Emerging Roles of RNA-Binding Proteins in Inner Ear Hair Cell Development and Regeneration.

RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.

The Adaptor Protein Lurap1 Is Required for Cell Cohesion during Epiboly Movement in Zebrafish.

Cell adhesion and polarized cellular behaviors play critical roles in a wide variety of morphogenetic events. In the zebrafish embryo, epiboly represents an important process of epithelial morphogenesis that involves differential cell adhesion and dynamic cell shape changes for coordinated movements of different cell populations, but the underlying mechanism remains poorly understood. The adaptor protein Lurap1 functions to link myotonic dystrophy kinase-related Rac/Cdc42-binding kinase with MYO18A for actomyosin retrograde flow in cell migration. We previously reported that it interacts with Dishevelled in convergence and extension movements during gastrulation. Here, we show that it regulates blastoderm cell adhesion and radial cell intercalation during epiboly. In zebrafish mutant embryos with loss of both maternal and zygotic Lurap1 function, deep cell multilayer of the blastoderm exhibit delayed epiboly with respect to the superficial layer. Time-lapse imaging reveals that these deep cells undergo unstable intercalation, which impedes their expansion over the yolk cell. Cell sorting and adhesion assays indicate reduced cellular cohesion of the blastoderm. These defects are correlated with disrupted cytoskeletal organization in the cortex of blastoderm cells. Thus, the present results extend our previous works by demonstrating that Lurap1 is required for cell adhesion and cell behavior changes to coordinate cell movements during epithelial morphogenesis. They provide insights for a further understanding of the regulation of cytoskeletal organization during gastrulation cell movements.

Syne2b/Nesprin-2 Is Required for Actin Organization and Epithelial Integrity During Epiboly Movement in Zebrafish.

Syne2b/nesprin-2 is a giant protein implicated in tethering the nucleus to the cytoskeleton and plays an important role in maintaining cellular architecture. Epiboly is a conserved morphogenetic movement that involves extensive spreading and thinning of the epithelial blastoderm to shape the embryo and organize the three germ layers. Dynamic cytoskeletal organization is critical for this process, but how it is regulated remains elusive. Here we generated a zebrafish syne2b mutant line and analyzed the effects of impaired Syne2b function during early development. By CRISPR/Cas9-mediated genome editing, we obtained a large deletion in the syne2b locus, predicted to cause truncation of the nuclear localization KASH domain in the translated protein. Maternal and zygotic syne2b embryos showed delayed epiboly initiation and progression without defects in embryonic patterning. Remarkably, disruption of Syne2b function severely impaired cytoskeletal organization across the embryo, leading to aberrant clustering of F-actin at multiple cell contact regions and abnormal cell shape changes. These caused disintegration of the epithelial blastoderm before the end of gastrulation in most severely affected embryos. Moreover, the migration of yolk nuclear syncytium also became defective, likely due to disorganized cytoskeletal networks at the blastoderm margin and in the yolk cell. These findings demonstrate an essential function of Syne2b in maintaining cytoskeletal architecture and epithelial integrity during epiboly movement.

Design and synthesis of novel xanthone-triazole derivatives as potential antidiabetic agents: α-Glucosidase inhibition and glucose uptake promotion.

Inhibiting the decomposition of carbohydrates into glucose or promoting glucose conversion is considered to be an effective treatment for type 2 diabetes. Herein, a series of novel xanthone-triazole derivatives were designed, synthesized, and their α-glucosidase inhibitory activities and glucose uptake in HepG2 cells were investigated. Most of the compounds showed better inhibitory activities than the parental compound a (1,3-dihydroxyxanthone, IC50 = 160.8 µM) and 1-deoxynojirimycin (positive control, IC50 = 59.5 µM) towards α-glucosidase. Compound 5e was the most potent inhibitor, with IC50 value of 2.06 µM. The kinetics of enzyme inhibition showed that compounds 5e, 5g, 5h, 6c, 6d, 6g and 6h were noncompetitive inhibitors, and molecular docking results were consistent with the noncompetitive property that these compounds bind to allosteric sites away from the active site (Asp214, Glu276 and Asp349). On the other hand, the glucose uptake assays exhibited that compounds 5e, 6a, 6c and 7g displayed high activities in promoting the glucose uptake. The cytotoxicity assays showed that most compounds were low-toxic to human normal hepatocyte cell line (LO2). These novel xanthone triazole derivatives exhibited dual therapeutic effects of α-glucosidase inhibition and glucose uptake promotion, thus they could be use as antidiabetic agents for developing novel drugs against type 2 diabetes.

Collagen triple helix repeat containing 1a (Cthrc1a) regulates cell adhesion and migration during gastrulation in zebrafish.

Cell adhesion and migration are key cell behaviours during gastrulation in early embryos and metastasis in cancers. Cthrc1 is a secreted protein highly conserved among vertebrates; it is upregulated in injured and diseased arteries, as well as in malignant cancers. There is increasing evidence showing that its expression and activity are associated with cancer progression and inflammatory diseases. However, the mechanism by which it regulates cell migration, and its implication during early development remains unclear. Here we show that zebrafish Cthrc1a is expressed in hypoblast cells, and is required for cell adhesion and migration during gastrulation. Knockdown of cthrc1a in whole embryo inhibits epiboly and convergent extension movements, and reduces the elongation of anteroposterior axis. Cell adhesion assay indicates that Cthrc1a is necessary for mesendoderm cells to interact with fibronectin-coated substratum, and to extend polarised cellular protrusions. Moreover, secreted Cthrc1a proteins diffuse efficiently between blastoderm cells and are recruited by neighbouring cells in an integrin-dependent manner. Consistently, there exists a functional interaction between Cthrc1a and integrin ß1 in anteroposterior axis elongation. These results provide insight into the function of Cthrc1a in the regulation of cell adhesion and migration during embryonic axis elongation.

Mutational analysis of dishevelled genes in zebrafish reveals distinct functions in embryonic patterning and gastrulation cell movements.

Wnt signaling plays critical roles in dorsoventral fate specification and anteroposterior patterning, as well as in morphogenetic cell movements. Dishevelled proteins, or Dvls, mediate the activation of Wnt/ß-catenin and Wnt/planar cell polarity pathways. There are at least three highly conserved Dvl proteins in vertebrates, but the implication of each Dvl in key early developmental processes remains poorly understood. In this study, we use genome-editing approach to generate different combinations of maternal and zygotic dvl mutants in zebrafish, and examine their functions during early development. Maternal transcripts for dvl2 and dvl3a are most abundantly expressed, whereas the transcript levels of other dvl genes are negligible. Phenotypic and molecular analyses show that early dorsal fate specification is not affected in maternal and zygotic dvl2 and dvl3a double mutants, suggesting that the two proteins may be dispensable for the activation of maternal Wnt/ß-catenin signaling. Interestingly, convergence and extension movements and anteroposterior patterning require both maternal and the zygotic functions of Dvl2 and Dvl3a, but these processes are more sensitive to Dvl2 dosage. Zygotic dvl2 and dvl3a double mutants display mild axis extension defect with correct anteroposterior patterning. However, maternal and zygotic double mutants exhibit most strongly impaired convergence and extension movements, severe trunk and posterior deficiencies, and frequent occurrence of cyclopia and craniofacial defects. Our results suggest that Dvl2 and Dvl3a products are required for the activation of zygotic Wnt/ß-catenin signaling and Wnt/planar cell polarity pathway, and regulate zygotic developmental processes in a dosage-dependent manner. This work provides insight into the mechanisms of Dvl-mediated Wnt signaling pathways during early vertebrate development.

Aucubin protects against lipopolysaccharide-induced acute pulmonary injury through regulating Nrf2 and AMPK pathways.

Aucubin (Ai), a natural compound isolated from plants, including Aucuba japonica and Eucommia ulmoides, shows significant anti-inflammatory and anti-oxidative bioactivities. Here, we attempted to explore the protect effects of Ai on LPS-induced acute lung injury (ALI). Our results indicated that Ai increased the survival rate and ameliorated pathogenic processes in lipopolysaccharide (LPS)-induced mice. However, nuclear factor erythroid 2-related factor 2 (Nrf2) deletion may impede protective effect of Ai. Additionally, Ai reduced oxidative stress by down-regulating malondialdehyde (MDA) and O2· activity, and enhancing Nrf2-targeted signals, including heme oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO-1). Also, Ai inhibited pro-inflammatory cytokines and phosphorylated-nuclear factor-κB (NF-κB) expression in LPS-administrated mice. However, these protective effects of Ai were suppressed in Nrf2-knockout mice. Importantly, Nrf2-deficiency showed no effects on phosphorylated AMP-activated protein kinase (p-AMPK) expression in mice treated with LPS and Ai. Similarly, in LPS-induced macrophages, Ai reduced reactive oxygen species (ROS) generation, elevated NQO-1 and HO-1 expression. LPS-stimulated pro-inflammatory cytokines and p-NF-κB were reversed by Ai. Of note, we found that Ai-induced Nrf2 activation was dependent on AMPK activation. Suppression of AMPK levels may inhibit Nrf2 activation, finally leading to up regulation of inflammatory response and oxidative stress. Thus, our findings indicated the crosstalk between Nrf2 and AMPK signaling pathways, and the interaction was essential for the anti-oxidant and anti-inflammatory effects of Ai in LPS-induced macrophages, which might be beneficial for finding new treatments against ALI.

Mutation of frizzled8a delays neural retinal cell differentiation and results in microphthalmia in zebrafish.

Eye formation in vertebrates involves highly coordinated processes, and the differentiation of various eye tissues is regulated by conserved transcription factors and signalling pathways. Mutations in key genes of the regulatory hierarchy lead to congenital disorders and ocular diseases. The Wnt signalling pathway plays a key role in different aspects of eye development, and several Wnt receptors of the Frizzled family are required for eye specification and differentiation. However, their precise function in these processes remains elusive. Here we show that mutation of the frizzled8a gene in zebrafish leads to microphthalmia. The differentiation of retinal layers is delayed, and retinal progenitor cells in microphthalmic embryos fail to normally exit the cell cycle to enter into the post-mitotic state. They exhibit delayed differentiation associated with enhanced apoptosis, which results in abnormal lamination of retinal layers, reduction in the number of retinal cells, and small eye phenotype. These findings suggest that Frizzled8a plays a specific role in regulating cell cycle progression during the differentiation of retinal progenitor cells.

Transplantation of Zebrafish Cells by Conventional Pneumatic Microinjector.

Generating chimeric zebrafish by transplantation is extremely useful for live imaging in developmental, stem cell, and cancer biology, and to answer the questions of how cells acquire, keep, and/or change their fate. However, as it is technically challenging, the use of transplantation approach remains very limited by the zebrafish community. In this study, we show that this cell grafting operation can be easily achieved by using a conventional pneumatic microinjector normally used for microinjections. Compared with previously published protocols, which need additional transplantation apparatus, this alternative transplantation method works well, but needs a simpler experimental setup, and is more accessible to all investigators.

Leucine repeat adaptor protein 1 interacts with Dishevelled to regulate gastrulation cell movements in zebrafish.

Gastrulation is a fundamental morphogenetic event that requires polarised cell behaviours for coordinated asymmetric cell movements. Wnt/PCP signalling plays a critical role in this process. Dishevelled is an important conserved scaffold protein that relays Wnt/PCP signals from membrane receptors to the modulation of cytoskeleton organisation. However, it remains unclear how its activity is regulated for the activation of downstream effectors. Here, we report that Lurap1 is a Dishevelled-interacting protein that regulates Wnt/PCP signalling in convergence and extension movements during vertebrate gastrulation. Its loss-of-function leads to enhanced Dishevelled membrane localisation and increased JNK activity. In maternal-zygotic lurap1 mutant zebrafish embryos, cell polarity and directional movement are disrupted. Time-lapse analyses indicate that Lurap1, Dishevelled, and JNK functionally interact to orchestrate polarised cellular protrusive activity, and Lurap1 is required for coordinated centriole/MTOC positioning in movement cells. These findings demonstrate that Lurap1 functions to regulate cellular polarisation and motile behaviours during gastrulation movements.

Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/ß-catenin and Wnt/planar cell polarity (PCP) signaling pathways.

Dishevelled (Dvl) is a key intracellular signaling molecule that mediates the activation of divergent Wnt pathways. It contains three highly conserved domains known as DIX, PDZ, and DEP, the functions of which have been well characterized in ß-catenin-dependent canonical and ß-catenin-independent noncanonical Wnt signaling. The C-terminal region is also highly conserved from invertebrates to vertebrates. However, its function in regulating the activation of different Wnt signals remains unclear. We reported previously that Dvl conformational change triggered by the highly conserved PDZ-binding C terminus is important for the pathway specificity. Here we provide further evidence demonstrating that binding of the C terminus to the PDZ domain results in Dvl autoinhibition in the Wnt signaling pathways. Therefore, the forced binding of the C terminus to the PDZ domain reduces the activity of Dvl in noncanonical Wnt signaling, whereas obstruction of this interaction releases Dvl autoinhibition, impairs its functional interaction with LRP6 in canonical Wnt signaling, and increases its specificity in noncanonical Wnt signaling, which is closely correlated with an enhanced Dvl membrane localization. Our findings highlight the importance of the C terminus in keeping Dvl in an appropriate autoinhibited state, accessible for regulation by other partners to switch pathway specificity. Particularly, the C-terminally tagged Dvl fusion proteins that have been widely used to study the function and cellular localization of Dvl may not truly represent the wild-type Dvl because those proteins cannot be autoinhibited.

Flexible, Luminescent Metal-Organic Frameworks Showing Synergistic Solid-Solution Effects on Porosity and Sensitivity.

Mixing molecular building blocks in the solid solution manner is a valuable strategy to obtain structures and properties in between the isostructural parent metal-organic frameworks (MOFs). We report nonlinear/synergistic solid-solution effects using highly related yet non-isostructural, phosphorescent CuI triazolate frameworks as parent phases. Near the phase boundaries associated with conformational diversity and ligand heterogeneity, the porosity (+150 %) and optical O2 sensitivity (410 times, limit of detection 0.07 ppm) can be drastically improved from the best-performing parent MOFs and even exceeds the records hold by precious-metal complexes (3 ppm) and C70 (0.2 ppm).

Identification of novel MYO18A interaction partners required for myoblast adhesion and muscle integrity.

The unconventional myosin MYO18A that contains a PDZ domain is required for muscle integrity during zebrafish development. However, the mechanism by which it functions in myofibers is not clear. The presence of a PDZ domain suggests that MYO18A may interact with other partners to perform muscle-specific functions. Here we performed double-hybrid screening and co-immunoprecipitation to identify MYO18A-interacting proteins, and have identified p190RhoGEF and Golgin45 as novel partners for the MYO18A PDZ domain. We have also identified Lurap1, which was previously shown to bind MYO18A. Functional analyses indicate that, similarly as myo18a, knockdown of lurap1, p190RhoGEF and Golgin45 by morpholino oligonucleotides disrupts dystrophin localization at the sarcolemma and produces muscle lesions. Simultaneous knockdown of myo18a with either of these genes severely disrupts myofiber integrity and dystrophin localization, suggesting that they may function similarly to maintain myofiber integrity. We further show that MYO18A and its interaction partners are required for adhesion of myoblasts to extracellular matrix, and for the formation of the Golgi apparatus and organization of F-actin bundles in myoblast cells. These findings suggest that MYO18A has the potential to form a multiprotein complex that links the Golgi apparatus to F-actin, which regulates muscle integrity and function during early development.

Phosphorescence doping in a flexible ultramicroporous framework for high and tunable oxygen sensing efficiency.

Doping very small amounts of Ru(II) into a flexible, ultramicroporous, fluorescent Zn(II) coordination polymer produced phosphorescent materials with very high and tunable oxygen quenching efficiency; and a simple color-changing ratiometric oxygen sensor has been constructed.

Magnetic variation induced by structural transformation from coordination chains to layers upon dehydration.

The reaction of CoBr(2), 1,2-di(4H-1,2,4-triazol-4-yl)diazene (bta) and KSCN yielded a one-dimensional coordination polymer [Co(SCN)(2)(bta)(H(2)O)(2)] with water molecules coordinated to the metal ions. After dehydration at 100 °C, the compound transformed into a layered coordination polymer [Co(SCN)(2)(bta)], whose structure was determined by powder X-ray diffraction based on the single-crystal structure of another layered coordination polymer [Cd(SCN)(2)(bta)]. Interestingly, a magnetic variation from a simple paramagnet to an antiferromagnetic ordered phase of a single-chain-magnet that exhibits both metamagnetic behaviour and slow magnetic relaxation was observed upon the dehydration process.

Solvent/additive-free synthesis of porous/zeolitic metal azolate frameworks from metal oxide/hydroxide.

Porous/zeolitic metal azolate frameworks may be efficiently prepared, with water as the only byproduct, by heating a mixture of metal oxide/hydroxide and azole ligand.

Porous ionic/molecular crystal composed of highly symmetric magnetic clusters.

A porous ionic/molecular crystal composed of discrete triakis tetrahedral Co(8) clusters with an ideal T(d) symmetry shows interesting magnetism and porosity.

Spin canting and/or metamagnetic behaviours of four isostructural grid-type coordination networks.

Four isostructural, two-dimensional (2D) grid-type coordination polymers bridged by carboxylate groups, namely [Co(8-qoac)(HCOO)] (1 x Co), [Mn(8-qoac)(HCOO)] (1 x Mn), [Co(8-qoac)(CH3COO)] (2 x Co) and [Mn(8-qoac)(CH3COO)] (2 x Mn) (8-qoacH = quinoline-8-oxy-acetate acid), were constructed to study the modulation effects of spin carriers and interlayer interactions on the magnetic behaviours. The grid-type layers in these compounds are composed of octahedral metal ions bridged by carboxylate groups of 8-qoac ligands in the anti-anti fashion and formate/acetate in the syn-anti fashion, and these 2D layers are further packed into 3D structures through pi-pi stacking and hydrogen-bonding interactions. The Mn(II) compounds show spin canting antiferromagnetic behaviours, while the Co(II) compounds exhibit not only spin canting but also metamagnetic behaviours. Moreover, the critical fields of 1 x Co and 2 x Co are changed from 200 Oe to 6000 Oe for the stronger interlayer interaction with the second ligand changing from formate to acetate.

Single crystal-to-single crystal transformation from ferromagnetic discrete molecules to a spin-canting antiferromagnetic layer.

A unique single crystal-to-single crystal transformation from 0D discrete molecules to a 2D coordination polymer exhibits magnetic property changes from a short-range ferromagnetic coupling to a long-range canting antiferromagnetic ordering.