BayesianSSA: a Bayesian statistical model based on structural sensitivity analysis for predicting responses to enzyme perturbations in metabolic networks.

BACKGROUND: Chemical bioproduction has attracted attention as a key technology in a decarbonized society. In computational design for chemical bioproduction, it is necessary to predict changes in metabolic fluxes when up-/down-regulating enzymatic reactions, that is, responses of the system to enzyme perturbations. Structural sensitivity analysis (SSA) was previously developed as a method to predict qualitative responses to enzyme perturbations on the basis of the structural information of the reaction network. However, the network structural information can sometimes be insufficient to predict qualitative responses unambiguously, which is a practical issue in bioproduction applications. To address this, in this study, we propose BayesianSSA, a Bayesian statistical model based on SSA. BayesianSSA extracts environmental information from perturbation datasets collected in environments of interest and integrates it into SSA predictions. RESULTS: We applied BayesianSSA to synthetic and real datasets of the central metabolic pathway of Escherichia coli. Our result demonstrates that BayesianSSA can successfully integrate environmental information extracted from perturbation data into SSA predictions. In addition, the posterior distribution estimated by BayesianSSA can be associated with the known pathway reported to enhance succinate export flux in previous studies. CONCLUSIONS: We believe that BayesianSSA will accelerate the chemical bioproduction process and contribute to advancements in the field.

Self-organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds.

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self-organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self-organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm-mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro-structured meshes with limited surface area for cell adhesion. We show that this adhesion-restriction strategy can trigger a two-stage self-organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8-10, then into spherical cysts following differentiation and self-organization of the sheet-forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two-photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E-cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late-stage blastocyst. Thus, the mesh culture microenvironment can initiate self-organization of hiPSCs into trophectoderm-mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.

From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase.

DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures.

Characteristic oxygen K-edge circular dichroism spectra of amino acid films by improved measurement technique.

Circular dichroism (CD) spectroscopy in the soft x-ray energy region is a new tool to study the local structure of chiral materials. In this paper, we introduce a method to measure high-quality CD spectra in the oxygen K-edge energy region. Characteristic CD spectra of thin films of the amino acids L-tyrosine and L-aspartic acid are reported and compared with those of films of L-alanine and L-serine. The signals from the oxygen 1s → π∗ transitions of COO-, which is a common moiety in these amino acids, reflect the local geometry of each amino acid.

The possible interplanetary transfer of microbes: assessing the viability of Deinococcus spp. under the ISS Environmental conditions for performing exposure experiments of microbes in the Tanpopo mission.

To investigate the possible interplanetary transfer of life, numerous exposure experiments have been carried out on various microbes in space since the 1960s. In the Tanpopo mission, we have proposed to carry out experiments on capture and space exposure of microbes at the Exposure Facility of the Japanese Experimental Module of the International Space Station (ISS). Microbial candidates for the exposure experiments in space include Deinococcus spp.: Deinococcus radiodurans, D. aerius and D. aetherius. In this paper, we have examined the survivability of Deinococcus spp. under the environmental conditions in ISS in orbit (i.e., long exposure to heavy-ion beams, temperature cycles, vacuum and UV irradiation). A One-year dose of heavy-ion beam irradiation did not affect the viability of Deinococcus spp. within the detection limit. Vacuum (10(-1) Pa) also had little effect on the cell viability. Experiments to test the effects of changes in temperature from 80 °C to -80 °C in 90 min (± 80 °C/90 min cycle) or from 60 °C to -60 °C in 90 min (± 60 °C/90 min cycle) on cell viability revealed that the survival rate decreased severely by the ± 80 °C/90 min temperature cycle. Exposure of various thicknesses of deinococcal cell aggregates to UV radiation (172 nm and 254 nm, respectively) revealed that a few hundred micrometer thick aggregate of deinococcal cells would be able to withstand the solar UV radiation on ISS for 1 year. We concluded that aggregated deinococcal cells will survive the yearlong exposure experiments. We propose that microbial cells can aggregate as an ark for the interplanetary transfer of microbes, and we named it 'massapanspermia'.

Structure-based mutational study of an archaeal DNA ligase towards improvement of ligation activity.

DNA ligases catalyze the joining of strand breaks in duplex DNA. The DNA ligase of Pyrococcus furiosus (PfuLig), which architecturally resembles the human DNA ligase I (hLigI), comprises an N-terminal DNA-binding domain, a middle adenylylation domain, and a C-terminal oligonucleotide-binding (OB)-fold domain. Here we addressed the C-terminal helix in the OB-fold domain of PfuLig by mutational analysis. The crystal structure of PfuLig revealed that this helix stabilizes a closed conformation of the enzyme by forming several ionic interactions with the adenylylation domain. The C-terminal helix is oriented differently in hLigI when DNA is bound; this suggested that disruption of its interaction with the adenylylation domain might facilitate the binding of DNA substrates. We indeed identified one of its residues, Asp540, as being critical for ligation efficiency. The D540R mutation improved the overall ligation activity relative to the wild-type enzyme, and at lower temperatures; this is relevant to applications such as ligation amplification reactions. Physical and biochemical analyses indicated that the improved ligation activity of the D540R variant arises from effects on the ligase adenylylation step and on substrate DNA binding in particular.

Crystallization and preliminary crystallographic study of DNA polymerase from Pyrococcus furiosus.

A new member of archaeal DNA polymerase from Pyrococcus furiosus was crystallized. Diffraction data to 3.1 A of the selenomethionine-derivatized crystal were collected, and preliminary crystallographic study has been completed. The crystal belongs to the space group C2 with unit cell parameters of a = 93.2 A, b = 124.9 A, c = 87.7 A, alpha = 90 degrees , beta = 109.7 degrees , and gamma = 90 degrees . Assuming the presence of one molecule in the asymmetric unit, the solvent content of the crystal is estimated to be 54%, corresponding to a Matthews coefficient V(M) of 2.7A (3) Da(-1).

Exonuclease processivity of archaeal replicative DNA polymerase in association with PCNA is expedited by mismatches in DNA.

Family B DNA polymerases comprise polymerase and 3' ->5' exonuclease domains, and detect a mismatch in a newly synthesized strand to remove it in cooperation with Proliferating cell nuclear antigen (PCNA), which encircles the DNA to provide a molecular platform for efficient protein-protein and protein-DNA interactions during DNA replication and repair. Once the repair is completed, the enzyme must stop the exonucleolytic process and switch to the polymerase mode. However, the cue to stop the degradation is unclear. We constructed several PCNA mutants and found that the exonuclease reaction was enhanced in the mutants lacking the conserved basic patch, located on the inside surface of PCNA. These mutants may mimic the Pol/PCNA complex processing the mismatched DNA, in which PCNA cannot interact rigidly with the irregularly distributed phosphate groups outside the dsDNA. Indeed, the exonuclease reaction with the wild type PCNA was facilitated by mismatched DNA substrates. PCNA may suppress the exonuclease reaction after the removal of the mismatched nucleotide. PCNA seems to act as a "brake" that stops the exonuclease mode of the DNA polymerase after the removal of a mismatched nucleotide from the substrate DNA, for the prompt switch to the DNA polymerase mode.

Vertical flow array chips reliably identify cell types from single-cell mRNA sequencing experiments.

Single-cell mRNA sequencing offers an unbiased approach to dissecting cell types as functional units in multicellular tissues. However, highly reliable cell typing based on single-cell gene expression analysis remains challenging because of the lack of methods for efficient sample preparation for high-throughput sequencing and evaluating the statistical reliability of the acquired cell types. Here, we present a highly efficient nucleic reaction chip (a vertical flow array chip (VFAC)) that uses porous materials to reduce measurement noise and improve throughput without a substantial increase in reagent. We also present a probabilistic evaluation method for cell typing depending on the amount of measurement noise. Applying the VFACs to 2580 monocytes provides 1967 single-cell expressions for 47 genes, including low-expression genes such as transcription factors. The statistical method can distinguish two cell types with probabilistic quality values, with the measurement noise level being considered for the first time. This approach enables the identification of various sub-types of cells in tissues and provides a foundation for subsequent analyses.

Coherent anti-Stokes Raman scattering hyperspectral imaging of cartilage aiming for state discrimination of cell.

Noninvasive cell analyses are increasingly important in the medical field. A coherent anti-Stokes Raman scattering (CARS) microscope is the noninvasive imaging equipment and enables to obtain images indicating molecular distribution. However, due to low-signal intensity, it is still challenging to obtain images of the fingerprint region, in which many spectrum peaks correspond to compositions of a cell. Here, to identify cell differentiation by using multiplex CARS, we investigated hyperspectral imaging of the fingerprint region of living cells. To perform multiplex CARS, we used a prototype of a compact light source generating both pump light and broadband Stokes light. Assuming application to regenerative medicine, we chose a cartilage cell, whose differentiation is difficult to be identified by change of the cell morphology. Because one of the major components of cartilage is collagen, we focused on distribution of proline, which accounts for approximately 20% of collagen. The spectrum quality was improved by optical adjustments of the power branching ratio and divergence of Stokes light. Periphery of a cartilage cell was highlighted in a CARS image of proline, and this result suggests correspondence with collagen generated as an extracellular matrix. The possibility of noninvasive analyses by using CARS hyperspectral imaging was indicated.

Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity.

The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.

Intravenous administration of 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (gamma-CEHC) to rats and determination of its plasma concentration and urinary sodium excretion.

A natriuretic hormone, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (gamma-CEHC) was administered intravenously to male Sprague-Dawley rats and the plasma concentration of gamma-CEHC along with urinary sodium (Na+) excretion was investigated. The plasma gamma-CEHC concentrations were fluorimetrically determined by a column-switching HPLC method consisting of both phenyl and octadecyl silica columns, following a pre-column fluorescence derivatization with a fluorescence reagent, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ). In rats fed with a high-NaCl (8.0%) diet, plasma gamma-CEHC concentrations rapidly decreased by 20% in 15-45 min after the administration of gamma-CEHC, while Na+ excretion gradually increased with time. Considering these results, the Na+ excretion effect appeared not to be associated with plasma gamma-CEHC concentration. In addition, attempts were made to examine a main urinary metabolite of gamma-CEHC, a large amount of 6-O-sulfated gamma-CEHC found to be present in the urine using an HPLC-tandem mass spectrometry. Thus, it is plausible that gamma-CEHC was easily metabolized to 6-O-sulfated metabolite and excreted into urine in rats.

Novel d-gamma-tocopherol derivative as a prodrug for d-gamma-tocopherol and a two-step prodrug for S-gamma-CEHC.

d-gamma-Tocopherol (gamma-Toc) and its major metabolite, 2, 7, 8-trimethyl-2S-(beta-carboxyethyl)-6-hydroxychroman (S-gamma-CEHC), are currently receiving attention concerning their unique pharmacological activities. In order to achieve the efficient delivery of gamma-Toc and S-gamma-CEHC in vivo, we synthesized d-gamma-tocopheryl N,N-dimethylglycinate hydrochloride (gamma-TDMG) as a water-soluble prodrug of gamma-Toc and a two-step prodrug of S-gamma-CEHC. gamma-TDMG is a solid (mp 161-163 degrees C) and is quite soluble in water over 50 mM. The hydrolysis of gamma-TDMG was effectively catalyzed by esterases in rat and human liver microsomes. The disposition of gamma-TDMG after iv administration in rats was compared with that of gamma-Toc solubilized with the surfactant, polyoxyethylene hydrogenated castor oil. The plasma and liver levels of gamma-Toc rapidly increased after the iv administration of the gamma-TDMG. The liver availability of gamma-Toc after the administration of gamma-TDMG was two times higher than that of the gamma-Toc administration. The relative systemic availability of S-gamma-CEHC after the gamma-TDMG administration was an equivalent value (102%), and the mean residence time of S-gamma-CEHC was eight times longer than the racemic gamma-CEHC administration. Based on these results, gamma-TDMG was identified as the most promising water-soluble prodrug of gamma-Toc and the two-step prodrug of S-gamma-CEHC.