Critical role of lattice vacancies in pressure-induced phase transitions of baroplastic diblock copolymers.

Baroplastic diblock copolymers exhibit order-disorder transitions and melt upon compression at low temperatures, in some cases even at ambient temperatures. Their unique low-temperature processability makes them promising candidates for sustainable polymeric materials. Despite their potential, however, the molecular mechanisms governing the pressure-induced phase transitions of these copolymers remain largely unexplored. This study develops a compressible self-consistent field theory for baroplastic copolymers based on a simple lattice vacancy model that explicitly incorporates voids to account for compressibility. The theory shows that the selective presence of voids in compressible domains stabilizes the ordered phase, while a reduction of voids under compression leads to the order-disorder transition. In addition, this work demonstrates for the first time the critical role of gas absorption rates in each segment in the pressure-induced order-disorder transition of baroplastic diblock copolymers. These findings have significant implications for the rational design of baroplastic polymers with tailored low-temperature processability.

Deep-Sea-Inspired Chemistry: A Hitchhiker's Guide to the Bottom of the Ocean for Chemists.

The ocean constitutes approximately 70% of Earth's surface. Its average depth is 3688 m, of which depths beyond 200 m are classified as the deep sea. The deep sea is distinct from the surface of the ocean in terms of pressure, temperature, and sunlight. The unique physicochemical processes under the extreme environment of the deep sea and the specialized biochemical mechanisms developed by organisms to survive in the deep sea can serve as a vast source of inspiration for scientific and technological advancements. In this Perspective, we discuss three examples of deep-sea-inspired chemistry: (1) soft materials that respond to high pressures such as those observed in the deep sea; (2) molecular self-assembly inspired by the chemistry of hot and compressed water in deep-sea hydrothermal vents; and (3) nanobiotechnology and biomimetics inspired by survival strategies of deep-sea organisms. Finally, we provide an outlook on deep-sea-inspired chemistry. This Perspective aims to promote the sustainable utilization of the ocean based on knowledge, as opposed to the conventional utilization of the ocean solely based on resources. We hope that this Perspective will encourage chemists to harness their inspiration gleaned from the deep sea.

Marinagarivorans cellulosilyticus sp. nov., a cellulolytic bacterium isolated from the deep-sea off Noma-misaki, Japan.

Cells from strain GE09T, isolated from an artificially immersed nanofibrous cellulose plate in the deep sea, were Gram-stain-negative, motile, aerobic cells that could grow with cellulose as their only nutrient. Strain GE09T was placed among members of Cellvibrionaceae, in the Gammaproteobacteria, with Marinagarivorans algicola Z1T, a marine degrader of agar, as the closest relative (97.4 % similarity). The average nucleotide identity and digital DNA-DNA hybridization values between GE09T and M. algicola Z1T were 72.5 and 21.2 %, respectively. Strain GE09T degraded cellulose, xylan and pectin, but not starch, chitin and agar. The different carbohydrate-active enzymes encoded in the genomes of strain GE09T and M. algicola Z1T highlights their differences in terms of target energy sources and reflects their isolation environments. The major cellular fatty acids of strain GE09T were C18 : 1 ω7c, C16 : 0 and C16 : 1 ω7c. The polar lipid profile showed phosphatidylglycerol and phosphatidylethanolamine. The major respiratory quinone was Q-8. Based on these distinct taxonomic characteristics, strain GE09T represents a new species in the genus Marinagarivorans, for which we propose the name Marinagarivorans cellulosilyticus sp. nov. (type strain GE09T=DSM 113420T=JCM 35003T).

Protocol for analyzing enzymatic hydrolysis of cellulose using surface pitting observation technology.

Assaying enzymatic degradation of water-insoluble substrates like cellulose is challenging because only the substrate surface is accessible to the enzymes resulting in low reaction rates. Here, we describe a protocol for surface pitting observation technology (SPOT), an ultra-sensitive quantitative assay for analyzing enzymatic hydrolysis of cellulose. We describe the use of a porous substrate to accelerate the hydrolysis rate of cellulose. We also detail the steps for combining inkjet patterning and optical profilometry to analyze volume loss upon hydrolysis. For complete details on the use and execution of this protocol, please refer to Tsudome et al. (2022).1.

An ultrasensitive nanofiber-based assay for enzymatic hydrolysis and deep-sea microbial degradation of cellulose.

Substrates for enzymatic reactions, such as cellulose and chitin, are often insoluble in water. The enzymatic degradation of these abundant organic polymers plays a dominant role in the global carbon cycle and has tremendous technological importance in the production of bio-based chemicals. In addition, biodegradation of plastics is gaining wide attention. However, despite the significance, assaying these degradation reactions remains technically challenging owing to the low reaction rate, because only the surface of the substrate is accessible to the enzymes. We developed a nanofiber-based assay for the enzymatic hydrolysis of cellulose. This assay facilitated the quantification of the enzymatic hydrolysis of <1 ng crystalline cellulose. Utilization of the assay for the functional screening of cellulolytic microorganisms revealed an unprecedented genetic diversity underlying the production of deep-sea cellulase. This study reiterates that interdisciplinary efforts, such as from nanotechnology to microbiology, are critical for solving sustainability challenges.

Two-step droplet formation in monodisperse nanodroplet generation in quenched hydrothermal solution as revealed by spontaneous transformation of nanodroplets to swollen micelles in octane­in­water nanoemulsions.

HYPOTHESIS: Monodisperse nanodroplet generation in quenched hydrothermal solution (MAGIQ) is a newly developed bottom-up process for preparing nanoemulsions. In this process, homogeneous solutions of oil in supercritical water are quenched by adding cold water containing a surfactant to induce rapid phase-separation, during which oil molecules self-assemble to form nano-sized oil droplets. The droplet size in MAGIQ is known to be influenced by the interplay of the phase-separation dynamics, coalescence kinetics of the droplets, and adsorption kinetics of the surfactant on the droplet surface; however, the primary stages of the droplet formation are still elusive. EXPERIMENTS: Octane­in­water nanoemulsions containing 0.5, 1, and 3 vol% octane were prepared by the MAGIQ method. Their ripening was studied by dynamic light scattering, and the phase diagram was established. FINDINGS: The nanoemulsions containing 0.5 and 1 vol% octane transformed to thermodynamically stable microemulsions containing swollen micelles, whereas the nanoemulsion containing 3 vol% octane underwent Ostwald ripening. The initial formation of the nano-sized droplets in the former was ascribed to a unique mechanism of droplet formation in MAGIQ-the droplets are first formed by the phase separation of homogeneous binary solutions of oil in supercritical water and then stabilized upon surfactant adsorption.

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies.

Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases' behaviors and functions in daily genetic engineering experiments.

Primary structure of gum arabic and its dynamics at oil/water interface.

Gum arabic (GA), an arabinogalactan-based gum, is a well-known powerful emulsifier. However, the poor stability of emulsion has often been pointed out. In order to clarify the origin, the structure-property relationship of GA, especially the interfacial property at oil/water interface, needs to be investigated. Here, we tried to correlate the primary structure with interfacial property at oil/water interface. A series of structural analyses by SEC-MALLS, SAXS, etc. showed that the primary structure of GA was a disk-like star shaped nanoparticle. The dynamic interfacial tension measurement showed that GA molecules adsorb onto oil surface in 2 steps: Firstly, the micron-aggregates of GA approach onto the oil surface, and then the aggregates are dissociated into nano-particles so that they cover the oil surface. Therefore, the emulsification and emulsion stability are controlled not by the property of the primary structure of GA but by the higher-order molecular network structure made of GA molecules.

The making of natural iron sulfide nanoparticles in a hot vent snail.

Biomineralization in animals exclusively features oxygen-based minerals with a single exception of the scaly-foot gastropod Chrysomallon squamiferum, the only metazoan with an iron sulfide skeleton. This unique snail inhabits deep-sea hot vents and possesses scales infused with iron sulfide nanoparticles, including pyrite, giving it a characteristic metallic black sheen. Since the scaly-foot is capable of making iron sulfide nanoparticles in its natural habitat at a relatively low temperature (∼15 °C) and in a chemically dynamic vent environment, elucidating its biomineralization pathways is expected to have significant industrial applications for the production of metal chalcogenide nanoparticles. Nevertheless, this biomineralization has remained a mystery for decades since the snail's discovery, except that it requires the environment to be rich in iron, with a white population lacking in iron sulfide known from a naturally iron-poor locality. Here, we reveal a biologically controlled mineralization mechanism employed by the scaly-foot snail to achieve this nanoparticle biomineralization, through δ34 S measurements and detailed electron-microscopic investigations of both natural scales and scales from the white population artificially incubated in an iron-rich environment. We show that the scaly-foot snail mediates biomineralization in its scales by supplying sulfur through channel-like columns in which reaction with iron ions diffusing inward from the surrounding vent fluid mineralizes iron sulfides.

Liquid Marbles in Nature: Craft of Aphids for Survival.

Some aphids that live in the leaf galls of the host plant are known to fabricate liquid marbles consisting of honeydew and wax particles as an inner liquid and a stabilizer, respectively. In this study, the liquid marbles fabricated by the galling aphids, Eriosoma moriokense, were extensively characterized with respect to size and size distribution, shape, nanomorphology, liquid/solid weight ratio, and chemical compositions. The stereo microscopy studies confirmed that the liquid marbles have a near-spherical morphology and that the number-average diameter was 368 ± 152 µm, which is 1 order of magnitude smaller than the capillary length of the honeydew. The field emission scanning electron microscopy studies indicated that micrometer-sized wax particles with fiber- and dumpling-like shapes coated the honeydew droplets, which rendered the liquid marbles hydrophobic and nonwetting. Furthermore, the highly magnified scanning electron microscopy images confirmed that the wax particles were formed with assemblage of submicrometer-sized daughter fibers. The contact angle measurements indicated that the wax was intrinsically hydrophobic and that the liquid marbles were stabilized by the wax particles in the Cassie-Baxter model. The weight ratio of the honeydew and the wax particles was determined to be 96/4, and the honeydew consisted of 19 wt % nonvolatile components and 81 wt % water. The 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy, and mass spectroscopy studies confirmed that the wax mainly consisted of triglycerides and that the honeydew mainly consisted of saccharides (glucose and fructose) and ribitol. The atomic force microscopy studies confirmed that honeydew is sticky in nature.

Unveiling the RNA virosphere associated with marine microorganisms.

The study of extracellular DNA viral particles in the ocean is currently one of the most advanced fields of research in viral metagenomic analysis. However, even though the intracellular viruses of marine microorganisms might be the major source of extracellular virus particles in the ocean, the diversity of these intracellular viruses is not well understood. Here, our newly developed method, referred to herein as fragmented and primer ligated dsRNA sequencing (flds) version 2, identified considerable genetic diversity of marine RNA viruses in cell fractions obtained from surface seawater. The RNA virus community appears to cover genome sequences related to more than half of the established positive-sense ssRNA and dsRNA virus families, in addition to a number of unidentified viral lineages, and such diversity had not been previously observed in floating viral particles. In this study, more dsRNA viral contigs were detected in host cells than in extracellular viral particles. This illustrates the magnitude of the previously unknown marine RNA virus population in cell fractions, which has only been partially assessed by cellular metatranscriptomics and not by contemporary viral metagenomic studies. These results reveal the importance of studying cell fractions to illuminate the full spectrum of viral diversity on Earth.

Free-Radical Polymerization of Acrylic Acid under Extreme Reaction Conditions Mimicking Deep-Sea Hydrothermal Vents.

Free-radical polymerization with a thermochemical initiator, which usually takes hours to complete, was dramatically accelerated under reaction conditions mimicking the deep-sea hydrothermal vents, where reaction mixtures were only briefly exposed to ultrahigh temperatures under pressure. In tests using acrylic acid and potassium persulfate, poly(acrylic acid) (M n = 2.1 × 104, D = 2.73) was obtained in 5.2 s with the monomer conversion of 60.3% in water at 200 °C and 25 MPa without using any catalysts. The process that we call heat-shock-induced polymerization may pave the way for an entirely new strategy in reaction engineering for developing extremely fast, green, and scalable processes for polymer synthesis.

Enantioselective Utilization of D-Amino Acids by Deep-Sea Microorganisms.

Microorganisms that utilize various D-amino acids (DAAs) were successfully isolated from deep-sea sediments. The isolates were phylogenetically assigned to Alphaproteobacteria, Gammmaproteobacteria, and Bacilli. Some of the isolates exhibited high enantioselective degradation activities to various DAAs. In particular, the Alphaproteobacteria Nautella sp. strain A04V exhibited robust growth in minimal medium supplemented with D-Val as a sole carbon and nitrogen source, whereas its growth was poor on minimal medium supplemented with L-Val instead of D-Val. Its growth was facilitated most when racemic mixtures of valine were used. In contrast, the Nautella strains isolated from shallow-sea grew only with L-Val. No significant differences were found among the strains in the genome sequences including genes possibly related to DAA metabolisms.

Biochemical and genetic characterization of ß-1,3 glucanase from a deep subseafloor Laceyella putida.

A ß-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80 °C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.

Distribution of eukaryotic serine racemases in the bacterial domain and characterization of a representative protein in Roseobacter litoralis Och 149.

Two distinct bacterial and eukaryotic serine racemases (SRs) have been identified based on phylogenetic and biochemical characteristics. Although some reports have suggested that marine heterotrophic bacteria have the potential to produce d-serine, the gene encoding bacterial SRs is not found in those bacterial genomes. In this study, using in-depth genomic analysis, we found that eukaryotic SR homologues were distributed widely in various bacterial genomes. Additionally, we selected a eukaryotic SR homologue from a marine heterotrophic bacterium, Roseobacter litoralis Och 149 (RiSR), and constructed an RiSR gene expression system in Escherichia coli for studying the properties of the enzyme. Among the tested amino acids, the recombinant RiSR exhibited both racemization and dehydration activities only towards serine, similar to many eukaryotic SRs. Mg2+ and MgATP enhanced both activities of RiSR, whereas EDTA abolished these enzymatic activities. The enzymatic properties and domain structure of RiSR were similar to those of eukaryotic SRs, particularly mammalian SRs. However, RiSR showed lower catalytic efficiency for L-serine dehydration (kcat/Km=0.094 min(-1) mM(-1)) than those of eukaryotic SRs reported to date (kcat/Km=0.6-21 min(-1) mM(-1)). In contrast, the catalytic efficiency for L-serine racemization of RiSR (kcat/Km=3.14 min(-1) mM(-1)) was 34-fold higher than that of l-serine dehydration. These data suggested that RiSR primarily catalysed serine racemization rather than dehydration.

In situ microscopic observation of chitin and fungal cells with chitinous cell walls in hydrothermal conditions.

Recent findings of intact chitin in fossil records suggest surprisingly high recalcitrance of this biopolymer during hydrothermal treatments. We also know in the experience of everyday life that mushroom, cells of which have chitinous cell walls, do not fall apart however long they are simmered. We used in situ optical microscopy to examine chitin and fungal cells with chitinous cell walls during hydrothermal treatments, and obtained direct evidence that they remained undegraded at temperatures well over 200 °C. The results show very hot and compressed water is needed to make mushrooms mushy.

Hyaluronate lyase of a deep-sea Bacillus niacini.

A hyaluronate lyase (BniHL) was purified to homogeneity from a culture of a deep-sea Bacillus niacin strain JAM F8. The molecular mass of purified BniHL was approximately 120 kDa. The purified enzyme degraded hyaluronan as well as chondroitin sulfates A and C by a ß-elimination mechanism. The optimal pH and temperature were around pH 6 and 45 °C for hyaluronan degradation. The enzyme required optimally 2, 50, and 100 mM calcium ions for degradation of hyaluronan, chondroitin sulfate C, and chondroitin sulfate A, respectively. Calcium ions slightly increased the thermal stability of the enzyme. In a genome analysis of strain JAM F8, a BniHL coding gene was identified on the bases of the molecular mass and N-terminal and internal amino acid sequences. The gene consisted of 3411 nucleotides and coded 1136 amino acids. The deduced amino acid sequence showed the highest similarity to the hyaluronate lyase of a Bacillus sp. A50 with 89 % identity.

Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan Trench and Nankai Trough seawater.

BACKGROUND: Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host. RESULTS: A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHB-4 (PHB-negative mutant). CONCLUSIONS: A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.

Anomalous long-range repulsion between silica surfaces induced by density inhomogeneities in supercritical ethanol.

Anomalous long-range repulsion, extending over several micrometres, emerged between silica surfaces around the ridge of density fluctuations in supercritical ethanol at temperatures and pressures near the gas/liquid critical point (T(c) = 241 °C, P(c) = 6.14 MPa). Analysis shows that augmentation of ethanol density around silica surfaces in the presence of density fluctuations facilitates dissociation of silanol groups, leading to long-range electrostatic repulsion in the nonpolar medium.

Non-engineered nanoparticles of C60.

We discovered that rubbing bulk solids of C60 between fingertips generates nanoparticles including the ones smaller than 20 nm. Considering the difficulties usually associated with nanoparticle production by pulverisation, formation of nanoparticles by such a mundane method is unprecedented and noteworthy. We also found that nanoparticles of C60 could be generated from bulk solids incidentally without deliberate engineering of any sort. Our findings imply that there exist highly unusual human exposure routes to nanoparticles of C60, and elucidating formation mechanisms of nanoparticles is crucial in assessing their environmental impacts.