Recombinant Spider Silk Fiber with High Dimensional Stability in Water and Its NMR Characterization.

Spider dragline silk has unique characteristics of strength and extensibility, including supercontraction. When we use it as a biomaterial or material for textiles, it is important to suppress the effect of water on the fiber by as much as possible in order to maintain dimensional stability. In order to produce spider silk with a highly hydrophobic character, based on the sequence of ADF-3 silk, we produced recombinant silk (RSSP(VLI)) where all QQ sequences were replaced by VL, while single Q was replaced by I. The artificial RSSP(VLI) fiber was prepared using formic acid as the spinning solvent and methanol as the coagulant solvent. The dimensional stability and water absorption experiments of the fiber were performed for eight kinds of silk fiber. RSSP(VLI) fiber showed high dimensional stability, which is suitable for textiles. A remarkable decrease in the motion of the fiber in water was made evident by 13C solid-state NMR. This study using 13C solid-state NMR is the first trial to put spider silk to practical use and provide information regarding the molecular design of new recombinant spider silk materials with high dimensional stability in water, allowing recombinant spider silk proteins to be used in next-generation biomaterials and materials for textiles.

Formylation of Recombinant Spider Silk in Formic Acid and Wet Spinning Studied Using Nuclear Magnetic Resonance and Infrared Spectroscopies.

We reported wet spinning of recombinant spider silk protein (RSSP) and formylation of RSSP in formic acid (FA). First, FA was selected as the spinning solvent and the detailed spinning condition was determined. Next, the mechanical property was compared between the RSSP fiber spun after allowing the spinning solution dissolved in FA to stand for 2 days and the fiber spun immediately after being dissolved in FA for 4 h. The tensile strength of the former fiber was lower than the strength of the latter fiber. This difference can be explained by the difference in the degree of formylation as follows. FA is a known formylating agent, although most researchers who prepared silk fiber by wet spinning with FA have not pointed out about formylation. The formylation of the Ser OH group was confirmed by 13C solution nuclear magnetic resonance (NMR), and the time course of formylation of the RSSP film prepared from the FA solution was tracked by Fourier transform infrared spectroscopy. The 13C solid-state NMR spectra were also compared between two kinds of the formylated RSSP fibers and indicated that the packing state was tighter for the latter fiber than the former one, which could explain higher tensile strength of the latter fiber in the dry state. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the RSSP sample decomposed gradually with storage time in FA and the decomposition has begun partly even at 2 h after dissolution in FA. The decomposition by formylation seems to have no significant effect on the backbone structure of the RSSP fiber, although the packing of the fiber becomes loose as a whole. Finally, preliminary trial of deformylation of the formylated RSSP fiber was performed.

Exploring the size of reference population for expected accuracy of genomic prediction using simulated and real data in Japanese Black cattle.

BACKGROUND: Size of reference population is a crucial factor affecting the accuracy of prediction of the genomic estimated breeding value (GEBV). There are few studies in beef cattle that have compared accuracies achieved using real data to that achieved with simulated data and deterministic predictions. Thus, extent to which traits of interest affect accuracy of genomic prediction in Japanese Black cattle remains obscure. This study aimed to explore the size of reference population for expected accuracy of genomic prediction for simulated and carcass traits in Japanese Black cattle using a large amount of samples. RESULTS: A simulation analysis showed that heritability and size of reference population substantially impacted the accuracy of GEBV, whereas the number of quantitative trait loci did not. The estimated numbers of independent chromosome segments (Me) and the related weighting factor (w) derived from simulation results and a maximum likelihood (ML) approach were 1900-3900 and 1, respectively. The expected accuracy for trait with heritability of 0.1-0.5 fitted well with empirical values when the reference population comprised > 5000 animals. The heritability for carcass traits was estimated to be 0.29-0.41 and the accuracy of GEBVs was relatively consistent with simulation results. When the reference population comprised 7000-11,000 animals, the accuracy of GEBV for carcass traits can range 0.73-0.79, which is comparable to estimated breeding value obtained in the progeny test. CONCLUSION: Our simulation analysis demonstrated that the expected accuracy of GEBV for a polygenic trait with low-to-moderate heritability could be practical in Japanese Black cattle population. For carcass traits, a total of 7000-11,000 animals can be a sufficient size of reference population for genomic prediction.

Robust induction of neural crest cells to derive peripheral sensory neurons from human induced pluripotent stem cells.

Because intractable itch reduces quality of life, understanding the fundamental mechanisms of itch is required to develop antipruritic treatments. Itch is mediated by peripheral sensory neurons, which originate from the neural crest (NC) during development. Itch-associated signaling molecules have been detected in genetically engineered animals and in cultures of peripheral neurons from dorsal root ganglia (DRG). Ethical difficulties collecting peripheral neurons from human DRG have limited analysis of itch in humans. This study describes a method of differentiating peripheral neurons from human induced pluripotent stem cells (hiPSCs) for physiological study of itch. This method resulted in the robust induction of p75 and HNK1 double-positive NC cells from hiPSCs. The expression of NC markers TFAP2A, SOX10 and SNAI1 increased during NC induction. The induction efficiency was nearly 90%, and human peripheral neurons expressing peripherin were efficiently differentiated from hiPSC-derived NC cells. Moreover, induced peripheral neurons expressed the sensory neuronal marker BRN3A and the itch-related receptors HRH1, MRGPRX1, IL31R and IL-4R. Calcium imaging analyses indicated that these peripheral neurons included sensory neurons responsive to itch-related stimuli such as histamine, BAM8-22, IL-31 and IL-4. These findings may enable detailed analyses of human DRG neurons and may result in new therapies for intractable itch.

Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography.

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focused-ion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, "ridge-like prominences" (RLPs).

Acetylation of Bombyx mori silk fibroin and their characterization in the dry and hydrated states using 13C solid-state NMR.

Acetylation of Bombyx mori silk fibroin (SF) was accomplished by chemical reaction of the side chain OH groups in Ser and Tyr residues with acetic anhydrate to increase hydrophobic character of SF. A combination of three kinds of 13C solid-state NMR techniques was used to elucidate the effect of acetylation in the dry and hydrated states for [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powder. The mobilities of Tyr and Ala residues in the amorphous region of acetylated 13C-labeled SF powder increased slightly and only very small amounts of the Ala residue with high mobility was observed by hydration. On the other hand, there are essentially no effect of water on the ß-sheet region of these residues and acetylated Ser residues. A similar tendency toward the effect of hydration for acetylated SF powder was observed for acetylated SF fiber. The remarkable improvement of the dimensional stability in water was attained although the tensile strengths of fibers were slightly lower by the acetylation of SF fiber. Thus, acetylation proved very effective to achieve water-proofing of SF materials and the effect of acetylation on the local structure and dynamics of SF could be clarified using 13C solid-state NMR.

KIF11 as a Potential Marker of Spermatogenesis Within Mouse Seminiferous Tubule Cross-sections.

The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development.

Conformational change of 13C-labeled 47-mer model peptides of Nephila clavipes dragline silk in poly(vinyl alcohol) film by stretching studied by 13C solid-state NMR and molecular dynamics simulation.

For determination of the conformation of irregular sequences in glycine-rich region of the Nephila clavipes spider dragline silk, the combination of 13C selectively labeled model peptides for the typical primary structure and their 13C solid-state NMR observations is very useful (T. Asakura et al. Macromolecules. 51 (2018) 3608-3619). However, spiders produce the fiber through the stretching process in nature and therefore, it is difficult to study conformational change by stretching as mimic using the model peptides because these are generally in the powder form. In this paper, 13C selectively labeled three model peptides, (Glu)4(Ala)6GlyGly12Ala13Gly14GlnGlyGlyTyrGlyGlyLeuGlySerGlnGly25Ala26Gly27ArgGly-GlyLeuGlyGlyGlnGly35Ala36Gly37(Ala)6(Glu)4 with three underlined 13C labeled blocks and their poly(vinyl alcohol) blend films were prepared and the conformational changes of these peptides were monitored by stretching of the films using 13C solid-state NMR. In addition, the molecular dynamics simulation was done to evaluate change in the conformation of the sequence by stretching theoretically. The fractions of ß-sheet of Ala36 and Gly37 residues in glycine-rich region adjacent to the C-terminal (Ala)6 sequence increased significantly by stretching compared with those of other 13C labeled Ala and Gly residues.

Emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers.

The conditions required for the emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers are assessed through an experimental approach that combines the spinning of regenerated fibers with controlled properties and their characterization by 13C solid-state nuclear magnetic resonance (NMR). Both supercontracting and non-supercontracting regenerated fibers are produced using the straining flow spinning (SFS) technique from 13C labeled cocoons. The short-range microstructure of the fibers is assessed through 13C CP/MAS in air and 13C DD/MAS in water, and the main microstructural features are identified and quantified. The mechanical properties of the regenerated fibers and their microstructures are compared with those of natural silkworm silk. The combined analysis highlights two possible key elements as responsible for the emergence of supercontraction: (1) the existence of an upper and a lower limit of the amorphous phase compatible with supercontraction, and (2) the existence of two ordered phases, ß-sheet A and B, which correspond to different packing arrangements of the protein chains.

Changes in the Local Structure of Nephila clavipes Dragline Silk Model Peptides upon Trifluoroacetic Acid, Low pH, Freeze-Drying, and Hydration Treatments Studied by 13C Solid-State NMR.

The conformational analysis of spider dragline silks is difficult because of the amorphous character of the silks. In this article, the fractions of several conformations were determined for three 47-mer peptides, (Glu)4(Ala)6GlyGly12Ala13Gly14GlnGlyGlyTyrGlyGlyLeuGlySerGlnGly25Ala26Gly27-ArgGlyGlyLeuGlyGlyGlnGly35Ala36Gly37(Ala)6(Glu)4, with three underlined 13C-labeled blocks using a 13C CP/MAS NMR method. The conformations of the 13C-labeled sites change significantly depending on the location of the labeled blocks when treated with trifluoroacetic acid, low pH, and freeze-drying. The conformations of Ala36 and Gly37 residues are strongly influenced by the specific conformation of the (Ala)6 sequence at the C-terminal side, but those of other residues, Ala13 and Gly14, and Ala 26 and Gly27, are basically not influenced by the conformations of (Ala)6. Through hydration of the ß-sheet peptide, sharp peaks with random coil could be observed depending on the position of the residue, and this result could be interpreted via the change in the Ramachandran map obtained from the molecular dynamics simulation.

Effect of Water on the Structure and Dynamics of Regenerated [3-13C] Ser, [3-13C] , and [3-13C] Ala-Bombyx mori Silk Fibroin Studied with 13C Solid-State Nuclear Magnetic Resonance.

The effects of water on the structure and dynamics of natural and regenerated silk fibroin (SF) samples were studied using 13C solid-state nuclear magnetic resonance (NMR) spectroscopy. We prepared different types of SF materials, sponges, and fibers with different preparation methods and compared their NMR spectra in the dry and hydrated states. Three kinds of 13C NMR techniques, r-INEPT, CP/MAS, and DD/MAS, coupled with 13C isotope labeling of Ser, Tyr, and Ala residues were used. In the hydrated sponges, several conformations, that is, Silk I* and two kinds of ß-sheets, A and B, random coil, and highly mobile hydrated random coil were observed, and the fractions were determined. The fractions were remarkably different among the three sponges but with only small differences among the regenerated and native fibers. The increase in the fraction of ß-sheet B might be one of the structural factors for preparing stronger regenerated SF fiber.

Involvement of µ-opioid Receptors and κ-opioid Receptors in Itch-related Scratching Behaviour of Imiquimod-induced Psoriasis-like Dermatitis in Mice.

The pathogenesis of psoriatic itch is poorly understood. The aim of this study was to investigate the involvement of opioid receptors in scratching behaviour of imiquimod-induced psoriasis-like dermatitis model mice. Topical application of 5% imiquimod cream to the rostral back skin of mice induced antihistamine-resistant scratching behaviour. The expression of µ-opioid receptor (MOR) protein increased in the epidermis, dorsal root ganglia (DRG) and spinal cord of imiquimod-treated mice. In contrast, the expression of κ-opioid receptor (KOR) protein decreased in the DRG and spinal cord of imiquimod-treated mice, and was undetectable in the epidermis of both groups. Topical or intraperitoneal administration of the MOR antagonist naloxone and oral administration of the centrally acting KOR agonist ICI-199,441 inhibited scratching behaviour, whereas oral administration of the peri-pherally-selective KOR agonist asimadoline did not. These results suggest that peripheral and central MOR and central KOR may be involved in the modulation of scratching behaviour in imiquimod-treated mice.

Accumulation of immunoglobulin G against Dermatophagoides farinae tropomyosin in dorsal root ganglia of NC/Nga mice with atopic dermatitis-like symptoms.

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as intractable itch, but its underlying mechanisms are poorly understood. This study assessed the relationship between immunoglobulin G (IgG) and dorsal root ganglia (DRG) in NC/Nga mice, a model of AD that manifests AD-like symptoms including itch. Immunohistochemical analysis showed large amounts of IgG in DRG extracts of NC/Nga mice with AD-like dermatitis, with a large fraction of the IgG distributed in satellite glial cells of the DRG. Proteomic analysis showed that this IgG was reactive against tropomyosin of Dermatophagoides farinae. These findings indicate that the accumulation of anti-tropomyosin IgG in DRG of atopic NC/Nga mice may be associated with the pathogenesis of AD-like symptoms, including itch.

Differences in therapeutic effects of topically applied corticosteroid and tacrolimus on atopic dermatitis-like symptoms in NC/Nga mice.

BACKGROUND: Topical corticosteroid and calcineurin inhibitor have similar therapeutic benefits in atopic dermatitis (AD), but the differences in therapeutic mechanisms of action of these agents against AD symptoms are not fully understood. OBJECTIVE: This study was performed to examine the different effects of topical betamethasone valerate (BMV), clobetasol propionate (CBP), and tacrolimus (TAC) on itch-related behavior and dermatitis in NC/Nga mice with AD-like symptoms. METHODS: AD-like dermatitis was induced in the dorsal skin of NC/Nga mice by repeated topical application of Dermatophagoides farinae body (Dfb) ointment twice weekly for three weeks. Mice with dermatitis scores over 5 were divided into five groups with equal dermatitis scores and treated with BMV, CBP, TAC, or Vaseline (Vas) once daily for two consecutive days, or were not treated (NT). Scratching behavior was analyzed using a SCLABA®-Real system. Transepidermal water loss (TEWL) before and after treatment was measured using a Tewameter® TM210. Skin collected from each group was analyzed histologically. RESULTS: After the second treatment, dermatitis showed significantly greater improvement in the CBP and TAC-treated groups than in the Vas-treated and NT groups. The numbers of scratching bouts were significantly lower in CBP and TAC-treated mice than in Vas-treated mice. TEWL was significantly lower in TAC-, but not in CBP-, treated mice than in Vas-treated mice. Immunohistochemical examination showed that BMV, CBP and TAC did not reduce the increased densities of epidermal protein gene product 9.5- and substance P-immunoreactive fibers. The numbers of dermal CD4-immunoreactive T cells were significantly lower in BMV and CBP-treated mice than in Vas-treated and NT mice. The numbers of dermal eosinophils were significantly lower in BMV, CBP and TAC-treated mice than in Vas-treated and NT mice, with CBP showing the strongest effect. CBP significantly reduced epidermal thickness compared with Vas and NT. There were no significant differences in the numbers of interleukin-31-immunoreactive cells and mast cells, or in expression of epidermal thymic stromal lymphopoietin among all five groups. CONCLUSION: The therapeutic potency of TAC against AD-like symptoms, including pruritus, is equal to that of the corticosteroid CBP. Epidermal innervation of sensory nerves itself might not be related to the therapeutic effects of topical tacrolimus and corticosteroids in its early phase.

Relationships among plasma granzyme B level, pruritus and dermatitis in patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by skin barrier dysfunction, allergic inflammation and intractable pruritus resistant to conventional antipruritic treatments, including H1-antihistamines. Granzymes (Gzms) are a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer cells that have been shown to modulate inflammation. However, the relationship between Gzms and pathology in AD remains unclear. OBJECTIVE: This study assessed the correlation between plasma GzmB levels and severity of pruritus and dermatitis, in AD patients. METHODS: Plasma was collected from 46 patients with AD, 24 patients with psoriasis, and 30 healthy controls. AD severity was assessed with the scoring atopic dermatitis (SCORAD) index, psoriasis severity with the psoriasis area and severity index (PASI), and degree of pruritus by visual analogue scale (VAS) score. GzmA, GzmB and gastrin releasing peptide (GRP) levels were measured by enzyme-linked immunosorbent assays. RESULTS: Plasma GzmB concentrations were significantly higher in patients with AD and psoriasis than in healthy controls. Correlation analyses showed that plasma GzmB concentrations positively correlated with SCORAD and serum levels of severity markers such as thymus and activation-regulated chemokine, and lactate dehydrogenase in AD patients. Moreover, plasma levels of GRP, an itch-related peptide, were higher in patients with AD, positively correlating with VAS score and plasma GzmB level. In addition, plasma GzmB concentration was significantly lower in the treatment group than the untreated group with AD. Meanwhile, there were no correlations among GzmB levels, VAS score and PASI score in patients with psoriasis. In contrast to the results of plasma GzmB, plasma GzmA levels were unchanged among AD, psoriasis and healthy groups, and showed no correlations with VAS score and SCORAD index in patients with AD. CONCLUSION: Plasma GzmB levels may reflect the degree of pruritus and dermatitis in patients with AD.

Possible Antipruritic Mechanism of Cyclosporine A in Atopic Dermatitis.

Cyclosporine A is an immunosuppressive agent that suppresses pruritus and is currently used in the treatment of patients with severe atopic dermatitis. The aim of this study was to elucidate the antipruritic mechanism of cyclosporine A using a mouse model of atopic dermatitis. Intraperitoneal injection of cyclosporine A (5 mg/kg) significantly reduced epidermal nerve density, number of scratching bouts, dermatitis scores, and transepidermal water loss, as well as decreasing the numbers of inflammatory cells in the dermis and decreasing epidermal thickness. Intraperitoneal injection of cyclosporine A dose-dependently inhibited increased itch-related receptor gene expression, such as interleukin-31 receptor A and neurokinin-1 receptor, in the dorsal root ganglion of atopic dermatitis model mice. Thus, the antipruritic efficacy of cyclosporine A may involve reduced epidermal nerve density and expression levels of itch-related receptor genes in the dorsal root ganglion, as well as improvement in acanthosis and reduction in cutaneous inflammatory cell number.