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melitensis 16M to mutant or overexpression strains. At 24?h post the inoculation, the infected mice were sacrificed by cervical www.selleckchem.com/products/PF-2341066.html dislocation and spleens were removed aseptically and homogenized with PBS containing 0.1% Triton X-100. Serial dilutions of spleen homogenates were prepared and plated in duplicate on TSA plates with or without kanamycin or ampicillin, and the CFU were counted after 4?days of infection at 37��C. The competitive index (CI) values were calculated from the ratios of total input and recovered wild-type and kanamycin-resistant ��BSR0602 mutant (or ampicillin-resistant BSR0602 overexpression strains) CFU as previously described (Shea et al., 1996). Statistical analysis Bacterial survivals under in vitro stresses and during in vivo infections were expressed as the mean percent of survival compared to untreated controls ��SD. Statistical analysis was performed using Student��s unpaired t test. For the CI assays, the data was analyzed by Rho kinase signaling pathway Student��s t test. For qRT-PCR experiments, significance was calculated by the Wilcoxon signed-rank test. In all cases, a P value of >0.05 was considered significant. Ethics statement All animal experiments were performed in strict accordance with experimental animal regulation ordinances defined by China National Science and Technology Commission. The protocol was approved by Animal Ethics Committee of Beijing Institute of Disease Control and Prevention. Animals are provided with humane care and health conditions during their stay in the facility. All individuals who use animals receive instruction in experimental methods and in the care, maintenance and handling of mice, and are under the committee��s supervision. Results Computational predictions of candidate sRNAS in B. melitensis Intergenic Non-specific serine/threonine protein kinase sequences were extracted from the B. melitensis 16M chromosome ("type":"entrez-nucleotide","attrs":"text":"NC_003317.1","term_id":"17986284","term_text":"NC_003317.1"NC_003317.1 and "type":"entrez-nucleotide","attrs":"text":"NC_003318.1","term_id":"17988344","term_text":"NC_003318.1"NC_003318.1) based on genome annotation. The intergenic regions (IGRs) with a minimal size of 80?bp were then subjected to BLAST against all Brucella spp. genomic sequences available at the NCBI web site. Hits with an E value