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Maize rough dwarf disease (MRDD) is one of the most serious viral diseases of maize in China. The object of this study is to develop a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of MRDD pathogen. Four RT-LAMP primers were designed on the basis of S10. Template RNA from infected leaves were used for RT-LAMP which were carried out under isothermal conditions at 61 °C for 90 min. RT-LAMP products were analyzed by electrophoresis in agarose gels or visual inspection of color change after staining with calcein. Sensitivity of the RT-LAMP assay was 100-fold higher than RT-PCR. This method can distinguish RBSDV from SRBSDV and the vectors carrying RBSDV from Rice stripe virus (RSV). The RT-LAMP assay shows excellent potential for application in the rapid and sensitive detection of MRDD pathogens in field samples or vector.

ARGONAUTE (AGO) proteins play crucial roles in plant defence against virus invasion. To date, the role of OsAGO2 in rice antiviral defence remains largely unknown. In this study, we determined that the expression of OsAGO2 in rice was induced upon rice black‐streaked dwarf virus (RBSDV) infection. Using transgenic rice plants overexpressing OsAGO2 and Osago2 mutants generated through transposon‐insertion or CRISPR/Cas9 technology, we found that overexpression of OsAGO2 enhanced rice susceptibility to RBSDV infection. Osago2 mutant lines exhibited strong resistance to RBSDV infection through the elicitation of an early defence response, including reprogramming defence gene expression and production of reactive oxygen species (ROS). Compared to Nipponbare control, the expression level of OsHXK1 (HEXOKINASE 1) increased significantly, and the methylation levels of its promoter decreased in the Osago2 mutant on RBSDV infection. The expression profile of OsHXK1 was the opposite to that of OsAGO2 during RBSDV infection. Overexpression of OsHXK1 in rice also induced ROS production and enhanced rice resistance to RBSDV infection. These results indicate that OsHXK1 controls ROS accumulation and is regulated by OsAGO2 through epigenetic regulation. It is noteworthy that the Osago2 mutant plants are also resistant to southern rice black‐streaked dwarf virus infection, another member of the genus Fijivirus. Based on the results presented in this paper, we conclude that OsAGO2 modulates rice susceptibility to fijivirus infection by suppressing OsHXK1 expression, leading to the onset of ROS‐mediated resistance. This discovery may benefit future rice breeding programmes for virus resistance., Expression of OsAGO2 is induced on rice black‐streaked dwarf virus (RBSDV) infection, which represses OsHXK1 expression via DNA methylation and controls reactive oxygen species‐mediated resistance in an inactivated mode to facilitate RBSDV infection.

Rice stripe virus (RSV) causes rice stripe disease, which is one of the most serious rice diseases in eastern Asian countries. It has been shown that overexpression of RSV coat protein (CP) in rice plants enhances resistance against virus infection. However, the detailed mechanism underlying RSV CP-mediated virus resistance remains to be determined. In this study, we show that both translatable and non-translatable RSV CP transgenic Arabidopsis plants exhibited immunity to virus infection. By using deep sequencing analysis, transgene-derived small interfering RNAs (t-siRNAs) from non-translatable CP transgenic plants and virus-derived small interfering RNAs (vsiRNAs) mapping in the CP region from RSV-infected wild-type plants showed similar sequence distribution patterns, except for a significant increase in the abundance of t-siRNA reads compared with that of CP-derived vsiRNAs. To further test the correlation of t-siRNAs with RSV immunity, we developed RSV CP transgenic Arabidopsis plants in an siRNA-deficient dcl2/3/4 mutant background, and these CP transgenic plants showed the same sensitivity to RSV infection as non-transgenic plants. Together, our data indicate that the expression of RSV CP protein from a transgene is not a prerequisite for virus resistance and RSV CP-mediated resistance is mostly associated with the RNA silencing mechanism in Arabidopsis plants.

Purpose Cancer stem cell-tumor microenvironment ecosystem is proposed to drive tumor heterogeneity. Tumor-infiltrating lymphocytes (TILs) in breast cancer ecosystem were demonstrated to indicate better prognosis and benefit from chemotherapy. This study sought to detect the association between breast cancer stem cells and TILs. Methods 92 patients with breast cancer were enrolled. Matched cancerous and paracancerous tissues were assembled in a tissue microarray and immunohistochemistry was employed to test expression of breast cancer stem cells (BCSCs) markers. TILs counts were estimated with global hematoxylin-eosin staining. The association between TILs and BCSCs phenotypes was analysed by multivariate analysis. Results Although it was unable to find direct significant association between BCSCs phenotypes and TILs, the BCSCs phenotype with CD44+CD24−ALDH1A1+EpCAM+CD49f+ was proved to be associated with worse DFS and OS (P = 0.037 and 0.001). This result was confirmed by cox proportional-hazards regression model (for DFS and OS respectively, HR = 2.438 and 3.383, P = 0.019 [95%CI 1.418–3.457] and 0.025 [95%CI 1.162–9.843]). Additionally, in results of TILs, plasma cell-predominant breast cancer (PPBC) was unexpectedly found to indicate worse OS and HR was 2.686 (P = 0.038 [95%CI 1.582–3.789]). Conclusions The BCSCs phenotype and PPBC may be helpful stratified factors in future clinical trials. The underlying mechanism needs further research.

Sepsis induced myocardial dysfunction (SIMD) results in high morbidity and mortality. However, the effective therapeutic strategies for SIMD treatment remain limited. Sirt3 is the main mitochondrial Sirtuin member and is a key modulator of mitochondrial metabolism and function. In this study, we aimed to investigate the effect and mechanism of Sirt3 on SIMD. SIMD was induced by 20 mg/kg Lipopolysaccharides (LPS) injection for 6 h in mice. Sepsis could induce the reduction of cardiac Sirt3 expression and global deficiency of Sirt3 exacerbated cardiac function. Quantitative acetyl-proteomics and cardiac metabolomics analysis revealed that loss of Sirt3 led to hyper-acetylation of critical enzymes within cardiac tricarboxylic acid (TCA) cycle and generation of lactate and NADH, subsequently promotion of cardiac dysfunction after sepsis. Additionally, to evaluate whether Emodin could be utilized as a potential Sirt3 modulator to treat SIMD, male wild type mice (WT mice) or global Sirt3 deficient mice (Sirt3-/- mice) were intraperitoneally injected with 40 mg/kg Emodin for 5 days followed by 20 mg/kg LPS administration for another 6 h and observed that exogenous administration of Emodin could attenuate myocardial dysfunction in septic WT mice. However, septic Sirt3-/- mice can not gain benefit on cardiac performance from Emodin infusion. In conclusion, this study presented the protective role of Sirt3 targeting SIMD, which may provide a potential novel approach to maintain normal cardiac performance after sepsis.

Rice black-streaked dwarf virus (RBSDV) infects rice plants, a major crop, and is transmitted via the small brown planthopper (SBPH: Laodelphax striatellus Fallen), causing significant economic loss in China. To rapidly diagnose RBSDV, a reverse transcription-recombinase polymerase amplification (RT-RPA) method was developed using P10 virus-specific primers and probes. Detection of terminally labeled amplification products was achieved with the lateral flow strip method. Our results demonstrate that RT-RPA and RT-PCR assays offer similar sensitivity and specificity in RBSDV detection using cDNA as template. The optimum RT-RPA reaction temperature and time was 37 °C and 20 min, respectively. By screening twenty-one field suspected rice plants, the RT-RPA assay was confirmed to be simple, rapid and reliable. Thus, the RBSDV RT-RPA assay developed here will be a successful tool for quick diagnosis of RBSDV-infected rice plants.

The exact effect of glycine pre-treatment on brain ischemic tolerance (IT) remains quite controversial. The objective of this study was to investigate the potential effects of glycine on IT. We used rat models of both in vitro ischemia (oxygen and glucose deprivation) and in vivo ischemia (transient middle cerebral artery occlusion). Low doses of glycine (L-Gly) significantly decreased hippocampal ischemic LTP (i-LTP), infarct volume, and neurological deficit scores which were administered before ischemia was induced in rats, whereas high doses of glycine exerted deteriorative effects under the same condition. These findings suggested that exogenous glycine may induce IT in a dose-dependent manner. Furthermore, L-Gly-dependent neuronal protection was inversed by L689, a selective NMDAR glycine site antagonist both in vitro (abolished i-LTP depression) and in vivo (increased infarct size reduction), but not glycine receptor (GlyR) inhibitor strychnine. Importantly, L-Gly-induced IT was achieved by NR2A-dependent cAMP-response element binding protein phosphorylation. These data imply that glycine pre-treatment may represent a novel strategy for inducing IT based on synaptic NMDAR-dependent neuronal transmission. A model of glycine induced dose-dependent bidirectional regulations in ischemic tolerance. (a) Under low dose of Gly pre-treatment, glycine induces NMDAR potentiation and CREB-dependent neuroprotection through the NMDAR co-agonist binding site. (b) Under high dose of Gly pre-treatment, the excessive glycine in synaptic cleft can activate neighboring extrasynaptic sites and combine to the GlyRs. Then, the deteriorative effects would be triggered after NMDAR endocytosis and synaptic depression. AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CREB, cAMP response element-binding protein; Gly, glycine; GlyR, glycine receptor; GlyT1, gycine transportor 1; NMDAR, N-methyl-d-aspartate receptor.

RNA interference (RNAi) is a conserved mechanism against viruses in plants and animals. It is thought to inactivate the viral genome by producing virus-derived small interfering RNAs (vsiRNAs). Rice black-streaked dwarf virus (RBSDV) is transmitted to plants by the small brown planthopper (Laodelphax striatellus), and seriously threatens production of rice in East Asia, particularly Oryza sativa japonica subspecies. Through deep sequencing, genome-wide comparisons of RBSDV-derived vsiRNAs were made between the japonica variety Nipponbare, and the indica variety 9311. Four small RNA libraries were constructed from the leaves and shoots of each variety. We found 659,756 unique vsiRNAs in the four samples, and only 43,485 reads were commonly shared. The size distributions of vsiRNAs were mostly 21- and 22-nt long, and A/U bias (66-68%) existed at the first nucleotide of vsiRNAs. Additionally, vsiRNAs were continuously but heterogeneously distributed along S1-S10 segments of the RBSDV genome. Distribution profiles of vsiRNA hotspots were similar in different hosts and tissues, and the 5'- and 3'-terminal regions of S4, S5, and S8 had more hotspots. Distribution and abundance of RBSDV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. Degradome analysis found 25 and 11 host genes appeared to be targeted by vsiRNAs in 9311 and Nipponbare. We report for the first time vsiRNAs derived from RBSDV-infected rice.

Trichinella spiralis(T. spiralis) or its excretory-secretory products (TsES) protect hosts from autoimmune diseases, which depend on inducing host T helper (Th) 2 immune response and inhibiting inflammatory factors. Sepsis is a systemic inflammatory response syndrome (SIRS) evoked by infection. Little is known about the effects of helminths or their excretory-secretory products on sepsis. Here, we investigated the effects of TsES in a mice model of polymicrobial sepsis. TsES improved survival, reduced organ injury, and enhanced bacterial clearance in septic mice. To investigate the molecular mechanism, macrophages from septic patients or the control group were incubated with TsES. TsES reduced sepsis-inducing inflammatory cytokines mediated by Toll-like receptors (TLR)in vitroby suppressing TLR adaptor-transducer myeloid differentiation factor 88 (MyD88) and nuclear factor- (NF-)-κB. Furthermore, TsES upregulated mannose receptor (MR) expression during sepsis. MR blocking attenuated the effects of TsES on MyD88 and NF-κB expression.In vivo, MR RNAi reduced the survival rate of septic mice treated with TsES, suggesting that TsES-mediated protection against polymicrobial sepsis is dependent on MR. Thus, TsES administration might be a potential therapeutic strategy for treating sepsis.

Background Virus infection induces and suppresses host gene expression on a global level. Rice stripe virus (RSV) is the type species of the genus Tenuivirus and infects rice and Arabidopsis plants. Microarray-based and next generation sequencing-based transcriptomic approaches have been used to study rice-RSV interactions. However, our knowledge of the response of Arabidopsis plants to RSV infection is limited, and it requires further investigation to determine the similarities (or differences) in virus-host interactions between monocot and dicot hosts infected with RSV. Methods We characterized transcriptome changes in Arabidopsis thaliana infected with rice stripe virus (RSV) with RNA-seq based digital gene expression (DGE) analysis. The transcriptomes of RSV-infected samples were compared to those of mock-treated samples at 14 and 21 days post-infection (dpi) during different stages of symptom development. Results We identified 624 differentially expressed genes (DEGs) in Arabidopsis influenced by RSV at 14 dpi and 21 dpi, among which at 14 dpi, 255 transcripts were induced, and 38 were repressed; at 21 dpi, 146 were induced, and 237 were repressed. Functional annotation indicated that these DEGs were related to multiple biological functions, including defense response, secondary metabolism, protein amino acid phosphorylation and response to abiotic stress. Conclusions Importantly, the transcription of genes related to host defense systems was activated by RSV infection at an early stage of symptom development (14 dpi), whereas over the infection period (21 dpi), the host defense response systems were suppressed. A total of 52 genes were continuously differentially expressed between the two time points, indicating that the majority of DEGs were transient and unique to a particular time point during symptom development. The DEGs, particularly the defense response genes, identified in this study are candidates suitable for further functional analysis during the RSV-Arabidopsis interaction. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0663-7) contains supplementary material, which is available to authorized users.

This study was designed to investigate the p38 mitogen-activated protein kinase (MAPK) signaling pathway involved in Aquaporin1 (AQP1) expression caused by staphylococcal peptidoglycan (PGN) in cultured rat pleural mesothelial cells (rPMCs) in vitro.RT-PCR and immunoblot analysis were used to determine the relative mRNA and protein levels of AQP1 by PGN in rPMCs. P38 kinase inhibitor SB203580, JNK inhibitor SP600125, and ERK1/2 inhibitor PD98059 were used to determine the effects of PGN-induced AQP1 expression by immunoblot. Activation of p38 by PGN was reflected by detecting the phosphorylation constituent of p38, using immunoblot. The shift of localization after activation of p38 by PGN was investigated by immunofluorescence assay.AQP1 transcription and protein expression were decreased by PGN in dose-dependent and time-dependent manners in rPMCs. Down-regulation of AQP1 by PGN was blocked only by SB203580, neither by SP600125 nor by PD98059. Furthermore, rPMCs exposed to PGN showed activation of p38 MAPK. Phospho-p38 protein production was increased by PGN stimulation in rPMCs. The localization of phospho-p38 was both in the cytosol and nuclei after PGN treatment, while its normal distribution is mainly in the cytosol in rPMCs.AQP1 expression was decreased by PGN in both dose-dependent and time-dependent manners in rPMCs. This down-regulation by PGN-induced AQP1 in rPMCs may be mediated by the activation of p38 MARK pathway.

Southern rice black-streaked dwarf virus (SRBSDV) causes southern rice black-streaked dwarf and maize rough dwarf diseases, which lead to severe yield losses of crops in Southeast Asia. We report here a SYBR Green I-based One-Step Real Time RT-PCR assay for quantifying SRBSDV in rice rapidly and accurately. Primers used for assay were designed from the conserved sequence in S9 RNA among SRBSDV isolates. The RNA standards targeting the S9 region were obtained by transcription in vitro for generation of a standard curve. The assay developed in this study was found to be 100 times more sensitive than the conventional RT-PCR for SRBSDV detection. The primers were very specific for SRBSDV. This study clearly demonstrated the potential usefulness of developed assay for detection and quantitation of SRBSDV in rice samples.

Background Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. Methods The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. Results UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. Conclusions We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0405-2) contains supplementary material, which is available to authorized users.

Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is transmitted by small brown planthoppers (Laodelphax striatellus Fallén, SBPH) and causes severe yield loss in epidemic years in China and other East Asian countries. Breeding for resistance to RBSDV is a promising strategy to control the disease. We identified Tetep that showed resistance to RBSDV using a field test and artificial inoculation test. An evaluation of the resistance mechanism revealed that Tetep was resistant to RBSDV but not to SBPH. Genetic analysis showed that the resistance of Tetep to RBSDV was controlled by quantitative trait loci (QTLs). Three new QTLs for RBSDV resistance were identified in this study, i.e., qRBSDV-3, qRBSDV-10 and qRBSDV-11. The LOD scores of qRBSDV-3, qRBSDV-10 and qRBSDV-11 were 4.07, 2.24 and 2.21, accounting for 17.5%, 0.3% and 12.4% of the total phenotypic variation, respectively. All the resistance loci identified in this study were associated with virus resistance genes. The alleles for enhancing resistance on chromosomes 3 and 11 originated from Tetep, whereas the other allele on chromosome 10 originated from a susceptible parent. The identified new resistance QTLs in this study are useful resources for efficiently breeding resistant rice cultivars to RBSDV.

The inheritance of resistance to white tip disease (WTDR) in rice (Oryza sativa L.) was analyzed with an artificial inoculation test in a segregating population derived from the cross between Tetep, a highly resistant variety that was identified in a previous study, and a susceptible cultivar. Three resistance-associated traits, including the number of Aphelenchoides besseyi (A. besseyi) individuals in 100 grains (NA), the loss rate of panicle weight (LRPW) and the loss rate of the total grains per panicle (LRGPP) were analyzed for the detection of the quantitative trait locus (QTL) in the population after construction of a genetic map. Six QTLs distributed on chromosomes 3, 5 and 9 were mapped. qNA3 and qNA9, conferring reproduction number of A. besseyi in the panicle, accounted for 16.91% and 12.54% of the total phenotypic variance, respectively. qDRPW5a and qDRPW5b, associated with yield loss, were located at two adjacent marker intervals on chromosome 5 and explained 14.15% and 14.59% of the total phenotypic variation and possessed LOD values of 3.40 and 3.39, respectively. qDRPW9 was considered as a minor QTL and only explained 1.02% of the phenotypic variation. qLRGPP5 contributed to the loss in the number of grains and explained 10.91% of the phenotypic variation. This study provides useful information for the breeding of resistant cultivars against white tip disease in rice.

Southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in Southeast Asia. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of SRBSDV using total RNA extracted from rice tissues and the insect pest, white-backed planthopper. The assay was based on a set of four primers matching a total of six sequences in the S9 region of SRBSDV genome. Presence of the virus could be detected in RT-LAMP reactions containing 1.2×10(-6)μg of a total RNA extract, which was ten times more sensitive than a classical RT-PCR assay. The SRBSDV could be distinguished from the closely related rice black-streaked dwarf virus (RBSDV) by this method, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay shows potential for detection of SRBSDV in field samples of hosts or vectors at a relatively low cost.