Language：English   Publishing type：Research paper (scientific journal)  

Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.

DOI： 10.1038/s41467-019-14181-3

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2019.10.155

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Language：English   Publishing type：Research paper (scientific journal)  

PURPOSE: To present phenotypic features of 22 patients with S-antigen (SAG) mutations. DESIGN: Retrospective cohort study. PARTICIPANTS: Twenty-one Japanese patients from 16 families with a homozygous c.924delA mutation and 1 patient with a homozygous c.636delT mutation in the SAG gene. METHODS: Clinical records on symptoms; best-corrected visual acuity; and Goldmann perimetry, fundus photography, fundus autofluorescence (FAF), OCT, and electroretinography results were reviewed. MAIN OUTCOME MEASURES: Best-corrected visual acuity, Goldmann perimetry results, imaging findings, and electroretinography results. RESULTS: Ten patients had Oguchi disease and 12 had retinitis pigmentosa (RP) with mean follow-up periods of 13.8 and 10.2 years, respectively. Retinitis pigmentosa patients were older (mean age, 56.0 years) than those with Oguchi disease (mean age, 22.1 years; P < 0.001) at the initial visit. Night blindness noted in childhood was the most common initial symptom for both Oguchi disease (80.0%) and RP (91.7%) patients. Best-corrected visual acuity in the logarithm of the minimum angle of resolution (logMAR) was well preserved in Oguchi disease patients (mean, 0.02 logMAR in both eyes) but reduced in most RP patients (mean, 1.32 logMAR [right eye] and 1.35 logMAR [left eye]). Similarly, the visual field in the retinal area was preserved in Oguchi disease patients (mean, 677 mm2 right eye and 667 mm2 left eye) and reduced in RP patients (mean, 369 mm2 right eye and 294 mm2 left eye). Fundus images revealed a characteristic golden sheen with no retinal degeneration in Oguchi disease patients, excluding 2 with macular degeneration detected by FAF, OCT, or both and 1 with mild retinal degeneration confirmed by OCT and fluorescein angiography. Pigmentary retinal degeneration most evident posteriorly was observed in RP patients, accompanied by a characteristic golden sheen in 12 of 14 patients undergoing ultra-widefield fundus imaging. OCT showed disrupted macular structure, and FAF revealed variable hypofluorescence. Electroretinography identified absent rod responses in both diseases, along with relative preservation of cone responses in Oguchi disease patients. Three patients showed progressive loss of the golden sheen based on fundus images, including 1 who demonstrated RP 26 years after the initial diagnosis of Oguchi disease. CONCLUSIONS: Retinitis pigmentosa with SAG mutations often shows a characteristic golden sheen surrounding posterior pigmentary retinal degeneration. Oguchi disease can show progressive degeneration in adulthood, rarely resulting in RP.

DOI： 10.1016/j.ophtha.2019.05.027

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Animal behavior is the final and integrated output of brain activity. Thus, recording and analyzing behavior is critical to understand the underlying brain function. While recording animal behavior has become easier than ever with the development of compact and inexpensive devices, detailed behavioral data analysis requires sufficient prior knowledge and/or high content data such as video images of animal postures, which makes it difficult for most of the animal behavioral data to be efficiently analyzed. Here, we report a versatile method using a hybrid supervised/unsupervised machine learning approach for behavioral state estimation and feature extraction (STEFTR) only from low-content animal trajectory data. To demonstrate the effectiveness of the proposed method, we analyzed trajectory data of worms, fruit flies, rats, and bats in the laboratories, and penguins and flying seabirds in the wild, which were recorded with various methods and span a wide range of spatiotemporal scales-from mm to 1,000 km in space and from sub-seconds to days in time. We successfully estimated several states during behavior and comprehensively extracted characteristic features from a behavioral state and/or a specific experimental condition. Physiological and genetic experiments in worms revealed that the extracted behavioral features reflected specific neural or gene activities. Thus, our method provides a versatile and unbiased way to extract behavioral features from simple trajectory data to understand brain function.

DOI： 10.3389/fnins.2019.00626

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DOI： 10.1111/ceo.13365

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DOI： 10.1111/ceo.13341

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-018-34413-8

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ymthe.2018.07.012

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DOI： 10.1167/iovs.18-23784

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/ceo.13099

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Language：English   Publishing type：Research paper (scientific journal)  

PURPOSE: The purpose of the study is to investigate the correlation between intraocular anti-retinal antibodies and clinical measurements in patients with rhegmatogenous retinal detachment (RRD) and proliferative vitreoretinopathy (PVR). MATERIAL AND METHODS: Aqueous humor and vitreous samples were collected from patients with RRD, PVR, and from control subjects with macular hole. The levels of total protein (TP), IgG, and anti-retinal antibodies were determined with a bicinchoninic acid assay, enzyme-linked immunosorbent assay, and dot blot, respectively. Correlations between these measurements were assessed using Pearson's correlation test. Analysis of variance followed by a post-hoc test or the Student t-test was used to compare differences between groups. RESULTS: The levels of anti-retinal antibodies and IgG were correlated with each other (P < 0.010). The IgG concentration was higher in patients with PVR than in controls in both the aqueous humor (P < 0.001) and the vitreous (P < 0.001), but not in patients with RRD. Conversely, TP levels and anti-retinal antibodies in both ocular fluids from RRD and PVR patients did not significantly differ from the controls. In a subgroup analysis, vitreal anti-retinal antibody levels were correlated with average macular thickness in the re-attached macula following surgery for macula-off RRD/PVR (P = 0.012). Furthermore, patients with post-operative cystoid macular edema had a higher level of vitreal anti-retinal antibodies than those without (P = 0.009). CONCLUSIONS: Intravitreal anti-retinal antibodies were increased in the eyes with maculopathy after surgical intervention for RRD/PVR.

DOI： 10.1080/02713683.2018.1451544

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

We found that hesperidin, a plant-derived bioflavonoid, may be a candidate agent for neuroprotective treatment in the retina, after screening 41 materials for anti-oxidative properties in a primary retinal cell culture under oxidative stress. We found that the intravitreal injection of hesperidin in mice prevented reductions in markers of the retinal ganglion cells (RGCs) and RGC death after N-methyl-D-aspartate (NMDA)-induced excitotoxicity. Hesperidin treatment also reduced calpain activation, reactive oxygen species generation and TNF-α gene expression. Finally, hesperidin treatment improved electrophysiological function, measured with visual evoked potential, and visual function, measured with optomotry. Thus, we found that hesperidin suppressed a number of cytotoxic factors associated with NMDA-induced cell death signaling, such as oxidative stress, over-activation of calpain, and inflammation, thereby protecting the RGCs in mice. Therefore, hesperidin may have potential as a therapeutic supplement for protecting the retina against the damage associated with excitotoxic injury, such as occurs in glaucoma and diabetic retinopathy.

DOI： 10.1038/s41598-017-06969-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Retinal ganglion cell degeneration triggered by axonal injury is believed to underlie many ocular diseases, including glaucoma and optic neuritis. In these diseases, retinal ganglion cells are affected unevenly, both spatially and temporally, such that healthy and unhealthy cells coexist in different patterns at different time points. Herein, we describe a temporally and spatially regulated adeno-associated virus gene therapy aiming to reduce undesired off-target effects on healthy retinal neurons. The Mcp-1 promoter previously shown to be activated in stressed retinal ganglion cells following murine optic nerve injury was combined with the neuroprotective intracellular transcription factor Nrf2. In this model, Mcp-1 promoter driven NRF2 expression targeting only stressed retinal ganglion cells showed efficacy equivalent to non-selective cytomegalovirus promoter-driven therapy for preventing cell death. However, cytomegalovirus promoter-mediated NRF2 transcription induced cellular stress responses and death of Brn3A-positive uninjured retinal ganglion cells. Such undesired effects were reduced substantially by adopting the Mcp-1 promoter. Combining a stress-responsive promoter and intracellular therapeutic gene is a versatile approach for specifically targeting cells at risk of degeneration. This strategy may be applicable to numerous chronic ocular and non-ocular conditions.

DOI： 10.1016/j.omtm.2017.04.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELIFE SCIENCES PUBLICATIONS LTD  

Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans. We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca2+](i)), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca2+](i) rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making.

DOI： 10.7554/eLife.21629

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Latanoprost was first developed for use in glaucoma therapy as an ocular hypotensive agent targeting the prostaglandin F2 alpha (FP) receptor. Subsequently, latanoprost showed a neuroprotective effect, an additional pharmacological action. However, although it is well-known that latanoprost exerts an ocular hypotensive effect via the FP receptor, it is not known whether this is also true of its neuroprotective effect. Klotho was firstly identified as the gene linked to the suppression of aging phenotype: the defect of klotho gene in mice results aging phenotype such as hypokinesis, arteriosclerosis, and short lifespan. After that, the function of klotho was also reported to maintain calcium homeostasis and to exert a neuroprotective effect in various models of neurodegenerative disease. However, the function of klotho in eyes including retina is still poorly understood. Here, we show that klotho is a key factor underlying the neuroprotective effect of latanoprost during post-axotomy retinal ganglion cell (RGC) degeneration. Importantly, a quantitative RT-PCR gene expression analysis of klotho in sorted rat retinal cells revealed that the highest expression level of klotho in the retina was in the RGCs. Latanoprost acid, the biologically active form of latanoprost, inhibits post-traumatic calpain activation and concomitantly facilitates the expression and shedding of klotho in axotomized RGCs. This expression profile is a good match with the localization, not of the FP receptor, but of organic anion transporting polypeptide 2B1, known as a prostaglandin transporter, in the ocular tissue. Furthermore, an organic anion transporting polypeptide 2B1 inhibitor suppressed latanoprost acid-mediated klotho shedding exvivo, whereas an FP receptor antagonist did not. The klotho fragments shed from the RGCs reduced the intracellular level of reactive oxygen species, and a specific klotho inhibitor accelerated and increased RGC death after axotomy. We conclude that the shed klotho fragments might contribute to the attenuation of axonal injury-induced calpain activation and oxidative stress, thereby protecting RGCs from post-traumatic neuronal degeneration.

DOI： 10.1111/jnc.13902

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

Purpose To test the genetic association between Japanese patients with primary open-angle glaucoma (POAG) and the previously reported POAG susceptibility loci and to perform genotype–phenotype analysis. Methods Genetic associations for 27 SNPs from 16 loci previously linked to POAG were assessed using genome-wide SNP data of the primary cohort (565 Japanese POAG patients and 1,104 controls). Reproducibility of the assessment was tested in 607 POAG cases and 455 controls (second cohort) with a targeted genotyping approach. For POAG-associated variants, a genotype–phenotype correlation study (additive, dominant, recessive model) was performed using the objective clinical data derived from 598 eyes of 598 POAG patients. Results Among 27 SNPs from 16 loci previously linked to POAG, genotypes for total of 20 SNPs in 13 loci were available for targeted association study. Among 8 SNPs in 3 loci that showed at least nominal association (P &lt
5.00E-02) in the primary cohort, a representative SNP for each loci (rs2157719 for CDKN2B-AS1, rs33912345 for SIX6, and rs9913911 for GAS7) were selected. For these SNPs the association was found significant in both the second cohort analysis and meta-analysis. The genotype–phenotype analysis revealed significant correlations between CDKN2B-AS1 (rs2157719) and decreased intraocular pressure (? = -6.89 mmHg, P = 1.70E-04
dominant model) after multiple corrections. In addition, nominal correlation was observed between CDKN2B-AS1 (rs2157719) and optic nerve head blood flow (? = -0.54 and -0.67 arbitrary units (AU), P = 2.00E-02 and 1.39E-02), between SIX6 (rs33912345) and decreased total peripapillary retinal nerve fiber layer thickness (? = -2.16 and -2.82 ?m, P = 4.68E-02 and 2.40E-02, additive and recessive model, respectively) and increased optic nerve head blood flow (? = 0.44 AU, P = 2.20E-02
additive model) and between GAS7 (rs9913911) and increased cup volume (? = 0.03 mm3, P = 4.60E-02) and mean cup depth (? = 0.03 mm3, P = 4.11E-02
additive model) and decreased pattern standard deviation (? = -0.87 dB, P = 2.44E-02
dominant model). Conclusion The association between SNPs near GAS7 and POAG was found in Japanese patients for the first time. Clinical characterization of the risk variants is an important step toward understanding the pathology of the disease and optimizing treatment of patients with POAG.

DOI： 10.1371/journal.pone.0186678

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Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

Background: Recently, topical dexamethasone-induced ocular hypertension and a consequent loss of retinal ganglion cells (RGCs) have been described in mice. This has been proposed as a model of steroid-induced glaucoma. In this study, we set up and evaluated a similar model in rats. Results: Ten-week old Sprague Dawley (SD) rats (N = 12) were used to evaluate the effect of topical 0.1 % dexamethasone (50 μl) administered 3 times daily for 4 weeks. Sodium chloride (0.9 %) was used in another group of rats (N = 12) that served as the controls. After 1 week, we observed a progressive decrease in body weight in the dexamethasone-treated rats compared both to the pre-treatment baseline and the vehicle-treated rats. In contrast to earlier work that showed elevated Intraocular pressure (IOP) following dexamethasone instillation in mice, IOP in the rats unexpectedly fell to 11.3 ± 1.3 mmHg in the treated eyes, compared to 14.8 ± 2.4 mmHg in the untreated eyes, after 3 weeks of topical dexamethasone (P = 0.032). Blood tests performed after 4 weeks of treatment showed a 3.3-fold increase in both plasma cholesterol (P &lt
0.001) and alanine transaminase (P = 0.019) in the dexamethasone-treated rats compared to the control rats. Meanwhile, topical steroid did not induce changes in either plasma blood glucose or glycated hemoglobin (HbA1c). We also did not detect changes in the expression of RGC markers (with real-time PCR) following the treatment. Conclusions: In contrast to mice, which previously showed increased IOP following the topical administration of dexamethasone, the rats displayed a paradoxical reduction in IOP following a similar treatment. This was accompanied by a loss of body weight without affecting the level of blood glucose.

DOI： 10.1186/s12952-016-0048-x

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Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The development of new treatments for intractable retinal diseases requires reliable functional assessment tools for animal models. In vivo measurements of neural activity within visual pathways, including electroretinogram (ERG) and visually evoked potential (VEP) recordings, are commonly used for such purposes. In mice, the ERG and VEPs are usually recorded under general anesthesia, a state that may alter sensory transduction and neurotransmission, but seldom in awake freely moving mice. Therefore, it remains unknown whether the electrophysiological assessment of anesthetized mice accurately reflects the physiological function of the visual pathway. Herein, we describe a novel method to record the ERG and VEPs simultaneously in freely moving mice by immobilizing the head using a custom-built restraining device and placing a rotatable cylinder underneath to allow free running or walking during recording. Injection of the commonly used anesthetic mixture xylazine plus ketamine increased and delayed ERG oscillatory potentials by an average of 67.5% and 36.3%, respectively, compared to unanesthetized mice, while having minimal effects on the a-wave and b-wave. Similarly, components of the VEP were enhanced and delayed by up to 300.2% and 39.3%, respectively, in anesthetized mice. Our method for electrophysiological recording in conscious mice is a sensitive and robust means to assess visual function. It uses a conventional electrophysiological recording system and a simple platform that can be built in any laboratory at low cost. Measurements using this method provide objective indices of mouse visual function with high precision and stability, unaffected by anesthetics.

DOI： 10.1371/journal.pone.0156927

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Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Axial spondylometaphyseal dysplasia (axial SMD) is an autosomal recessive disease characterized by dysplasia of axial skeleton and retinal dystrophy. We conducted whole exome sequencing and identified C21orf2 (chromosome 21 open reading frame 2) as a disease gene for axial SMD. C21orf2 mutations have been recently found to cause isolated retinal degeneration and Jeune syndrome. We found a total of five biallelic C21orf2 mutations in six families out of nine: three missense and two splicing mutations in patients with various ethnic backgrounds. The pathogenic effects of the splicing (splice-site and branch-point) mutations were confirmed on RNA level, which showed complex patterns of abnormal splicing. C21orf2 mutations presented with a wide range of skeletal phenotypes, including cupped and flared anterior ends of ribs, lacy ilia and metaphyseal dysplasia of proximal femora. Analysis of patients without C21orf2 mutation indicated genetic heterogeneity of axial SMD. Functional data in chondrocyte suggest C21orf2 is implicated in cartilage differentiation. C21orf2 protein was localized to the connecting cilium of the cone and rod photoreceptors, confirming its significance in retinal function. Our study indicates that axial SMD is a member of a unique group of ciliopathy affecting skeleton and retina.

DOI： 10.1371/journal.pone.0150555

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Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Age-dependent formation of macular drusen caused by the focal accumulation of extracellular deposits beneath the retinal pigment epithelium precede the development of age related macular degeneration (AMD), one of the leading causes of blindness worldwide. It is established that inflammation contributes to the pathogenesis of drusen and AMD. However, development of a preemptive therapeutic strategy targeting macular drusen and AMD has been impeded by the lack of relevant animal models because most laboratory animals lack macula, an anatomic feature present only in humans and a subset of monkeys. Reportedly, macular drusen and macular degeneration develop in monkeys in an age-dependent manner. In this study, we analyzed blood test results from 945 Macaca fascicularis, 317 with and 628 without drusen. First, a trend test for drusen frequency (the Cochran-Armitage test) was applied to the quartile data for each parameter. We selected variables with an increasing or decreasing trend with higher quartiles at P &lt; 0.05, to which multivariate logistic regression analysis was applied. This revealed a positive association of age (odds ratio [OR]: 1.10 per year, 95% confidence interval [CI]: 1.07-1.12) and white blood cell count (OR: 1.01 per 1 x 10(3)/mu l, 95% CI: 1.00-1.01) with drusen. When the monkeys were divided by age, the association between drusen and white blood cell count was only evident in younger monkeys (OR: 1.01 per 1 x 10(3)/mu l, 95% CI: 1.00-1.02). In conclusion, age and white blood cell count may be associated with drusen development in M. fascicularis. Systemic inflammation may contribute to drusen formation in monkeys.

DOI： 10.1371/journal.pone.0164899

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Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Glaucoma, a leading cause of blindness worldwide, is instigated by various factors, including axonal injury, which eventually leads to a progressive loss of retinal ganglion cells (RGCs). To study various pathways reportedly involved in the pathogenesis of RGC death caused by axonal injury, seven pathways were investigated. Pathway-specific fluorescent protein-coded reporters were each packaged into an adeno-associated virus (AAV). After producing axonal injury in the eye, injected with AAV to induce RGC death, the temporal activity of each stress-related pathway was monitored in vivo through the detection of fluorescent RGCs using confocal ophthalmoscopy. We identified the activation of ATF6 and MCP-1 pathways involved in endoplasmic reticulum stress and macrophage recruitment, respectively, as early markers of RGC stress that precede neuronal death. Conversely, inflammatory responses probed by NF-kappa B and cell-death-related pathway p53 were most prominent in the later phases, when RGC death was already ongoing. AAV-mediated delivery of stress/response reporters followed by in vivo cellular imaging is a powerful strategy to characterize the temporal aspects of complex molecular pathways involved in retinal diseases. The identification of promoter elements that are activated before the death of RGCs enables the development of pre-emptive gene therapy, exclusively targeting the early phases of diseased cells.

DOI： 10.1038/srep18141

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Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

An animal's behavior is modulated by learning; however, the behavioral component modulated by learning and the mechanisms of this modulation have not been fully understood. We show here that two types of neural signalings are required for the modulation of different behavioral components in non-associative odor learning in the nematode Caenorhabditis elegans. We have previously found that C. elegans avoid the repulsive odor 2-nonanone, and preexposure to the odor for 1 h enhances the avoidance behavior as a type of non-associative learning. Systematic quantitative analyses of behavioral components revealed that the odor preexposure caused increases in average duration of straight migration ("runs") only when the animals were migrating away from the odor source within a certain range of bearing, which likely corresponds to odor decrement. Further, genetic analyses revealed that the genes for neuropeptide or dopamine signalings are both required for the enhanced odor avoidance. Neuropeptide signaling genes were required for the preexposure-dependent increase in run duration. In contrast, dopamine signaling genes were required not for the increase in run duration but likely for maintenance of run direction. Our results suggests that multiple behavioral components are regulated by different neuromodulators even in non-associative learning in C. elegans. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2015.05.009

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Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The Ets family protein BmEts is assumed to be implicated in determination of diapause in the embryogenesis of Bombyx mori. In this study, we found that expression of BmEts was increased in the fat body and other tissues of the 5th instar larvae in response to Escherichia coli injection. Cotransfection experiments using a silkworm cell line revealed that overexpression of BmEts significantly elevated the activity of lebocin promoter but not of cecropin B1, cecropin D. attacin, and moricin promoters. Activation of the lebocin promoter by BmEts was dependent on at least two kappa B elements and the most proximal GGAA/T motif located on the 5'-upstream region. BmEts further synergistically enhanced E. coli or BmRelish1-d2 (active form)-stimulated lebocin promoter activation. Two kappa B elements were also found to be involved in promoter activation by BmRelish1-d2 and in synergistic promoter activation by BmEts and BmRelish1-d2 in the silkworm cells. Specific binding of recombinant BmEts to the proximal kappa B element and the most proximal GGAA/T motif and interaction between BmEts and BmRelish1 were also observed. To our knowledge, this is the first report of an Ets family protein directly regulating immune-related genes in invertebrates. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ibmb.2012.03.004

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Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.

DOI： 10.1007/s12033-011-9408-4

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Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

The enhancement of sensory responses after prior exposure to a stimulus is a fundamental mechanism of neural function in animals. Its molecular basis, however, has not been studied in as much depth as the reduction of sensory responses, such as adaptation or habituation. We report here that the avoidance behavior of the nematode Caenorhabditis elegans in response to repellent odors (2-nonanone or 1-octanol) is enhanced rather than reduced after preexposure to the odors. This enhancement effect of preexposure was maintained for at least 1 h after the conditioning. The enhancement of 2-nonanone avoidance was not dependent on the presence or absence of food during conditioning, which generally functions as a strong positive or negative unconditioned stimulus in the animals. These results suggest that the enhancement is acquired as a type of nonassociative learning. In addition, genetic and pharmacological analyses revealed that the enhancement of 2-nonanone avoidance requires dopamine signaling via D2-like dopamine receptor DOP-3, which functions in a pair of RIC interneurons to regulate the enhancement. Because dopamine signaling has been tightly linked with food-related information to modulate various behaviors of C. elegans, it may play different role in the regulation of the enhancement of 2-nonanone avoidance. Thus, our data suggest a new genetic and pharmacological paradigm for nonassociative enhancement of neural responses that is regulated by dopamine signaling.

DOI： 10.1523/JNEUROSCI.6023-09.2010

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Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

In this study, we have newly identified three bacteria-induced genes from the silkworm Bombyx mori by quantitative reverse transcriptase-polymerase chain reaction. One of these, eukaryotic initiation factor 4E-1 (eIF4E-1), is assumed to encode an eIF4E family, which plays a role in the initiation of translation as a mRNA cap-binding protein. The second gene is BmFOXG1, belonging to a family of forkhead transcription factors, FOXG1. The third gene is MBF2-related (MBF2-R) whose product has high homology to a co-activator protein MBF2 from B. mori. Although BmFOXG1 was up-regulated in the fat body in response to three kinds of bacteria, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, eIF4E-1 and MBF2-R were up-regulated by E. coli and B. subtilis, but not S. aureus, suggesting that bacteria possessing meso-diaminopimelic acid-containing peptidoglycan but not lysine-containing peptidoglycan activate eIF4E-1 and MBF2-R, probably through a conserved immune deficiency pathway. We further profiled the expression of three genes in different tissues and a silkworm cell line, NIAS-Bm-aff3, in response to bacteria, and at different times after bacterial challenge in the fat body. (C) 2010 Wiley Periodicals, Inc.

DOI： 10.1002/arch.20347

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Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The global transcriptional profile of host genes in the silkworm cell line during the early phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection was analyzed by oligonucleotide microarray. Our analysis showed 35 genes were significantly up-regulated and 17 genes were significantly down-regulated. This is the first report of changes in the expression of these genes in response to NPV infection. We further quantified the levels of mRNA expression by quantitative reverse transcriptase-polymerase chain reaction and confirmed that the expression of 13 (such as BmEts and BmToll10-3) genes significantly increased and 7 genes (such as Hsp20-1) significantly decreased after BmNPV infection. However, the expression levels of most genes were not dramatically changed except BmEts expression increased approximately 8.0-fold 12 h after BmNPV infection. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2009.10.015

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Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.

DOI： 10.1271/bbb.90223

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PubMed

Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

In the silkworm, Bombyx mori, antimicrobial peptide (AMP) genes are upregulated in the larval fat body by injection of bacteria and peptidoglycans (PGNs). The DAP-type PGN from Escherichia coli and Bacillus subtilis exhibited stronger elicitor activity for expression of AMP genes in B. mori than Lys-type PGN from Staphylococcus aureus, suggesting that differences in bacterial influence on the induction levels of these genes depend on the differences in types of PGN. BmRelish1 mRNA was more abundant than BmRel mRNAs in the larval fat bodies. Moreover, the ability of the BmRelish1 active form to enhance the promoter activity of AMP genes was higher than that of BmRels. The difference was related to the binding affinity of Rel family proteins to kappa B sites. Our results suggest that different amounts and different transcriptional activities of Rel family proteins result in differential activation of AMP genes by PGN type and bacterium species.

DOI： 10.1271/bbb.80685

Web of Science

PubMed

Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.

DOI： 10.1007/s12033-008-9108-x

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, was found to be unable to activate immune-related genes in Drosophila melanogaster. In contrast, highly purified LPS elicited immune-related gene expression in the fat body of Bombyx mori. However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune-related genes by LPS. Up-regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune-responsive cell line, NIAS-Bm-aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.

DOI： 10.1111/j.1365-2583.2009.00851.x

Web of Science

PubMed

Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mod. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mod has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ibmb.2008.09.001

Web of Science

PubMed

Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Two proteins (APAP I and II) of the glycoside hydrolase family 48 (Family GH48) were isolated from the active adults of the leaf beetle Gastrophysa atrocyanea. Full-length and cDNAs were sequenced. APAP I expression and function were examined in detail. The protein has a chitinase but not a glucanase and cellobiohydrolase activity. It is expressed in the feeding stages, including beetles whose diapause was terminated with a juvenile hormone agonist. Suppression of the APAP I expression by means of RNA interference prevented the hormonal termination of diapause. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.04.126

Web of Science

PubMed

Language：English   Publisher：The Japanese Society of Sericultural Science  

Small heat shock proteins (sHsps), which are constitutively expressed in the absence of stress during development in many organisms, have been correlated with not only enhanced thermoresistance but also the developmental, tissue-, and cell-specific induction and expression. This study demonstrates the constitutive sHsps 21 and 23 of an entomoresouce, the leaf beetle, <i>Gastrophysa atrocyanea</i>, display a double chaperone function <i>in vitro</i>. The RNAi suppression of two sHsp genes in adults resulted in decreased thermoresistance. A strong correlation was observed between the <i>in vitro</i> chaperone function and <i>in vivo</i> thermotolerance analysis, and the results support a possible double chaperone function critical for the survival of the leaf beetles against higher temperatures.<br>

DOI： 10.11416/jibs.75.15

Language：English   Publisher：Japanese Society of Applied Entomology and Zoology  

The leaf beetle, Gastrophysa atrocyanea Motschulsky (Coleoptera : Chrysomelidae) has been regarded as a bioagent suitable for the extermination of certain weeds, especially Rumex obtusifolius. We examined conditions for artificial mass rearing of this insect. An artificial diet based on the host-plant leaf powder and supplemented with feeding stimulants, was developed for laboratory rearing of the leaf beetles. In particular, the hatchability of eggs laid by the fourth generation and female longevity were equivalent to those for fresh host leaves. Consequently, the fifth generation was achieved by rearing on artificial diet.

DOI： 10.1303/aez.2005.119

CiNii Books

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.1785

Web of Science

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：日本蚕糸学会  

CiNii Books