Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.Molecular and Cellular Biolog

Molecular-scale surface structures of oligo(ethylene glycol)-terminated self-assembled monolayers investigated by frequency modulation atomic force microscopy in aqueous solution

The structure and protein resistance of oligo(ethylene glycol)-terminated self-assembled monolayers (OEG-SAMs) have been studied intensively using various techniques. However, their molecular-scale surface structures have not been well understood. In this study, we performed molecular-resolution imaging of OH-terminated SAMs (OH-SAMs) and hexa(ethylene glycol) SAMs (EG 6OH-SAMs) formed on a Au(111) surface in an aqueous solution by frequency modulation atomic force microscopy (FM-AFM). The results show that most of the ethylene glycol (EG) chains in an EG6OH-SAM are closely packed and well-ordered to present a molecularly flat surface even in an aqueous solution. In addition, we found that EG6OH-SAMs have nanoscale defects, where molecules take a disordered arrangement with their molecular axes parallel to the substrate surface. We also found that the domain size (50-200 nm) of an EG6OH-SAM is much larger than that of OH-SAMs (10-40 nm). These findings should significantly advance molecular-scale understanding about the surface structure of OEG-SAMs. © 2014 IOP Publishing Ltd

Preparation of Absorption-Resistant Hard Tissue Using Dental Pulp-Derived Cells and Honeycomb Tricalcium Phosphate

In recent years, there has been increasing interest in the treatment of bone defects using undifferentiated mesenchymal stem cells (MSCs) in vivo. Recently, dental pulp has been proposed as a promising source of pluripotent mesenchymal stem cells (MSCs), which can be used in various clinical applications. Dentin is the hard tissue that makes up teeth, and has the same composition and strength as bone. However, unlike bone, dentin is usually not remodeled under physiological conditions. Here, we generated odontoblast-like cells from mouse dental pulp stem cells and combined them with honeycomb tricalcium phosphate (TCP) with a 300 mu m hole to create bone-like tissue under the skin of mice. The bone-like hard tissue produced in this study was different from bone tissue, i.e., was not resorbed by osteoclasts and was less easily absorbed than the bone tissue. It has been suggested that hard tissue-forming cells induced from dental pulp do not have the ability to induce osteoclast differentiation. Therefore, the newly created bone-like hard tissue has high potential for absorption-resistant hard tissue repair and regeneration procedures

Geometrical Structure of Honeycomb TCP to Control Dental Pulp-Derived Cell Differentiation

Recently, dental pulp has been attracting attention as a promising source of multipotent mesenchymal stem cells (MSCs) for various clinical applications of regeneration fields. To date, we have succeeded in establishing rat dental pulp-derived cells showing the characteristics of odontoblasts under in vitro conditions. We named them Tooth matrix-forming, GFP rat-derived Cells (TGC). However, though TGC form massive dentin-like hard tissues under in vivo conditions, this does not lead to the induction of polar odontoblasts. Focusing on the importance of the geometrical structure of an artificial biomaterial to induce cell differentiation and hard tissue formation, we previously have succeeded in developing a new biomaterial, honeycomb tricalcium phosphate (TCP) scaffold with through-holes of various diameters. In this study, to induce polar odontoblasts, TGC were induced to form odontoblasts using honeycomb TCP that had various hole diameters (75, 300, and 500 mu m) as a scaffold. The results showed that honeycomb TCP with 300-mu m hole diameters (300TCP) differentiated TGC into polar odontoblasts that were DSP positive. Therefore, our study indicates that 300TCP is an appropriate artificial biomaterial for dentin regeneration

Significance of cancer stroma for bone destruction in oral squamous cell carcinoma using different cancer stroma subtypes

Stromal cells in the tumor microenvironment (TME) can regulate the progression of numerous types of cancer; however, the bone invasion of oral squamous cell carcinoma (OSCC) has been poorly investigated. In the present study, the effect of verrucous SCC‑associated stromal cells (VSCC‑SCs), SCC‑associated stromal cells (SCC‑SCs) and human dermal fibroblasts on bone resorption and the activation of HSC‑3 osteoclasts in vivo were examined by hematoxylin and eosin, AE1/3 (pan‑cytokeratin) and tartrate‑resistant acid phosphatase staining. In addition, the expression levels of matrix metalloproteinase (MMP)9, membrane‑type 1 MMP (MT1‑MMP), Snail, receptor activator of NF‑κB ligand (RANKL) and parathyroid hormone‑related peptide (PTHrP) in the bone invasion regions of HSC‑3 cells were examined by immunohistochemistry. The results suggested that both SCC‑SCs and VSCC‑SCs promoted bone resorption, the activation of osteoclasts, and the expression levels of MMP9, MT1‑MMP, Snail, RANKL and PTHrP. However, SCC‑SCs had a more prominent effect compared with VSCC‑SCs. Finally, microarray data were used to predict potential genes underlying the differential effects of VSCC‑SCs and SCC‑SCs on bone invasion in OSCC. The results revealed that IL1B, ICAM1, FOS, CXCL12, INS and NGF may underlie these differential effects. In conclusion, both VSCC‑SCs and SCC‑SCs may promote bone invasion in OSCC by enhancing the expression levels of RANKL in cancer and stromal cells mediated by PTHrP; however, SCC‑SCs had a more prominent effect. These findings may represent a potential regulatory mechanism underlying the bone invasion of OSCC

Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line‑derived xenograft model

The cancer stroma regulates bone invasion in oral squamous cell carcinoma (OSCC). However, data on normal stroma are limited. In the present study, the effects of gingival and periodontal ligament tissue‑derived stromal cells (G‑SCs and P‑SCs, respectively) and human dermal fibroblasts (HDFs) on bone resorption and osteoclast activation were assessed using hematoxylin and eosin and tartrate‑resistant acid phosphatase staining in a cell line‑derived xenograft model. The results demonstrated that G‑SCs promoted bone invasion and osteoclast activation and inhibited osteoclast proliferation following crosstalk with the human OSCC HSC‑3 cell line, whereas P‑SCs inhibited bone resorption and promoted osteoclast proliferation in vitro but had a minimal effect on osteoclast activation both in vitro and in vivo following crosstalk with HSC‑3 cells. Furthermore, the effects of G‑SCs, P‑SCs and HDFs on protein expression levels of matrix metalloproteinase (MMP)‑9, membrane type 1 MMP (MT1‑MMP), Snail, parathyroid hormone‑related peptide (PTHrP) and receptor activator of NF‑κB ligand (RANKL) in HSC‑3 cells in OSCC bone invasion regions were assessed using immunohistochemistry. The results demonstrated that G‑SCs had a more prominent effect on the expression of MMP‑9, MT1‑MMP, Snail, PTHrP, and RANKL, whereas P‑SCs only promoted RANKL and PTHrP expression and exerted a minimal effect on MMP‑9, MT1‑MMP and Snail expression. The potential genes underlying the differential effects of G‑SCs and P‑SCs on bone invasion in OSCC were evaluated using a microarray, which indicated that cyclin‑dependent kinase 1, insulin, aurora kinase A, cyclin B1 and DNA topoisomerase II alpha underlaid these differential effects. Therefore, these results demonstrated that G‑SCs promoted bone invasion in OSCC by activating osteoclasts on the bone surface, whereas P‑SCs exerted an inhibitory effect. These findings could indicate a potential regulatory mechanism for bone invasion in OSCC

Phosphatidylinositol-4,5-Biphosphate-Dependent Rearrangement of TRPV4 Cytosolic Tails Enables Channel Activation by Physiological Stimuli

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP$_2$), although the structural rearrangements occurring on PIP$_2$ binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP$_2$ binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence $^{121}$KRWRK$^{125}$, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP$_2$ from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP$_2$ facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP$_2$-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP$_2$ mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP$_2$ interacting site and conditions that deplete PIP$_2$ or restrict access of TRPV4 to PIP$_2$—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.Molecular and Cellular Biolog

A Study of Extracting Related Documents for Essay Evaluation Modules

本研究では，小論文採点システムにおいて必要となる小論文に関連した文書を取得する方法を開発した．本研究プロジェクトの自動採点の評価軸の1 つに「妥当性」がある．妥当性の評価手法として，小論文の内容がWikipediaの文書の内容と，どの程度一致しているかを基準に妥当性スコアを算出する方法を考えている．しかし，Wikipediaの文書は多様であり，小論文で取り上げていない議題に関する文書も多く存在する．そこで本論文では小論文ごとに適切な文書を取得する方法を提案する．いくつかの手法を試した結果，単語ベクトルを使用した方法が，関連した文書を獲得することができたことを報告する．We are developing an automatic Japanese essay-scoring system that is composed of 4 evaluation criteria, comprehensiveness, logical consistency, validity, spelling and grammar. In this paper, we discuss the most powerful approach to extract documents of Wikipedia that relates to the reference texts of the target essay theme for validity evaluation. The reason for using Wikipedia documents for evaluating validity of students’essays is that we assume that validity can be evaluated by the expanded discussions in Wikipedia documents that relates to the essay theme. Experimental results show that the skip-gram based word vector is the best approach to extract relating documents to reference texts among several keyword-based evaluation approaches

Proposing an Unsupervised Approach to Evaluate Essays Using IDF on Reference Data

大学入試において2020 年から記述式問題が導入されることから記述式の問題を自動で採点する手法の開発が求められている．本論では，エッセイタイプの小論文課題を対象に，課題に関連する参照データとWikipedia 全文から作成したidf を利用した事前採点不要な自動採点手法を提案する．先行研究において，日本語小論文を対象とした自動採点では，多くの事前採点が必要となり，実際の数百人規模の試験では利用することが難しいと考えられる．そこで本研究では，事前採点が不要な小論文採点手法を提案する．また，小論文の模擬試験を実施して小論文データを構築する．構築した小論文データに対して採点手法を用い，実験を行い評価する．また小論文データの人手による採点に対しても評価を行う．評価実験の結果neologd 辞書を利用した形態素解析器を用いて， idf 値を利用した形態素の一致数が，人手の評価値と相関が高いことを示す．In this paper, we describe an on-going study of developing an automatic essay-scoring system in Japanese. Essay scoring systems have already been developed and used mainly in English, while not many previous studies have been done on Japanese essay evaluations. Most of the methods and systems of automatic essay evaluation need not small number of previously human-graded essays for calibrating the parameter of regression functions or parameter of machine learning. The previous studies show the high performance for essay evaluation task, however, it must be not easy to assume large graded essays in, for example, actual tests or entrance examinations. Thus, we take a approach to evaluate Japanese essays without previously human-graded essays but with assuming reference data related to essay questions. The proposed method is a simple one, that is, evaluating the essays with co-occurrences with the reference data in their words or morphemes. In the method technical terms would be given high scores using neologd dictionary and idf values. Experimental results show that the proposed method works well in our developing Japanese mock trial writing tests. Key words automatic scoring of essays, human annotation, supportin

SOD3 Expression in Tumor Stroma Provides the Tumor Vessel Maturity in Oral Squamous Cell Carcinoma

Tumor angiogenesis is one of the hallmarks of solid tumor development. The progressive tumor cells produce the angiogenic factors and promote tumor angiogenesis. However, how the tumor stromal cells influence tumor vascularization is still unclear. In the present study, we evaluated the effects of oral squamous cell carcinoma (OSCC) stromal cells on tumor vascularization. The tumor stromal cells were isolated from two OSCC patients with different subtypes: low invasive verrucous squamous carcinoma (VSCC) and highly invasive squamous cell carcinoma (SCC) and co-xenografted with the human OSCC cell line (HSC-2) on nude mice. In comparison, the CD34+ vessels in HSC-2+VSCC were larger than in HSC-2+SCC. Interestingly, the vessels in the HSC-2+VSCC expressed vascular endothelial cadherin (VE-cadherin), indicating well-formed vascularization. Our microarray data revealed that the expression of extracellular superoxide dismutase, SOD3 mRNA is higher in VSCC stromal cells than in SCC stromal cells. Moreover, we observed that SOD3 colocalized with VE-cadherin on endothelial cells of low invasive stroma xenograft. These data suggested that SOD3 expression in stromal cells may potentially regulate tumor vascularization in OSCC. Thus, our study suggests the potential interest in SOD3-related vascular integrity for a better OSCC therapeutic strategy