bioRxiv (Cold Spring Harbor Laboratory), Jan 14, 2020
The establishment of polarity in cells and tissues is one of the first steps in multicellular dev... more The establishment of polarity in cells and tissues is one of the first steps in multicellular development. The 'eternal embryo' hydra can completely regenerate from a disorganized cell cluster or a small fragment of tissue of about 10, 000 cells. During regeneration, the cells first form a hollow cell spheroid, which then undergoes de-novo symmetry breaking to irreversibly polarize. Here, we address the symmetry-related shape changes. Prior to axis establishment, the spheroid of regenerating cells presents inflation oscillations on several timescales that are isotropic in space. There are transient periods of fluctuations in defined arbitrary directions, until these undergo a clearly identified, irreversible transition to directed fluctuations along the future main axis of the regenerating hydra. Stabilized cytosolic actin structures disappear during the de-novo polarization, while polymerized microtubules remain. In our observations applied drugs that depolymerize actin filaments accelerate the symmetry breaking process, while drug-stabilized actin filaments prevent it. Nocodazole-depolymerized microtubules prevent symmetry breaking, but regeneration can be rescued by the microtubule-stabilizing drug paclitaxel at concentrations where microtubular structures start to reappear. We discuss the possibility that mechanical fluctuations induce the orientation and position of microtubules, which contribute to β-catenin nuclear translocation, to increase the organizer-forming-potential of the cells. Our data suggest that in regenerating hydra spheroids, microtubules play a pivotal role in the cooperative polarization process of the self-organizing hydra spheroid.
Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. I... more Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example, CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a beadbased method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space.
In order to achieve coordinated migration through extracellular matrix and endothelial barriers d... more In order to achieve coordinated migration through extracellular matrix and endothelial barriers during metastasis, cancer cells must be endowed with specific structural and adhesive properties. In this context, comparison of the mechanical properties of transformed versus normal cells, on which little quantitative information is available, was the focus of this study. Normal human dermal fibroblasts and their SV40-transformed counterparts were analyzed using various manipulations. First, micropipet aspiration of suspended cells allowed calculation of a cortical tension (similar for normal and transformed cells), and an apparent viscosity (30% lower for transformed than for normal fibroblasts); in addition, transformed fibroblasts exhibited a more fragile surface than their normal counterparts. Second, tangential ultracentrifugation of adherent cells demonstrated cellular elongation in the direction of the centrifu~al field and the existence of critical forces for cell detachment, around 10-N: these were 1.6-fold greater for normal than for transformed cells. Finally, examination of the wrinkle patterns formed by cells plated on a deformable polydimethylsiloxane substrate, plus analysis of cell retraction caused by ATP treatment following detergent permeabilization showed that normal fibroblasts exhibited much more contractility than their transformed counterparts, which we characterized by a cell contraction rate. Such quantitative parameters which reveal differences in the mechanical behavior of normal and transformed cells may be used in the future as new markers of oncogenic transformation.
Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strai... more Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.
bioRxiv (Cold Spring Harbor Laboratory), May 8, 2020
While cell crawling on 2D substrate often show a spatially polarised shape with clear front and b... more While cell crawling on 2D substrate often show a spatially polarised shape with clear front and back, such polarisation is elusive for cells moving in 3D. However directional movement requires symmetry breaking and polarisation. Here, we show that cells embedded in 3D matrix form at both sides of the nucleus force-dipoles, driven by myosin, that locally and periodically pinch the matrix. Using a combination of 3D live cell imaging, traction force microscopy and a minimal model with multipolar expansion, we show that the existence of a phase shift between the two dipoles, mediated mainly by the microtubule network, is involved in directed cell motion. We confirm this mechanism by triggering local dipolar contractions with laser ablation leading to directed motion. Our study reveals that the cell controls its motility by synchronising dipolar forces distributed at front and back.
Theoretical analysis of periodic RNA modifications in a simple biochemical reactor reveals emerge... more Theoretical analysis of periodic RNA modifications in a simple biochemical reactor reveals emergent evolutionary properties.
The mechanical properties of cells are important for many biological processes, including wound h... more The mechanical properties of cells are important for many biological processes, including wound healing, cancers, and embryogenesis. Currently, our understanding of cell mechanical properties remains incomplete. Different techniques have been used to probe different aspects of the mechanical properties of cells, among them microplate rheology, optical tweezers, micropipette aspiration, and magnetic twisting cytometry. These techniques have given rise to different theoretical descriptions, reaching from simple Kelvin-Voigt or Maxwell models to fractional such as power law models, and their combinations. Atomic force microscopy (AFM) is a flexible technique that enables global and local probing of adherent cells. Here, using an AFM, we indented single retinal pigmented epithelium cells adhering to the bottom of a culture dish. The indentation was performed at two locations: above the nucleus, and towards the periphery of the cell. We applied creep compliance, stress relaxation, and os...
Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In... more Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In some cells, symmetry breaking manifests itself in a directional flow of actin from the front to the rear of the cell. Many cells, especially in physiological 3D matrices do not show such coherent actin dynamics and present seemingly competing protrusion/retraction dynamics at their front and back. How symmetry breaking manifests itself for such cells is therefore elusive. We take inspiration from the scallop theorem proposed by Purcell for micro-swimmers in Newtonian fluids: self-propelled objects undergoing persistent motion at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. We report similar observations for cells crawling in 3D. We quantified cell motion using a combination of 3D live cell imaging, visualization of the matrix displacement and a minimal model with multipolar expansion. We show that our cells embedded in a 3D matrix form myosin-d...
The static and dynamic properties of solutions of cetylpyridinium chlorate micelles in 0.1 and 1 ... more The static and dynamic properties of solutions of cetylpyridinium chlorate micelles in 0.1 and 1 M NaC103 brine are studied by light scattering, rheology, and fluorescence recovery after bleaching. The static results lead to a description of the Solutions of wormlike micelles as analogous to semidilute solutions of polymers in good solvent. However the dynamic properties are different from those predicted for solutions of equilibrium ('living") polymers. Comparison of the results in the two studied system lead us to propose that, with increasing salt content, the network of wormlike micelles has an increasing proportion of connections. It is argued that the existence of a certain proportion of connections modifies the reptation mechanism for the relaxation of stress and ultimately allows new mechanisms for the relaxation of stress to become operative.
We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachme... more We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosincoated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 pm/s at 300C) than attachment through anti-LC2 (4-5 pm/s at 300C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement.
The canonical set of amino acids leads to an exceptionally wide range of protein functionality. N... more The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.
The mechanical properties of cells are important for many biological processes, including wound h... more The mechanical properties of cells are important for many biological processes, including wound healing, cancers, and embryogenesis. Currently, our understanding of cell mechanical properties remains incomplete. Different techniques have been used to probe different aspects of the mechanical properties of cells, among them microplate rheology, optical tweezers, micropipette aspiration, and magnetic twisting cytometry. These techniques have given rise to different theoretical descriptions, reaching from simple Kelvin-Voigt or Maxwell models to fractional such as power law models, and their combinations. Atomic force microscopy (AFM) is a flexible technique that enables global and local probing of adherent cells. Here, using an AFM, we indented single retinal pigmented epithelium cells adhering to the bottom of a culture dish. The indentation was performed at two locations: above the nucleus, and towards the periphery of the cell. We applied creep compliance, stress relaxation, and os...
For the microarray MM-pairs (in and ) the probe base (DNA) is on the left and the target base (DN... more For the microarray MM-pairs (in and ) the probe base (DNA) is on the left and the target base (DNA or RNA) is on the right. The efficiency of RNA interference () (from [2]) is assumed to be determined by the stability of A-form RNA duplexes between the -bound and the complementary mRNA. The left base is part of the guide strand (at position 10) and the right base is part of the mRNA. Apart from the the base pair ·the mRNA and siRNA sequences remained fixed. In () to () purine bases are highlighted in blue. In () and () mismatches with respect to a perfect matching C·G base pair are highlighted in red. Details on the individual stability orders are provided in the text.<b>Copyright information:</b>Taken from "Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges"http://www.biomedcentral.com/1472-6750/8/48BMC Biotechnology 2008;8():48-48.Published...
The positional influence was eliminated by subtraction of the moving average MM profile. Subseque... more The positional influence was eliminated by subtraction of the moving average MM profile. Subsequent normalization was performed by division through the mean hybridization signal of the particular MM profile. (B) MM-type related influence in RNA/DNA oligonucleotide duplexes. Hybridization signal differences between the pairs of RNA/DNA- and analog DNA/DNA-duplexes are shown in Additional file .<b>Copyright information:</b>Taken from "Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges"http://www.biomedcentral.com/1472-6750/8/48BMC Biotechnology 2008;8():48-48.Published online 13 May 2008PMCID:PMC2435543.
Following solution-background correction of the raw intensity data (Methods section) hybridizatio... more Following solution-background correction of the raw intensity data (Methods section) hybridization signals were normalized with respect to the largest hybridization signal in each of the four insertion profiles. The probe motifs 1 to 4 hybridize at different sections of the target oligonucleotide. Mean profiles (thick lines) were obtained from the moving average of the particular insertion profiles (particular hybridization signal are shown as faint symbols – profile 4 is shown in detail in Fig. 5A). The mean profiles 1 to 3 have a distinct minimum between base positions 15 to 20. The stabilizing CG-rich region following after base position 20 results in increased hybridization signals in profile 4.<b>Copyright information:</b>Taken from "Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges"http://www.biomedcentral.com/1472-6750/8/48BMC Biotech...
bioRxiv (Cold Spring Harbor Laboratory), Jan 14, 2020
The establishment of polarity in cells and tissues is one of the first steps in multicellular dev... more The establishment of polarity in cells and tissues is one of the first steps in multicellular development. The 'eternal embryo' hydra can completely regenerate from a disorganized cell cluster or a small fragment of tissue of about 10, 000 cells. During regeneration, the cells first form a hollow cell spheroid, which then undergoes de-novo symmetry breaking to irreversibly polarize. Here, we address the symmetry-related shape changes. Prior to axis establishment, the spheroid of regenerating cells presents inflation oscillations on several timescales that are isotropic in space. There are transient periods of fluctuations in defined arbitrary directions, until these undergo a clearly identified, irreversible transition to directed fluctuations along the future main axis of the regenerating hydra. Stabilized cytosolic actin structures disappear during the de-novo polarization, while polymerized microtubules remain. In our observations applied drugs that depolymerize actin filaments accelerate the symmetry breaking process, while drug-stabilized actin filaments prevent it. Nocodazole-depolymerized microtubules prevent symmetry breaking, but regeneration can be rescued by the microtubule-stabilizing drug paclitaxel at concentrations where microtubular structures start to reappear. We discuss the possibility that mechanical fluctuations induce the orientation and position of microtubules, which contribute to β-catenin nuclear translocation, to increase the organizer-forming-potential of the cells. Our data suggest that in regenerating hydra spheroids, microtubules play a pivotal role in the cooperative polarization process of the self-organizing hydra spheroid.
Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. I... more Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example, CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a beadbased method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space.
In order to achieve coordinated migration through extracellular matrix and endothelial barriers d... more In order to achieve coordinated migration through extracellular matrix and endothelial barriers during metastasis, cancer cells must be endowed with specific structural and adhesive properties. In this context, comparison of the mechanical properties of transformed versus normal cells, on which little quantitative information is available, was the focus of this study. Normal human dermal fibroblasts and their SV40-transformed counterparts were analyzed using various manipulations. First, micropipet aspiration of suspended cells allowed calculation of a cortical tension (similar for normal and transformed cells), and an apparent viscosity (30% lower for transformed than for normal fibroblasts); in addition, transformed fibroblasts exhibited a more fragile surface than their normal counterparts. Second, tangential ultracentrifugation of adherent cells demonstrated cellular elongation in the direction of the centrifu~al field and the existence of critical forces for cell detachment, around 10-N: these were 1.6-fold greater for normal than for transformed cells. Finally, examination of the wrinkle patterns formed by cells plated on a deformable polydimethylsiloxane substrate, plus analysis of cell retraction caused by ATP treatment following detergent permeabilization showed that normal fibroblasts exhibited much more contractility than their transformed counterparts, which we characterized by a cell contraction rate. Such quantitative parameters which reveal differences in the mechanical behavior of normal and transformed cells may be used in the future as new markers of oncogenic transformation.
Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strai... more Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.
bioRxiv (Cold Spring Harbor Laboratory), May 8, 2020
While cell crawling on 2D substrate often show a spatially polarised shape with clear front and b... more While cell crawling on 2D substrate often show a spatially polarised shape with clear front and back, such polarisation is elusive for cells moving in 3D. However directional movement requires symmetry breaking and polarisation. Here, we show that cells embedded in 3D matrix form at both sides of the nucleus force-dipoles, driven by myosin, that locally and periodically pinch the matrix. Using a combination of 3D live cell imaging, traction force microscopy and a minimal model with multipolar expansion, we show that the existence of a phase shift between the two dipoles, mediated mainly by the microtubule network, is involved in directed cell motion. We confirm this mechanism by triggering local dipolar contractions with laser ablation leading to directed motion. Our study reveals that the cell controls its motility by synchronising dipolar forces distributed at front and back.
Theoretical analysis of periodic RNA modifications in a simple biochemical reactor reveals emerge... more Theoretical analysis of periodic RNA modifications in a simple biochemical reactor reveals emergent evolutionary properties.
The mechanical properties of cells are important for many biological processes, including wound h... more The mechanical properties of cells are important for many biological processes, including wound healing, cancers, and embryogenesis. Currently, our understanding of cell mechanical properties remains incomplete. Different techniques have been used to probe different aspects of the mechanical properties of cells, among them microplate rheology, optical tweezers, micropipette aspiration, and magnetic twisting cytometry. These techniques have given rise to different theoretical descriptions, reaching from simple Kelvin-Voigt or Maxwell models to fractional such as power law models, and their combinations. Atomic force microscopy (AFM) is a flexible technique that enables global and local probing of adherent cells. Here, using an AFM, we indented single retinal pigmented epithelium cells adhering to the bottom of a culture dish. The indentation was performed at two locations: above the nucleus, and towards the periphery of the cell. We applied creep compliance, stress relaxation, and os...
Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In... more Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In some cells, symmetry breaking manifests itself in a directional flow of actin from the front to the rear of the cell. Many cells, especially in physiological 3D matrices do not show such coherent actin dynamics and present seemingly competing protrusion/retraction dynamics at their front and back. How symmetry breaking manifests itself for such cells is therefore elusive. We take inspiration from the scallop theorem proposed by Purcell for micro-swimmers in Newtonian fluids: self-propelled objects undergoing persistent motion at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. We report similar observations for cells crawling in 3D. We quantified cell motion using a combination of 3D live cell imaging, visualization of the matrix displacement and a minimal model with multipolar expansion. We show that our cells embedded in a 3D matrix form myosin-d...
The static and dynamic properties of solutions of cetylpyridinium chlorate micelles in 0.1 and 1 ... more The static and dynamic properties of solutions of cetylpyridinium chlorate micelles in 0.1 and 1 M NaC103 brine are studied by light scattering, rheology, and fluorescence recovery after bleaching. The static results lead to a description of the Solutions of wormlike micelles as analogous to semidilute solutions of polymers in good solvent. However the dynamic properties are different from those predicted for solutions of equilibrium ('living") polymers. Comparison of the results in the two studied system lead us to propose that, with increasing salt content, the network of wormlike micelles has an increasing proportion of connections. It is argued that the existence of a certain proportion of connections modifies the reptation mechanism for the relaxation of stress and ultimately allows new mechanisms for the relaxation of stress to become operative.
We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachme... more We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosincoated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 pm/s at 300C) than attachment through anti-LC2 (4-5 pm/s at 300C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement.
The canonical set of amino acids leads to an exceptionally wide range of protein functionality. N... more The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.
The mechanical properties of cells are important for many biological processes, including wound h... more The mechanical properties of cells are important for many biological processes, including wound healing, cancers, and embryogenesis. Currently, our understanding of cell mechanical properties remains incomplete. Different techniques have been used to probe different aspects of the mechanical properties of cells, among them microplate rheology, optical tweezers, micropipette aspiration, and magnetic twisting cytometry. These techniques have given rise to different theoretical descriptions, reaching from simple Kelvin-Voigt or Maxwell models to fractional such as power law models, and their combinations. Atomic force microscopy (AFM) is a flexible technique that enables global and local probing of adherent cells. Here, using an AFM, we indented single retinal pigmented epithelium cells adhering to the bottom of a culture dish. The indentation was performed at two locations: above the nucleus, and towards the periphery of the cell. We applied creep compliance, stress relaxation, and os...
For the microarray MM-pairs (in and ) the probe base (DNA) is on the left and the target base (DN... more For the microarray MM-pairs (in and ) the probe base (DNA) is on the left and the target base (DNA or RNA) is on the right. The efficiency of RNA interference () (from [2]) is assumed to be determined by the stability of A-form RNA duplexes between the -bound and the complementary mRNA. The left base is part of the guide strand (at position 10) and the right base is part of the mRNA. Apart from the the base pair ·the mRNA and siRNA sequences remained fixed. In () to () purine bases are highlighted in blue. In () and () mismatches with respect to a perfect matching C·G base pair are highlighted in red. Details on the individual stability orders are provided in the text.<b>Copyright information:</b>Taken from "Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges"http://www.biomedcentral.com/1472-6750/8/48BMC Biotechnology 2008;8():48-48.Published...
The positional influence was eliminated by subtraction of the moving average MM profile. Subseque... more The positional influence was eliminated by subtraction of the moving average MM profile. Subsequent normalization was performed by division through the mean hybridization signal of the particular MM profile. (B) MM-type related influence in RNA/DNA oligonucleotide duplexes. Hybridization signal differences between the pairs of RNA/DNA- and analog DNA/DNA-duplexes are shown in Additional file .<b>Copyright information:</b>Taken from "Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges"http://www.biomedcentral.com/1472-6750/8/48BMC Biotechnology 2008;8():48-48.Published online 13 May 2008PMCID:PMC2435543.
Following solution-background correction of the raw intensity data (Methods section) hybridizatio... more Following solution-background correction of the raw intensity data (Methods section) hybridization signals were normalized with respect to the largest hybridization signal in each of the four insertion profiles. The probe motifs 1 to 4 hybridize at different sections of the target oligonucleotide. Mean profiles (thick lines) were obtained from the moving average of the particular insertion profiles (particular hybridization signal are shown as faint symbols – profile 4 is shown in detail in Fig. 5A). The mean profiles 1 to 3 have a distinct minimum between base positions 15 to 20. The stabilizing CG-rich region following after base position 20 results in increased hybridization signals in profile 4.<b>Copyright information:</b>Taken from "Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges"http://www.biomedcentral.com/1472-6750/8/48BMC Biotech...
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