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Previously we reported that a recombinant HSV-1 expressing murine IL-2 (HSV-IL-2) causes CNS demyelination in different strains of mice and in a T cell-dependent manner. Since TH17 cells have been implicated in CNS pathology, in the present study, we looked into the effects of IL-17A-/- and three of its receptors on HSV-IL-2-induced CNS demyelination. IL-17A-/- mice did not develop CNS demyelination, while IL-17RA-/-, IL-17RC-/-, IL-17RD-/- and IL-17RA-/-RC-/- mice developed CNS demyelination. Adoptive transfer of T cells from wild-type (WT) mice to IL-17A-/- mice or T cells from IL-17A-/- mice to Rag-/- mice induced CNS demyelination in infected mice. Adoptive T cell experiments suggest that both T cells and non-T cells expressing IL-17A contribute to HSV-IL-2-induced CNS demyelination with no difference in the severity of demyelination between the two groups of IL-17A producing cells. IL-6, IL-10, or TGFβ did not contribute to CNS demyelination in infected mice. Transcriptome analysis between IL-17A-/- brain and spinal cord of infected mice with and without T cell transfer from WT mice revealed that “neuron projection extension involved in neuron projection guidance” and “ensheathment of neurons” pathways were associated with CNS demyelination. Collectively, the results indicate the importance of IL-17A in CNS demyelination and the possible involvement of more than three of IL-17 receptors in CNS demyelination.

Hydrocephalus is a relatively common pediatric neurosurgical disease. Although congenital hydrocephalous is the most common cause, cerebral bleeding, infection of the central nervous system or brain tumors can also cause hydrocephalous in children. As timing of initial treatment varies depending on the cause of hydrocephalous, customized therapeutic strategy should be considered on a case-by-case basis to cope with growth and development. After the initiation of treatment such as shunt, most patients should be placed under clinical observation for life, as permanent devices which can adapt children's growth are not available, and shunt troubles such as occlusion, infection, disconnection, and inappropriate drainage are common during long term observation. Overall, ventriculoperitoneal shunt is a standard treatment. Other options such as ventriculoatrial or ventriculopleural shunt are selected to treat various shunt complications. In this article, we describe the characteristics of shunt complications in pediatric patients, as well as the symptoms, diagnosis, and treatments for various possible shunt complications. In addition, what kinds of attention should be paid for treating shunt trouble due to infection is discussed using a complicated case report.

Introduction In infants who have suffered head trauma there are two possible explanations for retinal hemorrhage (RH): direct vitreous shaking and occurrence in association with intracranial lesions. Which possibility is more plausible was examined. Material and methods This multicenter, retrospective study reviewed the clinical records of children younger than four years with head trauma who had been diagnosed with any findings on head computed tomography (CT) and/or magnetic resonance imaging (MRI). Of 452 cases, 239 underwent an ophthalmological examination and were included in this study. The relationships of RH with intracranial findings and the cause of injury were examined. Result Odds ratios for RH were significant for subdural hematoma (OR 23.41, p = 0.0004), brain edema (OR 5.46, p = 0.0095), nonaccidental (OR 11.26, p Conclusion Although nonaccidental, brain edema and self-inflicted falls were associated with RH, subdural hematoma was most strongly associated with RH.

Objective The medical history of injury given by parents of infants and toddlers with head trauma may not be accurate or completely true. The purpose of this study was to examine the relationship between subdural hematoma (SDH) due to nonaccidental injury and mechanisms of injury provided by caregivers. Methods Our multicenter study group retrospectively reviewed the clinical records of children younger than 4 years with head trauma who have been diagnosed with any finding on head computed tomography (CT) and/or magnetic resonance imaging (MRI). A total of 84 cases of subdural hematomas with retinal findings, including cases reported to the child guidance center and traffic and birth injuries, were included in the study. They were classified by the mechanism of injury provided by the caregivers. Clinical findings were reviewed and classified into nonaccidental and accidental groups. The mechanisms of the injuries were examined by multivariable analysis to identify which ones were statistically associated with nonaccidental injuries. Results Of the 84 patients with SDHs, 51 were classified into the nonaccidental group, and 33 children were classified into the accidental group. In 19 patients with a chief complaint of convulsion who had SDH but no episode of trauma, 18 were classified into the nonaccidental group. On multivariable analysis, unexplained convulsions (odds ratio: 12.04, 95% confidence interval: 1.44–100.49) were significantly associated with increased odds of nonaccidental injury. Conclusions In the present study, there was a relationship between nonaccidental injury and unexplained SDH with a chief complaint of convulsion.

We recently reported the role of type 2 innate lymphoid cells (ILC2s) in central nervous system (CNS) demyelination using a model of CNS demyelination involving recombinant herpes simplex virus 1 (HSV-1) that constitutively expresses mouse interleukin 2 (HSV-IL-2). In this investigation, we studied how ILC2s respond to HSV-IL-2 at the cellular level using cytokine and gene expression profiling. ILC2s infected with HSV-IL-2 expressed higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, IL-6, IL-13, IP-10, MIP-2, and RANTES, which include proinflammatory cytokines, than did those infected with parental control virus. In contrast, T(H)2 cytokines IL-4 and IL-9, which are typically expressed by ILC2s, were not induced upon HSV-IL-2 infection. Transcriptome sequencing (RNA-seq) analysis of HSV-IL-2 infected ILC2s showed significant upregulation of over 350 genes and downregulation of 157 genes compared with parental virus-infected ILC2s. Gene Ontology (GO) term analysis indicated that genes related to “mitosis” and “inflammatory response” were among the upregulated genes, suggesting that HSV-IL-2 infection drives the excessive proliferation and atypical inflammatory response of ILC2s. This change in ILC2 activation state could underlie the pathology of demyelinating diseases. IMPORTANCE Innate lymphocytes have plasticity and can change functionality; type 2 innate lymphoid cells (ILC2s) can convert to ILC1 or ILC3 cells or change their activation state to produce IL-17 or IL-10 depending on environmental cues. In this study, we investigated the gene and cytokine profiles of ILC2s, which play a major role in HSV-IL-2-induced CNS demyelination. ILC2s infected with HSV-IL-2 displayed a massive remodeling of cellular state. Additionally, ILC2s infected with HSV-IL-2 differed from those infected with parental HSV in cellular and viral gene expression profiles and in cytokine/chemokine induction, and they displayed enhanced activation and proinflammatory responses. These changes in ILC2 activation state could underlie the pathology of demyelinating diseases. These results also highlight the possible importance of pathogens as environmental cues to modify innate lymphocyte functionalities.

We recently reported the role of type 2 innate lymphoid cells (ILC2s) in central nervous system (CNS) demyelination using a model of CNS demyelination involving recombinant herpes simplex virus 1 (HSV-1) that constitutively expresses mouse interleukin 2 (HSV-IL-2). In this investigation, we studied how ILC2s respond to HSV-IL-2 at the cellular level using cytokine and gene expression profiling. ILC2s infected with HSV-IL-2 expressed higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, IL-6, IL-13, IP-10, MIP-2, and RANTES, which include proinflammatory cytokines, than did those infected with parental control virus. In contrast, TH2 cytokines IL-4 and IL-9, which are typically expressed by ILC2s, were not induced upon HSV-IL-2 infection. Transcriptome sequencing (RNA-seq) analysis of HSV-IL-2 infected ILC2s showed significant upregulation of over 350 genes and downregulation of 157 genes compared with parental virus-infected ILC2s. Gene Ontology (GO) term analysis indicated that genes related to "mitosis" and "inflammatory response" were among the upregulated genes, suggesting that HSV-IL-2 infection drives the excessive proliferation and atypical inflammatory response of ILC2s. This change in ILC2 activation state could underlie the pathology of demyelinating diseases.IMPORTANCE Innate lymphocytes have plasticity and can change functionality; type 2 innate lymphoid cells (ILC2s) can convert to ILC1 or ILC3 cells or change their activation state to produce IL-17 or IL-10 depending on environmental cues. In this study, we investigated the gene and cytokine profiles of ILC2s, which play a major role in HSV-IL-2-induced CNS demyelination. ILC2s infected with HSV-IL-2 displayed a massive remodeling of cellular state. Additionally, ILC2s infected with HSV-IL-2 differed from those infected with parental HSV in cellular and viral gene expression profiles and in cytokine/chemokine induction, and they displayed enhanced activation and proinflammatory responses. These changes in ILC2 activation state could underlie the pathology of demyelinating diseases. These results also highlight the possible importance of pathogens as environmental cues to modify innate lymphocyte functionalities.

Acetylcholine (ACh) promotes various cell migrations in vitro, but there are few investigations into this nonsynaptic role of ACh signaling in vivo. Here we investigate the function of a muscarinic receptor on an epithelial cell migration in Caenorhabditis elegans. We show that the migratory gonad leader cell, the linker cell (LC), uses an M1/M3/M5-like muscarinic ACh receptor GAR-3 to receive extrasynaptic ACh signaling from cholinergic neurons for its migration. Either the loss of the GAR-3 receptor in the LC or the inhibition of ACh release from cholinergic neurons resulted in migratory path defects. The overactivation of the GAR-3 muscarinic receptor caused the LC to reverse its orientation through its downstream effectors Gαq/egl-30, PLCβ/egl-8, and TRIO/unc-73. This reversal response only occurred in the fourth larval stage, which corresponds to the developmental time when the GAR-3::yellow fluorescent protein receptor in the membrane relocalizes from a uniform to an asymmetric distribution. These findings suggest a role for the GAR-3 muscarinic receptor in determining the direction of LC migration.

Background Aflatoxins (AFs) are carcinogenic mycotoxins. A simple, quick, and accurate method for the micro-analysis of AFs in foodstuffs, especially spices, is needed. Objective A sophisticated pretreatment method that combines solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization using immunoaffinity (IA) gel as the solid phase was developed to analyze AFs in spices simply, quickly, and sensitively by liquid chromatography with fluorescence detection. Method White and black pepper samples were extracted with a mixed solution of methanol/water (4:1) and then diluted with 7% aqueous solution of Triton-X. The solution was subjected to cleanup by SPDE using IA gel. Trifluoroacetic acid was added to the IA gel for on-site solid-phase fluorescence derivatization. Results Chromatograms containing well-separated peaks and few interference peaks from contaminants were obtained. The method detection limit of AFs in white and black pepper was 0.15–0.29 ng/g. Repeatability and intermediate precision were Conclusions The validity, reliability, practicality, and robustness of the developed method were verified. Highlights By using SPDE and solid-phase fluorescence derivatization in combination for AF analysis, fluorescence derivatization during cleanup was realized, leading to simplification of the pretreatment operation.

Objective Subdural hematoma in infants or toddlers has often been linked to abuse, but it is not clear how many cases actually occur and how many are suspected of abuse. The purpose of this study was to investigate subdural hematoma in infants and toddlers in Japan. Methods This multicenter, retrospective study reviewed the clinical records of children younger than 4 years with head trauma who were diagnosed with any finding on head computed tomography (CT) and/or magnetic resonance imaging (MRI), such as skull fracture and/or intracranial injury. A total of 452 children were included. The group suspected to have been abused was classified as nonaccidental, and the group considered to have been caused by an accident was classified as accidental. Subdural hematoma and other factors were examined on multivariate analysis to identify which factors increase the risk of nonaccidental injuries. Results Of the 452 patients, 158 were diagnosed with subdural hematoma. Subdural hematoma was the most common finding intracranial finding in head trauma in infants and toddlers. A total of 51 patients were classified into the nonaccidental group, and 107 patients were classified into the accidental group. The age of patients with subdural hematoma showed a bimodal pattern. The mean age of the accidental group with subdural hematoma was significantly older than that in the nonaccidental group (10.2 months vs 5.9 months, respectively. p < 0.001). Multivariate analysis showed that patients under 5 months old, retinal hemorrhage, and seizure were significant risk factors for nonaccidental injury (odds ratio (OR) 3.86, p = 0.0011; OR 7.63, p < 0.001; OR 2.49, p = 0.03; respectively). On the other hand, the odds ratio for subdural hematoma was 1.96, and no significant difference was observed (p = 0.34). Conclusions At least in Japanese children, infantile subdural hematoma was frequently observed not only in nonaccidental but also in accidental injuries. In infants with head trauma, age, the presence of retinal hemorrhage, and the presence of seizures should be considered when determining whether they were abused. Subdural hematoma is also a powerful finding to detect abuse, but care should be taken because, in some ethnic groups, such as the Japanese, there are many accidental cases.

Autism spectrum disorder (ASD) involves thousands of alleles in over 850 genes, but the current functional inference tools are not sufficient to predict phenotypic changes. As a result, the causal relationship of most of these genetic variants in the pathogenesis of ASD has not yet been demonstrated and an experimental method prioritizing missense alleles for further intensive analysis is crucial. For this purpose, we have designed a pipeline that uses Caenorhabditis elegans as a genetic model to screen for phenotype-changing missense alleles inferred from human ASD studies. We identified highly conserved human ASD-associated missense variants in their C. elegans orthologs, used a CRISPR/Cas9-mediated homology-directed knock-in strategy to generate missense mutants and analyzed their impact on behaviors and development via several broad-spectrum assays. All tested missense alleles were predicted to perturb protein function, but we found only 70% of them showed detectable phenotypic changes in morphology, locomotion or fecundity. Our findings indicate that certain missense variants in the C. elegans orthologs of human CACNA1D, CHD7, CHD8, CUL3, DLG4, GLRA2, NAA15, PTEN, SYNGAP1 and TPH2 impact neurodevelopment and movement functions, elevating these genes as candidates for future study into ASD. Our approach will help prioritize functionally important missense variants for detailed studies in vertebrate models and human cells.

Summary Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. Although viruses have been suggested to be a contributing environmental factor, conventional experimental MS mouse models do not account for this aspect. Here, we describe a mouse model to induce and evaluate demyelinating disease with both a viral and an immune component via ocular infection with a recombinant herpes simplex virus expressing murine interleukin-2. For complete details on the use and execution of this protocol, please refer to Hirose et al. (2020)., Graphical abstract, Highlights • We developed a model to induce demyelinating disease using HSV-1 expressing IL-2 • Recombinant HSV-1 incorporates environmental (HSV-1) and immune factors (IL-2) • We describe the steps to induce and evaluate demyelinating disease using our protocol, Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. Although viruses have been suggested to be a contributing environmental factor, conventional experimental MS mouse models do not account for this aspect. Here, we describe a mouse model to induce and evaluate demyelinating disease with both a viral and an immune component via ocular infection with a recombinant herpes simplex virus expressing murine interleukin-2.

Slit-ventricle syndrome (SVS) seems to encompass various pathophysiological abnormalities, including overdrainage of cerebrospinal fluid (CSF) and craniocerebral disproportion after extracranial CSF shunt placement. These pathologies result in small ventricle morphologically, and the ventricular catheter is obstructed by the collapsed ventricular walls. Patients with intermittent headaches, small ventricles on neuroimaging, and slow refill of the shunt reservoir are diagnosed with SVS. In this report, we present a case of SVS treated endoscopically. We detected bulges in a zigzag line on the ventricular walls according to side holes of the ventricular tube, and named them "steppingstone" phenomenon. It is a curious finding which directly shows that the intermittent obstruction of the ventricular tube occurs in patients with SVS. No previous articles have reported this phenomenon, and it indicates adequate treatment for SVS should be provided.

Lateral temporal encephalocele is an extremely rare clinical condition, with only 18 cases presented in the literature to date. No review articles have examined lateral temporal encephalocele in depth. We therefore reviewed past cases of lateral encephalocele to clarify the clinical characteristics of this extremely rare deformity. We also present a case of lateral encephalocele with arachnoid cyst which has never been reported in past reports. We identified 8 reports describing 18 cases of lateral temporal encephalocele. We therefore reviewed 19 cases of lateral temporal encephalocele, including our own experience, and discussed the clinical characteristics of this pathology. All the cases with lateral temporal encephalocele were detected at birth except for an occult case. The majority occurred at the pterion, and occurrence at the asterion appears much rarer. Due to the preference for the pterion, the ipsilateral orbital wall was also distorted in some cases. Lateral temporal encephalocele seems to have fewer associated malformations: only 3 cases of lateral temporal encephalocele had associated malformations, including our case which was associated with intracranial arachnoid cyst. The only case of lateral temporal encephalocele to have shown hydrocephalus was our own case. Patients with this deformity have relatively good prognoses: only 3 of the 19 cases showed delayed psychomotor development during follow-up. Provision of adequate treatment is likely to achieve a good prognosis in patients with lateral temporal encephalocele, so we should keep in mind this deformity when encountering pediatric patients with mass lesions on the temporal cranium.

We have developed an easy and specific enzyme-linked immunoassay (ELISA) for the simultaneous determination of serum metallothinein-1 (MT-1) and 2 (MT-2) in both humans and experimental animals. A competitive ELISA was established using a specific polyclonal antibody against rat MT-2. The antibody used for this ELISA had exhibited the same cross-reactivity with MT in humans and experimental animals. The NH2 terminal peptide of MT containing acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with the liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in an MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6ng/ml and the spiked MT-1was fully recovered from the plasma. We investigated the normal range of MT1/2 (25-75%tile) in 200 healthy human serum and found it to be 27-48ng/ml, and this was compared with the serum levels in various liver diseases. The serum MT1/2 levels in chronic hepatitis C (HCV) patients were significantly lower than healthy controls and also other liver diseases. In the chronic hepatitis cases, the MT1/I2 levels increased gradually, followed by the progression of the disease to liver cirrhosis and hepatocellular carcinoma. In particular, we found significantly elevated MT1/2 plasma levels in Wilson's disease patients, levels which were very similar to those in the Long-Evans Cinnamon (LEC) rat (model animal of Wilson's disease). Furthermore, a significantly elevated MT1/2 level was found in patients with Menkes disease, an inborn error of copper metabolism such as Wilson's disease.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

A sample preparation method was developed for determination of quinolones in honey using immunoaffinity resin. For this purpose, an immunoaffinity resin for quinolones was prepared by coupling a quinolone-specific monoclonal antibody to agarose resin. Honey samples diluted with phosphate buffer were reacted with immunoaffinity resin. After the resin was washed, quinolones were eluted with glycine-HCl. Quinolones in the eluate were determined by HPLC with fluorescence detection. No interfering peak was found on the chromatograms of honey samples. The recoveries of quinolones from samples were over 70% at fortification levels of 20 ng/g (for norfloxacin, ciprofloxacin and enrofloxacin) and 10 ng/g (for danofloxacin). The quantification limits of quinolones were 2 ng/g. This sample preprocessing method using immunoaffinity resin was found to be effective and suitable for determining residual quinolones in honey.

A simple and rapid assay method was developed for the detection of residual quinolones in contaminated milk. This assay uses specific anti-quinolones monoclonal antibody and can provide a simple, non-labour intensive method for on site detection.

Antibacterial reagents, especially quinolones, are widely used in animals and humans, and have caused serious problems to human health because of their residual contaminants in food. In order to screen for different kinds of newquinolones at the same time, a sensitive and specific enzyme-linked immunoassay (ELISA) has been developed. The anti-enrofloxacin monoclonal antibody was selected because of its ability to react with structurally related newquinolones in organic solvent. The antibody has 100% cross-reactivity with norfloxacin, ciprofloxacin and other newquinolones at 50% inhibition of control values IC50, but not with nitroflazone, sulphadimethoxine. The lowest detection limit of this ELISA was 0.7 ng/ml (ppb) when enrofloxacin was used as the calibrator. Eel extracts were spiked with enrofloxacin and the average recoveries at 10, 50, 100 ng/ml were 98, 102 and 91%, respectively. The proposed ELISA is a useful method for the practical microquantitation of various newquinolones in biologica...

This is a report based on a general survey given to undergraduate students and professors at Toyohashi University of Technology (TUT) about English education at TUT. Regarding students, the main purposes of the survey, conducted in the fall of 2005, were to find how students had studied English before enrolling in TUT, what the students thought of English teaching and learning at TUT, and how important they thought English was for their majors. As for the survey to professors, the main purposes were finding out instructor attitudes toward the teaching and learning of English at TUT and how necessary they felt the acquisition of English was for their students. In this study, attention is paid to the differences of awareness and attitudes between the professors and students toward English teaching and learning. Based on the results of this study, discussions on and refinements to the current English curriculum will be made in the near future at TUT with the aim of raising student English fluency, particularly in the areas of written and oral communication ability.

Distraction osteogenesis is a standard method for craniosynostosis. However, the technique using conventional devices still has some disadvantages, especially for anterior or posterior plagiocephaly with complex deformities. In the Nakajima’s angle-variable internal distraction (NAVID) system originally for maxillary surgeries, the cranial three-dimension (D) distractor with three dimensionally movable joint at the anterior arm has been developed recently in order to prevent the interference in the distraction process and excessive force. In this paper, we first reported two cases of anterior plagiocephaly, and one case of posterior plagiocephaly received distraction osteogenesis using new 3-D distractor in the NAVID system. In two cases of anterior plagiocephaly, the reshaping of supra-orbital bar in reference of simulating by the 3-D skull model was performed. In all cases, we fixed two paralleled 2-D distractors and a 3-D distractor in the upper frontal or parietal region. Within the limitations of this study, we believe that the NAVID system is suitable for infant plagiocephaly due to the simple and small joint arm. Furthermore, the usage of the 3-D distractor would reduce the interference with 2-D distractors and easily lead to attainment of targeted distracting distance.

SummaryMembrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

キノロン系抗菌剤は人や動物に対して治療を目的に幅広く使用されている．しかし，畜水産食品においてキノロン系抗菌剤の残留が数多く検出されていることから，薬物残留を評価するのに簡便且つ迅速に分析可能な市販ELISAキットによるスクリーニングが期待される．酵素免疫測定法（ELISA）法は多数の検体を一度に処理できるが，交差反応性に起因する同定能力等に課題を有する．そこで，ELISA法として開発されたNew Quinolone Kitの有用性を検証するために，高速液体クロマトグラフィー/蛍光検出法（HPLC/FL）を用いた高精度な機器分析法を構築し，比較検討した．HPLC/FLによる検出限界及び定量限界はエンロフロキサシンにおいて2 ng/g及び10 ng/gであった．エンロフロキサシン50 ng/gを添加したところ，回収率は107.8％ と良好な結果を得ることができた．ELISA法との相関性を検討するため，同一食肉を試料としてHPLC/FL及びELISA法をそれぞれ適用したところ，両者の値に相関性が認められた．HPLC/FLでは煩雑な前処理及び約120分の分析所要時間を必要とするため，ELISA法は1次スクリーニング法として有用であると考えられる．

Peroxisome proliferator-activated receptor gamma (PPARgamma) belongs to a superfamily of thyroid / steroid hormone receptors and regulates transcription of their target genes in a ligand-dependent manner. Recently, PPARgamma was reported to be expressed in several cell lines derived from breast, colon, stomach and lung cancers. Activation of PPARgamma by its ligand inhibits the growth of these tumor cells, suggesting that PPARgamma ligand is a potential anti-cancer agent in PPARgamma-expressing tumors. However, its expression in brain tumors has not been studied. We thus studied the expression in glioma samples with different pathological stages from 20 patients. It was demonstrated that 95% of the glioma tissue expressed PPARgamma mRNA. The results prompted us to study whether PPARgamma ligand affects the growth of cell lines derived from brain tumors. The receptor expression was studied in 9 cell lines either derived from malignant glioma or neuroblastoma. The expression was detected in a glioma cell line SK-MG-1 and in a neuroblastoma cell line NB-1. Addition of one of the PPARgamma ligands, troglitazone, induced growth inhibition in both cell lines. Further analyses revealed that this growth inhibition is caused by a PPARgamma-mediated induction of apoptosis. These results suggest that PPARgamma ligands could be a potential therapeutic agent for the treatment of the brain tumors expressing this receptor.

In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001, eLife digest In animals, cells can move from one place to another to shape tissues, heal wounds, or defend against invading microbes. A cell may move alone or it may be attached to others and move as part of a group. One member of the group leads this ‘collective migration’, but it is not known how the cells are able to stick to each other and move together. Collective migration takes place in the male gonad—the organ that makes sperm cells—in larvae of the nematode worm C. elegans. As the gonad matures, a group of cells form a simple chain that can move together. Kato et al. found that a protein called LINKIN must be present for this to happen. LINKIN is found in the membrane that surrounds animal cells. One section of the protein—called the β-propeller—sits on the outside surface of the membrane. The structure of the β-propeller is similar to a section of another protein—called α-integrin—that also allows cells to attach, suggesting LINKIN may work in a similar way. LINKIN is found in many animals, so Kato et al. searched for proteins that can interact with it in human cells. This search revealed three proteins that can interact with LINKIN and are required for the cells to move together. Two of the proteins control elements of the internal scaffolding of the cell: this scaffolding, which is known as the cytoskeleton, is involved in moving the cells. The experiments suggest that LINKIN coordinates the process of binding together with the changes in the cytoskeleton that are needed to allow the cells to move as one. The next challenge is to understand how LINKIN changes the internal program of the cells to achieve this. DOI: http://dx.doi.org/10.7554/eLife.04449.002

Desde que Moscú decidió albergar la cumbre APEC en Vladivostok, tanto decisores rusos como académicos han estado discutiendo sobre Rusia como una “potencia Euro-Pacífica”. Sin embargo, las prioridades regionales y bilaterales de Rusia en Asia, así como su estrategia para convertirse en un poder Pacífico siguen siendo inciertas. Con el aumento en importancia de un Océano Ártico libre de hielo, el área marítima de la región del Lejano Este gana en importancia para la armada rusa. Este artículo examina cómo el deshielo en el Océano Ártico podría influir la seguridad en torno a las Islas Kuriles.

This paper reports on a model surface that is inert in biological fluids and that is important for studies in biointerfacial science. A self-assembled monolayer (SAM) terminated in the mannitol group was found to prevent the adsorption of proteins and the attachment of cells. Surface plasmon resonance spectroscopy showed that the mannitol-terminated SAM prevented the adsorption of several proteins and was indistinguishable from a SAM presenting tri(ethylene glycol)groups. In a second set of experiments, monolayers were patterned into circular regions of hexadecanethiolate with the surrounding area terminated in the mannitol group in order to evaluate the inert surface for patterning cell attachment. 3T3 fibroblasts, attached to the circular regions, proliferated to occupy these regions completely and remained patterned on these regions for 25 days. The use of oligo(ethylene glycol)-terminated SAMs, by contrast, showed a loss in fidelity of the pattern after one week in culture. The mannitol-terminated mon...

An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-3 (MT-3) in experimental animals for the research of heavy metal and chemical toxicity has not been reported yet. Therefore, we have developed a competitive ELISA, using a specific monoclonal antibody raised against human recombinant MT-3 (rMT-3). The epitope mapping of the antibody was conducted using mouse, rat, and human MT-3s and peptide fragments of human MT-3. MT-1/2, MT-3 knock-out (KO) mice and human brain and liver were used for the evaluation of the ELISA. A pretreatment method of the tissue homogenates was also examined. The antibody used for the ELISA had the same cross-reactivity with MT-3 in humans and experimental animals. The human MT- 3 NH(2) terminal peptide (Fr. 1-17) was the demonstrated epitope of this antibody. The reactivity of this ELISA in brain homogenate of MT-3 KO mouse was significantly low compared with the wild type and MT-1/2 KO mice. The lowest detection limit of the ELISA was 10 ng/ml and over 80% of the spiked rMT-3 was recovered in the brain homogenate. The assay linearity was intact with a 5-fold dilution in the brain homogenate. The inter- and intra-assay CV was 6.5%, respectively. An effective pretreatment procedure of the tissue homogenate was also established for this MT-3 ELISA. In conclusion, this competitive ELISA is an easy and specific method for measuring the brain MT-3 level in experimental animals.

Here we report a low-cost and simple wide field-of-view (FOV) on-chip fluorescence-imaging platform, termed fluorescence Talbot microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly scalable FOV. The FTM prototype has a resolution of 1.2 μm and an FOV of 3.9 mm×3.5 mm. We demonstrate the imaging capability of FTM on fluorescently labeled breast cancer cells (SK-BR-3) and human embryonic kidney 293 (HEK) cells expressing green fluorescent protein.

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67 -RNAi–treated L4 larvae. We observed expression of 8,000–10,000 genes in the linker cell, 22–25% of which were up- or down-regulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67–dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans.

An intracellular polarity in activation of the Rac GTPase cooperates with an extracellular signal to ensure a robust pattern of stem cell division in Drosophila female germline stem cells., Background Drosophila female germline stem cells (GSCs) reside adjacent to a cellular niche that secretes Bone Morphogenetic Protein (BMP) ligands and anchors the GSCs through adherens junctions. The GSCs divide asymmetrically such that one daughter remains in the niche as a GSC, while the other is born away from the niche and differentiates. However, given that the BMP signal can be diffusible, it remains unclear how a local extracellular asymmetry is sufficient to result in a robust pattern of asymmetric division. Methods and Findings Here we show that GSCs are polarized with respect to the cellular niche. We first use a modified biosensor to demonstrate that the small GTPase Rac is asymmetrically activated within the GSC at the niche-GSC interface. Experiments using loss-of-function and gain-of-function mutations in Rac indicate that asymmetric Rac activity both localizes the microtubule binding protein Apc2 to orient one GSC centrosome at the niche-GSC interface during interphase and activates the Jun N-terminal kinase pathway to increase the ability of the GSC to respond to BMP ligands. Other processes act in concert with each function of Rac. Specifically, we demonstrate that the GSC cell cycle arrests at prometaphase if centrosomes are misoriented. Conclusions Thus, the GSCs, an adult stem cell present in a cellular niche, have a niche-associated polarity that couples control of the division plane with increased response to an extracellular maintenance signal. Other processes work in parallel with the Rac-mediated polarity to ensure a robust pattern of asymmetric division. We suggest that all adult stem cells likely employ multiple, independently acting mechanisms to ensure asymmetric division to maintain tissue homeostasis., Author Summary Adult stem cells are undifferentiated cells within an organism that undergo continual asymmetric division to produce two daughter cells that have different cell fates: one daughter remains a stem cell like its parent, while the other daughter differentiates. Thus the stock of stem cells is renewed while new cells are provided to ensure tissue maintenance. Often, an adult stem cell is present within a niche, a specific microenvironment that contains signals necessary to maintain the stem cell in an undifferentiated state. The plane of division of a niche-resident stem cell can be controlled so that one of its daughters arises outside of the niche. This daughter does not receive the maintenance signals and differentiates. However, because biological systems are inherently noisy, we postulated that there might be mechanisms that couple the control of the plane of stem cell division to the response to the extracellular maintenance signals to ensure a continued pattern of asymmetric division. In this study, we show that a well-characterized niche-resident stem cell, the Drosophila female germline stem cell (GSC), also has an intracellular polarity that contributes to the determination of daughter cell fate. We find that the small GTPase Rac is activated at the interface between the GSC and its niche and that this localized Rac activity has two functions: first, it orients the plane of GSC division to ensure that one GSC daughter is born outside the niche; and second, it increases the ability of the niche-resident GSC to respond to the maintenance signal. We propose that most stem cells integrate multiple mechanisms to ensure a robust pattern of asymmetric division.

Background An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-1 (MT-1) and 2 (MT-2) simultaneously in serum and other biological specimens in humans and experimental animals has not been developed yet. Methods We developed a competitive ELISA, a specific polyclonal antibody against rat MT-2. The epitope mapping of the antibody was conducted using MTs in mouse, rat, rabbit, human and the fragment peptides of human MT-2. MT1/2 and MT-3 knock-out mice and cadmium treated mice were used for the evaluation of the ELISA. Pretreatment method of serum was examined to deplete blocking factors for this assay. Results The antibody used for this ELISA had the same cross-reactivity with MT in humans and experimental animals. NH 2 terminal peptide of MT with acetylated methionine was proved to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the added MT-1 was fully recovered from serum. The mean MT concentration in our preliminary study was 23 ± 4.6 ng/ml in human serum. Cadmium treatment to mice induced significantly higher amount of MT in serum, liver, kidney and spleen as reported previously by different established methods. Conclusion The proposed competitive ELISA is an easy and specific method for practical use, determining total MT-1 and -2 simultaneously in serum and other biological specimens of human and experimental animals.

SignWriting is a practical writing system for sign languages. In this paper we present a software program, JSPad, for writing Japanese sign language (JSL) with SignWriting. SignWriting has a large set of visually iconic symbols that represent handshapes, movements, locations and facial expressions; it can take a lot of time to find and choose appropriate symbols from the set to compose signs, particularly for novice users of SignWriting. JSPad can generate SignWriting signs from JSL text written in the JJS notation, which is a gloss-based notation system we proposed. This facility allows users to write JSL sentences in SignWriting in shorter time.

Cell migration is a common event during organogenesis, yet little is known about how migration is temporally coordinated with organ development. We are investigating stage-specific programs of cell migration using the linker cell (LC), a migratory cell crucial for male gonadogenesis of C. elegans. During the L3 and L4 larval stages of wild-type males, the LC undergoes changes in its position along the migratory route, in transcriptional regulation of the unc-5 netrin receptor and zmp-1 zinc matrix metalloprotease, and in cell morphology. We have identified the tailless homolog nhr-67 as a cell-autonomous, stage-specific regulator of timing in LC migration programs. In nhr-67-deficient animals, each of the L3 and L4 stage changes is either severely delayed or never occurs, yet LC development before the early L3 stage or after the mid-L4 stage occurs with normal timing. We propose that there is a basal migration program utilized throughout LC migration that is modified by stage-specific regulators such as nhr-67.

The adhesion of cells is mediated by the binding of several cell-surface receptors to ligands found in the extracellular matrix. These receptors often have overlapping specificities for the peptide ligands, making it difficult to understand the roles for discrete receptors in cell adhesion, migration, and differentiation as well as to direct the selective adhesion of cell types in tissue-engineering applications. To overcome these limitations, we developed a strategy to rewire the receptor-ligand interactions between a cell and substrate to ensure that adhesion is mediated by a single receptor with unique specificity. The strategy combines a genetic approach to engineer the cell surface with a chimeric integrin receptor having a unique ligand binding domain with a surface chemistry approach to prepare substrates that present ligands that are bound by the new binding domain. We show that Chinese hamster ovary cells that are engineered with a chimeric beta1 integrin adhere, signal, and even migrate on a synthetic matrix.