Search Results (5,707)

Sodium p-perfluorous nonenoxybenzenesulfonate (OBS) has been proposed as a substitute for perfluorooctanesulfonic acid (PFOS), yet it has garnered increasing attention due to its environmental persistence and potential toxicity. Despite these concerns, the neurotoxic mechanisms of OBS remain unclear. This study investigates and compares the neurotoxic effects and mechanisms of OBS and PFOS in Caenorhabditis elegans. L4-stage worms were exposed to OBS (0.1–100 μM) or PFOS (100 μM) for 24 h. Neurobehavioral analysis showed that OBS exposure induced concentration-dependent neurobehavioral deficits, with 100 μM OBS significantly reducing pharyngeal pumping rate (29.8%), head swing frequency (23.4%), and body bending frequency (46.6%), surpassing the effects of PFOS. Both compounds decreased the fluorescence intensity of dopaminergic, glutamatergic, and γ-aminobutyric acid neurons and downregulated neurotransmitter-associated genes. They also increased ROS generation and inhibited antioxidant gene expression. Molecular docking revealed that OBS had a stronger binding affinity to p38 MAPK key protein (PMK-1) than PFOS. OBS and PFOS upregulated pmk-1 and skn-1, modulating oxidative stress and neuronal function. pmk-1 mutation differentially affected OBS-induced neurobehavioral changes and gene expression alterations. Our findings indicate that OBS exhibits stronger neurotoxicity than PFOS in Caenorhabditis elegans, mediated through the PMK-1 pathway. These results highlight the need for further investigation into the safety of OBS as a PFOS alternative. Full article

Reactive oxygen species (ROS) are often seen solely as harmful byproducts of oxidative metabolism, yet evidence reveals their paradoxical roles in both promoting and inhibiting cancer progression. Despite advances, precise context-dependent mechanisms by which ROS modulate oncogenic signaling, therapeutic response, and tumor microenvironment dynamics remain unclear. Specifically, the spatial and temporal aspects of ROS regulation (i.e., the distinct effects of mitochondrial versus cytosolic ROS on the PI3K/Akt and NF-κB pathways, and the differential cellular outcomes driven by acute versus chronic ROS exposure) have been underexplored. Additionally, the specific contributions of ROS-generating enzymes, like NOX isoforms and xanthine oxidase, to tumor microenvironment remodeling and immune modulation remain poorly understood. This review synthesizes current findings with a focus on these critical gaps, offering novel mechanistic insights into the dualistic nature of ROS in cancer biology. By systematically integrating data on ROS source-specific functions and redox-sensitive signaling pathways, the complex interplay between ROS concentration, localization, and persistence is elucidated, revealing how these factors dictate the paradoxical support of tumor progression or induction of cancer cell death. Particular attention is given to antioxidant mechanisms, including NRF2-mediated responses, that may undermine the efficacy of ROS-targeted therapies. Recent breakthroughs in redox biosensors (i.e., redox-sensitive fluorescent proteins, HyPer variants, and peroxiredoxin–FRET constructs) enable precise, real-time ROS imaging across subcellular compartments. Translational advances, including redox-modulating drugs and synthetic lethality strategies targeting glutathione or NADPH dependencies, further highlight actionable vulnerabilities. This refined understanding advances the field by highlighting context-specific vulnerabilities in tumor redox biology and guiding more precise therapeutic strategies. Continued research on redox-regulated signaling and its interplay with inflammation and therapy resistance is essential to unravel ROS dynamics in tumors and develop targeted, context-specific interventions harnessing their dual roles. Full article

Background: With the increasing shift in drug design away from classical drug targets towards the modulation of protein-protein interactions, synthetic peptides are gaining increasing relevance. The synthesis and purification of peptides via solid-phase peptide synthesis (SPPS) strongly rely on trifluoroacetic acid (TFA) as a cleavage agent and ion-pairing reagent, respectively, resulting in peptides being obtained as TFA salts. Although TFA has excellent properties for peptide production, numerous studies highlight the negative impact of using peptides from TFA⁻ salts in biological assays. Methods: Investigated peptides were synthesized via SPPS and the TFA⁻ counterion was exchanged for Cl⁻ via freeze-drying in different concentrations of HCl. Detection and quantification of residual TFA⁻ were carried out via FT-IR, ¹⁹F-NMR, and HPLC using an evaporative light-scattering detector (ELSD). A liposomal fluorescence assay was used to test for the influence of the counterion on the peptides’ passive membrane permeability. Results: All TFA⁻ detection methods were successfully validated according to ICH guidelines. TFA⁻ removal with 10 mM HCl was determined to be the optimal condition. No impact on peptide purity was observed at all HCl concentrations. Influences on permeability coefficients depending on peptide sequence and salt form were found. Conclusions: This study presents a systematic investigation of the removal of TFA⁻ counterions from synthetic peptides and their replacement with Cl⁻ counterions. Detected counterion contents were used to understand the impact of sequence differences, especially positive charges, on the amount and potential localization of counterions. Our findings emphasize the importance of counterion quantification and specification in assays with synthetic peptides. Full article

Conessine is a steroidal alkaloid found in many plants. The pharmacological efficacies of conessine on various ailments, including antiviral effects against Zika, Herpes, and Coronavirus, were reported. However, the effect of conessine on the influenza virus was still unknown. In this study, conessine exhibited a strong inhibitory effect against influenza A virus (IAV) infection. We examined the effect of conessine on IAV using green fluorescent protein (GFP)-expressing Influenza A/PR8/34 and wild-type A/PR8/34. The fluorescence-activated cell sorting, fluorescence microscopy, cytopathic effect analysis, and plaque assay demonstrated that conessine significantly inhibits IAV infection. Consistently, immunofluorescence results showed that conessine strongly reduces the expression of IAV proteins. The time-of-drug-addition assay revealed that conessine could affect the viral attachment and entry into the cells upon IAV infection. Further, conessine eradicated the virus before binding to the cells in the early stage of viral infection. Our results suggest that conessine has strong anti-viral efficacy against IAV infection and could be developed as an anti-influenza viral agent. Full article

Palindromic antimicrobial peptides (PAMs) constitute versatile scaffolds for the design and optimization of anticancer agents with applications in therapy, diagnosis, and/or monitoring. In the present study, fluorolabeled peptides derived from the palindromic sequence RWQWRWQWR containing fluorescent probes, such as 2-Aminobenzoyl, 5(6)-Carboxyfluorescein, and Rhodamine B, were obtained. RP-HPLC analysis revealed that the palindromic peptide conjugated to Rhodamine B (RhB-RWQWRWQWR) exhibited the presence of isomers, likely corresponding to the open-ring and spiro-lactam forms of the fluorescent probe. This equilibrium is dependent on the peptide sequence, as the RP-HPLC analysis of dimeric peptide (RhB-RRWQWR-hF-KKLG)₂K-Ahx did not reveal the presence of isomers. The antibacterial activity of the fluorescent peptides depends on the probe attached to the sequence and the bacterial strain tested. Notably, some fluorescent peptides showed activity against reference strains as well as sensitive, resistant, and multidrug-resistant clinical isolates of E. coli, S. aureus, and E. faecalis. Fluorolabeled peptides 1-Abz (MIC = 62 µM), RhB-1 (MIC = 62 µM), and Abz-1 (MIC = 31 µM) exhibited significant activity against clinical isolates of E. coli, S. aureus, and E. faecalis, respectively. The RhB-1 (IC₅₀ = 61 µM), Abz-1 (IC₅₀ = 87 µM), and RhB-2 (IC₅₀ = 35 µM) peptides exhibited a rapid, significant, and concentration-dependent cytotoxic effect on HeLa cells, accompanied by morphological changes characteristic of apoptosis. RhB-1 (IC₅₀ = 18 µM) peptide also exhibited significant cytotoxic activity against breast cancer cells MCF-7. These conjugates remain valuable for elucidating the possible mechanisms of action of these novel anticancer peptides. Rhodamine-labeled peptides displayed cytotoxicity comparable to that of their unlabeled analogues, suggesting that cellular internalization constitutes a critical early step in their mechanism of action. These findings suggest that cell death induced by both unlabeled and fluorolabeled peptides proceeds predominantly via apoptosis and is likely contingent upon peptide internalization. Functionalization at the N-terminal end of the palindromic sequence can be evaluated to develop systems for transporting non-protein molecules into cancer cells. Full article

Vaccination is the most effective method to counteract infectious diseases in farmed fish. It secures aquaculture production and safeguards the wild stock and aquatic ecosystem from catastrophic contagious diseases. In vaccine development, recombinant subunit vaccines are favorable candidates since they can be economically produced in large quantities without growing many pathogens, as in inactivated or attenuated vaccine production. However, recombinant subunit vaccines are often weak or deficient in immunogenicity, resulting in inadequate defenses against infections. Technologies that can increase the immunogenicity of recombinant subunit vaccines are in desperate need. Enhanced green fluorescence protein (EGFP) has a low antigenicity and is susceptible to folding changes and losing fluorescence after fusing with other proteins. Using these valuable features of EGFP, we comprehend two amphipathic alpha-helical peptides, AH1 and AH3, derived from Hepatitis C virus and Influenza A virus, respectively, that can induce high immune responses of their fused EGFP in fish without affecting their folding. AH3-EGFP has the most elevated cell binding, significantly 62% and 36% higher than EGFP and AH1-EGFP, respectively. Immunizations with AH1-EGFP or AH3-EGFP significantly induced higher anti-EGFP antibody levels 300–500-fold higher than EGFP immunization after the boost injection in rainbow trout. Our results suggest that AH1 and AH3 effectively increase the immunogenicity of EGFP without influencing its structure. Further validation of their value in other recombinant proteins is necessary to demonstrate their broader utility in enhancing the immunogenicity of subunit vaccines. We also suggest that EGFP and its variants are promising candidates for initially screening proper immunogenicity-enhancing peptides or proteins to advance recombinant subunit vaccine development. Full article

The objectives of this study were to prepare whey protein–quercetin–gellan gum conjugates using the pH-shift method and to evaluate the impacts of varying pH values and quercetin concentrations on the interaction mechanisms and functional characteristics of the complexes. Spectroscopic analyses (fluorescence, UV-vis, and FT-IR) revealed that new complexes formed under alkaline conditions. Notably, an increasing quercetin concentration led to a reduction in complex particle size and an increase in the zeta potential value, with these effects being more pronounced under alkaline conditions. The particle size was 425.7 nm, and the zeta potential value was −30.00 mV at a quercetin addition concentration of 15 umol/g protein. Additionally, the complexes formed under alkaline conditions exhibited superior foaming capacity, emulsification properties, and significantly enhanced free radical scavenging activity. The complex’s DPPH and ABTS radical scavenging rates rose by 41.57% and 57.69%, respectively. This study provides theoretical foundations and practical insights for developing protein—polyphenol systems, offering significant implications for the application of quercetin functional foods and supplements in the food science and pharmaceutical industries. Full article

Enhancer-promoter interactions occur in the chromatin loci delineated by the CCCTC-binding zinc-finger protein CTCF. CTCF binding is frequently perturbed in genetic disorders and cancer, allowing for misregulation of genes. Here, we developed a panel of chimeric proteins consisting of either full-length or truncated CTCF fused with programmable DNA-binding module dCas9 and fluorescent tracker EGFP. We found that the recruitment of a chimeric protein based on the CTCF N-terminal domain and two zinc-finger domains to the human HOXD locus leads to the de novo formation of a spatial contact with a nearby cohesin/CTCF-bound region, anchoring several chromatin loops. This chimeric protein did not show binding to CTCF motifs and did not affect the epigenetic and transcription profile of the locus. Recruitment of this chimeric protein is also able to restore chromatin loops, lost after deletion of an endogenous CTCF-binding site. Together, our data indicate that the ectopic recruitment of the CTCF N-terminal part could be an appropriate tool for 3D genome engineering. Full article

Maize–soybean intercropping is widely practised to improve land use efficiency, but shading from maize often limits soybean growth and productivity. Melatonin, a plant signaling molecule with antioxidant and growth-regulating properties, has shown potential in mitigating various abiotic stresses, including low light. This study investigated the efficacy of applying foliar melatonin (MT) to enhance shade tolerance and yield performance of soybean under intercropping. Four melatonin concentrations (0, 50, 100, and 150 µM) were applied to soybean grown under mono- and intercropping systems. The results showed that intercropping significantly reduced growth, photosynthetic activity, and yield-related traits. However, the MT application, particularly at 100 µM (MT₁₀₀), effectively mitigated these declines. MT₁₀₀ improved plant height (by up to 32%), leaf area (8%), internode length (up to 41%), grain yield (32%), and biomass dry matter (30%) compared to untreated intercropped plants. It also enhanced SPAD chlorophyll values, photosynthetic rate, stomatal conductance, chlorophyll fluorescence parameters such as Photosystem II efficiency (ɸPSII), maximum PSII quantum yield (Fv/Fm), photochemical quenching (qp), electron transport rate (ETR), Rubisco activity, and soluble protein content. These findings suggest that foliar application of melatonin, especially at 100 µM, can improve shade resilience in soybean by enhancing physiological and biochemical performance, offering a practical strategy for optimizing productivity in intercropping systems. Full article

Super-resolution microscopy (SRM) has revolutionized our understanding of subcellular structures, including cell organelles and viruses. For human immunodeficiency virus (HIV), SRM has significantly advanced knowledge of viral structural biology and assembly dynamics. This review analyzes how SRM techniques (particularly PALM, STORM, STED, and SIM) have been applied over the past decade to study HIV structural components and assembly. By categorizing and comparing studies based on SRM methods, HIV components, and labeling strategies, we assess the strengths and limitations of each approach. Our analysis shows that PALM is most commonly used for live-cell imaging of HIV Gag, while STED is primarily used to study the viral envelope (Env). STORM and SIM have been applied to visualize various components, including Env, capsid, and matrix. Antibody labeling is prevalent in PALM and STORM studies, targeting Env and capsid, whereas fluorescent protein labeling is mainly associated with PALM and focused on Gag. A recent emphasis on Gag and Env points to deeper investigation into HIV assembly and viral membrane dynamics. Insights from SRM studies of HIV not only enhance virological understanding but also inform future research in therapeutic strategies and delivery systems, including extracellular vesicles. Full article

Genetic transformation is an essential tool for investigating gene function and editing genomes. Kiwifruit, recognized as a significant global fresh fruit crop, holds considerable economic and nutritional importance. However, current genetic transformation techniques for kiwifruit are impeded by low efficiency, lengthy culture durations (a minimum of six months), and substantial labor requirements. In this research, we established an efficient system for shoot regeneration and the stable genetic transformation of the ‘Hayward’ cultivar, utilizing leaf explants in conjunction with two strains of Agrobacterium that harbor the expression vector pBI121-35S::GFP, which contains the green fluorescent protein (GFP) gene as a visible marker within the T-DNA region. Our results show that 93.3% of leaf explants responded positively to the regeneration medium, producing multiple independent adventitious shoots around the explants within a six-week period. Furthermore, over 71% of kanamycin-resistant plantlets exhibited robust GFP expression, and the entire transformation process was completed within four months of culture. Southern blot analysis confirmed the stable integration of GFP into the genome, while RT-PCR and fluorescence microscopy validated the sustained expression of GFP in mature plants. This efficient protocol for regeneration and transformation provides a solid foundation for micropropagation and the enhancement of desirable traits in kiwifruit through overexpression and gene silencing techniques. Full article

Icariin (ICA) is widely recognized for its health benefits. In this work, we examined the intermolecular interactions between ICA and proteinase K (PK) via multi-spectroscopic techniques and molecular simulations. The experimental findings revealed that ICA quenched the fluorescence emission of PK by forming a noncovalent complex. Both hydrogen bonding and van der Waals interactions are essential for the complex’s formation. Then Förster resonance energy transfer (FRET), competitive experiments, and synchronous fluorescence spectroscopy were adopted to verify the formation of the complex. Molecular docking studies demonstrated that ICA could spontaneously bind to PK by hydrogen bonding and hydrophobic interactions, which is consistent with the spectroscopic results. The PK-ICA complex’s dynamic stability was evaluated using a 50 ns molecular dynamics (MD) simulation. The simulation results revealed no significant structural deformation or positional changes throughout the entire simulation period. The complex appears to be rather stable, as seen by the average root-mean-square deviation (RMSD) fluctuations for the host protein in the PK-ICA complex of 1.08 Å and 3.09 Å. These outcomes of molecular simulations suggest that ICA interacts spontaneously and tightly with PK, consistent with the spectroscopic findings. The approach employed in this research presents a pragmatic and advantageous method for examining protein–ligand interactions, as evidenced by the concordance between empirical and theoretical findings. Full article

Due to widespread use of silver nanoparticles (AgNPs), the assessment of their potential harm to microalgal photosynthesis is crucial, as microalgae, together with cyanobacteria, contribute to approximately 50% of global oxygen production. This study investigated photosynthetic pigments, photosynthetic rate, chlorophyll a fluorescence, and the expression of photosynthesis-related genes and proteins in green alga Chlorella vulgaris after 72 h exposure to citrate- and cetyltrimethylammonium bromide (CTAB)-stabilized AgNPs, as well as silver ions (AgNO₃), at concentrations allowing 75% cell survival (EC₂₅). All treatments impaired photosynthetic performance. The most pronounced decreases in chlorophyll fluorescence parameters and photosynthetic rate, alongside elevated energy dissipation, were observed after exposure to AgNP-CTAB and AgNO₃. AgNP-citrate had milder effects and induced compensatory responses, reflected in an increased performance index and upregulation of photosynthesis-related proteins. AgNP-CTAB induced the strongest downregulation of gene and protein expression, likely due to its higher EC₂₅ concentration and cationic surface promoting interaction with photosynthetic structures. Although AgNO₃ caused fewer molecular changes, it significantly disrupted photosynthetic function, suggesting a direct effect of Ag⁺ ions on photosynthesis-related proteins. Overall, the results highlight the role of AgNPs’ surface coatings and dosage in determining their phytotoxicity, with photosystem disruption and oxidative stress emerging as key mechanisms of action. Full article

Tuberous sclerosis complex (TSC) is a systemic disease caused by mutations in either the TSC1 (encoding hamartin) or TSC2 (encoding tuberin) gene, with mutations in the TSC2 gene potentially leading to more severe clinical symptoms. Neurological symptoms are a common clinical manifestation of TSC, and neuroinflammation is thought to play an important role. Glial cells are a major source of neuroinflammation, but whether microglia are involved in the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and the expression of interleukin-1β (IL-1β) in TSC patients remains unclear. We used a transcriptome sequencing dataset for bioinformatics analysis to explore the differences in the expression of microglial inflammasome-associated hub genes. TSC2 knockdown (TSC2 KD) microglia (HMC3 cell line) were generated by lentivirus, and the expression of inflammasome-associated hub genes, microglial activation, and NLRP3 inflammasome activation were verified. In addition, experiments were performed to explore the regulatory effects of rapamycin. Bioinformatics analysis identified a total of eight inflammasome-associated hub genes. By detecting GFP fluorescence, TSC2 mRNA, TSC2 protein expression, and the phosphorylation of the mammalian target of rapamycin (p-mTOR)/mTOR, we confirmed that the TSC2 KD microglia model was successfully established. Compared with the control group, the TSC2 KD group presented higher mRNA levels and fluorescence intensities of microglia AIF1 and CD68, as well as greater reactive oxygen species (ROS) production. Eight inflammasome-associated hub gene mRNA assays revealed that the expression of the NLRP3 and IL1B genes was increased. Compared with the control group, the TSC2 KD group presented increased levels of NLRP3 and Pro-IL-1β proteins in cells and Cleaved-Caspase 1 and Cleaved-IL-1β proteins in the supernatant, suggesting NLRP3 inflammasome activation. Rapamycin intervention alleviated these changes, demonstrating that the TSC2 gene regulation of microglial activation and NLRP3 inflammasome activation are correlated with mTOR phosphorylation. In conclusion, microglia are activated in TSC patients and participate in the NLRP3 inflammasome-associated neuroinflammatory response, and rapamycin treatment can alleviate these changes. Full article

Methods such as AlphaFold have revolutionized protein structure prediction, making quantitative prediction of the thermodynamic stability of individual proteins and their complexes one of the next frontiers in computational protein modeling. Here, we develop methods for using protein language models (PLMs) with protein mutational datasets related to protein tertiary and quaternary stability. First, we demonstrate that fine-tuning of a ProtT5 PLM enables accurate prediction of the largest protein mutant stability dataset available. Next, we show that mutational impacts on protein function can be captured by fine-tuning PLMs, using green fluorescent protein (GFP) brightness as a readout of folding and stability. In our final case study, we observe that PLMs can also be extended to protein complexes by identifying mutations that are stabilizing or destabilizing. Finally, we confirmed that state-of-the-art simulation methods (free energy perturbation) can refine the accuracy of predictions made by PLMs. This study highlights the versatility of PLMs and demonstrates their application towards the prediction of protein and complex stability. Full article