Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay

The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26 µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H₂O₂ for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57–82 pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24 h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2′,3,4′-trihydroxy-4-methoxychalcone (THMC), and 2′,4′-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6 h incubation was found at 10 µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of lipopolysaccharide and the specific HO-1 inhibitor tin protoporphyrin IX. Taken together, we developed a convenient and highly sensitive ELISA-based HO-1 enzyme activity assay, allowing the identification and characterization of molecules potentially useful for the treatment of inflammatory and autoimmune diseases.     

Development of an automatic ROI setting program for input function determination in 99mTc-ECD non-invasive cerebral blood flow quantification

Non-invasive quantitative measurements are useful for clinical study as these are simple and pain-free procedures. A new non-invasive semi-automatic quantitative measurement method, the improved brain uptake ratio (IBUR) method using ^99mTc-ECD SPECT, has recently been reported. If an automatic ROI setting algorithm could be developed to determine the input function for the IBUR method, analysis of regional cerebral blood flow (rCBF) can be completed within a few min without recourse to complex techniques, through a fully automatic rCBF analysis program. The purpose of this study was to develop an automatic input function determination program for ^99mTc-ECD non-invasive cerebral blood flow quantification and to confirm the feasibility of use of this program.The images of 15 consecutive patients who underwent both ^99mTc-ECD chest RI angiography and SPECT examinations were used for development of the automatic arterial input function program. The images of 69 consecutive patients were used for validation of the program.The coincidence ratio between the ROI automatic method and the manual setting method was 98%. The mean difference in the ROI location was ±6.4 mm in the X direction and ±8.6 mm in the Y direction. Individual rCBF values obtained using these independent techniques were also reasonably well correlated (r = 0.95). The total time for the IBUR analysis using the automatic method is 2–3 min as compared to 20–30 min for the current analysis method. This technique improves the throughput of nuclear medical examinations.     

Poloxamer-chitosan-based Naringenin nanoformulation used in brain targeting for the treatment of cerebral ischemia

ObjectiveHere, the aim is to improve the bioavailability of Naringenin (NRG) in brain and to establish the highest remedial benefit from a novel anti-ischemic medicine i.e. NRG.MethodsA novel Naringenin-loaded-nanoemulsion (NE)-(in situ)-gel (i.e. thermoresponsive), was formulated with the help of Poloxamer-407 (20.0% w/v). Chitosan (CS, 0.50% w/v) was used to introduce the mucoadhesive property of NE-(in situ)-gel and finally called as NRG-NE-gel + 0.50%CS. A novel UHPLC-ESI-Q-TOF-MS/MS-method was optimized and used for NRG-NE-gel + 0.50%CS to quantify the Pharmacokinetic-(PK)-parameters in plasma as well as brain and to evaluate the cerebral ischemic parameters after MCAO i.e. locomotor activity, grip strength, antioxidant activity, and quantity the infarction volume in neurons with the safety/toxicity of NRG-NE-gel + 0.50%CS after i.n. administration in the rats.ResultsThe mucoadhesive potency and gelling temperature of NRG-NE-gel + 0.50%CS were observed 6245.38 dynes/cm² and 28.3 ± 1.0 °C, respectively. Poloxamer-407 based free micelles size was observed 98.31 ± 1.17 nm with PDI (0.386 ± 0.021). The pH and viscosity of NRG-NE-gel + 0.50%CS were found to be 6.0 ± 0.20 and 2447 ± 24cp (at 35.0 ± 1.0 °C temperature), respectively. An elution time and m/z NRG were observed 1.78 min and 270.97/150.96 with 1.22 min and m/z of 301.01/150.98 for Quercetin (IS) respectively. Inter and intra %precision and %accuracy was validated 1.01–3.37% and 95.10–99.30% with a linear dynamic range (1.00 to 2000.00 ng/ml). AUC_0-24 of plasma & brain were observed 995.60 ± 24.59 and 5600.99 ± 144.92 (ng min/ml g) in the rats after the intranasal (i.n.) administration of NRG-NE-gel + 0.50%CS. No toxicological response were not found in terms of mortalities, any-change morphologically i.e. in the microstructure of brain as well as nasal mucosa tissues, and also not found any visual signs in terms of inflammatory or necrosis.ConclusionIntranasally administered NRG-NE-gel + 0.50%CS enhanced the bioavailability of Naringenin in the brain. In the cerebral ischemic rats, significantly improved the neurobehavioral activity (locomotor & grip strength) followed by antioxidant activity as well as infarction volume. Finally, the toxicity studies carried out and established the safe nature of optimized-NRG-NE-gel + 0.50%CS.     

Direct quantification of in vitro cell growth through image analysis

Summary Objective, accurate, non-intrusive measurement of in vitro cell growth was realized through microcomputerized video image analysis. Recently-released video and digitizing hardware and software were incorporated into an analytical system which accurately quantified visual differences between cultures on a cell number or fresh mass basis. Sequential measurements during culture incubation further detected and quantified subtle changes in colony area and density resulting from growth. Each measurement was acquired rapidly, without encroaching on the in vitro environment, so cell growth was undisturbed. Custom software routines coordinated the quantification of this detailed record into precise cumulative growth curves.     

Promotion of cancer metastasis: candidate validation using an iTRAQ-based approach

Evaluation of: Ghosh D, Li Z, Tan XF, Lim TK, Mao Y, Lin Q. RAQ based quantitative proteomics approach validated the role of calcyclin binding protein (CacyBP) in promoting colorectal cancer metastasis. Mol. Cell Proteomics 12(7), 1865–1880 (2013).

The use of iTRAQ-based relative quantification is demonstrated by Ghosh et al. to analyze the downstream effectors of a calcyclin-binding protein, which is proposed to promote colorectal cancer progression. These findings are reviewed and discussed in relation to the need to establish robust in vitro model cell lines for identification of cancer-specific pathways and to confirm the leads generated by proteomic analysis.     

Colorimetric Evaluation of Cultured Osteoblast-like Cells (ROS 17/2.8)

We have developed a colorimetric method for evaluating the number of osteoblastic cells in culture without destroying the cells. This assay is based on the staining of basophilic cellular compounds with methylene blue. The dye bound by the cells is released at low pH and measured in a spectrophotometer at 662 nm. Linear correlations exist between the absorbance measured by the methylene blue assay and the number of cells seeded, the total cellular protein content, and thymidine labeling. This colorimetric method has the advantage of preserving cell integrity. After destaining, scanning electron microscopy can be performed on well preserved cell morphology.     

Subcellular Distribution of Tyrosine Hydroxylase in Some Catecholaminergic Rat Brain Areas Determined by a Quantitative Immunoblot Assay

The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (less than 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.     

Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.     

Image analysis - a simple method of algal culture growth assessment

A non-destructive method for the relative growth quantification of algae or cyanobacteria in the culture has been developed. It is based on image analysis and does not require any special equipment. Results provided by this method were compared with those provided by a chlorophyll a assessment, with a correlation factor of over 88%.