31,644 research outputs found
Persistence of recombinant bacteria to antimicrobial silver
No full textSilver, owing to its effective antimicrobial properties, has been used against a broad range of microorganisms. Silver is now utilized commonly in numerous consumer products, medical devices and clinical applications. However, the mechanism of action of the silver is not yet fully established and well-understood. In addition, it is also important to understand the biochemical and evolutionary pathways that give rise to resistance. Here, we report new genetic determinants for silver resistance in E. coli and explore aspects of their mechanism and laboratory evolution.
Initial exploration of the antimicrobial activity of silver showed that (1) antimicrobial ability of silver is time and dose-dependent; (2) Ag ions have much more antibiotic activity than silver nanoparticles (AgNPs) and (3) the antimicrobial ability of AgNPs is size-dependent. Further selection for resistance genes of E. coli using AgNO3 and AgNPs led to the identification of several candidates, including cysD and ycdB, which displayed cross-resistance to Ag ion and AgNPs as well as Cu+ and Cd2+. The genes cysD and ycdB conferred less resistance to metallic Ag(0) under anaerobic incubation than aerobic incubation. These results support that Ag+ ions are the main toxic agents of AgNPs. These novel anti-silver genes also endowed resistance to the antibiotics kanamycin and ampicillin; in these experiments, antibacterial synergy between kanamycin and silver, but not between ampicillin and silver, was also found. Quantification of oxygen radicals suggest that silver ion is bactericidal through production of reactive oxygen species and that silver-resistance genes prevent their generation.
The selected gene ycdB and control gene cueO, both of which led to increased silver resistance, encode Tat-dependent proteins, which are transported after folding from cytoplasm to periplasm. Chapter 2 focuses on several Tat-containing genes, which also gave more resistance to Ag ion. The 7 selected Tat sequence genes, including torA, yedY, sufI, ycdO and hybA, were recombinantly expressed in various truncated forms, showing that for ycdB and yedY deleting Tat sequences impaired export and silver-resistance ability, despite increased expression, but that for other Tat genes deleting Tat had little effect on either periplasmic translocation or resistance. In all cases, expression of the Tat export sequence alone or with the his-tag in absence of the gene led to suppression of resistance.
Finally, we explored the evolvability of selected genes, such as yeaO, ydgT, iscA and ycdB for silver-resistance. Evolved mutants of yeaO and ydgT were found that endowed increased resistance to silver compared to wildtypes. In these two cases, increased resistance to silver did not lead to increased antibiotic resistance. In short, several kinds of anti-silver genes were identified in our studies, showing various pathways rendering resistance to silver. Weak resistance functions for some genes were evolvable. Our studies provide a deeper insight into silver’s mechanism of action and of the possible resistance pathways in bacteria, which may in some cases lead also to cross-resistance to antibiotics
An in vitro model system for cytoskeletal confinement
No full textThe motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins -mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the complete contour of the filament which makes the technique applicable to a broad range of biological polymers, including those with a persistence length smaller than the optical resolution
X-ray studies of biological matter in microfluidic environments
No full textBiological systems such as cells and cellular components are governed by processes, which take place on nanometer to micrometer length scales. X-ray scattering, diffraction and imaging techniques are extremely well suited to study these processes as the spatial resolution extends well into the relevant length scales. At the same time, the investigation of physical and chemical properties and behavior of such systems requires well-defined and controllable sample environments. One successful way to establish such environments, including specified flow fields, concentration gradients and confinement regimes is by employing microfluidic technology tailored to the particular scientific question. This brief review focuses on microfluidic techniques that have been used to investigate biological matter by X-rays. In particular, we show how the characteristics of flow on the micron scale enable new scientific approaches as compared to macroscale experiments
Impact of Microscopic Motility on the Swimming Behavior of Parasites : Straighter Trypanosomes are More Directional
No full textMicroorganisms, particularly parasites, have developed sophisticated swimming mechanisms to cope with a varied range of environments. African Trypanosomes, causative agents of fatal illness in humans and animals, use an insect vector (the Tsetse fly) to infect mammals, involving many developmental changes in which cell motility is of prime importance. Our studies reveal that differences in cell body shape are correlated with a diverse range of cell behaviors contributing to the directional motion of the cell. Straighter cells swim more directionally while cells that exhibit little net displacement appear to be more bent. Initiation of cell division, beginning with the emergence of a second flagellum at the base, correlates to directional persistence. Cell trajectory and rapid body fluctuation correlation analysis uncovers two characteristic relaxation times: a short relaxation time due to strong body distortions in the range of 20 to 80 ms and a longer time associated with the persistence in average swimming direction in the order of 15 seconds. Different motility modes, possibly resulting from varying body stiffness, could be of consequence for host invasion during distinct infective stages
Characterization of single semiflexible filaments under geometric constraints
No full textConfinement effects on single semiflexible macromolecules are of central importance for a fundamental understanding of cellular processes involving biomacromolecules. To analyze the influence of confinement on the fluctuations of semiflexible macromolecules we study individual actin filaments in straight and curved microchannels. We experimentally characterize the segment distributions for fluctuating semiflexible filaments in microchannels as a function of the channel width. Moreover, the effect of channel curvature on the filament fluctuations is investigated. We find quantitative agreement between experimental results, Monte Carlo simulations, and the analytical description. This allows for determination of the persistence length of actin filaments, the deflection length, which characterizes the confinement effects, and the scaling exponents for the segment distribution of semiflexible macromolecules
Thomas Grisell letter to Thomas Rotch, 2nd mo 19th 1823
No full textThomas Grisell's letter reached the Rotch household several months before the unexpected death of Thomas Rotch in August, 1823. This is the last letter of the series and presumably the author learned of his friend's death before another letter was penned. 7.95" x 10" (20.2 by 25.5 cm
Mobility gradient induces cross-streamline migration of semiflexible polymers
No full textMany aspects of modern material science and biology rely on the strategic manipulation and understanding of polymer dynamics in confining micro- and nanoflow. We directly observe and analyze nonequilibrium structural and dynamic properties of individual semiflexible actin filaments in pressure-driven microfluidic channel flow using fluorescence microscopy. Different conformational shapes, such as filaments fluctuating in an elongated manner, parabolically bent, as well as tumbling, are identified. With increasing flow velocity, a strong center-of-mass migration toward the channel walls is observed. This significant migration effect can be explained by a shear rate dependent spatial diffusivity due to a gradient in chain mobility of the semiflexible polymers
Brownian motion of actin filaments in confining microchannels
No full textSince the cytoskeletal protein actin is one of the principal building blocks of mammalian cells, it has recently been arousing much interest. Here, we address questions concerning the mechanical and dynamic behaviour of individual actin filaments in confining geometries which mimic the dense cytoskeletal network in eukaryotic cells. Microfluidic devices fabricated by soft photolithography in combination with fluorescence microscopy are used to manipulate, observe and characterize these biopolymers. The polymer statistics is strongly dependent on the characteristics of the surroundings such as the degree of confinement and hydrodynamical flow. Besides this, the intrinsic mechanical properties of the filaments are dominated by the persistence length and the contour length. We analyse the tangent–tangent correlation and the radial distribution function in terms of a confining potential and the contour length of the filaments. In addition, we show that hydrodynamic flow can be successfully used to apply controlled local stress on actin filaments. Our results can be surprisingly well described by a straightforward model which approximates the confining energy of the microchannels using a parabolic potential
From ligand-stabilized gold nanoparticles to hybrid organic-inorganic superstructures
No full textGold nanoparticles (Au NPs) have many potential applications including nanoelectronics, catalysts and sensors. These future devices depend on stable and monodisperse NPs and their directed assembly.
Different macromolecular multidentate thioether ligands were synthesized and used for the stabilization of Au NPs with diameters of around 1.2 nm and narrow size distributions. The NPs were prepared from soluble gold (III) precursors in a two-phase process in presence of the respective ligand.
The properties of the thioether ligands concerning the sizes as well as the stability and dispersity of Au NPs were thereby investigated and it was found that the size and surface functionalization of NPs can be controlled by varying the size and shape of the thioether ligands. Bifunctional NPs were formed in the presence of linear octadentate ligands and their functionality, an oligo phenylene ethynylene (OPE) rod with a protected terminal acetylene, was used to form NP superstructures upon homocoupling. Switching from benzene to pyridine as anchor of the functionality induced a perpendicular arrangement of the rod on the NP surface. Theoretical calculations suggested that this controlled orientation was directed by coordination of the nitrogen’s lonepair to the gold surface. Thioether dendrimers were used to enlarge the ligand structure and thus its denticity. Their stabilizing ability strongly depended on the size of the protective ligand shell showing the importance of the tert-butyl functionalized benzene units. By using icosadentate dendrimers (20 thioether moieties) we were able to form monofunctionalized NPs.
These artificial molecules were used to form dumbbell structures with satisfying yields and controlled interparticle distances
Fluctuations of Single Confined Actin Filaments
No full textThermal fluctuations of individual actin filaments confined in rectangular microchannels with dimensions similar to the mesh size of the cytoskeleton in eukaryotic cells are studied experimentally using fluorescence microscopy and theoretically by a combination of analytical methods and Monte Carlo simulations. Compared to freely fluctuating filaments, long filaments confined in narrow channels exhibit enhanced tangent correlations and a characteristic shape of their correlation function. The tangent correlation function is calculated analytically by approximating the confining geometry by a parabolic potential. This approximation is validated by Monte Carlo simulations. For the quantitative analysis of experimental data additional corrections for image analysis effects have to be included, for which we provide a modified analytical approximation formula which is corroborated by simulations. This allows us to obtain both the persistence length LP describing the bending rigidity of the polymer and the deflection length λ characterizing confinement effects from fits to the experimental data. Our results confirm the scaling relation λ ∝ d2/3 between the deflection length and the channel width d
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