Novel biodegradable sandwich-structured nanofibrous drug-eluting membranes for repair of infected wounds: an in vitro and in vivo study

Dave Wei-Chih Chen1,2, Jun-Yi Liao3, Shih-Jung Liu2, Err-Cheng Chan41Department of Orthopedic Surgery, Chang Gung Memorial Hospital, 2Department of Mechanical Engineering, 3Graduate Institute of Medical Mechatronics, 4School of Medical Technology, Chang Gung University, Kwei-San, Tao-Yuan, TaiwanBackground: The purpose of this study was to develop novel sandwich-structured nanofibrous membranes to provide sustained-release delivery of vancomycin, gentamicin, and lidocaine for repair of infected wounds.Methods: To prepare the biodegradable membranes, poly(D, L)-lactide-co-glycolide (PLGA), collagen, and various pharmaceuticals, including vancomycin, gentamicin, and lidocaine, were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol. They were electrospun into sandwich-structured membranes with PLGA/collagen as the surface layers and PLGA/drugs as the core. An elution method and a high-pressure liquid chromatography assay were used to characterize in vivo and in vitro drug release from the membranes. In addition, repair of infected wounds in rats was studied. Histological examination of epithelialization and granulation at the wound site was also performed.Results: The biodegradable nanofibrous membranes released large amounts of vancomycin and gentamicin (well above the minimum inhibition concentration) and lidocaine in vivo for more than 3 weeks. A bacterial inhibition test was carried out to determine the relative activity of the antibiotics released. The bioactivity ranged from 40% to 100%. The nanofibrous membranes were functionally active in treating infected wounds, and were very effective as accelerators in early-stage wound healing.Conclusion: Using the electrospinning technique, we will be able to manufacture biodegradable, biomimetic, nanofibrous, extracellular membranes for long-term delivery of various drugs.Keywords: nanofibrous, sandwich-structured, drug-eluting membranes, electrospinning, release characteristics, repair, wound infectio

Cytotoxic Effect of Recombinant Mycobacterium tuberculosis CFP-10/ESAT-6 Protein on the Crucial Pathways of WI-38 Cells

To unravel the cytotoxic effect of the recombinant CFP-10/ESAT-6 protein (rCFES) on WI-38 cells, an integrative analysis approach, combining time-course microarray data and annotated pathway databases, was proposed with the emphasis on identifying the potentially crucial pathways. The potentially crucial pathways were selected based on a composite criterion characterizing the average significance and topological properties of important genes. The analysis results suggested that the regulatory effect of rCFES was at least involved in cell proliferation, cell motility, cell survival, and metabolisms of WI-38 cells. The survivability of WI-38 cells, in particular, was significantly decreased to 62% with 12.5 μM rCFES. Furthermore, the focal adhesion pathway was identified as the potentially most-crucial pathway and 58 of 65 important genes in this pathway were downregulated by rCFES treatment. Using qRT-PCR, we have confirmed the changes in the expression levels of LAMA4, PIK3R3, BIRC3, and NFKBIA, suggesting that these proteins may play an essential role in the cytotoxic process in the rCFES-treated WI-38 cells

Purification and Characterization of Neutral Sphingomyelinase from Helicobacter pylori †

ABSTRACT: Phospholipase activities of human gastric bacterium, Helicobacter pylori, are regarded as the pathogenic factors owing to their actions on epithelial cell membranes. In this study, we purified and characterized neutral sphingomyelinase (N-SMase) from the superficial components of H. pylori strains for the first time. N-SMase was purified 2083-fold with an overall recovery of 37%. The purification steps included acid glycine extraction, ammonium sulfate precipitation, CM-Sepharose, Mono-Q, and Sephadex G-75 column chromatography. Approximate molecular mass for the native N-SMase was around 32 kDa. When N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified enzyme exhibited a K m of 6.7 µM and a V max of 15.6 nmol of TNPAL-sphingosine/h/mg of protein at 37°C in 50 mM phosphate-buffered saline, pH 7.4. N-SMase reaches optimal activity at pH 7.4 and has a pI of 7.15. The enzyme activity is magnesium dependent and specifically hydrolyzed sphingomyelin and phosphatidylethanolamine. The enzyme also exhibits hemolytic activity on human erythrocytes. According to Western blot analysis, a rabbit antiserum against purified N-SMase from H. pylori cross-reacted with SMase from Bacillus cereus. Sera from individuals with H. pylori infection but not uninfected ones recognizing the purified N-SMase indicated that it was produced in vivo. In enzymelinked immunosorbent assays, the purified N-SMase used as an antigen was as effective as crude protein antigens in detecting human antibodies to H. pylori

D-xylose fermentation by Schizosaccharomyces pombe cloned with xylose isomerase gene

Renewable biomass, D-xylose, can be converted to ethanol by microbes which are screened from nature or mutagenesis. An alternative is to develop a genetically modified microorganism which possesses the gene for D-xylose utilization. The plasmid pDB248-XI containing the xylose isomerase gene from Escherichia coli has been cloned to the fission yeast Schizosaccharomyces pombe. A preliminary investigation showed that the modified yeast had the ability to produce ethanol by using D-xylose as substrate. Studies of environmental effects, fermentation limiting factors, and the xylose isomerase gene expression were performed in order to understand the biochemical and physiological characteristics of the modified yeast during D-xylose fermentation. The modified yeast was demonstrated to produce 0.42 g ethanol form 1 g D-xylose under the current situation. However, the studies also indicated that the ethanol yield could be increased through further genetic improvement

Bacterial luciferase activity in vivo

Abstract not availabl

Inhibition of enterovirus 71- induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis

Enterovirus 71 infection causes significant morbidity and mortality in children, yet there is no effective treatment. In this study, a protein-bound pigment, allophycocyanin purified from bluegreen algae is first reported to exhibit anti-enterovirus 71 activity. Allophycocyanin neutralized the enterovirus 71-induced cytopathic effect in both human rhabdomyosarcoma cells and African green monkey kidney cells. The 50% inhibitory concentration of allophycocyanin for neutralizing the enterovirus 71-induced cytopathic effect was approximately 0.045 AE 0.012 mM in green monkey kidney cells. The cytotoxic concentrations of allophycocyanin for rhabdomyosarcoma cells and African green monkey kidney cells were 1.653 AE 0.003 mM and 1.521 AE 0.012 mM, respectively. A plaque reduction assay showed that the concentrations of allophycocyanin for reducing plaque formation by 50% were approximately 0.056 AE 0.007 mM and 0.101 AE 0.032 mM, when allophycocyanin were added at the state of viral adsorption and post-adsorption, respectively. Antiviral activity was more efficient in cultures treated with allophycocyanin before viral infection compared with that in the cultures treated after infection. Allophycocyanin was also able to delay viral RNA synthesis in the infected cells and to abate the apoptotic process in enterovirus 71-infected rhabdomyosarcoma cells with evidence of characteristic DNA fragmentation, decreasing membrane damage and declining cell sub-G1 phase. It is concluded that allophycocyanin possesses antiviral activity and has a potential for development as an antienterovirus 71 agent