Design, synthesis, and biological evaluation of dihydroartemisinin-fluoroquinolone conjugates as a novel type of potential antitubercular agents.

Tuberculosis remains a global public health problem in recent years. To develop novel type of potential antitubercular agents, twelve novel dihydroartemisinin-fluoroquinolone (DHA-FQ) conjugates (three types of molecules) were gradually designed and conveniently synthesized. All the newly synthesized conjugates were well characterized and evaluated against different Mycobacterium tuberculosis strains in vitro. The screening results showed that five DHA-FQ conjugates were active toward M. tuberculosis H37Rv, and compound 3a exhibited the strongest inhibitory activity (MIC=0.0625 µg/mL), which was comparable to the positive control Moxifloxacin and even stronger than Ofloxacin. Conjugates 2a and 3a also displayed comparable activities against various clinically isolated sensitive and resistant M. tuberculosis strains (MIC=0.125-16 µg/mL) to Moxifloxacin. All target compounds possessed selective anti-M. tuberculosis ability. Preliminary structure-activity relationship demonstrated that short linker between DHA and FQ was favorable for strong antitubercular activity. This study provides a new clue for the development of novel antitubercular lead molecules.

Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine origin that harbours the multiresistance gene cfr.

OBJECTIVES: To determine the complete nucleotide sequence of the multidrug resistance plasmid pSCEC2, isolated from a porcine Escherichia coli strain, and to analyse it with particular reference to the cfr gene region. METHODS: Plasmid pSCEC2 was purified from its E. coli J53 transconjugant and then sequenced using the 454 GS-FLX System. After draft assembly, predicted gaps were closed by PCR with subsequent sequencing of the amplicons. RESULTS: Plasmid pSCEC2 is 135 615 bp in size and contains 200 open reading frames for proteins of ≥100 amino acids. Analysis of the sequence of pSCEC2 revealed two resistance gene segments. The 4.4 kb cfr-containing segment is flanked by two IS256 elements in the same orientation, which are believed to be involved in the dissemination of the rRNA methylase gene cfr. The other segment harbours the resistance genes floR, tet(A)-tetR, strA/strB and sul2, which have previously been found on other IncA/C plasmids. Except for these two resistance gene regions, the pSCEC2 backbone displayed >99% nucleotide sequence identity to that of other IncA/C family plasmids isolated in France, Chile and the USA. CONCLUSIONS: The cfr gene was identified on an IncA/C plasmid, which is well known for its broad host range and transfer and maintenance properties. The location on such a plasmid will further accelerate the dissemination of cfr and co-located resistance genes among different Gram-negative bacteria. The genetic context of cfr on plasmid pSCEC2 underlines the complexity of cfr transfer events and confirms the role that insertion sequences play in the spread of cfr.

[Zebrafish as a model animal for the study of blood-brain barrier permeability by biomolecules].

Blood-brain barrier (BBB) is the major obstacle for drug delivery into the central nervous system (CNS). However, there is no ideal model animal for the study of BBB permeability till now. Currently zebrafish (Danio rerio) has emerged as a powerful model organism for the study of vertebrate biology. In this study, the feasibility of using zebrafish as model animal was investigated for BBB permeability by comparing the results of administration of BBB-penetrating peptide and protein to mouse and zebrafish. The results showed that the BBBs of mouse and zebrafish were similar in molecular permeability. Additionally, zebrafish has advantageous features as a model animal, such as small size, fertile and easy to breed. Therefore, it is suggested that zebrafish may be a favored model for the study of BBB permeability.

[Replicative kinetics of classical swine fever virus in PK-15 cells].

In order to understand the replication kinetics of classical swine fever virus (CSFV) in in vitro cells PK-15 cells were seeded in 96-well tissues culture plates. After overnight incubation at 37 degrees C in 5% CO2 environment when growing to 80% confluence, the cells were infected with CSFV strain Shimen at 100 TCID50 per well. At various time post infection (p.i.) the replication of the virus in the cells were analyzed repectively by detection of viral antigen using indirect immunofluorescent assay (IFA), RNA replication using reverse transcription real-time PCR and viral production using titration of TCID50. In the results of the IFA the viral antigen could be detected as early as 8hrs p.i. and at 72h hrs p.i. almost all cells showed positive staining, the real-time PCR showed that the synthesis of viral genomic RNA was gradually increased between 8-24 hrs p.i. and reached its peak at 72 hrs p.i.. However, the synthesis of negative strand RNA was maintained at a low level for a whole period of culture although it could be detected at 8hrs p.i.. Titration of TCID50 demonstrated that the production of live virions increased at 8h and peaked between 48 - 72 hrs p.i. without significant lose of titer.

[Site-directed mutagensis of the major antigen E2 gene of CSFV, its high level expression in Escherichia coli and the immunonicity of recombinant E2 protein].

Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.