-
PloS One 2017Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino... (Comparative Study)
Comparative Study
Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos.
Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Base Sequence; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Hematopoietic System; In Situ Hybridization; Membrane Proteins; Morpholinos; Mutation; Phenotype; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Zebrafish; Zebrafish Proteins
PubMed: 28192479
DOI: 10.1371/journal.pone.0172227 -
Aquatic Toxicology (Amsterdam,... Apr 2017As an alternative to perfluorooctanesulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (commercial name: F-53B) has been used as a mist suppressant in...
As an alternative to perfluorooctanesulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (commercial name: F-53B) has been used as a mist suppressant in Chinese electroplating industries for over 30 years. It has been found in the environment and fish, and one acute assay indicated F-53B was moderately toxic. However, the toxicological information on this compound was incomplete and insufficient for assessment of their environment impact. The object of this study was to examine the developmental toxicity of F-53B using zebrafish embryos. Zebrafish embryos were incubated in 6-well plates with various concentrations of F-53B (1.5, 3, 6, and 12mg/L) from 6 to 132h post fertilization (hpf). Results showed that F-53B exposure induced developmental toxicity, including delayed hatching, increased occurrence of malformations, and reduced survival. Malformations, including pericardial and yolk sac edemas, abnormal spines, bent tails, and uninflated swim bladders, appeared at 84 hpf, and increased with time course and dose. A decrease in survival percentages was noted in the 6 and 12mg/L F-53B-treated groups at 132 hpf. Continuous exposure to 3mg/L F-53B resulted in high accumulation levels in zebrafish embryos, suggesting an inability for embryos to eliminate this compound and a high cumulative risk to fish. We also examined the cardiac function of embryos at specific developmental stages following exposure to different concentrations, and found that F-53B induced cardiac toxicity and reduced heart rate. Even under low F-53B concentration, o-dianisidine staining results showed significant decrease of relative erythrocyte number at 72 hpf before the appearance of observed effects of F-53B on the heart. To elucidate the underlying molecular changes, genes involved in normal cardiac development were analyzed using real-time qPCR in the whole-body of zebrafish embryos. F-53B inhibited the mRNA expression of β-catenin (ctnnb2) and wnt3a. The mRNA levels of β-catenin targeted genes (nkx2.5 and sox9b), which play critical roles in cardiogenesis, were also reduced after exposure. Thus, exposure to F-53B impaired the development of zebrafish embryos and disrupted cardiac development, which might be mediated by effects on the Wnt signaling pathway and decrease of erythrocyte numbers.
Topics: Alkanesulfonates; Alkanesulfonic Acids; Animals; Embryo, Nonmammalian; Erythrocytes; Fluorocarbons; Gene Expression Regulation, Developmental; Heart; Larva; RNA, Messenger; Water Pollutants, Chemical; Zebrafish; Zebrafish Proteins
PubMed: 28187362
DOI: 10.1016/j.aquatox.2017.02.002 -
Annals of Clinical Biochemistry Jan 2018Background The enzymatic method of caeruloplasmin measurement is based on copper-dependent oxidase activity. The advantage of the oxidase determination is that it has a...
Background The enzymatic method of caeruloplasmin measurement is based on copper-dependent oxidase activity. The advantage of the oxidase determination is that it has a much lower detection limit compared with immunoassay-based methods. It has found its application in both the diagnosis of Wilson's disease and also in the monitoring of patients' response to treatment. Methods The method previously described in literature was adapted for use on a 96-well plate. Caeruloplasmin oxidase activity results were derived from the equation: caeruloplasmin oxidase activity = (A-A) × 185 U/L. Results Repeatability (intra-batch) imprecision ranged from 6 to 15% and intermediate (inter-batch) imprecision varied from 7 to 16% for caeruloplasmin oxidative activities of 14, 29, 45 and 99 U/L. Between 3 and 92 U/L, the assay appeared linear with a regression coefficient R= 0.9958. The lower limit of quantification was 4 U/L. Samples were stable over a five-week period at 4℃ and for at least four freeze-thaw cycles. There was a statistically significant difference between the areas under ROC curve for copper-to-caeruloplasmin ratios between caeruloplasmin oxidative activity and immunoassay-based methods ( P < 0.0171). The reference interval for caeruloplasmin activity was determined to be 12-166 U/L. Conclusions Using the oxidative assay provides a cost-effective means of estimating caeruloplasmin concentrations. The method is easily adaptable to a 96-well plate format that facilitates high throughput of samples in a busy laboratory. The enzymatic method is more sensitive and specific for differentiating between Wilson's and non-Wilson's when compared with immunoassay-based methods.
Topics: Ceruloplasmin; Dianisidine; Hepatolenticular Degeneration; Humans; Limit of Detection
PubMed: 28166667
DOI: 10.1177/0004563217695350 -
Clinical Chemistry and Laboratory... Aug 2017Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer's disease (AD), comprising copper decreases within the brain and copper...
BACKGROUND
Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer's disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as "free" copper). The calculated non-Cp Cu (Walshe's) index has many limitations.
METHODS
A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o-dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe's index was also calculated.
RESULTS
The direct method was linear (0.9-5.9 μM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0-1.9 μM) was determined parametrically in 147 healthy controls (27-84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 μmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%-58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR-).
CONCLUSIONS
The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.
Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Copper; Female; Fluorescent Dyes; Humans; Male; Middle Aged; Spectrometry, Fluorescence
PubMed: 28076308
DOI: 10.1515/cclm-2016-0843 -
Journal of the Science of Food and... Aug 2017Peroxidase activity was increased during germination of green gram and such an increase may have benefits in many physiological processes. The present study aimed to...
BACKGROUND
Peroxidase activity was increased during germination of green gram and such an increase may have benefits in many physiological processes. The present study aimed to investigate the optimum conditions for the extraction, purification and characterization of peroxidase from the germinated green gram roots and also its application for the removal of phenols in water.
RESULTS
Peroxidase activity was increased by 300-fold in 5-day germinated green gram. Because the root was rich in peroxidase activity, peroxidase from roots was isolated and purified to homogeneity. The purified peroxidase showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular weight of 50 kDa, an optimum pH of 5.5 and a pH stability ranging from 5 to 9. The enzyme had 50% residual activity at 70 °C. It catalyzed the oxidation of a variety of substrates. The K value of the enzyme was 1.28 mmol L for o-dianisidine and 0.045 mmol L for H O . The enzyme lost 100% activity in the presence of dithiothreitol and cysteine. The addition of copper ion increased the enzyme activity by three-fold. Both soluble and immobilized peroxidases removed more phenol than p-chlorphenol, whereas horseradish peroxidase removed more p-chlorphenol. Thus, the green gram root peroxidase showed good pH and temperature stability, as well as the ability to remove phenolic compounds from effluent.
CONCLUSION
Peroxidase with good thermal and pH stability was purified from germinated green gram roots and has the ability to oxidize phenolic compounds from waste water. © 2016 Society of Chemical Industry.
Topics: Chlorophenols; Enzyme Stability; Enzymes, Immobilized; Hydrogen-Ion Concentration; Kinetics; Peroxidase; Phenol; Plant Proteins; Plant Roots; Vigna; Water Pollutants, Chemical
PubMed: 27976372
DOI: 10.1002/jsfa.8173 -
Journal of Pharmaceutics 2016Two simple methods are described for the determination of ethionamide (ETM) in bulk drug and tablets using cerium (IV) sulphate as the oxidimetric agent. In both...
Two simple methods are described for the determination of ethionamide (ETM) in bulk drug and tablets using cerium (IV) sulphate as the oxidimetric agent. In both methods, the sample solution is treated with a measured excess of cerium (IV) solution in HSO medium, and after a fixed standing time, the residual oxidant is determined either by back titration with standard iron (II) solution to a ferroin end point in titrimetry or by reacting with o-dianisidine followed by measurement of the absorbance of the orange-red coloured product at 470 nm in spectrophotometry. In titrimetry, the reaction proceeded with a stoichiometry of 1 : 2 (ETM : Ce (IV)) and the amount of cerium (IV) consumed by ETM was related to the latter's amount, and the method was applicable over 1.0-8.0 mg of drug. In spectrophotometry, Beer's law was obeyed over the concentration range of 0.5-5.0 g/mL ETM with a molar absorptivity value of 2.66 × 10 L/(mol·cm). The limits of detection (LOD) and quantification (LOQ) calculated according to ICH guidelines were 0.013 and 0.043 g/mL, respectively. The proposed titrimetric and spectrophotometric methods were found to yield reliable results when applied to bulk drug and tablets analysis, and hence they can be applied in quality control laboratories.
PubMed: 27818836
DOI: 10.1155/2016/5410573 -
Bioprocess and Biosystems Engineering Feb 2017In this study, laccase was immobilized on nylon 6,6/Fe composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The...
In this study, laccase was immobilized on nylon 6,6/Fe composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80 nm (diameter) and 2.70 MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11 weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33 V and the catalytic current was found to be 30 µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97 %) cell inhibition at 75 µM L within 24 h. The cytotoxicity of DMOB was significantly decreased to 78 % after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.
Topics: 3T3-L1 Cells; Animals; Caprolactam; Dianisidine; Enzymes, Immobilized; Ferric Compounds; Fungal Proteins; Laccase; Membranes, Artificial; Mice; Nanofibers; Polymers; Trametes
PubMed: 27757535
DOI: 10.1007/s00449-016-1686-6 -
Spectrochimica Acta. Part A, Molecular... Feb 2017Spectrophotometric method with three systems were developed here for the determination of gold(III) using o-dianisidine, aniline sulphate and catechol. Gold(III),in the...
Spectrophotometric method with three systems were developed here for the determination of gold(III) using o-dianisidine, aniline sulphate and catechol. Gold(III),in the system 1 it oxidizes o-dianisidine, in the system 2 it oxidizes catechol followed by its coupling with o-dianisidine, in the system 3 it oxidizes catechol followed by its coupling with aniline sulphate forming dye products with respective λ 446nm, 540nm, and 505nm. All the three systems were optimized and analytical parameters were calculated. The molar absorptivity values were 9.27×10, 1.97×10 and 1.62×10 respectively for the systems 1, 2 and 3 with the corresponding Sandell sensitivity values (μgcm), 0.0021, 0.0096 and 0.011. The optimized systems were used for the determination of gold present in some forensic jewellery and pharmaceutical samples and the results obtained were compared with the results of all samples determined by Inductively Coupled Plasma - Atomic Emission Spectrometric method and a few of them were also complemented by Energy Dispersive X-Ray Fluorescent spectral analysis.
Topics: Aniline Compounds; Calibration; Catechols; Color; Coloring Agents; Dianisidine; Forensic Sciences; Gold; Jewelry; Limit of Detection; Medicine, Ayurvedic; Oxidation-Reduction; Spectrometry, X-Ray Emission; Spectrophotometry, Atomic; Spectrophotometry, Ultraviolet; Tablets
PubMed: 27701047
DOI: 10.1016/j.saa.2016.09.045 -
Aquatic Toxicology (Amsterdam,... Aug 2016Silver_ nanoparticles (AgNPs), for their attractive antimicrobial properties, have become one of the most commercial nanomaterials used recently. AgNPs are reported to...
Silver_ nanoparticles (AgNPs), for their attractive antimicrobial properties, have become one of the most commercial nanomaterials used recently. AgNPs are reported to be toxic to blood cells of aquatic organisms and humans, however, few studies related to toxic effects of AgNPs in hematopoiesis using an in vivo model were available. Firstly, microarrays were applied to reveal transcriptional responses of zebrafish embryos to AgNPs at 24h post-fertilization (hpf)in this study, and hemoglobin genes were found to be down-regulated by AgNPs and to be enriched in the top 10 categories by Gene Ontology (GO) analysis. The reduced expressions of hemoglobin were further demonstrated by qRT-PCR detection, whole-mount in situ hybridization, and O-dianisidine staining at transcriptional and translational level. Next, the commitment of mesoderm, specification of hematopoietic progenitor cells and differentiation of erythroids were detected at different developmental stages in AgNPs-exposed embryos, and erythrogenesis were found to be inhibited by AgNPs in developmental-stage-specific and cell-specific manners. Finally, it was pointed out that AgNPs affected erythrogenesis mostly by their particles other than their releasing ions.
Topics: Animals; Down-Regulation; Embryo, Nonmammalian; Embryonic Development; Erythropoiesis; Female; Genetic Markers; Hemoglobins; Male; Metal Nanoparticles; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Silver; Water Pollutants, Chemical; Zebrafish
PubMed: 27340786
DOI: 10.1016/j.aquatox.2016.06.005 -
Zebrafish Aug 2016Thrombosis is a leading cause of death and the development of effective and safe therapeutic agents for thrombotic diseases has been proven challenging. In this study,...
Thrombosis is a leading cause of death and the development of effective and safe therapeutic agents for thrombotic diseases has been proven challenging. In this study, taking advantage of the transparency of larval zebrafish, we developed a larval zebrafish thrombosis model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days post fertilization) were treated with phenylhydrazine (PHZ) and a testing drug for 24 h. Tested drugs were administered into the zebrafish either by direct soaking or circulation microinjection. Antithrombotic efficacy was quantitatively evaluated based on our previously patented technology characterized as an image analysis of the heart red blood cells stained with O-dianisidine staining. Zebrafish at 2 dpf treated with PHZ at a concentration of 1.5 μM for a time period of 24 h were determined as the optimum conditions for the zebrafish thrombosis model development. Induced thrombosis in zebrafish was visually confirmed under a dissecting stereomicroscope and quantified by the image assay. All 6 human antithrombotic drugs (aspirin, clopidogrel, diltiazem hydrochloride injection, xuanshuantong injection, salvianolate injection, and astragalus injection) showed significant preventive and therapeutic effects on zebrafish thrombosis (p < 0.05, p < 0.01, & p < 0.001) in this zebrafish thrombosis model. The larval zebrafish thrombosis model developed and validated in this study could be used for in vivo thrombosis studies and for rapid screening and efficacy assessment of antithrombotic drugs.
Topics: Animals; Disease Models, Animal; Fibrinolytic Agents; Humans; Microinjections; Thrombosis; Zebrafish
PubMed: 27333081
DOI: 10.1089/zeb.2016.1263