Application of the cell-based RT-qPCR assay (C-QPA) for potency detection of the novel trivalent rotavirus vaccine in China.

BACKGROUND: Because of the deficiencies of traditional methods in multivalent rotavirus vaccine potency detection, a cell-based quantitative RT-qPCR assay (C-QPA) was established and validated for specificity, precision, and accuracy. METHODS: In order to further validate the robustness of this method in actual titer detection, the linear range and the practical application under different conditions were tested using monovalent and trivalent rotavirus samples and standards. RESULTS: Results showed that the linear range was 2.0-6.5, 3.9-8.3, and 3.5-8.1 UI (unit of infectivity) for G2, G3, and G4, respectively. Besides, unknown sample with high titer exceeding the linear range can be calculated by dilution. The UIs of serotypes G2, G3, and G4 in monovalent and trivalent rotavirus samples showed a relative deviation ≤4.10%, and the monovalent samples of the same serotype with or without protective agents showed a relative deviation ≤4.28%; the coefficient of variation (CV) of at least 176 tests (548 individual runs) of 3 in vitro-transcribed RNA standards with certain concentrations was not higher than 6.50%; the results of the trivalent samples tested by more than 149 times in 5 years (467 individual runs) showed the CVs lower than 12.66%; 15 samples detected by one laboratory showed a CV lower than 9.83%, while other three samples tested by two independent laboratories showed a CV lower than 6.90%. CONCLUSION: In summary, the C-QPA has good linearity, durability, repeatability, and reproducibility in practical application and has been proved by the authority to be widely used in the production, quality control and release of the recently licensed trivalent vaccine in China.

Feasibility study of vertical flow constructed wetland for tertiary treatment of nanosilver wastewater and temporal-spatial distribution of pollutants and microbial community.

Silver nanoparticles (AgNPs) have the potential to cause negative effects on nutrient removal in constructed wetlands (CWs), further leading to the deterioration of the water. The current work aimed to investigate the feasibility of vertical flow CW (VFCW) for tertiary treatment of AgNPs wastewater, temporal-spatial distribution of pollutants, and microbial community after 450-day exposure. Results reveal that the effluent of VFCW could still meet the discharge limits except the slightly excessive concentration of phosphorus (>0.5 mg/L) from day 390, with the average removal efficiencies of 83%, 61%, 42%, 70%, and 66% for the chemical oxygen demand, total nitrogen, ammonia nitrogen, total phosphorus, and soluble orthophosphate during 450 days, respectively. Results show that AgNPs removal was relatively stable over time, up to 96%. The temporal-spatial analysis reveals that all contaminants were mainly retained in the soil layer. The Ag concentrations in the upper soil layer and plant roots were higher than that in the lower soil layer and plant stems and leaves, respectively. Microbial sequencing analysis reveals the significant differences in the microbial community at different depths on day 450, with the dominant phyla of Proteobacteria, Acidobacteria, Chloroflexi and Bacteroidetes, and dominant genera of Halomonas and Pseudomonas. These results provide much needed knowledge for the implementation of ecological technologies for AgNPs and nutrient removal simultaneously.

Comparison of Iris pseudacorus wetland systems with unplanted systems on pollutant removal and microbial community under nanosilver exposure.

Rapidly developing industry raises concerns about the environmental risks of silver nanoparticles (AgNPs), but the effects of AgNPs on the performance and microbial community in the constructed wetlands remain unclear. In this study, long-term exposure of AgNPs in two VFCWs was conducted to determine the effects of AgNPs on the pollutant removal and microbial community structure. Before exposing AgNPs, the water quality of effluent was better in planted wetland (CW2), compared with unplanted wetland (CW1). After continuous exposure of 100µg/L AgNPs, the COD (chemical oxygen demand) removal of two CWs had no difference. However, addition of AgNPs reduced the nitrogen and phosphorus removal in two CWs, with decreasing average removal efficiencies of ammonia nitrogen from 46.31% to 32.09% and 59.66% to 51.06%, total nitrogen from 57.76% to 43.78% and 67.35 to 60.58%, total phosphorus from 71.29% to 59.31% and 67.35% to 60.58%, respectively. The vegetable wetlands showed higher resistances to AgNPs loading than unplanted wetlands. In addition, AgNPs accumulated in the wetland substrate, especially in the soil layer with the silver concentration of approximately 4.32µg/g. The small portion of silver was found in plant tissues, and plants played a minor role to remove the AgNPs from wastewater. Moreover, the constructed wetlands could effectively remove the AgNPs from the synthetic wastewater. The illumine high-throughput sequencing results demonstrated the variations of the bacterial community structure at the exposure of AgNPs. The results showed that the dominant phyla were Proteobacteria, Acidobacteria and Bacteroidetes. Compared with unplanted wetlands, the contents of several nitrifying bacteria such as Candidatus Nitrososphaera (AOA) and Nitrospira (NOB) at genus level increased, leading to the higher nitrogen removal in the planted wetlands.

Shifts of system performance and microbial community structure in a constructed wetland after exposing silver nanoparticles.

The increasing utilization of silver nanoparticles (Ag NPs) in industry and commerce inevitably raises its release into wastewater. In this work, effects of Ag NPs on system performance and microbial community along the way of a vertical flow constructed wetland (VFCW) were investigated, along with the removal and fate of Ag NPs within the system. Results showed that the performance of control wetland kept stable during the experimental period, and the top substrate layer (soil layer) of wetland could remove most of pollutants in the influent. The study also suggested that addition of Ag NPs did not significantly affect organic matters removal. However, adverse effects were observed on the nitrogen and phosphorus removal. Removal efficiencies of TN, NH4+-N and TP approximately obviously reduced by approximately 10.10%, 8.42% and 28.35% respectively in contrast to before dosing after exposing 100 µg/L Ag NPs for 94 d, while the no dosing wetland with the stable performance. It was found that Ag NPs accumulated in the upper soil layer more than in the lower soil layer, and Ag NPs could enter into the plant tissues. After continuous input of Ag NPs, removal efficiency of Ag NPs was measured as 95.72%, which showed that the CW could effectively remove Ag NPs from the wastewater. The high-throughput sequencing results revealed that Ag NPs caused the shifts in microbial community structures and changed the relative abundances of key functional bacteria, which finally resulted in a lower efficiency of biological nitrogen and phosphorus removal.

Impacts of silver nanoparticles on the nutrient removal and functional bacterial community in vertical subsurface flow constructed wetlands.

The widespread utilization of silver nanoparticles (AgNPs) in industrial and commercial products inevitably raises the release into wastewater. In this study, the influences of AgNPs (0, 50 and 200µg/L) on the performance and microbial community of CWs were evaluated. Compared with the control system (without AgNPs), the COD removal was not affected, whereas the TN, NH4+-N, and TP removals were evidently affected in presence of AgNPs, and the effects had a positive relation with AgNPs concentration. In addition, AgNPs were effectively removed from wastewater and mainly accumulated in the soil layer and plant tissues. The illumina high-throughput sequencing results indicated that there were obvious variations in the microbial community at different AgNPs concentrations, associated with the variations of CW performance. Sequencing analyses also showed that Proteobacteria, Acidobacteria, Bacteroidetes were the dominant phyla. These results may provide valuable information and reference for evaluating the effect of AgNPs on constructed wetlands.

A chimeric vacuolar Na(+)/H(+) antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast.

The vacuolar Na(+)/H(+) antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na(+) into the vacuole. However, the relatively low Na(+)/H(+) exchange efficiency of the identified Na(+)/H(+) antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na(+)/H(+) antiporters with improved transport activity. Two homologous Na(+)/H(+) antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na(+) and slightly more K(+) than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast.

Proteomic analysis shows that stress response proteins are significantly up-regulated in resistant diploid wheat (Triticum monococcum) in response to attack by the grain aphid (Sitobion avenae).

The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS-LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.

A GSHS-like gene from Lycium chinense maybe regulated by cadmium-induced endogenous salicylic acid and overexpression of this gene enhances tolerance to cadmium stress in Arabidopsis.

KEY MESSAGE: A GSHS gene, LcGSHS , was cloned from L. chinense for the first time. Evidence is presented here that endogenous SA accumulation maybe important for the regulation of LcGSHS expression level. Glutathione (GSH) plays a pivotal role in heavy metal detoxification. GSH synthetase (GSHS) catalyzes the rate-limiting step of GSH synthesis in plants. Salicylic acid (SA) is one of the important plant hormones, which plays a critical role in triggering plant responses to different stresses such as cadmium (Cd) stress. Until now, little has been done to explore the relationship among the accumulation of endogenous SA, GSHS transcript levels and the GSH content in plants under Cd treatment and we will investigate this link in this study. The chlorophyll content, transcripts level of LcGSHS gene, endogenous SA accumulation, GSH accumulation and Cd concentration in the leaves of Lycium chinense were studied under different treatment conditions. Endogenous SA, LcGSHS transcript expression and GSH content can be induced by Cd treatment in L. chinense, however, reduced by co-treatment with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of SA biosynthesis. Strong staining was observed in the leaves of Arabidopsis expressing ProLcGSHS::GUS under Cd stress and the staining was reduced by co-treatment with AIP. The transgenic Arabidopsis expressing ProLcGSHS::LcGSHS also showed greater tolerance to Cd stress than wild types. Evidence was presented here that under Cd stress, GSH accumulation occurred via enhanced LcGSHS gene expression and the SA signaling cascade was involved in this accumulation. Furthermore, the overexpression of LcGSHS in transgenic Arabidopsis resulted in greater tolerance to Cd stress than wild-type lines.

Positive feedback regulation of a Lycium chinense-derived VDE gene by drought-induced endogenous ABA, and over-expression of this VDE gene improve drought-induced photo-damage in Arabidopsis.

Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under light stress. We have cloned a VDE gene (LcVDE) from Lycium chinense, a deciduous woody perennial halophyte, which can grow in a large variety of soil types. The amino acid sequence of LcVDE has high homology with VDEs in other plants. Under drought stress, relative expression of LcVDE and the de-epoxidation ratio (Z+0.5A)/(V+A+Z) increased rapidly, and non-photochemical quenching (NPQ) also rose. Interestingly, these elevations induced by drought stress were reduced by the topical administration of abamine SG, a potent ABA inhibitor via inhibition of NCED in the ABA synthesis pathway. Until now, little has been done to explore the relationship between endogenous ABA and the expression of VDE genes. Since V serves as a common precursor for ABA, these data support the possible involvement of endogenous ABA in the positive feedback regulation of LcVDE gene expression in L. chinense under drought stress. Moreover, the LcVDE may be involved in modulating the level of photosynthesis damage caused by drought stress. Furthermore, the ratio of (Z+0.5A)/(V+A+Z) and NPQ increased more in transgenic Arabidopsis over-expressing LcVDE gene than the wild types under drought stress. The maximum quantum yield of primary photochemistry of PSII (Fv/Fm) in transgenic Arabidopsis decreased more slowly during the stressed period than that in wild types under the same conditions. Furthermore, transgenic Arabidopsis over-expressing LcVDE showed increased tolerance to drought stress.

Expression of cholera toxin B subunit-lumbrokinase in edible sunflower seeds-the use of transmucosal carrier to enhance its fusion protein's effect on protection of rats and mice against thrombosis.

Lumbrokinase (LK) is a group of serine proteases with strong fibrinolytic and thrombolytic activities and is useful for treating diseases caused by thrombus. Cholera toxin B subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. We investigate here the application of CTB as a transmucosal carrier in enhancing its fusion protein-LKs effect to protect rats against thrombosis. Thus, in this study, CTB-LK fusion gene separated by a furin cleavage site was expressed in seeds of Helianthus annuus L. The activity of recombinant protein in seeds of transgenic sunflower was confirmed by Western blot analysis, fibrin plate assays and GM1 -ganglioside ELISA. The thrombosis model of rats and mice revealed that the oral administration of peeled seeds of sunflower expressing CTB-LK had a more significant anti-thrombotic effect on animals compared with that administration of peeled seeds of sunflower expressing LK. It is possible to conclude that CTB can successfully enhance its fusion protein to be absorbed in rats or mice thrombosis model. The use of CTB as a transmucosal carrier in the delivery of transgenic plant-derived oral therapeutic proteins was supported. In addition, for the purpose of that recombinant CTB-LK was designed for oral administration, thus the expression of CTB-LK in edible sunflower seeds eliminated the need for downstream processing of proteins.

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco.

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H2O2) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (Pn) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.

Molecular interactions between wheat and cereal aphid (Sitobion avenae): analysis of changes to the wheat proteome.

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of wheat to infestation by cereal aphid (Sitobion avenae) were investigated in a proteomic analysis. Approximately, 500 protein spots were reproducibly detected in the extracts from leaves of wheat seedlings after extraction and 2-DE. Sixty-seven spots differed significantly between control and infested plants following 24 h of aphid feeding, with 27 and 11 up-regulated, and 8 and 21 down-regulated, in local or systemic tissues, respectively. After 8 days, 80 protein spots differed significantly between control and aphid treatments with 13 and 18 up-regulated and 27 and 22 down-regulated in local or systemic tissues, respectively. As positive controls, plants were treated with salicylic acid or methyl jasmonate; 81 and 37 differentially expressed protein spots, respectively, were identified for these treatments. Approximately, 50% of differentially expressed protein spots were identified by PMF, revealing that the majority of proteins altered by aphid infestation were involved in metabolic processes and photosynthesis. Other proteins identified were involved in signal transduction, stress and defence, antioxidant activity, regulatory processes, and hormone responses. Responses to aphid attack at the proteome level were broadly similar to basal non-specific defence and stress responses in wheat, with evidence of down-regulation of insect-specific defence mechanisms, in agreement with the observed lack of aphid resistance in commercial wheat lines.