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Gold nanoparticles (AuNPs) are widely used as an agent in photothermal therapy (PTT) against various cancers. However, a drug delivery system (DDS) is required for effective PTT using AuNPs as AuNPs accumulate passively in tumors. In the present study, we used polyhistidine peptide, a novel cell-penetrating peptide, which is efficiently internalized into tumor cells, as a DDS carrier for PTT using AuNPs. Polyhistidine peptide-modified AuNPs are efficiently internalized into RERF-LC-AI human lung squamous cancer cells and localized to the intracellular lysosome, which is based on the nature of the polyhistidine peptide. Furthermore, the polyhistidine peptide-modified AuNPs inhibited proliferation of RERF-LC-AI cells in a polyhistidine peptide modification-dependent manner under 660 nm laser irradiation. Quantitative real-time PCR showed increased expression levels of an apoptosis-related gene (bax) and heat stress-related gene (hsp70) in RERF-LC-AI cells treated with polyhistidine peptide-modified AuNPs and laser. Our findings highlight the efficacy of AuNPs modified with H16 peptide in PTT.

In hemophilia A, bleeding mostly correlates with factor VIII activity (FVIII:C), although some patients show discrepancy in bleeding severity and FVIII:C. We report a novel procoagulant mechanism associated with F8 p.H118R (c.353A G) in a young Japanese man with few bleeding episodes despite low levels of FVIII:C ( 1 IU/dL). Plasma FVIII:C was 1 IU/dL measured by one-stage clotting assay (OSA) and chromogenic substrate assay (CSA), whereas FVIII antigen (FVIII:Ag) was 9.7%. The global coagulation assay showed higher max speed in clot waveform analysis (CWA), shorter clotting time in rotation thromboelastometry (ROTEM) (1605 vs. 5000 s), shorter lag time (4.87 vs. 12.47 min) and larger ETP (207.9 vs. 53.3 nM*min) in thrombin generation assay, compared with FVIII-deficient control. Expressed recombinant H118R mutant in culture media showed low FVIII:C (1-5 IU/dL) by OSA, with non-hemophilia level of FVIII:Ag. Western blot analysis using recombinant H118R showed longer persistence of heavy-chain of H118R after incubation with α-thrombin, compared with wild-type. Incubation of H118R with activated protein C (APC) also showed longer persistence of A1-A2 domain. In conclusion, H118R showed prolonged activation by α-thrombin and delayed APC-related FVIII degradation. These properties may confer the procoagulant activity and few bleeding episodes despite low FVIII:C.

Natural enveloped viruses, in which nucleocapsids are covered with lipid bilayers, contain membrane proteins on the outer surface that are involved in diverse functions, such as adhesion and infection of host cells. Previously, we constructed an enveloped artificial viral capsid through the complexation of cationic lipid bilayers onto an anionic artificial viral capsid self-assembled from β-annulus peptides. Here we demonstrate the embedding of the membrane protein Connexin-43 (Cx43), on the enveloped artificial viral capsid using a cell-free expression system. The expression of Cx43 in the presence of the enveloped artificial viral capsid was confirmed by western blot analysis. The embedding of Cx43 on the envelope was evaluated by detection

Microtubules play important roles in biological functions by forming superstructures, such as doublets and branched structures, in vivo. Despite the importance, it is challenging to construct these superstructures in vitro. Here, we designed a tetrameric fluorescent protein Azami-Green (AG) fused with His-tag and Tau-derived peptide (TP), TP-AG, to generate the superstructures. Main binding sites of TP-AG can be controlled to the inside and outside of microtubules by changing the polymerization conditions. The binding of TP-AG to the inside promoted microtubule formation and generated rigid and stable microtubules. The binding of TP-AG to the outside induced various microtubule superstructures, including doublets, multiplets, branched structures, and extremely long microtubules by recruiting tubulins to microtubules. Motile microtubule aster structures were also constructed by TP-AG. The generation of various microtubule superstructures by a single type of exogenous protein is a new concept for understanding the functions of microtubules and constructing microtubule-based nanomaterials.

Microtubules (MTs) are hollow cytoskeletons with typically 15 nm inner diameter, which are crucial for various cellular activities including structural support, transport, and cell division. Becaus...

For light-induced stabilization of microtubules (MTs) to manipulate cells, a photo-reactive diazirine group was conjugated to a Tau-derived peptide, a motif binding on the inside of MTs. Ultraviolet (UV) light irradiation induced significant stabilization of MTs

Ribonuclease S (RNase S) is an enzyme that exhibits anticancer activity by degrading RNAs within cancer cells; however, the cellular uptake efficiency is low due to its small molecular size. Here we generated RNase S-decorated artificial viral capsids with a size of 70-170 nm by self-assembly of the β-annulus-S-peptide followed by reconstitution with S-protein at neutral pH. The RNase S-decorated artificial viral capsids are efficiently taken up by HepG2 cells and exhibit higher RNA degradation activity in cells compared with RNase S alone. Cell viability assays revealed that RNase S-decorated capsids have high anticancer activity comparable to that of standard anticancer drugs.

We have synthesized B-antigen-displaying dendrimers (16-mers) with different sizes and evaluated their affinity to their IgM antibody in order to investigate which design features lead to effective multivalency. Unexpectedly, the smallest dendrimer, which cannot chelate the multiple binding sites of IgM, clearly exhibited multivalency, together with an affinity similar to or higher than those of the larger dendrimers. These results indicate that the statistical rebinding model, which involves the rapid exchange of clustered glycans, significantly contributes to the multivalency of glycodendrimers. Namely, in the design of glycodendrimers, high-density glycan presentation to enhance statistical rebinding should be considered in addition to the ability to chelate multiple binding sites. This notion stands in contrast to the currently prevailing scientific consensus, which prioritizes the chelation model. This study thus provides new and important guidelines for molecular design of glycodendrimers.

Background Genetic characteristics and genetic carrier diagnosis in Japanese hemophilia female carriers have not been evaluated. Objectives To provide genetic information on Japanese hemophilia female carriers and demonstrate the advantages of genetic testing in carrier diagnosis. Methods DNA sequencing combined with long polymerase chain reaction for inversion and multiplex ligation‐dependent probe amplification for large mutations. Results Genetic analysis was performed in 69 male hemophiliac patients (48 hemophilia A [HA] and 21 hemophilia B [HB]) and 112 female family members (FFM) (80 from 50 families with HA and 32 from 22 families with HB). In 72 hemophiliac families, the identified F8 mutations were inversion (42%), missense (26%), and other variations (32%), while 74% of F9 mutations were point mutations. Among the 112 FFM, 53/80 (66%) with HA and 21/32 (66%) with HB were diagnosed genetically as carriers based on detection of heterozygous mutations. Low factor VIII activity (FVIII:C) levels (

Inspired by natural motors, synthetic motors powered by light have emerged as promising platforms for constructing artificial micro/nanorobots. As a concept of light-driven motors, we have previously reported propulsion of giant liposomes driven by light-induced peptide nanofiber growth on the surface, inspired by natural pathogens using external actin polymerization for their propulsion. However, their movement was nondirectional. Here, we used DNA microspheres (also known as nucleospheres) comprising DNA three-way junctions with self-complementary sticky ends as vehicles for directional propulsion by light-induced peptide nanofiber growth. By introducing a peptide-DNA conjugate connected by a photocleavage unit to the surface of nucleospheres, ultraviolet (UV) light irradiation induced the asymmetric peptide nanofiber growth on the surface. Nucleospheres exhibited directional movement away from the light source, showing negative phototaxis. This directional movement was maintained even after the light irradiation was ceased. Our phototactic system helps to better understand the mechanism of natural motors and construct bioinspired motors with controlled movement.

Microtubules (MTs) are tubular cytoskeletons, which are used for the various applications such as active matters and therapeutic targets. Although modification of the exterior surface of MTs is frequently used for functionalization of MTs, there was no approach to introduce molecules inside MTs. We previously developed a unique peptide binding to the inner surface of MT, which is derived from a MT-associated protein, Tau. The Tau-derived peptide (TP) can be used to introduce various nanomaterials inside MTs. Here we describe the TP-based encapsulation of fluorescent dye, gold nanoparticle, green fluorescent protein, and magnetic CoPt nanoparticles inside MTs.

The cyclization of peptides is a valuable strategy for the development of binding motifs to target proteins with improved affinity. Microtubules (MTs) are important targets for therapeutics, and a variety of MT-targeted drugs and peptides have recently been developed. We have previously designed a Tau-derived peptide (TP) that binds to the interior of MTs. In the present study, the development of a cyclic TP (TCP) for enhanced binding to tubulin and the stabilization of MTs are described. The fluorescently labeled cyclic peptide containing three glycine linkers (TCP3-TMR) exhibited a remarkably enhanced binding affinity to tubulin. The cyclic peptide was also demonstrated to stabilize MTs by enhancing polymerization and reducing depolymerization. Moreover, MTs were effectively formed by the treatment of cyclic peptides in the presence of guanosine triphosphate (GTP), while the linear peptide showed no such effect. These findings indicate that TCP is a useful binding motif that can stabilize MTs and is valuable for various therapeutic and material applications. Microtubules (MTs) consisting of tubulins are important targets of drugs for MT-related diseases. We have previously designed a linear Tau-derived peptide (TP) that binds to the interior of MTs. In this article, a cyclic TP (TCP) was developed for enhanced binding to tubulin and stabilization of MTs. The fluorescently labeled TCP exhibited a remarkably enhanced binding affinity to tubulin compared to the linear TP. The stabilization of MTs by binding of TCP was demonstrated, such as formation of typically unstable MTs in the presence of guanosine triphosphate.

Artificial virus-like capsids decorated with ribonuclease S (RNase S) on their exterior were constructed by the self-assembly of β-annulus-S-peptide and the interaction between S-peptide moiety and S-protein. The β-annulus-S-peptide was synthesized by native chemical ligation of β-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.

Anionic artificial viral capsids were self-assembled from β-annulus-EE peptide, then complexed with lipid-bilayer-containing cationic lipids via electrostatic interaction to form enveloped artificial viral capsids. The critical aggregation concentration of the enveloped artificial viral capsid was significantly lower than that of the uncomplexed artificial viral capsid, indicating that the lipid bilayer stabilised the capsid structure.

Patients with non-severe hemophilia A often show discrepancies in factor VIII (FVIII) activity. However, information on variant-specific coagulation assay characteristics in Japanese patients is limited. Pathogenic variants were classified into three groups, thrombin-cleavage site (TC), A1-A2-A3 interface (IF), and non-discrepant, with reference to previous studies. Cutoff values for the one-stage assay (OSA)/chromogenic substrate assay (CSA) ratio, which is suitable for distinguishing discrepancies, were determined for all five aPTT reagents. TGA and CWA parameters and bleeding scores were compared between groups. Two of the 39 patients with non-severe hemophilia A (5%) were classified as TC, 10 (26%) as IF, and 27 (69%) as non-discrepant. The OSA/CSA cutoff values between the groups varied widely by aPTT reagent and tended to be relatively low compared to previous studies. As an indicator of bleeding tendency, TGA had a low correlation coefficient for the IF variant, but this was not significant and was comparable to FVIII activity and CWA. Moreover, various parameters and bleeding tendency differed among patients with the same variants. Thus, our findings suggest that it is difficult to adequately assess the bleeding tendency of individual patients, even with the various assessments currently available.

We constructed GFP-encapsulated microtubules (MTs) using Tauderived peptide which binds to their interior. The encapsulation of GFP dramatically increased the rigidity of MTs, resulting in their enhanced velocity on a kinesin-coated substrate. Moreover, the GFP-encapsulated MTs were significantly more stable compared to normal MTs.