Arginine with leucine drives reactive oxygen species-mediated integrin α5ß1 expression and promotes implantation in mouse blastocysts.

The implantation rate of in vitro fertilization (IVF)-derived blastocysts after embryo transfer remains low, suggesting that the inadequate expression of specific proteins in culture-induced IVF-derived blastocysts contributes to low implantation rates. Therefore, treatment with appropriate regulation may improve the blastocyst implantation ability. This study demonstrated that the combination of l-arginine (Arg) and l-leucine (Leu) exerts distinct effects on IVF-derived mouse blastocysts. Arg with Leu promotes blastocyst implantation, whereas Arg alone decreases the blastocyst ability. Integrin α5ß1 expression was increased in blastocysts treated with Arg and Leu. Arg with Leu also increased reactive oxygen species (ROS) levels and showed a positive correlation with integrin α5ß1. Ascorbic acid, an antioxidant, decreased ROS and integrin α5ß1 levels, which were elevated by Arg with Leu. Meanwhile, the mitochondrial membrane potential (ΔΨm) in blastocysts did not differ between treatments. Glutathione peroxidase (GPx) is involved in ROS scavenging using glutathione (GSH) as a reductant. Arg with Leu decreased GPx4 and GSH levels in blastocysts, and blastocysts with higher ROS levels had lower GPx4 and GSH levels. In contrast, Arg alone increased the percentage of caspase-positive cells, indicating that Arg alone, which attenuated implantation ability, was associated with apoptosis. This study revealed that elevated ROS levels induced by Arg with Leu stimulated integrin α5ß1 expression, thereby enhancing implantation capacity. Our results also suggest that ROS were not due to increased production by oxidative phosphorylation, but rather to a reduction in ROS degradation due to diminished GPx4 and GSH levels.

Determination of Capillary Blood TSH and Free Thyroxine Levels Using Digital Immunoassay.

Background: The remote performance of thyroid function blood tests is complicated because it requires blood collection. Objective: To compare TSH and free thyroxine (FT4) levels between capillary and venous blood and assess the adequacy of measuring each value in capillary blood. Methods: This prospective intervention study was conducted at Ito Hospital and was based on the clinical research method. The participants were 5 healthy female volunteers and 50 patients (41 females and 9 males) between the ages of 23 and 81 years. To measure TSH and FT4 levels in capillary and venous blood, a digital immunoassay (d-IA) method capable of measuring trace samples was used. Chemiluminescence measurements were used as controls. Values obtained for each assay system were compared using Spearman's correlation analysis. Capillary blood was collected using an autologous device (TAP II; not approved in Japan). Results: Capillary plasma volume obtained using TAP II was 125 µL or more in 26 cases, 25 µL to 124 µL in 24 cases, and less than 25 µL in 5 cases. Strong correlations were noted in the TSH and FT4 levels between capillary and venous blood, with correlation coefficients of rs = 0.99 and rs = 0.97, respectively. Conclusion: Capillary TSH and FT4 levels strongly correlate with venous blood values. Trace samples can be used in high-precision d-IA methods. These results may promote telemedicine in assessing thyroid function.

Development of a quantitative thyroid-stimulating hormone assay system for a benchtop digital ELISA desktop analyzer.

Regular checkups for thyroid-stimulating hormone (TSH) levels are essential for the diagnosis of thyroid disease. The enzyme-linked immunosorbent assay (ELISA) technique is a standard method for detecting TSH in the serum or plasma of hospitalized patients. A recently developed next-generation ELISA, the digital immunoassay (d-IA), has facilitated detection of molecules with ultra-high-sensitivity. In this study, we developed a TSH assay system using the d-IA platform. By utilizing the ultrasensitivity of d-IA, we were able to use a sample volume of as little as 5 µL for each assay (the dead volume was 5 µL). The limits of blank, detection, and quantification (i.e., functional sensitivity), were 0.000346, 0.001953, and 0.002280 µIU/mL, respectively, and the precision of the total coefficient of variation did not exceed 10%. The correlation between serum and plasma levels indicated good agreement. Thus, our system successfully measured TSH using d-IA with a small sample volume and equal functional sensitivity to the current third generation like ARCHITECT TSH assay, which has a functional sensitivity of 0.0038 µIU/mL.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition.

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

Gsx2 is required for specification of neurons in the inferior olivary nuclei from Ptf1a-expressing neural progenitors in zebrafish.

Neurons in the inferior olivary nuclei (IO neurons) send climbing fibers to Purkinje cells to elicit functions of the cerebellum. IO neurons and Purkinje cells are derived from neural progenitors expressing the proneural gene ptf1a In this study, we found that the homeobox gene gsx2 was co-expressed with ptf1a in IO progenitors in zebrafish. Both gsx2 and ptf1a zebrafish mutants showed a strong reduction or loss of IO neurons. The expression of ptf1a was not affected in gsx2 mutants, and vice versa. In IO progenitors, the ptf1a mutation increased apoptosis whereas the gsx2 mutation did not, suggesting that ptf1a and gsx2 are regulated independently of each other and have distinct roles. The fibroblast growth factors (Fgf) 3 and 8a, and retinoic acid signals negatively and positively, respectively, regulated gsx2 expression and thereby the development of IO neurons. mafba and Hox genes are at least partly involved in the Fgf- and retinoic acid-dependent regulation of IO neuronal development. Our results indicate that gsx2 mediates the rostro-caudal positional signals to specify the identity of IO neurons from ptf1a-expressing neural progenitors.

Splicing- and demethylase-independent functions of LSD1 in zebrafish primitive hematopoiesis.

LSD1/KDM1A is a widely conserved lysine-specific demethylase that removes methyl groups from methylated proteins, mainly histone H3. We previously isolated the zebrafish LSD1 gene and demonstrated that it is required for primitive hematopoiesis. Recently, a neuron-specific splicing variant of LSD1 was found in mammals and its specific functions and substrate specificities were reported. To our surprise, zebrafish LSD1 cDNA, which we previously analyzed, was corresponded to the neuron-specific variant in mammals. In this study, we investigated the structures and expression of LSD1 splicing variants in zebrafish and found all 4 types of LSD1 isoforms: LSD1, LSD1+2al, LSD1+8al and LSD1+2al8al. Interestingly, LSD1+8al/LSD1+2al8al, which correspond to mammalian neuron-specific variants, expressed ubiquitously in zebrafish. We also performed phenotypic rescue experiments of a zebrafish LSD1 mutant (kdm1ait627) using human and zebrafish LSD1 variants to identify which variant is involved in primitive hematopoiesis. Unexpectedly, the overexpression of all types of human and zebrafish variants was able to rescue the hematopoietic phenotypes in LSD1 mutants. Furthermore, enzymatic-deficient LSD1K661A (human) and K638A (zebrafish) were also able to rescue the mutant phenotypes. These results suggest that the LSD1 functions in zebrafish primitive hematopoiesis are free from any splicing-dependent regulation or demethylation reaction.

Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.

Antibacterial Properties of Silicone Membranes after a Simple Two-Step Immersion Process in Iodine and Silver Nitrate Solutions.

　Silicone is widely used in packing materials, medical equipment, and separation membranes. Since microbial cells easily adhere to the surface of silicone materials and form biofilms, techniques for incorporating antimicrobial activity into silicone materials are in high demand. This study describes the preparation of silver (Ag)/silicone composite membranes through a simple two-step immersion process, utilizing an iodine solution followed by a silver nitrate solution at room temperature. Scanning electron microscopy (SEM) observations revealed that particles with sizes of several nanometers to several tens of nanometers were present on the silicone membrane surface; these particles were identified as silver iodide using energy-dispersive X-ray spectroscopy (EDS) . The Ag/silicone membrane possessed excellent antibacterial efficacy against Escherichia coli and Staphylococcus aureus, and the antibacterial efficacy (R) against both types of bacteria was R > 4, even after stomacher treatment or acidic treatment of pH 2-6 for 24 h. The mechanical strength of the silicone membrane was also maintained after antibacterial treatment, with Young's modulus values of 7.9±1.2 MPa and 8.3±1.5 MPa for the untreated membrane and Ag/silicone membrane, respectively (p > 0.05) . In addition, the reduction in permeation performance of the Ag/silicone membrane was only 20%, despite the antibacterial treatment on the membrane surface. This antibacterial treatment method of silicone membranes can be conducted at room temperature (25℃) without special equipment, and may be applied to other types of silicone materials.

Vector control efficacy of fly nets on preventing bovine leukemia virus transmission.

Bovine leukemia virus (BLV) is horizontally transmitted among cattle through infected blood. This 3-year field study (2013-2016) aimed to confirm the potential of the blood-sucking stable fly as a risk factor of BLV transmission and to determine the efficacy of vector control on preventing the transmission of BLV. The BLV-positive conversion rate during summer was higher than that during winter in a model dairy farm, where many stable flies were observed during the summer. After fly nets were fixed onto the barn to prevent fly invasion, the BLV-positive conversion rate during the summer was significantly decreased compared with that in the absence of fly nets (P<0.01). These findings suggest that vector control using a fly net may inhibit BLV transmission.

Madagascar ground gecko genome analysis characterizes asymmetric fates of duplicated genes.

BACKGROUND: Conventionally, comparison among amniotes - birds, mammals, and reptiles - has often been approached through analyses of mammals and, for comparison, birds. However, birds are morphologically and physiologically derived and, moreover, some parts of their genomes are recognized as difficult to sequence and/or assemble and are thus missing in genome assemblies. Therefore, sequencing the genomes of reptiles would aid comparative studies on amniotes by providing more comprehensive coverage to help understand the molecular mechanisms underpinning evolutionary changes. RESULTS: Herein, we present the whole genome sequences of the Madagascar ground gecko (Paroedura picta), a promising study system especially in developmental biology, and used it to identify changes in gene repertoire across amniotes. The genome-wide analysis of the Madagascar ground gecko allowed us to reconstruct a comprehensive set of gene phylogenies comprising 13,043 ortholog groups from diverse amniotes. Our study revealed 469 genes retained by some reptiles but absent from available genome-wide sequence data of both mammals and birds. Importantly, these genes, herein collectively designated as 'elusive' genes, exhibited high nucleotide substitution rates and uneven intra-genomic distribution. Furthermore, the genomic regions flanking these elusive genes exhibited distinct characteristics that tended to be associated with increased gene density, repeat element density, and GC content. CONCLUSION: This highly continuous and nearly complete genome assembly of the Madagascar ground gecko will facilitate the use of this species as an experimental animal in diverse fields of biology. Gene repertoire comparisons across amniotes further demonstrated that the fate of a duplicated gene can be affected by the intrinsic properties of its genomic location, which can persist for hundreds of millions of years.

Living alone is associated with an increased risk of institutionalization in older men: A follow-up study in Hamanaka Town of Hokkaido, Japan.

AIM: In order to clarify the risks of institutionalization or mortality among older adults living alone compared with those not living alone, we carried out a prospective study on older adults in Hamanaka Town in the far eastern part of Hokkaido, Japan. METHODS: All 978 community-dwelling residents aged 70-85 years in the town were chosen as study candidates between February and May of 2014. Written informed consent was obtained from 562 residents (57.5%), and a self-administered questionnaire, including a question about living arrangements, was mailed to them. They returned the completed questionnaire to us in 2014. A follow-up survey was carried out with a questionnaire mailed to each participant about institutionalization and mortality, three times, in February of 2015 and 2016, and in April of 2017. Hazard ratios and 95% confidence intervals were calculated with the Cox proportional hazards model. RESULTS: Living alone was significantly associated with an increased risk of institutionalization in the male participants, after adjusting for age, sex and having daily support from family around a participant (hazard ratio 5.71, 95% confidence interval 1.17-27.83), although it was not significant in the total participants or the female participants. Additional adjustments for a history of having common diseases did not change the results meaningfully. Living alone was not associated with the risk of mortality in the total participants, the male participants or the female participants. CONCLUSIONS: Poor social support in social networks for older men living alone may be etiologically associated with increased risk of institutionalization in rural area. Further study with a larger sample size is necessary to confirm this finding. Geriatr Gerontol Int 2018; 18: 867-872.

Improvement of implantation potential in mouse blastocysts derived from IVF by combined treatment with prolactin, epidermal growth factor and 4-hydroxyestradiol.

STUDY QUESTION: Can supplementation of medium with prolactin (PRL), epidermal growth factor (EGF) and 4-hydroxyestradiol (4-OH-E2) prior to embryo transfer improve implantation potential in mouse blastocysts derived from IVF? SUMMARY ANSWER: Combined treatment with PRL, EGF and 4-OH-E2 improves mouse blastocyst implantation rates, while alone, each factor is ineffective. WHAT IS KNOWN ALREADY: Blastocyst dormancy during delayed implantation caused by ovariectomy is maintained by continued progesterone treatment in mice, and estrogen injection rapidly activates blastocysts to implantation-induced status in vivo. While the expression of many proteins is upregulated in implantation-induced blastocysts, selective proteolysis by proteasomes, such as estrogen receptor α (ESR1), occurs in implantation-induced blastocysts to achieve implantation-competent status. It is worth evaluating the proteins expressed during these periods to identify humoral factors that might improve the implantation potential of IVF-derived blastocysts because the poor quality of embryos obtained by IVF is one of the major causes of implantation failure. STUDY DESIGN, SIZE, DURATION: Superovulated oocytes from ICR mice were fertilized with spermatozoa and then cultured in vitro in potassium simplex optimized medium (KSOM) without phenol red (KSOM-P) for 90-96 h. Blastocysts were treated with PRL (10 or 20 mIU/mL), EGF (5 or 10 ng/mL) or 4-OH-E2 (1 or 10 nM) in KSOM-P for 24 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Levels of breast cancer 1 (BRCA1), EGF receptor (EGFR, also known as ERBB1), ERBB4, tubulointerstitial nephritis antigen-like 1 (TINAGL1) and ESR1 protein were examined with immunohistochemical analysis using immunofluorescence methods and confocal laser scanning microscopy. For embryo transfer, six blastocysts were suspended in HEPES-buffered KSOM-P medium and transferred into the uteri of recipient mice on the morning of Day 4 (0900-1000 h) of pseudopregnancy (Day 1 = vaginal plug). The number of implantation sites was then recorded on Day 6 using the blue dye method. MAIN RESULTS AND THE ROLE OF CHANCE: PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the trophectoderm (TE). While PRL treatment resulted in an increase in EGFR, EGF increased both EGFR and ERBB4 in the blastocyst TE. TINAGL1 in the TE was enhanced by 4-OH-E2, which also increased localization of this protein to the basement membrane. Treatment with PRL, EGF or 4-OH-E2 alone did not improve blastocyst implantation rates. Combined treatment with PRL, EGF and 4-OH-E2 resulted in increased levels of EGFR, ERBB4, TINAGL1 and BRCA1 in the TE, whereas ESR1 was not upregulated in the treated blastocysts. Furthermore, combined treatment with PRL, EGF and 4-OH-E2 improved blastocyst implantation rates versus control (P = 0.009). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our studies were carried out in a mouse model, and the conclusions were drawn from limited results obtained from one species. Whether the increase in EGFR, ERBB4 and TINAGL1 protein in the TE improves implantation potential of blastocysts needs to be further studied experimentally by assessing other expressed proteins. The influence of combined supplementation in vitro of PRL, EGF and 4-OH-E2 on implantation also requires further examination and optimization in human blastocysts before it can be considered for clinical use in ART. WIDER IMPLICATIONS OF THE FINDINGS: Enhanced implantation potential by combined treatment with PRL, EGF and 4-OH-E2 appears to result in the upregulation of at least two distinct mechanisms, namely signaling via EGF receptors and basement membrane formation during the peri-implantation period in mice. While PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the TE, treatment with each alone did not improve blastocyst implantation. Therefore, BRCA1 protein appears to be unnecessary for the attachment reaction in blastocysts in mice Combined supplementation of PRL, EGF and 4-OH-E2 might also be of relevance for embryo transfer of human IVF-derived blastocysts for ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the JSPS KAKENHI [Grant numbers 22580316 and 25450390 (to H.M.)] and the Joint Research Project of Japan-U.S. Cooperative Science Program (to H.M.). The authors have no conflict of interest to declare.

Medaka and zebrafish contactin1 mutants as a model for understanding neural circuits for motor coordination.

A spontaneous medaka ro mutant shows abnormal wobbling and rolling swimming behaviors. By positional cloning, we mapped the ro locus to a region containing the gene encoding Contactin1b (Cntn1b), which is an immunoglobulin (Ig)-superfamily domain-containing membrane-anchored protein. The ro mutant had a deletion in the cntn1b gene that introduced a premature stop codon. Furthermore, cntn1b mutants generated by the CRISPR/Cas9 system and trans-heterozygotes of the CRISPR mutant allele and ro had abnormal swimming behavior, indicating that the cntn1b gene was responsible for the ro-mutant phenotype. We also established zebrafish cntn1a and cntn1b mutants by transcription activator-like effector nucleases (TALENs). Zebrafish cntn1b but not cntn1a mutants showed abnormal swimming behaviors similar to those in the ro mutant, suggesting that Cntn1b plays a conserved role in the formation or function of the neural circuits that control swimming in teleosts. Although Cntn1-deficient mice have abnormal cerebellar neural circuitry, there was no apparent histological abnormality in the cerebellum of medaka or zebrafish cntn1b mutants. The medaka cntn1b mutants had defective optokinetic response (OKR) adaptation and abnormal rheotaxis (body positioning relative to water flow). Medaka and zebrafish cntn1b mutants are effective models for studying the neural circuits involved in motor learning and motor coordination.

Evolutionary mechanisms that generate morphology and neural-circuit diversity of the cerebellum.

The cerebellum is derived from the dorsal part of the anterior-most hindbrain. The vertebrate cerebellum contains glutamatergic granule cells (GCs) and gamma-aminobutyric acid (GABA)ergic Purkinje cells (PCs). These cerebellar neurons are generated from neuronal progenitors or neural stem cells by mechanisms that are conserved among vertebrates. However, vertebrate cerebella are widely diverse with respect to their gross morphology and neural circuits. The cerebellum of cyclostomes, the basal vertebrates, has a negligible structure. Cartilaginous fishes have a cerebellum containing GCs, PCs, and deep cerebellar nuclei (DCNs), which include projection neurons. Ray-finned fish lack DCNs but have projection neurons termed eurydendroid cells (ECs) in the vicinity of the PCs. Among ray-finned fishes, the cerebellum of teleost zebrafish has a simple lobular structure, whereas that of weakly electric mormyrid fish is large and foliated. Amniotes, which include mammals, independently evolved a large, foliated cerebellum, which contains massive numbers of GCs and has functional connections with the dorsal telencephalon (neocortex). Recent studies of cyclostomes and cartilaginous fish suggest that the genetic program for cerebellum development was already encoded in the genome of ancestral vertebrates. In this review, we discuss how alterations of the genetic and cellular programs generated diversity of the cerebellum during evolution.

Gene expression profiling of granule cells and Purkinje cells in the zebrafish cerebellum.

The structure of the neural circuitry of the cerebellum, which functions in some types of motor learning and coordination, is generally conserved among vertebrates. However, some cerebellar features are species specific. It is not clear which genes are involved in forming these conserved and species-specific structures and functions. This study uses zebrafish transgenic larvae expressing fluorescent proteins in granule cells, Purkinje cells, or other cerebellar neurons and glial cells to isolate each type of cerebellar cells by fluorescence-activated cell sorting and to profile their gene expressions by RNA sequencing and in situ hybridization. We identify genes that are upregulated in granule cells or Purkinje cells, including many genes that are also expressed in mammalian cerebella. Comparison of the transcriptomes in granule cells and Purkinje cells in zebrafish larvae reveals that more developmental genes are expressed in granule cells, whereas more neuronal-function genes are expressed in Purkinje cells. We show that some genes that are upregulated in granule cells or Purkinje cells are also expressed in the cerebellum-like structures. Our data provide a platform for understanding the development and function of the cerebellar neural circuits in zebrafish and the evolution of cerebellar circuits in vertebrates. J. Comp. Neurol. 525:1558-1585, 2017. © 2016 Wiley Periodicals, Inc.

Multiple activation mechanisms of serotonin-mediated contraction in the carotid arteries obtained from spontaneously hypertensive rats.

Serotonin (5-hydroxytryptamine, 5-HT) is an important endogenous substance that regulates the vascular tone, and the abnormal signaling of 5-HT has been observed in the arteries under several pathophysiological conditions such as diabetes and hypertension. However, signaling pathways of 5-HT-mediated vasocontraction in hypertension remain unclear. Therefore, we tested the hypothesis that 5-HT-mediated contraction and contributions of various kinases such as mitogen-activated protein kinases (MAPKs), phosphoinositide 3-kinase (PI3K), Rho kinase (ROCK), and 3-phosphoinositide-dependent kinase 1 (PDK1) to the contraction would be altered in the carotid arteries obtained from spontaneously hypertensive rats (SHR) compared to control Wistar Kyoto (WKY) rats. In the carotid arteries from SHR (vs. those from WKY), (1) the 5-HT-mediated contraction was increased, whereas the norepinephrine-mediated contraction was not; (2) 5-HT-mediated contractions were partly inhibited by each kinase (extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase (JNK), PI3K, ROCK, and PDK1) inhibitor; and (3) 5-HT-stimulated phosphorylation of ERK1/2, p38 MAPK, JNK, myosin phosphatase target subunit 1 (MYPT1), and PDK1 was increased. The expression of ROCK2 but not ROCK1 was increased in the carotid arteries from SHR compared to WKY. The expression of 5-HT2A receptor, a major receptor of 5-HT-mediated contraction in rat carotid artery, was similar in carotid arteries between the two groups. These results suggest that 5-HT-mediated contraction was utilized multiple signaling pathways such as ERK1/2, p38 MAPK, JNK, PI3K, ROCK, and PDK1. Although 5-HT-mediated contraction was increased in the carotid arteries obtained from SHR, further studies are necessary to clarify how each kinase may integrate in the vascular smooth muscles under hypertension.

Impaired female fertility in tubulointerstitial antigen-like 1-deficient mice.

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert's membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1(-/-) embryos were not lethal during development to term, homologous matings of Tinagl1(-/-) females and Tinagl1(-/-) males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1(-/-) and Tinagl1(flox/flox). In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1(-/-) oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.

Influence of volunteer-led net step exercise class on older people's self-rated health in a depopulated town: A longitudinal study.

In a depopulated region where population aging is advancing, it is necessary to establish a method so local residents themselves can be actively involved in older people's health promotion. Net Step Exercise, a novel dual-task walking program, introduced residents to opportunities for physical activities and social participation without any health specialist support. In one depopulated town (Ikeda, Nakagawa-gun, Hokkaido, Japan), volunteer residents have held Net Step Exercise classes throughout the town since 2007. We longitudinally examined the influence of volunteer-led Net Step Exercise class participation on subsequent self-rated health in all individuals aged 70-79 years living in Ikeda. A total of 662 people who completed a baseline mail-in questionnaire survey in 2012 were followed until 2014. Logistic regression analysis was performed to examine the association with self-rated health after two years of class participation once a month or more at baseline, after controlling for confounds such as age, sex, years of education, living alone, baseline self-rated health, regular exercise, and other physical activities. The odds ratio of poor self-rated health in older people who participated in classes was 0.53 (95% confidence interval [CI]: 0.34-0.85) compared to older people not participating in classes. Even after confounding factors were adjusted, the odds ratio of class participation was 0.50 (95% CI: 0.29-0.85). This study showed that participation in volunteer-led Net Step Exercise might prevent poor self-rated health. Such Net Step Exercise classes are a feasible method for older people's health promotion in depopulated municipalities.

PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination.

The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.

Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.