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ACS Bio & Med Chem Au Jun 2024Synthetic modification of oligodeoxynucleotides (ODNs) via conjugation to nucleic acid binding small molecules can improve hybridization and pharmacokinetic properties....
Synthetic modification of oligodeoxynucleotides (ODNs) via conjugation to nucleic acid binding small molecules can improve hybridization and pharmacokinetic properties. In the present study, five Hoechst 33258 derived benzimidazoles were conjugated to T rich ODNs and their hybridization effectiveness was tested. Thermal denaturation studies revealed significant stabilization of complementary duplexes by ODN-benzimidazole conjugates, with the extent of stabilization being highly dependent on the length of the linker between DNA and benzimidazole. The increases in thermal stability were determined to be due to the binding of the benzimidazole moiety to the duplex. Circular dichroism and molecular modeling studies provided insights toward the influence of conjugation on duplex structure and how linker length impacts placement of the benzimidazole moiety in the minor groove. Furthermore, thermal denaturation studies with the complementary strand containing a single base mismatch or being RNA revealed that covalent conjugation of benzimidazoles to an ODN also enhances the sequence specificity. The fundamental studies reported herein provide a strategy to improve the stability and specificity properties of the ODN probes, which can be of use for targeting and diagnostics applications.
PubMed: 38911908
DOI: 10.1021/acsbiomedchemau.3c00074 -
Journal of Lipid Research Jun 2024Dramatic post-mortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an...
Dramatic post-mortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing post-mortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PG are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O. Based on this finding, we proposed a simple and reproducible alternative method to prevent post-mortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100 °C after ∼140 sec during boiling, though 3 min boiling was required to completely prevent post-mortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant post-mortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents post-mortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.
PubMed: 38909689
DOI: 10.1016/j.jlr.2024.100583 -
International Journal of Biological... Jun 2024Understanding how shear affects whey protein stability is crucial to deal with typical industrial issues occurring at the bulk solution/surface interface, such as...
Understanding how shear affects whey protein stability is crucial to deal with typical industrial issues occurring at the bulk solution/surface interface, such as fouling during heat treatments. However, at the state of the art, this effect remains unclear, contrary to that of temperature. This article presents a novel strategy to study the impact of shear rate and concentration on the accumulation of whey protein surficial deposits. It consists in applying a range of shear rates (0-200 s) at controlled temperature (65 °C) on whey protein solutions (5-10 wt%) by a parallel plate rheometer equipped with a glass disc, thus allowing the off-line characterization of the deposits by microscopy. Our results highlight an unequivocal effect of increasing shear stress. At 5 wt%, it fosters the formation of primary deposits (≈ 10 μm), whereas at 10 wt% it results in the development of complex branched structures (≈ 50 μm) especially for shear rates ranging from 140 s to 200 s. Based on the classification by size of the observed populations, we discuss possible hypotheses for the deposit growth kinetics, involving the interplay of different physico-chemical protein-surface interactions and paving the way to future further investigations.
PubMed: 38908625
DOI: 10.1016/j.ijbiomac.2024.133291 -
Scientific Reports Jun 2024Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars...
Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars represents the preferred strategy to combat these diseases. Moreover, achieving collaborative improvement between diseases resistance, abiotic stress, quality, and agronomic and yield traits is difficult in genetic breeding. In this study, the wheat cultivar, Guinong 29 (GN29), showed high resistance to powdery mildew and stripe rust at both seedling and adult plant stages, and was susceptible to leaf rust at the seedling stage but slow resistance at the adult-plant stage. Meanwhile, it has elite agronomic and yield traits, indicating promising coordination ability among multiple diseases resistance and other key breeding traits. To determine the genetic basis of these elite traits, GN29 was tested with 113 molecular markers for 98 genes associated with diseases resistance, stress tolerance, quality, and adaptability. The results indicated that two powdery mildew resistance (Pm) genes, Pm2 and Pm21, confirmed the outstanding resistance to powdery mildew through genetic analysis, marker detection, genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), and homology-based cloning; the stripe rust resistance (Yr) gene Yr26 and leaf rust resistance (Lr) genes Lr1 and Lr46 conferred the stripe rust and slow leaf rust resistance in GN29, respectively. Meanwhile, GN29 carries dwarfing genes Rht-B1b and Rht-D1a, vernalization genes vrn-A1, vrn-B1, vrn-D1, and vrn-B3, which were consistent with the phenotypic traits in dwarf characteristic and semi-winter property; carries genes Dreb1 and Ta-CRT for stress tolerance to drought, salinity, low temperature, and abscisic acid (ABA), suggesting that GN29 may also have elite stress-tolerance ability; and carries two low-molecular-weight glutenin subunit genes Glu-B3b and Glu-B3bef which contributed to high baking quality. This study not only elucidated the genetic basis of the elite traits in GN29 but also verified the capability for harmonious improvement in both multiple diseases resistance and other comprehensive traits, offering valuable information for breeding breakthrough-resistant cultivars.
Topics: Triticum; Disease Resistance; Plant Diseases; Ascomycota; Plant Breeding; Phenotype; Basidiomycota; Genes, Plant; Chromosome Mapping
PubMed: 38906938
DOI: 10.1038/s41598-024-64998-2 -
Frontiers in Nutrition 2024Biocatalysts (enzymes) play a crucial role in catalyzing specific reactions across various industries, often offering environmentally friendly and sustainable...
BACKGROUND
Biocatalysts (enzymes) play a crucial role in catalyzing specific reactions across various industries, often offering environmentally friendly and sustainable alternatives to chemical catalysts. However, their catalytic activities are susceptible to denaturation. In this study, we present the discovery of novel protein-based biocatalysts derived from processed foods, including skimmed milk, soy milk, cheese, and dried tofu. These food catalysts exhibit high availability, low cost, safety, and thermo-stability.
RESULTS
Focusing on the physiologically intriguing coenzyme pyrroloquinoline quinone (PQQ), we observed that the reaction with glycine to form imidazolopyrroquinoline (IPQ) did not proceed efficiently when PQQ was present at very low concentrations. Surprisingly, in the presence of protein-based foods, this reaction was significantly accelerated. Notably, skimmed milk enhanced the PQQ detection limit (600 times lower) during high-performance liquid chromatography (HPLC) following IPQ derivatization. Milk appears to facilitate the reaction between PQQ and various amino acids, primary amines, and secondary amines. Further investigations revealed that food catalysis operates through a non-enzymatic mechanism. Additionally, nuclear magnetic resonance spectroscopy demonstrated that milk components interacted with amino substrates due to the ability of amines to react with quinones on colloidal surfaces.
CONCLUSION
These practical food catalysts not only contribute to environmental safety but also hold significance across diverse scientific domains. Non-enzymatic protein catalysts find applications in biocatalysis, organic synthesis, food technology, analytical chemistry, and fundamental nutritional and evolutionary studies.
PubMed: 38903631
DOI: 10.3389/fnut.2024.1391681 -
Frontiers in Nutrition 2024The domestic silkworm, , has been widely used in silk production for centuries. It is also used as a bioreactor by the textile and pharmaceutical industries to mass...
The domestic silkworm, , has been widely used in silk production for centuries. It is also used as a bioreactor by the textile and pharmaceutical industries to mass produce recombinant bioactive proteins containing silk-based materials. Furthermore, silkworms are well-known as a source of food and have also been orally administered to prevent and treat several human disorders. In this study, we aimed to investigate the inherent bio-physicochemical properties of edible silkworms to accurately evaluate their clinical and nutritional potential. We prepared raw powder from whole larvae of silkworm. The yield rate of the powder derived from dried larvae was almost 100% (98.1-99.1% in replicates). As "percentage yield" translates to "Budomari" in Japanese, this raw powder was named "B100rw." We further prepared B100dn that was denatured through autoclaving. Thereafter, we examined whether B100rw sustained the original bio-physicochemical properties by comparing it with B100dn. There was no significant difference in nutritional content between B100rw and B100dn. B100rw contained proteins derived from silkworm larvae and mulberry leaves, whereas the proteins of B100dn were mostly degraded. On measuring the enzymatic activity of both powders using trehalase as an indicator enzyme, B100rw was found to maintain trehalase activity. B100rw also maintained a random coil conformation, similar to that of liquid silk. This suggested that B100rw sustained the unique bio-physicochemical properties of living larvae. These findings may facilitate the development of novel food products or orally administered vaccines.
PubMed: 38903626
DOI: 10.3389/fnut.2024.1404489 -
BioRxiv : the Preprint Server For... Jun 2024How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and...
UNLABELLED
How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from . Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiB's landscape. Firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this "Enigma" state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.
SIGNIFICANCE STATEMENT
One can conceptualize KaiB as an on-off switch to regulate circadian rhythms in bacteria, where the "On state" is the Fold-switched state that binds KaiC and other proteins, and the "Off state" is the Ground state. Our work exemplifies how evolution tuned the kinetics of interconversion to align with the hour-long timescale of its biological function. The Ground state is dramatically destabilized at cold temperatures, and the system contains an alternate "off" conformation that exchanges with the primary "off" conformation at faster timescales than the rate-limiting step. Our findings demonstrate a simple principle for evolving a protein switch: one part of a protein domain remains stably folded to serve as a scaffold for the rest of the protein to re-fold.
PubMed: 38895306
DOI: 10.1101/2024.06.03.597139 -
BioRxiv : the Preprint Server For... Jun 2024Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with...
Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with redox-active sulfur and function in prokaryotic restriction-modification and transcriptional regulation. Interest in PTs has grown due to the widespread distribution of the , and genes among bacteria and archaea, as well as the discovery of PTs in 5-10% of gut microbes. Efforts to map PTs in complex microbiomes using existing next-generation and direct sequencing technologies have failed due to poor sensitivity. Here we developed PT-seq as a high-sensitivity method to quantitatively map PTs across genomes and metagenomically identify PT-containing microbes in complex genomic mixtures. Like other methods for mapping PTs in individual genomes, PT-seq exploits targeted DNA strand cleavage at PTs by iodine, followed by sequencing library construction using ligation or template switching approaches. However, PT-specific sequencing reads are dramatically increased by adding steps to heat denature the DNA, block pre-existing 3'-ends, fragment DNA after T-tailing, and enrich iodine-induced breaks using biotin-labeling and streptavidin beads capture. Iterative optimization of the sensitivity and specificity of PT-seq is demonstrated with individual bacteria and human fecal DNA.
PubMed: 38895297
DOI: 10.1101/2024.06.03.597111 -
Anti-Inflammatory Activity of No-Ozone Cold Plasma in Lipopolysaccharide-Induced Periodontitis Rats.International Journal of Molecular... Jun 2024Periodontitis is an inflammatory disease caused by () in the oral cavity. This periodontal disease causes damage to the periodontal ligament and alveolar bone and can...
Periodontitis is an inflammatory disease caused by () in the oral cavity. This periodontal disease causes damage to the periodontal ligament and alveolar bone and can cause tooth loss, but there is no definite treatment yet. In this study, we investigated the possibility of using no-ozone cold plasma to safely treat periodontitis in the oral cavity. First, human gingival fibroblasts (HGFs) were treated with -derived lipopolysaccharide (PG-LPS) to induce an inflammatory response, and then the anti-inflammatory effect of NCP was examined, and a study was conducted to identify the mechanism of action. Additionally, the anti-inflammatory effect of NCP was verified in rats that developed an inflammatory response similar to periodontitis. When NCP was applied to PG-LPS-treated HGFs, the activities of inflammatory proteins and cytokines were effectively inhibited. It was confirmed that the process of denaturing the medium by charged particles of NCP is essential for the anti-inflammatory effect of NCP. Also, it was confirmed that repeated treatment of periodontitis rats with NCP effectively reduced the inflammatory cells and osteoclast activity. As a result, this study suggests that NCP can be directly helpful in the treatment of periodontitis in the future.
Topics: Animals; Porphyromonas gingivalis; Periodontitis; Lipopolysaccharides; Rats; Anti-Inflammatory Agents; Humans; Gingiva; Fibroblasts; Ozone; Plasma Gases; Male; Cytokines; Disease Models, Animal; Nitric Oxide; Cells, Cultured
PubMed: 38892350
DOI: 10.3390/ijms25116161 -
International Journal of Molecular... May 2024Dermatology and cosmetology currently prioritize healthy, youthful-looking skin. As a result, research is being conducted worldwide to uncover natural substances and...
Dermatology and cosmetology currently prioritize healthy, youthful-looking skin. As a result, research is being conducted worldwide to uncover natural substances and carriers that allow for controlled release, which could aid in the battle against a variety of skin illnesses and slow the aging process. This study examined the biological and physicochemical features of novel hydrogels containing cannabidiol (CBD) and α-terpineol (TER). The hydrogels were obtained from ε-caprolactone (CL) and poly(ethylene glycol) (PEG) copolymers, diethylene glycol (DEG), poly(tetrahydrofuran) (PTHF), 1,6-diisocyanatohexane (HDI), and chitosan (CHT) components, whereas the biodegradable oligomers were synthesized using the enzyme ring-opening polymerization (e-ROP) method. The in vitro release rate of the active compounds from the hydrogels was characterized by mainly first-order kinetics, without a "burst release". The antimicrobial, anti-inflammatory, cytotoxic, antioxidant, and anti-aging qualities of the designed drug delivery systems (DDSs) were evaluated. The findings indicate that the hydrogel carriers that were developed have the ability to scavenge free radicals and impact the activity of antioxidant enzymes while avoiding any negative effects on keratinocytes and fibroblasts. Furthermore, they have anti-inflammatory qualities by impeding protein denaturation as well as the activity of proteinase and lipoxygenase. Additionally, their ability to reduce the multiplication of pathogenic bacteria and inhibit the activity of collagenase and elastase has been demonstrated. Thus, the developed hydrogel carriers may be effective systems for the controlled delivery of CBD, which may become a valuable tool for cosmetologists and dermatologists.
Topics: Hydrogels; Cannabidiol; Skin; Humans; Cyclohexane Monoterpenes; Antioxidants; Regeneration; Polymers; Biocompatible Materials; Keratinocytes; HaCaT Cells; Drug Carriers; Drug Delivery Systems; Anti-Infective Agents
PubMed: 38892121
DOI: 10.3390/ijms25115934