Orchid Research Laboratories Ltd.

Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMECs) is a prerequisite for its traversal of the blood-brain barrier (BBB) and penetration into the brain. In the present study, we identified NlpI as a novel bacterial determinant contributing to E. coli K1 interaction with HBMECs. The deletion of nlpI did not affect the expression of the known bacterial determinants involved in E. coli K1-HBMEC interaction, such as type 1 fimbriae, flagella, and OmpA, and the contribution of NlpI to HBMECs binding and invasion was independent of those bacterial determinants. Previous reports have shown that the nlpI mutant of E. coli K-12 exhibits growth defect at 42°C at low osmolarity, and its thermosensitive phenotype can be suppressed by a mutation on the spr gene. The nlpI mutant of strain RS218 exhibited similar thermosensitive phenotype, but additional spr mutation did not restore the ability of the nlpI mutant to interact with HBMECs. These findings suggest the decreased ability of the nlpI mutant to interact with HBMECs is not associated with the thermosensitive phenotype. NlpI was determined as an outer membrane-anchored protein in E. coli , and the nlpI mutant was defective in cytosolic phospholipase A 2 α (cPLA 2 α) phosphorylation compared to the parent strain. These findings illustrate the first demonstration of NlpI's contribution to E. coli K1 binding to and invasion of HBMECs, and its contribution is likely to involve cPLA 2 α.

In order to know the different statistical tools used to analyze the data obtained from twenty-eight-day repeated dose oral toxicity studies with rodents and the impact of these statistical tools on interpretation of data obtained from the studies, study reports of 122 numbers of twenty-eight-day repeated dose oral toxicity studies conducted in rats were examined. It was found that both complex and easy routes of decision trees were followed for the analysis of the quantitative data. These tools include Scheffe's test, non-parametric type Dunnett's and Scheffe's tests with very low power. Few studies used the non-parametric Dunnett type test and Mann-Whitney's U test. Though Chi-square and Fisher's tests are widely used for analysis of qualitative data, their sensitivity to detect a treatment-related effect is questionable. Mann-Whitney's U test has better sensitivity to analyze qualitative data than the chi-square and Fisher's tests. We propose Dunnett's test for analysis of quantitative data obtained from twenty-eight-day repeated dose oral toxicity tests and for qualitative data, Mann-Whitney's U test. For both tests, one-sided test with p=0.05 may be applied.

A simple direct Chiral HPLC method was developed for the separation of the enantiomers of Frovatriptan Succinate[R-(+)-6-Carboxamido-3-N-methylamino-1,2,3,4-tetrahydrocarbazole hemisuccinate], an antimigraine drug.The enantioseparation was achieved on a Protein based Cellobiohydrolase column (Chiral-CBH) using an isocratic mobile phase consisting of 10 mM dihydrogen orthophosphate buffer and MeOH in the ratio of 92:8.Flow rate was kept at 0.6 mL/min.The resolution between the enantiomers was 4.4.The developed method was capable of detecting the S-enantiomer ≤ 6 ng/mL.The method was validated in terms of Selectivity, Linearity, Precision, Accuracy, Robustness, Limit of Detection (LOD), and Limit of Quantitation (LOQ).