Anticoagulation Therapy for Pregnancy-Associated Thrombosis: A Retrospective Observational Study.

Objectives: Pregnancy-associated deep vein thrombosis (DVT) is a rare disease, and data on anticoagulation therapy are lacking. The present study examined the treatment outcome with unfractionated heparin (UFH) subcutaneous injection in patients with pregnancy-associated DVT. Methods: This single-center, retrospective, observational study enrolled 15 patients with pregnancy-associated DVT treated from January 2014 to April 2021. Results: The median age was 35 years. The median gestation week at onset was 10 (interquartile range is 8-11). All patients presented with painful symptoms with edema. All patients had proximal DVT. Anticoagulation therapy using UFH was performed in 14 patients. The median continuous dose of heparin was 18,750 U/day, and the median subcutaneous dose was 20,000 U/day. During the outpatient period, the values of activated partial thromboplastin time fluctuated wildly, but the fibrin monomer complex level remained consistently low. There were two mild bleeding complications, but neither prevented the continuation of anticoagulation therapy. During delivery, thrombi were not detected in 10 of 13 patients (77%), whereas three patients (23%) exhibited regression without resolution of the thrombus. Conclusion: Anticoagulation using UFH subcutaneous injection was safely performed in patients with pregnancy-associated DVT without serious complications or progression of thrombosis.

Construction and Piezoelectric Properties of a Single-Peptide Nanotube Composed of Cyclic ß-peptides with Helical Peptides on the Side Chains.

To develop nanopiezoelectronics, it is necessary to investigate the relationship between the sizes and piezoelectric properties of the material. Peptide nanotubes (PNTs) composed of cyclic ß-peptides have been studied as leading candidates for nanopiezoelectric materials. The current drawback of PNTs is aggregation to form a PNT bundle structure due to strong dipole-dipole interactions between PNTs. Here, we report the construction and piezoelectric properties of single PNTs without nonspecific aggregation by side-chain modification of helical peptides. A cyclic tri-ß-peptide with a helical peptide was prepared by multiple-step liquid-phase peptide synthesis and assembled into PNTs by the vapor diffusion method. These nanotubes were characterized by polarized light microscopy and Fourier transform infrared (FTIR) spectroscopy. Additionally, atomic force microscopy (AFM) topographic images showed nanotubes with a height of 4 nm, which corresponds to the diameter of a PNT on a gold-coated mica substrate, indicating that a single PNT was prepared successfully. The converted piezoelectric response of a single PNT was determined to be 1.39 ± 0.12 pm/V. This value was consistent with that of a PNT bundle, which reveals that the piezoelectricity of PNTs is induced by deformation of their cyclic skeletons and is independent of the bundled structure. This finding not only demonstrates a new molecular design strategy to construct these smallest piezoelectric biomaterials by controlling the supramolecular hierarchical structures but also provides insights into the correlation between molecular assembly morphology and size-dependent piezoelectric properties.

A Novel Surface Modification and Immobilization Method of Anti-CD25 Antibody on Nonwoven Fabric Filter Removing Regulatory T Cells Selectively.

Anti-CD25 antibodies were immobilized on polypropylene (PP) nonwoven fabrics to specifically remove mouse regulatory T cells (Tregs) from mouse spleen cells. PP fibers were coated with peptide nanosheets, which were prepared by self-assembling of a mixture of X-poly(sarcosine)-b-(l-Leu-Aib)6 (X: glycolic acid or a phenylboronic acid) and Y-poly(sarcosine)-b-(d-Leu-Aib)6 (Y: glycolic acid or diazirine derivative). Anti-CD25 antibodies were immobilized by covalent linking between the sugar moiety of the antibody and the phenylboronic acid group on the peptide nanosheet. The removal rate of mouse Tregs from the mouse spleen cells was more than 95% only by passing the filters, while the nonspecific removal rates of other cells were less than 15%. The coating of peptide nanosheets on PP fibers was very effective to provide a suitable environment for the immobilized antibody to interact with the counterpart cells while the coating suppressed nonspecific adsorption of other cells.

Engineering pH-responsive switching of donor-π-acceptor chromophore alignments along a peptide nanotube scaffold.

A cyclic tri-ß-peptide cyclo(ß-Ala-ß-Ala-ß-Lys) having diethylaminonaphthalimide at the ß-Lys side chain (CP3Npi) self-assembled into a peptide nanotube in a solution of HFIP and water. CD spectra of the CP3Npi nanotubes show a negative Cotton effect at 441 nm and a positive Cotton effect at 393 nm, indicating that D-π-A naphthalimide chromophores are aligned in a left-handed chiral way along the nanotube. The CP3Npi nanotubes bear positive charges under acidic conditions retaining the nanotube structure but pH-responsive switching of D-π-A naphthalimide alignments along the nanotube between a left-handed chiral and random arrangement was observed. The peptide nanotube is a stable scaffold for attaining pH-responsive alignment switching of side-chain chromophores.

Sterical Recognition at Helix-Helix Interface of Leu-Aib-Based Polypeptides with and without a GxxxG-Motif.

An amphiphilic polypeptide, poly(sarcosine)- b-(l-Leu-Aib)8 (SL16), was reported to self-assemble into vesicles. A GxxxG motif, which is known to induce helix dimerization, is incorporated into the hydrophobic helical block of SL16 to synthesize poly(sarcosine)- b-(l-Leu-Aib)2-(Gly-Aib-l-Leu-Aib-Gly-Aib)-(l-Leu-Aib)3 (SG16). SG16 shows helix association in ethanol at a high concentration and low temperatures, which is not observed with SL16. SG16 self-assembles into vesicles, but are found to be more susceptible to rupture by the addition of Triton X-100 than SL16 vesicles. A mixture of SL16 and SG16 self-assembles into small sheets and micelles likely because of mismatch of the modes of helix association arising from sterical accommodation of iso-butyl groups at the helix-helix interface.

Chiral and random arrangements of flavin chromophores along cyclic peptide nanotubes on gold influencing differently on surface potential and piezoelectricity.

Two kinds of peptide nanotubes are prepared from cyclo(ß-Asp(flavin)-ß-alanine-ß-alanine) (C3FAA) and cyclo(ß-Asp(flavin)-ethylenediamine-succinic acid) (C3FES). The flavin chromophores are protruding on the C3FAA and C3FES peptide nanotube surfaces in random and chiral ways, respectively. The surface potentials of the C3FAA nanotube bundles on a gold substrate become larger than the C3FES nanotube bundles of the corresponding thicknesses. The converse piezoelectric coefficients are as small as less than 1 pm V-1. The peptide nanotube bundles are subjected to a thermal anneal treatment which raises up all the surface potentials and also the converse piezoelectricity of the C3FES nanotube bundles of 3 pm V-1. The macrodipole of the C3FAA nanotube and the chiral arrangement of the flavin groups in the C3FES nanotube are considered to contribute influentially to the surface potential and the piezoelectricity, respectively.

Piezoelectric property of bundled peptide nanotubes stapled by bis-cyclic-ß-peptide.

Cyclic tetra-ß-peptides (CP4s) and a bis-CP4 were synthesized to prepare peptide nanotubes (PNTs) by molecular stacking of cyclic peptides. The addition of bis-CP4 to the PNT preparation afforded PNT bundles increasing the direct and converse piezoelectiric coefficients, which is ascribable to bis-CP4 stapling PNTs into the parallel alignment of PNT dipoles.

Immune responses against Lewis Y tumor-associated carbohydrate antigen displayed densely on self-assembling nanocarriers.

Immune responses against Lewis y (LY) displayed on nanocarriers at different surface densities were studied. The high surface density of LY was obtained by the A2B-type amphiphilic polypeptides having LY at the two terminals [LY-poly(sarcosine)2-b-(l- or d-Leu-Aib)6]. The equimolar mixture of these two amphiphilic polypeptides formed interdigitated planar sheet-like molecular assemblies densely displaying LY (G4). G4 seemed to induce the anti-LY IgM upon immunization to BALB/c mice by only a single administration. However, the amount of anti-LY IgM produced was moderate and significantly less than that induced by two administrations of the other molecular assembly (G1) with the average surface density of LY at a 1/4 of that of G4. Further, the anti-LY IgM produced after two administrations of G4 lowered the avidity more than after one administration.

Compartmentalized host spaces accommodating guest aromatic molecules in a chiral way in a helix-peptide-aromatic framework.

A novel host molecular assembly of a free-standing flat nanosheet with compartmentalized spaces was prepared using a bolaamphiphilic peptide composed of two amphiphilic helical peptides and an oligo(naphthaleneethynylene) (ONE) unit at the center of the molecule. The nanosheet possesses void host spaces that can accommodate two mol-equivalent ONE groups to form columns of ONE groups in a right-handed helical way and ONE channels over a long distance. The present molecular system therefore can provide a chiral pore channel for relatively large molecules.

Two one-dimensional arrays of naphthyl and anthryl groups along peptide nanotubes prepared from cyclic peptides comprising α- and ß-amino acids.

A novel cyclic hexapeptide composed of l-α-naphthylalanine, d-α-anthrylalanine, and four ß-alanines (CP6) is synthesized and its molecular assembly into peptide nanotubes (PNTs) and the electronic properties arising from one-dimensional arrays of aromatic groups along the PNTs were investigated. CP6 with a combination of l- and d-α-amino acids is designed to self-assemble into PNTs with them stacking on top of each other under the constraint of maximizing the number of intermolecular hydrogen bonds between the cyclic peptides. Upon PNT formation, the respective side chains of l- and d-α-amino acids are aligned in line along the PNTs. The topological arrangement of the anthryl groups being in close proximity in the CP6 PNT is supported by higher photo-excited energy transfer, appearance of the induced Cotton effects, and the promoted photo-dimerization reaction upon PNT formation. AFM observations reveal that PNT bundles with diameters 5-15 nm are dielectric microcrystals having a piezoelectric coefficient of 2-6 pC N-1. Kelvin force microscopy observations show the generation of surface potentials over 100 mV owing to the one-dimensional array of the anthryl groups along PNTs. Incorporation of α-amino acids with opposite chirality into cyclic ß-peptides is therefore an effective molecular design for the nano-architecture of PNTs displaying one-dimensional arrays of chromophores along PNTs.

Temperature-Induced Phase Separation in Molecular Assembly of Nanotubes Comprising Amphiphilic Polypeptoid with Poly( N-ethyl glycine) in Water by a Hydrophilic-Region-Driven-Type Mechanism.

Two kinds of amphiphilic polypeptoids having different types of hydrophilic polypeptoids, poly(sarcosine)- b-(l-Leu-Aib)6 (ML12) and poly( N-ethyl glycine)- b-(l-Leu-Aib)6 (EL12), were self-assembled via two paths to phase-separated nanotubes. One path was via sticking ML12 nanotubes with EL12 nanotubes and the other was a preparation from a mixture of ML12 and EL12 in solution. In either case, nanotubes showed temperature-induced phase separation along the long axis, which was observed by two methods of labeling one phase with gold nanoparticles and fluorescence resonance energy transfer between the components. The phase separation was ascribed to aggregation of poly( N-ethyl glycine) blocks over the cloud point temperature. The addition of 5% trifluoroethanol was needed for the phase separation because the tight association of the helices in the hydrophobic region should be loosened to allow lateral diffusion of the components to be separated. The phase separation in molecular assemblies in water based on the hydrophilic-region-driven-type mechanism therefore requires sophisticated balances of association forces exerting among the hydrophilic and hydrophobic regions of the amphiphilic polypeptoids.

Effect of oscillation dynamics on long-range electron transfer in a helical peptide monolayer.

Electron transfer (ET) reactions via helical peptides composed of -(Aib-Pro)n- were studied in self-assembled monolayers and compared with -(Ala-Aib)n- peptides. Short Aib-Pro peptides showed slightly higher ET rates due to the better electronic coupling of the Pro residue. But, the 24mer Aib-Pro peptide showed a smaller ET rate than the corresponding Ala-Aib peptide. On the basis of DFT calculations, the deceleration of the ET rate of the longer Aib-Pro peptide is considered to be due to the smaller number of active modes of accordion-like oscillations than the Ala-Aib peptide, which has a strong influence on a long-range ET reaction.

Polymeric Micelle of A3B-Type Lactosome as a Vehicle for Targeting Meningeal Dissemination.

Polymeric micelle of the A3B-type lactosome comprising (poly(sarcosine))3-b-poly(l-lactic acid) was labeled with 111In. The 111In-labeled A3B-type lactosome was administered to the model mice bearing meningeal dissemination and bone metastasis at mandible. With single-photon emission computed tomography (SPECT) imaging, the meningeal dissemination was identified successfully by 111In-labeled A3B-type lactosome, which was superior to 201TlCl in regard of the imaging contrast. The 111In-labeled A3B-type lactosome was also potential in imaging selectively of bone metastasis at mandible, whilst a nonspecific imaging of the whole bone was obtained by the SPECT imaging using 99mTc-HMDP. The polymeric micelle of the A3B-type lactosome was therefore found to be effective as a vehicle of 111In to be targeted to meningeal dissemination and bone metastasis.

Phase-Separated Molecular Assembly of a Nanotube Composed of Amphiphilic Polypeptides Having a Helical Hydrophobic Block.

Amphiphilic block polypeptides of poly(sarcosine)-b-(l- or d-Leu-Aib)6 (SL12OMe or SD12OMe) and poly(sarcosine)-b-(l-Leu-Aib)7 (SL14OMe) were reported to self-assemble into a nanotube morphology. Herein, we tried to construct a phase-separated nanotube by sticking two different kinds of nanotubes. SD12OMe nanotubes were found to stick to SL14OMe nanotubes with a heat treatment at 50 °C, but the sticking yield was limited. The amphiphilic polypeptides were functionalized by replacement of methyl ester with aromatic groups of N-ethylcarbazole (SL12Ecz) and naphthalimide (SD12NpiTEG), but they lost the ability to form homogeneous nanotubes. A fraction of the functionalized amphiphilic polypeptides mixing in the nanotube-forming amphiphilic polypeptides, a mixture of SL12OMe and SL12Ecz (9:1) as well as a mixture of SD12OMe and SD12NpiTEG (9:1), allowed nanotube formation. These two kinds of nanotubes partly stuck together with a heat treatment at 15 °C to maintain a segregated state of two kinds of aromatic groups along the nanotube, resulting in the formation of a phase-separated nanotube.

Reaction specificity of keratanase II in the transglycosylation using the sugar oxazolines having keratan sulfate repeating units.

The reaction specificity of the transglycosylation catalyzed by keratanase II from Bacillus circulans KsT202 (KSase II) was studied by using the oxazoline derivatives having keratan sulfate repeating units. The addition of 10% organic cosolvent reduced the activity for the enzymatic transglycosylation. The oxazoline derivative of 6-O-sulfonato-N-acetyllactosamine (su-LacNAc) was processively oligomerized to the corresponding hexamer or longer by the enzyme. This result strongly implies that the enzyme has the large positively numbered subsites. In contrast, the transglycosylation of the su-LacNAc oxazoline donor with the 6-O-sulfonato-Lewis X (su-LeX) acceptor solely gave the su-LacNAc-su-LeX pentasaccharide. In addition, both the oxazoline derivatives of su-LeX and 6,6'-di-O-sulfonato-LacNAc have been exclusively oligomerized to the corresponding dimers respectively. These results strongly suggest that the steric hindrance exists around the (+3)(+4) subsites in KSase II. Furthermore, KSase II-catalyzed reaction of the excess su-LeX oxazoline with the su-LacNAc gave the su-LeX-su-LacNAc pentasaccharide as the sole transglycosylation product, also implying the steric hindrance at the catalytic center hampering processive shift of this pentasaccharide. Thus, KSase II has the sterically crowded structures at the catalytic center and around the (+3)(+4) subsites, which are all expected to be tunnel-like.

Joining Nanotubes Comprising Nucleobase-carrying Amphiphilic Polypeptides.

Three kinds of amphiphilic polypeptides, X-poly(sarcosine)-b-(L-Leu-Aib)6 (X = adenine, thymine, glycolic acid), were synthesized and self-assembled in a tris buffer to take on nanotube morphology. The nanotubes were joined together to extend the nanotube length with the addition of trifluoroethanol and heat treatment at 50 °C for 24 h. The length extension rate decreased in the order of adenine > glycolic acid > thymine depending on the N-terminal chromophores. Adenine-adenine interactions between the nanotubes were found to be more prevalent upon joining the nanotubes than adenine-thymine interactions. Further, adenines on the nanotube surface could chelate with Cu(ii) to thermodynamically stabilize the nanotube membrane. AFM imaging in liquid environment revealed that the membrane elasticity of the adenine nanotube was as high as ca. 1 MPa, which is considered to be strengthened as a result of the adenine-adenine interactions.

Modulation of immunogenicity of poly(sarcosine) displayed on various nanoparticle surfaces due to different physical properties.

Poly(sarcosine) displayed on polymeric micelle is reported to trigger a T cell-independent type2 reaction with B1a cells in the mice to produce IgM and IgG3 antibodies. In addition to polymeric micelle, three kinds of vesicles displaying poly(sarcosine) on surface were prepared here to evaluate the amounts and avidities of IgM and IgG3, which were produced in mice, to correlate them with physical properties of the molecular assemblies. The largest amount of IgM was produced after twice administrations of a polymeric micelle of 35 nm diameter (G1). On the other hand, the production amount of IgG3 became the largest after twice administrations of G3 (vesicle of 229 nm diameter) or G4 (vesicle of 85 nm diameter). The augmented avidity of IgG3 after the twice administrations compared with that at the single administration was the highest with G3. These differences in immune responses are discussed in terms of surface density of poly(sarcosine) chains, nanoparticle size, hydrophobic component of poly(L-lactic acid) or (Leu- or Val-Aib)n , and membrane elasticity of the nanoparticles. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Tuning the Viscoelasticity of Peptide Vesicles by Adjusting Hydrophobic Helical Blocks Comprising Amphiphilic Polypeptides.

Amphiphilic block polypeptides of poly(sarcosine)-b-(l-Val-Aib)6 and poly(sarcosine)-b-(l-Leu-Aib)6 and their stereoisomers were self-assembled in water. Three kinds of binary systems of poly(sarcosine)-b-(l-Leu-Aib)6 with poly(sarcosine)-b-poly(d-Leu-Aib)6, poly(sarcosine)-b-poly(l-Val-Aib)6, or poly(sarcosine)-b-(d-Val-Aib)6 generated vesicles of ca. 200 nm diameter. The viscoelasticity of the vesicle membranes was evaluated by the nanoindentation method using AFM in water. The elasticity of the poly(sarcosine)-b-(l-Leu-Aib)6/poly(sarcosine)-b-poly(d-Leu-Aib)6 vesicle was 11-fold higher than that of the egg yolk liposome but decreased in combinations of the Leu- and Val-based amphiphilic polypeptides. The membrane elasticity is found to be adjustable by a suitable combination of helical blocks in terms of stereocomplex formation and the interdigitation of side chains among helices in the molecular assemblies.

Immune activation with peptide assemblies carrying Lewis y tumor-associated carbohydrate antigen.

Molecular assemblies varying morphologies in a wide range from spherical micelle, nanosheet, curved sheet, nanotube and vesicle were prepared and loaded with Lewis y (Ley ) tumor-associated carbohydrate antigen on the assembly surface. The molecular assemblies were composed of poly(sarcosine)m -block-poly(L-lactic acid)30 (m = 15 or 50, Lactosome), poly(sarcosine)m -block-(D/L-Leu-Aib)n (m = 22 or 30, n = 6 or 8) and their combinations. The molecular assemblies carrying Ley on the surface were administered in BALB/c nu/nu mice. The major epitopes of the molecular assemblies are commonly Ley and poly(sarcosine). IgM productions upon administrations of the molecular assemblies were assayed by ELISA, showing that anti-poly(sarcosine) IgM was highly produced by Lactosome of spherical micelle but with a negligible amount of anti-Ley IgM. On the other hand, the nanosheet of the interdigitated monolayer triggered the production of anti-Ley IgM but with less anti-poly(sarcosine) IgM production. Taken together, IgM specificity differs according to the molecular environment of the epitopes in the molecular assemblies. The antigenicity of poly(sarcosine) was augmented in polymeric micelle providing loose environment for B cells to penetrate in, whereas a high density of Ley on the molecular assembly was required for anti-Ley IgM production. The antigenicity of Ley is therefore dependent on the molecular assemblies on which Ley is displayed on the surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Fusion and fission of molecular assemblies of amphiphilic polypeptides generating small vesicles from nanotubes.

Three amphiphilic block polypeptides, (sarcosine)m -b-(l- or d-Leu-Aib)n (L16, D16, D14), having different helical chain lengths or helicity are synthesized. A mixture of L16, D16, and D14 generates vesicles of diameters more than ca. 130 nm by injecting the ethanol solution into water and heating at 90°C for 1 h. On the other hand, when nanotubes composed of L16 and D14 having ca. 50 nm diameter are mixed with nanosheets composed of D16, smaller and homogeneous vesicles of ca. 60 nm diameter are obtained with the heat treatment. The time lapse TEM image analysis of the mixtures revealed some transient structures of nanotubes sticking a nanosheet or a vesicle at the open end of nanotubes. The precise size control of vesicles is therefore attainable by using nanotubes as a structural template regulating the size of vesicles near to the nanotube diameter upon the membrane fission processes.