Materials and Methods: The coagulometer for factor VIII assay is Sysmex CS-5100. All data were expressed as mean ± standard deviation (SD).
Results: A total of 135 cases were studied. Of these, 100 cases were of mild hemophilia A diagnosed by molecular genetics and, 15 cases were positive for LAC, which were confirmed by dilute Russell Viper venom test. Clot-based one-stage APTT assay showed 65% sensitivity and 80% specificity in diagnosing mild hemophilia A cases and out of 15 LAC cases, it showed false positivity in five cases. Chromogenic assay showed 85% sensitivity and 90% specificity in diagnosing mild hemophilia cases and was 100% specific in excluding LAC cases.
Conclusions: One-stage APTT assay is the most commonly used test for determining factor VIII levels but chromogenic assay are considered as the gold standard and recommended as the reference method by European Pharmacopoeia and ISTH subcommittee. Mild hemophilia A patients with missense mutations show discrepancy between the one-stage clot-based APTT assay and chromogenic assays for determination of factor VIII level and this can lead to misdiagnosis or misclassification of mild hemophilia A. Therefore, it is recommended that both the assays should be used in the evaluation of mild hemophilia cases.
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.
Materials and Methods: Dried root of P. glabra was extracted under reflux with methyl alcohol, fractionated through the vacuum liquid chromatography technique, and evaporated and then purified the compounds using column chromatography and preparative thin-layer chromatography. THP-1 cells were treated with amentoflavone, 5,7,4'-hydroxyflavonoid, and stigmasterol with various concentrations (0-30 µg/mL) and then incubated with MTS reagent for 2h. Treatment was done for 24, 48, and 72h. Then, effects of these compounds were also tested on PGE2, TNF-α, and IL-6 expression in human THP-1-derived macrophage cells for 24h.
Results: Three new compounds such as amentoflavone, 5,7,4'-hydroxyflavonoid, and stigmasterol were isolated. After 24h of incubation, a significant decrease in cell viability was reported with IC50 values of amentoflavone, 5,7,4'- hydroxyflavonoid, and stigmasterol (21 µg/mL ≡ 38 M), (18 µg/mL ≡ 66 M) and (20 µg/mL ≡ 48.5 M), respectively. Whereas for 48 and 72h treatment showed a less decreased cell viability compared with 24h treatment. These compounds also showed a significant reduction in the production of TNF-α, IL-6, and PGE2 in a dose-dependent manner.
Conclusions: The isolated new compounds showed significant cytotoxicity and anti-inflammatory effects.
Materials and Methods: Crude methanolic fraction of E. suberosa (Roxb) bark and its respective fractions were screened for the presence of different phytochemicals with different reagents. On the basis of increasing order of polarity, different organic solvents were used to obtain different fractions. Enzymatic studies were performed on crude methanolic extract of the plant. All the assays were performed under standard in vitro conditions.
Results: The phytochemical analysis shows the presence of alkaloids, phenols, triterpenoids, phytosterols, and flavonoids. Phenolic compounds and flavonoids are the major constituents of the plant. In anticholinesterase assay, the percent inhibition of standard drug (eserine) was 91.27 ± 1.17 and the half maximal inhibitory concentration (IC50) was 0.04 ± 0.0001. For α-glucosidase inhibition, the IC50 value for Dichloromethane fraction was 8.45 ± 0.13, for Methanol fraction it was 64.24 ± 0.15, and for aqueous fraction it was 42.62 ± 0.17 as compared with standard IC50 that is 37.42 (acarbose). Furthermore, results show that all fractions have potential against anti-urease enzyme, but DCM fraction of crude aqueous extract has significant IC50 value (45.26 ± 0.13) than other fractions.
Conclusion: Keeping in view all the results, it is evident that the plant can be used in future for formulating effective drugs against many ailments. Secondary metabolites and their derivatives possess different biological activities, for example, .g. flavonoids in cancer, asthma, and Alzheimer. Furthermore, the extracts of this plant can be used in their crude form, which is an addition to the complementary and alternative treatment strategies.
OBJECTIVE: Arene diazonium salts play an important role in organic synthesis as intermediate and a wide variety of aromatic compounds have been prepared using them. A serious drawback of arene diazonium salts is their instability in a dry state; therefore, they must be stored and handled carefully to avoid spontaneous explosion and other hazard events.
METHODS: The arene diazonium saccharin salts were prepared as active intermediates in situ through the reaction of various aryl amines with tert-butyl nitrite (TBN) in the presence of saccharin (Sac-H). Then, in situ obtained intermediates were used into the diazotization step without separation and purification in the current protocol.
RESULTS: A variety of aryl iodides were synthesized at a greener and low-cost method in the presence of TBN, Sac-H, glacial acetic acid, and TEAI.
CONCLUSION: In summary, a telescopic reaction is developed for the synthesis of aryl iodides. The current methodology is safe, cost-effective, broad substrate scope, and metal-free. All used reagents are commercially available and inert to moisture and air. Also, the saccharine and tetraethylammonium cation could be partially recovered from the reaction residue, which reduces waste generation, energy consumption, raw material, and waste disposal costs.
METHODOLOGY/PRINCIPAL FINDINGS: Two ORFeome phage display libraries of the entire Leptospira spp. genomes from five local strains isolated in Malaysia and seven WHO reference strains were constructed. Subsequently, 18 unique Leptospira peptides were identified in a screen using a pool of sera from patients with acute leptospirosis. Five of these were validated by titration ELISA using different pools of patient or control sera. The diagnostic performance of these five peptides was then assessed against 16 individual sera from patients with acute leptospirosis and 16 healthy donors and was compared to that of two recombinant reference proteins from L. interrogans. This analysis revealed two peptides (SIR16-D1 and SIR16-H1) from the local isolates with good accuracy for the detection of acute leptospirosis (area under the ROC curve: 0.86 and 0.78, respectively; sensitivity: 0.88 and 0.94; specificity: 0.81 and 0.69), which was close to that of the reference proteins LipL32 and Loa22 (area under the ROC curve: 0.91 and 0.80; sensitivity: 0.94 and 0.81; specificity: 0.75 and 0.75).
CONCLUSIONS/SIGNIFICANCE: This analysis lends further support for using ORFeome phage display to identify pathogen-associated immunogenic peptides, and it suggests that this technique holds promise for the development of peptide-based diagnostics for leptospirosis and, possibly, of vaccines against this pathogen.