Characterization of Edwardsiella piscicida CK108 flagellin genes and evaluation of their potential as vaccine targets in the zebrafish model.

The control of bacterial pathogens, including Edwardsiella piscicida, in the aquaculture industry has high economic importance. This study aimed to identify a potential live vaccine candidate against E. piscicida infection to minimize the side effects and elicit immunity in the host. This study evaluated the virulence factors of E. piscicida CK108, with a special focus on the flagella. E. piscicida has two important homologous flagellin genes, namely flagellin-associated protein (fap) and flagellin domain-containing protein (fdp). CK226 (Δfap), CK247 (Δfdp) and CK248 (Δfap, fdp) mutant strains were constructed. Both CK226 and CK247 displayed decreased length and thickness of flagellar filaments, resulting in reduced bacterial swimming motility, while CK248 was non-motile as it lacked flagella. The loss of flagella and decreased motility was expected to decrease the pathogenicity of CK248. However, the median lethal dose (LD50 ) of CK248 against zebrafish was lower than those of the wild-type, CK226 and CK247 strains. The protective immunity and cytokine gene expression levels in the CK248-infected zebrafish were lower than those in the wild type-infected zebrafish. In conclusion, Fap and Fdp are essential for flagella formation and motility, and for stimulating fish immune response, which can be utilized as a potential adjuvants for E. piscicida vaccination.

Edwardsiella piscicida lacking the cyclic AMP receptor protein (Crp) is avirulent and immunogenic in fish.

Edwardsiella piscicida is a Gram-negative pathogen that generally causes lethal septicemia in marine and freshwater fish. We generated a E. piscicida CK216 Δcrp mutant to investigate various biological roles related to this organism, including pathogenesis. Lack of Crp in CK216 was demonstrated by immunoblotting using a Crp-specific antibody. Compared to the parental strain, the mutant exhibited changes in three biochemical phenotypes, including ornithine decarboxylation, citrate utilization, and H2S production. Complementation of crp deletion in trans rescued the phenotype of the parental strain. This study proved that hemolytic activity in E. piscicida is controlled by Crp. In addition, significantly reduced motility of E. piscicida CK216 was observed, which resulted from a lack of flagella synthesis. To examine the virulence in fish, E. piscicida cells were injected into the goldfish (Carassius auratus) via intraperitoneal route. The LD50 of CK216 was 9.25 × 108 CFU, while that of the CK108 parental strain was 9.24 × 105 CFU, attenuated 1000 fold in goldfish. Fish immunized with CK216 elicited IgM responses. Moreover, 80% of goldfish immunized with 1 × 106 CFU survived after administration of a lethal dose (1 × 107 CFU) of virulent E. piscicida CK41, suggesting the potential for E. piscicida CK216 to serve as a live attenuated vaccine in aquaculture.

The RNA-binding protein HuD regulates autophagosome formation in pancreatic ß cells by promoting autophagy-related gene 5 expression.

Tight regulation of autophagy is critical for the fate of pancreatic ß cells. The autophagy protein ATG5 is essential for the formation of autophagosomes by promoting the lipidation of microtubule-associated protein LC3 (light chain 3). However, little is known about the mechanisms that regulate ATG5 expression levels. In this study, we investigated the regulation of ATG5 expression by HuD. The association of HuD with ATG5 mRNA was analyzed by ribonucleoprotein complex immunoprecipitation and biotin pulldown assays. HuD expression levels in pancreatic ß cells were knocked down via siRNA, elevated by overexpression of a HuD-expressing plasmid. The expression levels of HuD, ATG5, LC3, and ß-actin were determined by Western blot and quantitative RT-PCR analysis. Autophagosome formation was assessed by fluorescence microscopy in GFP-LC3-expressing cells and in pancreatic tissues from WT and HuD-null mice. We identified ATG5 mRNA as a post-transcriptional target of the mammalian RNA-binding protein HuD in pancreatic ß cells. HuD associated with the 3'-UTR of the ATG5 mRNA. Modulating HuD abundance did not alter ATG5 mRNA levels, but HuD silencing decreased ATG5 mRNA translation, and, conversely, HuD overexpression enhanced ATG5 mRNA translation. Through its effect on ATG5, HuD contributed to the lipidation of LC3 and the formation of LC3-positive autophagosomes. In keeping with this regulatory paradigm, HuD-null mice displayed lower ATG5 and LC3 levels in pancreatic ß cells. Our results reveal HuD to be an inducer of ATG5 expression and hence a critical regulator of autophagosome formation in pancreatic ß cells.

Analysis of zebrafish (Danio rerio) behavior in response to bacterial infection using a self-organizing map.

BACKGROUND: Animal behavioral responses have been recently established as a suitable tool for detecting contaminants in the environment for risk assessment in situ. In this study, we observed movement behavior of zebrafish (Danio rerio) before and after infection with Edwardsiella tarda CK41 for 3 days until death. METHODS: Infection status of zebrafish was confirmed through PCR and colonization assay as time progressed and lesion development in the tails of zebrafish was also examined. Movement behaviors in response to bacterial infection were patterned by self-organizing map (SOM) based on movement parameters, including speed (mm/s), acceleration (mm/s (2) ), stop duration (t), stop number (n), locomotory rate (mm/s), turning rate (rad/s), and meander (rad/mm). RESULTS: According to SOM result, clusters were identified firstly according to time and secondly according to infection. Two movement patterns were observed in the early period of infection: one group with minimum turning rate and meander (i.e., stiff movement) and the other group with maximum strop number. Late infection was characterized by long stop duration. CONCLUSION: SOM was suitable for extracting complex behavioral data and thus can serve as a referencing system for diagnosing disease development in order to reveal the mechanism of the infection process.

FDA-approved PDE4 inhibitors reduce the dominant toxicity of ALS-FTD-associated CHCHD10 S59L.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10( CHCHD10 ) have been identified as a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia(ALS-FTD). In our previous studies using in vivo Drosophila model expressing C2C10H S81L , and human cell models expressing CHCHD10 S59L , we have identified that the PINK1/Parkin pathway is activated and causes cellular toxicity. Furthermore, we demonstrated that pseudo-substrate inhibitors for PINK1 and mitofusin2 agonists mitigated the cellular toxicity of CHCHD10 S59L . Evidences using in vitro/ in vivo genetic and chemical tools indicate that inhibiting PINK1 would be the most promising treatment for CHCHD10 S59L -induced diseases. Therefore, we have investigated cellular pathways that can modulate the PINK1/Parkin pathway and reduce CHCHD10 S59L -induced cytotoxicity. Here, we report that FDA-approved PDE4 inhibitors reduced CHCHD10 S59L -induced morphological and functional mitochondrial defects in human cells and an in vivo Drosophila model expressing C2C10H S81L . Multiple PDE4 inhibitors decreased PINK1 accumulation and downstream mitophagy induced by CHCHD10 S59L . These findings suggest that PDE4 inhibitors currently available in the market may be repositioned to treat CHCHD10 S59L -induced ALS-FTD and possibly other related diseases.

FDA-approved PDE4 inhibitors alleviate the dominant toxicity of ALS-FTD-associated CHCHD10S59L by reducing the PINK1/Parkin pathway.

Background: Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) have been identified as a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia(ALS-FTD). In our previous studies using in vivo Drosophila model expressing CHCHD10S59L, and human cell models expressing CHCHD10S59L, we have identified that the PINK1/Parkin pathway is activated and causes cellular toxicity. Furthermore, we demonstrated that pseudo-substrate inhibitors for PINK1 and mitofusin2 agonists mitigated the cellular toxicity of CHCHD10S59L. Evidences using in vitro, in vivo genetic, and chemical tools indicate that inhibiting PINK1 would be the most promising treatment for CHCHD10S59L-induced diseases. Methods: An in vivo human cell culture and in vivo Drosophila models expressing CHCHD10S59L mutant were utilized in this study to evaluate the effect of PDE4 inhibitors in PINK-parkin mediated cytotoxicity through immunohistochemical and seahorse assays. Data were analysed using one-way ANOVA and post-hoc Dunnett's test for statistical significance. Results: We investigated cellular pathways that can modulate the PINK1/Parkin pathway and reduce CHCHD10S59L-induced cytotoxicity. Here, we report that FDA-approved PDE4 inhibitors reduced CHCHD10S59L-induced morphological and functional mitochondrial defects in human cells and an in vivo Drosophila model expressing C2C10HS81L. Multiple PDE4 inhibitors decreased PINK1 accumulation and downstream mitophagy induced by CHCHD10S59L. Conclusion: These findings suggest that PDE4 inhibitors currently available in the market may be repositioned to treat CHCHD10S59L-induced ALS-FTD and possibly other related diseases, and that disease treatment with PDE4 inhibitors should include careful consideration of the PINK1/Parkin pathway, as it is generally recognized as a protective pathway.

Heterotypic cell-in-cell structures between cancer and NK cells are associated with enhanced anticancer drug resistance.

The heterotypic CIC structures formed of cancer and immune cells have been observed in tumor tissues. We aimed to assess the feasibility of using heterotypic CICs as a functional biomarker to predict NK susceptibility and drug resistance. The heterotypic CIC-forming cancer cells showed a lower response to NK cytotoxicity and higher proliferative ability than non-CIC cancer cells. After treatment with anticancer drugs, cancer cells that formed heterotypic CICs showed a higher resistance to anticancer drugs than non-CIC cancer cells. We also observed the formation of more CIC structures in cancer cells treated with anticancer drugs than in the non-treated group. Our results confirm the association between heterotypic CIC structures and anticancer drug resistance in CICs formed from NK and cancer cells. These results suggest a mechanism underlying immune evasion in heterotypic CIC cancer cells and provide insights into the anticancer drug resistance of cancer cells.

TDP-43 and PINK1 mediate CHCHD10S59L mutation-induced defects in Drosophila and in vitro.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.

Goldfish, Carassius auratus, as an infection model for studying the pathogenesis of Edwardsiella piscicida.

This study demonstrates the feasibility of using goldfish as an infection model to investigate the pathogenesis of Edwardsiella piscicida. Goldfish were found to be susceptible to acute E. piscicida-induced disease and died in a dose-dependent manner. E. piscicida was further shown to replicate rapidly in the head kidneys and livers of infected goldfish from 1 d post-injection, and bacteria numbers were significantly decreased 5 d post-injection. Immune responses were successfully induced in goldfish injected with E. piscicida strains and 60% of goldfish inoculated with an attenuated E. piscicida strain were found to survive subsequent injection with a pathogenic strain. The results of differential leukocyte count experiments suggested that leukocytes were immediately recruited as an innate immune response against the infection. Thus, this well-characterized goldfish species is a suitable infection model for studying E. piscicida pathogenesis, and might be applicable to research on other fish diseases.

A small-molecule inhibitor targeting the AURKC-IκBα interaction decreases transformed growth of MDA-MB-231 breast cancer cells.

The Aurora kinases, Aurora A (AURKA), Aurora B (AURKB), and Aurora C (AURKC), are serine/threonine kinases required for the control of mitosis (AURKA and AURKB) or meiosis (AURKC). Several Aurora kinase inhibitors are being investigated as novel anticancer therapeutics. Recent studies demonstrated that AURKC activation contributes to breast cancer cell transformation. Therefore, AURKC is both a promising marker and therapeutic target for breast cancer; however, its signaling network has not been fully characterized. Using translocation-based cellular assays, we identified IκBα as a binding partner of AURKC, and found that AURKC phosphorylates IκBα at Ser32, thereby activating it. In silico modeling and computational analyses revealed a small-molecule inhibitor (AKCI) that blocked the AURKC-IκBα interaction and exerted antitumor activity in MDA-MB-231 breast cancer cells. Specifically, AKCI induced G2/M cell-cycle arrest through modulation of the p53/p21/CDC2/cyclin B1 pathways. In addition, the drug significantly inhibited MDA-MB-231 cell migration and invasion, as well as decreasing colony formation and tumor growth. Via its interaction with IκBα, AURKC indirectly induced NF-κB activation; accordingly, AKCI decreased PMA-induced activation of NF-κB. Thus, the small-molecule inhibitor AKCI represents a first step towards developing targeted inhibitors of AURKC protein binding, which may lead to further advances in the treatment of breast cancer.

Nutlin-3 induces HO-1 expression by activating JNK in a transcription-independent manner of p53.

A recent study reported that p53 can induce HO-1 by directly binding to the putative p53 responsive element in the HO-1 promoter. In this study, we report that nutlin-3, a small molecule antagonist of HDM2, induces the transcription of HO-1 in a transcription-independent manner of p53. Nutlin-3 induced HO-1 expression at the level of transcription in human cancer cells such as U2OS and RKO cells. This induction of HO-1 did not occur in SAOS cells in which p53 was mutated and was prevented by knocking down the p53 protein using p53 siRNA transfection, but not by PFT-α, an inhibitor of the transcriptional activity of p53. Accompanying HO-1 expression, nutlin-3 stimulated the accumulation of ROS and the phosphorylation of MAPKs such as JNK, p38 MAPK and ERK1/2. Nutlin-3-induced HO-1 expression was suppressed by TEMPO, a ROS scavenger, and chemical inhibitors of JNK and p38 MAPK but not ERK1/2. In addition, nutlin­3-induced phosphorylation of JNK but not p38 MAPK was inhibited by TEMPO. Notably, the levels of nutlin-3-induced ROS were correlated with the mitochondrial translocation of p53 and this induction was prevented by PFT-µ, an inhibitor of the mitochondrial translocation of p53. Consistent with the effect of the ROS scavenger and MAPK inhibitors, PFT-µ reduced HO-1 expression and the phosphorylation of JNK induced by nutlin-3. In the experiments of analyzing cell death, the knockdown of HO-1 augmented nutlin-3-induced apoptosis. Collectively, these results suggest that nutlin-3 induces HO-1 expression via the activation of both JNK which is dependent on ROS generated by p53 translocated to the mitochondria and p38 MAPK which appears to be stimulated by a ROS-independent mechanism, and this HO-1 induction may inhibit nutlin-3-induced apoptosis, constituting a negative feedback loop of p53-induced apoptosis.

Wilms' tumor gene 1 enhances nutlin-3-induced apoptosis.

Nutlin-3, a human double minute 2 (HDM2) antagonist, induces cell cycle arrest or apoptosis by upregulating p53 in cancer cells. WT1, the product of Wilms' tumor gene 1, has been shown to interact with p53, but the effect of WT1 on nutlin-3-induced apoptosis has yet to be examined. To address this issue, we analyzed the inhibitory effect of nutlin-3 on cell growth as a function of Wt1 expression status using a Wt1-inducible U2OS cell line. In the absence of Wt1 expression, nutlin-3 induced cell cycle arrest with marginal cytotoxicity. Furthermore, upon Wt1 expression, nutlin-3 exerted a marked degree of cell death, as evidenced by the accumulation of hypo-diploid cells and LDH release. During cell death induction, cytochrome c was released into the cytosol, and caspase-9 and -3 were activated, suggesting that an intrinsic apoptotic pathway may be involved in this cell death. Consistent with this, z-VAD-Fmk, a pan-caspase inhibitor and the overexpression of BCL-XL attenuated the cell death. Nutlin-3 caused an increase in the mRNA levels of both BCL-XL and BAK, as well as their corresponding protein levels in mitochondria. In the presence of Wt1, nutlin-3-induced BCL-XL expression was attenuated while the expression of nutlin-3-induced BAK was potentiated. Collectively, these results suggest that WT1 potentiates nutlin-3-induced apoptosis by downregulating the expression of BCL-XL while upregulating that of BAK, which leads to the activation of an intrinsic apoptotic pathway.

Nutlin-3 induces BCL2A1 expression by activating ELK1 through the mitochondrial p53-ROS-ERK1/2 pathway.

Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-µ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited nutlin-3-induced ERK1/2 such as U0126, PFT-µ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.

Anthocyanins in the black soybean (Glycine max L.) protect U2OS cells from apoptosis by inducing autophagy via the activation of adenosyl monophosphate-dependent protein kinase.

Anthocyanins (ATCs) have been reported to induce apoptosis in various types of cancer cells, stimulating the development of ATCs as a cancer chemotherapeutic or chemopreventive agent. It was recently reported that ATCs can induce autophagy, however, the mechanism for this remains unclear. In the present report, we carried out mechanistic studies of the mechanism involved in ATC-induced autophagy using ATCs extracted from black soybeans (cv. Cheongja 3, Glycine max L.). ATCs clearly induced hallmarks of autophagy, including LC3 puncta formation and the conversion of LC3-I to LC3-II in U2OS human osteosarcoma cells. The induction of autophagy was accompanied by the phosphorylation of multiple protein kinases including extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), protein kinase B (AKT) and adenosyl mono-phosphate-dependent protein kinase (AMPK). While chemical inhibitors against ERK1/2, p38 MAPK, JNK and AKT failed to inhibit ATC-induced autophagy, the suppression of AMPK by compound C (CC) as well as siRNA against AMPK reduced ATC-induced autophagy. The treatment of ATCs resulted in a decrease in intracellular ATP contents and the activation of AMPK by AICAR treatment also induced autophagy. It is noteworthy that the reduction of autophagy via the inhibition of AMPK resulted in enhanced apoptosis in ATC-treated cells. In addition, siRNA against forkhead box O3A (FOXO3a), a downstream target of AMPK, suppressed ATC-induced autophagy and p27KIP1 siRNA increased apoptosis in ATC-treated cells. Collectively, it can be concluded that ATCs induce autophagy in U2OS cells via activation of the AMPK-FOXO3a pathway and protect cells from ATC-induced apoptosis via the AMPK-p27KIP1 pathway. These results also suggest that autophagy-modulating agents could contribute to the efficient development of ATCs as anticancer therapy.

Induction of the Intrinsic Apoptotic Pathway by 3-Deazaadenosine Is Mediated by BAX Activation in HL-60 Cells.

3-Deazaadenosine (DZA), a potent inhibitor of S-adenosylhomocysteine hydrolase, was previously proposed to induce intrinsic apoptosis in human leukemic cells. In the present study, we analyzed the mechanism underlying the DZA-induced intrinsic apoptotic pathway. DZA activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an accumulation of hypo-diploidic cells, the processing of multiple procaspases and an inhibitory effect of z-VAD-Fmk on this cell death. During DZA-induced apoptosis, cytochrome c (cyt c) was released into the cytosol. This was neither prevented by z-VAD-Fmk and nor was it associated with the dissipation of mitochondrial membrane potential (ΔΨ(m)). Prior to the release of cyt c, BAX was translocated from the cytosol to mitochondria and underwent oligomerization. Finally, the overexpression of BCL-XL protected HL-60 cells from apoptosis by blocking both the cyt c release and BAX oligomerization. Collectively, these findings suggest that DZA may activate intrinsic apoptosis by stimulating BAX activation and thereby the release of cyt c.