text
string | predicted_class
string | confidence
float16 |
|---|---|---|
To examine whether the inoculation with SA03 improved photosynthetic capacity in plants, several parameters associated with photosynthesis were examined. Under non-stress condition, there was indistinct difference between the non-inoculated and inoculated plants (Figures 2A–D). After 2 and 4 weeks of the stress treatment, total chlorophyll content was markedly decreased in the non-inoculated plants. However, the inoculated plants displayed higher chlorophyll levels under the stress compared with the non-inoculated plants [Figure 2A; F(5,54) = 84.31, P < 0.05]. In accordance with this, soil inoculation significantly increased photosynthetic efficiency in the stress-treated plants. The values of Fv/Fm, a pivotal index for the efficiency of PSII photochemistry, were also remarkably increased in the inoculated plants under the stress compared with the non-inoculated plants [Figure 2B; F(5,54) = 124.74, P < 0.05]. Similar results were observed for ΦPSII [Figure 2C; F(5,54) = 143.80, P < 0.05] and Pn [Figure 2D; F(5,54) = 118.43, P < 0.05]. After 4 weeks of saline–alkaline treatment, the non-inoculated plants displayed fully swollen chloroplasts and more rudimentary grana lamellae in plastids of mesophyll cells, but the number of normal grana stacking and grana lamellae was greater in the inoculated plants (Figures 2E–L).
|
study
| 100.0 |
Effects of B. licheniformis SA03 on the photosynthesis of Chrysanthemum plants under saline–alkaline stress. After 10 days of bacterial inoculation, the non-inoculated (NI) and inoculated (I) were subjected to saline–alkaline treatment for 2 and 4 weeks, respectively. The treated plants were used to measure (A) total chlorophyll content; (B) Fv/Fm; (C) ΦPSII; and (D) Pn. In addition, transmission electron micrographs of chloroplast ultrastructure in mesophyll cells of plants. Chloroplast ultrastructure of the non-inoculated (E) and inoculated plants (G) before the stress treatment, and (F,H) indicated locally amplified view in (E,G), respectively. Chloroplast ultrastructure of the non-inoculated (I) and inoculated plants (K) after the stress treatment, and (J,L) indicated locally amplified view in (I,K), respectively. Scale bar = 1 μm. BS, before the stress treatment; AS, after the stress treatment. Data are expressed as the mean values of three replicates (±SE) with 10 plants each. Different letters indicate significant differences using two-way ANOVA followed by the Duncan’s multiple range test at P < 0.05.
|
study
| 100.0 |
The availability of Fe is exceedingly low in alkaline soils due to its immobilization (Römheld and Marschner, 1986). This often becomes a key limiting factor for plant growth and development. Thus, plants that are more tolerant to alkaline stress may equip with efficient systems to mine Fe from soils. To verify this hypothesis, shoot and root Fe concentrations were measured. Under non-stress condition, no striking difference in shoot Fe concentrations was observed between the non-inoculated and inoculated plants (Table 1). However, a marked decrease in shoot Fe concentrations was found in the non-inoculated plants after 2 and 4 weeks of the stress treatment [Table 1; F(5,54) = 57.42, P < 0.05]. Furthermore, shoot Fe concentrations of the inoculated plants were relatively higher than that of the non-inoculated plants. A similarly changing tendency of Fe concentrations was found in roots [Table 1; F(5,54) = 86.68, P < 0.05].
|
study
| 100.0 |
Considering that plants experiencing saline–alkaline stress have to counteract excess toxic Na+ (Zhang et al., 2015), the effects of saline–alkaline stress on Na+ and K+ concentrations as well as Na+/K+ ratios in plants were investigated. Before saline–alkaline treatment, no significant difference was observed between the non-inoculated and inoculated plants (Figure 3). After 2 and 4 weeks of the stress treatment, shoot K+ concentrations of the inoculated plants were 33 and 45% higher than that of the non-inoculated plants [Figure 3A; F(5,54) = 122.69, P < 0.05], and root K+ concentrations were 67 and 75% higher [Figure 3B; F(5,54) = 171.30, P < 0.05], respectively. On the contrary, shoot Na+ concentrations of the inoculated plants were 35 and 42% lower than those of the non-inoculated plants [Figure 3C; F(5,54) = 290.86, P < 0.05], and root Na+ concentrations of the inoculated plants were 30 and 56% lower [Figure 3D; F(5,54) = 510.83, P < 0.05], respectively. These further led to higher ratio of Na+/K+ in shoots [Figure 3E; F(5,54) = 426.03, P < 0.05] and roots [Figure 3F; F(5,54) = 640.31, P < 0.05] the non-inoculated plants than that of the inoculated plants under saline–alkaline stress, respectively.
|
study
| 100.0 |
Effects of B. licheniformis SA03 on (A,B) K+ concentration, (C,D) Na+ concentration, and (E,F) Na+/K+ ratio of shoots and roots in Chrysanthemum plants before (BS) or after (AS) saline–alkaline treatments. Treatments and statistical analysis were as described in Figure 2.
|
study
| 100.0 |
To inspect whether the inoculated of plants with SA03 could confront oxidative stress imposed by saline–alkaline treatment, we measured the content of two major types of ROS including O2•– and H2O2 in leaves of plants. Before saline–alkaline treatment, a slight increase of O2•– [F(5,54) = 160.58, P < 0.05] and H2O2 [F(5,54) = 213.56, P < 0.05] levels was observed in the inoculated plants compared with the non-inoculated plants (Figures 4A,B). However, the values of MDA and EL, important indicators of oxidative damage, in both the non-inoculated and inoculated plants did not differ significantly (Figures 4C,D). After 2 and 4 weeks of the stress treatment, the content of O2•– and H2O2 was markedly increased, especially in the 4-week treatment. By contrast, the inoculated plants displayed markedly lower content of O2•– and H2O2. Consistent with this, saline–alkaline treatment considerably induced a great increase of MDA in plants. However, the leaves of inoculated plants had 36 and 32% lower MDA content than that of the non-inoculated plants under the stress, respectively [Figure 4C; F(5,54) = 311.48, P < 0.05]. Similarly, soil inoculation markedly decreased the EL levels in the leaves of inoculated plants under the stress compared with the non-inoculated plants [Figure 4D; F(5,54) = 253.81, P < 0.05].
|
study
| 100.0 |
In plants, antioxidant enzymatic systems play essential roles in modulating dynamic homeostasis of cellular ROS, and thus detoxifying ROS effectively (Liu and Pang, 2010; Zhou et al., 2016b). Here, we investigated several antioxidant enzymatic activities in plants. The activities of antioxidant enzymes tested were no evident difference between the non-inoculated and inoculated plants before saline–alkaline treatment (Figure 5). After 2 and 4 weeks of the stress treatment, the activities of SOD, the first cellular defensive line converting O2•– into H2O2, were significantly increased in the inoculated plants [Figure 5A; F(5,54) = 63.87, P < 0.05]. In contrast to the non-inoculated plants, the activities of CAT converting H2O2 to H2O and O2 were greatly higher in the inoculated plants [Figure 5B; F(5,54) = 48.78, P < 0.05]. The AsA-GSH cycle is an important metabolic pathway that eliminates H2O2 through multiple enzyme catalyzing reactions (Wei L. et al., 2015). The activities of APX [Figure 5C; F(5,54) = 42.93, P < 0.05], DHAR [Figure 5D; F(5,54) = 61.64, P < 0.05], MDHAR [Figure 5E; F(5,54) = 116.41, P < 0.05], and GR [Figure 5F; F(5,54) = 77.76, P < 0.05] were significantly enhanced in the inoculated plants under the stress compared with the non-inoculated plants. Similar results were also observed for the activities of GSH metabolizing enzymes including GPX [Figure 5G; F(5,54) = 79.77, P < 0.05] and GST [Figure 5H; F(5,54) = 26.36, P < 0.05].
|
study
| 100.0 |
Effects of B. licheniformis SA03 on antioxidant enzymatic activities including (A) SOD, (B) CAT, (C) APX, (D) DHAR, (E) MDHAR, (F) GR, (G) GPX, and (H) GST in Chrysanthemum plants before (BS) or after (AS) saline–alkaline treatment. Treatments and statistical analysis were as described in Figure 2.
|
study
| 100.0 |
To clarify the mechanisms underlying SA03-induced saline–alkaline tolerance in plants, gene transcriptional profiles were analyzed by RNA-Seq. A total of 28,837,670 and 29,075,976 raw reads were generated in both the non-inoculated and inoculated plants by 454 sequencing, respectively (Supplementary Table S2), and the raw reads data were submitted into the NCBI SRA database (accession No. SRR5388903). After filtering out low quality reads, 23,021,019 (79.82%) and 22,541,540 (77.52%) clean reads were remained in both the NI and I library, respectively. Moreover, 22,003,842 clean reads (76.3%) in the NI library and 21,924,113 clean reads (75.4%) in the I library were uniquely mapped. We further compared analyses of gene expression between the non-inoculated and inoculated plants for screening DEGs with an FDR-adjusted p-value < 0.05 as the threshold (Supplementary Table S3).
|
study
| 100.0 |
Compared with the non-inoculated plants, there were 693 up-regulated unigenes and 2456 down-regulated unigenes in the inoculated plants (Figure 6). Moreover, up- and down-regulated unigenes were aligned to the GO and COG database for classifying and predicting gene functions. GO term annotations for these up-regulated unigenes were classified to three major categories including molecular function, cellular component and biological process. Of these, assignments to biological process constituted the majority, followed by cellular component and molecular function (Figure 7). The most overrepresented categories in the up-regulated DEGs were mainly related to some important pathways such as ‘response to salt stress,’ ‘response to water deprivation,’ ‘response to iron ion,’ and ‘hydrogen peroxide catabolic process’ in biological processes; ‘plasma membrane,’ ‘plant-type vacuole,’ ‘plastide,’ and ‘peroxisome’ in cellular component; ‘peroxidase activity,’ ‘glutathione transferase activity,’ ATPase activity,’ and ‘ferric-chelate reductase activity’ in molecular function. Among these up-regulated DEGs, some genes involved in Fe acquisition, Na+ transport and antioxidant systems were observably activated in the inoculated plants (Supplementary Table S4). Thus, the enhanced stress tolerance of plants by SA03 was closely associated with multiple signaling pathways involving Fe uptake and stress adaption. Moreover, qRT-PCR was used to confirm gene expression profiles that were found in the DEGs (eight unigenes: IRT1, FRD3, NHX1, AHA2, ZEP1, YSL1, SAUR21, and NAS1). The changing patterns of gene expression were in accordance with that detected by RNA-Seq (Supplementary Figure S1), indicating a high reliability of RNA-Seq data.
|
study
| 100.0 |
Distribution of differentially expressed genes between the non-inoculated (NI) and inoculated (I) libraries. (A) Scatter plot of gene expression difference. Green and red dots indicate up- and down-regulated genes under saline–alkaline stress, respectively. Blue dots indicate genes without significant differential expression, and gray dots indicate genes that were filtered out low quality reads. (B) Statistics of DEGs. Different color column indicated DEGs with diverse fold changes.
|
study
| 99.94 |
List of top 10 significant GO terms for up-regulated differentially expressed genes (DEGs) of SA03-inoculated plants based on GO classifications. GO terms were categorized into three groups: biological process, cellular component and molecular function. The P-value indicates the significance of the comparison between the non-inoculated and inoculated plants.
|
study
| 100.0 |
Some hormone signaling pathways in host plants are tightly regulated by some PGPR strains, and thus affecting various physiological processes (Poupin et al., 2016; Zhou et al., 2016a). ABA plays a cardinal role in abiotic stress responses in plants (Zhang et al., 2006). In this study, transcriptomic analyses indicated that the inoculation with SA03 might affect cellular ABA levels in plants, and further regulate stress-related signaling pathways. To test the hypothesis, the ABA content in plants was measured. The roots of inoculated plants exhibited slightly higher ABA content than that of non-inoculated plants before saline–alkaline treatment, whereas no significant difference was observed in shoots (Table 2). After 2 and 4 weeks of saline–alkaline treatment, the ABA content was significantly higher in shoots [F(5,54) = 369.14, P < 0.05] and roots [F(5,54) = 123.34, P < 0.05] of the inoculated plants than that of the non-inoculated plants (Table 2). Furthermore, we explored the effects of fluridone (FLU), an inhibitor of ABA biosynthesis, on the inoculated plants under saline–alkaline stress. It was observed that treatment with 10 μM FLU abolished the SA03-induced stress tolerance of plants (Figure 8A). Many studies have indicated that NO serves as an important signal molecule to regulate abiotic stress responses, Fe uptake and remobilization in plants (Graziano and Lamattina, 2007; Tanou et al., 2009; Chen et al., 2010; Wang et al., 2017). For this reason, we examined if NO was involved in the SA03-mediated stress responses in plants. Intriguingly, the inoculation with SA03 could not increase the tolerance of plants to saline–alkaline stress after treatment with 150 μM c-PTIO, a scavenger of NO. That was similar to the phenotypes observed for the FLU-treated plants (Figure 8A).
|
study
| 100.0 |
Treatment with FLU or c-PTIO abrogated the effects of B. licheniformis SA03 on saline–alkaline tolerance of Chrysanthemum plants. After 10 days of bacterial inoculation, plants were subjected to saline–alkaline stress for 4 weeks with or without FLU or c-PTIO treatments. These plants were used to analyze (A) growth phenotypes, shoot and root (B) K+ or (C) Na+ concentration, shoot (D) and root (E) Na+/K+ ratio, shoot (F) and root (G) Fe concentrations. I, inoculated plants; NI, non-inoculated plants; BS, before the stress treatment; AS, after the stress treatment. Data are expressed as the mean values of three replicates (±SE) with 10 plants each. Different letters indicate significant differences using one-way ANOVA followed by the Duncan’s multiple range test at P < 0.05.
|
study
| 100.0 |
In addition, some physiological parameters were determined in the stress-treated plants with FLU or c-PTIO treatment. After 4 weeks of FLU exposure, shoot and root Na+ concentrations [Figure 8B; F(7,72) = 333.37, P < 0.05] were significantly increased in the inoculated plants under saline–alkaline stress, but shoot and root K+ concentrations [Figure 8C; F(7,72) = 94.66, P < 0.05] was evidently decreased in the inoculated plants under the stress, thereby leading to higher shoot [Figure 8D; F(3,36) = 89.93, P < 0.05] and root [Figure 8E; F(3,36) = 231.41, P < 0.05] Na+/K+ ratios. However, there was no significant difference in these parameters between the non-inoculated and FLU-treated inoculated plants. Furthermore, shoot Fe concentrations of non-inoculated plants did not differ significantly from the FLU-treated inoculated plants (Figure 8F). Similarly, root Fe concentrations were no significant difference between the non-inoculated and FLU-treated inoculated plants (Figure 8G). Moreover, the Na+ and K+ concentrations, Na+/K+ ratios as well as Fe concentrations were also determined in c-PTIO-treated plants. The changing tendency of physiological parameters in the c-PTIO-treated inoculated plants displayed the similarities with the results observed for the FLU-treated inoculated plants, whereas these negative effects was notably aggravated by c-PTIO treatment. Concomitantly, either FLU or c-PTIO treatment significantly reduced total chlorophyll content [Figure 9A; F(3,36) = 147.13, P < 0.05] and photosynthetic parameters including Fv/Fm [Figure 9B; F(3,36) = 422.56, P < 0.05], ΦPSII [Figure 9C; F(3,36) = 166.62, P < 0.05] and Pn [Figure 9D; F(3,36) = 107.68, P < 0.05] in the inoculated plants under the stress compared with the untreated inoculated plants. As the photosynthetic apparatus, fully swollen chloroplasts occurred in the leaves of non-inoculated plants under the stress, whereas chloroplast ultrastructure of the inoculated leaves was not severely damaged by saline–alkaline stress (Figures 9E–L). When plants were treated with FLU or c-PTIO, the number of grana stacking was evidently decreased in the inoculated plants under the stress. However, the damages were even further increased in the c-PTIO-treated inoculated plants. These results implied the roles of ABA and NO in the SA03-induced stress tolerance of plants.
|
study
| 100.0 |
Treatment with FLU or c-PTIO affected (A) total chlorophyll content, (B) Fv/Fm, (C) ΦPSII, and (D) Pn. In addition, chloroplast ultrastructure of the non-inoculated and inoculated plants: (E) the inoculated plants, (G) the non-inoculated plants, and (I) FLU- or (K) c-PTIO-treated inoculated plants. (F,H,J,L) Indicated locally amplified views in (E,G,I,K), respectively. Scale bar = 1 μm. Treatments and statistical analysis were as described in Figure 8.
|
study
| 100.0 |
In this study, both ABA and NO seemed to participate in SA03-induced saline–alkaline tolerance of plants. This raised the question how ABA interacted with NO to regulate the adaptive responses of SA03-inoculated plants to saline–alkaline stress. Since ABA has been shown to regulate the NO accumulation in plants under salt stress (Zhang et al., 2009), we wondered if SA03-induced a great increase of ABA resulted in promoting NO biosynthesis. Indeed, we found that cellular NO levels were markedly higher in the inoculated roots compared with the non-inoculated plants under non-stress condition. Moreover, saline–alkaline treatment strikingly enhanced the NO biosynthesis in plants, whereas the inoculated plants accumulated more NO than the non-inoculated plants. Intriguingly, FLU exposure did not remarkably affect endogenous NO content in the inoculated plants under non-stress condition, whereas a marked decrease of NO levels was observed in the FLU-treated inoculated plants under the stress compared with the untreated inoculated plants (Supplementary Figure S2).
|
study
| 100.0 |
Enhanced Fe acquisition and reduced Na+ toxicity are required for improving the tolerance of plants to saline–alkaline stress (Li et al., 2016). In this study, the SA03-inoculated plants exhibited higher Fe accumulation and lower Na+ levels under saline–alkaline stress. qRT-PCR analyses showed that the transcription levels of Fe acquisition-related genes and Na+/H+ antiporter genes were significantly up-regulated in the inoculated plants compared with the non-inoculated plants. To clarify how microbial induction of ABA and NO regulated these adaptive responses, the transcription of genes associated with Fe acquisition and Na+/H+ antiporters were investigated in plants treated with FLU or c-PTIO. The expression levels of Fe uptake-related (IRT1, FRO2, and AHA2) [Figure 10; F(11,24) = 202.47, P < 0.05], Fe transport-related (YSL1, YSL2, FRD3 and NAS1) [Figure 10; F(15,32) = 71.20, P < 0.05], and Na+/H+ antiporter (NHX1, NHX2 and NHX5) [Figure 10; F(11,24) = 251.41, P < 0.05] genes were remarkably increased in the inoculated plants under the stress compared with the non-inoculated plants. However, their transcription levels were markedly down-regulated in the inoculated plants after FLU treatment. Upon exposure to c-PTIO, the transcription of Fe acquisition-related and Na+/H+ antiporter genes was greatly repressed in the inoculated plants, but no significant difference was observed for the transcription of Fe transport-related genes. Moreover, either FLU or c-PTIO treatment reduced the expression of Na+/H+ antiporter genes in the inoculated plants under the stress.
|
study
| 100.0 |
Treatment with FLU or c-PTIO affected the expression of Fe acquisition and Na+/H+ antiporter genes in SA03-inoculated Chrysanthemum plants. After 10 days of SA03-inoculation, plants were subjected to saline–alkaline stress for 4 weeks with FLU or c-PTIO treatment. These plants were used to examine the transcription of (A) Fe uptake-related (IRT1, FRO2, and AHA2), (B) Fe transport-related (YSL1, YSL2, FRD3, and NAS1), and (C) Na+/H+ antiporter (NHX1, NHX2, and NHX5) genes by qRT-PCR. Data are expressed as the mean values of three replicates (±SE). Different letters indicate significant differences using two-way ANOVA followed by the Duncan’s multiple range test at P < 0.05.
|
study
| 100.0 |
Along long-term evolution, plants have developed some flexible mechanisms to adapt to adverse environments (Marschner and Römheld, 1994; Chen et al., 2010; Li et al., 2016). It is one of important strategies to attract colonization of diverse beneficial microbes in the rhizosphere of host plants (Lebeis et al., 2015). It is widely recognized that the complex mutualistic interactions can assist plants to cope with unfavorable conditions (Ait Barka et al., 2006; Mishra et al., 2014; Sukweenadhi et al., 2015). Recently, numerous studies have indicated that PGPR confers increased tolerance of plants to various abiotic stresses including drought, salinity, and nutrient deficiency (Dey et al., 2004; Scholz et al., 2011; Zhou et al., 2016a,b). However, whether PGPR could induce saline–alkaline tolerance in plants and the underlying mechanisms remain elusive. We reported here for the first time that Chrysanthemum plants inoculated with B. licheniformis SA03 were greater resistant to saline–alkaline conditions, as evidenced by lower biomass loss and higher survival rates. Moreover, transcriptomic, biochemical, and pharmacological analyses were combined to unravel the mechanisms behind SA03 activated the adaptive responses of plants to saline–alkaline conditions. Our results revealed that the SA03-induced ABA accumulation was required for the tolerance of plants to saline–alkaline stress, indicating that the increased ABA level was a primarily acting mode of SA03 to regulate saline–alkaline stress response in plants.
|
study
| 99.94 |
Plant growth and productivity is tightly associated with adverse conditions, which cause a marked decrease in plant photosynthesis, thereby affecting its productivity (Li et al., 2016; Zhou et al., 2016b). In this study, in addition to greater biomass, the SA03-inoculated plants displayed higher chlorophyll content under saline–alkaline stress, which possibly led to stronger photosynthetic capacity. This positive effect may improve the growth performance of inoculated plants under saline–alkaline stress. A great photosynthetic efficiency in plants indicates that the photosynthetic apparatus is not considerably damaged by saline–alkaline treatment (Gong et al., 2014). It has recently been shown that the structural and functional integrity of chloroplasts are seriously destroyed by saline–alkaline stress (Li et al., 2016). In this study, the stress-treated plants displayed leaf chlorosis, indicating a serious disturbance of normal chloroplast development. Saline–alkaline stress has recently been shown to reduce the contents of chlorophyll and affect normal chloroplast development (Gong et al., 2014). Here, the inoculation with SA03 conspicuously lessened oxidative damages to chloroplast ultrastructure caused by saline–alkaline stress to a large extent, thereby leading to high photosynthetic efficiency. Furthermore, the non-inoculated plants exhibited the increased values of ROS and MDA under saline–alkaline stress, whereas their values were markedly lower in the inoculated plants. Additionally, the EL levels were relatively lower in the inoculated plants than the non-inoculated plants. Consistently, the inoculated plants owned higher ROS-detoxifying enzymatic activities than the non-inoculated plants. Ample evidence has indicated that the enhanced activities of antioxidant enzymes play important roles in modulating ROS levels in plants under adverse stresses (Gong et al., 2014; Wei L. et al., 2015; Zhou et al., 2016b). Therefore, these findings indicated that the inoculation with SA03 can alleviate oxidative damage imposed by saline–alkaline stress.
|
study
| 99.94 |
In fact, plants have to cope with two inevitable challenges including low availability of Fe and Na+ toxicity under saline–alkaline stress (Li et al., 2016). Fe often forms exceedingly insoluble hydroxides and oxides under alkaline conditions, thereby reducing its availability for plants (Romera and Alcántara, 2004). In this study, the mixtures of NaHCO3 and Na2CO3 were added into soils to mimic saline–alkaline conditions with high levels of Na+ and soil pH values. Overproduction of cellular ROS can be triggered by either Fe deficiency or high concentrations of Na+, which causes peroxidation of membrane lipid and proteins, and even cell death (Gong et al., 2014). Here, the SA03-inoculated plants experienced less ROS-mediated oxidative injury under saline–alkaline stress compared with the non-inoculated plants. This allowed us to conclude that SA03 conferred more efficient systems of plants to regulate the accumulation of Fe and Na+. To prove these assumptions, the Fe concentrations in both the non-inoculated and inoculated plants were firstly examined under saline–alkaline stress. A significant decrease of Fe concentrations was observed in shoots of the non-inoculated plants grown under the stress. However, the Fe concentrations remained relatively high in shoots of the inoculated plants. The results demonstrated that the SA03-inoculated plants equipped with an efficient system of Fe acquisition, thereby enhancing the adaptation of plants to Fe deficiency induced by saline–alkaline stress.
|
study
| 100.0 |
Besides low Fe bioavailability, high Na+ concentrations seriously disrupt plant growth in saline–alkaline soils, and thus exposing plants to saline stress (Wang et al., 2012). Saline-tolerant plants can effectively maintain a high K+ concentration and a concurrent low Na+ in their shoots, indicating that the Na+/K+ ratio is an index of salt tolerance in plants (Cui et al., 2016). Transgenic plants with high tolerance to salt stress display lower Na+/K+ ratio compared with wild-type plants (Cui et al., 2016; Zhang et al., 2016). Takahashi et al. (2007) have reported that the saline-tolerant reed plants have lower shoot Na+/K+ ratio than plants that are the most sensitive to saline stress. We observed here that shoots and roots of SA03-inoculated plants displayed relatively lower Na+/K+ ratio than non-inoculated plants under saline–alkaline stress, indicating that the enhanced saline–alkaline tolerance by SA03 was partially attributable to minimize Na accumulation. Exposure to high concentrations of Na+ has been shown to severely damage various enzymatic activities in plants (Gong et al., 2014). It is well documented that the increased ABA levels remarkably increases root net Na+ efflux and H+ influx, and decreases net K+ efflux in transgenic maize by activation of Na+/H+ antiporters and K+ channels (Zhang et al., 2016). Hence, the induced expression of Na+/H+ antiporter genes is a crucial strategy for plants to tolerate salt stress.
|
study
| 100.0 |
To elucidate the mechanisms underlying the SA03-induced stress tolerance of plants, comparative transcriptomic analyses were used to identify DEGs associated with the adaptive responses of plants to the stress. Here, the inoculation with SA03 activated several major biological processes associated with salt and drought stress, Fe acquisition, wounding, and response to ABA under saline–alkaline stress. It is well known that ABA plays a cardinal role in the regulation of abiotic stress responses and tolerance in plants. The increased ABA levels can activate a wide array of many stress-responsive genes in plants, which contributes to abiotic stress tolerance. In addition, ABA has been demonstrated to regulate Fe deficiency responses in Arabidopsis plants (Lei et al., 2014). Intriguingly, ABA can also enhance the activity of plasma membrane H+-ATPase to release protons along root tips (Xu et al., 2013). In this study, transcriptomic analyses showed that the ATPase activity was significantly increased in the SA03-inoculated plants under saline–alkaline stress. Recent studies have indicated that plasma membrane H+-ATPase plays a vital role in the adaptation of plant roots to alkaline conditions by modulating proton secretion (Fuglsang et al., 2007; Yang et al., 2010), indicating that the ABA-induced H+-ATPase activity can increase the release of protons into plant rhizosphere for counteracting adverse impacts imposed by alkaline pH conditions. More recently, some PGPR strains markedly induce ABA accumulation in host plants under abiotic stress (Salomon et al., 2014; Cohen et al., 2015; Park et al., 2017). Salomon et al. (2014) have reported that B. licheniformis Rt4M10 reduces water losses in drought-treated grapevine plants by induction of ABA synthesis. Azospirillum brasilense ameliorates drought stress in Arabidopsis thaliana mainly through enhancement of ABA levels (Cohen et al., 2015). Moreover, the inoculation with Bacillus aryabhattai SRB02 induces a great increase of ABA level and further activates ABA-mediated stomatal closure in soybean, which contributes to better heat stress tolerance (Park et al., 2017). These results have indicated that application of PGPR can enhance the tolerance of host plants to various abiotic stresses by activating ABA-mediated signaling pathways. Thus, the increased tolerance of SA03-inoculated plants against saline–alkaline stress may result from alteration of ABA levels.
|
study
| 99.94 |
To verify this hypothesis, the changing patterns of ABA content in plants were examined. As expectedly, a marked increase in ABA levels was observed in leaves and roots of non-inoculated plants as a consequence of saline–alkaline treatment. Also, the inoculated plants exhibited a great rise in ABA levels, displaying a similar pattern for the non-inoculated plants. However, more ABA accumulation was observed in the inoculated plants than the non-inoculated plants. Wei L.X. et al. (2015) have reported that ABA treatment can improve the tolerance of rice plants to alkaline stress. Hence, the SA03-induced ABA accumulation may be responsible for enhancing the adaptation of plants to saline–alkaline stress. We further examined if the inhibited ABA biosynthesis affected the SA03-induced stress responses in plants. When plants were treated with FLU, the inoculated plants exhibited similar phenotypes with the non-inoculated plants under saline–alkaline stress. Consistently, several physiological parameters such as photosynthesis and the accumulation of Na+, K+ and Fe were markedly altered in the inoculated plants under the stress, which indistinctly differed from the non-inoculated plants. Similar results are recently reported by Wei L.X. et al. (2015) in which FLU exposure markedly increases the degree of cell membrane injury and reduces relative water content under alkaline stress, indicating that ABA plays a vital role in mediating the adaptive responses of plants to alkaline stress. Additionally, qRT-PCR analyses revealed that FLU exposure pronouncedly down-regulated the transcription of some Fe acquisition- and Na+ transport-related genes, which was in accordance with the physiological parameters observed.
|
study
| 100.0 |
Interestingly, the inoculation with SA03 notably promoted the NO accumulation in the stress-treated plants accompanied by the increased cellular ABA levels. However, FLU exposure notably repressed the NO biosynthesis under saline–alkaline stress. It has previously been indicated that NO plays crucial roles in abiotic stress response and tolerance in plants, which is combined with ABA and other hormones (León et al., 2014). Moreover, NO acts as downstream signals of ABA to regulate salt tolerance in plants by activating antioxidant enzymatic activities and Na+/H+ antiporters (Zhang et al., 2009). Recently, NO has also been found to regulate Fe deficiency responses by remobilizing cell wall Fe and provoking the FIT1-mediated signaling pathways (Graziano and Lamattina, 2007; Chen et al., 2010; Wang et al., 2017). Thus, NO may function as a secondary messenger of ABA to activate diverse adaptive mechanisms that alleviate adverse effects caused by saline–alkaline stress. In this study, the inoculated plants treated with c-PTIO shared the resemblance in phenotypic traits and alteration of physiological parameters with the FLU-treated inoculated plants under the stress. Intriguingly, c-PTIO exposure did not markedly down-regulate the expression of Fe transport-related genes in the inoculated plants compared with the FLU-treated inoculated plants, although no significant difference in shoot Fe concentrations was observed between the c-PTIO- and FLU-treated plants under saline–alkaline stress. Previous studies have indicated that NO not only regulates FIT1-mediated IRT1 and FRO2, but also can chelate Fe from cell wall (Chen et al., 2010). It is well known that about 75% of Fe is deposited in root cell walls, which are severed as largest reservoir for apoplastic Fe (Bienfait et al., 1985). Fe deficiency can trigger rapid accumulation of NO to invoke Fe uptake and reutilize cell wall Fe (Graziano and Lamattina, 2007). These indicated that inhibition of NO biosynthesis blocked plant uptake of Fe from rhizosphere soils and remobilization of apoplastic Fe from roots to shoots under Fe deficient conditions. This may explain the reason that the FLU-treated inoculated plants had high-level expressions of Fe transport-related genes under saline–alkaline stress, which could not lead to the increase in shoot Fe concentrations. Therefore, the SA03-induced stress tolerance of plants mainly attributed to the ABA-mediated NO signaling pathways.
|
study
| 99.94 |
The inoculation of Chrysanthemum plants with B. licheniformis SA03 ameliorated the detrimental impacts caused by saline–alkaline stress by enhancement of the ABA levels. Soil inoculation sufficiently activated a series of adaptive mechanisms such as increased antioxidant enzymatic activities, enhanced Fe acquisition, and decreased Na+ accumulation in host plants, which were in correlation with the actions of NO. The findings confirmed the roles of SA03 in assisting host plants to saline–alkaline stress and its use as a potential strategy in sustainable agriculture.
|
study
| 100.0 |
Characterized by a median survival ranging from 5 to 59 months and accounting for 70% of adult primary central nervous system tumors, glioma has been regarded as the most deadly brain malignancy that is essentially incurable [1, 2]. Currently, TMZ is widely used in glioma treatment and has been proved to benefits patients [3, 4]. Unfortunately, drug resistance restricts the clinical application of TMZ and therefore contributes to the dismal outcome of glioma patient [5, 6]. This challenging problem attributes to multitudes of aberrant molecular changes, such as MGMT promoter methylation, α5β1 integrin expression, sonic hedgehog and notch pathway activation [7–10]. However, none of them has been successfully translated into clinical application and resultantly improved TMZ efficacy [11–14]. Thus, comprehensive understanding of TMZ resistance in glioma is urgently needed.
|
review
| 99.9 |
DNA-PKcs is a nuclear protein serine/threonine kinase which plays a pivotal role in the non-homologous end-joining (NHEJ) pathway for DNA double-strand break (DSBs) repair . Recently, series of studies have characterized that DNA-PKcs can also act as molecular promoter to control a wide array of cellular functions, such as cell cycle, metabolism, hypoxia, inflammatory response [16–19]. Activation of DNA-PKcs regulates various tumor-promoting molecules, such as maintaining the stability of Snail1, Chk1–Claspin complex, or enhancing the transcriptional activity of p53 and androgen receptor (AR), and resultantly mediates the progression of colon cancer, prostate adenocarcinoma, hepatocellular carcinomas (HCC) and other human cancers [20–23]. However, the role of DNA-PKcs in glioma progression remains to be elucidated.
|
review
| 99.1 |
As a result of its functional diversity, DNA-PKcs has been documented to play a critical role in the development of chemoresistance. Ganesh R. Panta et al noted that DNA-PKcs activated prosurvival NF-κB pathway through MEK/ERK signaling and opposed the apoptotic response following chemotherapeutic agent . Furthermore, Suk-Bin Seo et al reported that TRAIL inhibited the activation of DNA-PKcs/AKT/GSK-3β pathway and thereby sensitized tumor cells to vinblastine and doxorubicin . Thus, exploring the biologic function of DNA-PKcs and its mediated signaling may provide an ideal therapeutic target for overcoming TMZ resistance in glioma treatment.
|
study
| 99.25 |
In this study, we surveyed the p-DNA-PKcs (Ser 2056) level in human glioma samples and observed that hyperactivation of DNA-PKcs was closely associated with both malignant progression and poor clinical outcome of glioma patients. We further explored the potential correlation between inhibition of DNA-PKcs and TMZ efficacy in glioma. The results demonstrate a striking synergistic effect between DNA-PKcs inhibitor KU0060648 and TMZ in glioma cells. Inhibition of DNA-PKcs enhances TMZ sensitivity mainly via suppression of AKT activation. This study provides a potential target for evaluating glioma progression and improving TMZ efficacy in glioma therapy.
|
study
| 100.0 |
To investigate the activated status of DNA-PKcs in glioma progression, we first evaluated the expression levels of phosphorylated-DNA-PKcs (Ser 2056, p-DNA-PKcs S2056) in human gliomas and their paired adjacent nontumorous tissues or normal human brain tissues using immunoblotting. As shown in Figure 1A, p-DNA-PKcs was significantly higher in 7 human glioma specimens than their respective adjacent nontumorous tissues or 2 normal brains. Immunohistochemistry (IHC) analysis in a cohort of 217 paraffin-embedded glioma samples further confirmed the overexpression of p-DNA-PKcs in 57.2% of gliomas (124/217) as compared with corresponding non-tumor tissues (62/217, 28.6%; Figure 1B - 1C, Supplementary Table S1). We then assessed the relationship between p-DNA-PKcs levels and the clinical features of glioma. Strong expressions of p-DNA-PKcs were positively correlated with higher grade tumor status (Figure 1D - 1E, Supplementary Figure S1), and also closely associated with worse survival of glioma as determined by the Kaplan-Meier and log-rank tests for survival analysis (OS, p < 0.0001; Figure 1F). More importantly, multi-variate analysis through Cox regression model with all 6 parameters (p-DNA-PKcs level, age, gender, tumor location, debulking degree, tumor grade) identified the independent prognostic significance of p-DNA-PKcs (hazard ratio: 3.052; p < 0.001; 95% CI: 2.204 - 4.572), which was not linked to known prognostic factors such as ages and tumor grades (Supplementary Table S2).
|
study
| 100.0 |
A. Immunoblotting analysis of p-DNA-PKcs (S2056) expression in 2 normal human brains from trauma, 7 paired primary glioma tissues (T) and matched adjacent nontumorous tissues (ANT) from the same patient (Patients No.1,2: WHO grade IV; No.3,4: WHO grade III; No.5,6: WHO grade II; No.7: WHO grade I). Actin was used as a loading control. B, C. Immunohistochemistry (IHC) study on p-DNA-PKcs expressions between gliomas and paired normal tissues. Representative IHC images (B) (magnification, ×40 as indicated) and statistical analysis (C) (p < 0.001, t test). D. IHC staining of p-DNA-PKcs in different grades of gliomas and normal brain tissues (magnification, ×10 and ×40 as indicated). E. Correlation between p-DNA-PKcs expression and tumor grade in surveyed cohort. (Bars, median expression values of IHC scores; ★, p < 0.05; ★★, p < 0.001; Wilcoxon rank sum test). F. Kaplan-Meier curves of glioma patients with low vs. high level of p-DNA-PKcs (n=217; p < 0.0001, log-rank test).
|
study
| 100.0 |
Aiming to comprehend if the activated DNA-PKcs arose from DSBs, we selected 155 patients with primary glioma occurrence and null chemo- or radiotherapy before surgery from our glioma cohort, then surveyed the expression of γH2AX. On the contrary to that p-DNA-PKcs levels were positively associated with glioma grades, γH2AX did not appear to be discriminatingly expressed among different grades of glioma tissues (Supplementary Figure S2A). Further analysis certified that there was not correlation between expression of γH2AX and p-DNA-PKcs, suggesting that activation of DNA-PKcs in glioma was not exclusively in response to DSBs (Supplementary Figure S2B). Taken together, these results indicated that p-DNA-PKcs expression was abnormally overexpressed in gliomas and dysregulated expression of p-DNA-PKcs correlated with malignant development and poor prognosis in clinical glioma patients.
|
study
| 100.0 |
Next we sought to address the expression of p-DNA-PKcs in glioma cell lines. Data in Figure 2A revealed that, in contrast to normal human astrocyte (NHA) which possessed an undetectable level of activated DNA-PKcs, p-DNA-PKcs were expressed in a panel of glioma cells. We also examined the γH2AX level in these cells. However, none of them demonstrated an obvious band of γH2AX (Supplementary Figure S3). Notably, cell lines with high levels of p-DNA-PKcs (U87 and M059K) demonstrated higher IC50 values of TMZ compared with A172 and H4. We then determined whether DNA-PKcs contributed to glioma cell growth and TMZ sensitivity. Strikingly, DNA-PKcs siRNA transfection effectively suppressed the proliferation of U87 and M059K cells and enhanced the cytotoxic effect of TMZ using MTS assays (Figure 2B and Supplementary Figure S4).
|
study
| 100.0 |
A. Assessment of p-DNA-PKcs/DNA-PKcs expression in human glioma cell lines by immunoblotting. Temozolomide (TMZ) IC50 values were presented in right table. B. siRNA-mediated knockdown of DNA-PKcs reduced glioma cell lines U87 and M059K proliferation. Immunoblotting confirmed the knockdown efficacy, and cell growth curve was measured by MTS assay. C. Pharmacologic characteristic of KU0060648 on p-DNA-PKcs inhibition following 6-hour incubation in U87 and M059K cells (left). Proliferations of two cell lines were investigated by MTS assay after 72-hours treatment (right). D. TMZ IC50 of U87 and M059K in the absence or presence of KU0060648. Two cell lines were incubated for 3 days in a range of concentrations of TMZ with or without of 1 or 10 μM KU0060648 and IC50 value was then calculated by MTS assay (Bars, SD; ★, p < 0.05; ★★, p < 0.001, one-way ANOVA test). E. Colony formation assay evaluating the growth of U87 and M059K cells in treatment of TMZ (100 μM) and KU0060648 (1 or 10 μM) either alone or in combination for 14 days. Cells were stained with 0.1% crystal violet (Bars, SD; ★, p < 0.05; ★★, p < 0.001; one-way ANOVA test).
|
study
| 100.0 |
We further characterized if inhibition of DNA-PKcs could suppress glioma cell growth or enhance TMZ sensitivity using KU0060648, a novel DNA-PKcs inhibitor which was applied in previous researches [26, 27]. As shown in Figure 2C, KU0060648 inhibited the activation of DNA-PKcs (left panel of Figure 2C) and the proliferation of glioma cells (right panel of Figure 2C) in a dose-dependent manner. Moreover, KU0060648-mediated DNA-PKcs inhibition led to sensitization of TMZ in glioma cells. Addition of 1 or 10 μM of KU0060648 increased TMZ efficacy remarkably with the IC50 values dropping from 487 ± 24 μM to 154 ± 16 μM or 44 ± 12 μM, respectively in U87 (Figure 2D). Similar results were observed in M059K (Figure 2D). To better understand the effect of KU0060648 on TMZ sensitivity, glioma cell lines with lower levels of p-DNA-PKcs, namely H4 and U373, were also investigated. In contrast to U87 and M059K, KU0060648 only mildly restored the sensitivity to TMZ in H4 and U373 (Supplementary Figure S5), which supported the idea that KU0060648-sensitized TMZ depended on the suppression of p-DNA-PKcs levels.
|
study
| 100.0 |
We subsequently assessed the long-term influence of KU0060648 on tumor growth and TMZ sensitivity using anchorage-dependent colony formation assay. Figure 2E elucidated that co-administration of KU0060648 and TMZ substantially reduced colony formation compared with each single agent. Collectively, our data revealed that inhibition of DNA-PKcs by KU0060648 could suppress proliferation and enhance the TMZ cytotoxicity in glioma cells.
|
study
| 100.0 |
To investigate the effect of KU0060648, TMZ or KU0060648/TMZ on apoptosis of glioma cells, U87 cells were treated with these agents alone or their combination for 48 hours and then cell apoptosis was assessed by flow cytometry (FCM). As shown in Figure 3A, 1 μM or 10 μM KU0060648 alone was able to increase the apoptotic rates to 14.8% and 56.9% respectively in U87 cells. 100 μM TMZ exerted minimal effect, while KU0060648 dose-dependently enhanced the apoptotic rate of TMZ treatment, which was certified in TUNEL assay (Figure 3B). To further classify the proapoptotic effect of combination treatment, morphological changes of U87 cells were evaluated using Hoechst staining (Figure 3C). 100 μM TMZ alone did not induce evident morphological alternations in U87 cells. 1 or 10 μM KU0060648 alone, or in combination with 100 μM TMZ could arouse the distinct morphology features of apoptosis, including the formation of apoptotic body and early coalescence of nuclear chromatin, margination and nuclear shrinkage. Similar results were obtained in M059K (Figure 3A - 3C). Notably, we discovered that treatment of KU0060648 increased the caspase 3/7 activity-promoting effect of TMZ in both U87 and M059K (Figure 3D), suggesting the synergistic augment of apoptosis caused by KU0060648 and TMZ majorly depended on the activation of caspases.
|
study
| 100.0 |
U87 (up) and M059K (down) cells were treated with 100 μM TMZ, 1 or 10 μM KU0060648, and their combinations for 48-hours, cell apoptosis was evaluated by Annexin V/PI FCM assay A. TUNEL assay B. and Hoechst 33258 staining C. Caspase-3/7 activity was detected in the above treatments D. (Scale bars: 100 μm; bars, SD; ★, p < 0.05; ★★, p < 0.001; one-way ANOVA test).
|
study
| 100.0 |
In order to determine whether KU0060648 inhibited glioma growth and sensitized temozolomide through suppression of DNA damage repair, we detected γH2AX foci, a frequently used assay visualizing the occurrence of DSBs, in KU0060648 single agent group or co-administrated with TMZ (Supplementary Figure S6). Indeed, there was undetectable level of background foci formation per cell in DMSO-treated control group, which was not increased by sole KU0060648 (data not shown). Such a finding prompted that KU0060648 exerted its anti-malignancy effects on glioma cells via a DNA damage repair independent manner, which consisted with the effect of NU7441, another well-known DNA-PKcs specific inhibitor, in a previous study . Exposure of 100 μM temozolomide alone did form γH2AX foci in both U87 and M059K cells. However, it declined rapidly, as only 11% (U87) and 16% (M059K) remained respectively after 120 hours. Additional KU0060648 did not affect the level of TMZ-induced foci formation but significantly retarded the loss of γH2AX foci. On the basis of these results, we postulated that suppressing glioma malignancies and enhancing TMZ efficacy by DNA-PKcs inhibitor might not merely depend on inhibition of DSBs repair, and thus, DNA-PKcs inhibitors can be used as single or synergized with TMZ against glioma.
|
study
| 100.0 |
Highly invasive and angiogenic nature of glioma contributes to the dismal outcome. However, instead of fighting against those aggressive characteristics, TMZ efficacy could be compromised by glioma invasion and angiogenesis. To determine whether KU0060648 inhibited invasive properties of glioma, U87 cells were treated with KU0060648 alone or in combination with TMZ for 16 hours and then assessed by Matrigel-coated Transwell assay. As shown in Figure 4A, 100 μM TMZ could not inhibit the invasiveness of U87, but rather slightly increased cells invasion. In contrast, 1 or 10 μM KU0060648 alone significantly decreased the invasive ability of U87 cells and profoundly prompted the anti-invasive ability of TMZ.
|
study
| 100.0 |
A. Cell invasion was quantified by Matrigel coated Transwell assay in U87 and M059K cells with 100 μM TMZ, 1 or 10 μM KU0060648, and their combinations treatment. Penetrated cell were counted using NIH ImageJ (Scale bars: 100 μm; bars, SD; ★, p < 0.05; ★★, p < 0.001; one-way ANOVA test). B. Treatment of TMZ (100 μM) and KU0060648 (1 or 10 μM) either alone or in combination inhibited glioma angiogenesis. Tube length and branch were quantified using NIH ImageJ (Scale bars: 100 μm; bars, SD; ★, p < 0.05; ★★, p < 0.001; one-way ANOVA test).
|
study
| 100.0 |
Furthermore, HUVEC tube formation assay indicated that schedule of KU0060648 treatment dose-dependently inhibited in vitro angiogenesis. 1 or 10 μM KU0060648 alone reduced the tube formation index of HUVECs, and dose-dependently enhanced the anti-angiogenic ability of 100 μM TMZ (Figure 4B) which was ineffective while promoted angiogenesis as single agent in U87 cells. Similar results were also obtained in M059K cells (Figure 4A and 4B).
|
study
| 100.0 |
To dissect the potential mechanism by which DNA-PKcs inhibition mediated antiproliferative effect and synergized with TMZ, we treated U87 and M059K cells with AKT inhibitor MK-2206 or MEK inhibitor PD98059 either alone or in combination with TMZ. After 3-day administration, both single and combination strategies of MK-2206 treatment resulted in a substantial decrease in cell viability. However, PD98059 did not induce additional growth inhibition in glioma cells, suggesting that AKT signaling contributed to the biological effect of KU0060648 (Figure 5A). We further observed that KU0060648 caused a strong and dose-dependent decrease in AKT phosphorylation (p-AKT Ser 473) of U87 and M059K cells using immunoblotting (Figure 5B), which indicated that inhibition of DNA-PKcs could suppress the activation of AKT in glioma cells.
|
study
| 100.0 |
A. Cell proliferation/viability of U87 and M059K was determined by MTS assay after 3-day incubation with 2 or 10 μM MK2206, 5 or 25 μM PD98059, or in combination with 100 μM TMZ. (Bars, SD; ★, p < 0.05; ★★, p < 0.001; N.S, no significance; one-way ANOVA test). B. KU0060648 dose response (6 hours) in disrupting DNA-PKcs/AKT interaction. Whole cell lysates (WCL) were immunoprecipitated with the antibodies against the indicated proteins. Immunocomplexes were then immunoblotted using antibodies against the indicated proteins, which is accompanied with p-AKT (S473) expression. C. Immunoblotting (left) and correlation analyses (right) of p-DNA-PKcs expression with the levels of p-AKT in 8 freshly collected human glioma samples (Pearson's correlation coefficients). Actin was used as loading controls. D. The expression levels of p-AKT were associated with the expression of p-DNA-PKcs in 42 primary human glioma specimens, which was quantified by IHC. Two representative cases with serial sections staining were shown (left) (magnification, ×10 and ×40 as indicated). Percentage of samples showed low or high p-AKT expression relative to the levels of p-DNA-PKcs (right). (★★, p < 0.001; chi-square test).
|
study
| 100.0 |
To exclude the off-target effect of KU0060648, especially its nature of ATP competitive inhibitor which can suppress the AKT activity, we investigated the inhibition of DNA-PKcs-AKT axis by KU0060648 after depleting DNA-PKcs in U87 and M059K (Supplementary Figure S7). RNAi specifically repressed the expression of DNA-PKcs and the activity of AKT. After knockdown of the DNA-PKcs, KU0060648 did not exert additional inhibitory effect on AKT phosphorylation. These findings indicated that KU0060648 was a specific inhibitor of DNA-PKcs rather than PI3K-AKT signaling, which was consistent with a previous study .
|
study
| 100.0 |
We then performed immunoprecipitation to evaluate if dephosphorylation of DNA-PKcs by KU006048 led to an immediate dissociation of AKT from DNA-PKcs. As expected, KU0060648 disrupted the formation of DNA-PKcs/ AKT complex in a dose dependent manner and correspondingly inhibited the activation of AKT (Figure 5B).
|
study
| 100.0 |
Especially, analyses of 8 freshly collected glioma specimens clarified a positively clinical relevance between p-DNA-PKcs expression and p-AKT (Figure 5C), which was confirmed by a validate cohort of 42 glioma patients with IHC staining (Figure 5D). These data further unearthed a functional link between p-DNA-PKcs and p-AKT in glioma patients, supporting the notion that DNA-PKcs contributed to glioma aggressiveness via activating AKT signaling.
|
study
| 100.0 |
To identify which downstream effectors of AKT signaling were involved in mediating KU0060648 enhanced TMZ efficacy, we extracted total proteins from U87 and M059K cells after treatment with control solvent, 1 or 10 μM KU0060648, 100 μM TMZ or their combination. KU0060648 dose-dependently inhibited AKT signaling downstream effectors, such as pro-proliferative factor c-Myc, anti-apoptotic proteins survivin or Mcl-1, invasion related molecule MMP-9 and angiogenesis promoting factor VEGF (Figure 6A). Besides, activity of GSK-3β, the acknowledged direct substrates of AKT, was also suppressed by KU0060648 treatment (Figure 6A). It was noteworthy that TMZ alone did increase, although not enormously, the activities of DNA-PKcs and its downstream AKT, leading to elevate expression of pro-invasion and pro-angiogenesis effectors. Significantly, combination of KU0060648 enhanced the inhibitory effect on the DNA-PKcs/AKT signaling compared with each agent alone (Figure 6A), which suggested an underlying machinery of synergy between KU0060648 and TMZ. Similar trends were also obtained in mRNA level using qPCR (Figure 6B).
|
study
| 100.0 |
A. Immunoblotting analysis of p-AKT (S473), p-GSK3β (S9), survivin, Mcl-1, c-Myc, MMP9 and VEGF expressions in U87 and M059K cells treated with TMZ (100 μM) and KU0060648 (1 or 10 μM) either alone or in combination for 24 hours. B. Real time PCR examined the mRNA level of survivin, Mcl-1, c-Myc, MMP9 and VEGF in the treated U87 and M059K cells. (Bars, SD; ★, p < 0.05; one-way ANOVA test).
|
study
| 100.0 |
On the basis of the in vitro data, it was pivotal to determine if KU0060648 could synergistically promote the antitumor effect of TMZ in vivo. U87 cells were injected subcutaneously into the flank of nude mice, and once tumors grew to approximately 50-75 mm3, mice were randomly treated with KU0060648 (low dose group, 10 mg/kg; high dose group, 50 mg/kg), TMZ (10 mg/kg), or their combination. As shown in Figure 7A, KU0060648 dose-dependently inhibited the growth of U87 tumor. However, 10 mg/kg TMZ alone did not make tumor size regress. Especially, the co-administration of low (10 mg/kg) or high dose (50 mg/kg) of KU0060648 with TMZ resulted in a substantial tumor growth inhibition compared with each agent alone, respectively, confirming that the synergistic effect between KU0060648 and TMZ in vivo. We further address the long-term survival prolonging effect of synergistic KU0060648/TMZ treatment. As shown in Figure 7B, the survival rate of U87 tumor-beard nude mice treated with co-administrated KU0060648/TMZ was profoundly increased compared with each agent alone.
|
study
| 100.0 |
A. Tumor volume (expressed as the mean ± SEM) of U87-beared mice in treatment of KU0060648 (10 or 50 mg/kg, once daily, i.p.) and TMZ (10 mg/kg, thrice a week, i.p.) alone, or co-administrated KU0060648 (10 or 50 mg/kg, once daily, i.p.) and TMZ (10 mg/kg, thrice a week, i.p.). Tumor size was measured every three days for the indicated period starting at day 14. The observation period was 30 days. Control mice were received the 0.9% saline. n=5 per group. B. Kaplan-Meier curves for illustration of the survival periods of xenograft-bearing mice in the treatment. n=5 per group C. IHC staining of subcutaneous xenografts samples from each treatment group. Representative IHC staining of p-DNA-PKcs (S2056), p-AKT (S473), Ki-67, c-Myc, MMP-9, VEGF, Survivin, cleaved caspase-3 and γH2AX in serial sections (magnification, ×10).
|
study
| 100.0 |
We next determined the effect of the combination of KU0060648 and TMZ on the regulation of downstream effectors of AKT signaling and malignant markers in tumorous tissues. IHC analysis revealed that KU0060648 or KU0060648/TMZ treatment effectively inhibited the expression of p-DNA-PKcs and p-AKT (Figure 7C). In addition, staining of γH2AX was surveyed. Consistent with our in vitro study, significant increases of γH2AX levels only emerged in comparison between KU0060648/TMZ and TMZ groups, while not in KU0060648 treatment alone. KU0060648 or KU0060648/TMZ treatment also significantly suppressed the expression of Ki-67 (the biomarker of proliferation), c-Myc, survivin, MMP-9 and VEGF and enhanced the expression of apoptotic promoting biomarker caspase-3 in U87 tumors (Figure 7C). Collectively, these results suggested that combination of KU0060648 and TMZ was more efficient in inhibition of the U87 tumors than either of the single agents.
|
study
| 100.0 |
In this study, we reported that the p-DNA-PKcs (S2056) was significantly elevated in glioma compared with adjacent normal brain, and such a high level of phosphorylated DNA-PKcs tightly associated with malignancy of glioma, including clinical stage and the survival time of glioma patients, collectively demonstrating a pivotal role of DNA-PKcs in the progression of glioma. This study also investigated the role of DNA-PKcs in TMZ sensitivity in glioma cells. The results indicated that inhibition of DNA-PKcs activation decreased glioma cell malignancies and promoted TMZ efficacy. Specifically, we demonstrated that inhibitor of DNA-PKcs, namely the KU0060648, functioned mainly due to suppression of AKT signaling.
|
study
| 100.0 |
The most well-characterized factor that controls DNA-PKcs kinase activity is the broken ends of DNA double-strand breaks (DSBs), which is generated either endogenously or exogenously . Binding to DNA initiates the activation of DNA-PKcs kinase. Once activated, DNA-PKcs kinase phosphorylates and alters the activity of proteins that mediate NHEJ involved in DNA damage response, including phosphorylating histone variant H2AX (γH2AX) at Ser139 either directly or indirectly through AKT/GSK3β signaling . In order to see if the hyperactivated DNA-PKcs was derived from DSBs, we investigated the expression of γH2AX in paired glioma samples using IHC. Our data revealed a poor correlation between p-DNA-PKcs and γH2AX, suggesting that activation of DNA-PKcs in glioma was not necessarily dependent on DSBs. Recently, growing evidences have characterized that, in addition to DSBs, several molecules might also perform as key modulators of DNA-PKcs activity in human cells. For example, Liccardi G et al. reported that nuclear translocated EGFR could bind to DNA-PKcs and enhance DNA-PKcs activity . Besides, Olsen BB et al. showed a critical role of protein kinase CK2 in DNA-PKcs activation in glioblastoma cells . Combined with these studies, our data that the upregulated p-DNA-PKcs levels in glioma samples did not correlate with DSB load indicated multiple underlying mechanisms might account for activation of DNA-PKcs in various tumors.
|
study
| 100.0 |
DNA-PKcs plays a great part in classic DNA damage repair pathway. Inhibition of DNA-PKcs is therefore an attractive approach to modulating resistance to therapeutically induced DNA insults. Current cancer therapy invariably utilizes a combination of chemotherapeutic agents and numerous reports have documented the in vitro antiproliferative effect of DNA-PKcs inhibitors [33 - 35]. NU7026 and NU7441, two DNA-PKcs inhibitors, have been clarified to improve the efficacy of various chemotherapeutic drugs, such as etoposide, cisplatin and doxorubicin in some types of solid tumors, including colon cancer, non-small cell lung cancer (NSCLC), leukemia or HCC [36 - 38]. We described here that inhibition of DNA-PKcs by newly-designed inhibitor KU0060648 dose-dependently enhanced the cytotoxic effect of TMZ in glioma cells. Furthermore, the combination of KU0060648 and TMZ could also increase the apoptotic rates. Notably, KU0060648 alone did not enhance the DNA damage in glioma cell lines, suggesting that inhibition of DNA damage repair might not account for, at least not predominant, mechanism that KU0060648 suppressed glioma malignancies.
|
study
| 99.94 |
Poor prognosis and high lethality of glioma is largely attributed to the high invasion and angiogenesis property of glioma cells [39, 40], which is clinically linked to chemotherapy resistance [41, 42]. These malignant phenotypes essentially support glioma cell proliferation and spreading. Our data demonstrates that KU0060648 effectively inhibited the glioma invasion and angiogenesis in vitro. However, we noticed that administration of 100 μM TMZ slightly promoted these malignant properties of glioma. Such a phenomenon of “chemotherapy induced tumor invasion and angiogenesis” is not a new story in the preclinical literatures, but is often overlooked as a mechanism that may contribute to eventual resistance and disease progression following therapy failure [43, 44]. Several studies have revealed that activation of DNA-PKcs might be a key tone to facilitate cancer cell invasion and angiogenesis, through coordinately regulating the expressions of related effectors or activities of signals [45, 46]. In our study, we discovered that TMZ treatment alone induced the activation of DNA-PKcs, then upregulated the level of downstream effectors. When KU0060648 was added, those adverse effects of TMZ were eliminated and even brought about a synergistic inhibition of glioma invasion and angiogenesis.
|
study
| 100.0 |
The synergistic effect of KU0060648 and TMZ was also observed in the treatment of glioma in vivo. High dose of KU0060648 addition to TMZ had a more remarkable anti-tumor effect than the low dose of KU0060648 in the presence of TMZ as well as KU0060648 at its high dose alone, further suggesting the feasibility of combination of DNA-PKcs inhibitor and TMZ on the glioma growth. 100 μM TMZ alone could not upregulate the survival time in animal harboring U87 tumors. However, with increased dose, KU0060648 sufficiently extended the survival time of TMZ treatment group, confirming that the combination of KU0060648 and TMZ had a synergistic effect for prolonging the survival period of nude mice harboring U87 xenograft tumors. Collectively, these data expanded the feasibility of the combination of the DNA-PKcs inhibitor with chemotherapeutic agents in solid tumor treatment.
|
study
| 100.0 |
The present study implies that the molecular mechanism underlying DNA-PKcs inhibition-enhanced TMZ efficacy can be majorly related to the inhibition of AKT signaling. Constitutive activation of AKT exists in a variety of malignances including glioma, and is closely correlated with cancer development and progression [47, 48]. Specifically, hyperactivation of AKT also confers cell resistance to many chemotherapy agents [49, 50]. As we found, allosteric AKT inhibitor MK2206 mimicked the effect of KU0060648 that substantially decreased cell growth and sensitized TMZ efficacy, suggesting KU0060648 functioned mainly through suppression of AKT. Further investigations confirmed the inhibitory effect of KU0060648 on DNA-PKcs/AKT signal. More importantly, we disclosed that KU0060648 induced inhibition of AKT activation may be, at least partly, derived from its disruption of the interaction between DNA-PKcs and AKT in glioma cells. Consistently, the downstream molecules of AKT, such as c-Myc, and AKT-related anti-apoptotic, metastatic or angiogenic molecules could be efficiently suppressed by KU0060648 treatment. Many studies have shown that these substrates participate in the resistance of certain cancers to chemotherapy [51 - 55]. Taken together, these data revealed that the single agent of KU0060648 or in combination with TMZ in glioma treatment mainly depended on inhibition of AKT signaling.
|
study
| 100.0 |
A series of work from Brian Hemmings groups has identified DNA-PKcs, as a central node, amplifies and conveys signals from the damaged DNA to DSBs repair and anti-apoptosis machineries, for example through AKT pathway, thus promoting survival [56, 57]. However, role of activated DNA-PKcs/AKT axis without chemo- or radiotherapy triggered has been completely ignored, which is certainly existed as our result illustrated. In fact, mountains of researches have observed that DNA-PKcs promotes tumor malignancies through stimulating and integrating an extensive network of cellular signaling which is not involving DNA damage repair [20, 23, 46]. Actually, in vitro assay has demonstrated that synthetic peptides of DNA-PKcs can phosphorylate AKT on Ser 473 , which provided a fundament that activated DNA-PKcs, whatever DSBs induced or non-DSBs induced, could potentially play an additional role in enhanced AKT signaling. This was confirmed by our clinical investigation that a linear correlation between p-DNA-PKcs and p-AKT expression was discovered. Such a finding shed light on the overlooked function of non-DSBs stimulated DNA-PKcs/AKT signal, which potentially promotes glioma progression.
|
study
| 100.0 |
In conclusion, our findings suggest that overactivation of DNA-PKcs is clinically and functionally relevant to the progression of human glioma, and mediates TMZ resistance in glioma treatment. Disrupting the DNA-PKcs/AKT interaction and consequently regulating the downstream effectors of AKT may provide a potential mechanism by which suppression of DNA-PKcs activity can sensitize the response to TMZ and possibly several other chemotherapeutic agents.
|
study
| 100.0 |
The human glioma cell lines U373, A172, M059K were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA); H4, U87, U251 were purchased from the Cell Culture Center (Chinese Academy of Medical Sciences, Beijing, China). All these cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and at 37°C in 5% CO2.
|
other
| 95.06 |
KU0060648, Temozolomide, MK-2206, PD98059 were obtained from Merck Millipore. Anti-total DNA-PKcs, anti-phospho-DNA-PKcs (p-DNA-PKcs, Ser 2056) and anti-VEGF antibodies were purchased from Abcam. Anti-AKT, anti-p-AKT (Ser 473), anti-p-histone H2AX (γH2AX, Ser 139), anti-GSK3β, anti-p-GSK3β (Ser 9), anti-survivin, anti-Mcl-1, anti-c-Myc, anti-MMP9, anti-Ki-67, anti-cleaved caspase-3 antibodies, anti-Actin, anti-mouse and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology.
|
other
| 99.94 |
In total, 217 paraffin-embedded, archived glioma samples and their respective adjacent non-cancerous tissues were obtained from the Sanbo Brain Hospital, Capital Medical University (Beijing, China). Fresh gliomas and adjacent non-tumor tissues were collected using protocols approved by the Ethics Committee of Sanbo Brain Hospital, and informed consent was obtained from all patients. Normal brain tissues (2 fresh and 21 paraffin-embedded samples) were obtained from car accident patients who received craniotomy in our hospital, and had been ethnically approved for scientific applications as mentioned before . The clinical and pathological classification and stage were determined according to the WHO classification of brain tumors criteria. The clinical information for the patient samples is summarized in Supplementary Tables S1 and S2 (see Supplementary material).
|
study
| 100.0 |
Briefly, tissue sections were deparaffinized, soaked in Tris-EDTA buffer (pH 8.0) and boiled in the microwave, then incubated with the primary antibodies at 4°C overnight. Next day, slides were washed and stained by the secondary antibody and DAB disclosure, counterstained with hematoxylin, dehydrated and mounted. The sections were reviewed and scored independently by two observers. Degree of immunostaining was determined based on both the proportion of positively stained tumor cells and the intensity of staining. The proportion of positive tumor cells was scored as follows: 0, no positive tumor cells; 1, < 10%; 2, 10%–35%; 3, 35%–75%; 4, > 75%. The intensity of staining was graded according to the following criteria: 1, weak staining (light yellow); 2, moderate staining (yellow–brown); 3, strong staining (brown). The IHC score was calculated as staining intensity score × proportion of positive tumor cells. Using this method of assessment, the expression of p-DNA-PKcs was scored as 0, 1, 2, 3, 4, 6, 8, 9, and 12. High expression of p-DNA-PKcs and p-AKT referred to IHC score ≥ 6 and low expression of p-DNA-PKcs and p-AKT was defined as IHC score < 6.
|
study
| 100.0 |
Specific siRNA targeting DNA-PKcs was designed and provided by Ribobio (Cat#: siG0812181317001 and siG0812181317002). Transfection of siRNA was done according to the manufacturer's protocol. Briefly, cells were plated in 60-mm culture plates at 5×105 cells per well, grown for 12 h, then transfected with siRNA using Lipofectamine 2000 (Invitrogen). Transfected cells were incubated at 37°C with 5% CO2 for 24 h.
|
study
| 99.9 |
Proliferation/viability of cells was determined using MTS assay as previously described with minor modifications . Briefly, a total of 3 × 103 cells in 100 μL of 10% FBS culture medium were seeded in 96-well plates. Once confluent, cells were cultured for 72 h before analysis. Then, the medium was aspirated and incubated with MTS solution (Promega, Madison, WI, USA) for 1 h. The viable cell number was reflected as the MTS absorbance which was measured spectrophotometrically at 490 nm. For evaluating the long-term proliferation of cells (colony formation assay), 1 × 103 tumor cells were plated into 60-mm dishes in 10% FBS culture medium. After 14 days, the cells were washed with PBS, fixed with methanol and 0.1% crystal violet. The colonies were counted and then photographed. All experiments were carried out in triplicate.
|
study
| 100.0 |
The Annexin V-FITC early apoptosis detection kit (Neobioscience, Shenzhen, China) was used to identify the apoptotic cells. Briefly, approximate 105 cells were harvested, washed with cold PBS twice and resuspended with 350 μL 1 × Binding Buffer. Then, 5 μL of the Annexin V-FITC conjugate was added. After 20 minutes' light-prevented incubation at room temperature, cell suspension was diluted to a final volume of 500 μL/assay with ice cold 1 × Binding Buffer. Next, 10 μL of the Propidium Iodide (PI) solution were added to each sample tube, and the samples were analyzed by FACS Canto™II cell analyzer (BD Biosciences, San Jose, CA, USA).
|
study
| 99.94 |
As previously described , One-Step TUNEL Apoptosis Assay Kit (Beyotime, Jiangsu, China) was applied in TUNEL assay according to the manuscript. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Staining was evaluated using fluorescence microscopy.
|
other
| 99.8 |
Hoechst 33258 staining was performed as described previously with minor modifications . Briefly, cells were fixed with 4% formaldehyde solution for 30 min at room temperature and washed twice with PBS. Fixed cells were stained with Hoechst 33258 of 50 ng/mL and incubated for 30 min at room temperature and washed with PBS. Apoptotic cells were identified by condensation and fragmentation of nuclei examined by an Olympus IX71 fluorescence microscope. The apoptotic rate of cell population was calculated as the ratio of apoptotic cells to total cells counted ×100. A minimum of 500 cells were counted for each treatment.
|
study
| 99.94 |
After cells being treated with reagents either alone or in a combination for 48 hours, the activity of caspase-3/7 was evaluated using Caspase-Glo 3/7 Assay Kit (Promega Corporation) according to manufacturer's instruction. Results were expressed as relative luminescence units (RLU).
|
study
| 99.94 |
Cells were grown on coverslips at a density of 1×105 cells per well. After treatment with TMZ (100 μM) or additional KU0060648 (1 μM and 10 μM) for distinct time periods, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and incubated in 0.1% Triton X-100 for 20 min, then blocked with 5% normal goat serum (Sigma-Aldrich). The cells were reacted with anti-γH2AX (1:500) at 4°C overnight. After that, cells were incubated with FITC- (fluorescein isothiocyanate) conjugated secondary antibodies for 1 h at 37°C. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) at a final concentration of 0.1 μg/mL. Images were visualized and recorded with a Zeiss LSM780 confocal microscope (Carl Zeiss Inc., Oberkochen, Germany).
|
study
| 99.94 |
The transwell invasion assay was performed using the transwell chamber with a Matrigel-coated filter. A total of 5 × 104 cells to be tested were starved in serum and growth factor-free medium overnight and then plated on the top chamber with or without agents as indicated for 18 h, followed by removal of cells inside the upper chamber with cotton swabs, and the invasive cells on the lower side were fixed, stained with 0.1% crystal violet solution and counted using light microscope. The experiment was repeated three times.
|
study
| 99.94 |
Matrigel (50 μL) was pipetted into each well of a 12-well plate and polymerized for 2 hours at 37°C. HUVECs (1 × 104) in 150 μL of conditioned medium from each treatment group were added to each well and incubated at 37°C in 5% CO2 for 24 h. Pictures were taken under a × 100 bright-field microscope. The experiment was repeated three times.
|
study
| 99.94 |
Total RNA from cells was extracted with TRIzol (Invitrogen). First-strand cDNA was synthesized by using the Superscript II-reverse transcriptase kit (Invitrogen) according to the manufacturer's instructions. Real-time PCR (qPCR) was conducted using SYBR Premix Ex Taq (Takara) on an ABI 7300 Real-Time PCR System (Applied Biosystems). All samples were normalized to GAPDH. Gene-specific qPCR primer pairs are provided in as below.
|
study
| 99.56 |
Total cell protein extracts were separated on 10% or 15% SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were subsequently probed with indicated primary antibodies and anti-mouse or anti-rabbit secondary antibodies, respectively. All of the first antibodies were diluted at 1:1000 except for Actin at 1:5000. The chemiluminescence signal was detected with Luminescent Image Analyzer LAS-4000 (Fujifilm). Blotting membranes were stripped and reprobed with anti-Actin as a loading control.
|
study
| 99.94 |
For immunoprecipitation assays, cells were washed with cold PBS and lysed with cold lysis buffer (50mM Tris-Cl, pH 7.4, 150mm NaCl, 1mm EDTA, 1% NP-40, 0.25% sodium deoxycholate and protease inhibitor mixture) at 4°C for 30 min. Cellular extracts were incubated with appropriate primary antibodies on rotator at 4°C overnight, followed by the addition of protein A/G sepharose beads for 2 h at 4°C. Beads were then washed five times with lysis buffer. The immune complexes were subjected to SDS–PAGE followed by IB with secondary antibodies.
|
study
| 99.94 |
Female, 5 weeks old, Nu/Nu mice were purchased from Vital River laboratories (Beijing, China). All animal care and experiments were carried out according to the Institutional Animal Welfare Guidelines of Chinese Academy of Medical Sciences. A total of 1×106 U87 cells were injected subcutaneously into mice. To assess tumor growth, treatment began 2 weeks after injection of tumor cells. Mice were randomly divided into 6 groups (n = 5 per group): Control group: normal saline intraperitoneal (i.p.) injection once day; single-agent KU0060648 group: 10 mg/kg (low dosage) or 50 mg/kg (high dosage) KU0060648 i.p. once daily for 30 days; TMZ group: 10 mg/kg i.p. once daily for 30 days, and KU0060648/TMZ combination group: KU0060648 was administered i.p. once daily for 5 days with the first dose immediately before 10 mg/kg TMZ. At the end of each experiment, animals were sacrificed, and tumors were calculated and paraffin-embedded. Sections of 5.0 μm were cut and subjected to IHC staining.
|
study
| 99.94 |
Statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5.0 (GraphPad software Inc., La Jolla, CA, USA). Survival curves were plotted using Kaplan–Meier estimates. Wilcoxon rank sum test was used for statistical analysis of clinical scores; χ2 (chi-square) test was applied in studying the correlation between γH2AX and p-DNA-PKcs, and p-DNA-PKcs and p-AKT. Comparisons between 2 groups were performed using the Student's t test. Bivariate correlations between study variables were calculated by Pearson's correlation coefficients. One-way ANOVA test was used for statistical analysis of remaining data. All tests were two-tailed Data are presented as means ± SD. P < 0.05 was considered statistically significant.
|
study
| 100.0 |
Bovine enterovirus (BEV) is a single positive-stranded RNA virus belonging to the genus Enterovirus within family Picornaviridae. The viral particle is composed of a small, non-enveloped and icosahedral virion and 7.5 k-base genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at the 5′ and 3′ ends. The ORF encodes a single long polyprotein containing structural proteins (VP1, VP2, VP3 and VP4 encoded in P1) and non-structural proteins (2A, 2B and 2C encoded in P2 as well as 3A, 3B, 3C and 3D encoded in P3) [1, 2].
|
study
| 72.6 |
Genus Enterovirus is divided into 12 species defined as Enterovirus A–H and J (EV-A, B, C, D, E, F, G, H and J) and Rhinovirus A–C (RV-A, B and C) . BEVs belong to EV-E and EV-F (formerly known as BEV-A and BEV-B, respectively) and can be distinguished from other EVs by the unique secondary structure of their RNA genome: a 5′-cloverleaf and internal ribosome entry site (IRES) linked by additional nucleotide sequences at the 5′UTR [3–5]. Since the isolation of BEVs from cattle in the late 1950s [6–8], studies worldwide have detected BEVs not only in cattle but also in other animal species including possums, bottlenose dolphins, camels and alpacas [8–12]. Although BEVs have been classified based on virus antigenicity determined by cross neutralization testing [13–16], the genotype based on the capsid protein (particularly in VP1) amino acid sequences are also used to classify BEVs [4, 10–12, 17, 18]. BEVs are classified into 4 sero-/genotypes and 6 sero-/genotypes in EV-E (E1, E2, E3 and E4) and EV-F (F1, F2, F3, F4, F5 and F6), respectively.
|
study
| 84.7 |
Although most EVs cause only mild symptoms, including hand-foot-and-mouth disease, herpangina, pleurodynia and rashes [19, 20], some members belonging to the genus Enterovirus can cause severe diseases. The most well known pathogen is poliovirus affecting humans. Poliovirus and some of other EVs, including coxsackie virus and echovirus, can invade the central nervous system causing neurological diseases, including aseptic meningitis, encephalitis and ataxia [21, 22]. In other animals, although porcine teschovirus, formerly classified as porcine enterovirus, can cause a neurological disorder known as Teschen/Talfan disease , the pathogenicity of EVs infecting animals are still unclear. In case of cattle, foot-and-mouth disease virus belonging to the genus Aphthovirus of the family Picornaviridae can cause vesicular diseases leading to a serious economic impact for farmers ; the pathogenicity of viruses belonging to the genus Enterorovirus is still unclear. Several reports have claimed that BEVs can cause diarrhea, respiratory diseases, reproductive diseases and infertility in cattle [25–27]; however, BEVs have also been widely detected in asymptomatic cattle and their environment, and experimental infection trials of BEV have failed to produce clinical signs [28–30]. Therefore, whether BEV infection is clinically important remains unclear.
|
review
| 99.44 |
It is widely known that most viruses belonging to genus Enterovirus utilize “canyon” as their binding site to cells surface receptors, which is formed by outer capsid proteins including VP1, VP2 and VP3 . Several studies of other enteroviruses revealed that sequences of the VP1 coding region are responsible for the phenotype of viruses; some amino acid substitutions in this region altered the pathogenicity and cell tropism of the viruses [32–34]. Although the cell surface receptor to BEV has not been identified, it is likely that the capsid proteins, including VP1, may be responsible for the phenotype of BEVs, as their capsid proteins also form a “canyon” on the outer side of the virion, and a strain isolated from cattle with severe symptoms contained specific amino acid substitutions in the capsid regions [27, 35]. To elucidate the determinants of BEV virulence in hosts, genomic information of BEVs must be determined.
|
study
| 100.0 |
Recently, deep sequencing techniques using high-throughput sequencers have been used to evaluate virome including novel viruses in clinical samples without viral isolation to determine total genomic information within samples [36, 37]. We previously identified novel viruses infecting the intestinal tracts of livestock using high-throughput sequencers to study enterovirus, picornavirus and astrovirus in the feces of goat, swine and cattle, respectively [38–41].
|
study
| 100.0 |
Previously, we reported the detection of novel kobu-like virus in Japanese Black cattle, using feces of calf, by metagenomics analysis. In the present study, we identified a novel BEV in feces collected for that survey . To characterize the genomic features of this virus, complete genome sequences were determined and phylogenetic trees were constructed. In addition, secondary RNA structures in the 5′UTR and pairwise identity were analyzed.
|
study
| 100.0 |
Previously, we reported the detection of a novel kobu-like virus in Japanese black cattle by deep sequencing method . During the metagenomics surveillance, nucleotide sequences with high similarity to BEVs were identified in feces collected from a calf with diarrhea. This feces was collected from a 1-month-old calf with diarrhea in Kagoshima prefecture (Kagoshima sample) in 2014. No other clinical sign was observed except diarrhea. Feces was collected directly from the rectum on the onset day. One gram feces was diluted with 9 mL PBS (−) to prepare a 10% fecal suspension and centrifuged at 10,000 × g for 10 min. The supernatant was collected and stored at −80 °C before RNA extraction and virus isolation.
|
study
| 99.94 |
The supernatant of the Kagoshima sample was subjected to virus isolation. The fecal supernatant was filtered through a 0.45-μm pore size membrane and treated with 10 μg/mL acetylated trypsin (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at room temperature before virus isolation. Treated samples were inoculated into Mardin-Darby bovine kidney cells. Blind passage was subsequently conducted three times. Minimum Essential Medium was used as negative control (Sigma-Aldrich).
|
study
| 99.94 |
In this study, three BEVs isolated in Japan, BEV IS1/Bos taurus/JPN/1990 (BEV-IS1) and IS2/Bos taurus/JPN/1990 (BEV-IS2), were additionally sequenced and analyzed. These viruses were isolated from a fecal sample collected from one cow at the same time in 1990 in the Ishikawa prefecture (The clinical features of cattle infected with BEV-IS1 and IS2 have not been recorded). In addition, BEV Ho12/Bos taurus/JPN/2009 (BEV-Ho12) was isolated from diarrheic feces collected in Hokkaido in 2009 by as described above .
|
study
| 100.0 |
Total RNA was extracted from 0.25-mL supernatants of isolated viruses and 10% fecal samples using TRIzol LS Reagent (Life Technologies, Carlsbad, CA, USA). RNA samples were normalized to 10–100 ng of RNA per reaction, using a Qubit_2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). The cDNA library of sample RNA was constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina version 2.0 (New England Biolabs, Ipswich, MA, USA) as described previously and sequenced using MiSeq (Illumina, San Diego, CA, USA) with the MiSeq reagent kit V2 (300 cycles) (Illumina). Briefly, all reads were generated as 151 paired end reads. Each sample was multiplexed with other 23 samples prepared from diarrheal feces of other calves (data not shown). 5′-Full RACE Core Set (TaKaRa Bio, Shiga, Japan) and 3′Full RACE Core Set (TaKaRa Bio) were used to complement virus sequences of the 5′ end and 3′ end, respectively.
|
study
| 100.0 |
All nucleotide sequences determined by Miseq (referred to as “reads”) were converted to FASTAQ format on MiSeq reporter V2.3 and subsequently analyzed using CLC Genomics Workbench 6.0 (CLC bio, Cambridge, MA, USA). Briefly, the ends of all reads were trimmed to remove adaptor sequences located at both ends of each read. Trimmed reads were assembled into contigs using a de novo assembly algorithm. Contigs generated by de novo assembly algorithm were analyzed using BlastN.
|
study
| 99.9 |
Hypothetical polyprotein cleavage sites of the viruses were predicted by multiple alignments with other BEVs and confirmed by the NetPicoRNA . Nucleotide (nt) sequences or amino acid (aa) sequences were aligned using ClustalW. Phylogenetic trees were constructed by maximum likelihood (ML) methods on MEGA5.2.2 . The mtREV24 + G + F model (5′UTR), rtREV + F model (3′UTR), rtREV + G + F model (P1), rtREV + G + I (P2 and P3), and WAG + G + I (VP1) were employed as evolutionary models for ML method. Pairwise identity was analyzed on CLC Genomics Workbench and the secondary RNA structure of the 5′UTR was predicted by Mfold .
|
study
| 100.0 |
RT-PCR was performed by using PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa Bio) to confirm the sequences of the contigs obtained from the Kagoshima sample. Three primer sets were designed based on the contig sequences of this sample. Primer sequences are given in Additional file 1: Table S1. PCR products were sequenced using a 3130xl Genetic analyzer (Applied Biosystems, Foster City, CA, USA).
|
study
| 99.94 |
To confirm the presence of other pathogens in the Kagoshima sample, detection of agents causing diarrhea using our real-time PCR system, referred to as “Dembo-PCR,” was performed . This system can identify 19 species of pathogens, including virus, bacteria and protozoa. Briefly, viral DNA and RNA were extracted by high pure viral nucleic acid extraction kit (Roche Diagnostics GmbH, Mannheim, Germany) and bacteria and protozoa DNA were extracted by QIAamp Fast DNA stool mini kit (QIAGEN, Hilden, Germany). Nucleic acids extracted by each kit were subjected to Dembo-PCR, according to a previous report .
|
study
| 100.0 |
Although virus isolation using supernatants of the Kagoshima feces was repeated three times, no cytopathic effect could be detected. Therefore, RNA extracted from the Kagoshima sample collected in 2014 and virus stocks of BEV-IS1, BEV-IS2 and BEV-Ho12 were subjected to deep sequencing. The Kagoshima sample was sequenced twice, and all reads obtained from the two runs were used to generate contigs (the first and second deep sequencing yielded 1,304,032 and 929,976 reads, respectively). The results of BlastN analysis revealed that bovine enterovirus F, group A rotavirus (RVA), bovine kobu-like virus and bovine picornavirus were identified with E value = 0. However, RVA was not detected in feces by Dembo-PCR.
|
study
| 100.0 |
The total BEV read counts (percentages indicate BEV reads per total reads of the first and second deep sequencing) of the Kagoshima sample were 1202 reads (0.05%), and an approximately 7400 nt contig was obtained from the integrated result with a 24.24 average sequence read depth (maximum read depth was 46). The complete genome was determined using 5′ and 3′ end RACE methods. Because amino acid sequences of VP1 were not similar to those of other enteroviruses by homology analysis as described below, the VP1 genome sequence was confirmed by directly sequencing the PCR product. As a result, sequences obtained from direct sequencing agreed with the results of deep sequencing. The genome length of BEV from the Kagoshima sample was 7414 nt, excluding the poly (A) tail. We named this BEV as BEV AN12/Bos taurus/JPN/2014 (BEV-AN12).
|
study
| 100.0 |
Viral genomes of isolated viruses including complete ORFs were also determined. The genome lengths of BEV-IS1, BEV-IS2 and BEV-Ho12 were 7413 nt (P1: 2517 nt, P2: 1737 nt, and P3: 2271 nt), 7394 nt (P1: 2496 nt, P2: 1734 nt, and P3: 2271 nt), and 7350 nt (P1: 2496 nt, P2: 1734 nt, and P3: 2271 nt), respectively. The sequences of BEV-AN12, BEV-Ho12, BEV-IS1 and BEV-IS2 were deposited in the DDBJ/EMBL/GenBank database under the accession numbers LC038188, LC150008, LC150009 and LC150010, respectively.
|
study
| 100.0 |
Table 1 shows the pairwise aa (polyprotein, 2C + 3CD, P1-P3, VP1-VP4 and 3D) or nt (5′UTR and 3′UTR) identity of BEV-AN12 to representative strains of each species belonging to BEVs and other Japanese BEVs. Deduced aa sequences encoding polyprotein, 2C + 3CD, P1, P2, P3, 3D and four capsid proteins (encoding VP4, VP2, VP3 and VP1) were compared to each EV-E and F. BEV-AN12 possessed showed identity to EV-Fs in polyprotein, 2C + 3CD, P2, P3 and 3D than to those of EV-Es. However, low aa identity (aa identity <70%) was observed in P1 to EV-Es and EV-Fs. Particularly, the VP1 region of BEV-AN12 encoded in P1 showed a significantly low aa identity to other BEVs (54.7% ≤ aa identity ≤ 58.6%). As a result of multiple alignment analysis, several motifs conserved among the genomes of genus Enterovirus were detected in the genome of BEV-AN12. In particular, the [PS] ALXAAXETG motif in VP1, GXCG motif in 2A, GXXGXGKS motif for NTP-binding in 2C, GXCG motif forming part of the catalytic active site in 3C, and KDE [LI] R in 3D were identified [47–50]. However, the putative cleavage site at the junction of VP3/VP1 was a glutamine/serine for BEV-AN12. In addition, a 6-aa insertion in the 2A region was identified at position 835–840 aa (PLRTTG) in the BEV-AN12 genome. Multiple alignment using aa sequences encoding polyprotein is supplemented as Additional file 2: Table S2.Table 1Pairwise nucleotide (5′UTR and 3′UTR) and amino acid (polyprotein, 2C + 3CD, P1-P3, 3D and VP1-4) identity (%) of BEV-AN12 to Japanese BEVs, EV-Es and EV-FsBEV AN12/Bos Taurus/JPN/2014 (LC038188)Bovine enterovirus in JapanEV-E1EV-E2EV-E3EV-E4EV-F1EV-F2EV-F3EV-F4IS1/Bos taurus/JPN/1990(LC150009)IS2/Bos taurus/JPN/1990(LC150010)Ho12/Bos taurus/JPN/2009 (LC150008)LC-R4 (DQ092769)K2577 (AF123432)HY12 (KF748290)PAK-NIH-21E5 (AFK92921)BEV-261 (NC_021220)3A (AY508697)PS87/Belfast (DQ092794)Possum enterovirus W6 (AY462107)5′UTR76.179.880.875.874.271.1N/A81.880.481.378.0Polyprotein73.686.686.273.273.672.7N/A85.084.685.682.42C + 3CD80.998.898.680.780.980.2N/A98.198.193.697.9P165.868.768.365.166.064.5N/A68.267.267.667.1VP475.485.585.575.479.775.4N/A82.685.585.582.6VP274.272.673.072.272.673.4N/A72.671.471.070.2VP365.071.671.265.865.864.6N/A70.070.070.872.8VP156.957.956.854.756.556.254.756.158.655.056.8P277.896.495.778.077.677.3N/A92.391.694.989.7P379.098.898.578.679.178.3N/A98.098.398.393.73D84.099.198.783.783.783.5N/A98.798.998.995.03′UTR71.686.1N/A71.671.673.0N/A84.786.186.181.9N/A: Sequences are not available
|
study
| 100.0 |
The putative secondary 5′UTR RNA structure of BEV-AN12 is shown in Fig. 1. Zell et al. reported that the 5′UTR of BEVs form additional cloverleaf structures and small stem loops between the 5′-cloverleaf structure and IRES, which clearly distinguish BEVs from other EVs . Similarly, our analysis revealed that all Japanese BEVs had BEV-specific structures (domains I* and I**). Domains II, III, IV, V and VI, which are the main domains of type 1 IRES directing cap-independent translation , were also observed in all Japanese BEVs.Fig. 1Secondary RNA structure of 5′UTR of BEV-AN12. Putative secondary RNA structure of 5′UTR of BEV-AN12 was predicted by Mfold. Domains I, I*, I**, II, III, IV, V and VI domain were predicted. Domains I* and I** conserved among BEVs are shown with a bold line. Yn-Xm-AUG motif conserved in domain VI is indicated by a bold line. Kozak consensus sequences with start codon are also indicated by a bold line
|
study
| 100.0 |
Secondary RNA structure of 5′UTR of BEV-AN12. Putative secondary RNA structure of 5′UTR of BEV-AN12 was predicted by Mfold. Domains I, I*, I**, II, III, IV, V and VI domain were predicted. Domains I* and I** conserved among BEVs are shown with a bold line. Yn-Xm-AUG motif conserved in domain VI is indicated by a bold line. Kozak consensus sequences with start codon are also indicated by a bold line
|
study
| 100.0 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.