Synthesis of All-Peptide-Based Rotaxane from a Proline-Containing Cyclic Peptide.

Rotaxane cross-linkers enhance the toughness of the resulting rotaxane cross-linked polymers through a stress dispersion effect, which is attributed to the mobility of the interlocked structure. To date, the compositional diversity of rotaxane cross-linkers has been limited, and the poor compatibility of these cross-linkers with peptides and proteins has made their use in such materials challenging. The synthesis of a rotaxane composed of peptides may result in a biodegradable cross-linker that is compatible with peptides and proteins, allowing the fortification of polypeptides and proteins and ultimately leading to the development of innovative materials that possess excellent mechanical properties and biodegradability. However, the chemical synthesis of all-peptide-based rotaxanes has remained elusive because of the absence of strong binding motifs in peptides, which prevents an axial peptide from penetrating a cyclic peptide. Here, we synthesized all-peptide-based rotaxanes using an active template method for proline-containing cyclic peptides. The results of molecular dynamics simulations suggested that cyclic peptides with an expansive inner cavity and carbonyl oxygens oriented toward the center are favorable for rotaxane synthesis. This rotaxane synthesis method is expected to accelerate the synthesis of peptides and proteins with mechanically interlocked structures, potentially leading to the development of peptide- and protein-based materials with unprecedented functionalities.

Photoacoustic Imaging Using Polysaccharide-Porphyrin Complexes by Photoirradiation at Long Wavelengths.

Photoacoustic (PA) imaging is a novel biological imaging technique with superior depth resolution compared to fluorescence imaging. The efficacy of PA imaging depends on contrast agents that possess considerable absorbance at longer wavelengths, coupled with high permeability in biological tissue and minimal fluorescence, achieved through mitigating aggregation-caused quenching (ACQ) that attenuates PA intensity. Despite the successful transfer of porphyrin 2 featuring amino moieties from polysaccharides to liposomes, most of 2 incorporated within λ-carrageenan (CGN-2 complex) remained in CGN under acidic lysosomal conditions (pH 5.0). Consequently, the CGN-2 complex exhibited a strong PA signal under 680 nm photoirradiation in Colon26 cells owing to the ACQ of 2. Moreover, the PA intensity of the CGN-2 complex was further enhanced under 780 nm photoirradiation owing to the increased absorbance at 780 nm facilitated by the redshift of the Q-band at pH 5.0.

Prevalence and associated factors of locomotive syndrome in young Japanese adults: a cross-sectional study.

BACKGROUND: The onset of locomotive syndrome (LS) precedes that of frailty. Therefore, the first step in extending healthy life expectancy is to implement measures against LS in young adults. The aim of this study was to investigate the prevalence of LS and its associated factors in young adults for early detection and prevention of LS. METHODS: The participants of this study comprised 413 university students specializing in health sciences (192 males and 221 females) with an average age of 19.1 ± 1.2 years. All participants voluntarily participated in the study and reported no serious health problems. The presence or absence of LS was evaluated using the stand-up test, two-step test, and the 25-question Geriatric Locomotive Function Scale. Additionally, musculoskeletal assessment (one-leg standing, squatting, shoulder elevation, and standing forward bend), body composition analysis (weight, body mass index, body fat mass, body fat percentage, skeletal muscle mass index (SMI), and phase angle), handgrip strength test, physical activity assessment, and nutritional assessment were conducted. Sex-stratified analyses were performed, comparing groups with and without LS. Factors associated with LS were explored using binomial logistic regression. RESULTS: Of the 413 young adults studied, 86 individuals (20.8%) were found to have LS. When stratified by sex, LS was observed to have a considerably higher prevalence in females (55, 24.9%) than in males (31, 16.1%). In males, the notable differences between the groups with and without LS were observed in one-leg standing and phase angle, whereas in females, differences were identified in body fat mass, body fat percentage, SMI, musculoskeletal pain, and handgrip strength. Two types of binomial logistic regression analysis revealed that the inability to perform one-leg standing was associated with LS in males, while the presence of musculoskeletal pain and a high body fat percentage were identified as factors associated with LS in females. CONCLUSIONS: One in five young adults were found to have LS in this study, underscoring the necessity for early intervention and LS health education. Furthermore, effective management of musculoskeletal pain is also crucial.

Plant Mitochondrial-Targeted Gene Delivery by Peptide/DNA Micelles Quantitatively Surface-Modified with Mitochondrial Targeting and Membrane-Penetrating Peptides.

Plant mitochondria play essential roles in metabolism and respiration. Recently, there has been growing interest in mitochondrial transformation for developing crops with commercially valuable traits, such as resistance to environmental stress and shorter fallow periods. Mitochondrial targeting and cell membrane penetration functions are crucial for improving the gene delivery efficiency of mitochondrial transformation. Here, we developed a peptide-based carrier, referred to as Cytcox/KAibA-Mic, that contains multifunctional peptides for efficient transfection into plant mitochondria. We quantified the mitochondrial targeting and cell membrane-penetrating peptide modification rates to control their functions. The modification rates were easily determined from high-performance liquid chromatography chromatograms. Additionally, the gene carrier size remained constant even when the mitochondrial targeting peptide modification rate was altered. Using this gene carrier, we can quantitatively investigate the relationships between various peptide modifications and transfection efficiency and optimize the gene carrier conditions for mitochondrial transfection.

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts.

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

A Photoresponsive Artificial Viral Capsid Self-Assembled from an Azobenzene-Containing ß-Annulus Peptide.

Photoinduced structural changes in peptides can dynamically control the formation and dissociation of supramolecular peptide materials. However, the existence of photoresponsive viral capsids in nature remains unknown. In this study, we constructed an artificial viral capsid possessing a photochromic azobenzene moiety on the peptide backbone. An azobenzene-containing ß-annulus peptide derived from the tomato bushy stunt virus was prepared through solid-phase synthesis using Fmoc-3-[(3-aminomethyl)-phenylazo]phenylacetic acid. The azobenzene-containing ß-annulus (ß-Annulus-Azo) peptide showed a reversible trans/cis isomerization property. The ß-annulus-azo peptide self-assembled at 25 µM into capsids with the diameters of 30-50 nm before UV irradiation (trans-form rich), whereas micrometer-sized aggregates were formed after UV irradiation (cis-form rich). The artificial viral capsid possessing azobenzene facilitated the encapsulation of fluorescent-labeled dextrans and their photoinduced release from the capsid.

All-Peptide-Based Polyion Complex Vesicles: Facile Preparation and Encapsulation of the Protein in Active Form.

The polyion complex vesicle (PICsome) is a promising platform for bioactive molecule delivery as well as nanoreactor systems. In addition to anionic and cationic charged blocks, a hydrophilic poly(ethylene glycol) (PEG) block is mostly employed for PICsome formation; however, the long-term safety of the PEG component in vivo is yet to be clarified. In this study, we developed novel PEG-free PICsome comprising all peptide components. Instead of the PEG block, we selected the sarcosine (Sar) oligomer as a hydrophilic block and fused it with anionic oligo(l-glutamic acid). Mixing the Sar-containing anionic peptide with cationic oligo(l-lysine) resulted in the formation of stable vesicles. The peptide-based PICsome was able to encapsulate a model protein in its hollow structure. After modification of the surface with a cell-penetrating peptide, the protein-encapsulated PICsome was successfully delivered into plant cells, indicating its promised for application as a biocompatible carrier for protein delivery.

Peptide-Based Polyion Complex Vesicles That Deliver Enzymes into Intact Plants To Provide Antibiotic Resistance without Genetic Modification.

Direct delivery of enzymes into intact plants using cell-penetrating peptides (CPPs) is an attractive approach for modifying plant functions without genetic modification. However, by conventional methods, it is difficult to maintain the enzyme activity for a long time because of proteolysis of the enzymes under physiological conditions. Here, we developed a novel enzyme delivery system using polyion complex vesicles (PICsomes) to protect the enzyme from proteases. We created PICsome-bearing reactive groups at the surface by mixing an anionic block copolymer, alkyne-TEG-P(Lys-COOH), and a cationic peptide, P(Lys). The PICsome encapsulated neomycin phosphotransferase II (NPTII), a kanamycin resistance enzyme, and protected NPTII from proteases in vitro. A CPP-modified PICsome delivered NPTII into the root hair cells of Arabidopsis thaliana seedlings and provided kanamycin resistance in the seedlings that lasted for 7 days. Thus, the PICsome-mediated enzyme delivery system is a promising method for imparting long-term transient traits to plants without genetic modification.

Construction of Ribonuclease-Decorated Artificial Virus-like Capsid by Peptide Self-assembly.

Artificial virus-like capsids decorated with ribonuclease S (RNase S) on their exterior were constructed by the self-assembly of ß-annulus-S-peptide and the interaction between S-peptide moiety and S-protein. The ß-annulus-S-peptide was synthesized by native chemical ligation of ß-annulus-SBz peptide with Cys-containing S-peptide that self-assembled into artificial virus-like capsids of approximately 47 nm in size. Reconstruction of RNase S on the artificial virus-like capsids afforded spherical assembly attached small spheres on the surface, which retained ribonuclease activity.

Self-assembled artificial viral capsids bearing coiled-coils at the surface.

In order to construct artificial viral capsids bearing complementary dimeric coiled-coils on the surface, a ß-annulus peptide bearing a coiled-coil forming sequence at the C-terminus (ß-annulus-coiled-coil-B) was synthesized by a native chemical ligation of a ß-annulus-SBn peptide with a Cys-containing coiled-coil-B peptide. Dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) images revealed that the ß-annulus-coiled-coil-B peptide self-assembled into spherical structures of about 50 nm in 10 mM Tris-HCl buffer. Circular dichroism (CD) spectra indicated the formation of the complementary coiled-coil structure on the spherical assemblies. Addition of 0.25 equivalent of the complementary coiled-coil-A peptide to the ß-annulus-coiled-coil-B peptide showed the formation of spherical assemblies of 46 ± 14 nm with grains of 5 nm at the surface, whereas addition of 1 equivalent of the complementary coiled-coil-A peptide generated fibrous assemblies.

Inclusion of Zinc Oxide Nanoparticles into Virus-Like Peptide Nanocapsules Self-Assembled from Viral ß-Annulus Peptide.

A viral ß-annulus peptide connected with a zinc oxide (ZnO)-binding sequence (HCVAHR) at its N-terminal was synthesized, and the inclusion behavior of quantum-sized ZnO nanoparticles into the peptide nanocapsules formed by self-assembly of the peptide in water was investigated. Dynamic light scattering (DLS) measurements showed that ZnO nanoparticles (approximately 10 nm) in the presence of the peptide (0.1 mM) formed assemblies with an average size of 48 ± 24 nm, whereas ZnO nanoparticles in the absence of the peptide formed large aggregates. Transmission electron microscopy (TEM) observations of the ZnO nanoparticles in the presence of the peptide revealed that ZnO nanoparticles were encapsulated into the peptide nanocapsules with a size of approximately 50 nm. Fluorescence spectra of a mixture of the peptide and ZnO nanoparticles suggested that the ZnO surface and the peptide interact. Template synthesis of ZnO nanoparticles with the peptide nanocapsules afforded larger nanoparticles (approximately 40 nm), which are not quantum-sized ZnO.

Specific interactions and binding energies between thermolysin and potent inhibitors: molecular simulations based on ab initio molecular orbital method.

Biochemical functions of the metalloprotease thermolysin (TLN) are controlled by various inhibitors. In a recent study we identified 12 compounds as TLN inhibitors by virtual screening and in vitro competitive binding assays. However, the specific interactions between TLN and these inhibitors have not been clarified. We here investigate stable structures of the solvated TLN-inhibitor complexes by classical molecular mechanics simulations and elucidate the specific interactions between TLN and these inhibitors at an electronic level by using ab initio fragment molecular orbital (FMO) calculations. The calculated binding energies between TLN and the inhibitors are qualitatively consistent with the experimental results, and the FMO results elucidate important amino acid residues of TLN for inhibitor binding. Based on the calculated results, we propose a novel potent inhibitor having a large binding affinity to TLN.

Specific interactions and binding free energies between thermolysin and dipeptides: molecular simulations combined with ab initio molecular orbital and classical vibrational analysis.

Thermolysin (TLN) is a metalloprotease widely used as a nonspecific protease for sequencing peptide and synthesizing many useful chemical compounds by the chemical industry. It was experimentally shown that the activity and functions of TLN are inhibited by the binding of many types of amino acid dipeptides. However, the binding mechanisms between TLN and dipeptides have not been clarified at the atomic and electronic levels. In this study, we investigated the binding mechanisms between TLN and four dipeptides. Specific interactions and binding free energies (BFEs) between TLN and the dipeptides were calculated using molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital (FMO) methods. The molecular systems were embedded in solvating water molecules during calculations. The calculated BFEs were qualitatively consistent with the trend of the experimentally observed inhibition of TLN activity by binding of the dipeptides. In addition, the specific interactions between the dipeptides and each amino acid residue of TLN or solvating water molecules were elucidated by the FMO calculations.