Effects of polycaprolactone degradation products on the water flea, Daphnia magna: Carbodiimide additives have acute and chronic toxicity.

Plastics have benefited our lives in many ways, but their long persistence in the environment causes serious problems. Rapid decomposition and detoxification of plastics after use are significant challenges. As a possible solution, biodegradable plastics have attracted attention, and for environmental risk assessment research on polymer toxicity, use of indicator organisms, like water fleas and fish, has increased globally. However, such research often focuses on standardized substances without considering changes in toxicity due to plastic degradation products. Additionally, tests generally focus on acute toxicity, while long-term effects on organismal reproduction and lifespan are largely unknown. Understanding the impact of degraded polymers on biological activities is crucial for accurate risk assessment. In this study, we investigated the biological toxicity of substances generated during degradation of polycaprolactone (PCL), a common biodegradable plastic, using the indicator organism, Daphnia magna. We examined PCL, oligocaprolactones (OCLs), and monomers resulting from polymer cleavage, as well as carbodiimides, added during polyester synthesis. As a result, PCL, which is insoluble in water, reduced individual survival and total number of offspring at an exposure concentration of 100 mg/L, while no toxicity was observed for water-soluble degradation products, OCLs, and monomers. Furthermore, carbodiimides, which are expected to be released during PCL degradation, showed strong toxicity, significantly reducing individual survival and total number of offspring at 0.1-10 mg/L. These findings suggest that changes in physical properties due to polymer degradation and release of additives can significantly alter their toxicity.

Effect of Oligomers Derived from Biodegradable Polyesters on Eco- and Neurotoxicity.

Biodegradable polymers are eco-friendly materials and have attracted attention for use in a sustainable society because they are not accumulated in the environment. Although the characteristics of biodegradable polymers have been assessed well, the effects of their degradation products have not. Herein, we comprehensively evaluated the chemical toxicities of biodegradable polyester, polycaprolactone (PCL), and synthetic oligocaprolactones (OCLs) with different degrees of polymerization. While the PCL did not show any adverse effects on various organisms, high levels of shorter OCLs and the monomer (1 µg/mL for freshwater microorganisms and 1 mg/mL for marine algae and mammalian cells) damaged the tested organisms, including freshwater microorganisms, marine algae, and mammalian cells, which indicated the toxicities of the degradation products under unnaturally high concentrations. These results highlight the need for a further understanding of the effects of the degradation products resulting from biodegradable polyesters to ensure a genuinely sustainable society.

1000 spider silkomes: Linking sequences to silk physical properties.

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.

Relaxation of the Plant Cell Wall Barrier via Zwitterionic Liquid Pretreatment for Micelle-Complex-Mediated DNA Delivery to Specific Plant Organelles.

Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle-targeted DNA delivery in plants. However, nanocarrier-mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall-loosening zwitterionic liquid (ZIL) with a peptide-displaying micelle complex for organelle-specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier-mediated cargo delivery to specific organelles in living plants.

Polymer-coated carbon nanotube hybrids with functional peptides for gene delivery into plant mitochondria.

The delivery of genetic material into plants has been historically challenging due to the cell wall barrier, which blocks the passage of many biomolecules. Carbon nanotube-based delivery has emerged as a promising solution to this problem and has been shown to effectively deliver DNA and RNA into intact plants. Mitochondria are important targets due to their influence on agronomic traits, but delivery into this organelle has been limited to low efficiencies, restricting their potential in genetic engineering. This work describes the use of a carbon nanotube-polymer hybrid modified with functional peptides to deliver DNA into intact plant mitochondria with almost 30 times higher efficiency than existing methods. Genetic integration of a folate pathway gene in the mitochondria displays enhanced plant growth rates, suggesting its applications in metabolic engineering and the establishment of stable transformation in mitochondrial genomes. Furthermore, the flexibility of the polymer layer will also allow for the conjugation of other peptides and cargo targeting other organelles for broad applications in plant bioengineering.

Chemoenzymatic Synthesis of Sialyl Sulfo-Oligosaccharides as Potent Siglec-8 Ligands via Transglycosylation Catalyzed by Keratanase II.

Sialyl type-II sulfo-oligosaccharides are gaining much attention as bioactive ligands for Siglecs. In this study, we have achieved the first synthesis of sialyl type-II sulfo-oligosaccharides chemoenzymatically by utilizing the transglycosylation activity of keratanase II. The oxazoline derivative of α(2→3)-sialylated 6,6'-di-sulfo-LacNAc (3) was newly designed as the glycosyl donor for enzymatic transglycosylation. Keratanase II efficiently catalyzed the transglycosylation of 3 with two kinds of glycosyl acceptors, 6-sulfo-Lewis X and 6,6'-di-sulfo-LacNAc derivatives, providing sialyl sulfo-hexasaccharide (1) and sialyl sulfo-pentasaccharide (2) with 86 and 95% yields, respectively. The products 1 and 2 showed higher affinity to Siglec-8 with KD 70 and 25 µmol·L-1, respectively, compared to the known ligand of the α(2→3)-sialylated 6,6'-di-sulfo-Lewis X with KD 185 µmol·L-1. Thus, this study will advance not only the study of Siglec-8 biology but also the exploration of functions of sialyl sulfo-oligosaccharides having various microstructures.

Three-dimensional nanoscale analysis of light-dependent organelle changes in Arabidopsis mesophyll cells.

Different organelles function coordinately in numerous intracellular processes. Photorespiration incidental to photosynthetic carbon fixation is organized across three subcellular compartments: chloroplasts, peroxisomes, and mitochondria. Under light conditions, these three organelles often form a ternary organellar complex in close proximity, suggesting a connection with metabolism during photorespiration. However, due to the heterogeneity of intercellular organelle localization and morphology, organelles' responses to changes in the external environment remain poorly understood. Here, we used array tomography by field emission scanning electron microscopy to image organelles inside the whole plant cell at nanometer resolution, generating a three-dimensional (3D) spatial map of the light-dependent positioning of chloroplasts, peroxisomes, nuclei, and vacuoles. Our results show, in light-treated cells, the volume of peroxisomes increased, and mitochondria were simplified. In addition, the population of free organelles decreased, and the ternary complex centered on chloroplasts increased. Moreover, our results emphasized the expansion of the proximity area rather than the increase in the number of proximity sites interorganelles. All of these phenomena were quantified for the first time on the basis of nanoscale spatial maps. In summary, we provide the first 3D reconstruction of Arabidopsis mesophyll cells, together with nanoscale quantified organelle morphology and their positioning via proximity areas, and then evidence of their light-dependent changes.

Imaging of the Entry Pathway of a Cell-Penetrating Peptide-DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves.

Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide-DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.

The balance of crystalline and amorphous regions in the fibroin structure underpins the tensile strength of bagworm silk.

Protein-based materials are considered versatile biomaterials, and their biodegradability is an advantage for sustainable development. Bagworm produces strong silk for use in unique situations throughout its life stages. Rigorous molecular analyses of Eumeta variegata suggested that the particular mechanical properties of its silk are due to the coexistence of poly-A and GA motifs. However, little molecular information on closely related species is available, and it is not understood how these properties were acquired evolutionarily or whether the motif combination is a conserved trait in other bagworms. Here, we performed a transcriptome analysis of two other bagworm species (Canephora pungelerii and Bambalina sp.) belonging to the family Psychidae to elucidate the relationship between the fibroin gene and silk properties. The obtained transcriptome assemblies and tensile tests indicated that the motif combination and silk properties were conserved among the bagworms. Furthermore, our analysis showed that C. pungelerii produces extraordinarily strong silk (breaking strength of 1.4 GPa) and indicated that the cause may be the C. pungelerii -specific balance of crystalline/amorphous regions in the H-fibroin repetitive domain. This particular H-fibroin architecture may have been evolutionarily acquired to produce strong thread to maintain bag stability during the relatively long development period of Canephora species relative to other bagworms.

Surface Analysis of Native Spider Draglines by FE-SEM and XPS.

Although the physical and biological functions of the skin layer of spider dragline have been studied and partially clarified, the morphology and elemental contents of the skin layer of silk fibers have not been investigated in detail to date. Here, the surface of Nephila clavata spider dragline was evaluated by field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) to obtain clear surface morphological and molecular information. The FE-SEM images of the spider dragline indicate that the spider dragline forms a bundle of microfibrils. This hierarchical structure might induce faint fibrilar and network-like patterns on the surface of the dragline. XPS analysis revealed the presence of Na, P, and S, which are reasonably explained by considering the biological components of the major ampullate gland of spiders. The results obtained here are preliminary but will be important to consider the molecular transition of silk proteins to form excellent hierarchical structures during the spider dragline spinning process.

Computational study on the polymerization reaction of d-aminopeptidase for the synthesis of d-peptides.

Almost all natural proteins are composed exclusively of l-amino acids, and this chirality influences their properties, functions, and selectivity. Proteases can recognize proteins composed of l-amino acids but display lower selectivity for their stereoisomers, d-amino acids. Taking this as an advantage, d-amino acids can be used to develop polypeptides or biobased materials with higher biostability. Chemoenzymatic peptide synthesis is a technique that uses proteases as biocatalysts to synthesize polypeptides, and d-stereospecific proteases can be used to synthesize polypeptides incorporating d-amino acids. However, engineered proteases with modified catalytic activities are required to allow the incorporation of d-amino acids with increased efficiency. To understand the stereospecificity presented by proteases and their involvement in polymerization reactions, we studied d-aminopeptidase. This enzyme displays the ability to efficiently synthesize poly d-alanine-based peptides under mild conditions. To elucidate the mechanisms involved in the unique specificity of d-aminopeptidase, we performed quantum mechanics/molecular mechanics simulations of its polymerization reaction and determined the energy barriers presented by the chiral substrates. The enzyme faces higher activation barriers for the acylation and aminolysis reactions with the l-stereoisomer than with the d-substrate (10.7 and 17.7 kcal mol-1 higher, respectively). The simulation results suggest that changes in the interaction of the substrate with Asn155 influence the stereospecificity of the polymerization reaction.

Two Birds with One Stone: Spontaneous Size Separation and Growth Inhibition of Femtosecond Laser-Generated Surfactant-Free Metallic Nanoparticles via ex Situ SU-8 Functionalization.

Laser ablation in liquids (LAL) offers a facile technique to develop a large variety of surfactant-free nanomaterials with high purity. However, due to the difficulty in the control of the particle synthesis process, the as-prepared nanomaterials always have a broad size distribution with a large polydispersity (σ). Surfactant-free properties can also cause problems with particle growth, which further increases the difficulty in size control of the colloids. Therefore, searching for strategies to simultaneously unify the sizes of colloids and inhibit particle growth has become significantly important for LAL-synthesized nanomaterials to be extensively used for biological, catalytic, and optical applications, in which fields particle size plays an important role. In this work, we present a facile way to simultaneously realize these two goals by ex situ SU-8 photoresist functionalization. Ag nanoparticles (NPs) synthesized by femtosecond laser ablation of silver in acetone at laser powers of 300 and 600 mW were used as starting materials. The synthesized Ag NPs have a broad size distribution between 1 and 200 nm with an average size of ca. 5.9 nm and σ of 127-207%. After ex situ SU-8 functionalization and 6 months storage, most particles larger than 10 nm become aggregates and precipitate, which makes the size distribution narrow with an average diameter of 4-5 nm and σ of 48-78%. The precipitation process is accompanied by the decrease in colloid mass from the initial ∼0.2 to 0.10-0.11 mg after ex situ SU-8 functionalization and 6 months colloid storage. Morphology analysis indicates that ex situ SU-8 functionalization inhibits the particle growth into polygonal nanocrystals. Radical polymerization of SU-8 on Ag NPs is considered to be the reason for both spontaneous size separation and growth inhibition phenomena. Benefiting from Ag NPs embedment and acetone dissolution, the glass-transition temperature of SU-8 photoresist increased from 314 to 331 °C according to thermogravimetric analysis. The universality of ex situ SU-8 functionalization-induced growth inhibition and size separation behaviors is further proved using the Au colloids generated by LAL in acetone. This work is expected to provide a new route for better size control of LAL-synthesized colloids via ex situ photoresist functionalization, although a half of colloidal mass is wasted due to radical polymerization-induced colloidal precipitation.

The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization.

The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease.