Evaluation of factors related to Anaesthesia-induced Lens opacity in experimental mice.

To investigate conditions that cause temporal lens opacity, we tested chemical and physical factors, such as anaesthesia dose, ocular surface dryness, and infrared (IR) light exposure in anaesthetised C57BL/6 N mice. Mice were anaesthetised with a low (80%; tiletamine/zolazepam 32 mg/kg and xylazine 8 mg/kg, intraperitoneal injection) or high (120%; 48 mg/kg and 12 mg/kg) dose of anaesthetic and examined every 5 min from 10 to 30 min after anaesthesia was induced. Lens opacity levels were assessed and graded (1-6) using the standard classification system. Regardless of the anaesthetic dose, lens opacity grade was 1-2 in moisturised eyes with application of 0.5% carboxymethylcellulose, and 5-6 in dry ocular surface conditions. Lens opacity in mice with high-dose anaesthetic in the dry ocular surface condition was not different from that of mice with low-dose anaesthetic. Lens opacity grade 1-2 was noted in eyes in the wet ocular surface condition, regardless of IR light exposure. During IR light exposure in eyes in the dry ocular surface condition, lens opacity (grade 6) in mice with high-dose anaesthetic was not different from that (grade 6) in mice with low-dose anaesthetic. We demonstrated that ocular surface dryness might be a relevant factor for the formation and progression of lens opacity in anesthetized C57BL/6 N mice. Anaesthesia dose and IR light exposure did not strongly influence lens opacity formation. Furthermore, eyes with corneal dryness-induced lens opacity recovered to normal status without additional intervention.

Biallelic Deletion of Pxdn in Mice Leads to Anophthalmia and Severe Eye Malformation.

Peroxidasin (PXDN) is a unique peroxidase containing extracellular matrix motifs and stabilizes collagen IV networks by forming sulfilimine crosslinks. PXDN gene knockout in Caenorhabditis elegans (C. elegans) and Drosophila results in the demise at the embryonic and larval stages. PXDN mutations lead to severe eye disorders, including microphthalmia, cataract, glaucoma, and anterior segment dysgenesis in humans and mice. To investigate how PXDN loss of function affects organ development, we generated Pxdn knockout mice by deletion of exon 1 and its 5' upstream sequences of the Pxdn gene using the CRISPR/Cas9 system. Loss of both PXDN expression and collagen IV sulfilimine cross-links was detected only in the homozygous mice, which showed completely or almost closed eyelids with small eyes, having no apparent external morphological defects in other organs. In histological analysis of eye tissues, the homozygous mice had extreme defects in eye development, including no eyeballs or drastically disorganized eye structures, whereas the heterozygous mice showed normal eye structure. Visual function tests also revealed no obvious functional abnormalities in the eyes between heterozygous mice and wild-type mice. Thus, these results suggest that PXDN activity is essential in eye development, and also indicate that a single allele of Pxdn gene is sufficient for eye-structure formation and normal visual function.

Development of a Pde6b Gene Knockout Rat Model for Studies of Degenerative Retinal Diseases.

Purpose: To describe the phenotypes of a newly developed Pde6b-deficient rat model of retinal degeneration. Methods: Pde6b knockout rats were produced by CRISPR-Cpf1 technology. Pde6b knockout rats were evaluated for ocular abnormalities by comparison with wild-type eyes. Eyes were imaged using fundus photography and optical coherence tomography (OCT), stained by hematoxylin and eosin (H&E), and examined by TUNEL assay. Finally, eyes were functionally assessed by electroretinograms (ERGs). Results: Pde6b knockout rats exhibited visible photoreceptor degeneration at 3 weeks of postnatal age. The fundus appearance of mutants was notable for pigmentary changes, vascular attenuation with an irregular vascular pattern, and outer retinal thinning, which resembled retinitis pigmentosa (RP) in humans. OCT showed profound retinal thinning in Pde6b knockout rats; the outer nuclear layer (ONL) was significantly thinner in Pde6b knockout rats, with relative preservation of the inner retina at 3 weeks of postnatal age. H&E staining confirmed extensive degeneration of the ONL, beginning at 3 weeks of postnatal age; no ONL remained in the retina by 16 weeks of postnatal age. Retinal sections of Pde6b knockout rats were highly positive for TUNEL, specifically in the ONL. In ERGs, Pde6b knockout rats showed no detectable a- or b-waves at 8 weeks of postnatal age. Conclusions: The Pde6b knockout rat exhibits photoreceptor degeneration. It may provide a better model for experimental therapy for RP because of its slower progression and larger anatomic architecture than the corresponding mouse model. Further studies in this rat model may yield insights into effective therapies for human RP.

EEG dataset and OpenBMI toolbox for three BCI paradigms: an investigation into BCI illiteracy.

BACKGROUND: Electroencephalography (EEG)-based brain-computer interface (BCI) systems are mainly divided into three major paradigms: motor imagery (MI), event-related potential (ERP), and steady-state visually evoked potential (SSVEP). Here, we present a BCI dataset that includes the three major BCI paradigms with a large number of subjects over multiple sessions. In addition, information about the psychological and physiological conditions of BCI users was obtained using a questionnaire, and task-unrelated parameters such as resting state, artifacts, and electromyography of both arms were also recorded. We evaluated the decoding accuracies for the individual paradigms and determined performance variations across both subjects and sessions. Furthermore, we looked for more general, severe cases of BCI illiteracy than have been previously reported in the literature. RESULTS: Average decoding accuracies across all subjects and sessions were 71.1% (± 0.15), 96.7% (± 0.05), and 95.1% (± 0.09), and rates of BCI illiteracy were 53.7%, 11.1%, and 10.2% for MI, ERP, and SSVEP, respectively. Compared to the ERP and SSVEP paradigms, the MI paradigm exhibited large performance variations between both subjects and sessions. Furthermore, we found that 27.8% (15 out of 54) of users were universally BCI literate, i.e., they were able to proficiently perform all three paradigms. Interestingly, we found no universally illiterate BCI user, i.e., all participants were able to control at least one type of BCI system. CONCLUSIONS: Our EEG dataset can be utilized for a wide range of BCI-related research questions. All methods for the data analysis in this study are supported with fully open-source scripts that can aid in every step of BCI technology. Furthermore, our results support previous but disjointed findings on the phenomenon of BCI illiteracy.

Newly developed method for mouse olfactory behavior tests using an automatic video tracking system.

OBJECTIVE: The mouse is the most popular animal model in olfactory research. Behavior tests with odorants are essential for determining olfactory phenotype. To the best of our knowledge, the mouse olfactory behavior test has not been standardized, making the results vulnerable to inter-observer variation. We sought to develop a new mouse olfactory behavior test assessed by an automatic video tracking system with minimal inter-observer variation. METHODS: A video-tracking system was used to automatically track mouse behavior in standard breeding cages with C57BL/6N mice. We tested two odorants (peanut butter for the preference test, 2MB acid for the avoidance test) and distilled water (for a control). Mouse behavior was recorded for 3min and analyzed. For the preference test, investigation time was measured. For the avoidance test, time spent in sectors away from the odorant zone was measured. To confirm our experimental settings, we also evaluated an anosmia mouse model prepared with intranasal administration of ZnSO4. RESULTS: All strains of mice showed reproducible behavior patterns of preference or avoidance for the odorants. The anosmia mouse model, as expected, failed to show an olfactory ability for preference or avoidance, and this was well-matched by histologic changes caused by the ZnSO4 treatment. The automatic video tracking system successfully tracked and automatically calculated mouse behavior with good reproducibility. CONCLUSION: Our olfactory behavior test offers a simple and accurate method to evaluate olfactory function in mice. This test can be utilized as a possible standard method to search for features of olfactory phenotypes in mice.

Identification of a cell cycle-dependent duplicating complex that assembles basal bodies de novo in Naegleria.

During the differentiation of the amoeba Naegleria pringsheimi into a flagellate, a transient complex containing γ-tubulin, pericentrin-like protein, and myosin II (GPM complex) is formed, and subsequently a pair of basal bodies is assembled from the complex. It is not understood, however, how a single GPM is formed nor how the capability to form this complex is acquired by individual cells. We hypothesized that the GPM is formed from a precursor complex and developed an antibody that recognizes Naegleria (Ng)-transacylase, a component of the precursor complex. Immunostaining of differentiating cells showed that Ng-transacylase is concentrated at a site in the amoeba and that γ-tubulin is transiently co-concentrated at the site, suggesting that the GPM is formed from a precursor, GPMp, which contains Ng-transacylase and is already present in the amoeba. Immunostaining of growing N. pringsheimi with Ng-transacylase antibody revealed the presence of one GPMp in interphase cells, but two GPMps in mitotic cells, suggesting that N. pringsheimi maintains one GPMp per cell by duplicating and segregating the complex according to its cell cycle. Our results demonstrate the existence of a cell cycle-dependent duplicating complex that provides a site for the de novo assembly of the next generation of basal bodies.

Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key regulator of cilia length and Hedgehog signaling.

Endocrine-cerebro-osteodysplasia (ECO) syndrome is a recessive genetic disorder associated with multiple congenital defects in endocrine, cerebral, and skeletal systems that is caused by a missense mutation in the mitogen-activated protein kinase-like intestinal cell kinase (ICK) gene. In algae and invertebrates, ICK homologs are involved in flagellar formation and ciliogenesis, respectively. However, it is not clear whether this role of ICK is conserved in mammals and how a lack of functional ICK results in the characteristic phenotypes of human ECO syndrome. Here, we generated Ick knockout mice to elucidate the precise role of ICK in mammalian development and to examine the pathological mechanisms of ECO syndrome. Ick null mouse embryos displayed cleft palate, hydrocephalus, polydactyly, and delayed skeletal development, closely resembling ECO syndrome phenotypes. In cultured cells, down-regulation of Ick or overexpression of kinase-dead or ECO syndrome mutant ICK resulted in an elongation of primary cilia and abnormal Sonic hedgehog (Shh) signaling. Wild-type ICK proteins were generally localized in the proximal region of cilia near the basal bodies, whereas kinase-dead ICK mutant proteins accumulated in the distal part of bulged ciliary tips. Consistent with these observations in cultured cells, Ick knockout mouse embryos displayed elongated cilia and reduced Shh signaling during limb digit patterning. Taken together, these results indicate that ICK plays a crucial role in controlling ciliary length and that ciliary defects caused by a lack of functional ICK leads to abnormal Shh signaling, resulting in congenital disorders such as ECO syndrome.

Developmentally regulated GTP-binding protein 2 ameliorates EAE by suppressing the development of TH17 cells.

Developmentally regulated GTP-binding protein 2 (DRG2) represents a novel subclass of GTP-binding proteins. We here report that transgenic overexpression of DRG2 in mice ameliorates experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The protective effect of DRG2 in EAE was mediated by the inhibition of the development of T(H)17 cells. DRG2 enhanced the activity of PPARγ, which led to an inhibition of the nuclear factor kappa B (NF-κB) activity and IL-6 production in antigen presenting cells and an inhibition of the development of T(H)17 cells. Our results demonstrate that DRG2 is an essential modulator of EAE.

Transcription factor Sp1 regulates basal transcription of the human DRG2 gene.

Developmentally regulated GTP-binding protein 2 (DRG2) is an evolutionarily conserved GTP-binding protein. DRG2 mRNA expression has been confirmed in many animal and human tissues. DRG2 is thought to play an essential role in the control of cell growth and differentiation. However, transcriptional regulation of DRG2 is largely unknown. To investigate the mechanisms controlling DRG2 expression, we cloned 1509bp of the 5'-flanking sequence of this gene. Deletion analysis showed that the region between -113 and -70 is essential for the basal level expression of the DRG2 gene in K562 human erythroleukemic cells. Mutation of a putative stimulating protein 1 (Sp1) regulatory site located at position -108 resulted in a significant decline in DRG2 promoter activity. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis revealed that Sp1 binds to this site. Knockdown of Sp1 expression using siRNA inhibited the promoter activation as well as the endogenous DRG2 transcriptional level. Taken together, these results demonstrate that basal expression level of DRG2 is regulated by the Sp1 transcription factor.

The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1.

The mRNA-binding protein Ssd1 is a substrate for the Saccharomyces cerevisiae LATS/NDR orthologue Cbk1, which controls polarized growth, cell separation, and cell integrity. We discovered that most Ssd1 localizes diffusely within the cytoplasm, but some transiently accumulates at sites of polarized growth. Cbk1 inhibition and cellular stress cause Ssd1 to redistribute to mRNA processing bodies (P-bodies) and stress granules, which are known to repress translation. Ssd1 recruitment to P-bodies is independent of mRNA binding and is promoted by the removal of Cbk1 phosphorylation sites. SSD1 deletion severely impairs the asymmetric localization of the Ssd1-associated mRNA, SRL1. Expression of phosphomimetic Ssd1 promotes polarized localization of SRL1 mRNA, whereas phosphorylation-deficient Ssd1 causes constitutive localization of SRL1 mRNA to P-bodies and causes cellular lysis. These data support the model that Cbk1-mediated phosphorylation of Ssd1 promotes the cortical localization of Ssd1-mRNA complexes, whereas Cbk1 inhibition, cellular stress, and Ssd1 dephosphorylation promote Ssd1-mRNA interactions with P-bodies and stress granules, leading to translational repression.

De novo formation of basal bodies in Naegleria gruberi: regulation by phosphorylation.

The de novo formation of basal bodies in Naegleria gruberi was preceded by the transient formation of a microtubule (MT)-nucleating complex containing gamma-tubulin, pericentrin, and myosin II complex (GPM complex). The MT-nucleating activity of GPM complexes was maximal just before the formation of visible basal bodies and then rapidly decreased. The regulation of MT-nucleating activity of GPM complexes was accomplished by a transient phosphorylation of the complex. Inhibition of dephosphorylation after the formation of basal bodies resulted in the formation of multiple flagella. 2D-gel electrophoresis and Western blotting showed a parallel relationship between the MT-nucleating activity of GPM complexes and the presence of hyperphosphorylated gamma-tubulin in the complexes. These data suggest that the nucleation of MTs by GPM complexes precedes the de novo formation of basal bodies and that the regulation of MT-nucleating activity of GPM complexes is essential to the regulation of basal body number.