30 research outputs found
RNA-Seq gene expression profiling of HepG2 cells: the influence of experimental factors and comparison with liver tissue
Pure Gauge Configurations and Tachyon Solutions to String Field Theories Equations of Motion
In constructions of analytical solutions to open string field theories pure
gauge configurations parameterized by wedge states play an essential role.
These pure gauge configurations are constructed as perturbation expansions and
to guaranty that these configurations are asymptotical solutions to equations
of motions one needs to study convergence of the perturbation expansions. We
demonstrate that for the large parameter of the perturbation expansion these
pure gauge truncated configurations give divergent contributions to the
equation of motion on the subspace of the wedge states. We perform this
demonstration numerically for the pure gauge configurations related to tachyon
solutions for the bosonic and the NS fermionic SFT. By the numerical
calculations we also show that the perturbation expansions are cured by adding
extra terms. These terms are nothing but the terms necessary to make valued the
Sen conjectures.Comment: 30 pages, 9 figures, references added and conclusion extende
Genetically Encoded Red Photosensitizers with Enhanced Phototoxicity
Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures
Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore.
Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers
KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light.
Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity
Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia
Stereoview of the water channel (residues shown in yellow) with a chain of seven water molecules (red spheres) and the pore (residues shown in blue) filled with four water molecules.
<p>Residues mutated in this work are labeled in red. (Figure created with <i>PYMOL</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145740#pone.0145740.ref037" target="_blank">37</a>])</p
Schematic representation of the chromophore.
<p>Trp66 isomers described by χ1 and χ2 torsional angles around C<sup>α</sup>-C<sup>β</sup> and C<sup>β</sup>-C<sup>γ</sup> bonds, respectively (the corresponding dihedral angles are defined by atoms N-C<sup>α</sup>-C<sup>β</sup>- C<sup>γ</sup> and C<sup>α</sup>-C<sup>β</sup>-C<sup>γ</sup>-C<sup>δ</sup>; shown in orange). (A) <i>trans-cis</i> (KillerOrange and mKillerOrange structures; present work), (B) <i>cis-cis</i> (Cerulean structure at pH 7; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145740#pone.0145740.ref018" target="_blank">18</a>]), (C) <i>cis-trans</i> (Cerulean structure at pH 5; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145740#pone.0145740.ref017" target="_blank">17</a>]). (Figure was created with ChemDraw [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145740#pone.0145740.ref039" target="_blank">39</a>]).</p
Alignment of the amino acid sequences of photosensitizers (overall identity ~95%).
<p>The residues in the chromophore nearest environment are shown in bold red and the chromophore forming triad is highlighted in yellow.</p
The nearest chromophore environment of KillerOrange/mKillerOrange.
<p>The crystal structures of two proteins have a difference in position 177 –(Leu and Phe, respectively) and in the presence of glycerol (GOL—component of the cryo-protecting solution) in mKillerOrange structure. Hydrogen bonds (≤3.3 Å) are shown as blue dashed lines, water molecules (W) as red spheres, and van der Waals contacts (≤3.9 Å) as red “eyelashes”. (Figure prepared with <i>LIGPLOT/HBPLUS</i>, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145740#pone.0145740.ref038" target="_blank">38</a>]).</p