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In JoVE (1)
Other Publications (7)
- Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
- Applied Microbiology and Biotechnology
- Proceedings of the National Academy of Sciences of the United States of America
- Folia Microbiologica
- Natural Product Research
- Proceedings of the National Academy of Sciences of the United States of America
- Natural Product Research
Articles by Siyuan Wang in JoVE
JoVE General
Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap
1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Department of Physics, Lewis-Sigler Institute for Integrative Genomics, Princeton University
We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.
Other articles by Siyuan Wang on PubMed
Stochastic Model of Coliphage Lambda Regulatory Network
The dynamic properties of the regulatory network governing the choice between lytic and lysogenic growths of coliphage lambda is studied using a Markov chain stochastic model. Our computer simulation confirms the finding by Li et al. [Proc. Natl. Acad. Sci. USA 101, 4781 (2004)] on the dynamics of budding yeast: that the biological stationary states are global attractors; the biological pathways of lytic and lysogenic growths are attracting trajectories; and the network functions are robustly designed against structural perturbations. In addition, our model shows the stochastic switch from lysogen to lytic growth, which has been observed in experiments. A definition of pseudoenergy is introduced in the network dynamics to reveal a transitionlike behavior in the system.
Permeabilization of Microbacterium Oxylans Shifts the Conversion of Puerarin from Puerarin-7-O-glucoside to Puerarin-7-O-fructoside
The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 +/- 31.9 microM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 +/- 48.0 microM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, (13)C NMR, (1)H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 +/- 5.4 microM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 +/- 27.7 microM puerarin-7-O-glucoside and 187.8 +/- 29.5 microM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 +/- 24.0 microM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product's composition.
Actin-like Cytoskeleton Filaments Contribute to Cell Mechanics in Bacteria
A filamentous cytoskeleton largely governs the physical shape and mechanical properties of eukaryotic cells. In bacteria, proteins homologous to all three classes of eukaryotic cytoskeletal filaments have recently been discovered. These proteins are essential for the maintenance of bacterial cell shape and have been shown to guide the localization of key cell-wall-modifying enzymes. However, whether the bacterial cytoskeleton is stiff enough to affect the overall mechanical rigidity of a cell has not been probed. Here, we used an optical trap to measure the bending rigidity of live Escherichia coli cells. We find that the actin-homolog MreB contributes nearly as much to the stiffness of a cell as the peptidoglycan cell wall. By quantitatively modeling these measurements, our data indicate that the MreB is rigidly linked to the cell wall, increasing the mechanical stiffness of the overall system. These data are the first evidence that the bacterial cytoskeleton contributes to the mechanical integrity of a cell in much the same way as it does in eukaryotes.
Hydroxylation Modification and Free Radical Scavenging Activity of Puerarin-7-O-fructoside
Puerarin-7-O-fructoside was transformed by Trichoderma harzianum CGMCC 1523 into 3'-hydroxypuerarin-7-O-fructoside; this was identified by MS and NMR. However, puerarin-7-O-glucoside was not directly hydroxylated but hydrolyzed back into puerarin, which was transformed into 3'-hydroxypuerarin by the same fungi. Comparative analysis of free radical scavenging activity of DPPH showed that the free radical scavenging activity of puerarin-7-O-glucoside was reduced to approximately 1/2 of that of puerarin, while the free radical scavenging activity of puerarin-7-O-fructoside was increased to approximately 1.5 times of that of puerarin. The free radical scavenging activity of 3'-hydroxypuerarin-7-O-fructoside was further increased by 2.2 times of that of puerarin-7-O-fructoside, which was close to that of 3'-hydroxypuerarin.
LC Determination and Pharmacokinetic Study of Vitexin-4″-O-glucoside in Rat Plasma After Oral Administration
A simple and specific high-performance liquid chromatography (HPLC) method was developed for the pharmacokinetic study of vitexin-4″-O-glucoside (VOG) in rats after oral administration. The plasma samples were deproteinised with methanol after the addition of an internal standard, hesperidin. HPLC analysis was performed on a Diamonsil C(18) analytical column, using methanol -0.5% aqueous phosphoric acid (45:55, v/v) as the mobile phase with ultraviolet detection at 330 nm. The calibration curve was linear over the range of 5-450 µg mL(-1) in rat plasma. The average extraction recovery of VOG was 98.74% ± 0.44%, and the relative standard deviations of the intra- and inter-day precisions were not greater than 4.1% and 2.0%, respectively. The validated method was successfully applied during a pharmacokinetic study in rats after oral administration of VOG at different doses, and all the results indicated that the pharmacokinetics of VOG in rats obeyed nonlinear processes.
The Bacterial Actin MreB Rotates, and Rotation Depends on Cell-wall Assembly
Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.
Simultaneous Determination of Three Polyphenols in Rat Plasma After Orally Administering Hawthorn Leaves Extract by the HPLC Method
A simple and sensitive HPLC method was developed to simultaneously determine three active compounds, vitexin-4″-O-glucoside (VG), vitexin-2″-O-rhamnoside (VR) and hyperoside (HP), in rat plasma after administering the hawthorn leaves extract (HLE). An HPLC assay with baicalin as the internal standard was carried out using a Phenomsil C(18) analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6 : 1.5 : 18.5 : 74, v/v/v/v). The calibration curves were linear over the range of 2.5-500, 0.2-25 and 0.25-12.5 µg mL(-1) for VG, VR and HP, respectively. The method was reproducible and reliable, with relative standard deviations of the intra- and inter-day precision between 1.2% and 13.2% for the analysis of the three analytes. The validated HPLC method herein described was successfully applied to the pharmacokinetic study of VG, VR and HP after oral administration of HLE to rats over the dose range of 2.5-10 mL kg(-1).
