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Bio-protocol Jul 2023Myeloperoxidase (MPO) is an enzyme contained in lysosomal azurophilic granules of neutrophils. MPO activity has been shown to correlate with the number of neutrophils in...
Myeloperoxidase (MPO) is an enzyme contained in lysosomal azurophilic granules of neutrophils. MPO activity has been shown to correlate with the number of neutrophils in histological sections of the gastrointestinal tract and is therefore accepted as a biomarker of neutrophil invasion in the gut. This protocol describes an easy, cost-effective kinetic colorimetric assay to quantify myeloperoxidase activity in intestinal tissue samples. It is explained using tissue collected in mice but can also be used for other laboratory animals. In a first step, tissue specimens are homogenized using a phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB), which extracts MPO from neutrophils. The obtained supernatant is added to a reagent solution containing o-dianisidine dihydrochloride, which is a peroxidase substrate. Finally, the change in absorption is measured via spectrophotometry and converted to a standardized unit of enzyme activity. The assay is illustrated and compared to a commercially available enzyme-linked immunoassay (ELISA), demonstrating that MPO activity does not necessarily correlate with MPO protein expression in tissue samples. Key features Optimized for use in mice and rats but can also be used for samples of other species. Measures enzymatic activity instead of mRNA or protein expression. Requires a spectrophotometer. Can be performed in duplo using 10 mg of (dry-blotted) gut tissue or more. Graphical overview.
PubMed: 37456337
DOI: 10.21769/BioProtoc.4758 -
ChemPlusChem Jul 2023A mechanochemical method was used to obtain four new quercetin (QUE) co-crystals. Three co-formers have the systems of the heterocyclic rings with the oxygen and...
A mechanochemical method was used to obtain four new quercetin (QUE) co-crystals. Three co-formers have the systems of the heterocyclic rings with the oxygen and nitrogen atoms and they form co-crystals at the stoichiometric ratio of 1 : 2. In contrast, the QUE : o-dianisidine co-crystal represents the 1 : 1 stoichiometry and the former molecule is the aniline derivative. The X-ray crystallography and FT-IR and FT-Raman spectra revealed formation of the intermolecular O-H…N or N-H…O hydrogen bonds. The dynamics of the hydrogen bonds was investigated using the XPS method. The N 1s XPS spectra showed no proton transfer in the QUE : FEN and QUE : O-DIA co-crystal systems. The QUE : BZFP and QUE : EBZFP show the two-site static disorder across the proton transfer pathway to the pyridine ring, with the occupancies (C=N : C=NH ) of 72 : 28 and 77 : 23, respectively.
PubMed: 37337973
DOI: 10.1002/cplu.202300166 -
Analytica Chimica Acta Aug 2023In this work, copper oxide nanoparticles (CuONPs) nanozymes paper-based analytical device was designed for the rapid detection of organophosphate pesticides in fruits...
In this work, copper oxide nanoparticles (CuONPs) nanozymes paper-based analytical device was designed for the rapid detection of organophosphate pesticides in fruits and vegetables. The paper-based analytical device was modified with silica oxide nanoparticles to enhance the assay sensitivity. CuO nanozymes displayed peroxidase-like activity and catalyzed the oxidation of o-dianisidine in the presence of HO from the hydrolysis of acetylthiocholine. This results in the formation of a brown-colored product. In the presence of organophosphate pesticides such as malathion, acetylcholinesterase activity was inhibited, resulting in reduced color intensity production, and which was measured with a smartphone. The proposed nanozymes paper-based analytical device exhibited a good linear detection range (0.1-5 mg L), a low detection limit of 0.08 mg L, and the analysis time was only about 10 min for malathion detection under optimal conditions. Moreover, the CuONPs had excellent catalytic activity and higher stability than peroxidase. Finally, this device can be applied to detect organophosphate pesticides in fruits and vegetables with rapidity, accuracy, portability, and ease of handling in the field.
Topics: Vegetables; Pesticides; Malathion; Fruit; Acetylcholinesterase; Hydrogen Peroxide; Organophosphorus Compounds; Insecticides; Peroxidases; Biosensing Techniques
PubMed: 37257977
DOI: 10.1016/j.aca.2023.341377 -
Comparative Biochemistry and... Jul 2023Sesamin, the major lignan in sesame seeds (Sesamum indicum L.), is known to have several pharmaceutical activities. However, its toxicological profile is still limited,...
Sesamin, the major lignan in sesame seeds (Sesamum indicum L.), is known to have several pharmaceutical activities. However, its toxicological profile is still limited, especially regarding embryotoxicity. This study aimed to evaluate the developmental toxicity of sesamin in zebrafish embryos. After 72 h exposure, sesamin did not affect the survival and hatching rates, nor did it cause malformation in zebrafish embryos. Cardiotoxicity was also evaluated by monitoring embryo heartbeats and erythrocyte staining using o-dianisidine. The results showed that sesamin did not affect heart morphology, heart rate, or cardiac output in zebrafish embryos. The present study also evaluated sesamin's anti-angiogenesis, antioxidant and anti-inflammation activities. Sesamin significantly decreased the sub-intestinal vessel plexus as revealed by alkaline phosphatase staining indicating the compound exhibited anti-angiogenesis activity. For the antioxidant and anti-inflammatory assays, oxidative stress and inflammation in zebrafish embryos were induced by hydrogen peroxide and lipopolysaccharide, respectively. The reactive oxygen species (ROS) and nitric oxide (NO) production were detected using a fluorescent dye. Sesamin significantly decreased ROS and NO production in zebrafish embryos. In addition, the transcription examination by qRT-PCR of oxidative- and inflammation-related genes showed that sesamin affected the genes in a manner that correlated with results from the efficacy assays. In conclusion, the present study revealed that sesamin did not cause embryotoxicity and cardiotoxicity in zebrafish embryos. In addition, it exhibited evidence of anti-angiogenesis, antioxidant and anti-inflammatory activities.
Topics: Animals; Zebrafish; Antioxidants; Reactive Oxygen Species; Cardiotoxicity; Oxidative Stress; Lignans; Inflammation; Anti-Inflammatory Agents; Embryo, Nonmammalian
PubMed: 37098389
DOI: 10.1016/j.cbpc.2023.109637 -
International Journal of Nanomedicine 2023Curcumin (CUR) and piperine (PP) are bioactive compounds with prominent pharmacological activities that have been investigated for the treatment of various diseases. The...
Development of Curcumin and Piperine-Loaded Bio-Active Self-Nanoemulsifying Drugs and Investigation of Their Bioactivity in Zebrafish Embryos and Human Hematological Cancer Cell Lines.
PURPOSE
Curcumin (CUR) and piperine (PP) are bioactive compounds with prominent pharmacological activities that have been investigated for the treatment of various diseases. The aim of the present study is to develop Bio-SNEDDS for CUR and PP as a combined delivery system for cancer therapy.
METHODS
CUR and PP loaded Bio-SNEDDSs with varying compositions of bioactive lipid oils, surfactants, and cosolvents were prepared at room temperature. Bio-SNEDDSs were characterized using a Zetasizer Nano particle size analyzer and further examined by transmission electron microscopy (TEM) for morphology. The in vivo toxicity of the preparations of Bio-SNEDDS was investigated in wild-type zebrafish embryos and cytotoxicity in THP-1 (human leukemia monocytic cells), Jurkat (human T lymphocyte cells) and HUVEC (non-cancerous normal) cells.
RESULTS
Bio-SNEDDSs were successfully developed with black seed oil, Imwitor 988, Transcutol P and Cremophor RH40 at a ratio of 20/20/10/50 (%w/w). The droplet size, polydispersity index and zeta potential of the optimized Bio-SNEDDS were found to be 42.13 nm, 0.59, and -19.30 mV, respectively. Bio-SNEDDS showed a spherical structure evident by TEM analysis. The results showed that Bio-SNEDDS did not induce toxicity in zebrafish embryos at concentrations between 0.40 and 30.00 μg/mL. In TG (fli1: EGFP) embryos treated with Bio-SNEDDS, there was no change in the blood vessel structure. The O-dianisidine staining of Bio-SNEDDS treated embryos at 48 h post-fertilization also showed a significant reduction in the number of blood cells compared to mock (DMSO 0.1% V/V) treated embryos. Bio-SNEDDS induced significant levels of cytotoxicity in the hematological cell lines THP-1 and Jurkat, while low toxicity in normal HUVEC cell lines was observed with IC50 values of 18.63±0.23 μg/mL, 26.03 ± 1.5 μg/mL and 17.52 ± 0.22 μg/mL, respectively.
CONCLUSION
Bio-SNEDDS exhibited enhanced anticancer activity and could thus be an important new pharmaceutical formulation to treat leukemia.
Topics: Animals; Humans; Pharmaceutical Preparations; Curcumin; Zebrafish; Drug Delivery Systems; Solubility; Administration, Oral; Surface-Active Agents; Hematologic Neoplasms; Leukemia; Emulsions; Nanoparticles; Biological Availability
PubMed: 37051315
DOI: 10.2147/IJN.S400330 -
Bioprocess and Biosystems Engineering Apr 2023Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests,...
Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40 kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04 M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.
Topics: Recombinant Proteins; Isoenzymes; Horseradish Peroxidase; Escherichia coli; Pichia
PubMed: 36527454
DOI: 10.1007/s00449-022-02837-2 -
Bioprocess and Biosystems Engineering Mar 2023Dye-contaminated wastewater discharge from textile and dye manufacturing industries is reported as a world worse water polluter due to the toxic and mutagenic behavior...
Dye-contaminated wastewater discharge from textile and dye manufacturing industries is reported as a world worse water polluter due to the toxic and mutagenic behavior of dyes. Peroxidase, one of the key enzymes of oxidoreductases, is widely distributed in nature and has been currently exploited in industries for various applications. Widespread applications of peroxidases are associated with their nonspecific nature towards a wide spectrum of substrates such as phenols, aromatic amines, pesticides, antibiotics, and synthetic dyes. The present study explored the potential of ammonium sulfate precipitated partially purified Brassica oleracea L. var. botrytis leaves peroxidase for degradation of reactive textile dyes Remazol Turquoise Blue 133 G and Drim Red CL4BN. Various physico-chemical parameters such as pH (2-9), temperature (20-70 ℃), enzyme activity (3-24 U/mL), concentrations of HO (0.4-1.4 Mm) and dye (10-100 mg/L) were optimized for enzymatic decolorization of both dyes' solution. Studies revealed that maximum degradation (95%) of Remazol Turquoise Blue 133 G with peroxidase was achieved with 25 mg/L of initial dye concentration, in the presence of 0.8 mM hydrogen peroxide with 45 min of incubation time, at pH 3, 4, and 5, and 70 °C. Maximal decolorization (97%) of Drim Red CL4BN was obtained at pH 2.0, in 10 min of incubation time at 45 ℃ using o-dianisidine hydrochloride as a redox mediator. In conclusion, the findings illustrate the prospect of Brassica oleracea peroxidase to remediate dye pollutants and dye-based industrial effluents in a green technology theme.
Topics: Peroxidase; Botrytis; Hydrogen Peroxide; Peroxidases; Coloring Agents; Textile Industry; Textiles; Plant Leaves; Brassica; Biodegradation, Environmental
PubMed: 36454313
DOI: 10.1007/s00449-022-02820-x -
Spectrochimica Acta. Part A, Molecular... Jan 2023In this paper, we report a novel surface-enhanced Raman spectroscopy (SERS) substrate based on hierarchical β-BiO/AuAg microspheres for rapid, sensitive and selective...
In this paper, we report a novel surface-enhanced Raman spectroscopy (SERS) substrate based on hierarchical β-BiO/AuAg microspheres for rapid, sensitive and selective detection of environment pollutants including o-dianisidine (o-diASD) and Hg in environmental samples. The sheet-like β-BiO not only provides large specific surface areas for adsorption of molecules and AuAg, but also emerges as semiconductor matrix with chemical enhancement combined with AuAg with electromagnetic enhancement, making promising SERS activity. Particularly, the β-BiO/AuAg shows high SERS performance for 4-mercaptobenzoic acid and TMB with minimum detectable concentration of 0.1 μg/L with enhancement factor of 3.1 × 10 and 6.3 × 10, respectively. The density functional theory simulations were further adopted to explain the high SERS activity and selectivity for o-diASD and TMB. Finally, the β-BiO/AuAg was applied to direct detection of o-diASD, and indirect detection of Hg by TMB marking in environmental samples. The linearity range of 0.5-200.0 and 0.2-500.0 μg/L with limit of detection of 0.2 and 0.07 μg/L for o-diASD and Hg ions can be achieved, respectively. This method provides a novel strategy in designing and fabricating SERS substrates with high performance for rapid, sensitive and accurate analysis of environmental pollutants.
Topics: Spectrum Analysis, Raman; Metal Nanoparticles; Environmental Pollutants; Microspheres; Mercury
PubMed: 36179562
DOI: 10.1016/j.saa.2022.121907 -
Journal of Microbiology (Seoul, Korea) Aug 2022Hydrogen peroxide (HO) is produced by alpha-hemolytic streptococci in aerobic conditions. However, the suitable method for detection of HO-producing streptococci in oral...
Hydrogen peroxide (HO) is produced by alpha-hemolytic streptococci in aerobic conditions. However, the suitable method for detection of HO-producing streptococci in oral microbiota has not been setup. Here we show that o-dianisidine dye and horseradish peroxidase were useful in tryptic soy agar medium to detect and isolate HO-producing bacteria with the detection limit of one target colony in > 10 colony-forming units. As a proof, we isolated the strain HP01 (KCTC 21190) from a saliva sample using the medium and analyzed its characteristics. Further tests showed that the strain HP01 belongs to Streptococcus oralis in the Mitis group and characteristically forms short-chain streptococcal cells with a high capacity of acid tolerance and biofilm formation. The genome analysis revealed divergence of the strain HP01 from the type strains of S. oralis. They showed distinctive phylogenetic distances in their ROS-scavenging proteins, including superoxide dismutase SodA, thioredoxin TrxA, thioredoxin reductase TrxB, thioredoxin-like protein YtpP, and glutaredoxin-like protein NrdH, as well as a large number of antimicrobial resistance genes and horizontally transferred genes. The concatenated ROS-scavenging protein sequence can be used to identify and evaluate Streptococcus species and subspecies based on phylogenetic analysis.
Topics: Hydrogen Peroxide; Phylogeny; Reactive Oxygen Species; Saliva; Streptococcus; Streptococcus oralis; Thioredoxins
PubMed: 35835959
DOI: 10.1007/s12275-022-2261-2 -
The Science of the Total Environment Oct 2022Ethylparaben (EP), one of the parabens, a ubiquitous food and cosmetic preservatives, has caused widespread concern due to its health risks. Recently, studies have found...
Ethylparaben (EP), one of the parabens, a ubiquitous food and cosmetic preservatives, has caused widespread concern due to its health risks. Recently, studies have found that parabens exposure during pregnancy is negatively correlated with fetal and early childhood development. However, studies about EP on embryo development are few. In this study, the cardiotoxicity effects of EP concentrations ranging from 0 to 20 mg/L on zebrafish embryo development were explored. Results showed that EP exposure induce abnormal cardiac function and morphology, mainly manifested as pericardial effusion and abnormal heart rate in early-stage development of zebrafish embryos. Through transcriptome sequencing followed by Gene Ontology enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, we further confirmed that EP exposure ultimately leads to cardiac morphologic abnormalities via the following three mechanisms: 1. Disruption of the retinoic acid signaling pathway related to original cardiac catheter development; 2. Inhibition of gene expression related to myocardial contraction; 3. Orientation development disturbance of heart tube. Moreover, O-Dianisidine staining, whole-mount in situ hybridization at 30 and 48 hours post fertilization (hpf) and hematoxylin-eosin staining results all confirmed the decreased heart's return blood volume, misoriented heart tubes toward either the right or the middle side, and heart loop defects. For the first time, we explored the mechanism by which EP exposure causes abnormal heart development in zebrafish embryos, laying the foundation for further revealing of the EP toxicity on embryonic development.
Topics: Animals; Cardiotoxicity; Child, Preschool; Embryo, Nonmammalian; Gene Expression Profiling; Humans; Parabens; Transcriptome; Zebrafish
PubMed: 35752233
DOI: 10.1016/j.scitotenv.2022.156785