Sucrose non-fermenting1-related protein kinase VcSnRK2.3 promotes anthocyanin biosynthesis in association with VcMYB1 in blueberry.

Sucrose non-fermenting1-related protein kinase-2 (SnRK2) is a plant-specific protein kinase family and an important component of the abscisic acid (ABA) signaling pathway. However, there is a lack of relevant studies in blueberry (Vaccinium corymbosum). In this study, we identified six SnRK2 family members (from VcSnRK2.1 to VcSnRK2.6) in blueberries for the first time. In addition, we found that VcSnRK2.3 expression was not only positively correlated with fruit ripening but was also induced by ABA signaling. Transient expression in blueberry fruits also proved that VcSnRK2.3 promoted anthocyanin accumulation and the expression of anthocyanin synthesis-related genes such as VcF3H, VcDFR, VcANS, and VcUFGT. Transgenic Arabidopsis thaliana seeds and seedlings overexpressing VcSnRK2.3 showed anthocyanin pigmentation. Yeast two-hybrid assays (Y2H) and Bimolecular fluorescence complementation assays (BiFC) demonstrated that VcSnRK2.3 could interact with the anthocyanin positive regulator VcMYB1. Finally, VcSnRK2.3 was able to enhance the binding of VcMYB1 to the VcDFR promoter. Via regulation transcription of anthocyanin biosynthesis genes, VcSnRK2.3 promoted anthocyanin accumulation in blueberry. The above results suggest that VcSnRK2.3 plays an important role in blueberry anthocyanin synthesis, is induced by ABA, and can interact with VcMYB1 to promote anthocyanin biosynthesis in blueberry.

Randomized, sham-controlled trial of acupuncture for post-cataract surgery dry eye disease.

PURPOSE: To determine the efficacy of acupuncture in patients with post-cataract surgery dry eye disease (DED). METHODS: Ninety participants with post-cataract surgery DED were randomly assigned (1:1) to receive true acupuncture (n = 44) or non-penetrating sham acupuncture (n = 46) twice weekly for 8 weeks. The primary outcome was the change from baseline to week 8 in the noninvasive tear film break-up time (TFBUT). Participants were followed until week 12. Secondary outcomes included Ocular Surface Disease Index (OSDI) score, tear meniscus height (TMH), corneal fluorescein staining (CFS) score, meiboscore and corrected distance visual acuity (CDVA). RESULTS: The estimated mean change from baseline in the noninvasive TFBUT was 1.52 for true acupuncture versus 0.77 for sham acupuncture (adjusted difference -0.75 [95% CI -1.39 to -0.12]; P = 0.02) at week 8 and 1.49 for true acupuncture versus 0.81 for sham acupuncture (adjusted difference -0.68 [95% CI, -1.29 to -0.07]; P = 0.029) at week 12. The mean change in the OSDI score was -20.13 for true acupuncture versus -13.44 for sham acupuncture (adjusted difference 6.69 [95% CI, 0.64 to 12.74]; P = 0.031) at week 8, but revealed no statistically between-group differences at week 12. Mean changes in TMH, CFS score, meiboscore and CDVA did not differ significantly between the two groups. CONCLUSIONS: Among patients with post-cataract surgery DED, acupuncture compared with sham acupuncture resulted in statistically significant benefit on tear film stability. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR1800020132).

Regulation of autophagy and lipid accumulation under phosphate limitation in Rhodotorula toruloides.

Background: It is known that autophagy is essential for cell survival under stress conditions. Inorganic phosphate (Pi) is an essential nutrient for cell growth and Pi-limitation can trigger autophagy and lipid accumulation in oleaginous yeasts, yet protein (de)-phosphorylation and related signaling events in response to Pi limitation and the molecular basis linking Pi-limitation to autophagy and lipid accumulation remain elusive. Results: Here, we compared the proteome and phosphoproteome of Rhodotorula toruloides CGMCC 2.1389 under Pi-limitation and Pi-repletion. In total, proteome analysis identified 3,556 proteins and the phosphoproteome analysis identified 1,649 phosphoproteins contained 5,659 phosphosites including 4,499 pSer, 978 pThr, and 182 pTyr. We found Pi-starvation-induced autophagy was regulated by autophagy-related proteins, but not the PHO pathway. When ATG9 was knocked down, the engineered strains produced significantly less lipids under Pi-limitation, suggesting that autophagy required Atg9 in R. toruloides and that was conducive to lipid accumulation. Conclusion: Our results provide new insights into autophagy regulation under Pi-limitation and lipid accumulation in oleaginous yeast, which should be valuable to guide further mechanistic study of oleaginicity and genetic engineering for advanced lipid producing cell factory.

Single-Molecule Real-Time and Illumina Sequencing to Analyze Transcriptional Regulation of Flavonoid Synthesis in Blueberry.

Blueberries (Vaccinium corymbosum) contain large amounts of flavonoids, which play important roles in the plant's ability to resist stress and can also have beneficial effects on human health when the fruits are eaten. However, the molecular mechanisms that regulate flavonoid synthesis in blueberries are still unclear. In this study, we combined two different transcriptome sequencing platforms, single-molecule real-time (SMRT) and Illumina sequencing, to elucidate the flavonoid synthetic pathways in blueberries. We analyzed transcript quantity, length, and the number of annotated genes. We mined genes associated with flavonoid synthesis (such as anthocyanins, flavonols, and proanthocyanidins) and employed fluorescence quantitative PCR to analyze the expression of these genes and their correlation with flavonoid synthesis. We discovered one R2R3 MYB transcription factor from the sequencing library, VcMYB1, that can positively regulate anthocyanin synthesis in blueberries. VcMYB1 is mainly expressed in colored (mature) fruits. Experiments showed that overexpression and transient expression of VcMYB1 promoted anthocyanin synthesis in Arabidopsis, tobacco (Nicotiana benthamiana) plants and green blueberry fruits. Yeast one-hybrid (Y1H) assay, electrophoretic mobility shift assay, and transient expression experiments showed that VcMYB1 binds to the MYB binding site on the promoter of the structural gene for anthocyanin synthesis, VcMYB1 to positively regulate the transcription of VcDFR, thereby promoting anthocyanin synthesis. We also performed an in-depth investigation of transcriptional regulation of anthocyanin synthesis. This study provides background information and data for studying the synthetic pathways of flavonoids and other secondary metabolites in blueberries.

Jamming Strategy Optimization through Dual Q-Learning Model against Adaptive Radar.

Modern adaptive radars can switch work modes to perform various missions and simultaneously use pulse parameter agility in each mode to improve survivability, which leads to a multiplicative increase in the decision-making complexity and declining performance of the existing jamming methods. In this paper, a two-level jamming decision-making framework is developed, based on which a dual Q-learning (DQL) model is proposed to optimize the jamming strategy and a dynamic method for jamming effectiveness evaluation is designed to update the model. Specifically, the jamming procedure is modeled as a finite Markov decision process. On this basis, the high-dimensional jamming action space is disassembled into two low-dimensional subspaces containing jamming mode and pulse parameters respectively, then two specialized Q-learning models with interaction are built to obtain the optimal solution. Moreover, the jamming effectiveness is evaluated through indicator vector distance measuring to acquire the feedback for the DQL model, where indicators are dynamically weighted to adapt to the environment. The experiments demonstrate the advantage of the proposed method in learning radar joint strategy of mode switching and parameter agility, shown as improving the average jamming-to-signal radio (JSR) by 4.05% while reducing the convergence time by 34.94% compared with the normal Q-learning method.

Synthesis and characterization of magnetic molecularly imprinted polymers for effective extraction and determination of kaempferol from apple samples.

The specific of magnetic molecularly imprinted polymers (Fe3O4@SiO2-MIPs) for kaempferol were fabricated by using acrylamide (AM) as the functional monomer, azobisisobutyronitrile (AIBN) as the initiator and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The Fe3O4@SiO2-MIPs showed high adsorption capacity (3.84 mg/g) for kaempferol, and the adsorption equilibrium was achieved within 50 min. The specific recognition capacity of Fe3O4@SiO2-MIPs was 3.02 times as high as that of Fe3O4@SiO2-NIPs. The Fe3O4@SiO2-MIPs showed high selectivity towards kaempferol over structural analogues. The recoveries of proposed method at three spiked levels analysis were ranged from 90.5% to 95.4% with the relative standard deviations (RSD) less than 5%. The obtained Fe3O4@SiO2-MIPs were successfully applied for the extraction and determination of kaempferol from apple samples. The established method was simple and feasible, which showed high selectivity, fast separation and satisfactory recoveries for real sample analysis.

RNA interference in the oleaginous yeast Rhodosporidium toruloides.

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development.

Synthesis and characterization of core-shell magnetic molecularly imprinted polymers for selective recognition and determination of quercetin in apple samples.

Magnetic molecularly imprinted polymers (M-MIPs) with core-shell structure were prepared through the surface molecular imprinting technique by using quercetin as the template molecule. The resultant Fe3O4@MIPs were characterized using X-ray diffraction, Fourier transform infrared spectrum, transmission electron microscopy, and vibrating sample magnetometer. The Fe3O4@MIPs possessed high adsorption capacity and specific recognition for quercetin. The maximum adsorption capacity of Fe3O4@MIPs towards quercetin was 10.52 mg/g, and could reach binding equilibrium within 30 min. The selectivity and specific recognition ability of Fe3O4@MIPs were evaluated according to their recognition to quercetin and its analogues. The Fe3O4@MIPs were successfully applied as selective sorbents to extract and determine quercetin from apple samples with a relatively high recoveries (89.2-93.6%) and relative standard deviations of <5%. The obtained Fe3O4@MIPs have potential application for quercetin determination in real samples.

Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation.

Metabolic engineering of Rhodosporidium toruloides, a robust lipid and caroteinoid producer, is of great importance for oleochemicals and carotenoids production. However, the Agrobacterium-mediated gene transformation is tedious and time consuming. Here, we described a fast and efficient genetic transformation of R. toruloides using electroporation with linear DNA fragments, and the process was optimized. The results showed that 2 × 103 transformants can be obtained at 0.7 kV/µg linear DNA by using hygromycin and bleomycin as selection markers after the competent cells pretreated with 25 mM DTT and 100 mM LiAc. Our results would facilitate mutant library construction and metabolic engineering of R. toruloides for production of oleochemicals and carotenoids. We further demonstrated that all transformants arose due to illegitimate integration of transforming DNA fragments by colony PCR.

De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.

BACKGROUND: Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. RESULTS: In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. CONCLUSIONS: Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the cranberry and related species.

Identification and expression analysis of MATE genes involved in flavonoid transport in blueberry plants.

Multidrug and toxic compound extrusion (MATE) proteins are the most recently identified family of multidrug transporters. In plants, this family is remarkably large compared to the human and bacteria counterpart, highlighting the importance of MATE proteins in this kingdom. Here 33 Unigenes annotated as MATE transporters were found in the blueberry fruit transcriptome, of which eight full-length cDNA sequences were identified and cloned. These proteins are composed of 477-517 residues, with molecular masses ~54 kDa, and theoretical isoelectric points from 5.35 to 8.41. Bioinformatics analysis predicted 10-12 putative transmembrane segments for VcMATEs, and localization to the plasma membrane without an N-terminal signal peptide. All blueberry MATE proteins shared 32.1-84.4% identity, among which VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8, and VcMATE9 were more similar to the MATE-type flavonoid transporters. Phylogenetic analysis showed VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8 and VcMATE9 clustered with MATE-type flavonoid transporters, indicating that they might be involved in flavonoid transport. VcMATE1 and VcMATE4 may be involved in the transport of secondary metabolites, the detoxification of xenobiotics, or the export of toxic cations. Real-time quantitative PCR demonstrated that the expression profile of the eight VcMATE genes varied spatially and temporally. Analysis of expression and anthocyanin accumulation indicated that there were some correlation between the expression profile and the accumulation of anthocyanins. These results showed VcMATEs might be involved in diverse physiological functions, and anthocyanins across the membranes might be mutually maintained by MATE-type flavonoid transporters and other mechanisms. This study will enrich the MATE-based transport mechanisms of secondary metabolite, and provide a new biotechonology strategy to develop better nutritional blueberry cultivars.

[The Characteristic Research of ·OH Induced by Water on an Argon Plasma Jet].

·OH plays a crucial role in many fields, having aroused wide public concern in the world. Atmospheric Pressure Plasma Jet, which can be achieved by portable device due to working without the vacuum environment, has the advantages of high concentration of reactive species, high electron temperature and low gas temperature. It has become an important research topic in the field of gas discharge with a strong prospect. Especially, how to induce plasma jet to produce ·OH has become a new hotpot in the field of low-temperature plasma. It has been reported that mass ·OH can be induced successfully when water vapor is added to the working gas, but it will be unstable when the concentrate of water reaches a certain degree. Thus, a device of argon plasma jet with a Ring-to-Ring Electrode Configuration has been designed to interact with water in the surrounding air to generate ·OH under atmospheric pressure. In order to increase the production of ·OH, ultrasonic atomizing device is introduced to promote water concentration around the plasma plume. The generating rule of OH(A2J) induced by water has been extensively studied under different voltages and flow rate. ·OH output induced by the plasma has been tested by emission spectrometry, and at the meanwhile, Ar atomic spectral lines at 810.41 and 811.48 nm are also recorded in order to calculate the electron temperature in argon plasma plume. The results show that the water surrounding the plasma plume can be induced to produce ·OH, and OH(A2 ∑+) output increases with the electrode voltage rising from 20 to 28 kV. When the flow rate increases from 100 to 200 L x h(-1), the OH(A2∑+) output increases, but from 200 to 600 L x h(-1), it decreases. The production rules of OH(A2∑+) is the same as that of electron temperature. Therefore, the presumption is proved that ·OH output mainly affected by electron temperature.

[Detection of Lead in Water by Electrolyte Cathode Atmospheric Glow Discharge Emission Spectroscopy].

A device based on electrolyte cathode atmospheric glow discharge atomic emission spectroscopy (ELCAD-AES) has been developed to determine the metal ion Pb in water. The emission intensity of Ph was significantly enhanced with the increase concentration of Pb, and the emission intensity has a linear relationship with concentration while the concentration of Pb in the range of 10-80 mg x L(-1). The effects of discharge current and easily ionizable elements on the emission spectral of Pb were investigated, and the emission intensity reached greatest when the discharge current increased to 70 mA, and the easily ionizable elements generated weak effect on the emission spectral of Pb. The effect of acidification regent on emission spectral of Pb was discussed. It was found that it perform best when acidified with HNO3, and reducing the pH can improve the emission intensity of Pb effectively. The emission intensity of Pb at different region was detected near cathode region, thus obtained the best detection position. Under the optimized experimental parameters, the detection limit of Ph was 0.7 mg x L(-1) and relative standard deviation was 1.7%. The recovery of samples was 95%-106%, result and shows that this method has better accuracy. These results provide an available method for further research of detection trace heavy mental elements in water using ELCAD-AES.

Glycotope sharing between snail hemolymph and larval schistosomes: larval transformation products alter shared glycan patterns of plasma proteins.

Recent evidence supports the involvement of inducible, highly diverse lectin-like recognition molecules in snail hemocyte-mediated responses to larval Schistosoma mansoni. Because host lectins likely are involved in initial parasite recognition, we sought to identify specific carbohydrate structures (glycans) shared between larval S. mansoni and its host Biomphalaria glabrata to address possible mechanisms of immune avoidance through mimicry of elements associated with the host immunoreactivity. A panel of monoclonal antibodies (mABs) to specific S. mansoni glycans was used to identify the distribution and abundance of shared glycan epitopes (glycotopes) on plasma glycoproteins from B. glabrata strains that differ in their susceptibilities to infection by S. mansoni. In addition, a major aim of this study was to determine if larval transformation products (LTPs) could bind to plasma proteins, and thereby alter the glycotopes exposed on plasma proteins in a snail strain-specific fashion. Plasma fractions (< 100 kDa/> 100 kDa) from susceptible (NMRI) and resistant (BS-90) snail strains were subjected to SDS-PAGE and immunoblot analyses using mAB to LacdiNAc (LDN), fucosylated LDN variants, Lewis X and trimannosyl core glycans. Results confirmed a high degree of glycan sharing, with NMRI plasma exhibiting a greater distribution/abundance of LDN, F-LDN and F-LDN-F than BS-90 plasma (< 100 kDa fraction). Pretreatment of blotted proteins with LTPs significantly altered the reactivity of specific mABs to shared glycotopes on blots, mainly through the binding of LTPs to plasma proteins resulting in either glycotope blocking or increased glycotope attachment to plasma. Many LTP-mediated changes in shared glycans were snail-strain specific, especially those in the < 100 kDa fraction for NMRI plasma proteins, and for BS-90, mainly those in the > 100 kDa fraction. Our data suggest that differential binding of S. mansoni LTPs to plasma proteins of susceptible and resistant B. glabrata strains may significantly impact early anti-larval immune reactivity, and in turn, compatibility, in this parasite-host system.

Cloning and Sequencing of cDNA Encoding the Diazepam Binding Inhibitor (DBI).

The anti-morphine peptide PPC from dog brain has an inhibitory effect on morphine analgesia. It is highly homologous to DBI. The analgesic effect of bovine DBI was measured, showing the potentiation effect on morphine analgesia. In order to study the relationship between PPC and DBI, cDNA library derived from dog liver had been screened using partial gene of rat DBI as a probe. A positive clone was obtained. The entire sequence of the insert of this positive clone provided complete sequence of a peptide which was identified as the DBI peptide. It shows 88.5% homology with pig DBI, 87.4% with bovine, 83.9% with human, 46.5% with yeast, respectively. The sequence of N-terminal 50 amino acid residues except 55th, 58th site and C-terminal 17 amino acids is the same to DBI from dog. PPC has 23 amino acids more than DBI inside.