Search Results (161)

A convenient one-pot strategy was adopted to fabricate Cu-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) nanosheets through the coordination of HEPES with copper ions under alkaline conditions, and the obtained nanosheets possessed dual laccase-mimetic and oxidase-mimetic activities for phenolic pollutant analysis and show potential for dye decolorization/removal. The Cu-HEPES nanosheets displayed significant dual enzyme-mimetic activities, effectively catalyzing the oxidation conversion for phenolic compounds, specifically 2,4-dichlorophenol (2,4-DCP), to yield quinone imine products characterized by a unique absorption signal at 500 nm. Meanwhile, Cu-HEPES nanosheets also facilitated the conversion of 3,3′,5,5′-tetramethylbenzidine to yield a yellow-green substance with a distinctive absorption signal at 458 nm. The Cu-HEPES nanosheets displayed a satisfactory affinity (K_m = 23.2 μM) for 2,4-DCP, facilitating its use in accurately detecting 2,4-DCP (linear range from 3.3 to 16.6 µM) with a detection limit of 0.3 µM. Subsequently, the devised colorimetric sensing platform was applied to measure 2,4-DCP levels in real tap water samples, yielding satisfactory spiked recoveries ranging from 92.7% to 106.9%. Finally, the synthesized Cu-HEPES nanosheets were preliminarily evaluated for the simultaneous decolorization/removal of malachite green and Congo red, suggesting their potential applicability in aqueous systems containing multiple organic dyes. Full article

Singlet oxygen is produced by light-dependent excitation of molecular oxygen using photosensitizers. Using blue light and riboflavin (RF) or riboflavin phosphate (RFP) as photosensitizers, we studied the reaction of singlet oxygen with tertiary amines in aqueous solution because many are used as biochemical buffers and as electron donors in photoreduction reactions. The reactions of singlet oxygen with multiple tertiary amines, including ethylenediamine tetraacetic acid (EDTA), bicine, and triethanolamine (TEOA), produced micromolar hydrogen peroxide (H₂O₂) as the stable end product. For bicine and TEOA, but not EDTA, H₂O₂ yield increased as pH increased due to their higher amine pKa values. A white LED used in conjunction with riboflavin and tertiary amines also produced H₂O₂, a contaminant likely to form during tissue culture manipulations. Direct photoreduction of 2,6-dichlorophenolindophenol and 2,3-dimethoxy-5-methyl-p-benzoquinone was achieved using blue light, RF or RFP, and tertiary amines as electron donors. With RF, EDTA was the optimal electron donor for both substrates, whereas with RFP, bicine and TEOA were superior to EDTA. Photochemical redox cycling of both quinones produced H₂O₂ via singlet oxygen-dependent re-oxidation of reduced quinols. Several amine buffers, including HEPES and PIPES, reacted with singlet oxygen to produce H₂O₂ but did not function as electron donors in quinone photoreduction assays. Full article

In protein solutions, an additive that increases protein–protein attractive interactions is expected to decrease protein crystal solubility and raise the temperature at which liquid–liquid phase separation (LLPS) occurs. In contrast, addition of 0.10 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) to lysozyme–NaCl aqueous solutions at constant pH (7.4) and ionic strength (0.20 M) decreases solubility but lowers the LLPS temperature. This leads to the broadening of the LLPS metastability gap in the phase diagram and an enhancement of protein crystallization yield from LLPS. We theoretically examine the effect of HEPES on both solubility and LLPS boundaries using a colloid model. Under the hypothesis that HEPES stabilizes protein–protein contacts in the crystal lattice by physical cross-linking, we apply cell theory to describe the thermodynamic behavior of the crystalline phase and use solubility data to show that HEPES increases protein–protein attraction energy by 2.7%. Since an increase in attraction incorrectly predicts a rise in the LLPS temperature, we consider that HEPES also enhances the anisotropic character of protein–protein interactions. To describe the thermodynamic behavior of the solution phase, we start from Barker–Henderson second-order perturbation theory on the hard-sphere reference fluid with square-well potential and local-compressibility approximation. We modify this model so that it can reproduce the correct mathematical expression of the second virial coefficient. This also leads to better agreement with Monte Carlo simulations. We then approximately incorporate anisotropy by assuming that the square-well attraction energy is a temperature-dependent average over all the surface of a particle with a given fractional coverage of attractive spots. The attraction energy of the attractive spots is set to be the same as that of the protein–protein contacts in the crystal. Only fractional coverage (anisotropy) was varied to successfully fit the effect of HEPES on the LLPS boundary. Full article

Capacitation is a key maturation process that enables spermatozoa to acquire fertilizing ability and can be induced in vitro using capacitation media. Because capacitation protocols differ markedly among laboratories, we compared three compositionally distinct Hepes-, Tris-, and TALP-based media. This study was performed in boar spermatozoa using 3–6 biological replicates of pooled ejaculates depending on the assay, with 46 ejaculate samples from 12 boars in total. The aim was to determine whether such non-standardized conditions differentially affect signaling pathways leading to capacitation and thereby influence the detection of commonly used capacitation markers. We found clear differences among the tested media. All three induced capacitation-associated events, but their functional and molecular effects were not equivalent. The Hepes-based medium supported sperm motility most effectively, increasing total and progressive motility to 60.0% and 48.7%, respectively, after 1 h of incubation and maintaining the highest motility throughout the incubation period. In contrast, the Tris-based medium maintained lower but relatively stable motility, whereas the TALP-based medium showed a rapid decline in total motility from 53.1% to 15.2% during the first hour. The TALP-based medium induced the highest and most sustained protein kinase A (PKA) activity, reaching 0.047 U/mL at 0 h and 0.040 U/mL after 3 h, whereas the Hepes- and Tris-based media showed lower and less sustained activity ranging from 0.003 to 0.030 U/mL during incubation. In addition, distinct patterns of protein tyrosine phosphorylation were observed depending on the medium used. In particular, the TALP-based medium containing bicarbonate and bovine serum albumin (BSA) and the Hepes-based medium with the highest BSA concentration were associated with the highest levels of total protein tyrosine phosphorylation. Phosphoproteomic analysis further revealed condition-specific phosphorylation events, indicating that sperm maturation is dynamically regulated by the surrounding molecular environment. In contrast, no significant differences were detected in oxidative phosphorylation or in electron transport system complexes among the tested media. These findings show that differences in capacitation media composition, particularly in bicarbonate and BSA content, can markedly alter signaling outcomes and the interpretation of capacitation markers, with important implications for reproductive technologies and experimental standardization. Full article

Macrosteles fascifrons, a representative aster leafhopper frequently detected in rice-growing environments, is an economically significant insect that inhabits rice fields and plays a role in the ecology of crop pests and disease transmission. To expand the understanding of viral diversity associated with the aster leafhopper, we analyzed its virome using deep transcriptome sequencing. In addition to several previously reported viruses, we identified two previously unreported RNA viruses, tentatively designated as Macrosteles fascifrons hepe-like virus 1 (MfHV1) and Macrosteles fascifrons rhabdovirus 1 (MfRV1). The complete genome sequences of both genomes were obtained using overlapping RT-PCR and rapid amplification of cDNA ends. Excluding the poly(A) tail, the genome of MfHV1 is 6688 nucleotides in length and exhibits a genomic organization characteristic of the family Hepeviridae, comprising three major open reading frames (ORFs) that encode a putative nonstructural polyprotein, a capsid protein, and a small accessory protein. The ORF encoding the capsid protein partially overlaps with the ORF encoding the small accessory protein, a genomic feature commonly observed in hepe-like viruses. The genome of MfRV1 is 14,984 nucleotides in length and displays the canonical genomic organization of the family Rhabdoviridae. An additional accessory ORF was identified between the putative M and G genes. Phylogenetic analysis based on polyprotein sequences placed MfHV1 within the Hepeviridae, most closely related to insect-associated hepe-like viruses, whereas MfRV1 clustered within the subfamily Deltarhabdovirinae. According to ICTV guidelines, virus classification is based on a combination of sequence divergence, phylogenetic relationships, and genome organization. MfHV1 and MfRV1 share low amino acid sequence identities with known viruses (maximum 36.07% for the MfHV1 polyprotein and 47.7% for the MfRV1 RNA-dependent RNA polymerase). Based on sequence divergence, genome organization, and phylogenetic placement, these viruses are classified as putative novel members of their respective families. This study expands the diversity of virus-associated sequences detected in M. fascifrons and provides additional genomic resources for understanding insect-associated RNA viruses. Full article

Chitosan is non-toxic, harmless, biocompatible, and antimicrobial, and can be readily modified. These properties have made it widely used in carrier research. Based on this, a fluorescent probe P was synthesized in high yield from naphthalimide derivatives and carboxymethyl chitosan (CMCS). The probe exhibited enhanced fluorescence in the presence of Al³⁺ and quenched fluorescence in the presence of Mg²⁺ in different media. Among common metal ions and anions, the probe demonstrated good selectivity and sensitivity toward Al³⁺ and Mg²⁺. Under optimal conditions (ethanol–water solution, 1:9, v:v, pH 6.0, 20 mM HEPES), a significant linear relationship was observed for Al³⁺ in the concentration range of 0–90 µM. In ethanol, the fluorescence intensity of the probe at 427 nm decreased regularly with increasing Mg²⁺ concentration, also showing a clear linear relationship within the 5–90 µM range. Full article

This study compared the bioenergetic profiles of immature and in vitro–matured bovine cumulus–oocyte complexes (COCs) using Seahorse extracellular flux technology, with the aim of establishing standardized conditions for real-time metabolic assessment during in vitro maturation (IVM). Groups of five COCs were analysed prior to maturation and after 22 h of IVM using the Seahorse XFp Analyzer to measure oxygen consumption rate (OCR, pmoL/min) and extracellular acidification rate (ECAR, mpH/min), providing dynamic readouts of oxidative phosphorylation and glycolysis that extend beyond conventional endpoint assays. To optimize assay performance, three media were first evaluated: TCM199, DMEM/F12, and HEPES-buffered synthetic oviductal fluid (HSOF). HSOF yielded the most reliable readings for immature COCs, whereas TCM199 provided superior conditions for mature COCs. Adhesion strategies were then tested by comparing uncoated wells with wells coated with fibronectin, concanavalin A, or Matrigel^®. Sequential injections of oligomycin and rotenone plus antimycin A enabled partitioning of mitochondrial and glycolytic contributions to ATP production. COC maturation was associated with a clear metabolic shift from glycolysis toward oxidative metabolism. Immature COCs displayed a predominantly glycolytic phenotype, while mature COCs showed increased active mitochondrial ATP production. Adhesion conditions markedly affected the detected metabolic profile: concanavalin A and fibronectin supported effective attachment and were associated with robust energy metabolism, whereas Matrigel^® and poor adhesion were linked to quiescent profiles with low OCR and ECAR signals. Together, these data define practical assay parameters for extracellular flux analysis of COCs and highlight the increasing reliance on mitochondrial function as a hallmark of oocyte maturation, supporting improved metabolic phenotyping for IVM optimization. Full article

A non-targeted metabolomics technique was employed to investigate the dynamic metabolite changes of scallop (Patinopecten yessoensis) adductor muscle (SAM) and scallop mantle (SM) during the early (E), middle (M), and late (L) harvest period from June to July. Many more metabolites were identified from SM (831) than from SAM (231), and 24 and 12 significantly differential metabolites were screened, respectively. Organic acids and derivatives, lipids and lipid-like molecules, and organoheterocyclic compounds were the primary metabolite classes in both SAM and SM. In SAM, the levels of most altered metabolites, such as butyryl carnitine, (±)8-HEPE, 14(S)-HDHA, l-Kynurenine, and l-(−)-Methionine), decreased with the extended harvest time, whereas pipecolic acid and oleamide increased. Conversely, in SM, linoleamide, oleamide, dimethyl citrate, kynurenine, and pipecolic acid declined, while 5,6-Dihydrothymidine, dimethylsulfoniopropionate, and 13(S)-HOTrE increased with a longer harvest time. Pipecolic acid, exhibiting an obvious up-regulated response during the whole harvest period, was found to be the sole differential metabolite shared by SAM and SM. Annotation analysis showed that five metabolites were respectively identified in SAM and SM, and these metabolites were separately related to five metabolic pathways with slight differences among the two tissues. Amino acid metabolism/degradation and fatty acid metabolism were the primary pathways. These findings could provide new insights into the dynamic quality changes of scallops during the harvest period and may play a potential guiding role in the aquaculture and harvest of scallops. Full article

Background: Self-amplifying mRNA (sa-mRNA) represents a promising platform for vaccines and gene therapies, offering sustained protein expression at low doses through self-replication. For vaccines targeting respiratory pathogens, pulmonary delivery of sa-mRNA lipid nanoparticles (LNPs) is particularly advantageous, enabling direct delivery to the infection site and induction of mucosal immunity. Objective: In this study, we evaluated the stability of sa-mRNA–LNPs under refrigerated and frozen conditions and developed a dry powder formulation suitable for inhalation, produced by freeze-drying followed by cryomilling with leucine. Methods: sa-mRNA–LNPs formulated in HEPES buffer with 20% (w/v) sucrose were stored for up to 8 weeks as liquid or freeze-dried samples at various temperatures (−80 °C, −20 °C, 4 °C, and 20 °C). Biological stability was assessed by transfection efficiency in HeLa cells, while physical stability was characterized by encapsulation efficiency, zeta potential, particle size, and polydispersity index. Results: Liquid formulations remained stable for at least 8 weeks at −80 °C and −20 °C but rapidly lost stability at 4 °C and 20 °C. Freeze-drying effectively preserved sa-mRNA–LNP functionality and structural integrity for up to 8 weeks at 4 °C, with only minor structural changes. Subsequent cryomilling in the presence of 4 wt-% leucine produced a respirable dry powder while retaining approximately 60% of the original sa-mRNA–LNP functionality. Although cryomilling induced some structural alterations, the remaining functional fraction remained stable during storage. The resulting powders displayed favorable aerosol performance for deep lung delivery, as demonstrated by cascade impaction (MMAD = 4.13 ± 0.26 µm). Conclusions: In conclusion, freeze-drying effectively preserved sa-mRNA–LNP integrity at 4 °C, whereas cryomilling with leucine produced a respirable dry powder suitable for pulmonary delivery, providing a foundation for globally accessible, needle-free sa-mRNA vaccines against respiratory diseases. Full article

Oxylipins are specialized lipid mediators that can have dual functions, either promoting inflammation or supporting resolution. Exposure to air pollution is associated with systemic inflammation that may be modified by oxylipins derived from polyunsaturated fatty acids (FA). In this study, we examined whether short-term air pollution exposure is associated with changes in circulating oxylipins in healthy adults, who were on high- or low-dietary omega-3 fatty acid (n-3 FA) intakes. We measured 56 oxylipin species from participants’ plasma samples and employed mixed-effects models to assess the associations, stratified by n-3 FA groups. Plasma concentrations of oxylipins derived from n-3 FA [e.g., 14-hydroxydocosahexaenoic acid (14-HDHA) & 11-hydroxydocosahexaenoic acid (11-HDoHE), and 12-hydroxyeicosapentaenoic acid (12-HEPE)] were significantly higher in the high n-3 FA group compared to the low group. Conversely, selected oxylipins derived from n-6 FA [e.g., 15-hydroxyeicosatetraenoic acid (15-HETE) and 14,15-Dihydroxyeicosatrienoic acid (14,15-DiHETrE)] were significantly lower in the high n-3 group. Exposure to PM_2.5, O₃, and NO₂ was associated with reductions in pro-inflammatory oxylipins produced by lipoxygenase in the high n-3 FA group, but not in the low group; for example, 12-HETE. Furthermore, participants in the high n-3 group exposed to PM_2.5, O₃, and NO₂ had elevated levels of n-3 FA-derived pro-resolving oxylipins compared to those in the low n-3 group; for instance, 12-HEPE and 14-HDHA & 11-HDoHE. In conclusion, short-term air pollution exposure was associated with lower pro-inflammatory and higher pro-resolving oxylipin levels in the high n-3 FA group. These findings suggest n-3-derived lipid metabolites may promote inflammation resolution induced by air pollution. Full article

Lactobacillus acidophilus is a widely researched probiotic bacterium with broad applications in health and biotechnology; however, its protoplast formation has not been extensively investigated. This study aimed to optimize conditions for L. acidophilus protoplast formation. Freeze-dried cells were suspended in 20 mM HEPES buffer (pH 7) supplemented with sucrose (1.0 M, 1.5 M, and 2.0 M) to induce hyperosmotic conditions, yielding a final cell density of 10⁸ cells/mL. The suspensions were treated with 125 µg/mL lysozyme and incubated at 37 °C for 30 min, 1 h, or 2 h. Prior to enzymatic treatment, the buffer, lysozyme, and cell suspensions were equilibrated at either 22 °C (room temperature) or 37 °C. Phase contrast microscopy was used to evaluate protoplast formation across all treatment combinations, and a three-way ANOVA was conducted to assess the effects of buffer molarity, incubation time, and temperature. Protoplasts are valuable tools for genetic manipulation, cell fusion, and cell wall studies, yet optimized protocols for their generation in L. acidophilus are lacking. The highest protoplast yield with minimal lysis was observed under 2.0 M sucrose conditions after 2 h of incubation, particularly when all components were equilibrated at 37 °C. Prolonged lysozyme exposure increased lysis, especially at lower buffer molarities. Elevated buffer molarity conferred a protective effect by maintaining cell integrity during enzymatic digestion. These findings highlight the importance of osmotic strength and thermal equilibration in optimizing protoplast formation and provide a reproducible framework for controlled enzymatic treatments in L. acidophilus. Full article

Oxylipins are tightly linked to ovarian function. However, the roles and mechanisms of oxylipins in ovarian aging of laying ducks remain poorly understood. In this study, 72-week-old laying Jinyun ducks were categorized into high-laying (HL) and low-laying (LL) groups. Plasma and ovarian tissues were analyzed for antioxidant indices, transcriptomic, and targeted lipidomic. Results demonstrated that, compared to the LL group, the HL group ducks exhibited lower levels of MDA in both plasma and ovarian tissues, while exhibiting higher levels of SOD, GSH-Px, and T-AOC. Differentially expressed genes were primarily enriched in fatty acid oxidation and inflammation-related pathways, with FOXM1 identified as a pivotal gene involved in delaying ovarian aging. Furthermore, oxylipin profiles in plasma and ovary exhibit marked distinctions between the HL and LL group ducks. Notably, 5-hydroxy-eicosapentaenoic acid (5-HEPE) was substantially upregulated in both plasma and ovaries of the HL group ducks. Meanwhile, 5-HEPE significantly enhanced FOXM1 expression and mitigated oxidative stress in granulosa cells. Collectively, this study provides the first comprehensive analysis of oxylipin changes in the ovaries and plasma of ducks with differing laying performances at the late-laying stage. These findings offer novel insights into the prevention and alleviation of ovarian oxidative stress in ducks during this period. Full article

Residential radon is a leading environmental cause of lung cancer, but circulating biomarkers linking home exposure to pathogenic biology are not well defined. We conducted an exposure-contrast study in Hang Dong District, Chiang Mai, measuring indoor radon in 48 homes and enrolling adults from <50 Bq/m³ (low) and ≥100 Bq/m³ (high) households for serum profiling. Mean indoor radon was 61.8 ± 18.4 Bq/m³ (range 34–126), with 6.2% of homes ≥100 Bq/m³. Small RNA sequencing identified 55 differentially expressed miRNAs (12 up, 43 down) in high-radon serum. Notably, miR-200b-3p, miR-200c-3p, and miR-194-5p were increased, while miR-3913-5p, miR-584-5p, miR-30a-3p, miR-22-3p, and miR-125a-5p were decreased. Target enrichment (KEGG/GO) implicated PI3K–Akt and MAPK hubs with Ras/Wnt/VEGF alongside focal adhesion/ECM–receptor/actin–cytoskeleton and immune-regulatory modules. Untargeted LC–MS metabolomics showed exposure-aligned shifts: higher PUFAs and oxylipins (e.g., AA, EPA; 9-HEPE, 8-HETE, 5,12-DiHETE), elevated acyl-carnitines (β-oxidation), and increased inosine/hypoxanthine, consistent with lipid/steroid remodeling, mitochondrial fuel reprogramming, oxidative stress, and nucleotide turnover. Integrated interpretation supports DDR/ATM → PI3K/Akt–MAPK activation with EMT/adhesion remodeling, angiogenic signaling, and immune modulation—linking residential radon to lung cancer mechanisms. Given the small sample size (n = 10), these findings should be interpreted as preliminary and hypothesis-generating, warranting validation in larger cohorts. Nevertheless, findings support household testing, remediation at ≥100 Bq/m³, and integrated exposure studies considering PM2.5 co-exposures. Full article

A novel fluorescent probe (PYB) for selective and sensitive detection of Cu²⁺ ions was rationally designed and synthesized via a multi-step organic reaction using pyrene as the fluorophore and salicylaldehyde-diethylenetriamine Schiff base as the recognition moiety. The structural characterization of PYB was confirmed by ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (HRMS). Photophysical properties investigation revealed that the probe exhibited strong fluorescence emission at 362 nm in DMF/HEPES-NaOH buffer solution (v:v = 1:1, pH 7.4), which underwent a significant fluorescence quenching response (quenching efficiency up to 77%) upon the addition of Cu²⁺, attributed to the formation of a 1:1 PYB-Cu²⁺ complex (binding constant K = 799.65 M⁻¹). The probe showed excellent selectivity for Cu²⁺ over other common metal ions (Ba²⁺, Na⁺, Mg²⁺, Zn²⁺, Cd²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Hg²⁺, Fe³⁺, Co²⁺), with a low detection limit of 8.35 × 10⁻⁷ M, which is well below the maximum allowable concentration of Cu²⁺ in drinking water specified by the World Health Organization (WHO). Furthermore, a portable fluorescent test strip based on PYB was successfully fabricated, enabling rapid and visual detection of Cu²⁺ under UV light. Fluorescence imaging experiments in living HepG2 cells demonstrated that PYB could penetrate cell membranes efficiently and realize the intracellular detection of exogenous Cu²⁺. These results collectively indicate that PYB holds great potential as a practical tool for Cu²⁺ detection in environmental monitoring, food safety, and biological systems. Full article

Fumarate, succinate, maleate, dihydroxyfumarate, D–tartarate, L–tartarate, DL–tartarate, L-malate, D-malate, oxaloacetate, citrate, and DL-isocitrate in the 5–100 μM concentration range were incubated in 12.5 mM HEPES/25 mM TRIS base containing 200 μM Eu³⁺–tetracycline and 60% (v/v) formamide (pH unadjusted). After 30 min of incubation, they were separated at 4 °C by capillary electrophoresis utilizing laser-induced luminescence detection with 12.5 mM HEPES/25 mM TRIS base containing 60% formamide as the running buffer. All analytes yielded peaks, with the exception of fumarate, succinate, and maleate. L-Malate was detected down to 100 nM. The main component of this study was the analysis of malate. The objective was to develop a stereoselective methodology for the detection of L-malate. This was achieved by varying the formamide concentration and separation temperature. When the temperature was increased to 22 °C and the formamide concentration decreased to 40%, the sensitivity for L-malate was diminished about 10-fold, but that for D-malate was eliminated. This combination of conditions allowed for the stereospecific analysis of L-malate. Full article