Browsing by Author "Yew, Tri-Rung"

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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
;
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.
Flexible UV-Ozone-Modified Carbon Nanotube Electrodes for Neuronal Recording
(2010-05-18)
Hsu, Hui-Lin
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Teng, I-Ju
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Chen, Yung-Chan
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Hsu, Wei-Lun
;
Lee, Yu-Tao
Carbon nanotubes were grown on flexible polyimide substrates at temperatures below 400 degrees C as electrodes for extracellularly neuronal recording. The electrical charge-transfer and electrochemical properties of such CNT electrodes were enhanced by UV-ozone exposure, which induced the formation of C-O, C=O, and O-C=O bonds and reduced the CNT/electrolyte interfacial impedance while increasing the interfacial capacitance.
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
;
Chen, Feng-Jung
;
Huang, Ying-Jung
;
Chen, Shin-Yu
;
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.
Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue
(2013)
Ho, Chen-Ta
;
Lin, Ruei-Zeng
;
Chen, Rong-Jhe
;
Chin, Chung-Kuang
;
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.
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
;
Huang, Ying-Jung
;
Chan, Chia-Han
;
Hsu, Long
;
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
;
Peng, Hwei-Ling
;
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
;
Chan, Chia-Han
;
Huang, Ying-Jung
;
Liu, Kuo-Liang
;
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.
Surface Rigidity Change of Escherichia coli after Filamentous Bacteriophage Infection
(2009-04-21)
Chen, Yi-Yang
;
Wu, Chien-Chen
;
Hsu, Jye-Lin
;
Peng, Hwei-Ling
;
Chang, Hwan-You
In this study, the feasibility using atomic force microscopy (AFM) to study the interaction between bacteriophages (phages) and bacteria in situ was demonstrated here. Filamentous phage M 13 specifically infects the male Escherichia coli, which expresses F-pili. After infection, E. coli become fragile and grows at a slower rate. AFM provides a powerful tool for investigating these changes in a near-physiological environment. Using high-resolution AFM in phosphate-buffered saline, the damage to the lipopolysaccharide (LPS) layer on the outer membrane of the M13 phage-infected E. coli was observed. The membrane became smoother and more featureless compared to those that were not infected. Besides, the force-distance (f-d) curves were measured to reveal the surface rigidity change in E. coli after M13 phage infection. The effective spring constant and Young's modulus of E. coli decreased after M13 phage infection. Furthermore, the AFM tip was pressed against E. coli to stud), the response of E. coli under load before and after M 13 phage infection. The results showed that after infection E. coli became less rigid and the membrane was also damaged. However, the stiffness changes, including the spring constant and Young's modulus of E. coli, are negligible after M 13 phage infection compared with those in previous reports, which may be one of the reasons that E. coli still can maintain its viability after filamentous phage infection.
Uncoiling mechanism of Klebsiella pneumoniae type 3 pili measured by using optical tweezers
(2007-01-01)
Chen, Feng-Jung
;
Chan, Chia-Han
;
Liu, Kuo-Liang
;
Huang, Ying-Jung
;
Peng, Hwei-Ling
Pili are bacterial appendages that play many important roles in bacterial behaviors, physiology and interaction with hosts. Via pili, bacteria are able to adhere to, migrate onto, and colonize on host cells, mechanically. Different from the most studied type I and P type pili, which are rigid and thick with an average of 6 similar to 7 nm in diameter, type 3 pili are relatively tiny (3-5 nm in diameter) and flexible, and their biophysical properties remains unclear. By using optical tweezers, we found that the elongation processes of type 3 pili are divided into three phases: (1) elastic elongation, (2) uncoiling-elongation, and (3) intrinsic elongation, separately. Besides, the uncoiling force of the recombinant pili displayed on the surface of E. coli [pmrkABCD(VI)F] is measured 20 pN in average stronger than that of E. coli [pmrkABCD(VI)]. This suggests that pilin MrkF is involved in determining the mechanical properties of the type 3 pili.
Uncoiling mechanism of Klebsiella pneumoniae type 3 pili measured by using optical tweezers - art. no. 66441W
(2007)
Chen, Feng-Jung
;
Chan, Chia-Han
;
Liu, Kuo-Liang
;
Huang, Ying-Jung
;
Peng, Hwei-Ling
Pili are bacterial appendages that play many important roles in bacterial behaviors, physiology and interaction with hosts. Via pili, bacteria are able to adhere to, migrate onto, and colonize on host cells, mechanically. Different from the most studied type I and P type pili, which are rigid and thick with an average of 6 similar to 7 nm in diameter, type 3 pili are relatively tiny (3-5 nm in diameter) and flexible, and their biophysical properties remains unclear. By using optical tweezers, we found that the elongation processes of type 3 pili are divided into three phases: (1) elastic elongation, (2) uncoiling-elongation, and (3) intrinsic elongation, separately. Besides, the uncoiling force of the recombinant pili displayed on the surface of E. coli [pmrkABCD(VI)F] is measured 20 pN in average stronger than that of E. coli [pmrkABCD(VI)]. This suggests that pilin MrkF is involved in determining the mechanical properties of the type 3 pili.