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Background: Alopecia areata (AA) is an autoimmune disease resulting in non-scarring hair loss. Animal models are useful means to identify candidates for therapeutic agents. The C3H/HeJ mouse AA model induced via transferring cultured lymphoid cells isolated from AA-affected mice is widely used for AA research. However, this conventional method requires the continuous breeding of AA mice., Objective: We aimed to establish a new method to generate AA model using the transfer of cryopreserved cells, which allows the rapid induction of a large number of AA mice when needed., Methods: We cryopreserved lymph node cells soon after isolation from AA-affected mice and injected thawed-cultured cells into recipient mice. H&E staining, immunohistochemical staining, quantitative real-time PCR and ELISA were conducted to identify pathological characteristics. Flow cytometry was performed to reveal the profile of transferred cells., Results: More than 90 % of recipient mice developed AA-like hair loss and showed inflammatory cell infiltration around anagen hair follicles, markedly increased mRNA expressions of interferon-γ, CXCL11, and granzyme B, and elevated interferon-α protein levels in the skin compared with naïve mice. Higher percentages of effector memory T cells and dendritic cells in transferred cells resulted in a higher incidence of AA., Conclusion: This is the first report to establish a method for generating AA mice using cryopreserved lymphocytes. These AA mice have similar pathological characteristics to AA mice generated with the conventional method and AA patients. This convenient and reproducible method is expected to be valuable for AA study., Competing Interests: Declaration of Competing Interest All authors are employees of Maruho Co., Ltd., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Canine degenerative myelopathy (DM) is an adult-onset fatal disease characterized by progressive degeneration of the spinal cord. Affected dogs have homozygous mutations in superoxide dismutase 1, and thus DM is a potential spontaneous animal model of human familial amyotrophic lateral sclerosis (ALS). Neuroinflammation is the pathological hallmark of ALS, whereby proinflammatory cytokines and chemokines are overproduced by activated glial cells such as astrocytes and microglia. However, the detailed pathogenesis of spinal cord degeneration in DM remains unknown. To further characterize the pathological mechanism of DM, we analyzed the caudal cervical cords of ten Pembroke Welsh Corgis pathologically diagnosed with DM by quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry (IHC), and double immunofluorescence. Compared to control spinal cord tissues, we found significantly enhanced transcriptions of interleukin-1β, tumor necrosis factor-α, CC motif chemokine ligand (CCL) 2 and vascular cell adhesion molecule -1 mRNA in the spinal cords of DM dogs. Moreover, IHC for the class II major histocompatibility complex molecules HLA-DR and CCL2 indicated that the immunopositive areas of activated macrophages/microglia and CCL2 protein were significantly increased in DM, and CCL2 protein was mainly overproduced by astrocytes. Our results suggest a proinflammatory state of the microenvironment in the DM spinal cord in which activated microglia and astrocytes play important roles by secreting a set of cytokines, chemokines, and expressing adhesion molecules., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Motile human-skin fibroblasts form macroscopic swirling patterns when grown to confluence on a culture dish. In this paper, we investigate the effect of coating the culture-dish surface with collagen on the resulting pattern, using human-skin fibroblast NB1RGB cells as the model system. The presence of the collagen coating is expected to enhance the adherence of the fibroblasts to the dish surface, and thereby also enhance the traction that the fibroblasts have as they move. We find that, contrary to our initial expectation, the coating does not significantly affect the motility of the fibroblasts. Their eventual number density at confluence is also unaffected. However, the coherence length of cell orientation in the swirling pattern is diminished. We also find that the fibroblasts cultured in collagen-coated dishes are rounder in shape and shorter in perimeter, compared with those cultured in uncoated polystyrene or glass culture dishes. We hypothesise that the rounder cell-shape which weakens the cell-cell nematic contact interaction is responsible for the change in coherence length. A simple mathematical model of the migrating fibroblasts is constructed, which demonstrates that constant motility with weaker nematic interaction strength does indeed lead to the shortening of the coherence length.

Aberrant aggregation of the RNA-binding protein TDP-43 in neurons is a hallmark of frontotemporal lobar degeneration caused by haploinsufficiency in the gene encoding progranulin 1,2 . However, the mechanism leading to TDP-43 proteinopathy remains unclear. Here we use single-nucleus RNA sequencing to show that progranulin deficiency promotes microglial transition from a homeostatic to a disease-specific state that causes endolysosomal dysfunction and neurodegeneration in mice. These defects persist even when Grn -/- microglia are cultured ex vivo. In addition, single-nucleus RNA sequencing reveals selective loss of excitatory neurons at disease end-stage, which is characterized by prominent nuclear and cytoplasmic TDP-43 granules and nuclear pore defects. Remarkably, conditioned media from Grn -/- microglia are sufficient to promote TDP-43 granule formation, nuclear pore defects and cell death in excitatory neurons via the complement activation pathway. Consistent with these results, deletion of the genes encoding C1qa and C3 mitigates microglial toxicity and rescues TDP-43 proteinopathy and neurodegeneration. These results uncover previously unappreciated contributions of chronic microglial toxicity to TDP-43 proteinopathy during neurodegeneration.

Mechanically tough solid polymer electrolytes (SPEs) are required to meet the demand for flexible and stretchable electrochemical devices for diverse applications, especially for wearable devices. It is well known that the inhomogeneity of a polymer network greatly affects its mechanical properties, but the evaluation of its effect on electrolyte properties including mechanical properties has not been accomplished yet because of the coexistence of various inhomogeneities (e.g., dangling bonds, loops, chain entanglements, and inhomogeneous distribution of cross-linking points). Herein, we discuss the effect of distribution of cross-linking densities in SPEs on its electrolyte properties by employing a model polymer network composed of a homogeneous 4-arm poly(ethylene glycol) (tetra-PEG) network and Li[TFSA] ([TFSA]: bis(trifluoromethanesulfonyl)amide). Tetra-PEGs having different molecular weights (Mn = 5, 10, 20, and 40 kDa) are subjected to the Michael addition reaction to induce network inhomogeneity while the average cross-linking densities are matched. It was found that thermal and ion transport properties of tetra-PEG SPEs do not depend on network inhomogeneity but on the average network size, which indicates that these properties reflect the averaged thermal fluctuation of polymer chains in terms of spatial and temporal dimensions. On the other hand, the mechanical toughness was largely dependent on the network homogeneity, and fracture strain, energy, and Young's modulus decreased by introducing network inhomogeneity. Rheological measurements showed that a transient cross-linking between Li cations and oxygens of tetra-PEG as well as the homogeneous distribution of the chemical cross-linking points contribute to the excellent mechanical properties of SPEs.

Heterophylly, or phenotypic plasticity in leaf form, is a remarkable feature of amphibious plants. When the shoots of these plants grow underwater, they often develop surprisingly different leaves from those that emerge in air. Among aquatic plants, it is typical for two or more distinct leaf development processes to be observed in the same individual exposed to different environments. Here, we analyze the developmental processes of heterophylly in the amphibious plant Callitriche palustris L. (Plantaginaceae). First, we reliably cultured this species under laboratory conditions and established a laboratory strain. We also established a framework for molecular-based developmental analyses, such as whole-mount in situ hybridization. We observed several developmental features of aerial and submerged leaves, including changes in form, stomata and vein formation, and transition of the meristematic zone. Then we defined developmental stages for C. palustris leaves. We found that in early stages, aerial and submerged leaf primordia had similar forms, but became discriminable through cell divisions with differential direction, and later became highly distinct via extensive cell elongation in submerged leaf primordia., (Copyright © 2020 Koga, Doll, Hashimoto, Toyooka and Tsukaya.)

Brain- and testis-specific immunoglobulin superfamily (BT-IgSF) (also known as IgSF11), one of the immunoglobulin superfamily proteins, is a cell adhesion molecule, expressed in the developing cerebellum. We hypothesized that BT-IgSF might have some function in the development of cerebellum, although the physiological roles of BT-IgSF in the cerebellum remain unclear. To investigate the role of BT-IgSF in the development of mouse cerebellum, we first determined the presence of BT-IgSF in the newborn mouse cerebellum; its expression level was found to be much higher than that in the adults. BT-IgSF was abundantly expressed in the molecular layer, where cerebellar granule cell precursors (CGCPs) are in the differentiation stage during migration. We subsequently analyzed the effects of BT-IgSF-knockdown and -overexpression on the proliferation and differentiation of primary cultured CGCPs. BT-IgSF suppressed the proliferation of CGCPs, and promoted their differentiation into cerebellar granule cells. Taken together, our results suggested that BT-IgSF is one of the important cell adhesion molecules that regulate the developmentof mouse cerebellum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vitronectin (VN), one of the extracellular matrix proteins, controls the maturation of cerebellar granule cells (CGCs) through the promotion of the initial differentiation stage progress. However, the receptors of VN in the initial differentiation stage of CGC precursors (CGCPs) have not been clarified. In this study, we characterized the receptor candidates for VN in CGCPs. First, we confirmed that αvβ3 and αvβ5 integrins, which are receptor candidates for VN, were co-localized with VN in the developing cerebellum and primary cultured CGCPs. Next, the knockdown (KD) of αv, β3, and β5 integrins with small interference RNA (siRNA) for each integrin reduced the ratio of Tuj1, a final differentiation marker, -positive CGCPs. We further studied whether αvβ3 and αvβ5 integrins control the initial differentiation stage. The KD of αv and β5, but not β3, integrins significantly increased the ratio of transient axonal glycoprotein 1 (TAG1), an initial differentiation marker, -positive CGCPs, whereas the KD of αv and β3 integrins, not β5 integrin, stimulated the proliferation of CGCPs. Overexpression of β5 integrin stimulated the progress of the initial differentiation stage as well. To confirm the interaction between αvβ5 integrin and VN, VN was added to β5 integrin-KD CGCPs. The promotion of the progress of initial differentiation by VN was abrogated by β5 integrin KD using small hairpin RNA (shRNA). Taken together, our results indicated that αvβ5 integrin, as the very receptor of VN, is responsible for the progress of the initial differentiation stage in mouse CGCPs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Traumatic brain injury (TBI) is caused by physical damage to the brain and it induces blood-brain barrier (BBB) breakdown and inflammation. To diminish the sequelae of TBI, it is important to decrease haemorrhage and alleviate inflammation. In this study, we aimed to determine the effects of 2-carba-cyclic phosphatidic acid (2ccPA) on the repair mechanisms after a stab wound injury as a murine TBI model. The administration of 2ccPA suppressed serum immunoglobulin extravasation after the injury. To elucidate the effects of 2ccPA on inflammation resulting from TBI, we analysed the mRNA expression of inflammatory cytokines. We found that 2ccPA prevents a TBI-induced increase in the mRNA expression of Il-1β, Il-6, Tnf-α and Tgf-β1. In addition, 2ccPA reduces the elevation of Iba1 levels. These data suggest that 2ccPA attenuates the inflammation after a stab wound injury via the modulation of pro-inflammatory cytokines release from microglial cells. Therefore, we focused on the function of 2ccPA in microglial polarisation towards M1 or M2 phenotypes. The administration of 2ccPA decreased the number of M1 and increased the number of M2 type microglial cells, indicating that 2ccPA modulates the microglial polarisation and shifts them towards M2 phenotype. These data suggest that 2ccPA treatment suppresses the extent of BBB breakdown and inflammation after TBI.

Patients with tenascin-X (TNX)-deficient type Ehlers-Danlos syndrome (EDS) do not exhibit delayed wound healing, unlike classic type EDS patients, who exhibit mutations in collagen genes. Similarly, in TNX-knockout (KO) mice, wound closure of the skin is normal even though these mice exhibit a reduced breaking strength. Therefore, we speculated that the wound healing process may be affected in the absence of TNX. In this study, to investigate the effects of TNX absence on wound healing-related properties, we performed collagen gel contraction assays with wild-type (WT) and TNX-KO mouse embryonic fibroblasts (MEFs). Collagen gels with embedded TNX-KO MEFs showed significantly greater contraction than those containing WT MEFs. Subsequently, we assessed collagen gel contraction-related properties, such as the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and the protein and mRNA expression levels of transforming growth factor β1 (TGF-β1) in the collagen gels. The activities of MMP-2 and MMP-9 and the expression level of TGF-β1 were elevated in the absence of TNX. Furthermore, filopodia-like protrusion formation, cell proliferation, migration, and collagen expression in MEFs were promoted in the absence of TNX. These results indicate that these wound healing-related properties are affected in a TNX-deficient extracellular environment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Feeding systems such as grazing affect the fatty acid profile of bovine milk fat. In addition, milk fat is formed as the product of fatty acid metabolism in cow bodies before being secreted into milk. However, how grazing influences milk fatty acid profile through the metabolism has not been completely characterized. When fatty acid concentrations in Holstein milk were compared between grazing and non-grazing periods, α-linolenic acid was significantly higher in the grazing period than in the non-grazing period. This could be explained with an increase in α-linolenic acid feeding with grazing. α-linolenic acid had a linear positive correlation with conjugated linoleic acid (9c,11t-18:2) (CLA) and vaccenic acid (VA) during the grazing period, whereas CLA had higher correlation with linoleic acid rather than with α-linolenic acid during the non-grazing period. These data indicate that the high content of dietary α-linolenic acid affects CLA and VA formation in milk of grazing periods via α-linolenic acid metabolism into VA., (© 2016 Japanese Society of Animal Science.)

Fibroblasts are the cells within the dermis layer of skin which are responsible for generating connective tissue, where collagen is the main component. However, the effects of collagen on the motion behavior of human skin fibroblasts were not studied well. We therefore propose an image analysis method to quantify the effects of collagen on the motion characteristics of fibroblasts. Images are divided into disjoint blocks and three gradient based descriptors, namely histogram of oriented gradients (HOG), the modified HOG (modiHOG), and the gradient direction histogram (GDH) are then applied to each block. These features can extract the orientation and shape or orientation only information of the skin fibroblasts from images. An orientation similarity function and a quantitative index, called first minimum similarity distance, are defined to quantify the orientation behavior of fibroblasts. An experiment with two sets of fibroblasts images, obtained from cell culture with and without collagen coating, demonstrates that the skin fibroblasts produce smaller orientation patterns, when they are cultured in the presence of collagen proteins. A statistical t-test is then performed, which validates the significant differences in the orientation behavior of the skin fibroblasts in the two cases mentioned.

A haustorium is the unique organ that invades host tissues and establishes vascular connections. Haustorium formation is a key event in parasitism, but its underlying molecular basis is largely unknown. Here, we use Phtheirospermum japonicum, a facultative root parasite in the Orobanchaceae, as a model parasitic plant. We performed a forward genetic screen to identify mutants with altered haustorial morphologies. The development of the haustorium in P. japonicum is induced by host-derived compounds such as 2,6-dimethoxy-p-benzoquinone. After receiving the signal, the parasite root starts to swell to develop a haustorium, and haustorial hairs proliferate to densely cover the haustorium surface. We isolated mutants that show defects in haustorial hair formation and named them haustorial hair defective (hhd) mutants. The hhd mutants are also defective in root hair formation, indicating that haustorial hair formation is controlled by the root hair development program. The internal structures of the haustoria in the hhd mutants are similar to those of the wild type, indicating that the haustorial hairs are not essential for host invasion. However, all the hhd mutants form fewer haustoria than the wild type upon infection of the host roots. The number of haustoria is restored when the host and parasite roots are forced to grow closely together, suggesting that the haustorial hairs play a role in stabilizing the host-parasite association. Thus, our study provides genetic evidence for the regulation and function of haustorial hairs in the parasitic plant., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Brachypodium distachyon is an emerging model plant for studying biological phenomena in temperate grasses. Study of the growth scale is essential to analyse spatio-temporal changes in molecular factors throughout the life cycle. For sensitive and robust staging based on morphology in B. distachyon, we demonstrated the utility of the BBCH (Biologische Bundesanstalt, Bundessortenamt and CHemical industry) scale, which is comparable to the Zadoks scale conventionally used for Triticeae crops. We compared the chronological progression of B. distachyon accessions Bd21 and Bd3-1, in addition to the progression of Chinese Spring wheat. The comparison of growth stages illustrates the morphological similarities and differences in the timing of life cycle events. Furthermore, we compared metabolite accumulation patterns across different growth stages and across different stress conditions using a widely targeted metabolome analysis. Metabolic profiling determined commonalities and specificities in chemical properties that were dependent on organisms, growth stages and/or stress conditions. Most metabolites accumulated equivalently in B. distachyon and wheat. This qualitative similarity indicated the superiority of B. distachyon as a model for Triticeae crops. The growth scale of B. distachyon should provide a conceptual framework for comparative analysis and for knowledge integration between this model grass and crops in the Pooideae subfamily., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147, a membrane-bound glycoprotein with two extracellular loop domains (termed loops I and II), progresses tumor invasion and metastasis by increasing the production of matrix metalloproteinase (MMP) in peritumoral stoma cells. EMMPRIN has also been associated with the control of migration activity in some tumor cells, but little is known about how EMMPRIN regulates tumor cell migration. In the present study, EMMPRIN siRNA suppressed the gene expression and production of EMMPRIN in human uterine cervical carcinoma SKG-II cells. An in vitro scratch wound assay showed enhancement of migration of EMMPRIN-knockdown SKG-II cells. In addition, the SKG-II cell migration was augmented by adding an E. coli-expressed human EMMPRIN mutant with two extracellular loop domains (eEMP-I/II), which bound to the cell surface of SKG-II cells. However, eEMP-I/II suppressed the native EMMPRIN-mediated augmentation of proMMP-1/procollagenase-1 production in a co-culture of the SKG-II cells and human uterine cervical fibroblasts, indicating that the augmentation of SKG-II cell migration resulted from the interference of native EMMPRIN functions by eEMP-I/II on the cell surface. Furthermore, a systematic peptide screening method using nine synthetic EMMPRIN peptides coding the loop I and II domains (termed EM1-9) revealed that EM9 (170HIENLNMEADPGQYR184) facilitated SKG-II cell migration. Moreover, SKG-II cell migration was enhanced by administration of an antibody against EM9, but not EM1 which is a crucial site for the MMP inducible activity of EMMPRIN. Therefore, these results provide novel evidence that EMMPRIN on the cell surface limits the cell migration of human uterine cervical carcinoma cells through 170HIENLNMEADPGQYR184 in the loop II domain. Finally, these results should provide an increased understanding of the functions of EMMPRIN in malignant cervical carcinoma cells, and could contribute to the development of clinical strategies for cervical cancer therapy.