Optogenetics on Zebrafish.

Optogenetics brought noninvasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models that receive the full benefit of this technique and provides behavioral studies based on intact individual nervous system. In this chapter, we describe methods to introduce optogenetic genes into zebrafish, and desirable apparatus for photostimulation and motion analysis with an example from our studies.

Recurrent de novo MAPK8IP3 variants cause neurological phenotypes.

c-Jun-amino-terminal kinase-interacting protein 3 (JIP3), encoded by MAPK8IP3, is an adaptor protein of the kinesin-1 complex and essential for axonal transport in neurons. However, an association between MAPK8IP3 variants and human disease has not been established. We identified 5 individuals from four families with recurrent de novo variants c.1732C>T (p.Arg578Cys) and c.3436C>T (p.Arg1146Cys) in MAPK8IP3. The core phenotype includes spastic diplegia, intellectual disability, cerebral atrophy, and corpus callosum hypoplasia. Zebrafish embryos overexpressing human mutant JIP3 showed axon varicosities of the posterior lateral line nerve, suggesting an adverse effect on the developing axons. Our results suggest that MAPK8IP3 variants cause a neurodevelopmental disease. ANN NEUROL 2019;85:927-933.

Tissue regeneration during lower jaw restoration in zebrafish shows some features of epimorphic regeneration.

Zebrafish have the ability to regenerate skeletal structures, including the fin, skull roof, and jaw. Although fin regeneration proceeds by epimorphic regeneration, it remains unclear whether this process is involved in other skeletal regeneration in zebrafish. Initially in epimorphic regeneration, the wound epidermis covers the wound surface. Subsequently, the blastema, an undifferentiated mesenchymal mass, forms beneath the epidermis. In the present study, we re-examined the regeneration of the zebrafish lower jaw in detail, and investigated whether epimorphic regeneration is involved in this process. We performed amputation of the lower jaw at two different positions; the proximal level (presence of Meckel's cartilage) and the distal level (absence of Meckel's cartilage). In both manipulations, a blastema-like cellular mass was initially formed. Subsequently, cartilaginous aggregates were formed in this mass. In the proximal amputation, the cartilaginous aggregates were then fused with Meckel's cartilage and remained as a skeletal component of the regenerated jaw, whereas in the distal amputation, the cartilaginous aggregates disappeared as regeneration progressed. Two molecules that were observed during epimorphic regeneration, Laminin and msxb, were expressed in the regenerating lower jaw, although the domain of msxb expression was out of the main plain of the aggregate formation. Administration of an inhibitor of Wnt/ß-catenin signaling, a pathway associated with epimorphic regeneration, showed few effects on lower jaw regeneration. Our finding suggests that skeletal regeneration of the lower jaw mainly progresses through tissue regeneration that is dependent on the position in the jaw, and epimorphic regeneration plays an adjunctive role in this regeneration.

From neuron to behavior: Sensory-motor coordination of zebrafish turning behavior.

Recent development of optogenetics brought non-invasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models for this technique, which enables us to investigate neural circuits for behaviors based on a whole individual nervous system. In this article we review our recent finding that suggests sensory-motor coordination in larval zebrafish escape behavior. When water vibration stimulates mechanosensory Rohon-Beard (RB) neurons, intra-spinal reflex circuit launches contralateral trunk muscle contraction that makes rapid body curvature for turning. In addition, positional information of the stimulus is conveyed to supra-spinal circuits, and then regulates the curvature strength for appropriate escape pathway from the threat. Sensory-motor coordination is a fundamental feature to adapt behaviors to environment, and zebrafish larvae would be an excellent model for elucidating its neural backbones.

Morphologies of Bacillus subtilis communities responding to environmental variation.

Bacterial communities exhibit a variety of growth morphologies in constructing robust systems under different environmental conditions. We review the diverse morphologies of Bacillus subtilis communities and their mechanisms of self-organization. B. subtilis uses different cell types to suit environmental conditions and cell density. The subpopulation of each cell type exhibits various environment-sensitive properties. Furthermore, division of labor among the subpopulations results in flexible development for the community as a whole. We review how B. subtilis community morphologies and growth strategies respond to environmental perturbations.

NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3ß resulting in the stabilization of MYCN in human neuroblastomas.

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3ß, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3ß, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3ß inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3ß activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Membrane Translocation and Organelle-Selective Delivery Steered by Polymeric Zwitterionic Nanospheres.

The majority of nanoparticles designed for cellular delivery of drugs and imaging agents enter the cell via endocytotic pathways leading to their entrapment in endosomes that present a robust barrier to further trafficking of the nanoparticles within the cells. A few materials, such as the cell penetrating peptides (CPPs), are known to enter cells not only via endocytosis, but also via translocation through the cell membrane into the cytoplasm, successfully bypassing the endosomes. We report here that random copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate and poly(ethylene glycol) methacrylate, p(DMAPS-ran-PEGMA), are internalized in cells primarily via translocation through the cell membrane rather than endocytosis. The properties of the polymers and their modes of uptake were investigated systematically by dynamic light scattering, confocal fluorescence microscopy, and flow cytometry. Using specific inhibitors of the cellular uptake machinery in a human cervical carcinoma cell line (HeLa), we show that these nontoxic synthetic polyzwitterions exist in cell media as self-assembled nanospheres that unravel as they adsorb on the plasma membrane and translocate through it. Conjugates of p(DMAPS-ran-PEGMA) with rhodamine B were delivered selectively to the mitochondria, whereas doxorubicin (Dox)-p(DMAPS-ran-PEGMA) conjugates were accumulated in both the nucleus and the mitochondria, effectively inducing apoptosis in HeLa cells. These findings suggest that the noncytotoxic and readily synthesized p(DMAPS-ran-PEGMA) can find applications as bioimaging tools and drug nanocarriers.

NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells.

NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas.

Application of infrared laser to the zebrafish vascular system: gene induction, tracing, and ablation of single endothelial cells.

OBJECTIVE: Infrared laser-evoked gene operator is a new microscopic method optimized to heat cells in living organisms without causing photochemical damage. By combining the promoter system for the heat shock response, infrared laser-evoked gene operator enables laser-mediated gene induction in targeted cells. We applied this method to the vascular system in zebrafish embryos and demonstrated its usability to investigate mechanisms of vascular morphogenesis in vivo. APPROACH AND RESULTS: We used double-transgenic zebrafish with fli1:nEGFP to identify the endothelial cells, and with hsp:mCherry to carry out single-cell labeling. Optimizing the irradiation conditions, we finally succeeded in inducing the expression of the mCherry gene in single targeted endothelial cells, at a maximum efficiency rate of 60%. In addition, we indicated that this system could be used for laser ablation under certain conditions. To evaluate infrared laser-evoked gene operator, we applied this system to the endothelial cells of the first intersegmental arteries, and captured images of the connection between the vascular systems of the brain and spinal cord. CONCLUSIONS: Our results suggest that the infrared laser-evoked gene operator system will contribute to the elucidation of the mechanisms underlying vascular morphogenesis by controlling spatiotemporal gene activation in single endothelial cells, by labeling or deleting individual vessels in living embryos.

Dynamic changes in the gene expression of zebrafish Reelin receptors during embryogenesis and hatching period.

The brain morphology of vertebrates exhibits huge evolutionary diversity, but one of the shared morphological features unique to vertebrate brain is laminar organization of neurons. Because the Reelin signal plays important roles in the development of the laminar structures in mammalian brain, investigation of Reelin signal in lower vertebrates will give some insights into evolution of vertebrate brain morphogenesis. Although zebrafish homologues of Reelin, the ligand, and Dab1, a cytoplasmic component of the signaling pathway, have been reported, the Reelin receptor molecules of zebrafish are not reported yet. Here, we sought cDNA sequence of zebrafish homologue of the receptors, vldlr and apoer2, and examined their expression patterns by in situ hybridization. Developmental gene expression pattern of reelin, dab1, vldlr, and apoer2 in the central nervous system of zebrafish was compared, and their remarkable expression was detected in the developing laminar structures, such as the tectum and the cerebellum, and also non-laminated structures, such as the pallium. The Reelin receptors exhibited different spatial and temporal gene expression. These results suggest a possibility that duplication and subsequent functional diversity of Reelin receptors contributed to the morphological and functional evolution of vertebrate brain.

Islet1 selectively promotes peripheral axon outgrowth in Rohon-Beard primary sensory neurons.

We isolated a novel zebrafish mutant, lullaby (llb), and showed that the llb locus encodes the zebrafish orthologue of isl1. Rohon-Beard (RB) primary sensory neurons are multipolar neurons that extend their central axons longitudinally within the spinal cord and also extend their peripheral axons under the skin. In llb embryos, the outgrowth of the peripheral axons of RB neurons was selectively impaired, which correlated with down-regulation of the expression of dihydropyrimidinase-like 3 (dpysl3, also known as collapsin response mediator protein 4, crmp4). Antisense morpholino oligonucleotide (AMO)-mediated knockdown of dpysl3 inhibited the outgrowth of the peripheral axons of RB neurons, and semaphorin 3d (sema3d) AMO enhanced this effect. These data indicate that Dpysl3 is cooperating with Sema3d in the peripheral axon outgrowth, and Isl1 is required for the selective outgrowth of the peripheral axons of RB neurons by maintaining the expression of dpysl3.

A sublethal ATP11A mutation associated with neurological deterioration causes aberrant phosphatidylcholine flipping in plasma membranes.

ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.

Trajectories for Post-traumatic Stress Disorder Symptoms Among Local Disaster Recovery Workers Following the Great East Japan Earthquake: Group-based Trajectory Modeling.

BACKGROUND: As many local municipality and medical workers were involved in disaster recovery duties following the Great East Japan Earthquake (GEJE) on March 11, 2011, the aim of this work was to elucidate the distinct trajectories for post-traumatic stress disorder (PTSD) symptoms and associated factors among these personnel. They confronted a diverse range of stressors both as survivors and as relief workers; however, little is known about their longitudinal PTSD symptoms. METHODS: The participants were 745 local municipality and hospital medical workers [average age: 43.6 ± 9.5 years, range: 20 - 66 years; 306 (59%) women] involved in disaster recovery duties following the GEJE. PTSD symptoms were measured using the Japanese version of the PTSD Checklist Specific Version (PCL-S) at four time points: 14, 30, 43, and 54 months after the GEJE. Using group-based trajectory modeling, distinct trajectories were elucidated. RESULTS: We identified five distinct PTSD symptoms profiles: resistance (n = 467, 62.7%), subsyndromal (n = 181, 24.3%), recovery (n = 47, 6.3%), fluctuating (n = 26, 3.5%), and chronic (n = 24, 3.2%). The trajectories differed according to the post-disaster working conditions and personal disaster experiences. LIMITATIONS: Potential selection bias resulting from the limited number of participants who completed all waves. The survey was conducted in one region of the disaster area. CONCLUSIONS: The majority of participants remained stable, with a relatively small group classified as chronic and fluctuating. Our results highlight the importance of improved working conditions and sustained monitoring of workers responding to natural disasters.

Dataset on mechanical, thermal and structural characterization of plant fiber-based biopolymers prepared by hot-pressing raw coconut coir, and milled powders of cotton, waste bagasse, wood, and bamboo.

This article presents experimental data on visual, mechanical, thermal, and structural characterization by hot-pressing four sources of milled plant powders and coconut fibers. It correlates chemical composition obtained by (FTIR), particle size, and reports bending strength, water resistance morphological (SEM) and thermal stability, structural properties (FTIR and XRD). It further supplements findings of the influence of microfibrillation and chemical composition on hot-pressing plant fibers as presented in the research article "Effects of Chemical Composition, Mild Alkaline Pretreatment and Particle Size on Mechanical, Thermal, and Structural Properties of Binderless Lignocellulosic Biopolymers Prepared by Hot-Pressing Raw Microfibrillated Phoenix Dactylifera and Cocos Nucifera Fibers and Leaves" [1]. For more insights into the difference among non-lignin-, lignin-, and semi lignin-based adhesion refer to the research article [1]. This dataset is made publicly available for potential reuse in recycling agricultural waste fibers for value-added materials.

Diabetes care providers' manual for disaster diabetes care.

To ensure that experiences and lessons learned from the unprecedented 2011 Great East Japan Earthquake are used to improve future disaster planning, the Japan Diabetes Society (JDS) launched the "Research and Survey Committee for Establishing Disaster Diabetes Care Systems Based on Relevant Findings from the Great East Japan Earthquake" under the supervision of the Chairman of the JDS. The Committee conducted a questionnaire survey among patients with diabetes, physicians, disaster medical assistance teams (DMATs), nurses, pharmacists, and nutritionists in disaster areas about the events they saw happening, the situations they found difficult to handle, and the needs that they felt required to be met during the 2011 Great East Japan Earthquake. A total of 3,481 completed questionnaires were received. Based on these and other experiences and lessons reported following the 2011 Great East Japan Earthquake and the 2004 Niigata-Chuetsu Earthquakes, the current "Manual for Disaster Diabetes Care" has been developed by the members of the Committee and other invited authors from relevant specialties. To our knowledge, the current Manual is the world's first to focus on emergency diabetes care, with this digest English version translated from the Japanese original. It is sincerely hoped that patients with diabetes and healthcare providers around the world will find this manual helpful in promoting disaster preparedness and implementing disaster relief.

Diabetes Care Providers' Manual for Disaster Diabetes Care.

To ensure that experiences and lessons learned from the unprecedented 2011 Great East Japan Earthquake are used to improve future disaster planning, the Japan Diabetes Society (JDS) launched the "Research and Survey Committee for Establishing Disaster Diabetes Care Systems Based on Relevant Findings from the Great East Japan Earthquake" under the supervision of the Chairman of the JDS. The Committee conducted a questionnaire survey among patients with diabetes, physicians, disaster medical assistance teams (DMATs), nurses, pharmacists, and nutritionists in disaster areas about the events they saw happening, the situations they found difficult to handle, and the needs that they felt required to be met during the 2011 Great East Japan Earthquake. A total of 3,481 completed questionnaires were received. Based on these and other experiences and lessons reported following the 2011 Great East Japan Earthquake and the 2004 Niigata-Chuetsu Earthquakes, the current "Manual for Disaster Diabetes Care" has been developed by the members of the Committee and other invited authors from relevant specialties. To our knowledge, the current Manual is the world's first to focus on emergency diabetes care, with this digest English version translated from the Japanese original. It is sincerely hoped that patients with diabetes and healthcare providers around the world will find this manual helpful in promoting disaster preparedness and implementing disaster relief.

Crucial role of peroxiredoxin III in placental antioxidant defense of mice.

We observed frequent stillbirth in peroxiredoxin III (PrxIII) knockout maternal mice. Quantitative real time PCR (qRT-PCR) and Western-blot analysis revealed increased oxidative stress in placentas that were deficient in PrxIII. We did not find significant difference between PrxIII knockout maternal mice and wild-type littermates in hematological parameters, fetal number, and embryonic development. Nevertheless, we noticed enhanced expression of PrxI in erythrocytes of pregnant knockout mice. Our results provided in vivo evidence that PrxIII played a crucial role in placental antioxidant defense. Up-regulation of PrxI might provide a compensation that protected erythrocytes against oxidative damage.

Application of heat shock promoter in transgenic zebrafish.

The heat shock promoter is useful for regulating transgene expression in small water-living organisms. In zebrafish embryos, downstream gene expression can be greatly induced throughout the body by raising the temperature from 28.5 degrees C to 38.0 degrees C. By manipulating the local temperature within an embryo, spatial control of transgene expression is also possible. One such way for inducing heat shock response in targeted cells is by using a laser microbeam under the microscope. In addition, random mosaic expression by transient gene expression and transplantation of the transgenic embryo into a wild type host can be considered a powerful tool for studying gene functions using this promoter. In this paper, we review the applications of the zebrafish heat shock protein promoter as a gene expression tool and for lineage labeling and transcription enhancer screening.

[Assessment of competence of predialysis chronic kidney disease stage 5 patients to make treatment decisions: preliminary report].

The competence to consent to treatment of 26 adults with stage 5 predialysis chronic kidney disease (CKD) (16 males, 10 females, age; 58 +/- 11 years, creatinine clearance; 10.1 +/- 3.9 mL/min)was assessed using two kinds of format: the MacArthur Competence Assessment Tool-Treatment (MacCAT-T) and mini-mental-state examination (MMSE). The MacCAT-T revealed poor ability for understanding(3.72 +/- 1.11 points; perfect score, 6 points), appreciating (2.88 +/- 0.88 points; perfect score, 4 points)and reasoning(4.30 +/- 2.11 points; perfect score, 8 points). The MMSE revealed poor performance on the attentional task. The level of attentional deficit was significantly related to both poor ability for understanding and reasoning (r = 0.432, p = 0.031 and r = 0.542, p = 0.014, respectively). These results suggest that the competence of predialysis CKD stage 5 patients to consent to treatment is impaired partly via an attentional deficit.

The reliability and validity for Japanese type 2 diabetes patients of the Japanese version of the acceptance and action diabetes questionnaire.

BACKGROUND: The purpose of this study was to determing which psychological traits of Japanese type 2 diabetes patients would provide reliability and validity to the Japanese version of the Acceptance and Action Diabetes Questionnaire (AADQ-J). METHODS: Various questionnaires were administered to type 2 diabetes patients who were registered on the database of the research service provider; data from a total of 600 patients (mean ± SD age was 57.50 ± 9.87 years, female 21.83%) were analyzed. RESULTS: Three items were excluded because of psychometric concerns related to the original 11-item AADQ. Confirmation factor analyses revealed that the eight-item version demonstrated the best indicators of a goodness of fit. The questionnaire showed adequate internal consistency. The questionnaire demonstrated high measurement accuracy in broad trait values by the test information function of Item Response Theory. The questionnaire showed stronger positive correlations with self-care activities and HbA1c than with diabetes distress and depressive mood. CONCLUSIONS: The eight-item Japanese version of AADQ has reliability and validity for type 2 diabetes patients.