A Microfluidic Channel Method for Rapid Drug-Susceptibility Testing of Pseudomonas aeruginosa

The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h. The drug susceptibility testing microfluidic (DSTM) device was prepared using soft lithography. It consisted of five sets of four microfluidic channels sharing one inlet slot, and the four channels are gathered in a small area, permitting simultaneous microscopic observation. Antimicrobials were pre-introduced into each channel and dried before use. Bacterial suspensions in cation-adjusted Mueller–Hinton broth were introduced from the inlet slot and incubated for 3 h. Susceptibilities were microscopically evaluated on the basis of differences in cell numbers and shapes between drug-treated and control cells, using dedicated software. The results of 101 clinically isolated strains of P. aeruginosa obtained using the DSTM method strongly correlated with results obtained using the ordinary microbroth dilution method. Ciprofloxacin, meropenem, ceftazidime, and piperacillin caused elongation in susceptible cells, while meropenem also induced spheroplast and bulge formation. Morphological observation could alternatively be used to determine the susceptibility of P. aeruginosa to these drugs, although amikacin had little effect on cell shape. The rapid determination of bacterial drug susceptibility using the DSTM method could also be applicable to other pathogenic species, and it could easily be introduced into clinical laboratories without the need for expensive instrumentation.     

Immunotherapy with dendritic cells pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8+ cytotoxic T lymphocytes and natural killer cells

Background and purpose Immunization with heat shock proteins, gp96, elicits specific protective immunity against parent tumors. However, it is marginally effective as a therapeutic tool against established tumors. In the present study, we evaluated the efficacy and mechanism of immunotherapy with bone marrow-derived dendritic cells (DCs) pulsed with tumor-derived gp96 against murine lung cancer. Methods Mice were transplanted subcutaneously with ovalbumin (OVA)-transfected Lewis Lung Cancer (LLC-OVA) cells and immunized with gp96 derived from LLC-OVA, DCs, or DCs pulsed with gp96 derived from LLC-OVA. Results The antitumor effect was significantly enhanced in the mice immunized with DCs pulsed with gp96 derived from LLC-OVA, compared to mice immunized with gp96 or DCs (P < 0.05). The antitumor effect was significantly dependent on natural killer (NK) cells and CD8⁺ cells and partially dependent on CD4⁺ cells. Analysis by laser confocal microscopy demonstrated that gp96 was shown on the cell surface at 15 min, and after 30 min internalized in the endosomes and not in the endoplasmic reticulum or lysosomes. OVA-specific⁺ CD8⁺ cells were more readily recruited into the draining lymph nodes and higher CD8⁺ cytotoxic T cell activity against LLC-OVA was observed in splenocytes from mice immunized with DCs pulsed with gp96 derived from LLC-OVA. Re-challenge of the surviving mice with LLC-OVA tumors after the initial tumor inoculation showed dramatic retardation in tumor growth. Conclusion In conclusion, immunotherapy of DCs pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8⁺ cytotoxic T lymphocytes and NK cells.     

Comparison of the mesophilic cellulosome‐producing Clostridium cellulovorans genome with other cellulosome‐related clostridial genomes

Clostridium cellulovorans, an anaerobic and mesophilic bacterium, degrades native substrates in soft biomass such as corn fibre and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Recently, we have reported the whole‐genome sequence of C. cellulovorans comprising 4220 predicted genes in 5.10 Mbp [Y. Tamaru et al., (2010) J. Bacteriol., 192: 901–902]. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome‐producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, and they coded for not only carbohydrate‐degrading enzymes but also a lipase, peptidases and proteinase inhibitors. Interestingly, two novel genes encoding scaffolding proteins were found in the genome. According to KEGG metabolic pathways and their comparison with 11 Clostridial genomes, gene expansion in the C. cellulovorans genome indicated mainly non‐cellulosomal genes encoding hemicellulases and pectin‐degrading enzymes. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced cellulosome‐producing Clostridium strains for industrial applications such as biofuel production.     

Overexpression of human WNK1 increases paracellular chloride permeability and phosphorylation of claudin-4 in MDCKII cells

Pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder characterized by hypertension, hyperkalemia, and hyperchloremic acidosis, is reportedly due to mutations in WNK1 and WNK4 kinase genes. However, the pathogenesis of the disease remains unknown. Mutations in the WNK1 gene are the deletions in the first intron, which reportedly increases WNK1 mRNA expression. Thus, we generated WNK1 over-expressing stable cell lines using MDCKII cells to model the distal nephron of PHAII patients. Using these cell lines, we investigated whether increased WNK1 expression might affect paracellular chloride permeability and claudin phosphorylation, since we previously observed an increase in both with a disease-causing mutant WNK4. WNK1 expression in MDCKII cells increased chloride permeability two to threefold. Co-expression of wild-type WNK4 did not further increase WNK1-enhanced chloride permeability. WNK1 expression also induced phosphorylation of endogenous claudin-4 in MDCKII cells, as well as over-expressed claudin-4. Combined, these results suggest that increased WNK1 expression has the same effect on chloride permeability and claudin phosphorylation as the mutant WNK4. Thus, increased chloride shunt may be involved in the pathogenesis of PHAII caused by WNK1 mutations.     

Shisa2 promotes the maturation of somitic precursors and transition to the segmental fate in Xenopus embryos

In vertebrate somitogenesis, FGF and Wnt signals constitute a morphogenetic gradient that controls the maturation of the presomitic mesoderm (PSM) as well as the transition to segmental units. It remains unclear, however, whether there is a regulatory mechanism that promotes the transition by a direct regulation of FGF and Wnt signaling in the PSM. Here we show that Shisa2, a member of a novel Shisa gene family, plays an essential role in segmental patterning during Xenopus somitogenesis. Shisa2 encodes an endoplasmic reticulum (ER) protein that cell-autonomously inhibits FGF and Wnt signaling by preventing the maturation and the cell-surface expression of their receptors. Shisa2 is expressed in the PSM and its knockdown caused a reduction in somite number by the delayed maturation of PSM and anterior shift of the transition; however, the phase of the segmental clock remained intact. These phenotypes were abolished by the inhibition of both FGF and Wnt signals, but by neither alone. We therefore propose that the individual inhibition of both types of signaling by the regulation of receptor maturation in the ER plays an essential role in the establishment of proper segmental patterning.     

Megakaryoblastic leukemia factor-1 gene in the susceptibility to coronary artery disease

Coronary artery disease (CAD) is based on the atherosclerosis of coronary artery and may manifest with myocardial infarction or angina pectoris. Although it is widely accepted that genetic factors are linked to CAD and several disease-related genes have been reported, only a few could be replicated suggesting that there might be some other CAD-related genes. To identify novel susceptibility loci for CAD, we used microsatellite markers in the screening and found six different candidate CAD loci. Subsequent single nucleotide polymorphism (SNP) association studies revealed an association between CAD and megakaryoblastic leukemia factor-1 gene (MKL1). The association with a promoter SNP of MKL1, ?184C > T, was found in a Japanese population and the association was replicated in another Japanese population and a Korean population. Functional analysis of the MKL1 promoter SNP suggested that the higher MKL1 expression was associated with CAD. These findings suggest that MKL1 is involved in the pathogenesis of CAD.