Monitoring residue levels and dietary risk assessment of thiamethoxam and its metabolite clothianidin for Chinese consumption of Chinese kale.

BACKGROUND: Thiamethoxam is widely used to control pests in Chinese kale, popularly consumed leafy vegetables. The potential risk to the environment and human health has aroused much public concern. Therefore, it is important to investigate the degradation behavior, residue distribution and dietary risk assessment of thiamethoxam in Chinese kale. RESULTS: A sensitive analytical method for determination of thiamethoxam and its metabolite clothianidin residue in Chinese kale was established and validated through a quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The recoveries were 85.4-101.2% for thiamethoxam and 79.5-108.1% for clothianidin, with the relative standard deviations (RSDs) of 0.9-10.2% and 1.8-6.0%, respectively. For the dissipation kinetics, the data showed that thiamethoxam in Chinese kale was degraded with the half-lives of 4.1 to 4.5 days. In the terminal residue experiments, the residues of thiamethoxam were 0.017-0.357 mg kg-1 after application 2-3 times with a preharvest interval (PHI) of 7 days under the designed dosages. The chronic and acute dietary exposure assessment risk quotient (RQ) values of thiamethoxam in Chinese kale for different Chinese consumers were 0.08-0.19% and 0.05-0.12%, respectively, and those of clothianidin were 0.01-0.04% and 0.02-0.04%, respectively, all of the RQ values were lower than 100%. CONCLUSION: Thiamethoxam in Chinese kale was rapidly degraded following first-order kinetics models. The dietary risk of thiamethoxam and clothianidin through Chinese kale was negligible to consumers. The results from this study are important reference for Chinese governments to developing criteria for the safe and rational use of thiamethoxam, setting maximum residue levels (MRLs), monitoring the quality safety of agricultural products and protecting consumer health. © 2021 Society of Chemical Industry.

Aphid endosymbiont facilitates virus transmission by modulating the volatile profile of host plants.

BACKGROUND: Most plant viruses rely on vectors for their transmission and spread. One of the outstanding biological questions concerning the vector-pathogen-symbiont multi-trophic interactions is the potential involvement of vector symbionts in the virus transmission process. Here, we used a multi-factorial system containing a non-persistent plant virus, cucumber mosaic virus (CMV), its primary vector, green peach aphid, Myzus persicae, and the obligate endosymbiont, Buchnera aphidicola to explore this uncharted territory. RESULTS: Based on our preliminary research, we hypothesized that aphid endosymbiont B. aphidicola can facilitate CMV transmission by modulating plant volatile profiles. Gene expression analyses demonstrated that CMV infection reduced B. aphidicola abundance in M. persicae, in which lower abundance of B. aphidicola was associated with a preference shift in aphids from infected to healthy plants. Volatile profile analyses confirmed that feeding by aphids with lower B. aphidicola titers reduced the production of attractants, while increased the emission of deterrents. As a result, M. persicae changed their feeding preference from infected to healthy plants. CONCLUSIONS: We conclude that CMV infection reduces the B. aphidicola abundance in M. persicae. When viruliferous aphids feed on host plants, dynamic changes in obligate symbionts lead to a shift in plant volatiles from attraction to avoidance, thereby switching insect vector's feeding preference from infected to healthy plants.

Silencing of Odorant-Binding Protein Gene OBP3 Using RNA Interference Reduced Virus Transmission of Tomato Chlorosis Virus.

Tomato chlorosis virus (ToCV) is widespread, seriously impacting tomato production throughout the world. ToCV is semi-persistently transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Currently, insect olfaction is being studied to develop novel pest control technologies to effectively control B. tabaci and whitefly-borne virus diseases. Despite current research efforts, no report has been published on the role of odorant-binding proteins (OBPs) in insect preference under the influence of plant virus. Our previous research showed that viruliferous B. tabaci preferred healthy plants at 48 h after virus acquisition. In this study, we determined the effect of OBPs on the host preference interactions of ToCV and whiteflies. Our results show that with the increase in acquisition time, the OBP gene expressions changed differently, and the OBP3 gene expression showed a trend of first rising and then falling, and reached the maximum at 48 h. These results indicate that OBP3 may participate in the host preference of viruliferous whiteflies to healthy plants. When the expression of the OBP3 gene was knocked down by an RNA interference (RNAi) technique, viruliferous Mediterranean (MED) showed no preference and the ToCV transmission rate was reduced by 83.3%. We conclude that OBP3 is involved in the detection of plant volatiles by viruliferous MED. Our results provide a theoretical basis and technical support for clarifying the transmission mechanism of ToCV by B. tabaci and could provide new avenues for controlling this plant virus and its vectors.

Analysis of EPA and DHA in the viscera of marine fish using gas chromatography.

The viscera of 10 kinds of marine fishes were collected for fish oil extraction and detection of DHA and EPA, two most important polyunsaturated fatty acids. The fish oil extraction ratio for the evaluated fishes varied from 0.95% to 10.18% (wt%). Pseudosciaena crocea presented the highest fish oil yield, followed by Mustelus manazo, Hippoglossus and Sciaenopsocellatus. A gas chromatography method was then established for analysis of EPA/DHA. The EPA concentration (in methyl ester form) in the fish oil varied from 1.39 to 10.65(mg/g). Epinephelus awoara presented the highest EPA concentration (p<0.05), followed by Epinephelussp, Sciaenopsocellatus and Hippoglossus. The DHA concentration (in methyl ester form) in the fish oil varied from 0.58 to 37.02 (mg/g). Epinephelus awoara presented the highest DHA concentration (p<0.05), followed by Sciaenopsocellatus, Pseudosciaena crocea and Hippoglossus. No strict positive correlation between the EPA/DHA concentration and the sea depth where the fish live was observed. The fishes living in middle depth presented highest EPA/DHA concentration.

Analysis of apoptosis induced by perfluorooctane sulfonates (PFOS) in mouse Leydig cells in vitro.

To explore the possible mechanism of perfluorooctane sulfonates (PFOS's) reproductive toxicity, mouse Leydig cells cultured in vitro were exposed to a serial concentration of PFOS for four more days of culture. Apoptosis during the process was checked. After 24 h, apoptosis occurred to all of the groups ≥ 50 µg/mL PFOS. After 72 h, 37.5 µg/mL dose also showed apoptosis, and the most apoptosis signals, averagely 18 per well, were observed in 62.5 µg/mL dose group. An increase in ROS (p < 0.05) and a decrease of mitochondrial membrane potential (p < 0.01) was confirmed in those groups with ≥ 12.5 µg/mL dose. ROS levels peaked in 50 µg/mL and 62.5 µg/mL groups, nearly two-folds higher than control. PFOS was also observed to down-regulate the protein expression of Bcl-2 and to up-regulate that of Bax. The apoptosis induced by PFOS in mouse Leydig cells was shown to be related to mitochondrially mediated pathways and to involve oxidative stress.

Co-infection of TYLCV and ToCV increases cathepsin B and promotes ToCV transmission by Bemisia tabaci MED.

Tomato disease is an important disease affecting agricultural production, and the combined infection of tomato chlorosis virus (ToCV) and tomato yellow leaf curl virus (TYLCV) has gradually expanded in recent years, but no effective control method has been developed to date. Both viruses are transmitted by Bemisia tabaci Mediteranean (MED). Previously, we found that after B. tabaci MED was fed on ToCV-and TYLCV-infected plants, the transmission efficiency of ToCV was significantly higher than that on plants infected only with ToCV. Therefore, we hypothesize that co-infection could enhance the transmission rates of the virus. In this study, transcriptome sequencing was performed to compare the changes of related transcription factors in B. tabaci MED co-infected with ToCV and TYLCV and infected only with ToCV. Hence, transmission experiments were carried out using B. tabaci MED to clarify the role of cathepsin in virus transmission. The gene expression level and enzyme activity of cathepsin B (Cath B) in B. tabaci MED co-infected with ToCV and TYLCV increased compared with those under ToCV infection alone. After the decrease in cathepsin activity in B. tabaci MED or cathepsin B was silenced, its ability to acquire and transmit ToCV was significantly reduced. We verified the hypothesis that the relative expression of cathepsin B was reduced, which helped reduce ToCV transmission by B. tabaci MED. Therefore, it was speculated that cathepsin has profound research significance in the control of B. tabaci MED and the spread of viral diseases.

Genistein and tyrphostin AG556 inhibit inwardly-rectifying Kir2.1 channels expressed in HEK 293 cells via protein tyrosine kinase inhibition.

Previous studies reported the controversial effects that protein tyrosine kinase (PTK) inhibition could induce an up-regulation or down-regulation of Kir2.1 current. The present study investigates how the recombinant human Kir2.1 channels are regulated by PTKs using whole-cell patch voltage-clamp, immunoprecipitation and Western blot, and mutagenesis approaches. We found that hKir2.1 current was reversibly inhibited by the broad spectrum PTK inhibitor genistein and the highly selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 in a concentration-dependent manner. The inhibition of hKir2.1 channels by genistein or AG556 was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of Kir2.1 channels was reduced by genistein or AG556, and the reduction was significantly antagonized by orthovanadate. The mutation of Y242 dramatically reduced the inhibitory response to AG556. The results obtained in this study demonstrate that hKir2.1 channels are down-regulated by PTK inhibition, suggesting that EGFR kinase participates in the modulation of human cardiac excitability.

Metabolomics and transcriptomics reveal the effect of hetero-chitooligosaccharides in promoting growth of Brassica napus.

The hetero-chitooligosaccharide (HTCOS) is a naturally occurring biopolymer in the exoskeleton of crustaceans and insects. Although some studies have been carried out on HTCOS in inducing plant resistance and promoting growth, the molecular mechanism of HTCOS in plants is not clear. In this study, an integrated analysis of metabolomics and transcriptomics was performed to analyze the response of Brassica napus to hetero-chitooligosaccharides treatment. The levels of 26 metabolites in B. napus were significantly changed under the HTCOS treatment. Amongst these metabolites, 9 metabolites were significantly up-regulated, including pentonic acid, indole-3-acetate, and γ-aminobutyric acid. Transcriptome data showed that there were 817 significantly up-regulated genes and 1064 significantly down-regulated genes in B. napus under the HTCOS treatment. Interestingly, the indole-3-acetate (IAA) content under the HTCOS treatment was about five times higher than that under the control condition. Moreover, four genes related to plant hormone signal transduction, three AUX/IAA genes, and one ARF gene, were significantly up-regulated under the HTCOS treatment. Furthermore, the plant height, branching number, and biomass of B. napus under the HTCOS treatment were significantly increased compared to that in the control condition. This evidence indicated that the HTCOS treatment contributed to accumulating the content of plant hormone IAA in the B. napus, up-regulating the expression of key genes in the signaling pathway of plant growth and improving the agronomic traits of B. napus.

Metagenomic analysis of the dynamical conversion of photosynthetic bacterial communities in different crop fields over different growth periods.

Photosynthetic bacteria are beneficial to plants, but knowledge of photosynthetic bacterial community dynamics in field crops during different growth stages is scarce. The factors controlling the changes in the photosynthetic bacterial community during plant growth require further investigation. In this study, 35 microbial community samples were collected from the seedling, flowering, and mature stages of tomato, cucumber, and soybean plants. 35 microbial community samples were assessed using Illumina sequencing of the photosynthetic reaction center subunit M (pufM) gene. The results revealed significant alpha diversity and community structure differences among the three crops at the different growth stages. Proteobacteria was the dominant bacterial phylum, and Methylobacterium, Roseateles, and Thiorhodococcus were the dominant genera at all growth stages. PCoA revealed clear differences in the structure of the microbial populations isolated from leaf samples collected from different crops at different growth stages. In addition, a dissimilarity test revealed significant differences in the photosynthetic bacterial community among crops and growth stages (P<0.05). The photosynthetic bacterial communities changed during crop growth. OTUs assigned to Methylobacterium were present in varying abundances among different sample types, which we speculated was related to the function of different Methylobacterium species in promoting plant growth development and enhancing plant photosynthetic efficiency. In conclusion, the dynamics observed in this study provide new research ideas for the detailed assessments of the relationship between photosynthetic bacteria and different growth stages of plants.

[Response of Photosynthetic Bacterial Community to Cadmium Contamination in Paddy Soil].

Heavy metal pollution in rice fields leads to huge losses in rice yield every year and is thus of increasing concern. Therefore, it is important to understand the changes in the microecology and physicochemical properties of paddy soil under different levels of cadmium pollution. The purpose of this study was to investigate the response of the photosynthetic bacterial community in paddy soil to different cadmium pollution levels using 16S sequencing technology. The results showed that pH, total cadmium, and available cadmium content decreased gradually with the increase in cadmium pollution. The soil α diversity was slightly different in the high cadmium (Cd), medium Cd, and low Cd groups; however, the enriched photosynthetic populations and photosynthetic bacterial communities were significantly different among these groups. The effective connections between photosynthetic bacterial species in the high Cd group were significantly greater than those in the medium and low Cd groups, the connections were closer, and the density was higher. Alkaline nitrogen, pH, available (P/K), total (N/P), organic matter, total cadmium, and available cadmium were important factors affecting the photosynthetic bacterial community and were significantly correlated with the photosynthetic bacterial community, explaining 59.90% of the variation in the photosynthetic bacterial community. Effective Cd content was significantly positively correlated with Methylorubrum populi, Methylorubrum extorquens, Methylobacterium sp. Leaf125, and Rhodopseudomonas sp. AAP120 (R>0.05, P<0.05). This study will provide a theoretical basis for the microbial remediation of cadmium contamination in paddy fields. This study is important for understanding the effects of cadmium pollution on specific functional microbial populations in paddy soils.

Integrated Analysis of microRNA and mRNA Transcriptome Reveals the Molecular Mechanism of Solanum lycopersicum Response to Bemisia tabaci and Tomato chlorosis virus.

Tomato chlorosis virus (ToCV), is one of the most devastating cultivated tomato viruses, seriously threatened the growth of crops worldwide. As the vector of ToCV, the whitefly Bemisia tabaci Mediterranean (MED) is mainly responsible for the rapid spread of ToCV. The current understanding of tomato plant responses to this virus and B. tabaci is very limited. To understand the molecular mechanism of the interaction between tomato, ToCV and B. tabaci, we adopted a next-generation sequencing approach to decipher miRNAs and mRNAs that are differentially expressed under the infection of B. tabaci and ToCV in tomato plants. Our data revealed that 6199 mRNAs were significantly regulated, and the differentially expressed genes were most significantly associated with the plant-pathogen interaction, the MAPK signaling pathway, the glyoxylate, and the carbon fixation in photosynthetic organisms and photosynthesis related proteins. Concomitantly, 242 differentially expressed miRNAs were detected, including novel putative miRNAs. Sly-miR159, sly-miR9471b-3p, and sly-miR162 were the most expressed miRNAs in each sample compare to control group. Moreover, we compared the similarities and differences of gene expression in tomato plant caused by infection or co-infection of B. tabaci and ToCV. Taken together, the analysis reported in this article lays a solid foundation for further research on the interaction between tomato, ToCV and B. tabaci, and provide evidence for the identification of potential key genes that influences virus transmission in tomato plants.

NSs, the Silencing Suppressor of Tomato Spotted Wilt Orthotospovirus, Interferes With JA-Regulated Host Terpenoids Expression to Attract Frankliniella occidentalis.

Tomato spotted wilt orthotospovirus (TSWV) causes serious crop losses worldwide and is transmitted by Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). NSs protein is the silencing suppressor of TSWV and plays an important role in virus infection, cycling, and transmission process. In this research, we investigated the influences of NSs protein on the interaction of TSWV, plants, and F. occidentalis with the transgenic Arabidopsis thaliana. Compared with the wild-type Col-0 plant, F. occidentalis showed an increased number and induced feeding behavior on transgenic Arabidopsis thaliana expressing exogenous NSs. Further analysis showed that NSs reduced the expression of terpenoids synthesis-related genes and the content of monoterpene volatiles in Arabidopsis. These monoterpene volatiles played a repellent role in respect to F. occidentalis. In addition, the expression level of plant immune-related genes and the content of the plant resistance hormone jasmonic acid (JA) in transgenic Arabidopsis were reduced. The silencing suppressor of TSWV NSs alters the emission of plant volatiles and reduces the JA-regulated plant defenses, resulting in enhanced attractiveness of plants to F. occidentalis and may increase the transmission probability of TSWV.

The NIa-Protease Protein Encoded by the Pepper Mottle Virus Is a Pathogenicity Determinant and Releases DNA Methylation of Nicotiana benthamiana.

It is well documented that the canonical function of NIa-protease (NIa-Pro) of the potyviruses is responsible for cleaving the viral polyprotein into functional proteins. Although NIa-Pro is vital for the infection cycle of potyviruses, the function of NIa-Pro in the interaction of the potyvirus host is not clear. In this study, NIa-Pro is ectopically expressed from a potato virus X (PVX) vector and infiltrates Nicotiana benthamiana wild type and 16-TGS. The pathogenicity and inhibition of host transcriptional gene silencing (TGS) are characterized. Ectopic expression of NIa-Pro from a PVX vector resulted in severe mosaic symptoms followed by a hypersensitive-like response in N. benthamiana. Furthermore, PepMoV NIa-Pro was able to reverse established TGS of a green fluorescent protein transgene by reducing methylation of promoter sequences in N. benthamiana and possessed the capacity to interfere with the global methylation of N. benthamiana. Taken together, the results of this study likely suggest that PepMoV NIa-Pro is a pathogenicity determinant and a potent suppressor of host TGS and suggest that NIa-Pro may employ novel mechanisms to suppress host antiviral defenses. To the best of our knowledge, this is the first report of a plant RNA virus modulating host TGS in a novel manner by interfering with the establishment of the methylation step of the plant DNA methylation pathway.

Human Kir2.1 channel carries a transient outward potassium current with inward rectification.

We have previously reported a depolarization-activated 4-aminopyridine-resistant transient outward K(+) current with inward rectification (I (to.ir)) in canine and guinea pig cardiac myocytes. However, molecular identity of this current is not clear. The present study was designed to investigate whether Kir2.1 channel carries this current in stably transfected human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp technique. It was found that HEK 293 cells stably expressing human Kir2.1 gene had a transient outward current elicited by voltage steps positive to the membrane potential (around -70 mV). The current exhibited a current-voltage relationship with intermediate inward rectification and showed time-dependent inactivation and rapid recovery from inactivation. The half potential (V (0.5)) of availability of the current was -49.4 +/- 2.1 mV at 5 mM K(+) in bath solution. Action potential waveform clamp revealed two components of outward currents; one was immediately elicited and then rapidly inactivated during depolarization, and another was slowly activated during repolarization of action potential. These properties were similar to those of I (to.ir) observed previously in native cardiac myocytes. Interestingly, inactivation of the I (to.ir) was strongly slowed by increasing intracellular free Mg(2+) (Mg(2+) ( i ), from 0.03 to 1.0, 4.0, and 8.0 mM). The component elicited by action potential depolarization increased with the elevation of Mg(2+) ( i ). Inclusion of spermine (100 muM) in the pipette solution remarkably inhibited both the I (to.ir) and steady-state current. These results demonstrate that the Mg(2+) ( i )-dependent current carried by Kir2.1 likely is the molecular identity of I (to.ir) observed previously in cardiac myocytes.

Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels.

Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 microM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 microM) and the Src-family kinase inhibitor PP2 (10 microM) remarkably inhibited hERG channel current (I(hERG)), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556. Our results demonstrate the novel information that I(hERG) is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.

Overexpression of microRNA-495 improves the intestinal mucosal barrier function by targeting STAT3 via inhibition of the JAK/STAT3 signaling pathway in a mouse model of ulcerative colitis.

We aim to investigate the role of microRNA-495 (miR-495) in the intestinal mucosal barrier by indirectly targeting signal transducer and activator of transcription 3 (STAT3) through the Janus kinase-signal transducer and activator of transcription (JAK)/STAT3 signaling pathway in a mouse model of ulcerative colitis (UC). BALB/c mice were selected for establishing mice model of UC, and intestinal tissues of normal and UC mice were collected. ELISA was conducted for detecting levels of TNF-α, IL-6, IFN-γ and IL-10. The levels of SOD, MPO, MDA and NO were tested in the intestinal tissues. Dual luciferase reporter gene assay was applied to determine whether miR-495 directly targets STAT3. Cells were cultured, transfected and assigned into: normal group, blank group, NC group, miR-495 mimic group, miR-495 inhibitor group, siRNA-STAT3 group and miR-495 inhibitor+siRNA-STAT3 group. MTT was used for testing cell proliferation, flow cytometry for cell cycle and apoptosis. Northern blotting and Western blotting were performed to detect miR-495 expression and expressions of STAT3, JAK and Claudin-1. Results show that the UC group had higher expression levels of TNF-α, IL-6, IFN-γ, MPO, MDA, NO, STAT3 and JAK and lower expression levels of IL-10, SOD, miR-495 and Claudin-1, compared to the normal group. Dual luciferase reporter gene assay confirmed that STAT3 was the target gene of miR-495. The miR-495 mimic and siRNA-STAT3 groups had higher expressions of Claudin-1, higher cell proliferation and increased amount of cells in S phase, but lower expressions of STAT3 and JAK, decreased amount of cells in G0/G1 phase and cell apoptotic rate compared with the blank, NC groups. We also found that the miR-495 inhibitor+siRNA-STAT3 group had reduced miR-495 expression. No significant differences were found in mRNA and protein expressions of STAT3, JAK and Claudin-1, cell proliferation, apoptosis and cycle amongst the miR-495 inhibitor+siRNA-STAT3 groups. Our study provides evidence that miR-495 improves the intestinal mucosal barrier function by targeting STAT3 through inhibiting the JAK/STAT3 signaling pathway in UC mice.

[Cloning and prokaryotic expression of Rhodoblastus acidophilus 5-aminolevlinate synthase gene].

5-aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (ALAS). However, the fidelity of ALAS gene among species is low. The ALAS gene of photosynthetic bacteria Rhodoblastus acidophilus was cloned from its genomic DNA by conventional PCR and Veterette PCR and further sequenced. The identity of ALAS gene among photosynthetic bacteria species is from 64.0% to 95.1% according to phylogenic analysis. Furthermore, the ALAS gene was subcloned into an expression vector pQE30. For the overproduction of ALA, the recombinant ALAS was overexpressed in Escherichia coli strains JM109, M15 and BL21 (DE3), respectively. The expected 44kD protein was detected by SDS-PAGE in three E. coli strains after IPTG induction and further purified by affinity purification on Ni-NTA. The conditions including strain, medium, substrate of ALA synthesize (glycine and succinic acid), and ALA dehydratase inhibitor (levulinic acid) were optimized for attainning the maximum yield of ALA in E. coli. The ALA production was established on E. coli M15, medium 1 supplied with 100mmol/L glycine and 50mmol/L succinic acid, and 40mmol/L levulinic acid. The activity of ALAS was up to 333U/min x mg of protein. Meanwhile, the output of ALA was reached to 5.379g/L, which is the highest yield of ALA up to date by biofermentation. ALA has a variety of agricultural applications not only as an herbicide, insecticide, and growth promoting factor, but also based on its ability to confer salt and cold temperature tolerance in plants. Our recombinant bacteria are of great potential in the production of ALA. Our results offer an easy and simple ALA mass production method and may stimulate the application of ALA in agriculture.

Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil.

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10-2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10-1 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

High mobility group box 1 protein attenuates myocardial ischemia reperfusion injury via inhibition of the p38 mitogen-activated protein kinase signaling pathway.

The present study aimed to determine the effects of high mobility group box 1 protein (HMGB1) on myocardial ischemia reperfusion (I/R) injury in rats following acute myocardial ischemia and investigate the underlying molecular mechanisms of these effects. Male Wistar rats were randomly divided into the following groups (n=10/group): Sham operation; I/R; HMGB50 (50 ng/kg HMGB1 before I/R); HMGB100 (100 ng/kg HMGB1 before I/R); and HMGB200 (200 ng/kg HMGB1 before I/R). Serum cardiac troponin I (cTnI), interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels were subsequently measured. Myocardial levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were also determined. Myocardial infarction size (IS) was determined by 2,3,5-triphenyltetrazolium chloride staining. Myocardial expression of hypoxia inducible factor (HIF)-1α and phosphorylated p38 mitogen-activated protein kinase (P-p38 MAPK) protein was measured using western blotting. The results demonstrated that HMGB1 significantly decreased serum levels of cTnI, IL-6 and TNF-α and myocardial IS in I/R rats compared with the sham group (all P<0.05). HMGB1 also significantly decreased and increased myocardial levels of MDA and SOD, respectively (both P<0.05). HMGB1 significantly increased myocardial expression of HIF-1α and decreased expression of P-p38 MAPK following I/R (both P<0.05). These effects of HMGB1 occurred in a dose-dependent manner. The results of the current study indicate that the cardioprotective effects of intravenous HMGB1 are associated with increased myocardial expression of HIF-1α via inhibition of P-p38 MAPK expression, leading to inhibition of the P-p38 MAPK signaling pathway.

[Effects of Rhodopseudomonas palustris PSB06 on Pepper Rhizosphere Microbial Community Structure].

The use of biological pesticide can greatly reduce the soil pollution in the environment. Exploring the effect of biological pesticide on community diversity and distribution of pathogenic bacteria will provide theoretic basis for subsequent researches on biological pesticide micro-ecological control. In order to explore the microbial ecological mechanism of pepper phytophthora blight, this research compared the difference of microbial diversity between rhizosphere soil of infected and healthy plants, and the effects of Rhodopseudomonas palustris PSB06 on microbial diversities of plant rhizosphere soil were investigated using Illumina MiSeq sequencing technology. The results showed that there was less difference in the microbial diversity from the same soil between the seventh day and the fourteenth day. The microbial diversity of rhizosphere soil of healthy plants was higher than that of rhizosphere soil of infected plants. The soil sprayed with Rhodopseudomonas palustris PSB06 exhibited the highest diversity. Moreover, the abundance of Actinomycetes in the rhizosphere soil of healthy plants was higher than that of infected plants, and the highest abundance of Actinomycetes was observed in the soil sprayed with Rhodopseudomonas palustris PSB06. The microbial diversity between rhizosphere soil of infected and healthy plants was significantly different. Spraying Rhodopseudomonas palustris PSB06 could significantly alter the microbial community structure of the soil. It could also increase the diversity of microorganism and the abundance of Actinomycetes in the soil.