Outer Membrane Vesicles Derived from Escherichia coli Up-Regulate Expression of Endothelial Cell Adhesion Molecules In Vitro and In Vivo

Escherichia coli, as one of the gut microbiota, can evoke severe inflammatory diseases including peritonitis and sepsis. Gram-negative bacteria including E. coli constitutively release nano-sized outer membrane vesicles (OMVs). Although E. coli OMVs can induce the inflammatory responses without live bacteria, the effect of E. coli OMVs in vivo on endothelial cell function has not been previously elucidated. In this study, we show that bacteria-free OMVs increased the expression of endothelial intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1, and enhanced the leukocyte binding on human microvascular endothelial cells in vitro. Inhibition of NF-κB and TLR4 reduced the expression of cell adhesion molecules in vitro. OMVs given intraperitoneally to the mice induced ICAM-1 expression and neutrophil sequestration in the lung endothelium, and the effects were reduced in ICAM-1^-/- and TLR4^-/- mice. When compared to free lipopolysaccharide, OMVs were more potent in inducing both ICAM-1 expression as well as leukocyte adhesion in vitro, and ICAM-1 expression and neutrophil sequestration in the lungs in vivo. This study shows that OMVs potently up-regulate functional cell adhesion molecules via NF-κB- and TLR4-dependent pathways, and that OMVs are more potent than free lipopolysaccharide.     

Cultivation of the two perennial brown algae Ecklonia cava and E. stolonifera for abalone feeds in Korea

Ecklonia cava and Ecklonia stolonifera are perennial brown algae that form sea forests off the coast of Korea. Both species are cultured to supply a summer feed for the abalone industry. Recent expansion of the abalone industry in Korea has been bringing an increase in demand for fresh algal supply. Zoospores of the two algae were seeded in October 2006 on seed frames coiled with 100 m of seed fibers. After 2 months of indoor culture and 2 months of intermediate culture, growth and production of the two algae were compared during their main cultivation period from March 2007 to June 2008, in the culture ground in Wando, Korea (34°26′18.68″ N, 127°05′43.88″ E), in situ. Growth rate of E. cava and E. stolonifera was 1.058 and 3.089 mm day^?1, respectively. The mean production of E. stolonifera obtained from the culture ropes was ca. 12 kg wet wt. m^?1 of culture rope while production of E. cava was ca. 3 kg wet wt. m^?1 of culture rope. The difference in production was attributed from the different growth strategies of the two algae, with only E. stolonifera being able to regenerate blades from the holdfast. The ability to regenerate blades from the holdfast therefore makes E. stolonifera the preferred species for biomass production for abalone feeds. In a 120-day feeding experiment, growth rate, weight gain, and survival rate of abalone showed that E. cava and E. stolonifera feeds could provide an alternative feed to Saccharina japonica during summer months.     

Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp.

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonas reinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropia tenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropia tenera/Chlamydomonas reinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.     

Biological activity in traditional Alaska pollack sikhae during low temperature fermentation

Biological activity was examined on Alaska pollack sikhae produced with 4 treatments (by irradiating at 5 or 10 kGy, or by adding either 0.1 or 0.3% of chitooligosaccharide), compared with control (2-step fermentation only) during fermentation at -2 degrees C. The extracts (500 ppm level) of sikhae had antimicrobial activities against 4 different strains of food poisoning bacteria such as Staphy. aureus, B. subtilis, B. cereus, and L. monocytogenes. Antioxidative activity (EDA(50), 11.55 mg/mL) in control group increased with time up to 60 days of fermentation but decreased thereafter, while those levels in other products were kept within 10.60-18.30 mg/mL ranges during fermentation. Inhibitory activity of angiotensin-I converting enzyme (ACE) (IC(50), 1.51-2.89 mg/mL) in all products was observed during fermentation except at 0 day. Inhibitory activity of xanthine oxidase (XO) (IC(50), 0.65-0.87 mg/mL) in all products also increased with time up to 30 days of fermentation. Without irradiating or adding of chitooligosaccharide, Alaska pollack sikhae showing biological activities was enough by 2-step fermentation and storage at -2 degrees C only.     

A multiplex polymerase chain reaction for a differential diagnosis of Plasmodium falciparum and Plasmodium vivax

A multiplex PCR was designed for the differential diagnosis of the two parasite species by targeting the 18S rRNA gene with a set of primer combinations, amplifying DNA fragments of 1451-bp and 833-bp for P. falciparum and P. vivax, respectively. The sensitivity of this PCR test was high, as minimal as 0.1 parasite per one microliter of blood sample and a minimum of four copies of the target gene could be detected. For the diagnosis of mixed infection of two Plasmodium spp., there were no apparent competition or cross-reaction between the majority and minority Plasmodium species. The multiplex PCR was evaluated on 210 clinical samples and 60 normal controls. The PCR test yielded highly concordant results with microscopic examination, with the only one exception of a mixed (P. falciparum plus P. vivax) infection case, which was diagnosed as a single infection of P. falciparum by microscopy. We propose that the multiplex PCR is a sensitive, specific, and rapid tool that can serve as a useful differential diagnostic tool for detecting P. falciparum and P. vivax.     

MICB Allele Genotyping on Microarrays by Improving the Specificity of Extension Primers

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8⁺ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles (*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024) present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations.