Distribution of Kakugo Virus and Its Effects on the Gene Expression Profile in the Brain of the Worker Honeybee Apis mellifera L.▿ †

We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), obtained from the brains of aggressive honeybee worker bees that had counterattacked giant hornets. Here we examined the tissue distribution of KV and alterations of gene expression profiles in the brains of KV-infected worker bees to analyze possible effects of KV infection on honeybee neural and physiological states. By use of in situ hybridization, KV was broadly detected in the brains of the naturally KV-infected worker bees. When inoculated experimentally into bees, KV was detected in restricted parts of the brain at the early infectious stage and was later detected in various brain regions, including the mushroom bodies, optic lobes, and ocellar nerve. KV was detected not only in the brain but also in the hypopharyngeal glands and fat bodies, indicating systemic KV infection. Next, we compared the gene expression profiles in the brains of KV-inoculated and noninoculated bees. The expression of 11 genes examined was not significantly affected in KV-infected worker bees. cDNA microarray analysis, however, identified a novel gene whose expression was induced in the periphery of the brains of KV-infected bees, which was commonly observed in naturally infected and experimentally inoculated bees. The gene encoded a novel hypothetical protein with a leucine zipper motif. A gene encoding a similar protein was found in the parasitic wasp Nasonia genome but not in other insect genomes. These findings suggest that KV infection may affect brain functions and/or physiological states in honeybees

Clinical features of a toddler with bilateral bullous retinoschisis with a novel RS1 mutation

Purpose: To report the clinical and genetic findings of a male toddler who presented bilateral bullous retinoschisis with a novel RS1 mutation. Observations: This is an observational case report of a patient referred to our hospital with esotropia. A comprehensive ophthalmic examination was performed with the boy (age, 1 year 4 months) under general anesthesia that included fundus examinations, fluorescein angiography (FA), swept-source optical coherence tomography (SS-OCT), and full-field electroretinography (FF-ERG). Genetic analysis of the coding region in the RS1 gene was performed by Sanger sequencing for the patient and mother. There was a family history of X-linked retinoschisis (XLRS). Fundus examinations and FA showed bullous retinoschisis bilaterally in the inferior retina. The SS-OCT images showed two kinds of schisis in the inner nuclear layer (INL) and more proximally. In general, the inner plexiform layer, ganglion cell layer, and retinal nerve fiber layer are in the proximal INL; however, in this case there was hyperreflective tissue with a rough surface instead of normal retinal layers. In addition, in the schisis cavity between the hyperreflective tissue and separated retina, a number of hyperreflective fiber-like strands arose from the hyperreflective tissue and extended to the schisis cavity. During the follow-up period, the bullous retinoschisis collapsed spontaneously in the right eye. FF-ERG showed a reduced b-wave and relatively preserved a-wave in all components. Genetic analysis showed a novel RS1 mutation (c.185_186insT, p.E62DfsX24 in exon 4) in the patient and mother. Conclusions and importance: We report the detailed retinal structure in a genetically identified case of bullous retinoschisis. The notable finding was that the cavity of bullous retinoschisis contained a number of fiber-like strands as observed in the cavity of typical retinoschisis

Screening of Drugs to Treat 8p11 Myeloproliferative Syndrome Using Patient-Derived Induced Pluripotent Stem Cells with Fusion Gene CEP110-FGFR1

<div><p>Induced pluripotent stem (iPS) cells provide powerful tools for studying disease mechanisms and developing therapies for diseases. The 8p11 myeloproliferative syndrome (EMS) is an aggressive chronic myeloproliferative disorder (MPD) that is caused by constitutive activation of <i>fibroblast growth factor receptor 1</i>. EMS is rare and, consequently, effective treatment for this disease has not been established. Here, iPS cells were generated from an EMS patient (EMS-iPS cells) to assist the development of effective therapies for EMS. When iPS cells were co-cultured with murine embryonic stromal cells, EMS-iPS cells produced more hematopoietic progenitor and hematopoietic cells, and CD34⁺ cells derived from EMS-iPS cells exhibited 3.2–7.2-fold more macrophage and erythroid colony forming units (CFUs) than those derived from control iPS cells. These data indicate that EMS-iPS cells have an increased hematopoietic differentiation capacity, which is characteristic of MPDs. To determine whether a tyrosine kinase inhibitor (TKI) could suppress the increased number of CFUs formed by EMS-iPS-induced CD34⁺ cells, cells were treated with one of four TKIs (CHIR258, PKC 412, ponatinib, and imatinib). CHIR258, PKC 412, and ponatinib reduced the number of CFUs formed by EMS-iPS-induced CD34⁺ cells in a dose-dependent manner, whereas imatinib did not. Similar effects were observed on primary peripheral blood cells (more than 90% of which were blasts) isolated from the patient. This study provides evidence that the EMS-iPS cell line is a useful tool for the screening of drugs to treat EMS and to investigate the mechanism underlying this disease.</p></div

Formation of hematopoietic colonies by CD34⁺ cells that were derived from EMS-iPS cells or control iPS cells co-cultured with AGM-S3 cells.

<p>EMS-iPS cells and control iPS cells (control 1: 201B7, control 2: TkDA3-1) were co-cultured with AGM-S3 cells. After 12 days, cells derived from iPS cells were collected. CD34⁺ cells were isolated for clonal hematopoietic culture. After 12 days, colonies were examined. The mean numbers of colonies ± SD of triplicate cultures are showed. G, granulocyte; M, macrophage; GM, granulocyte-macrophage; E, erythroid; and Mix, mixed-lineage colonies.</p><p>*p < 0.05 compared with the number of colonies formed by CD34⁺ cells derived from control iPS cells (Student’s t-test).</p><p>Formation of hematopoietic colonies by CD34⁺ cells that were derived from EMS-iPS cells or control iPS cells co-cultured with AGM-S3 cells.</p

EMS-iPS cells exhibit augmented hematopoiesis.

<p>(a) Cobblestone morphology of iPS cells. Control human 201B7 iPS cells (B7) or EMS-iPS cells were co-cultured with murine AGM-S3 cells. After 12 days of co-culture, cells with a cobblestone morphology were detected at the peripheries of colonies. Images on the left are at ×100 magnification. Images on the right show higher magnification (×200 magnification) images of the boxed regions. (b) Flow cytometric analysis of cells cocultured with AGMS-3 on day 12. Cells were stained with antibodies specific CD34 and CD45. The percentages of CD34+ and /or CD45+ cells were shown (n = 3; bars represent SDs, p<0.05).</p

Generation of iPS cells from an EMS patient with the t(8;9)(p12;q33) translocation.

<p>(<b>a</b>) Morphology of EMS-iPS cells (×40 magnification). (<b>b</b>) Expression of the pluripotency markers Nanog (A), SOX2 (B), OCT3/4 (C), and PODXL (D) in EMS-iPS cells (×10 magnification). (<b>c</b>) Semi-quantitative RT-PCR analysis of the expression levels of endogenous (end) and viral-derived (Tg) reprogramming factors. Primary BM cells from the patient and control 201B7 human iPS cells (B7) are included as controls. (<b>d</b>) Bisulfite sequencing analyses of the <i>OCT3/4</i> and <i>NANOG</i> promoter regions in patient BM cells and EMS-iPS cells. White and black circles represent unmethylated and methylated (Me) CpG dinucleotides, respectively. (<b>e</b>) Hematoxylin and eosin staining of a teratoma derived from EMS-iPS cells (×20 magnification). The teratoma is composed of gut-like epithelium (endoderm), skeletal muscle (mesoderm), and melanocytes (ectoderm). (<b>f</b>) EMS-iPS cells exhibit the 46, XY, t(8;9)(p12;q33) karyotype, as determined by G-banding analysis. (<b>g</b>) PCR analysis of the expression level of the CEP110-FGFR1 fusion transcript in primary BM cells from the patient and EMS-iPS cells.</p