Search Results (624)

Mitochondrial electron transport chain (ETC) dysfunction is a major driver of bioenergetic failure, redox imbalance, and drug toxicity, yet strategies to restore oxidative phosphorylation under ETC blockade remain limited. Redox-active small molecules could, in principle, shuttle electrons from NADH to distal ETC components and oxygen, thereby modulating both respiration and reactive oxygen species (ROS) formation. Here, we show that the enzyme-independent redox cycler phenazine methosulfate (PMS) rewires mitochondrial redox circuits and restores respiration in human glioblastoma cells and cell-free systems under ETC inhibition. At subtoxic concentrations, PMS acutely increased oxygen consumption and mitochondrial superoxide generation via NADH–PMS–O₂ redox cycling, while restoring mitochondrial membrane potential and ATP synthesis under ETC blockade, and shifting metabolism away from glycolytic lactate production. This profile is consistent with a protective redox-bypass role, distinct from the pro-apoptotic effects reported following high-dose, prolonged PMS exposure. The PMS-driven restoration of electron flow, mitochondrial membrane potential, and respiratory ATP synthesis under inhibition of Complex I (rotenone), III (antimycin A and myxothiazol), and/or IV (cyanide) is consistent with direct cytochrome c reduction, as demonstrated herein, and engagement of multiple ETC redox centers, including coenzyme Q₁₀. In metformin-treated cells, PMS reversed suppression of respiration and lactate accumulation, outperforming existing redox-bypass strategies. These findings identify PMS-driven redox cycling as a previously unrecognized chemical redox-bypass mechanism that both regenerates mitochondrial bioenergetics and reshapes ROS production, suggesting a potential approach to counteract drug- and toxin-induced mitochondrial dysfunction and to exploit redox vulnerabilities in cancer. Full article

Staphylococcus aureus is a major cause of foodborne intoxication through the production of heat-stable enterotoxins (SEs) and is also an important opportunistic pathogen of humans and livestock. Meat and meat products are major vehicles for this pathogen because their protein-rich composition supports bacterial growth and toxin production. However, the combined effects of temperature and nutrient composition on S. aureus growth and virulence expression under food-relevant conditions remain unclear. In this study, we investigated the interactive effects of temperature and nutritional context on the growth and virulence-associated phenotypes under model food-relevant environments with the reference strain S. aureus FRI-S6. Bacterial growth, biofilm formation, staphylococcal enterotoxins A and B (SEA, SEB), and hemolytic activity were evaluated at 25 °C and 37 °C in brain heart infusion (BHI) medium supplemented with NaCl, glucose, or tryptone to simulate diverse food-relevant conditions. Growth was generally faster at 37 °C, whereas glucose-supplemented cultures at 25 °C reached higher cell densities during prolonged incubation. Biofilm formation increased at 37 °C in BHI and glucose conditions. SEA production was enhanced at 37 °C under NaCl and tryptone, but at 25 °C in glucose-rich conditions. In contrast, SEB production and hemolytic activity were consistently higher at 37 °C, particularly in the presence of tryptone and glucose. These findings demonstrate the strong interaction between temperature and nutrient composition in shaping S. aureus virulence in food environments and provide important insights for food safety risk assessment and highlight practical implications for controlling enterotoxin production in meat products and other foods during storage and processing. Full article

The neurotoxin botulinum toxin type A (BoNT/A), produced by the bacteria Clostridium botulinum, is commonly injected intramuscularly (IM) for the management of chronic muscle hyperactivity, such as post-stroke spasticity. New indications such as peripheral neuropathic pain require alternative routes of BoNT/A administration, such as subcutaneous (SC) or intradermal (ID). While IM BoNT/A injections may elicit anti-drug-antibodies (ADAs), their occurrence following SC or ID administration is unknown. Therefore, we investigated whether repeated SC or ID injections of 150 kDa BoNT/A can elicit ADAs in a dose-dependent manner, and whether these differ depending upon the route of administration. Mice were injected 5 times ID or SC with 150 kDa BoNT/A or, for higher doses, inactive mutant BoNT/A (DRBoNT/A) or inactivated toxoid (IA-BoNT/A). Total ADAs were analyzed by an immunoassay and the subgroup of neutralizing ADAs by an in vivo digit abduction score (DAS) assay following a challenge of 0.6 U BoNT/A IM. DRBoNT/A and IA-BoNT/A injections elicit ADAs (22.7 U/mL vs. 460.5 U/mL for ID; 4.7 U/mL vs. 339.4 U/mL for SC), while therapeutic doses of 150 kDa BoNT/A do not. Whereas mice with repeated 150 kDa BoNT/A injections at therapeutic dose show an unrestricted DAS of 3.7 (ID) or 3.4 (SC), mice injected repeatedly with 1.8 µg/kg DRBoNT/A or 500 µg/kg IA-BoNT/A show only a minimal DAS of ≤0.7, indicating a high titer of neutralizing ADAs. No differences were observed between administration routes. Accordingly, repeated ID or SC injections of pure 150 kDa BoNT/A at therapeutic doses fail to induce ADA formation in mice. On the other hand, DRBoNT/A ID injections induce higher ADA concentrations than SC, but generate similar amounts of neutralizing ADAs. IA-BoNT/A injections induce ADAs and neutralizing ADAs similarly after ID and SC injections. ADA development at intermediate BoNT/A doses can be higher after ID injection, but does not lead to differences in neutralizing ADAs. Our data demonstrate that the antibody response to botulinum toxin depends predominantly on the protein load, and less on the administration route. Full article

Chitosan-based interpenetrating networks (IPNs) have become highly attractive as advanced super-adsorbent materials due to their ability to combine a high density of functional adsorption sites with enhanced structural stability under physiological conditions. While chitosan offers intrinsic advantages such as biocompatibility, biodegradability, and chemical functionality, its adsorption efficiency, mechanical strength, and long-term stability may offer limited performance in complex biomedical environments. The formation of interpenetrating networks provides an effective strategy to overcome these limitations by interlacing chitosan with other polymer networks, resulting in a synergistic enhancement of physicochemical and adsorption properties. The formation of chitosan-based IPNs offers tunable control of network structure, porosity, swelling behaviour, and adsorption kinetics, which in turn results in enhanced retention and controlled interaction of drugs, biomolecules, toxins, and other therapeutic agents. Variations in polymer composition, crosslinking density, and network interactions further facilitate the controlled tailoring of adsorption properties for targeted biomedical applications. This review presents a comprehensive and critical assessment of recent progress in the fabrication, functionalization, and structure–property relationships of chitosan-based IPNs, with a main emphasis on their super-adsorbent behaviour. Furthermore, this review highlights key biomedical applications of IPNs, including controlled drug delivery, wound healing systems, tissue engineering scaffolds, detoxification platforms, and biosensing devices. Current issues in scalability, stability, and clinical translation are discussed, as well as future perspectives that highlight the potential of chitosan-based IPNs as high-performance, sustainable super-adsorbent materials for advanced biomedical technologies. Full article

Antibiotic resistance is a growing global health threat. However, its evolution cannot be fully understood without considering antibiotic tolerance and persistence. Persister cells are phenotypic variants that survive lethal antibiotic exposure without heritable resistance, primarily through growth arrest, metabolic slowdown, and stress-adaptive states. Although persistence has been viewed as a transient survival phenomenon, increasing evidence suggests that it may also have a genetic basis by preserving populations during antibiotic-induced bottlenecks and enabling regrowth, mutation, and selection under certain conditions. This review examines the molecular mechanisms underlying persister formation, including toxin–antitoxin systems, stringent-response signaling, ATP depletion, translational arrest, and stress-response networks. We discuss how persistence contributes to antibiotic tolerance in biofilms, host environments, and recurrent infections, and how repeated antibiotic exposure may promote stepwise evolution from phenotypic survival to stable resistance in specific contexts. Evidence from experimental evolution, clinical observations, and system-level analyses supports a potential but context-dependent link between persistence and resistance. We also highlight therapeutic strategies targeting persister cells, including antipersister compounds, metabolic activation, combination therapies, bacteriophages, and alternative approaches. Finally, we outline future research directions, emphasizing single-cell technologies, systems biology, longitudinal clinical studies, and evolution-informed treatment design. A comprehensive understanding of persistence and its evolutionary implications is essential for improving treatment efficacy and limiting the emergence of long-term antibiotic resistance. Full article

The appressorium is a specialized infection structure formed by Metarhizium anisopliae during host invasion. To investigate the correlation between appressorium formation and fungal pathogenicity, as well as its impact on insect cuticular metabolism, different concentrations of sulforaphane were used to inhibit the appressorium formation rate of M. anisopliae. The relationship between appressorium formation rate and pathogenicity against Opisina arenosella larvae was evaluated, and LC-MS-based metabolomics was employed to characterize changes in cuticular compounds during the appressorium formation stage, thereby elucidating the chemical responses of the insect cuticle to appressorium formation. The appressorium formation rate of M. anisopliae was significantly and positively correlated with its pathogenicity (p ≤ 0.05). As the appressorium formation rate increased, pathogenicity against O. arenosella larvae increased and the killing speed accelerated. LC-MS metabolomics revealed that after appressorium formation, 102 differential cuticular compounds unique to O. arenosella larvae were identified, mainly including benzenes and substituted derivatives, amino acids and derivatives, and heterocyclic compounds. In addition, metabolic pathways associated with immune defense (tyrosine metabolism), antifungal defense (histidine metabolism), and toxin degradation (flavonoid degradation) in the larval cuticle were activated. These results demonstrate that the appressorium plays an important role in host infection by M. anisopliae, markedly alters cuticular metabolism, and activates defense- and detoxification-related metabolic pathways in the host. This study provides a theoretical basis for further investigations into fungus–insect cuticle interactions. Full article

Eryptosis is a programmed cellular death that leads to the removal of defective red blood cells (RBCs). It is driven by convergent intracellular pathways centered on cytosolic Ca²⁺ overload, ceramide formation, caspase and calpain activation, disruption of membrane phospholipid asymmetry, and the externalization of phosphatidylserine on the cell surface, which marks the cell for clearance by macrophages. In hemodialysis (HD), intermittent extracorporeal circulation exposes erythrocytes to mechanical stress, bio-incompatible membrane surfaces, and rapid osmotic and ionic shifts. Experimental evidence indicates that osmotic shock induces eryptosis through synergistic Ca²⁺ influx and sphingomyelinase-dependent ceramide generation, providing a mechanistic framework for intradialytic erythrocyte injury. Clinical studies report heterogeneous eryptotic responses during HD, reflecting the balance between toxin removal and procedure-related stress. In contrast, peritoneal dialysis (PD) imposes sustained exposure to hyperosmolar, glucose-based solutions and is strongly influenced by inflammation and residual kidney function. Clinical and experimental data consistently demonstrate increased eryptosis in PD patients, with marked amplification during peritonitis and close associations with inflammatory mediators. This review integrates mechanistic and clinical evidence on eryptosis in HD and PD, highlights modality-specific triggers converging on shared downstream pathways and discusses translational implications and research priorities for improving dialysis biocompatibility and anemia management. Full article

Globally, the rise in MDR P. aeruginosa infections poses a serious threat to public health, as these strains frequently exhibit extensive resistance to conventional antibiotics, prompting interest in bacteriophages as alternative treatments. In this study, we isolated and characterized a lytic P. aeruginosa phage, vB_Pae_YuaWU01, from hospital sewage in southern Thailand. Morphological analysis revealed Siphovirus-like characteristics. The phage demonstrated efficient host adsorption, with approximately 85.9% of particles attached within 15 min, and exhibited a latent period of 50 min with a burst size of 17.2 PFU/cell. It showed strong lytic activity, consistently suppressing bacterial growth without no regrowth observed over 72 h. Notably, the phage significantly inhibited biofilm formation by up to 59.9% and reduced pre-established biofilms by 39.78% at the highest tested concentration (10⁹ PFU/mL). Genome analysis revealed a 61,824 bp double-stranded DNA genome with 64.48% GC content and 88 predicted genes. Bioinformatic analysis suggests that the genome is organized into structural, replication, and lysis modules. Importantly, no toxin, antimicrobial resistance, lysogeny, or tRNA genes were identified, suggesting a favorable safety profile. The phage was classified within the genus Yuavirus, showing 97.4% genomic similarity to Sphaerotilus phage SN1, which infects a different host strain. The findings highlight its potential as a genetically safe therapeutic agent; however, its limited host range indicates that it may be best positioned as a strategic component of phage cocktails or as a synergistic partner with antibiotics to maximize therapeutic efficacy. Full article

Bacterial persistence has been extensively studied as a possible explanation for strain survival under stress; however, in Enterococcus spp., this ability is still an understudied phenomenon. In this study, 40 Enterococcus spp. isolates of human clinical (n = 10), veterinary commensal (n = 10), veterinary clinical (n = 10) and veterinary environmental (hospital surfaces) (n = 10) origins, were exposed to a high concentration of ciprofloxacin. Time–kill curves were established, after which antimicrobial susceptibility profiles were reassessed. Subsequently, the only presumptive persister was selected for Whole-Genome Sequencing, together with one isolate showing no evidence of persister formation. Comparative genomic analyses were conducted to identify genetic variations between exposed and non-exposed isolates and to explore potential genetic determinants associated with persistence. Observed genetic features present in the persister isolate included toxin–antitoxin systems, a cold-shock protein and the tyrosine-type recombinase/integrase XerC, which may represent putative candidates for further investigation. Interestingly, the majority of toxin–antitoxin system-associated genes were found in plasmids. This study represents an important step towards a better understanding of persistence development in Enterococcus spp.; however, validation using other methodologies such as RNA-sequencing is an important next step. Full article

Bacterial β-barrel pore-forming toxins, including Staphylococcus aureus α-toxin (Hla) and Bacillus cereus toxins hemolysin II (HlyII) and cytolytic toxin K2 (CytK-2), are secreted by bacterial cells as water-soluble monomers. These monomers assemble within lipid bilayers to form cylindrical pores, leading to lysis of target eukaryotic cells. We created mutant forms of these toxins that, based on the results of X-ray structural analysis of Hla and the prediction of the 3D structure of HlyII and CytK2, can form intramolecular disulfide bonds in monomers. The substitutions were made in the region responsible for toxin insertion into the target membrane. The mutant forms reversibly altered their hemolytic activity depending on the presence of reducing reagents and were non-toxic when injected into experimental animals. The immune response to injection of the mutant forms of Hla and CytK-2 toxins resulted in higher antibody titers against the wild-type toxins and a higher level of immunological memory than with injection of the HlyII mutant. The mutant form of CytK-2 demonstrates the properties of a prototype vaccine, as immunization with this protein protects animals against the effects of the wild-type toxin. Full article

Toxin A and B from Clostridioides difficile are the main pathogenicity factors for clinical symptoms of C. difficile infections. Receptor-mediated endocytosis and endosomal escape are required for targeting substrate proteins of the Rho-GTPase family. We previously reported that Toxin B (TcdB) affects endo-lysosomal transport and autophagic flux of target cells. These effects are independent from pathogenic Rho inhibition. Here, we aimed at further characterization of this event by immunofluorescent characterization of the vesicular structures that are affected. We found large aggregates of damaged endolysosomal structures positive for EEA1, LAMP1, CHMP4B and TcdB, as well as an increase in perinuclear concentration of non-mature autophagosomes (amphisomes) positive for SQSTM, Rab7, and LC3B. We investigated whether Rab7, a regulator of late endosome transport, is causative for decreased lysosome function. Although TcdB induced an increase in active Rab7, as tested by an RILP pull-down assay, inhibition of Rab7 did not prevent TcdB-induced decrease in cathepsin D as a surrogate for lysosome dysfunction. It also indicates that the observed increase in Rab7 positive amphisomes is secondary to lysosomal dysfunction. By applying an autoproteolytic deficient mutant of TcdB we proved that the release of the glucosyltransferase domain is mandatory for triggering all of these effects. This suggests that after membrane perforation the toxin remnants leave an open leak in endolysosomes affecting ion homeostasis. Investigation of all large clostridial glucosyltransferases and other toxins revealed lysosomal dysfunction as a general effect of many but not of all toxins that integrate into the endosome membrane. Full article

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (K_D = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, K_D = 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems. Full article

Introduction: Bacteriophages are emerging as viable food safety tools, yet their global implementation is hindered by regulatory fragmentation and a lack of harmonized data standards. This review addresses the gap between scientific maturity and governance readiness by evaluating manufacturing quality, safety requirements, and international oversight frameworks. Methods: A narrative review was conducted through a structured search of databases including PubMed, Scopus, Web of Science, and Google Scholar (up to December 2025). We analyzed scientific research and publicly available regulatory documents from agencies such as the FDA, EFSA, USDA, Health Canada, and FSANZ to identify authorization routes and manufacturing standards. Results: Commercial phage products are primarily approved as processing aids in jurisdictions like the United States, Canada, and Australia/New Zealand. We identified convergent technical requirements across these regions, including genomic integrity (absence of toxins and antimicrobial resistance genes), purity, potency, and matrix-validated efficacy. However, significant gaps remain in unified terminology, environmental risk assessment, and post-market monitoring for resistance emergence. Conclusions: To facilitate global adoption, a One Health-oriented governance cycle is proposed. This includes establishing interoperable phage seed banks, standardized dossier formats, and adaptive lifecycle controls (phagovigilance) to ensure long-term efficacy and public trust. Full article

Aspergillus flavus (A. flavus) is a common contaminant of food and feed due to the production of aflatoxin B₁, which is susceptible to environmental signals. Nevertheless, how red light plays a role in A. flavus remains unclear. Here, we identified the uncharacterized hypothetical protein G4B84_010091 as a red-light sensor, defined as fungal phytochrome A (FphA), in A. flavus. The fphA knockout strain (ΔfphA) and complementary strain (fphA-com) were successfully constructed to characterize the function of FphA. Our results indicated that aflatoxin B₁ biosynthesis was promoted, while the development of conidia and sclerotia as well as the infection of peanuts were impaired in ΔfphA when compared with WT or fphA-com. The FphA^ΔRR domain deletion mutant exhibited all the phenotypes observed in the ΔfphA strain, indicating that the RR domain is indispensable for the function of FphA. In summary, FphA is involved not only in the formation of spores and sclerotia, but also in aflatoxin B₁ biosynthesis and the pathogenicity of A. flavus, which offers a potential target for novel approaches to controlling the dispersal and toxin production of this fungus. Full article

Bacillus cereus is a major foodborne pathogen responsible for food spoilage and foodborne illness, including strains producing emetic toxins. In this study, two bacteriophages, PBC_MG88 and PBC_MG99, were isolated from wastewater using emetic B. cereus strains as hosts and were comprehensively characterized. Both phages formed clear plaques with halos and exhibited siphovirus morphology. Host range analysis against 172 B. cereus strains showed that PBC_MG88 and PBC_MG99 infected 50 and 60 strains, respectively. One-step growth experiments revealed efficient lytic activity, with latent periods of 20–25 min and burst sizes of 59–63 PFU per infected cell. More than 90% of phage particles adsorbed to host cells within 15 min. Both phages were stable across a wide temperature range (4–55 °C) and pH values (4–11). Genome sequencing revealed ~37 kb double-stranded DNA genomes lacking antibiotic resistance or virulence genes; however, the presence of lysogeny-related genes suggests a temperate lifestyle. Comparative genomic analyses indicated that both phages represent novel species within the genus Lwoffvirus. Biofilm assays demonstrated significant inhibition of B. cereus biofilm formation and reduction of pre-established biofilms. Overall, this study expands knowledge of B. cereus phage diversity and highlights the importance of genomic characterization in phage-based biocontrol research. Full article