell morphology and alleviated cell shrinkage. Additionally, the dipeptides decreased intracellular reactive oxygen species (ROS) levels, with AH and FC exhibiting the most significant reductions of 9.69% and 11.01%, respectively, compared to the control. Moreover, antioxidant dipeptides significantly increased the ethanol content and wort fermentability of the beer. Beer aroma analysis revealed that dipeptide supplementation enhanced both the content and diversity of ester compounds without adversely affecting beer flavor. Therefore, antioxidant dipeptides represent a promising additive for enhancing the stress tolerance and fermentation performance of lager yeast during high-gravity brewing.

Bladder carcinoma (BLCA) is a prevalent urological malignancy with limited therapeutic options. Interferon α inducible protein 6 (IFI6) is implicated in various cancers, but its role in BLCA remains unexplored. qRT-PCR and western blot were used for expression analysis. Functional assays (cell counting kit 8, 5-ethynyl-2'-deoxyuridine staining, flow cytometry, wound healing, transwell, and 2', 7'-dichlorodihydrofluorescein diacetate fluorescent probe) and xenograft mouse models were used to assess the effects of IFI6 and RNA binding motif protein 15 (RBM15) on BLCA. The N6-methyladenosine (m6A) modification sites on IFI6 were predicted by the sequence-based RNA adenosine methylation site predictor website and the combination of RBM15 was validated via RNA-binding proteins site prediction websites, methylated RNA immunoprecipitation, Actinomycin D, dual-luciferase reporter assay, and RNA binding protein immunoprecipitation assays. IFI6 was elevated in BLCA tissues and cells. IFI6 knockdown could inhibit proliferation, migration, and invasion, while enhancing apoptosis and oxidative stress of BLCA cells. Besides, silencing of IFI6 blocked BLCA tumor growth. RBM15, an m6A writer, directly bound IFI6 to stabilize its mRNA through m6A modification. Rescue experiments confirmed that IFI6 overexpression reversed RBM15 knockdown-mediated the suppression effect on BLCA cell proliferation and metastasis. Our study identifies that RBM15/IFI6 axis is a novel m6A-dependent pathway driving BLCA progression, offering a potential therapeutic target for BLCA.