A lesson from polybutylene succinate plastisphere to the discovery of novel plastic degrading enzyme genes in marine vibrios.

Polybutylene succinate (PBS) is an eco-friendly green plastic. However, PBS was shown as being non-biodegradable in marine environments, and up until now, only a limited number of PBS-degrading marine microbes have been discovered. We first set up in vitro PBS- and PBSA (polybutylene succinate adipate)-plastispheres to characterize novel PBS-degrading marine microbes. Microbial growth and oxygen consumption were observed in both PBS- and PBSA-plastispheres enriched with natural seawater collected from Usujiri, Hokkaido, Japan, and Vibrionaceae and Pseudoalteromonadaceae were significantly enriched on these films. Further gene identification indicated that vibrios belonging to the Gazogenes clade possess genes related to a PBS degrading enzyme (PBSase). The PBS degradation assay for six Gazogenes clade vibrios identified Vibrio ruber, Vibrio rhizosphaerae, and Vibrio spartinae as being capable of degrading PBS. We further identified the gene responsible for PBSase from the type strain of V. ruber, and the purified recombinant vibrio PBSase was found to have low-temperature adaptation and was active under high NaCl concentrations. We also provided docking models between the vibrio PBSase and PBS and PBSA units to show how vibrio PBSase interacts with each substrate compared to the Acidovorax PBSase. These results could contribute to a more sustainable society through further utilization of PBS in marine environments and plastic recycling.

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

Estimating available unused dead wood materials for heat generation in Mongolia: how much coal can unused dead wood materials substitute?

In this study, we estimated the technoeconomic availability of three types of dead trees, namely commercially fallen, normally fallen, and standing, in Mongolian subgroups. For dead trees of three major Mongolian tree species (Siberian larch, Scotch pine, and Asian white birch), lumber, firewood, and unused material as sources of energy were quantified and operational costs (marking, felling, skidding, transporting, loading/unloading, processing, and stumpage prices) were estimated. As a result, most Mongolian subgroups were profitable and suitable for the harvest of unused materials. Moreover, unused materials of dead trees can substitute nearly 5.45 million tons of coal, although it is not sufficient to meet the 0.6 years of coal demand across Mongolia. However, in some places, unused dead wood materials can completely substitute coal for decades. Therefore, although the heat utilization of woody biomass is not suitable for Mongolia as a whole, it is feasible in specific regions where forest resources are abundant, such as some Mongolian sub-provinces. Promoting the use of dead trees can reduce the risk of damage caused by wildfires and pests, which are serious problems in Mongolia, and keep the forests healthy. Furthermore, unused materials can generate new income opportunities, and the forest land can be well prepared by removing dead trees to promote planting for sustainable forest management. Finally, dead trees can be used as a resource until transition to sustainable forests with living trees.

Evaluation of Contact Force between Aneurysm Model and Coil for Embolization of Intracranial Aneurysms.

Objective: To ensure safe coil embolization for intracranial aneurysms, it is important to investigate the contact force between the coil and the aneurysm wall. However, it is unclear how the catheter tip position and the diameter of the secondary loop of the coil influence the contact force. In this study, we measured the contact force between a coil and an aneurysm biomodel under different conditions. Methods: A commercially available coil was inserted through a microcatheter into a silicone rubber aneurysm model at a constant speed (1 mm/s) using an automatic stage, and the contact force between the coil and the aneurysm wall was measured by a force sensor attached on the aneurysm model. The inner diameter of the spherical aneurysm was 5 mm. The effects of varying the position of the catheter tip (near dome, center, near neck) and the diameter of the secondary coil (4.5 mm) were evaluated. Results: When the catheter tip was inserted more deeply into the aneurysm (especially near the dome), the contact force increased. The contact force also increased as the secondary coil diameter was increased with the catheter tip near and in the center of the dome. Conclusion: These results suggest that the catheter tip position and the secondary coil diameter affect the contact force. In particular, the contact force should be considered large with the catheter tip near the dome to ensure safe coil deployment.

Local surface plasmon resonance of gold nanoparticles as a correlative light and electron microscopy (CLEM) tag for biological samples.

In this study, we investigated use of local surface plasmon resonance (LSPR) of metal nanoparticles (NPs) as a correlative light and electron microscopy (CLEM) tag for biological samples. Gold NPs in ultra-thin sections for TEM revealed that LSPR could be observed by optical microscopy at sizes of 20 nm or larger. Gold NPs at sizes less than 20 nm could be observed using the gold enhancement method. Therefore, this CLEM tag could be applied to immunoelectron microscopy using this gold enhancement method.

Development of novel correlative light and electron microscopy linkage system using silicon nitride film.

In this study, we investigated the optical properties of a silicon nitride (SiN) film. The thin SiN film (30 nm thick) exhibited good light transmittance and little autofluorescence and could be used as a microscope slide for optical microscopy (OM). In addition, we developed a novel correlative light and electron microscopy (CLEM) that combines OM with transmission electron microscopy (TEM) using an SiN thin film. In this system, CLEM was performed by replacing a detachable retainer with a holder for TEM and an adaptor for OM. The advantage of this method is that the same specimens can be sequentially observed using suitable OM and TEM.

Direct Determination of Cross-Link Density and Its Correlation with the Elastic Modulus of a Gel with Slidable Cross-Links.

The universal relationship between the elastic modulus and the cross-link density of a conventional rubber/gel has been demonstrated experimentally to be inapplicable to gels with slidable cross-links. Herein, we describe the synthesis of slide-ring (SR) gel networks devoid of intramolecular cross-links by the cross-coupling of two differently functionalized polyrotaxanes. The cross-link density was determined from the characteristic UV absorption attributed to the asymmetric cross-linked moiety. The cross-link density was shown to correlate considerably more weakly with the Young's modulus than conventional gels and rubbers that follow a universal proportional dependence. In addition, even at a similar cross-link density, the modulus appeared to be lower due to a lower density of cyclic components along the threading chain, i.e., the "coverage", though the data were limited in the narrow cross-link density range. These results might suggest a considerably lower contribution from the conformational entropy of chains associated with sliding through the cross-links and the counteracting entropy attributed to ring arrangement, though effects of the different persistence length due to the coverage difference could affect the modulus.

Fabrication and characterization of sample-supporting film made of silicon nitride for large-area observation in transmission electron microscopy.

In transmission electron microscopy (TEM), silicon nitride (SiN) films are widely used as sample-supporting films owing to their robustness. We fabricated large-scale SiN films deposited by low-pressure chemical vapor deposition (LPCVD). This preparation method is advantageous for large window areas, since it yields films with control over properties such as tension and thickness. We fabricated large SiN windows for mounting large ultrathin sections and for acquiring large-area TEM images. Thus, sample sections sliced by conventional sample preparation techniques were successfully mounted on these sample-supporting films. We successfully obtained a 680 × 250 µm2 TEM montage image of a whole Drosophila embryo.