A simple and rapid assay of lysosomal-targeting CDy6 for long-term real-time viability assessments in 2D and 3D in vitro culture models.

CDy6, a BODIPY-derived compound, is used to label lysosomes and visualize proliferating cells. However, its effectiveness in long-term, real-time cell viability assays using 2D or 3D cell culture models is unclear. We evaluated the suitability of CDy6 by assessing cell health using human keratinocyte and fibroblast cell lines in both models. Cells were stained with CDy6 or other dyes and fluorescent images were obtained with confocal microscopy. CLV extracts derived from CDy6-stained HaCaT cells were also dissolved with DMSO and analyzed using a spectrometer. Furthermore, we added CDy6-stained collagen hydrogels to CCD-986sk cells, loaded them into a frame construction to establish a 3D dermal layer for long-term culture, and analyzed the status of the CLVs. The CLV method, also measured using a spectrometer, yielded results similar to MTT assay for validating viability. In contrast to calcein AM staining, the CLV method allows for both absorbance measurement and imaging under short-term and long-term culture conditions with less cytotoxicity. In conclusion, the CLV method provides a simple and sensitive tool for assessing the status of live cells in 2D and 3D in vitro cell culture models and can be used as an alternative to animal testing, such as with 3D artificial skin models.

UBAP2 plays a role in bone homeostasis through the regulation of osteoblastogenesis and osteoclastogenesis.

Osteoporosis is a condition characterized by decreased bone mineral density (BMD) and reduced bone strength, leading to an increased risk of fractures. Here, to identify novel risk variants for susceptibility to osteoporosis-related traits, an exome-wide association study is performed with 6,485 exonic single nucleotide polymorphisms (SNPs) in 2,666 women of two Korean study cohorts. The rs2781 SNP in UBAP2 gene is suggestively associated with osteoporosis and BMD with p-values of 6.1 × 10-7 (odds ratio = 1.72) and 1.1 × 10-7 in the case-control and quantitative analyzes, respectively. Knockdown of Ubap2 in mouse cells decreases osteoblastogenesis and increases osteoclastogenesis, and knockdown of ubap2 in zebrafish reveals abnormal bone formation. Ubap2 expression is associated with E-cadherin (Cdh1) and Fra1 (Fosl1) expression in the osteclastogenesis-induced monocytes. UBAP2 mRNA levels are significantly reduced in bone marrow, but increased in peripheral blood, from women with osteoporosis compared to controls. UBAP2 protein level is correlated with the blood plasma level of the representative osteoporosis biomarker osteocalcin. These results suggest that UBAP2 has a critical role in bone homeostasis through the regulation of bone remodeling.

FIR-preconditioning promotes Akt-mTOR-exosome manufacture in cooperation with MITF to boost resilience of rat bone marrow-derived stem cells.

A previous study from our laboratory observed the protective effects of far-infrared irradiation (FIR) on bone marrow-derived stem cells (BMSCs) against oxidative stress. However, it remains unknown precisely how FIR influences BMSC survival. We identify an unexpected route among the expression of MITF, BCL2, mTOR, and exosome in FIR-preconditioned BMSCs. MITF siRNA demonstrated that loss of MITF expression not only inhibited cell proliferation but also reduced the FIR-mediated expression of mTOR, BCL2, and exosome. mTOR signaling pathways have been implicated in cell growth, proliferation, and survival. We also found that rapamycin, a potent and selective inhibitor of mTOR, when combined with MITF siRNA, repressed FIR-mediated CD63 and BCL2 expression. In addition, FIR-preconditioned BMSCs demonstrated more tolerance in multiple stressful environments than untreated BMSCs. The elevated exosomes in conditioned medium derived from FIR-preconditioned BMSCs also repaired H9c2 cells that sustained cellular damage after subjected to an array of environmental stress conditions. Taken together, these results reveal a possible mechanism about how FIR-preconditioned BMSCs and its conditioned media could contribute to cellular resilience during environmental changes via MITF-Akt-mTOR associated with exosome manufacture. FIR preconditioning could thus complement and improve therapeutic applications of BMSCs on outcomes of various disorders.

Low-molecular-weight fucoidan inhibits the proliferation of melanoma via Bcl-2 phosphorylation and PTEN/AKT pathway.

Fucoidan, a sulfate polysaccharide obtained from brown seaweed, has various bioactive properties, including anti-inflammatory, anti-cancer, anti-viral, anti-oxidant, anti-coagulant, anti-thrombotic, anti-angiogenic, and anti-Helicobacter pylori properties. However, the effects of low-molecular-weight fucoidan (LMW-F) on melanoma cell lines and three dimensional (3D) cell culture models are not well understood. This study aimed to investigate the effects of LMW-F on A375 human melanoma cells and cryopreserved biospecimens derived from patients with advanced melanoma. Ultrasonic wave was used to fragment fucoidan derived from Fucus vesiculosus into smaller LMW-F. MTT and live/dead assays showed that LMW-F inhibited cell proliferation in both A375 cells and patient-derived melanoma explants in a 3D-printed collagen scaffold. The PTEN/AKT pathway was found to be involved in the anti-melanoma effects of fucoidan. Western blot analysis revealed that LMW-F reduced the phosphorylation of Bcl-2 at Thr 56, which was associated with the prevention of anti-apoptotic activity of cancer cells. Our findings suggested that LMW-F could enhance anti-melanoma chemotherapy and improve the outcomes of patients with melanoma resistance.

Transwell-Hypoxia Method Facilitates the Outgrowth of 3D-Printed Collagen Scaffolds Loaded with Cryopreserved Patient-Derived Melanoma Explants.

A previous study from our laboratory demonstrated the effects of in vitro three-dimensional (3D)-printed collagen scaffolds on the maintenance of cryopreserved patient-derived melanoma explants (PDMEs). However, it remains unknown whether 3D-printed collagen scaffolds (3D-PCSs) can be harmonized with any external culture conditions to increase the growth of cryopreserved PDMEs. In this study, 3D-PCSs were manufactured with a 3DX bioprinter. The 3D-printed collagen scaffold-on-frame construction was loaded with fragments of cryopreserved PDMEs (approximately 1-2 mm). 3D-PCSs loaded with patient-derived melanoma explants (3D-PCS-PDMEs) were incubated using two types of methods: (1) in transwells in the presence of a low concentration of oxygen (transwell-hypoxia method) and (2) using a traditional adherent attached to the bottom flat surface of a standard culture dish (traditional flat condition). In addition, we used six different types of media (DMEM high glucose, MEM α, DMEM/F12, RPMI1640, fibroblast basal medium (FBM), and SBM (stem cell basal medium)) for 7 days. The results reveal that the culture conditions of MEM α, DMEM/F12, and FBM using the transwell-hypoxia method show greater synergic effects on the outgrowth of the 3D-PCS-PDME compared to the traditional flat condition. In addition, the transwell-hypoxia method shows a higher expression of the MMP14 gene and the multidrug-resistant gene product 1 (MDR1) than in the typical culture method. Taken together, our findings suggest that the transwell-hypoxia method could serve as an improved, 3D alternative to animal-free testing that better mimics the skin's microenvironment using in vitro PDMEs.

Differential Clearance of Aß Species from the Brain by Brain Lymphatic Endothelial Cells in Zebrafish.

The failure of amyloid beta (Aß) clearance is a major cause of Alzheimer's disease, and the brain lymphatic systems play a crucial role in clearing toxic proteins. Recently, brain lymphatic endothelial cells (BLECs), a non-lumenized lymphatic cell in the vertebrate brain, was identified, but Aß clearance via this novel cell is not fully understood. We established an in vivo zebrafish model using fluorescently labeled Aß42 to investigate the role of BLECs in Aß clearance. We discovered the efficient clearance of monomeric Aß42 (mAß42) compared to oligomeric Aß42 (oAß42), which was illustrated by the selective uptake of mAß42 by BLECs and peripheral transport. The genetic depletion, pharmacological inhibition via the blocking of the mannose receptor, or the laser ablation of BLECs resulted in the defective clearance of mAß42. The treatment with an Aß disaggregating agent facilitated the internalization of oAß42 into BLECs and improved the peripheral transport. Our findings reveal a new role of BLECs in the differential clearance of mAß42 from the brain and provide a novel therapeutic strategy based on promoting Aß clearance.

Production of Multiple Cell-Laden Microtissue Spheroids with a Biomimetic Hepatic-Lobule-Like Structure.

The construction of an in vitro 3D cellular model to mimic the human liver is highly desired for drug discovery and clinical applications, such as patient-specific treatment and cell-based therapy in regenerative medicine. However, current bioprinting strategies are limited in their ability to generate multiple cell-laden microtissues with biomimetic structures. This study presents a method for producing hepatic-lobule-like microtissue spheroids using a bioprinting system incorporating a precursor cartridge and microfluidic emulsification system. The multiple cell-laden microtissue spheroids can be successfully generated at a speed of approximately 45 spheroids min-1 and with a uniform diameter. Hepatic and endothelial cells are patterned in a microtissue spheroid with the biomimetic structure of a liver lobule. The spheroids allow long-term culture with high cell viability, and the structural integrity is maintained longer than that of non-structured spheroids. Furthermore, structured spheroids show high MRP2, albumin, and CD31 expression levels. In addition, the in vivo study reveals that structured microtissue spheroids are stably engrafted. These results demonstrate that the method provides a valuable 3D structured microtissue spheroid model with lobule-like constructs and liver functions.

Optogenetic Manipulation of Olfactory Responses in Transgenic Zebrafish: A Neurobiological and Behavioral Study.

Olfaction is an important neural system for survival and fundamental behaviors such as predator avoidance, food finding, memory formation, reproduction, and social communication. However, the neural circuits and pathways associated with the olfactory system in various behaviors are not fully understood. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of neuronal circuits have created new opportunities to understand such neural circuits. Here, we generated a transgenic zebrafish to manipulate olfactory signal optically, expressing the Channelrhodopsin (ChR2) under the control of the olfactory specific promoter, omp. We observed light-induced neuronal activity of olfactory system in the transgenic fish by examining c-fos expression, and a calcium indicator suggesting that blue light stimulation caused activation of olfactory neurons in a non-invasive manner. To examine whether the photo-activation of olfactory sensory neurons affect behavior of zebrafish larvae, we devised a behavioral choice paradigm and tested how zebrafish larvae choose between two conflicting sensory cues, an aversive odor or the naturally preferred phototaxis. We found that when the conflicting cues (the preferred light and aversive odor) were presented together simultaneously, zebrafish larvae swam away from the aversive odor. However, the transgenic fish with photo-activation were insensitive to the aversive odor and exhibited olfactory desensitization upon optical stimulation of ChR2. These results show that an aversive olfactory stimulus can override phototaxis, and that olfaction is important in decision making in zebrafish. This new transgenic model will be useful for the analysis of olfaction related behaviors and for the dissection of underlying neural circuits.

Genetic Approaches Using Zebrafish to Study the Microbiota-Gut-Brain Axis in Neurological Disorders.

The microbiota-gut-brain axis (MGBA) is a bidirectional signaling pathway mediating the interaction of the microbiota, the intestine, and the central nervous system. While the MGBA plays a pivotal role in normal development and physiology of the nervous and gastrointestinal system of the host, its dysfunction has been strongly implicated in neurological disorders, where intestinal dysbiosis and derived metabolites cause barrier permeability defects and elicit local inflammation of the gastrointestinal tract, concomitant with increased pro-inflammatory cytokines, mobilization and infiltration of immune cells into the brain, and the dysregulated activation of the vagus nerve, culminating in neuroinflammation and neuronal dysfunction of the brain and behavioral abnormalities. In this topical review, we summarize recent findings in human and animal models regarding the roles of the MGBA in physiological and neuropathological conditions, and discuss the molecular, genetic, and neurobehavioral characteristics of zebrafish as an animal model to study the MGBA. The exploitation of zebrafish as an amenable genetic model combined with in vivo imaging capabilities and gnotobiotic approaches at the whole organism level may reveal novel mechanistic insights into microbiota-gut-brain interactions, especially in the context of neurological disorders such as autism spectrum disorder and Alzheimer's disease.

3D-Printed Collagen Scaffolds Promote Maintenance of Cryopreserved Patients-Derived Melanoma Explants.

The development of an in vitro three-dimensional (3D) culture system with cryopreserved biospecimens could accelerate experimental research screening anticancer drugs, potentially reducing costs and time bench-to-beside. However, minimal research has explored the application of 3D bioprinting-based in vitro cancer models to cryopreserved biospecimens derived from patients with advanced melanoma. We investigated whether 3D-printed collagen scaffolds enable the propagation and maintenance of patient-derived melanoma explants (PDMEs). 3D-printed collagen scaffolds were fabricated with a 3DX bioprinter. After thawing, fragments from cryopreserved PDMEs (approximately 1-2 mm) were seeded onto the 3D-printed collagen scaffolds, and incubated for 7 to 21 days. The survival rate was determined with MTT and live and dead assays. Western blot analysis and immunohistochemistry staining was used to express the function of cryopreserved PDMEs. The results show that 3D-printed collagen scaffolds could improve the maintenance and survival rate of cryopreserved PDME more than 2D culture. MITF, Mel A, and S100 are well-known melanoma biomarkers. In agreement with these observations, 3D-printed collagen scaffolds retained the expression of melanoma biomarkers in cryopreserved PDME for 21 days. Our findings provide insight into the application of 3D-printed collagen scaffolds for closely mimicking the 3D architecture of melanoma and its microenvironment using cryopreserved biospecimens.

Substance P enhances the local activation of NK1R-expressing c-kit+ cardiac progenitor cells in right atrium of ischemia/reperfusion-injured heart.

BACKGROUND: Localization of neurokinin 1 receptor (NK1R), the endogenous receptor for neuropeptide substance P (SP), has already been described for the right atrium (RA) of the heart. However, the biological role of SP/NK1R signal pathways in the RA remains unclear. Sprague-Dawley rats were randomly divided into 4 groups (n = 22 each); subjected to sham, ischemia/reperfusion-injury (I/R), I/R with 5 nmole/kg SP injection (SP + I/R), and SP + I/R with 1 mg/kg RP67580 injection (RP, a selective non-peptide tachykinin NK1R antagonist) (RP/SP + I/R). The left anterior descending coronary artery was occluded for 40 min followed by 1 day reperfusion with SP or SP + RP or without either. After 1 day, both atria and ventricles as well as the heart apexes were collected. RESULTS: SP promoted the expression of c-Kit, GATA4, Oct4, Nanog, and Sox2 in only the RA of the SP + I/R rats via NK1R activation. In agreement with these observations, NK1R-expressing c-Kit+ Nkx2.5+GATA4+ cardiac progenitor cells (CPCs) in the ex vivo RA explant outgrowth assay markedly migrated out from RA1 day SP + I/R approximately 2-fold increase more than RA1 day I/R. Treatment of SP promoted proliferation, migration, cardiosphere formation, and potential to differentiate into cardiomyocytes. Using RP inhibitor, NK1R antagonist not only inhibited cell proliferation and migration but also reduced the formation of cardiosphere and differentiation of c-Kit+ CPCs. CONCLUSION: SP/NK1R might play a role as a key mediator involved in the cellular response to c-Kit+ CPC expansion in RA of the heart within 24 h after I/R.

Substance P Administered after Myocardial Infarction Upregulates Microphthalmia-Associated Transcription Factor, GATA4, and the Expansion of c-Kit+ Cells.

Microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper transcription factor, can govern gene expression by binding to E box elements in the promoter region of its target gene. Although high levels of MITF have been observed in cardiomyocytes and the heart, the role of MITF after myocardial infarction (MI) remains unclear. We investigated the association between substance P (SP)/neurokinin-1 receptor (NK1R) signaling and MITF expression after MI. Male Sprague-Dawley rats (8 weeks) were randomly divided in two groups: ischemia/reperfusion injury (I/R) and SP injection (5 nmol/kg, SP+I/R). At the end of 7 days, the left ventricle (LV; LV7daysI/R, LV7daysSP+I/R) and infarct-related areas (IA; IA7daysI/R, IA7daysSP+I/R) from the hearts were collected. Immunofluorescence staining demonstrated that the LV7daysSP+I/R had a larger population of c-Kit+ GATA4high cells, which markedly upregulated MITF, c-Kit, and GATA4. c-Kit+ cells in the explant-derived cells (EDCs) derived from IA7daysSP+I/R migrated more widely than EDCs IA7daysI/R. Immunofluorescence staining, western blot analysis, and qRT-PCR assay showed that SP-treated c-Kit+ cells exhibited a high expression of c-Kit, GATA4, and MITF. FTY720 (a MITF inhibitor), RP67580 (NK1R inhibitor), or both inhibited the migration and proliferation of c-Kit+ cells increased by SP and blocked the upregulation of c-Kit, GATA4, and MITF. Overall, we suggest that MITF might be a potential regulator in SP-mediated c-Kit+ cell expansion post-MI via c-Kit and GATA4.

Targeted knockout of a chemokine-like gene increases anxiety and fear responses.

Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Preconditioning with far-infrared irradiation enhances proliferation, cell survival, and migration of rat bone marrow-derived stem cells via CXCR4-ERK pathways.

Far-infrared radiation (FIR) has been shown to exert positive effects on the cardiovascular system. However, the biological effects of FIR on bone marrow-derived stem cells (BMSCs) are not understood. In the present study, BMSCs were isolated from rat femur bone marrow and cultured in vitro. To investigate the effects of an FIR generator with an energy flux of 0.13 mW/cm2 on rat BMSCs, survival of BMSCs was measured by crystal violet staining, and cell proliferation was additionally measured using Ez-Cytox cell viability, EdU, and Brd U assays. FIR preconditioning was found to significantly increase BMSC proliferation and survival against H2O2. The scratch and transwell migration assays showed that FIR preconditioning resulted in an increase in BMSC migration. qRT-PCR and Western blot analyses demonstrated that FIR upregulated Nanog, Sox2, c-Kit, Nkx2.5, and CXCR4 at both the mRNA and protein levels. Consistent with these observations, PD98059 (an ERK inhibitor) and AMD3100 (a CXCR4 inhibitor) prevented the activation of CXCR4/ERK and blocked the cell proliferation and migration induced by FIR. Overall, these findings provide the first evidence that FIR confers a real and significant benefit on the preconditioning of BMSCs, and might lead to novel strategies for improving BMSC therapy for cardiac ischemia.

Zebrafish knockout of Down syndrome gene, DYRK1A, shows social impairments relevant to autism.

BACKGROUND: DYRK1A maps to the Down syndrome critical region at 21q22. Mutations in this kinase-encoding gene have been reported to cause microcephaly associated with either intellectual disability or autism in humans. Intellectual disability accompanied by microcephaly was recapitulated in a murine model by overexpressing Dyrk1a which mimicked Down syndrome phenotypes. However, given embryonic lethality in homozygous knockout (KO) mice, no murine model studies could present sufficient evidence to link Dyrk1a dysfunction with autism. To understand the molecular mechanisms underlying microcephaly and autism spectrum disorders (ASD), we established an in vivo dyrk1aa KO model using zebrafish. METHODS: We identified a patient with a mutation in the DYRK1A gene using microarray analysis. Circumventing the barrier of murine model studies, we generated a dyrk1aa KO zebrafish using transcription activator-like effector nuclease (TALEN)-mediated genome editing. For social behavioral tests, we have established a social interaction test, shoaling assay, and group behavior assay. For molecular analysis, we examined the neuronal activity in specific brain regions of dyrk1aa KO zebrafish through in situ hybridization with various probes including c-fos and crh which are the molecular markers for stress response. RESULTS: Microarray detected an intragenic microdeletion of DYRK1A in an individual with microcephaly and autism. From behavioral tests of social interaction and group behavior, dyrk1aa KO zebrafish exhibited social impairments that reproduce human phenotypes of autism in a vertebrate animal model. Social impairment in dyrk1aa KO zebrafish was further confirmed by molecular analysis of c-fos and crh expression. Transcriptional expression of c-fos and crh was lower than that of wild type fish in specific hypothalamic regions, suggesting that KO fish brains are less activated by social context. CONCLUSIONS: In this study, we established a zebrafish model to validate a candidate gene for autism in a vertebrate animal. These results illustrate the functional deficiency of DYRK1A as an underlying disease mechanism for autism. We also propose simple social behavioral assays as a tool for the broader study of autism candidate genes.

Ottogi Inhibits Wnt/ß-catenin Signaling by Regulating Cell Membrane Trafficking of Frizzled8.

Wnt signaling controls critical developmental processes including tissue/body patterning. Here we report the identification of a novel regulator of Wnt signaling, OTTOGI (OTG), isolated from a large-scale expression screening of human cDNAs in zebrafish embryos. Overexpression of OTG in zebrafish embryos caused dorso-anteriorized phenotype, inhibited the expression of Wnt target genes, and prevented nuclear accumulation of ß-catenin. Conversely, knockdown of zebrafish otg using specific antisense morpholino promoted nuclear accumulation of ß-catenin and caused ventralization. However, OTG failed to rescue headless-like phenotype induced by inhibition of GSK-3ß activity, suggesting that OTG acts upstream of GSK-3ß. OTG bound specifically to Frizzled8 (Fz8) receptor and caused retention of Fz8 in the endoplasmic reticulum possibly by preventing N-linked glycosylation of Fz8. Taken together, our data indicate that OTG functions as a novel negative regulator of Wnt signaling during development by the modulation of cell surface expression of Fz receptor.

Corrigendum to "Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish".

[This corrects the article DOI: 10.1155/2016/1473578.].

Cardioprotective effects of PKG activation by soluble GC activator, BAY 60-2770, in ischemia-reperfusion-injured rat hearts.

Soluble guanylate cyclase (sGC) has been suggested as a therapeutic target for cardiac ischemia-reperfusion (IR) injury. Until now, the molecular mechanism of BAY 60-2770, a sGC activator, in cardiac IR injury has not been assessed. To identify the cardioprotective effects of BAY 60-2770 in IR-injured rat hearts, IR injury was established by occlusion of LAD for 40 min and reperfusion for 7 days, and the effects of BAY 60-2770 on myocardial protection were assessed by echocardiography and TTC staining. 5 nM and 5 µM of BAY 60-2770 were perfused into isolated rat hearts in a Langendorff system. After 10- or 30-min reperfusion with BAY 60-2770, cGMP and cAMP concentrations and PKG activation status were examined. Hearts were also perfused with 1 µM KT5823 or 100 µM 5-HD in conjunction with 5 nM Bay 60-2770 to evaluate the protective role of PKG. Mitochondrial oxidative stress was investigated under hypoxia-reoxygenation in H9c2 cells. In IR-injured rat hearts, BAY 60-2770 oral administration reduced infarct size by TTC staining and improved left ventricular function by echocardiography. Tissue samples from BAY 60-2770-perfused hearts had approximately two-fold higher cGMP levels. BAY 60-2770 increased PKG activity in the myocardium, and the reduced infarct area by BAY 60-2770 was abrogated by KT-5823 in isolated myocardium. In H9c2 cardiac myoblasts, hypoxia-reoxygenation-mediated mitochondrial ROS generation was diminished with BAY 60-2770 treatment, but was recovered by pretreatment with KT-5823. BAY 60-2770 demonstrated a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. BAY 60-2770 has a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. These results demonstrated that BAY 60-2770 may be used as a therapeutic agent for cardiac IR injury.

Mind Bomb-Binding Partner RanBP9 Plays a Contributory Role in Retinal Development.

Ran-binding protein family member, RanBP9 has been reported in various basic cellular mechanisms and neuropathological conditions including schizophrenia. Previous studies have reported that RanBP9 is highly expressed in the mammalian brain and retina; however, the role of RanBP9 in retinal development is largely unknown. Here, we present the novel and regulatory roles of RanBP9 in retinal development of a vertebrate animal model, zebrafish. Zebrafish embryos exhibited abundant expression of ranbp9 in developing brain tissues as well as in the developing retina. Yeast two-hybrid screening demonstrated the interaction of RanBP9 with Mind bomb, a component of Notch signaling involved in both neurogenesis and neural disease autism. The interaction is further substantiated by co-localization studies in cultured cells. Knockdown of ranbp9 resulted in retinal dysplasia with defective proliferation of retinal cells, downregulation of neuronal differentiation marker huC, elevation of neural proliferation marker her4, and alteration of cell cycle marker p57kip2. Expression of the Müller glial cell marker glutamine synthase was also affected in knockdown morphants. Our results suggest that Mind bomb-binding partner RanBP9 plays a role during retinal cell development of zebrafish embryogenesis.

Enhanced-autophagy by exenatide mitigates doxorubicin-induced cardiotoxicity.

OBJECTIVES: Exenatide is a glucagon-like peptide-1 analogue that mitigates myocardial injury caused by ischemia-reperfusion injury via the survival signaling pathway. We hypothesized that exenatide would provide a protective effect in doxorubicin-induced cardiotoxicity. METHODS: H9c2 cardiomyocytes were pre-treated with exenatide followed by doxorubicin (DOX), and cell viability and intracellular reactive oxygen species (ROS) were subsequently measured. In order to determine the role of autophagy, we performed western blot as well as TUNEL and autophagosome staining. Additionally, rats were treated with exenatide 1h prior to every DOX treatment. Left ventricular (LV) function and performance were then assessed by echocardiography. Myocardial and serum ROS was measured with DHE fluorescence and ROS/RNS assay. RESULTS: DOX-induced caspase-3 activation decreased after pre-treatment with exenatide both in vivo and in vitro. Oxidative stress was attenuated by exenatide in H9c2 cells, as well as in cardiac tissue and serum. The number of autophagosomes and autophagic markers were further increased by exenatide in the DOX-treated H9c2 cells, which mediated AMPK activation. Suppression of the autophagosome abolished exenatide-induced anti-apoptotic effect. Echocardiography showed that pre-treatment with exenatide significantly improved LV dysfunction that is induced by DOX treatment. Exenatide inhibits the DOX-induced production of intracellular ROS and apoptosis in the myocardium. The autophagic markers increased in exenatide pre-treated cardiac tissue. CONCLUSION: Exenatide reduces DOX-induced apoptosis of cardiomyocytes by upregulating autophagy and improving cardiac dysfunction. These novel results highlight the therapeutic potential of exenatide to prevent doxorubicin cardiotoxicity.