Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species.     

Garlic Oil Suppressed Nitrosodiethylamine-Induced Hepatocarcinoma in Rats by Inhibiting PI3K-AKT-NF-κB Pathway

To explore the underlying mechanisms for the protective effects of garlic oil (GO) against nitrosodiethylamine (NDEA)-induced hepatocarcinoma, 60 male Wistar rats were randomized into 4 groups (n=15): control group, NDEA group, and two GO plus NDEA groups. The rats in GO plus NDEA groups were pretreated with GO (20 or 40 mg/kg) for 7 days. Then, all rats except those in control group were gavaged with NDEA for 20 weeks, and the rats in GO plus NDEA groups were continuously administered with GO. The results showed that GO co-treatment significantly suppressed the NDEA-induced increases of alpha fetal protein (AFP) level in serum, nuclear atypia in H&E staining, sirius red-positive areas and proliferating cell nuclear antigen (PCNA) expression. The molecular mechanisms exploration revealed that the protein levels of phosphatidylinositol 3 kinase (PI3K)-p85, PI3K-p110, total AKT, p-AKT (Ser473) and p-AKT (Thr308) in the liver of NDEA group rats were higher than those in control group rats. In addition, NDEA treatment induced IκB degradation and NF-κB p65 phosphorylation, and up-regulated the protein levels of downstream pro-inflammatory mediators. GO co-treatment significantly reversed all the above adverse effects induced by NDEA. These results suggested that the protective effects of GO against NDEA-induced hepatocarcinoma might be associated with the suppression of PI3K- AKT-NF-κB pathway.     

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.     

MMP-12-mediated by SARM-TRIF signaling pathway contributes to IFN-γ-independent airway inflammation and AHR post RSV infection in nude mice

Background

Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection.

Methods

Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression.

Results

RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice.

Conclusions

MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0176-8) contains supplementary material, which is available to authorized users.     

Inhibition of integrin β3, a binding partner of kallistatin,leads to reduced viability,invasion and proliferation in NCI-H446 cells

Background

Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.

Methods

Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.

Results

Interestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.

Conclusions

These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

     

Comprehensive analysis of the long noncoding RNA HOXA11-AS gene interaction regulatory network in NSCLC cells

Background

Long noncoding RNAs (lncRNAs) are related to different biological processes in non-small cell lung cancer (NSCLC). However, the possible molecular mechanisms underlying the effects of the long noncoding RNA HOXA11-AS (HOXA11 antisense RNA) in NSCLC are unknown.

Methods

HOXA11-AS was knocked down in the NSCLC A549 cell line and a high throughput microarray assay was applied to detect changes in the gene profiles of the A549 cells. Bioinformatics analyses (gene ontology (GO), pathway, Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses) were performed to investigate the potential pathways and networks of the differentially expressed genes. The molecular signatures database (MSigDB) was used to display the expression profiles of these differentially expressed genes. Furthermore, the relationships between the HOXA11-AS, de-regulated genes and clinical NSCLC parameters were verified by using NSCLC patient information from The Cancer Genome Atlas (TCGA) database. In addition, the relationship between HOXA11-AS expression and clinical diagnostic value was analyzed by receiver operating characteristic (ROC) curve.

Results

Among the differentially expressed genes, 277 and 80 genes were upregulated and downregulated in NSCLC, respectively (fold change ≥2.0, P < 0.05 and false discovery rate (FDR) < 0.05). According to the degree of the fold change, six upregulated and three downregulated genes were selected for further investigation. Only four genes (RSPO3, ADAMTS8, DMBT1, and DOCK8) were reported to be related with the development or progression of NSCLC based on a PubMed search. Among all possible pathways, three pathways (the PI3K-Akt, TGF-beta and Hippo signaling pathways) were the most likely to be involved in NSCLC development and progression. Furthermore, we found that HOXA11-AS was highly expressed in both lung adenocarcinoma and squamous cell carcinoma based on TCGA database. The ROC curve showed that the area under curve (AUC) of HOXA11-AS was 0.727 (95% CI 0.663–0.790) for lung adenocarcinoma and 0.933 (95% CI 0.906–0.960) for squamous cell carcinoma patients. Additionally, the original data from TCGA verified that ADAMTS8, DMBT1 and DOCK8 were downregulated in both lung adenocarcinoma and squamous cell carcinoma, whereas RSPO3 expression was upregulated in lung adenocarcinoma and downregulated in lung squamous cell carcinoma. For the other five genes (STMN2, SPINK6, TUSC3, LOC100128054, and C8orf22), we found that STMN2, TUSC3 and C8orf22 were upregulated in squamous cell carcinoma and that STMN2 and USC3 were upregulated in lung adenocarcinoma. Furthermore, we compared the correlation between HOXA11-AS and de-regulated genes in NSCLC based on TCGA. The results showed that the HOXA11-AS expression was negatively correlated with DOCK8 in squamous cell carcinoma (r = ?0.124, P = 0.048) and lung adenocarcinoma (r = ?0.176, P = 0.005). In addition, RSPO3, ADAMTS8 and DOCK8 were related to overall survival and disease-free survival (all P < 0.05) of lung adenocarcinoma patients in TCGA.

Conclusions

Our results showed that the gene profiles were significantly changed after HOXA11-AS knock-down in NSCLC cells. We speculated that HOXA11-AS may play an important role in NSCLC development and progression by regulating the expression of various pathways and genes, especially DOCK8 and TGF-beta pathway. However, the exact mechanism should be verified by functional experiments.

     

Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens

Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.     

Nitrous oxide emissions from surface flow and subsurface flow constructed wetland microcosms: Effect of feeding strategies

The effects of continuous and intermittent feeding strategies on nitrogen removal and N₂O emission from surface flow and subsurface flow constructed wetlands were evaluated in this study. Microcosm wetlands planted with Phragmites australis were constructed and operated with different feeding strategies for the 4-month experiment. Results showed the intermittent feeding strategy could enhance the removal of ammonium effectively in the subsurface flow constructed wetlands, although it had no significant effect for the surface flow wetlands. And the intermittent feeding mode could promote the emission of N₂O. The amount of N₂O-N emission from the subsurface flow constructed wetlands with intermittent feeding mode was about 5 times higher than that with continuous feeding strategy and the emission rate ranged from 0.09 ± 0.03 to 7.33 ± 1.49 mg/m²/h. Compared with the surface flow constructed wetlands, the N₂O emission in the subsurface flow constructed wetlands was affected significantly by the intermittent feeding mode.