Herein, we introduce a simple route to fabricating hydrophilic microfluidic chips with an alternative material, a UV‐cured polyurethane‐related polymer, known as Norland Optical Adhesive (NOA 63). ...Conventionally, polydimethylsiloxane (PDMS) is widely used to fabricate microfluidic chips as an alternative to glass or SiO2 because PDMS is easily molded and relatively cheap. However, despite these advantages, the hydrophobicity of PDMS entails critical problems when it is used in microfluidic chips because microchannels inside the microfluidic chips, which have extremely low surface tension, are difficult to fill with aqueous solution without an extra pumping system. To overcome these problems, significant efforts have been focused on developing procedures to change the PDMS surface to be hydrophilic. However, the resulting hydrophilicity is generally short‐lived and the modification procedures require cumbersome multi‐steps. In the present study, we demonstrate that microchannel‐molding and microfluidic chip construction are easier using NOA 63 than when using PDMS and that the hydrophilicity of the NOA surface, which is induced by treatment with O2 plasma, lasts longer, for at least one month. Due to the longer lasting hydrophilicity, microchannels in NOA 63 microfluidic chips are spontaneously filled with solution by capillary reaction without any extra pumping over the period. The feasibility of NOA 63‐based microfabrication is verified by demonstrating NOA 63 microfluidic platforms with antibody‐immobilized beads for immunoassays.
Capillary forces can be used to fill this commercially available UV‐cured photopolymer with aqueous solutions (see figure) as opposed to the more commonly used polydimethylsiloxane. The beauty of this is that microfluidic chips created from this material can easily be produced by a simple oxygen‐plasma treatment. The chips in their turn can then be used for assays and other optical detection systems.
Zn
2+ uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficient conditions.
ykgM and
zinT (formerly
yodA), regulated by Zur (zinc uptake regulator), have ...been reported as being highly induced during zinc shortage. This work reports that
ykgM and
zinT in enterohemorrhagic
Escherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those in stationary-phase planktonic cells and a mutation of either
ykgM or
zinT genes led to the inhibition of curli biosynthesis. Inductively coupled plasma mass spectroscopy showed that the
ykgM and
zinT mutants contained lower concentrations of Zn
2+ than the wild type. Both mutants were less attached to both the glass surface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR data indicated that the expression of
csgA, which encodes the major curli subunit, was inhibited in both mutants with a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficient condition were covered with a lower amount of curli compared to the wild type and often became filamentous. Zn
2+ supplementation restored curli production and prevented filamentation in the mutants. Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zinc concentration in EHEC and intracellular zinc deficiency inhibits curli production.
► ykgM and zinT genes were highly expressed in biofilms of EHEC. ► The proteins are required for maintaining optimal intracellular zinc concentration. ► Intracellular zinc deficiency inhibits curli production. ► These results suggest that the genes play important roles in surface attachment.
Quorum sensing (QS) is defined as a cellto- cell signaling process that collectively regulates the gene expression of bacteria via small signaling molecules called autoinducers (AIs). It was reported ...that QS-regulated gene expression in
Pseudomonas aeruginosa
failed to occur at a high Reynolds number (Re= 3,000), since AI-2, a secreted interspecies signaling molecule, was washed away and so could not reach the minimum concentration required for QS. In this study, we describe the effects of flow speed on QS-stimulated biofilm formation in
Escherichia coli
O157:H7 inside a very thin microchannel (3 cm×1 cm×45 μm). In microtiter plates, the wild-type strain produced high amounts of exopolysaccharide, whereas its QS mutants
ΔluxS
and
ΔlsrK
, defective in AI-2 production and phosphorylation, made less exopolysaccharide. Confocal laser scanning microscopy showed that at a flow rate of 1 μL/min the wild-type strain formed rounded biofilms, whereas such biofilms formed by the QS mutants were fewer and thinner. At a flow rate of 10 μL/min, none of the tested strains formed mature biofilms. Our results suggest that QS is essential in the biofilm maturation under static or very slow laminar flow conditions where the biofilm signal can be easily accumulated and transported to the sessile cells.
Structural and Functional Importance of Outer Membrane Proteins in Vibrio cholerae Flagellum Bari, Wasimul, Yonsei University College of Medicine, Seoul, Republic of Korea; Lee, K.M., Yonsei University College of Medicine, Seoul, Republic of Korea; Yoon, S.S., Yonsei University College of Medicine, Seoul, Republic of Korea
The journal of microbiology,
08/2012, Letnik:
50, Številka:
4
Journal Article
Recenzirano
Vibrio cholerae has a sheath-covered monotrichous flagellum that is known to contribute to virulence. Although the structural organization of the V. cholerae flagellum has been extensively studied, ...the involvement of outer membrane proteins as integral components in the flagellum still remains elusive. Here we show that flagella produced by V. cholerae O1 El Tor strain C6706 were two times thicker than those from two other Gram-negative bacteria. A C6706 mutant strain (SSY11) devoid of two outer membrane proteins (OMPs), OmpU and OmpT, produced thinner flagella. SSY11 showed significant defects in the flagella-mediated motility as compared to its parental strain. Moreover, increased shedding of the flagella-associated proteins was observed in the culture supernatant of SSY11. This finding was also supported by the observation that culture supernatants of the SSY11 strain induced the production of a significantly higher level of IL-8 in human colon carcinoma HT29 and alveolar epithelial A549 cells than those of the wild-type C6706 strain. These results further suggest a definite role of these two OMPs in providing the structural integrity of the V. cholerae flagellum as part of the surrounding sheath.
A gene encoding a γ-butyrolactone autoregulator receptor, which has a common activity as DNA-binding transcriptional repressors controlling secondary metabolism and/or morphological differentiation ...in Streptomyces, was cloned from a natamycin producer, Streptomyces natalensis. PCR using the primers designed for the two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA) gave a 102-bp band. The sequence of this band had a high similarity to the expected region of a receptor gene. By genomic Southern hybridization with the 102-bp insert as a probe, a 687-bp intact receptor gene (sngR) was obtained from S. natalensis. To clarify the in vivo function of sngR, a sngR-disrupted strain was constructed, and the phenotypes were compared with those of the wild-type strain. The sngR-disruptants started natamycin production 6 h earlier and showed a 4.6-fold higher production of natamycin than the wild-type strain. In addition, the sporulation began earlier and the number of spores was tenfold more abundant than that of the wild-type strain. All the phenotypes were restored back to the original phenotypes of the wild-type strain by complementation with the intact sngR, indicating that the autoregulator receptor protein of S. natalensis acts as a primary negative regulator both on the biosynthesis of natamycin and sporulation.
Velvet deer antler (VDA) is well known oriental medicine claimed to have tonic activities as improving bone mineral density (BMD), immune-enhancing, rejuvenating and many other medicinal activities. ...Ossified deer antler (ODA) is bony product produced by over-calcification of deer antler due to late harvesting. The extraction efficiency of ODA by conventional boiling in water must be very poor due to bony nature, hence the reputations for the medicinal efficacies of ODA has been highly under-evaluated compared to that of VDA without any experimental evidences. Employing our new efficient water extraction process (135 oC), the extracts of ODA and VDA were analysed to compare the contents of bioactive components and the potencies of pharmacological activities. The results showed that; 1) The 135 oC extraction (autoclaving) of ODA gave highly increased amount of biomass, 120% more than the conventional extraction by 100-boiling, whereas the same treatment for VDA showed only 15% increased amount of biomass. 2) Feeding the ODA- or VDA-extracts to oophorectomized rats showed very potent BMD-recovering activity. 3) During the ossification of deer antler, the total collagen content was found to be increased by addition of type-1 to pre-existing type-2 collagen, but not replacement of type-2 to type-1 collagen. High titer of peptide hormones like growth hormone and IGF-1 were detected in the ODA- and VDA-extracts and also in the serum of ODA- or VDA-treated oophorectomized animals dose-dependently. Present experimental data will give a conclusion that folkloric poor reputations on ODA must be concerned only with poor extraction efficiency of conventional 100 oC water extraction and not based on the composition of bioactive substances of ODA.
Although the ciliate Tetrahymena is a good model for the study of chemotaxis, its profound motility makes it difficult to monitor intracellular calcium (Ca(2+)) changes induced by chemotactic ...stimuli. In this study, we report a microfluidic-based chemotaxis system generating directional chemotactic gradients under highly viscous conditions, suppressing T. pyriformis motility, and allowing for the stable confocal imaging of changes in intracellular Ca(2+) in the ciliate. Once the viscous condition was achieved, directional chemical gradients were formed inside the center chamber via the release of N-methyl-d-aspartate (NMDA), a known chemoattractant, from the surrounding chemical reservoirs into the center chamber. As a result, intracellular Ca(2+) in the ciliate increased up to three-fold, and its distribution was skewed in the direction of NMDA stimulation. However, the Ca(2+) in ciliates pretreated with phospholipase C (PLC) or phosphatidylinositol-3-kinase (PI3K) blockers did not increase even after stimulation. Additionally, the PI3K blocker induced the secretion of granules, the size of which was dependent on the concentration of the blocker. Collectively, the results indicate that both PLC and PI3K perform pivotal roles in controlling the levels of intracellular Ca(2+) in T. pyriformis during chemotaxis.
The prior sequencing of the upstream region of the γ-butyrolactone autoregulator receptor gene (sngR) in Streptomyces natalensis revealed the presence of a 972-bp gene encoding a BarX homologue ...(SngA), which acts as a pleiotropic regulator controlling secondary metabolism and morphological differentiation. In this study, we investigated the in vivo function of SngA in S. natalensis, by comparing the natamycin production, morphology, and transcription of genes related to natamycin biosynthesis in a wild-type strain and a sngA-deleted mutant. The disruption of sngA resulted in a decrease in natamycin production, and in the induction of pigment production that had not been previously observed from S. natalensis. On the other hand, the insertion of the intact sngA with its own promoter, into the wild-type strain, resulted in a 1.7-fold increase in natamycin production. Spore formation decreased in comparison to that of the wild-type strain when the sngA-deleted mutant was grown on YEME agar, MS medium, and ISP4 medium. All phenotypes were restored to the original wild-type phenotypes upon complementation with the intact sngA, suggesting that SngA has pleiotropic functions in controlling both morphological differentiation and secondary metabolite production.
PDF
