Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling

Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as ＂bait＂： Fusobacterium nucleatum （F. nucleatum） whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans （S. mutans） whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the ＂bait＂ oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The ＂prey＂ bacterial mixtures （including known species or natural saliva samples） were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.

Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin- eosin (HE) staining. RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues. CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.

The anti-angiogenic and antibacterial effect of Tinomiscium philippinense Miers.(Menispermaceae)leaf extract

Objective:To determine the toxicity profile,anti-angiogenic and antibacterial activity of the crude and semi-crude leaf extracts of Tinomiscium philippinense(T.philippinense).Methods:The leaves of T.philippinense were extracted with methanol and partitioned with solvents of different polarities,namely,hexane,dichloromethane and butanol.The extracts were subjected to duck chorioallantoic membrane assay to establish its anti-angiogenic property.Microwell assay was utilized to determine the minimum inhibitory concentration and minimum bactericidal concentration of the different extracts of the plant.Results:The dichloromethane leaf extract of T.philippinense at 1000μg/disc showed the highest anti-angiogenic activity with 37.46%inhibition.All the fractions exhibited a bacteriostatic and bactericidal effect on the three bacterial strains with Pseudomonas aeruginosa,a Gram negative lactose fermenter exhibiting a higher sensitivity to dichloromethane semi-crude extract among the treatment groups.For the toxicity test,no mortality and no change in behavior were observed in the Sprague-Dawley rats 14 days after the oral administration of the plant extracts.The methanolic leaf extract of T.philippinense is non-toxic at a maximum dose of 5000 mg/kg.Conclusions:The dichloromethane leaf extract of T.philippinense is a potential antiangiogenic endemic plant species.This plant extract is also a potential antibacterial candidate as determined by microwell assay.The anti-angiogenic and antibacterial activity of the plant may be attributed to the essential oil,steroid,flavonoid,sterol and triterpene content of the plant.