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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 -
Integrative Medicine (Encinitas, Calif.) May 2024This literature review explores the role of dietary oxalate in the development of chronic inflammatory kidney disease in middle-aged and older individuals. The authors... (Review)
Review
This literature review explores the role of dietary oxalate in the development of chronic inflammatory kidney disease in middle-aged and older individuals. The authors pose the following questions: Is oxalate produced endogenously? If food sources contribute to chronic kidney disease and inflammation, what are those foods? What role do cultural food preparation and cooking play in denaturing food oxalates? The concentration of oxalates found within the body at any particular time is not limited to edible plants; normal human metabolic processes of breaking down ascorbic acid may create up to 30 mg of oxalate daily. Research supports urolithiasis as a common urologic disease in industrialized societies. Approximately 80% of kidney stones are composed of calcium oxalate, resulting in hyperoxaluria. Exogenous (originating outside the cell or organism) oxalate sources include ascorbic acid, amino acids, and glyoxal metabolism. Additional research estimates the daily endogenous (produced within the cell or organism) production of oxalate to be 10-25 mg. Suboptimal colonization of oxalate-degrading bacteria and malabsorptive disease are also contributing factors to the development of chronic kidney disease. Oxalate transcellular processes, though poorly understood, rely on multifunctional anion exchangers, and are currently being investigated. A review of research showed that normal human metabolic processes, including the breakdown of ascorbic acid, account for 35-55% of circulating oxalates and can create ≤30 mg of circulating serum oxalate daily. Glyoxylic acid accounts for 50-70% of circulating urinary oxalate in compromised individuals with liver glycation, bacterial insufficiencies, malabsorption, and anion exchange challenges. For persons with a family history of kidney stones, consumption of foods high in oxalates may be consumed in moderation, provided there is adequate calcium intake in the diet to decrease the absorption of oxalates from the meal ingested.
PubMed: 38911445
DOI: No ID Found -
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 -
Methods in Molecular Biology (Clifton,... 2024We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass...
We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.
Topics: Proteomics; Single-Cell Analysis; Chromatography, Liquid; Humans; Mass Spectrometry; Proteome; Proteins
PubMed: 38907148
DOI: 10.1007/978-1-0716-3934-4_7 -
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 -
Archives of Biochemistry and Biophysics Jun 2024One of important characteristics of Alzheimer's disease is a persistent oxidative/nitrosative stress caused by pro-oxidant properties of amyloid-beta peptide (Aβ) and... (Review)
Review
One of important characteristics of Alzheimer's disease is a persistent oxidative/nitrosative stress caused by pro-oxidant properties of amyloid-beta peptide (Aβ) and chronic inflammation in the brain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is easily oxidized under oxidative stress. Numerous data indicate that oxidative modifications of GAPDH in vitro and in cell cultures stimulate GAPDH denaturation and aggregation, and the catalytic cysteine residue Cys152 is important for these processes. Both intracellular and extracellular GAPDH aggregates are toxic for the cells. Interaction of denatured GAPDH with soluble Aβ results in mixed insoluble aggregates with increased toxicity. The above-described properties of GAPDH (sensitivity to oxidation and propensity to form aggregates, including mixed aggregates with Aβ) determine its role in the pathogenesis of Alzheimer's disease.
PubMed: 38906311
DOI: 10.1016/j.abb.2024.110065 -
Molecular Biology Reports Jun 2024Protein nanocages resemble natural biomimetic carriers and can be engineered to act as targeted delivery systems, making them an attractive option for various drug...
Protein nanocages resemble natural biomimetic carriers and can be engineered to act as targeted delivery systems, making them an attractive option for various drug delivery and biomedical applications. Our research investigated the genetic link of a specific anti-HER2 peptide (LTVSPWY) to the exposed N-terminal region of the maize (Zea mays) ferritin 1 (ZmFer1) protein nanocage, employing either a 7-amino acid (for LTVS-ZmFer1) or 16-amino acid (for LTVS-L-ZmFer1) linker. We utilized a heat treatment method to load the chemotherapeutic drug doxorubicin into the protein nanocage. The construct with the longer linker (LTVS-L) produced a greater amount of soluble protein nanocage and was selected for further experiments. The average size, polydispersity index, and zeta potential of the engineered protein nanocage were 19.01 nm, 0.168, and - 2.13 mV, respectively. The LTVS-L-ZmFer1 protein nanocage exhibited excellent thermal stability, withstanding temperatures up to 100 °C with only partial denaturation. Furthermore, we observed that cellular uptake of the LTVS-L-ZmFer1 protein nanocages in HER2-positive breast cancer cells was significantly higher compared to ZmFer1 after labeling with FITC (fluorescein isothiocyanate) (P-value = 0.0001). In addition, we observed a significant decrease in the viability of SKBR3 cells when treated with DOX-loaded LTVS-L-ZmFer1 protein nanocages compared to cells treated with DOX-loaded ZmFer1 protein nanocages. Therefore, this new treatment strategy may prove to be an effective way to reduce both the side effects and toxicity associated with conventional cancer treatments in patients with HER2-positive breast cancer.
Topics: Humans; Receptor, ErbB-2; Doxorubicin; Drug Delivery Systems; Cell Line, Tumor; Breast Neoplasms; Recombinant Proteins; Ferritins; Zea mays; Protein Engineering; Female; Drug Carriers; Plant Proteins
PubMed: 38904710
DOI: 10.1007/s11033-024-09636-w -
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