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Background Cantharidin is an inhibitor of protein phosphatase 2 A (PP2A), and has been frequently used in clinical practice. In our previous study, we proved that cantharidin could arrest cell cycle in G2/M phase. Since cells at G2/M phase are sensitive to radiotherapy, in the present study, we investigated the radiotherapy-sesitization effect of cantharidin and the potential mechanisms involved. Methods Cell growth was determined by MTT assay. Cell cycle was evaluated by flow cytometry. DNA damage was visualized by phospho-Histone H2A.X staining. Expression of mRNA was tested by microarray assay and real-time PCR. Clinical information and RNA-Seq expression data were derived from The Cancer Genome Atlas (TCGA) pancreatic cancer cohort. Survival analysis was obtained by Kaplan-Meier estimates. Results Cantharidin strengthened the growth inhibition effect of irradiation. Cantharidin drove pancreatic cancer cells out of quiescent G0/G1 phase and arrested cell cycle in G2/M phase. As a result, cantharidin strengthened DNA damage which was induced by irradiation. Moreover, cantharidin repressed expressions of several genes participating in DNA damage repair, including UBE2T, RPA1, GTF2HH5, LIG1, POLD3, RMI2, XRCC1, PRKDC, FANC1, FAAP100, RAD50, RAD51D, RAD51B and DMC1, through JNK, ERK, PKC, p38 and/or NF-κB pathway dependent manners. Among these genes, worse overall survival for pancreatic cancer patients were associated with high mRNA expressions of POLD3, RMI2, PRKDC, FANC1, RAD50 and RAD51B, all of which could be down-regulated by cantharidin. Conclusion Cantharidin can sensitize pancreatic cancer cells to radiotherapy. Multiple mechanisms, including cell cycle regulation, enhanced DNA damage, and inhibited DNA damage repair, may be involved.

Establishment of a stable analytical methodology with high-quality results is an urgent need for screening cancer biomarkers in early diagnosis of cancer. In this study, we incorporate holographic optical tweezers with upconversion luminescence encoding to design an imageable suspension array and apply it to conduct the detection of two liver cancer related biomarkers, carcinoembryonic antigen and alpha fetal protein. This bead-based assay is actualized by forming a bead array with holographic optical tweezers and synchronously exciting the upconversion luminescence of corresponding trapped complex beads fabricated with a simple one-step sandwich immunological recognition. Owing to the fact that these flowing beads are stably trapped in the focal plane of the objective lens which tightly converges the array of the laser beams by splitting a 980 nm beam using a diffraction optical element, a fairly stable excitation condition is achieved to provide reliable assay results. By further taking advantage of the eminent encoding capability of upconversion nanoparticles and the extremely low background signals of anti-Stokes luminescence, the two targets are well-identified and simultaneously detected with quite sound sensitivity and specificity. Moreover, the potential on-demand clinical application is presented by employing this approach to respond the targets toward complex matrices such as serum and tissue samples, offering a new alternative for cancer diagnosis technology.

A novel nanosilica/polydimethylsiloxane (SiO2/PDMS) coated stirring bar was adopted in the sorption extraction (SBSE) of phenols in water, and the high performance liquid chromatography-fourier transform infrared spectroscopy (HPLC-FTIR) was subsequently used to determination of phenol concentration. The SiO2/PDMS coating was prepared by sol-gel method and characterized with respect to morphology and specific surface area. The results of field-emission scanning electron microscope (FE-SEM) and N2 adsorption-desorption as well as phenol adsorption experiments denoted that SiO2/PDMS has larger surface area and better adsorption capacity than commercial PDMS. The extraction efficiency of phenol with SiO2/PDMS coated stirring bar was optimized in terms of ion strength, flow rate of phenol-involved influent, type of desorption solvent and desorption time. More than 75% of phenol desorption efficiency could be kept even after 50 cycles of extraction, indicating the high stability of the SiO2/PDMS coated stirring bar. Approximately 0.16 mg/L 2, 5-dimethylphenol (2, 5-DMP), which was 34-fold more toxic than phenol, was detected in water through HPLC-FTIR. However, 2, 5-DMP could be oxidized to 5-methy-2-hydroxy benzaldehyde after disinfection in drinking water treatment process. Therefore, the proposed method of SiO2/PDMS-SBSE-HPLC-FTIR is successfully applied in the analysis of phenols isomers in aqueous environment.

Three hydrogen-bonded organic salts (HTBA)2(H4ATMP)·(H2O)3 (1), (HTBA)6(H3O)(H2.5ATMP)2·(H2O)2 (2) and (HTBA)6(H3ATMP)2·H2O (3) were synthesized by combining different molar ratios of amino trimethylene phosphonic acid (H6ATMP) and tert-butylamine (TBA). There are three-dimensional hydrogen-bonded networks in 1–3. The crystallization water molecules that are loosely trapped in the supramolecular hydrogen-bonded networks in 1 and 2 undergo order–disorder transitions whose dynamic changes are likely to be responsible for dielectric transitions and relaxations.

Rictor is a key regulatory/structural subunit of the mammalian target of rapamycin complex 2 (mTORC2) and is required for phosphorylation of Akt at serine 473. It plays an important role in cell survival, actin cytoskeleton organization and other processes in embryogenesis. However, the role of Rictor/mTORC2 in the embryonic cardiac differentiation has been uncovered. In the present study, we examined a possible link between Rictor expression and cardiomyocyte differentiation of the mouse embryonic stem (mES) cells. Knockdown of Rictor by shRNA significantly reduced the phosphorylation of Akt at serine 473 followed by a decrease in cardiomyocyte differentiation detected by beating embryoid bodies. The protein levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein) and α-Actinin (cardiomyocyte biomarker) decreased in Rictor knockdown group during cardiogenesis. Furthermore, knockdown of Rictor specifically inhibited the ventricular-like cells differentiation of mES cells with reduced level of ventricular-specific protein, MLC-2v. Meanwhile, patch-clamp analysis revealed that shRNA-Rictor significantly increased the number of cardiomyocytes with abnormal electrophysiology. In addition, the expressions and distribution patterns of cell-cell junction proteins (Cx43/Desmoplakin/N-cadherin) were also affected in shRNA-Rictor cardiomyocytes. Taken together, the results demonstrated that Rictor/mTORC2 might play an important role in the cardiomyocyte differentiation of mES cells. Knockdown of Rictor resulted in inhibiting ventricular-like myocytes differentiation and induced arrhythmias symptom, which was accompanied by interfering the expression and distribution patterns of cell-cell junction proteins. Rictor/mTORC2 might become a new target for regulating cardiomyocyte differentiation and a useful reference for application of the induced pluripotent stem cells.

Cancer, as the most invasive disease in the world, has led to an increasing amount of death year by year, so it is highly desired to develop a portable device to monitor the aberrant expression of biomarker in cancer patient. Here, we present a bio-photonic periodic nanostructures sensor chip assisted cyclic enzymatic amplification method to detect miRNA-21 with a detection limit of 55 fM. By employing biocompatible polydopamine nanospheres (PDANs) and DNaseⅠto construct an target-recycling amplification process on the photonic crystals, the output fluorescence signal can be strengthened selectively and short amplification time is needed. Benefiting from the synergy of the enhancement of photonic crystals and enzymatic cycle amplification, we realize high sensitivity detection of miRNA-21 with a detection range of 1 pM–10 nM and a detection limit of about four orders of magnitudes lower than the method employs no amplification, showing an expectable prospect in the early diagnosis of cancer.

Tartary buckwheat is widely accepted as its nutritional value. Some allergic reactions hinder its utilization. This research focused on evaluating the core epitope of 16 kDa allergen (Fag t 2) in tartary buckwheat. Six B- and seven T cell epitopes of Fag t 2 were predicted, and six B cell epitope-mutants were expressed in Pichia pastoris. Bioinformatics analysis and SDS-PAGE demonstrated that the molecular weight, isoelectric point and spatial structures of six mutant allergens were similar with Fag t 2, with the same signal peptide sequences and α-amylase inhibitor domain. There was no significant change in mutants' spatial conformation confirmed by Circular Dichroism. The position K132N and peptides at 108–117 and 132–141 were the core B- and T cell epitopes of Fag t 2 confirmed by competitive inhibition ELISA and dot blot. This result was of great significance on the study of allergen epitopes in prevention and treatment of hypersensitivity.

Research has shown that FMS-like tyrosine kinase 3 (FLT3) may be a vital drug target for acute myeloid leukemia (AML). However, even though the clinically relevant F691L gatekeeper mutation conferred resistance to current FLT3 drug quizartinib, PLX3397 remained unaffected. In this study, the protein–ligand interactions between FLT3 kinase domain (wild-type or F691L) and quizartinib or PLX3397 were compared via an integrated computational approach. The classical molecular dynamics (MD) simulations in conjunction with dynamic cross-correlation (DCC) analysis, solvent-accessible surface area (SASA), and free energy calculations indicated that the resistant mutation may induce the conformational change of αC-helix and A-loop of the FLT3 protein. The major variations were controlled by the electrostatic interaction and SASA, which were allosterically regulated by residues Glu-661 and Asp-829. When FLT3-F691L was bound to quizartinib, a large conformational change was observed via combination of accelerated MD simulations (aMDs), principal component analysis (PCA), and free energy landscape (FEL) calculations. The umbrella sampling (US) simulations were applied to investigate the dissociation processes of the quizartinib or PLX3397 from FLT3-WT and FLT3-F691L. The calculated results suggested that PLX3397 had similar dissociation processes from both FLT3-WT and FLT3-F691L, but quizartinib dissociated more easily from FLT3-F691L than from FLT3-WT. Thus, reduced residence time was responsible for the FLT3-F691L resistance to inhibitors. These findings indicated that both the conformational changes of αC-helix and A-loop and the drug residence time should be considered in the design of drugs so that rational decisions can be made to overcome resistance to FLT3-F691L.

Exonuclease Ⅷ (Exo Ⅷ), an ATP-independent dsDNA 5'-3' exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ (tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×10⁵ U/mg. In a 10 μL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×10⁶ cfu/μg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×10⁴ recombinant clones per μg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.核酸外切酶Ⅷ (Exonuclease Ⅷ，Exo Ⅷ) 是一种不依赖于ATP 的dsDNA 5ʹ-3ʹ核酸外切酶，可作为体外DNA 重组反应极具应用价值的候选蛋白。目前关于Exo Ⅷ在体外DNA 重组反应中的应用尚未有文献报道。本研究构建了保留完整外切活性的截短Exo Ⅷ (Truncated exonuclease Ⅷ，tExo Ⅷ) 的重组表达载体pET28a-tExo Ⅷ，实现了tExo Ⅷ在大肠杆菌中的高效表达，在纯化获得高纯度蛋白的基础上，对体外重组反应的温度、反应时间、同源臂长度等因素进行了优化分析。研究结果表明，tExo Ⅷ在大肠杆菌中以可溶性形式高效表达，每升可纯化92.40 mg tExo Ⅷ，比活力为1.21×10⁵ U/mg；在10 μL 的重组体系中，2.5 U 的tExo Ⅷ于25 ℃反应12.5 min随后50 ℃保温50 min 时重组效率最高。添加Pfu DNA 聚合酶的体外同源臂延伸策略可以有效提高重组克隆的效率。以转化效率为2.2×10⁶ CFU/μg 的Mach1T1 为受体细胞，对于含有21 bp 同源臂的1 kb 片段与5.8 kb 线性载体的重组，每微克载体可形成1.1×10⁴ 个重组克隆，且阳性率大于80%。同源臂长度在8–21 bp 范围内，重组反应效率随着同源臂长度增加而提高。在最佳反应条件下，同源臂长度仅为8 bp 仍可实现有效的重组反应。Exo Ⅷ介导的体外重组体系具有酶制备方法简单、对DNA 的克隆无酶切位点限制及高重组克隆效率等显著优点，是分子生物学领域具有潜在应用价值的高效基因克隆新体系。.

Background Lead (Pb) pollution is a widespread environmental problem that is harmful to living organisms. Tartary buckwheat (Fagopyrum tataricum), a member of the family Polygonaceae, exhibits short growth cycles and abundant biomass production, could be an ideal plant for phytoremediation due to its high Pb tolerance. Here, we aimed to explore the molecular basis underlying the responses of this plant to Pb stress. Results In our study, ultrastructural localization assays revealed that Pb ions primarily accumulate in leaf vacuoles. RNA deep sequencing (RNA-Seq) of tartary buckwheat leaves was performed on two Pb-treated samples, named Pb1 (2000 mg/kg Pb (NO3)2) and Pb2 (10,000 mg/kg Pb (NO3)2), and a control (CK). A total of 88,977 assembled unigenes with 125,203,555 bases were obtained. In total, 2400 up-regulated and 3413 down-regulated differentially expressed genes (DEGs) were identified between CK and Pb1, and 2948 up-regulated DEGs and 3834 down-regulated DEGs were generated between CK and Pb2, respectively. Gene Ontology (GO) and pathway enrichment analyses showed that these DEGs were primarily associated with ‘cell wall’, ‘binding’, ‘transport’, and ‘lipid and energy’ metabolism. The results of quantitative real-time PCR (qRT-PCR) analyses of 15 randomly selected candidate DEGs and 6 regulated genes were consistent with the results of the transcriptome analysis. Heterologous expression assays in the yeast strain Δycf1 indicated that overexpressing CCCH-type zinc finger protein 14 (ZFP14) enhanced sensitivity to Pb2+, while 5 other genes, namely, metal transporter protein C2 (MTPC2), phytochelatin synthetase-like family protein (PCSL), vacuolar cation/proton exchanger 1a (VCE1a), natural resistance-associated macrophage protein 3 (Nramp3), and phytochelatin synthetase (PCS), enhanced the Pb tolerance of the mutant strain. Conclusion Combining our findings with those of previous studies, we generated a schematic model that shows the metabolic processes of tartary buckwheat under Pb stress. This study provides important data for further genomic analyses of the biological and molecular mechanisms of Pb tolerance and accumulation in tartary buckwheat.

A finite element model was established and modal analysis was performed on a wind turbine main shaft. By adjusting the length of the expansion sleeve, the effect of the length of the expansion sleeve on the natural frequency of the wind turbine shaft was summarized. The results show that the greater the length of the expansion sleeve, the greater the natural frequency of the wind turbine shaft.

With the vigorous development of wind power generation worldwide, the problem of stress concentration at the position of the main shaft shoulder of the wind turbine has become increasingly prominent. Therefore, a finite element model was established for the main shaft of a wind turbine, and the stress field of the main shaft of the wind turbine under the limit load condition was simulated and calculated, and the influence of the position of the shoulder on the flange side on the stress concentration under different radii of curvature was analyzed. It provides important guidance for the manufacture and inspection of the main shaft of the wind turbine.

Summary Objectives In the post-pandemic period 2010–2015, seasonal influenza A(H3N2) virus predominated in Hangzhou, southeast of China, with an increased activity and semi-annual seasons. This study utilized HA virus gene segment sequences to analyze the divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance in Hangzhou. Methods Virological and serological analyses of 124 representative A(H3N2) viruses from prospective studies of systematic surveillance samples were conducted to quantify the genetic and antigenic characteristics and their vaccine strain match. Results Bayesian phylogenetic inference showed that two separate subgroups 3C.3 and 3C.2 probably diverged from group 3C in early 2012 and then evolved into groups 3C.3a and 3C.2a, respectively, in the 2014/15 influenza season. Furthermore, high amino acid substitution rates of the HA1 subunit were found in A(H3N2) group 3C.2a variants, indicating that increased antigenic drift of A(H3N2) group 3C.2a virus is associated with a vaccine mismatch to the 2015/16 vaccine reference strain Switzerland/9715293/2013 (group 3C.3a). Conclusions A portion of the group 3C.2a isolates are not covered by the current A(H3N2) vaccine strain. These findings offer insights into the emergence of group 3C.2a variants with epidemic potential in the imminent influenza seasons.

Herein, we demonstrate a novel approach to detect intracellular adenosine triphosphate (ATP) from “fluorescence” to “enhanced fluorescence” by metal-enhanced fluorescence (MEF), which is an activatable means to amplify fluorescence signal. In the meantime, hybridization chain reaction (HCR) is employed to aggregate DNA on the substrate of MEF, and the length of generated DNA strands is controllable. ATP target molecules specifically bind to aptamer, which is designed as the recognition part of the nanoswitch, and the sticky ends are released. Then, HCR is triggered, bringing Cy7 modified on H1 and H2 close to the surface of gold nanobipyramids (AuNBPs) substrate. The fluorescence signal is amplified with the ATP concentration increasing owing to MEF. Thus, the strategy based on structure-switching aptamer triggering metal-enhanced fluorescence (SATMEF) for Cy7 is designed to detect ATP via “enhanced fluorescence” signals, and the maximum fluorescence enhancement by the approach of SATMEF reaches 5.63-fold. We applied this DNA nanoswitch for quantitative analysis to ATP, and the results show that the intensity of enhanced Cy7 fluorescence is positively correlated with the ATP concentration in the range of 0.2−10 μM, with a detection limit of 35 nM. We believe that the SATMEF for quantitative detection by signal from “fluorescence” to “enhanced fluorescence” provides a new approach for those fluorophores which are superior but difficult to be quenched to construct DNA nanoswitches.

To optimize the preparation of total saponins of Aralia taibaiensis phospholipid complex( TSAT-PC) by the central composite design-response surface method.Total saponins of Aralia taibaiensis phospholipid complex was prepared by using solvent evaporation method, five factors including reaction solvent, reaction time, reaction temperature, ratio of reactants on this reaction, and the concentration of the drug were investigated, then to optimize the preparation of TSAT-PC by the central composite design response surface method, and to study its physicochemical properties.The optimal process conditions were as follows, the reaction time was 1 h, the reaction temperature was 45 ℃,the ratio of soya lecithine ( SL) and TSAT was 3∶ 1, the reaction concentration was16 mg / m L, the complexing rate was 97. 23%,it was less than 5% with the predicted deviation; IR analysis proved the formation of TSAT-PC, and the solubility in the octyl alcohol was higher than the original drug.TSAT-PC was successfully developed by the optimized process, enhance the solubility in octyl alcohol, which provide the reference for the further development and utilization of Chinese materia medica preparation.

Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may affect diverse systems in animals and humans, including the cardiovascular system. However, little is known about the mechanism by which it affects the biological systems. Herein, we used embryonic stem cell test procedure as a tool to assess the developmental cardiotoxicity of PFOS. The differentially expressed proteins were identified by quantitative proteomics that combines the stable isotope labeling of amino acids with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Results of the embryonic stem cell test procedure suggested that PFOS was a weak embryotoxic chemical. Nevertheless, a few marker proteins related to cardiovascular development (Brachyury, GATA4, MEF2C, α-actinin) were significantly reduced by exposure to PFOS. In total, 176 differential proteins were identified by proteomics analysis, of which 67 were upregulated and 109 were downregulated. Gene ontology annotation classified these proteins into 13 groups by molecular functions, 12 groups by cellular locations and 10 groups by biological processes. Most proteins were mainly relevant to either catalytic activity (25.6%), nucleus localization (28.9%) or to cellular component organization (19.8%). Pathway analysis revealed that 32 signaling pathways were affected, particularly these involved in metabolism. Changes in five proteins, including L-threonine dehydrogenase, X-ray repair cross-complementing 5, superoxide dismutase 2, and DNA methyltransferase 3b and 3a were confirmed by Western blotting, suggesting the reliability of the technique. These results revealed potential new targets of PFOS on the developmental cardiovascular system.

Tartary buckwheat (Fagopyrum tataricum, TB) is an important functional food containing proteins with balanced amino acid composition and more flavonoids than common buckwheat (Fagopyrum esculentum, CB). Buckwheat contains highly potent allergens that trigger an allergic reaction via an IgE mediated response. In this work, the full-length cDNA encoding Fag t 2 from tartary buckwheat seeds was cloned by screening the cDNA library of seed-filling period. The recombinant Fag t 2 (rFag t 2) expressed in Pichia pastoris SMD1168H was purified by purified by immobilized metal affinity chromatography. It demonstrated that Fag t 2 was a major allergen in tartary buckwheat with the activity of IgE binding and pepsin resistance, along with the thermal stability. The identification of natural Fag t 2 (n Fag t 2) confirmed that the Fag t 2 protein belongs to the 2S albumin family, only existing in embryo. Most interesting, we discovered that Fag t 2 had a α-amylase inhibitor domain near the end of C-terminal. The possible activity of α-amylase inhibitor of Fag t 2 will be detected in subsequent studies.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase and functions through two distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2), with their key components Raptor and Rictor, to play crucial roles in cellular survival and growth. However, the roles of mTORC1 and mTORC2 in regulating cardiomyocyte differentiation from mouse embryonic stem (mES) cells are not clear. In this study, we performed Raptor or Rictor knockdown experiments to investigate the roles of mTORC1 and mTORC2 in cardiomyocyte differentiation. Ablation of Raptor markedly increased the number of cardiomyocytes derived from mES cells with well-organized myofilaments. Expression levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein), andalpha;-Actinin (cardiomyocyte marker) were increased in Raptor knockdown cells. In contrast, loss of Rictor prevented cardiomyocyte differentiation. The dual ablation of Raptor and Rictor also decreased the number of cardiomyocytes. The two complexes exerted a regulatory mechanism in such a manner that knockdown of Raptor/mTORC1 resulted in a decreased phosphorylation of Rictor (Thr1135), which subsequently activated Rictor/mTORC2 in the differentiation of mES cells into cardiomyocytes. In conclusion, mTORC1 and mTORC2 played different roles in cardiomyocyte differentiation from mES cells in vitro. The activation of Rictor/mTORC2 was critical for facilitating cardiomyocyte differentiation from mES cells. Thus, this complex may be a promising target for regulating myocardial differentiation from embryonic stem cells or induced pluripotent stem cells.

We show that octylamine-modified poly-(acrylic acid) is adopted to wrap hydrophobic upconversion nanoparticles (UCNPs), and the UCNPs are further biofunctionalized and applied to integrate luminescence imaging with optical tweezers for detecting microRNA-21 sequences.

Comprehensive and quantitative proteomic information on citrus floral bud is significant for understanding male sterility of the cybrid pummelo (G1+HBP) with nuclear genome of HBP and foreign mitochondrial genome of G1. Scanning electron microscopy and transmission electron microscopy analyses of the anthers showed that the development of pollen wall in G1+HBP was severely defective with a lack of exine and sporopollenin formation. Proteomic analysis was used to identify the differentially expressed proteins between male sterile G1+HBP and fertile type (HBP) with the aim to clarify their potential roles in anther development and male sterility. On the basis of iTRAQ quantitative proteomics, we identified 2235 high-confidence protein groups, 666 of which showed differentially expressed profiles in one or more stages. Proteins up- or down-regulated in G1+HBP were mainly involved in carbohydrate and energy metabolism (e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, ATP synthase, and malate dehydrogenase), nucleotide binding (RNA-binding proteins), protein synthesis and degradation (e.g., ribosome proteins and proteasome subunits). Additionally, the proteins located in mitochondria also showed changed expression patterns. These findings provide a valuable inventory of proteins involved in floral bud development and contribute to elucidate the mechanism of cytoplasmic male sterility in the cybrid pummelo.

One pair of primers was designed according to Gallus and Meleagris gallopavo interferon β (IFN-β) sequences published in GenBank. The primers and RNA extraction from the spleen of Coturnix were used to amplify Coturnix IFN-β cDNA by real-time polymerase chain reaction (RT-PCR). The product was cloned into pEasy-T1 vector. Evaluating recombinant plasmid by PCR and restriction enzyme digestion. Sequence the cloning sequences, comparing the sequencing results by NCBI. We successfully got a Coturnix IFN-β partial sequence. The sequence was subtyped and put to homologous analysis. The results suggested the homology of IFN-β gene of Coturnix and gene of Coturnix and chicken (88.7%), the homology of IFN-β gene of Coturnix and chicken (88.7%), the homology of IFN-β gene of Coturnix and Anas platyrhynchos (72.5%), the homology of IFN-β sequence registered in GenBank. The analysis of the genetic tree showed that the relationship of Coturnix and chicken IFN-β had a high homology. It can be seen that in this study we successfully got a partial sequence of IFN-β of quail.

Objectives We investigated the chemo-sensitization of a ligustrazine derivate, (E)-2-(2, 4-dimethoxystyryl)-3, 5, 6-trimethylpyrazine (DLJ14) on Adriamycin (Adr, Wanle, Shenzhen, China)-resistant human breast cancer (MCF-7/A) cells both in vivo and in vitro. Methods The antitumour effects of DLJ14 and Adr was observed in MCF-7/A cells by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in vitro and was evaluated by MCF-7/A xenografts in nude mice. The intracellular Adr accumulation was assessed by mean fluorescence intensity of Adr. The messenger RNA level of glutathione (GSH) S-transferase (GST)π in MCF-7/A cells was determined by real-time reverse transcription PCR assay. The expression of GSTπ, c-jun NH2-terminal kinase (JNK) and phosphor-JNK (p-JNK) was detected by Western blotting method. Key findings The MTT results showed that DLJ14 exhibited a weak inhibition on proliferation of both MCF-7 and MCF-7/A cells, in contrast with the strong inhibition of verapamil. When DLJ14 is combined with Adr, the inhibitory effect on MCF-7/A cells and MCF-7/A xenografts was enhanced significantly through increasing intracellular accumulation of Adr by inhibition of GSH level and the activity of GSH peroxidase and GST. Moreover, DLJ14 could downregulate the expression of GSTπ and increase the expression of JNK and p-JNK in MCF-7/A cells or in xenografts. Conclusion DLJ14 is a promising chemo-sensitization candidate for the reversal of multidrug resistance in cancers.

Access control through restricting users to access to resources, prevents invasion of illegal users and legal users access to unauthorized resources to ensure integrity and privacy of workflow data, which is the key of workflow security mechanism. According to the identity authentication and access authority control, the paper analyzes the safety management entrusted mechanism of Acegi which is Spring security framework, makes full use of its characteristics of depended injection (DI), and realizes statement and configuration of all kinds of safety components such as authentication management and access decision management.

To detect the pollution level of insulators in operation, based on light energy loss theory of medium optical wave guide and mathematical modeling of least squares supporting vector machine, by a lot of contrast tests, this paper designed and developed monitoring terminal of optical sensor. Collecting live temperature, humidity luminous flux attenuation etc. and sending the collected data to data monitoring centre through GSM data communication module, the workstation of data monitoring centre could calculate to get equivalent salt deposit density (ESDD) using mathematical model, so that insulators pollution level can be detected on-line automatically and continuously.

Fluopsin C, an antibiotic isolated from Pseudomonas jinanesis, has shown antitumor effects on several cancer cell lines. In the current study, the oncotic cell death induced by fluopsin C was investigated in human breast adenocarcinoma cells in vitro. Human breast adenocarcinoma cell lines MCF-7 and MD-MBA-231 were used. The cytotoxicity was evaluated using MTT assay. Time-lapse microscopy and transmission electron microscopy were used to observe the morphological changes. Cell membrane integrity was assessed with propidium iodide (PI) uptake and lactate dehydrogenase (LDH) assay. Flow cytometry was used to measure reactive oxygen species (ROS) level and mitochondrial membrane potential (Δψm). A multimode microplate reader was used to analyze the intracellular ATP level. The changes in cytoskeletal system were investigated with Western blotting and immunostaining. Fluopsin C (0.5-8 μmol/L) reduced the cell viability in dose- and time-dependent manners. Its IC50 values in MCF-7 and MD-MBA-231 cells at 24 h were 0.9 and 1.03 μmol/L, respectively. Fluopsin C (2 μmol/L) induced oncosis in both the breast adenocarcinoma cells characterized by membrane blebbing and swelling, which was blocked by pretreatment with the pan-caspase inhibitor Z-VAD-fmk. In MCF-7 cells, fluopsin C caused PI uptake into the cells, significantly increased LDH release, induced cytoskeletal system degradation and ROS accumulation, decreased the intracellular ATP level and Δψm. Noticeably, fluopsin C exerted comparable cytotoxicity against the normal human hepatocytes (HL7702) and human mammary epithelial cells with the IC50 values at 24 h of 2.7 and 2.4 μmol/L, respectively. Oncotic cell death was involved in the anticancer effects of fluopsin C on human breast adenocarcinoma cells in vitro. The hepatoxicity of fluopsin C should not be ignored.

Telekin, a eudesmane-type sesquiterpene lactone compound isolated from Chinese folk medicine Carpesium divaricatum, has been reported to strongly inhibit the proliferation of cancer cells. In this study, the involvement of a mitochondria-mediated pathway in the pro-apoptotic action of telekin was investigated in human hepatocellular carcinoma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that telekin exhibited excellent anti-proliferation activity in hepatocellular carcinoma cells and low cytotoxicity to normal hepatocyte cells. Telekin-induced apoptosis was characterized by chromatin condensation, formation of apoptotic bodies, and exposure of phosphatidylserine on the extracellular surface, as revealed by 4,6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry. Flow cytometry analysis showed that telekin induced the loss of mitochondrial membrane potential (MMP), as well as increased the levels of intracellular free calcium and reactive oxygen species (ROS). Additionally, Western blot results demonstrated that telekin induced the decrease in Apaf-1 and Bcl-2 expression, increase in Bax expression, release of cytochrome C, and activation of caspase-9 and caspase-3 in HepG-2 cells. These findings indicate that telekin activates the mitochondria-mediated apoptotic pathway in hepatocellular carcinoma cells and may merit further investigation as a potential therapeutic agent for the treatment of hepatocellular carcinoma.

This paper proposes to establish a CNC machine tool security system using artificial intelligence-based, and discusses the key technologies: the dynamic fuzzy neural network(DFNN), and based on this, describes an established security system of CNC machine tool.

A two-chambered MFC packed with activated carbon and used acrylic fiber wastewater as carbon source was constructed to study the electrical and anolyte change characteristics of the cell, the influence of activated carbon packing materials on electricity generation performance was analysed. The results indicated that microbial fuel cell could be upstart successfully when anaerobic sludge taken from wastewater treatment plants of acrylic fiber companies was used as inoculation; the maximum power density output of MFC was 211W/m3, and the internal resistance was 1311; N-contained aromatic compounds in anolyte was absorbed by activated carbon which made the cell could stably operated for a long time without renewing anolyte; anaerobic metabolism process was occurred in anode chamber, and during which N-contained aromatic compounds was degraded to amide, carboxylic acid, ammonia, etc.

To investigate the effects of 1-oxoeudesm-11(13)eno-12,8a-lactone (OEL), a novel eudesmane-type sesquiterpene isolated from Aster himalaicus, on the cell cycle and apoptosis in human glioblastoma cells in vitro.Human malignant glioblastoma cell lines U87 and A172 were used. The cytotoxicity of OEL was examined using the MTT assay. Cell apoptosis was assessed with DAPI staining and flow cytometry. DNA damage was determined by measuring the phosphorylation of H2AX using immunofluorescence staining and Western blotting. Cell cycle profiles were measured with flow cytometry. The mRNA expression of p53 and p21Waf1/Cip1 was investigated using real-time PCR. The protein expression of γ-H2AX, caspase-9, caspase-3, p53, p21Waf1/Cip1, cyclin B1, and cdc2 was analyzed with Western blotting.Treatment of the malignant glioblastoma cells with OEL inhibited the cell growth in dose- and time-dependent manners (the values of IC(50) at 48 and 72 h were 29.5 and 16.99 μmol/L, respectively, in U87 cells; 7.2 and 9.5 μmol/L, respectively, in A172 cells). OEL (10-30 μmol/L) induced apoptosis and G(2)/M phase arrest in both U87 and A172 cells. OEL induced the phosphorylation of cdc2, a G(2)/M phase cyclin-dependent kinase, and decreased the expression of cyclin B1 required for progression through the G(2)/M phase in U87 cells. The compound remarkably increased the phosphorylation of H2AX in U87 cells. Moreover, OEL increased the mRNA and protein levels of p53 and its target gene p21(Waf1/Cip1) in U87 cells. The compound also induced p53 phosphorylation. Pretreatment with PFT-α, a specific inhibitor of p53 transcriptional activity, could partially reverse the inhibition of OEL on the viability of U87 and A172 cells.OEL suppresses the growth of human glioblastoma cells in vitro via inducing DNA damage, p53-mediated cell cycle arrest and apoptosis, thus warrants further studies as a lead compound of anti-glioblastoma drug.

The stability of the karst area transmission towers pile foundation and engineering deal with the problem, the success or failure related to the electric power system operation, the karst area transmission towers pile foundation stability of the subject, the power sector is currently no in-depth case studies, qualitative analysis has been used method and semi-quantitative analysis of the reliability of the method of evaluation results is restricted, and thus how to seek a more rational analysis method and its application in theory worthy of further exploration. In this paper, numerical simulation conducted in-depth study and exploration of a karst area transmission tower pile stability evaluation, design, construction and operation of the project has a certain reference value, has certain reference significance for similar construction projects.

The components for connecting high-performance liquid chromatography (HPLC) with Fourier-transform infrared spectroscopy (FTIR) were investigated to determine estrogen in the water environment, including heating for atomization, solvent removal, sample deposition, drive control, spectrum collection, chip swap, cleaning and drying. Results showed that when the atomization temperature was increased to 388 K, the interference of mobile phase components (methanol, H2O, acetonitrile, and NaH2PO4) were completely removed in the IR measurement of estrogen, with 0.999 of similarity between IR spectra obtained after separation and corresponding to the standard IR spectra. In experiments with varying HPLC injection volumes, high similarity for IR spectra was obtained at 20 ul injection volume at 0.01 mg/L BPA while a useful IR spectrum for 10 ng/L BPA was obtained at 80 ul injection volume. In addition, estrogen concentrations in the natural water samples were calculated semi-quantitatively from the peak intensities of IR spectrum in the mid-infrared region.

Background G1 + HBP is a male sterile cybrid line with nuclear genome from Hirado Buntan pummelo (C. grandis Osbeck) (HBP) and mitochondrial genome from “Guoqing No.1” (G1, Satsuma mandarin), which provides a good opportunity to study male sterility and nuclear-cytoplasmic cross talk in citrus. High-throughput sRNA and degradome sequencing were applied to identify miRNAs and their targets in G1 + HBP and its fertile type HBP during reproductive development. Results A total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2) and transport inhibitor response 1 (TIR1). Eight target genes were confirmed to be sliced by corresponding miRNAs using 5’ RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between sterile line G1 + HBP and fertile line HBP were identified. Differential expression of miRNAs and their target genes between two lines was validated by quantitative RT-PCR, and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulatory mechanism of miR167a was investigated by yeast one-hybrid and dual-luciferase assays that one dehydrate responsive element binding (DREB) transcription factor binds to miR167a promoter and transcriptionally repress miR167 expression. Conclusion Our study reveals the altered expression of miRNAs and their target genes in a male sterile line of pummelo and highlights that miRNA regulatory network may be involved in floral bud development and cytoplasmic male sterility in citrus. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2882-0) contains supplementary material, which is available to authorized users.

The release and transport of heavy metals (Ni, Cr, Cu and Pb) from Liangshui River sediments into the overlying water column during the resuspension event were determined using an annular flume with a velocity ranging from 0.15 to 0.35 m/s. It is shown that the suspended particulate matters (SPM) increased as much as nearly 25 times from 165 to 4220 mg/L as the velocity increased. Heavy metals showed an increase in dissolved phase as the velocity increased due to their desorption from the SPM. Acid-soluble heavy metals increased as the velocity increased, indicating that part of the heavy metals transformed from stable phase to labile phase during resuspension. Heavy metal concentrations in the SPM on volume normalization increased by approximately 2–6 times. However, on the mass weighted basis they decreased, approaching the bulk-sediment contents at high velocity, due to the “particle concentration effect”. The distribution coefficients (KD) of heavy metals were higher at slower velocity during the sediment resuspension, which could be attributed to the decrease of fine particles (silt/clay fraction) during resuspension.

Recycling high speed counter-current chromatography (HSCCC) was successfully applied to resolution of (R, S)-naproxen (NAP) using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phase solvent system composed of n-hexane–ethyl acetate–0.1 mol L−1 phosphate buffer solution with pH = 2.67 (8:2:10, v/v/v) was selected. Influence factors for the chiral separation process were investigated, including concentration of HP-β-CD, equilibrium temperature and pH of aqueous phase. Suitable elution mode was selected for HSCCC enantioseparation of (R, S)-NAP. Under optimum separation conditions, 29 mg of (R, S)-NAP was separated using preparative recycling HSCCC with the molar ratio HP-β-CD/NAP racemate 83:1. Technical details for recycling elution mode were discussed as for chiral HSCCC separation. The purities of both (S)-NAP and (R)-NAP were over 99.5% as determined by HPLC. Enantiomeric excess of (S)-NAP and (R)-NAP reached 99.4%. Recovery for NAP enantiomers from HSCCC fractions was 82–89%, yielding 13 mg of (S)-NAP and 12 mg of (R)-NAP.

By using the existing research experience and through the in-depth study of the harmonious society theory, this paper sets up a harmonious society index system. Moreover, based on this result and by making use of the method of AHP, this paper carries positive analysis on the construction of harmonious society of Beijing City.

Intergranular corrosion in heat-affected zone of low carbon 12Cr-Ni ferritic stainless steel was investigated by the method of practice Z in ASTM A 763-93. The microstructure of welded joint was characterized using an optical microscope and a scanning electronic microscope. Severe intergranular corrosion (IGC) was only observed in low temperature heat-affected zone (LTHAZ) adjacent to base metals at the back of the welded joints in unstabilized steel. On the other hand, IGC was not observed in the welded joints of stabilized steels. According to the analysis of microstructure, the severe IGC is attributed to both the precipitated phase along the grain boundaries inducing the presence of chromium-depleted zone and the welding residual tensile stress promoting the corrosion. Therefore, stabilized elements addition such as Ti and Nb is required in order to avoid the IGC in welded joint of these steels. Relief residual tensile stress by post weld heat treatment would also be an effective method to avoid the IGC in the welded joints of these steels.

ADP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis in plants, is composed of two small and two large subunits, and has plastidial and cytosolic isoforms. In kernels of wheat ( Triticum aestivum L . ) and barley ( Hordeum vulgare L.), transcripts for cytosolic ( Ta.AGP.S1a ) and plastidial ( Ta.AGP.S1b ) small subunits of AGPase were encoded by the same gene ( Ta.AGP.S.1 ) by use of the alternative first exons. In this study, a cDNA sequence (1631 bp) [NCBI: EU586278] encoding a novel Ta.AGP.S1b transcript was isolated in kernels of Chinese common wheat cultivars. Compared with another Ta.AGP.S1b transcript [NCBI: FJ643609] isolated in kernels of non-Chinese wheat cultivars, EU586278 lacked a long fragment (117 bp) at its 5′terminal, resulting in a shorten transit peptide. The lacked fragments of Ta.AGP.S1b (EU586278) were universally found in surveyed 22 Chinese common wheat cultivars. Partial genomic DNA sequence [NCBI: FJ907395 ] of Ta.AGP.S.1 gene, which was corresponded to 5′terminal of EU586278 transcript, was also isolated in Chinese common cultivars and sequencing indicated that FJ907395 contained the corresponding lacked fragment of EU586278 transcript, inferring the lacked fragment in EU586278 transcript was not present in the genome, but possibly occurred at transcription level. Using TargetP software, the predicated transit peptide of putative plastidial SSU encoded by EU586278 contained merely 25 amino acids, considerably shorter than those of other plant AGP. S.1bs (54–70 amino acids). Phylogenetic tree analysis indicated that the amino acid sequence of EU586278 transit peptide was not clustered together with those of other wheat Ta.AGP.S1bs [NCBI: AF536819 and FJ643609] and barley AGP.S1b [NCBI: Z48563]. These implied that EU586278 could be a novel Ta.AGP.S1b transcript. Semi-quantitative PCR analysis indicated that transcripts of EU586278 were abundantly expressed in leaf, moderately in endosperm and stem, and weakly in root.

Efficient and divergent synthesis of 2-substituted quinolines and isoxazolo[4,3-c]quinolines, was achieved from N-formyl-1,2-dihydroquinolines with the aid of sodium azide. The synthesis is substituent-dependent. Thus N-formyl-1,2-dihydroquinolines with a hydrogen atom at the 3-position afforded 2-substituted quinolines in good to excellent yields, while with a 3-formyl group, N-formyl-1,2-dihydroquinolines unexpectedly gave isoxazolo[4,3-c]quinolines in high yields.

The rates of starch accumulation, the activities of ADP-glucose pyrophosphorylase (AGPase, the key enzyme of starch synthesis), and the transcript levels of its four subunits were measured and compared between two large-spike winter wheat cultivars (Yujiao 2 and Lankao Aizao 8) during the periods of grain filling. During the first 10 days of grain filling periods, there were no differences in the rates of starch accumulation and activities of AGPase between the two wheat cultivars. In subsequent filling periods, however, the rates of starch accumulation and activities of AGPase in grains of cv. Yujiao 2 were significantly higher than those in cv. Lankao Aizao 8. cDNA sequences for four subunits of AGPase (cytosolic small subunit, SSU I; plastidial small subunit, SSU II; cytosolic large subunit, LSU I and plastidial large subunit, LSU II) were amplified, and their transcript levels during the whole grain filling were measured using semi-quantitative RT–PCR. No difference in transcript levels of SSU I, SSU II and LSU II was found between the two wheat cultivars during the whole grain filling period. However, the transcript levels of LSU I showed an obvious difference since 15 days of anthesis between two wheat cultivars, and LSU I transcripts were higher in cv. Yujiao 2 than those in cv. Lankao Aizao 8. This was clearly consistent with those in the starch accumulation rates and AGPase activities, implying that LSU I transcript levels could be related to the differences in the activities of AGPase and rates of starch accumulation.

A combined fixed-film system composed of anaerobic biofilter, aerobic biofilter, and intermittent aerated biofilter (IABF) is developed for enhanced biological phosphorus removal (EBPR) treating domestic wastewater. This work presents details on the performance of IABF under aerobic condition, where phosphorus-accumulating organisms are accumulated. Analysis on distribution of phosphorus in both the bulk and the biofilm indicates that the PAOs-rich biofilm is characterized by a high activity, a strong P capacity, and a good adaptability of fluctuations in aerobic continuous loading. An innovative means for P removal slows down accumulation of P in biofilm. As a result, removal of P-rich biomass is no longer a key limitation of EBPR performance in the biofilm system. Long backwashing interval is practicable in IABF under aerobic continuous feeding regime.

The aim of the study was to develop a multiplex PCR-based DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA microarrays. The detection limits were 10(1) copies/microl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses.

Farfugium japonicum (L.) KITAM, named "Lian-Peng-Cao" in China, has been traditionally used in Chinese folk medicine to treat sore throat, cold and cough due to its anti-inflammatory properties. In this study, the anti-inflammatory action of 3β-angeloyloxy-8β,10β-dihydroxyeremophila-7(11)-en-12,8α-lactone (FJ1) isolated from Farfugium japonicum and its molecular mechanism in RAW264.7 cells were investigated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that FJ1 with or without 3 µg/mL lipopolysaccharide (LPS) had no significant cytotoxicity in RAW264.7 cells. The production of nitric oxide (NO) was identified with a Griess reagent kit. The mRNA expression of inducible nitric oxide synthase (iNOS) and cytokines, including tumor necrosis factor α (TNF-α) and cyclooxygenase-2 (COX-2), was measured by real-time polymerase chain reaction (PCR). Reactive oxygen species (ROS) production was detected by flow cytometry analysis. Western blot was used to examine the protein expression of nuclear factor-kappa B (NF-κB)/p65, inhibitor of kappa B (IκB)-α, phosphorylated IκB-α (p-IκB-α), mitogen-activated protein kinase (MAPK) molecules, iNOS, and TNF-α. We discovered that FJ1 possesses anti-inflammatory effects that inhibit the release of LPS-stimulated pro-inflammatory cytokines, including NO and ROS. The molecular mechanism of FJ1-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules, including extracellular signal-related kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), FJ1 also reverses IκB degradation and attenuates the mRNA and protein expression of NF-κB-related downstream inducible enzymes and cytokines, such as iNOS, TNF-α in RAW264.7 cells. The results suggest that FJ1 has anti-inflammatory properties, which indicates that F. japonicum can be utilized to treat inflammatory diseases. The potential mechanism is associated with the NF-κB and MAPK activation pathways in LPS-stimulated macrophages.