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International Journal of Molecular... May 2024The wide use of mono- or bis-styryl fluorophores in biomedical applications prompted the presented design and study of a series of trimeric and tetrameric...
The wide use of mono- or bis-styryl fluorophores in biomedical applications prompted the presented design and study of a series of trimeric and tetrameric homo-analogues, styryl moieties arranged around a central aromatic core. The interactions with the most common biorelevant targets, ds-DNA and ds-RNA, were studied by a set of spectrophotometric methods (UV-VIS, fluorescence, circular dichroism, thermal denaturation). All studied dyes showed strong light absorption in the 350-420 nm range and strongly Stokes-shifted (+100-160 nm) emission with quantum yields () up to 0.57, whereby the mentioned properties were finely tuned by the type of the terminal cationic substituent and number of styryl components (tetramers being red-shifted in respect to trimers). All studied dyes strongly interacted with ds-DNA and ds-RNA with 1-10 nM affinity, with dye emission being strongly quenched. The tetrameric analogues did not show any particular selectivity between ds-DNA or ds-RNA due to large size and consequent partial, non-selective insertion into DNA/RNA grooves. However, smaller trimeric styryl series showed size-dependent selective stabilization of ds-DNA vs. ds-RNA against thermal denaturation and highly selective or even specific recognition of several particular ds-DNA or ds-RNA structures by induced circular dichroism (ICD) bands. The chiral (ICD) selectivity was controlled by the size of a terminal cationic substituent. All dyes entered efficiently live human cells with negligible cytotoxic activity. Further prospects in the transfer of ICD-based selectivity into fluorescence-chiral methods (FDCD and CPL) is proposed, along with the development of new analogues with red-shifted absorbance properties.
Topics: Circular Dichroism; Humans; DNA; Fluorescent Dyes; RNA, Double-Stranded; Cations; Spectrometry, Fluorescence; Styrenes; Nucleic Acid Denaturation
PubMed: 38891911
DOI: 10.3390/ijms25115724 -
Foods (Basel, Switzerland) Jun 2024Soy remains the legume protein of excellence for plant-based meat alternatives due to its fiber-forming potential. In this study, protein-rich powders from soy protein...
Soy remains the legume protein of excellence for plant-based meat alternatives due to its fiber-forming potential. In this study, protein-rich powders from soy protein isolate (SPI), concentrate (SPC), and their mixture (SPM) were thoroughly characterized for their proximate composition, nutritional quality, and physicochemical properties to understand their structuring behavior during high-moisture extrusion. SPI presented higher degrees of protein denaturation and aggregation, least gelation concentration and lower essential amino acid contents. Thus, an SPI:SPC combination (1:9 ratio, 70% protein) was extruded at three different screw speeds (300, 350, and 400 rpm) and two temperature profiles (120 and 140 °C maximum temperature). The effects of the processing parameters on the extrudates were evaluated for their appearance (fibrousness), texture (TPA, cutting force, and anisotropy), color, protein structure (FTIR), and trypsin inhibitors. Higher temperatures resulted in softer and darker extrudates, with increased visual and instrumental anisotropy. Increasing screw speeds led to softer and lighter extrudates, without a clear fibrousness effect. β-sheet structures decreased and intermolecular aggregates (A1) increased after extrusion, especially at 140 °C, together with the formation of intramolecular aggregates (A2). Extrusion also significantly decreased the amount of trypsin inhibitors (>90%). This study demonstrates that extrusion parameters need to be carefully selected to achieve meat analogs with optimal textural and nutritional characteristics.
PubMed: 38890977
DOI: 10.3390/foods13111748 -
Nature Protocols Jun 2024Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking... (Review)
Review
Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples.
PubMed: 38890499
DOI: 10.1038/s41596-024-01004-z -
Frontiers in Nutrition 2024Composite natural emulsifiers such as whey protein isolate (WPI) and chitosan (CS) are commonly used in Pickering emulsions to address the effect of thermal deformation...
Composite natural emulsifiers such as whey protein isolate (WPI) and chitosan (CS) are commonly used in Pickering emulsions to address the effect of thermal deformation of proteins before complexation with CS and heating after complexation. In this study, the properties of WPI and CS composites were investigated by complexing CS with either unmodified WPI or thermally denatured WPI (DWPI). Three types of composite particles were prepared, WPI-CS, DWPI-CS, and D(WPI-CS). Atomic force microscopy revealed that the composite particles formed larger aggregates with increased contour size and surface roughness compared to CS and WPI, whereas the interfacial tension decreased, indicating improved emulsifying abilities. Fourier-transform infrared analysis revealed differences in the hydrogen bonds between CS and WPI/DWPI. All three composite particles formed stable emulsions with droplet sizes of 20.00 ± 0.15, 27.80 ± 0.35, and 16.77 ± 0.51 μm, respectively. Thermal stability experiments revealed that the curcumin emulsion stabilized with WPI-CS and DWPI-CS exhibited relatively better thermal stability than that stabilized with D(WPI-CS). experiments results indicated that the bioaccessibility of the curcumin emulsion stabilized with WPI-CS was 61.18 ± 0.16%, significantly higher than that of the emulsions prepared with the other two composite particles ( < 0.05). This study will enable the customized design of WPI composite-based Pickering emulsions for application in the food and nutrition industries.
PubMed: 38887503
DOI: 10.3389/fnut.2024.1418120 -
Cell & Bioscience Jun 2024Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated...
BACKGROUND
Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics.
RESULTS
In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins.
CONCLUSIONS
Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology.
PubMed: 38886783
DOI: 10.1186/s13578-024-01265-x -
The Biochemical Journal Jun 2024Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and...
Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper fruit ripening and after exposure to NO enriched atmosphere, a broad analysis has allowed to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was downregulated by 50% in ripe fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by peroxynitrite, occurring near the catalase active center. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of fruits, with activity significantly downregulated in ripe fruits. Nitration plays a key role in this downregulation, favoring an increase in H2O2 during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is downregulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.
PubMed: 38884605
DOI: 10.1042/BCJ20240247 -
Journal of Pharmacy & Bioallied Sciences Apr 2024Considerable focus has been directed toward green synthesis as a dependable, sustainable, and environmentally friendly approach for synthesizing various nanomaterials. ,...
BACKGROUND
Considerable focus has been directed toward green synthesis as a dependable, sustainable, and environmentally friendly approach for synthesizing various nanomaterials. , a quickly grown pantropical weed, has been used widely for its anti-inflammatory and antimicrobial activity in traditional medicine. The development of strontium-based nanoparticles and nanoparticles linked with strontium has garnered attention in recent years due to their established utility in diverse domains such as effective drug distribution, bioimaging, cancer treatment, and advancements in bone engineering.
AIMS AND OBJECTIVES
To examine the green synthesise of strontium nanoparticles using Mimosa pudica and its anti-inflammatory activity.
MATERIAL AND METHODS
-mediated strontium nanoparticles' anti-inflammatory activity was tested using bovine serum albumin denaturation assay, egg albumin denaturation assay, and membrane stabilization assay with diclofenac sodium as the standard. Result: In all three assays, increasing concentration of -mediated strontium nanoparticles exhibited an increasing anti-inflammatory effect, which was similar to the standard diclofenac sodium.
CONCLUSION
Consequently, this holds promise as a new potential anti-inflammatory agent in forthcoming applications.
PubMed: 38882793
DOI: 10.4103/jpbs.jpbs_586_23 -
Journal of Pharmacy & Bioallied Sciences Apr 2024contains andrograpanin, which is both anti-inflammatory and anti-infective. comprises over 150-200 species from the family . exerts various properties, including...
contains andrograpanin, which is both anti-inflammatory and anti-infective. comprises over 150-200 species from the family . exerts various properties, including anti-inflammatory property. Herbal mouthwash was made using and extract. The anti-inflammatory effect was evaluated using an albumin denaturation assay and egg albumin denaturation. The percentage of protein denaturation that is inhibited by the formulation of and indicates that it has strong anti-inflammatory effect. According to the findings, as concentration is raised, the formulation's anti-inflammatory activity rises. The formulation's percentage inhibition values are also equivalent to those of a typical anti-inflammatory medicine, indicating that it may be effective as a natural anti-inflammatory agent.
PubMed: 38882775
DOI: 10.4103/jpbs.jpbs_581_23 -
Peptide Science (Hoboken, N.J.) Mar 2024Coiled coils are one of most common protein quaternary structures and represent the best understood relationship between amino acid sequence and protein conformation....
Coiled coils are one of most common protein quaternary structures and represent the best understood relationship between amino acid sequence and protein conformation. Whereas the roles of residues at the canonical heptad positions the , , , and are understood in precise detail, conventional approaches often assume that the solvent-exposed -, -, and -positions can be varied broadly for application-specific purposes with minimal consequences. However, a growing body of evidence suggests that interactions among these , , and residues can contribute substantially to coiled-coil conformational stability. In the trimeric coiled coil described here, we find that -position Glu10 engages in a stabilizing long-range synergistic interaction with -position Lys18 (ΔΔΔG = -0.65 ± 0.02 kcal/mol). This favorable interaction depends strongly on the presence of two nearby -position residues: Lys 7 and Tyr14. Extensive mutational analysis of these residues in the presence of added salt vs. denaturant suggests that this long-range synergistic interaction is primarily electrostatic in origin, but also depends on the precise location and acidity of a side-chain hydrogen-bond donor within -position Tyr14.
PubMed: 38882551
DOI: 10.1002/pep2.24336 -
GeroScience Jun 2024Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly...
Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age.For precision analysis, three different plasma pools were analysed over 26 days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3 months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10 years, respectively. Samples were collected every 3 months and 3 weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits.The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits.The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths.
PubMed: 38877341
DOI: 10.1007/s11357-024-01239-4