Browsing by Author "Chang, Hwan-You"

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A microfluidic chip with a U-shaped microstructure array for multicellular spheroid formation, culturing and analysis
(2014-03-01)
Fu, Chien-Yu
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Tseng, Sheng-Yang
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Yang, Shih-Mo
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Hsu, Long
;
Liu, Cheng-Hsien
Multicellular spheroids (MCS), formed by self-assembly of single cells, are commonly used as a three-dimensional cell culture model to bridge the gap between in vitro monolayer culture and in vivo tissues. However, current methods for MCS generation and analysis still suffer drawbacks such as being labor-intensive and of poor controllability, and are not suitable for high-throughput applications. This study demonstrates a novel microfluidic chip to facilitate MCS formation, culturing and analysis. The chip contains an array of U-shaped microstructures fabricated by photopolymerizing the poly(ethylene glycol) diacrylate hydrogel through defining the ultraviolet light exposure pattern with a photomask. The geometry of the U-shaped microstructures allowed trapping cells into the pocket through the actions of fluid flow and the force of gravity. The hydrogel is non-adherent for cells, promoting the formation of MCS. Its permselective property also facilitates exchange of nutrients and waste for MCS, while providing protection of MCS from shearing stress during the medium perfusion. Heterotypic MCS can be formed easily by manipulating the cell trapping steps. Subsequent drug susceptibility analysis and long-term culture could also be achieved within the same chip. This MCS formation and culture platform can be used as a micro-scale bioreactor and applied in many cell biology and drug testing studies.
Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1
(2008-04-11)
Hsu, Jye-Lin
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Chen, Hsuan-Cheng
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Peng, Hwei-Ling
;
Chang, Hwan-You
;
生物科技學系
Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346-PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.
Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1
(2007-06-15)
Hung, Ruei-Jiun
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Chien, Han-Sheng
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Lin, Ruei-Zeng
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Lin, Ching-Ting
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Vatsyayan, Jaya
UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDPglucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP- glucose as a substrate to generate UDP- glucuronic acid. The Km values of PA2022 and PA3559 for UDP-glucose are similar to 0.1 and 0.4 mM, whereas the K-m values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP- glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg2+, which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.
EMULATED CIRCULATORY SYSTEM INTEGRATED WITH LOBULE-MIMETIC LIVER TO ENHANCE THE LIVER FUNCTION
(2012)
Sivashankar, Shilpa
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Puttaswamy, Srinivasu Valagerahally
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Dai, Tz-Shuian
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Lin, Ciou-Wun
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Yang, Shih-Mo
In the proposed paper, we report dielectrophoresis (DEP) liver lab chip integrated with Poly ethylene glycol diacrylate (PEGDA) based blood vessel within the microfluidic system for reconstructing the engineered liver tissue with the feature of liver function enhancement. The titanium (Ti) electrodes are well designed to provide positive dielectrophoresis (pDEP) and negative dielectrophoresis (nDEP) force to pattern HepG2 and 3T3 fibroblast cells. To mimic the blood flow direction of the liver lobule in vivo, we have integrated the reconstructed liver lobule with hydrogel encapsulated blood vessel. As smooth muscle cells and endothelial cells are the main components of blood vessel, we have utilized these cells and encapsulated within PEGDA hydrogel to mimic blood vessels. When assayed for urea concentration in cultured medium for five time points, the trend illustrated the enhanced metabolism of hepatocytes via blood vessel integrated lobule-mimetic pattern.
Enhancing ATP-based bacteria and biofilm detection by enzymatic pyrophosphate regeneration
(2010-04-15)
Lee, Hui-Ju
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Ho, Ming-Rong
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Bhuwan, Manish
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Hsu, Ching-Yi
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Huang, Meng-Shun
The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PP(i) in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5'-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PPi and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudo monas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines. (C) 2009 Elsevier Inc. All rights reserved.
Evidence of DNA transfer through F-pilus channels during Escherichia coli conjugation
(2008-07-01)
Shu, An-Chi
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Wu, Chien-Chen
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Chen, Yi-Yang
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Peng, Hwei-Ling
;
Chang, Hwan-You
The mechanism of DNA transfer from Escherichia coli (E. coli) Hfr donor strain AT2453 to recipient strain AB1157 during the conjugation process has been investigated by liquid atomic force microscopy (AFM). With the success of immobilizing both E. coli strains on gelatin-treated glass under aqueous solution, the F-pilus between an E. coli mating pair could be clearly imaged and dissected by an AFM probe. Another AFM probe functionalized with an anti-single-stranded DNA (ssDNA) antibody was then applied to detect transferring ssDNA. According to the AFM force spectrum, the transferring ssDNA could be detected only in the dissected area with a binding force of 109 5 pN measured. Our results provide direct evidence indicating that the DNA was transferred through the F-pilus channel between an E. coli mating pair during their conjugation.
Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria
(2011-11-01)
Lee, Hui-Ju
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Ho, Min-Rong
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Tseng, Chih-Sian
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Hsu, Ching-Yi
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Huang, Meng-Shun
Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia cob is approximately 10(4) colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP(i) and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP(i)-recycling loop was completed using ATP sulfurylase and adenosine 5' phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water. (C) 2011 Elsevier Inc. All rights reserved.
Fabrication of PEG Hydrogel Microstructures in Microfluidic System for Multicellular Spheroid Formation, Culturing and Analysis
(2011)
Fu, Chien-Yu
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Tseng, Sheng-Yang
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Yang, Shih-Mo
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Hsu, Long
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Liu, Cheng-Hsien
This study presents a novel microfluidic device for 3 dimension cell culturing and analysis. Through defining the exposure to ultra-violet light by a photomask, localized photopolymerization of poly (ethylene glycol) diacrylate hydrogels form biocompatible and permselective microstructures within a microfluidic device. The designed geometry of microstructures allowed cells to be trapped into structures and to form spheroids in situ. Subsequent analysis and long-term three-dimension cell culture can achieve within the same device. This technique may apply as a microscale bioreactor or a platform for spheroid-based drug screening and study.
Finger-powered agglutination lab chip with CMOS image sensing for rapid point-of-care diagnosis applications
(2020-01-21)
Lu, Chung Hsiang
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Shih, Ting-Sheng
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Shih, Po-Chen
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Pendharkar, Gaurav Prashant
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Liu, Cheng-En
Agglutination is an antigen-antibody reaction with visible expression of aggregation of the antigens and their corresponding antibodies. Applications extend to the identification of acute bacterial infection, hemagglutination, such as blood grouping, and diagnostic immunology. Our finger-powered agglutination lab chip with external CMOS image sensing was developed to support a platform for inexpensive, rapid point-of-care (POC) testing applications related to agglutination effects. In this paper, blood grouping (ABO and Rh grouping) was utilized to demonstrate the function of our finger-powered agglutination lab chip with CMOS image sensing. Blood antibodies were preloaded into the antibody reaction chamber in the lab chip. The blood sample was pushed through the antibody reaction chamber using finger-powered pressure actuation to initiate a hemagglutination reaction to identify the blood type at the on-chip detection area using our homemade CMOS image sensing mini-system. Finger-powered actuation without the need for external electrical pumping is excellent for low-cost POC applications, but the pumping liquid volume per finger push is hard to control. In our finger-powered agglutination lab chip with CMOS image sensing, we minimized the effects of different finger push depths and achieved robust performance for the test results with different push depths. The driving sample volume per finger push is about 0.79 mm(3). For different chips and different pushes, the driven sample volume per finger push was observed to vary in the range of 0.64 to 1.18 mm(3). The red blood cells were separated from the plasma on-chip after the whole blood sample was finger pumped and before the red blood cells reached the antibody chamber via an embedded plasma-separation membrane. Our homemade CMOS image mini-system robustly read and identified the agglutination results on our agglutination lab chip.
Genetic Requirements for Klebsiella pneumoniae-Induced Liver Abscess in an Oral Infection Model
(2009-07-01)
Tu, Ya-Chun
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Lu, Min-Chi
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Chiang, Ming-Ko
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Huang, Shu-Ping
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Peng, Hwei-Ling
Klebsiella pneumoniae is the predominant pathogen of primary liver abscess. However, our knowledge regarding the molecular basis of how K. pneumoniae causes primary infection in the liver is limited. We established an oral infection model that recapitulated the characteristics of liver abscess and conducted a genetic screen to identify the K. pneumoniae genes required for the development of liver abscess in mice. Twenty-eight mutants with attenuated growth in liver or spleen samples out of 2,880 signature-tagged mutants that produced the wild-type capsule were identified, and genetic loci which were disrupted in these mutants were identified to encode products with roles in cellular metabolism, adhesion, transportation, gene regulation, and unknown functions. We further evaluated the virulence attenuation of these mutants in independent infection experiments and categorized them accordingly into three classes. In particular, the class I and II mutant strains exhibited significantly reduced virulence in mice, and most of these strains were not detected in extraintestinal tissues at 48 h after oral inoculation. Interestingly, the mutated loci of about one-third of the class I and II mutant strains encode proteins with regulatory functions, and the transcript abundances of many other genes identified in the same screen were markedly changed in these regulatory mutant strains, suggesting a requirement for genetic regulatory networks for translocation of K. pneumoniae across the intestinal barrier. Furthermore, our finding that preimmunization with certain class I mutant strains protected mice against challenge with the wild-type strain implied a potential application for these strains in prophylaxis against K. pneumoniae infections.
Histidine-containing Phosphotransfer Protein-B (HptB) Regulates Swarming Motility through Partner-switching System in Pseudomonas aeruginosa PAO1 Strain
(2012-01-13)
Bhuwan, Manish
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Lee, Hui-Ju
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Peng, Hwei-Ling
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Chang, Hwan-You
;
生物科技學系
The histidine-containing phosphotransfer protein-B (HptB; PA3345) is an intermediate protein involved in transferring a phosphoryl group from multiple sensor kinases to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. The objective of this study was to elucidate the biological significance of the HptB-PA3346 interaction and the regulatory mechanisms thereafter. The transcription profiling analysis of an hptB knock-out mutant showed that the expression of a number of motility-related genes was altered consistent with the non-swarming phenotype observed for the mutant. Domain analysis indicated that the PA3346 C-terminal region (PA3346C) exhibits similar to 30% identity with the anti-sigma factor SpoIIAB of Bacillus subtilis. The presence of Ser/Thr protein kinase activity targeting an anti-sigma antagonist, PA3347, at Ser-56 was confirmed in PA3346C using an in vitro phosphorelay assay. Furthermore, PA3346C and the anti-sigma(28) factor FlgM were found to interact with PA3347 individually both in vivo and in vitro. FlgM displaced PA3346C in binding of PA3347 and was then competitively displaced by sigma(28) from the PA3347-FlgM complex, forming a phosphorylation-dependent partner-switching system. The significance of PA3347 phosphorylation in linking the partner-switching system and swarming motility was established by analyzing the swarming phenotype of the PA3347 knock-out mutant and its complement strains.
Identification of amino acid residues important for the phosphomannose isomerase activity of Ps1B in Pseudomonas aeruginosa PAO1
(2008-10-15)
Lee, Hui-Ju
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Chang, Hwan-You
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Venkatesan, Nandinin
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Peng, Hwei-Ling
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生物科技學院
Phosphomannose isomerase (PMI) plays a pivotal role in biosynthesis of GDP-mannose, an important precursor of many polysaccharides. We demonstrate in this study that Pseudomonas aeruginosa pslB encodes a protein with GDP-mannose pyrophosphorylase/PMI dual activities. The PMI activity is Co2+-dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the KM approximately 8-20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. Overall, these results provide a basis for understanding the catalytic mechanism of PMI.
Identification of Protein Domains on Major Pilin MrkA That Affects the Mechanical Properties of Klebsiella pneumoniae Type 3 Fimbriae
(2012-05-15)
Chan, Chia-Han
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Chen, Feng-Jung
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Huang, Ying-Jung
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Chen, Shin-Yu
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Liu, Kuo-Liang
The Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helixlike filament. This study determined the biomechanical properties of the fimbriae and analyzed 11 site-directed MrkA mutants to identify domains that are critical for the properties. Escherichia coli strains expressing type 3 fimbriae with an Ala substitution at either F34, V45, C87, G189, T196, or Y197 resulted in a significant reduction in biofilm formation. The E. coli strain expressing MrkAG189A remained capable of producing a normal number of fimbriae. Although F34A, V45A, T196A, and Y197A substitutions expressed on E. coli strains produced sparse quantities of fimbriae, no fimbriae were observed on the cells expressing MrkAC87A. Further investigations of the mechanical properties of the MrkAG189A fimbriae with optical tweezers revealed that, unlike the wild-type fimbriae, the uncoiling force for MrkAG189A fimbriae was not constant. The MrkAG189A fimbriae also exhibited a lower enthalpy in the differential scanning calorimetry analysis. Together, these findings indicate that the mutant fimbriae are less stable than the wildtype. This study has demonstrated that the C-terminal beta strands of MrkA are required for the assembly and structural stability of fimbriae.
Integration of organic opto-electrowetting and poly(ethylene) glycol diacrylate (PEGDA) microfluidics for droplets manipulation
(2013-04-01)
Yu, Tung-Ming
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Yang, Shih-Mo
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Fu, Chien-Yu
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Liu, Ming-Huei
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Hsu, Long
This paper reports a fabrication technology which integrates organic opto-electrowetting (OEW) with PEGDA-based microfluidics between stacked ITO glass chips for droplets generation and manipulation. Organic OEW can be realized by using titanium oxide phthalocyanine (TiOPc) as a photoconductive layer. Optical images can be projected on an organic OEW area to induce the local virtual electrodes which reduce the surface energy for actuating droplets. Low-molecular-weight (LMW) PEGDA (poly(ethylene) glycol diacrylate) material is used to form stable and biocompatible microchannels between the stacked ITO glass chips by UV photopolymerization process. PEGDA-based microfluidics provides a simpler way to enhance droplets applications in organic OEW. We demonstrate (1) the droplet formation in PEGDA microstructure, (2) the motion of droplet-in-air and droplet-in-oil operated by TiOPc OEW in this platform. This technology requires only spin-coating and UV exposure and is more cost-effective than previous methods. (C) 2011 Elsevier B.V. All rights reserved.
Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue
(2013)
Ho, Chen-Ta
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Lin, Ruei-Zeng
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Chen, Rong-Jhe
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Chin, Chung-Kuang
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Gong, Song-En
A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.
Localization and characterization of VVA0331, a 489-kDa RTX-like protein, in Vibrio vulnificus YJ016
(2009-05-01)
Chou, Li-Fang
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Peng, Hwei-Ling
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Yang, Yu-Chung
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Kuo, Min-Chieh
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Chang, Hwan-You
Vibrio vulnificus YJ016 contains three genes encoding proteins homologous to repeats-in-toxin proteins. One of these genes, vva0331, possesses a long open reading frame of 13,971 bp in length and resides on the small chromosome between two gene clusters encoding a type I secretion system and several regulatory proteins, respectively. Bioinformatic analysis revealed that VVA0331 consist of nineteen 87-amino acid repeats, two Arg-Gly-Asp motifs, four cysteine residues, an outer membrane protein domain, a polysaccharide-binding site and several motifs related to cell adhesions. These features are distinct from those of typical repeat-in-toxins and autotransporter adhesins. Real-time quantitative PCR analysis indicates that vva0331 gene expression is activated at 30A degrees C and regulated by iron. In addition, VVA0331 is present primarily in a secreted form as determined by cell fractionation assay and Western blot analysis. No significant difference in Hep2 cell adherence, cytotoxicity, and virulence was observed between the wild type and vva0331 mutant strains. In contrast, these strains exhibited apparently different outer membrane protein profiles, and antiserum raised against C-terminal region of VVA0331 reacted with an 85-kDa outer membrane protein of V. vulnificus YJ016.
Magnetic bead-based DNA detection with multi-layers quantum dots labeling for rapid detection of Escherichia coli O157:H7
(2008-12-01)
Liu, Yi-Ju
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Yao, Da-Jeng
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Chang, Hwan-You
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Liu, Chien-Ming
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Chen, Chih
This work demonstrated the feasibility of detecting 250 zM Escherichia coli O157:H7 eaeA target DNA by using a magnetic bead-based DNA detection assay with designed labeling strategy within 40-60 min. The magnetic beads were used as the solid support for the binding probe and isolated the target DNA from the sample. The detection signals could be amplified from the multi-layers biotin-streptavidin conjugated quantum dots based on binding with specific designed biotinlyted linker. This assay method would provide a simple, rapid, and ultra-sensitive detection method for DNA or other biomolecular analysis. (C) 2008 Elsevier B.V. All rights reserved.
Measurement of the adhesive force between a single Klebsiella pneumoniae type 3 fimbria and collagen IV using optical tweezers
(2006-11-10)
Chang, Bo-Jui
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Huang, Ying-Jung
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Chan, Chia-Han
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Hsu, Long
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Peng, Hwei-Ling
Type 3 fimbriae are important adhesive filaments that assist Klebsiella pneumoniae to establish an infection. Different MrkD adhesin variants on the fimbriae are known to display distinct adherence capability for the bacteria to bind extracellular matrix proteins, although the difference has not been determined physically. For this reason, the adhesive force between type 3 fimbriae and collagen IV were measured using optical tweezers. The measured force data displayed a periodic histogram thus Fourier analysis was applied to group it to extract the adhesive force of a single molecular pair. Specifically, we showed that grouping should begin with an offset at the first half of the period. Finally, we first present the adhesive force between each mrkD(V2)-, mrkD(V3)-, and mrkD(V4)-expressed fimbriae and collagen IV is 2.03, 3.79, and 2.87 pN, respectively, This result can be referred to further research on mrkD allelic effect on bacteria infection. (c) 2006 Elsevier Inc. All rights reserved.
Novel microchip for in situ TEM imaging of living organisms and bio-reactions in aqueous conditions
(2008)
Liu, Kuo-Liang
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Wu, Chien-Chen
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Huang, Ying-Jung
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Peng, Hwei-Ling
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Chang, Hwan-You
A novel and disposable microchip (K-kit) with SiO(2) nano-membranes was developed and used as a specimen kit for in situ imaging of living organisms in an aqueous condition using transmission electron microscopy (TEM) without equipment modi. cation. This K-kit enabled the successful TEM observation of living Escherichia coli cells and the tellurite reduction process in Klebsiella pneumoniae. The K. pneumoniae and Saccharomyces cerevisiae can stay alive in K-kit after continuous TEM imaging for up to 14 s and 42 s, respectively. Besides, different tellurite reduction profiles in cells grown in aerobic and anaerobic environments can be clearly revealed. These results demonstrate that the K-kit developed in this paper can be useful for observing living organisms and monitoring biological processes in situ.
Structural and Mechanical Properties of Klebsiella pneumoniae Type 3 Fimbriae
(2011-04-01)
Chen, Feng-Jung
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Chan, Chia-Han
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Huang, Ying-Jung
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Liu, Kuo-Liang
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Peng, Hwei-Ling
This study investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium. Transmission electron microscopy and optical tweezers were used to understand the ability of the bacterium to survive flushes. An individual K. pneumoniae type 3 fimbria exhibited a helix-like structure with a pitch of 4.1 nm and a three-phase force-extension curve. The fimbria was first nonlinearly stretched with increasing force. Then, it started to uncoil and extended several micrometers at a fixed force of 66 +/- 4 pN (n +/- 22). Finally, the extension of the fimbria shifted to the third phase, with a characteristic force of 102 +/- 9 pN (n = 14) at the inflection point. Compared with the P fimbriae and type 1 fimbriae of uropathogenic Escherichia coli, K. pneumoniae type 3 fimbriae have a larger pitch in the helix-like structure and stronger uncoiling and characteristic forces.