Ensemble Bayesian Analysis of Bistability in a Synthetic Transcriptional Switch

An overarching goal of synthetic and systems biology is to engineer and understand complex biochemical systems by rationally designing and analyzing their basic component interactions. Practically, the extent to which such reductionist approaches can be applied is unclear especially as the complexity of the system increases. Toward gradually increasing the complexity of systematically engineered systems, programmable synthetic circuits operating in cell-free <i>in vitro</i> environments offer a valuable testing ground for principles for the design, characterization, and analysis of complex biochemical systems. Here we illustrate this approach using <i>in vitro</i> transcriptional circuits (“genelets”) while developing an activatable transcriptional switch motif and configuring it as a bistable autoregulatory circuit, using just four synthetic DNA strands and three essential enzymes, bacteriophage T7 RNA polymerase, <i>Escherichia coli</i> ribonuclease H, and ribonuclease R. Fulfilling the promise of predictable system design, the thermodynamic and kinetic constraints prescribed at the sequence level were enough to experimentally demonstrate intended bistable dynamics for the synthetic autoregulatory switch. A simple mathematical model was constructed based on the mechanistic understanding of elementary reactions, and a Monte Carlo Bayesian inference approach was employed to find parameter sets compatible with a training set of experimental results; this ensemble of parameter sets was then used to predict a test set of additional experiments with reasonable agreement and to provide a rigorous basis for confidence in the mechanistic model. Our work demonstrates that programmable <i>in vitro</i> biochemical circuits can serve as a testing ground for evaluating methods for the design and analysis of more complex biochemical systems such as living cells

Optical Absorption of Armchair MoS₂ Nanoribbons: Enhanced Correlation Effects in the Reduced Dimension

We carry out first-principles calculations of the quasi-particle band structure and optical absorption spectra of H-passivated armchair MoS₂ nanoribbons (AMoS₂NRs) by employing the approach combining the Green’s function perturbation theory (<i>GW</i>) and the Bethe-Salpeter equation (BSE), i.e., <i>GW</i>+BSE. Optical absorption spectra of AMoS₂NRs show the exciton multibands (their binding energies are close to or less than 1 eV) which are much stronger than a single layer of MoS₂. However, they are absent in the spectra by the approach of <i>GW</i> and the random phase approximation (RPA), i.e., <i>GW</i>+RPA. This signifies that the excitonic correlation effects are strongly enhanced in the reduced dimensional structure of MoS₂. We also calculate the exciton wave functions for the few lowest energy excitons, which are found to have non-Frenkel character

Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System

<div><p>We present a simple and rapid method to isolate extracellular vesicles (EVs) by using a polyethylene glycol/dextran aqueous two-phase system (ATPS). This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs.</p></div

TEM, western blot and RT-PCR for comparison of ATPS, ATPS-Batch and ultra-centrifugation.

<p>(a) TEM image of EVs from ATPS and ultra-centrifugation method. The image did not show morphological difference between both methods. (b) The pellet after ultra-centrifugation was resuspended in 70 μl of PBS which was the same as the volume of the bottom phase of ATPS used to isolate EVs. Using the prepared samples, CD81 western blot was performed for the same sample volume (5 μl). Protein samples (5 μl) from standard mixture, ultra-centrifugation (25 μl for Ultra*5), ATPS method, and ATPS combined with Batch number 2 and 4 (ATPS-Batch #2 and #4) were used to confirm recovery efficiency. The band was brighter than that of the ultra-centrifugation method. (c) Purity of EVs was analyzed by western blot using CD81 antibody with the same protein amount (0.4 μg) from standard mixture, ultra-centrifugation, ATPS method, and ATPS-Batch #2 and #4. (d) RT-PCR was performed with 4.5 μl of isolated RNA from ultra-centrifugation, ATPS and ATPS combined with batch procedure. Bands of the house-keeping gene GAPDH and melanoma tumor marker Melan A were stronger after ATPS and ATPS-Batch than after ultra-centrifugation.</p

Recovery efficiency of EVs and proteins in batch procedure.

<p>After batch procedure, the recovery efficiency of EVs was almost unchanged while the recovery efficiency of proteins decreased.</p

Reliure de Voyage autour de mon jardin

La reliure industrielle apparaît tôt au XIX e siècle mais c'est surtout dans la deuxième moitié du siècle qu'elle connaît un véritable essor. Il s'agit de cartonnages recouverts le plus souvent de papier ou de percaline (toile de coton lustrée qui apparaît vers 1830). Ces cartonnages d'éditeur étaient réalisés à faible coût mais devaient cependant copier l'esthétique des reliures précieuses : dorures, décors d'entrelacs, de semis, etc. À partir de 1840, ils s'ornent de médaillons lithographiés en couleur dans le but de séduire le public (alors même que l'ouvrage reste en noir et blanc). Destinés surtout à un public jeune, ces livres servaient notamment de cadeaux lors des distributions de prix scolaires ou pour des étrennes. Certaines maisons d’édition étaient renommées pour leurs cartonnages : Martial Ardant, Mame, Mégard, Hachette, Hetzel, etc. Ici Voyage autour de mon jardin d’Alphonse Karr s’orne d’un cartonnage d’éditeur en toile gaufrée bleue, à décor floral doré et polychrome, par plaque.téléchargeabl

Partitioning studies.

<p>(a) Image of partitioned B16BL6 melanoma cells and polystyrene beads (hydrophobic) after phase separation. (b) Contact angle of melanoma and polystyrene beads with DEX-phase. The contact angle between melanoma and DEX-phase was ~40°, indicating that the cell membranes prefer DEX-phase to PEG-phase. Meanwhile, the contact angle between the polystyrene bead and DEX-phase was ~150°, indicating that polystyrene beads prefer PEG-phase.</p

Comparison of ATPS, ATPS-Batch #4 and ultra-centrifugation method.

<p>(a) Recovery efficiency. (b) Enrichment ratio.</p

Scheme and phase diagram for PEG/DEX ATPS.

<p>(a) Scheme of ATPS separation. EVs prefer DEX-phase to PEG-phase after phase separation using centrifugation (~1000ⅹg). (b) Phase diagram of PEG/DEX ATPS. The two-phase forms when system concentration is above the binodal curve.</p

Controllable Preparation of Monodisperse Microspheres Using Geometrically Mediated Droplet Formation in a Single Mold

We present a surfactant-free fabrication method for simultaneous generation of monodisperse microspheres with controllable size manner. Droplets that become microspheres by solidification processes are made in a two-step process: capillary rising-induced fluid division and wetting of immiscible fluid in a micromold. Design of the mold geometry and the monomer concentration primarily determines the microsphere size and the size distribution. Furthermore, the synergistic effect of two parameters is able to efficiently manipulate the microsphere sizes from submicrometers to a few hundred micrometers