Methods: In this present study, two protein extractions methods were performed to analyze female Ae. aegyti proteome, via TCA acetone precipitation extraction method and a commercial protein extraction reagent CytoBusterTM. Then, protein identification was performed by LC-ESI-MS/MS and followed by functional protein annotation analysis.
Results: The CytoBusterTM reagent gave the highest protein yield with a mean of 475.90 µg compared to TCA acetone precipitation extraction showed 283.15 µg mean of protein. LC-ESI-MS/MS identified 1,290 and 890 proteins from the CytoBusterTM reagent and TCA acetone precipitation, respectively. When comparing the protein class categories in both methods, there were three additional categories for proteins identified using CytoBusterTM reagent. The proteins were related to scaffold/adaptor protein (PC00226), protein binding activity modulator (PC00095) and intercellular signal molecule (PC00207). In conclusion, the CytoBusterTM protein extraction reagent showed a better performance for the extraction of proteins in term of the protein yield, proteome coverage and extraction speed.
OBJECTIVE: In the present study, we investigated the cytotoxic effect of 80% ethanol extract of P. amarus and its marker constituents (phyllanthin, hypophyllanthin, gallic acid, niranthin, greraniin, phyltetralin, isolintetralin, corilagin and ellagic acid) on HCT116 and their underlying mechanisms of action.
METHODS: Their antiproliferative and apoptotic effects on HCT 116 were performed using MTT assay and flow cytometric analysis, respectively, while caspases 3/7, 8 and 9 activities were examined using the colorimetric method. The expression of cleaved poly ADP ribose polymerase enzyme (PARP) and cytochrome c proteins was investigated by the immune-blot technique.
RESULTS AND DISCUSSION: HPLC and LC-MS/MS analyses demonstrated that the extract contained mainly lignans and polyphenols. The plant samples markedly suppressed the growth and expansion of HCT116 cells in a concentration- and time-dependent manner with no toxicity against normal human fibroblast CCD18 Co. P. amarus extract, phyllanthin and gallic acid induced mode of cell death primarily through apoptosis as confirmed by the exteriorization of phosphatidylserine. Caspases 3/7, 8, and 9 activities increased in a concentration-dependent manner following 24h treatment. The expressions of cleaved PARP (Asp 214) and cytochrome c were markedly upregulated.
CONCLUSION: P. amarus extract, phyllanthin and gallic acid exhibited an apoptotic effect on HCT116 cells through the caspases-dependent pathway.
OBJECTIVE: In this presented work, an analytical method by gas chromatography coupled with flame ionization detection (GC-FID) has been developed to determine organic solvents in radiopharmaceutical samples. The effect of injection holding time, temperature variation in the injection port, and the column temperature on the analysis time and resolution (R ≥ 1.5) of ethanol and acetonitrile was studied extensively.
METHODS: The experimental conditions were optimized with the aid of further statistical analysis; thence, the proposed method was validated following the International Council for Harmonisation (ICH) Q2 (R1) guideline.
RESULTS: The proposed analytical method surpassed the acceptance criteria including the linearity > 0.990 (correlation coefficient of R2), precision < 2%, LOD, and LOQ, accuracy > 90% for all solvents. The separation between ethanol and acetonitrile was acceptable with a resolution R > 1.5. Further statistical analysis of Oneway ANOVA revealed that the increment in injection holding time and variation of temperature at the injection port did not significantly affect the analysis time. Nevertheless, the variation in injection port temperature substantially influenced the resolution of ethanol and acetonitrile peaks (p < 0.05).
CONCLUSION: The proposed analytical method has been successfully implemented to determine the organic solvent in the [18F]fluoro-ethyl-tyrosine ([18F]FET), [18F]fluoromisonidazole ([18F]FMISO), and [18F]fluorothymidine ([18F]FLT).