Bayesian-based decipherment of in-depth information in bacterial chemical sensing beyond pleasant/unpleasant responses.

Chemical sensing is vital to the survival of all organisms. Bacterial chemotaxis is conducted by multiple receptors that sense chemicals to regulate a single signalling system controlling the transition between the direction (clockwise vs. counterclockwise) of flagellar rotation.

Silinanyl Rhodamines and Silinanyl Fluoresceins for Super-Resolution Microscopy.

Single-molecule localization microscopy (SMLM) enables the visualization of biomolecules at unprecedented resolution and requires control of the fluorescent blinking (ON/OFF) states of fluorophores to detect single-molecule fluorescence without overlapping of the signals. Although SMLM probes based on the intramolecular spirocyclization of Si-xanthene fluorophores have been developed, fluorophores with lower ON/OFF ratios are required for SMLM visualization of high-density structures. Here, we describe a silinane structure that lowers the ON/OFF ratio of Si-xanthene fluorophores.

Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qβ replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system.

Evolutionary Consequence of a Trade-Off between Growth and Maintenance along with Ribosomal Damages.

Microorganisms in nature are constantly subjected to a limited availability of resources and experience repeated starvation and nutrition. Therefore, microbial life may evolve for both growth fitness and sustainability. By contrast, experimental evolution, as a powerful approach to investigate microbial evolutionary strategies, often targets the increased growth fitness in controlled, steady-state conditions.

A transcription and translation-coupled DNA replication system using rolling-circle replication.

All living organisms have a genome replication system in which genomic DNA is replicated by a DNA polymerase translated from mRNA transcribed from the genome. The artificial reconstitution of this genome replication system is a great challenge in in vitro synthetic biology. In this study, we attempted to construct a transcription- and translation-coupled DNA replication (TTcDR) system using circular genomic DNA encoding phi29 DNA polymerase and a reconstituted transcription and translation system.

Development of a reporter peptide that catalytically produces a fluorescent signal through α-complementation.

In α-complementation, inactive N-terminal (α-domain) and C-terminal (ω-domain) fragments of β-galactosidase associate to reconstitute the active protein. To date, the effect of α-domain size on α-complementation activity has not been systematically investigated. In this study, we compared the complementation activities of α-domains of various sizes using an in vitro system.

Design, synthesis, and structure-activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists.

Herein we describe the design, synthesis, and structure-activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33 b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33 b ((-)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments.

The evolutionary enhancement of genotype-phenotype linkages in the presence of multiple copies of genetic material.

Genetic evolutionary mechanisms employed by protolife developed without accompanying regulatory mechanisms for the amounts of genetic material in protocells. When many copies of genetic material are present, inactive copies generated by mutations are not effectively excluded through phenotypic selection. We demonstrate a model of gene evolution initiated with different amounts of DNA inside artificial protocells.

Adaptive evolution of an artificial RNA genome to a reduced ribosome environment.

The reconstitution of an artificial system that has the same evolutionary ability as a living thing is a major challenge in the in vitro synthetic biology. In this study, we tested the adaptive evolutionary ability of an artificial RNA genome replication system, termed the translation-coupled RNA replication (TcRR) system. In a previous work, we performed a study of the long-term evolution of the genome with an excess amount of ribosome.

Liposome display for in vitro selection and evolution of membrane proteins.

Liposome display is a novel method for in vitro selection and directed evolution of membrane proteins. In this approach, membrane proteins of interest are displayed on liposome membranes through translation from a single DNA molecule by using an encapsulated cell-free translation system. The liposomes are probed with a fluorescence indicator that senses membrane protein activity and selected using a fluorescence-activated cell sorting (FACS) instrument.

Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.

In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.

Effects of ribosomes on the kinetics of Qβ replication.

Bacteriophage Qβ utilizes some host cell translation factors during replication. Previously, we constructed a kinetic model that explains replication of long RNA molecules by Qβ replicase. Here, we expanded the previous kinetic model to include the effects of ribosome concentration on RNA replication.

Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment.

The ability to evolve is a key characteristic that distinguishes living things from non-living chemical compounds. The construction of an evolvable cell-like system entirely from non-living molecules has been a major challenge. Here we construct an evolvable artificial cell model from an assembly of biochemical molecules.

In vitro evolution of α-hemolysin using a liposome display.

In vitro methods have enabled the rapid and efficient evolution of proteins and successful generation of novel and highly functional proteins. However, the available methods consider only globular proteins (e.g.

Kinetic model of double-stranded RNA formation during long RNA replication by Qβ replicase.

Qβ replicase is an RNA-dependent RNA polymerase, which synthesizes the complementary RNA using a single-stranded RNA as a template. The formation of non-replicable double-stranded RNA (dsRNA) by hybridization between newly synthesized RNA and the template RNA hinders the broader application of Qβ replicase. Here, we developed a kinetic model of Qβ RNA replication consisting of two reaction pathways of dsRNA formation, which quantitatively explains the dynamics of dsRNA formation of three template RNAs.

Effects of compartment size on the kinetics of intracompartmental multimeric protein synthesis.

The cell contents are encapsulated within a compartment, the volume of which is a fundamental physical parameter that may affect intracompartmental reactions. However, there have been few studies to elucidate whether and how volume changes alone can affect the reaction kinetics. It is difficult to address these questions in vivo, because forced cell volume changes, e.

A controllable gene expression system in liposomes that includes a positive feedback loop.

We introduced a positive feedback loop into a LacI-dependent gene expression system in lipid vesicles, producing a cell-like system that senses and responds to an external signal with a high signal-to-noise ratio. This fully reconstituted system will be a useful tool in future applications in in vitro synthetic biology.

α-Complementation in an artificial genome replication system in liposomes.

Genome size is considered one of the limiting factors for the replication of primitive life forms. However, the relationship between genome size and replication efficiency has not been tested experimentally. In this study, we examined the effect of genome size on genome replication by using an artificial cell model: a self-replicating RNA genome encapsulated in a liposome.

Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: modeling of ligand-dependent and -independent expression.

Aptazymes are useful as RNA-based switches of gene expression responsive to several types of compounds. One of the most important properties of the switching ability is the signal/noise (S/N) ratio, i.e.

Cell-free protein synthesis from a single copy of DNA in a glass microchamber.

To achieve a cell-mimetic reaction environment, we fabricated and tested quartz microchambers for conducting protein synthesis using an in vitro transcription and translation system, the PURE system. By introducing a glass microchamber and blocking the surface of the chamber with amino acids, the concentration of the synthesized marker protein (green fluorescent protein, GFP) was significantly improved compared to that in the poly(dimethylsiloxane) (PDMS) microchamber. The concentration was below the detection limit in the PDMS microchambers, whereas the glass microchambers yielded 700 nM GFP, representing 41% of the bulk reaction.

Importance of parasite RNA species repression for prolonged translation-coupled RNA self-replication.

Increasingly complex reactions are being constructed by bottom-up approaches with the aim of developing an artificial cell. We have been engaged in the construction of a translation-coupled replication system of genetic information from RNA and a reconstituted translation system. Here a mathematical model was established to gain a quantitative understanding of the complex reaction network.

Sudden death with massive hemoptysis from rupture of a thoracic inflammatory aortic aneurysm: an autopsy case report.

An 84-year-old-man was admitted to the Department of Neurosurgery for a sudden episode of fainting. Brain computed tomography and magnetic resonance imaging demonstrated no fresh lesions. Anorexia, fever and elevation of C-reactive protein and creatine phosphokinase were observed, and the patient was transferred to the Department of Internal Medicine for further examination and treatment.

Kinetic analysis of β-galactosidase and β-glucuronidase tetramerization coupled with protein translation.

Both β-galactosidase (GAL) and β-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction.

In vitro selection of proteins that undergo covalent labeling with small molecules by thiol-disulfide exchange by using ribosome display.

There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond.