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The need for high-energy batteries has driven the development of binder-free electrode architectures. However, the weak bonding between the electrode particles and the current collector cannot withstand the severe volume change of active materials upon battery cycling, which largely limit the large-scale application of such electrodes. Using tin nanoarrays electrochemically deposited on copper substrate as an example, here we demonstrate a strategy of strengthening the connection between electrode and current collector by thermally alloying tin and copper at their interface. The locally formed tin-copper alloys are electron-conductive and meanwhile electrochemically inactive, working as an ideal “glue” robustly bridging tin and copper to survive harsh cycling conditions in sodium ion batteries. The working mechanism of the alloy “glue” is further characterized through a combination of electrochemical impedance spectroscopy, atomic structural analysis and in situ X-ray diffraction, presenting itself as a promising strategy for engineering binder-free electrode with endurable performance., The authors here report a binder-free electrode based on tin nanoarrays deposited on copper substrate. It is found that the locally formed electrochemically inactive tin-copper alloys work as a glue that bridges tin and copper to survive harsh cycling conditions in sodium ion batteries.

In this paper, combined with the actual engineering, a modular transportation status monitoring system is designed, which can be used to measure environmental conditions such as vibration, inclination, temperature and humidity during the process of transportation. The system consists of a main control module, a storage module, a data acquisition module, a real-time clock module, a power management module and an alarm module. The design scheme has high development efficiency and strong practicability. The measurement device used in the experiment for environmental monitoring during transportation process performs well, and can be a typical application case of innovative practice teaching

There are five elements in group IVA of the periodic table, i.e., carbon (C), silicon (Si), germanium (Ge), tin (Sn) and lead (Pb), of which Si, Ge, and Sn can be used as alloying-type electrode materials for sodium-ion batteries. Pb is also capable of alloying with sodium, but it is generally ruled out as the cause of toxicity. In recent years, materials based on Si, Ge, and Sn have been intensively exploited as sodium anodes because of their abundant resource and large capacity with reasonable working voltages. However, successful deployment of these anode materials needs to overcome kinetic and thermodynamic issues related to poor electrochemical activity, particle pulverization associated with large volume swelling, and formation of unstable solid-electrolyte interphase. A diversity of material strategies has been employed to address these difficulties, mainly leveraging on the knowledge recently advanced for lithium anodes. This review highlights such issues and provides valuable insights for possible solutions, which serves as a guide and inspiration for future material innovation for rechargeable batteries.

According to the characteristics of high-dimensional imbalance distribution of motor bearing fault data, a design scheme of classification model is proposed for the high-dimensional data reduction problem in the classification algorithm. For details: Combining standard particle swarm optimization algorithm and random forest algorithm, a new high-dimensional data reduction algorithm is proposed. Aiming at the imbalance problem of data categories in the classification algorithm, we proposes to use machine learning under the sum of squares of dynamic deviations criterion to divide the minority sample data set into mixed regions, high-purity minority sample regions and outlier regions, and then use smote algorithm to complete the data equalization processing, so as to make the sample data equalization processing more reasonable, Focusing on the task of motor bearing fault classification, a design scheme of using standard particle swarm optimization algorithm to improve the least squares support vector machine model is proposed.

Background Platelet activation and subsequent accumulation at sites of vascular injury perform a central role in thrombus formation, which is believed to be the trigger of several cardiovascular diseases, such as atherosclerosis, myocardial infarction and strokes. In this sense, the search for agents that are capable of blocking platelets aggregation has important implications for these diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicine herb for eliminating stasis to subdue swelling and hemostasis. Our previous study found several compounds extracted from this herb, including 1, 6-di-O-caffeoyl-β-D-glucopyranoside (CGP), showed inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation. Purpose The aim of current study is confirmation of the anti-platelet effects and elucidation of the probable mechanisms. Methods The experiments were performed on platelet rich plasma freshly isolated from SD rat. ADP, U46619 or arachidonic acid (AA) induced platelet aggregation assay were performed to evaluate the anti-platelet properties of CGP. Activated αIIbβ3 integrin abundance, serotonin (5-HT) secretion, thromboxane A2 (TXA2) synthesis was determined to assess the effects of CGP on platelet activation. Furthermore, RhoA and PI3K/Akt/GSK3β signal transduction were analyzed by Western Blotting assay. In addition, radiolabelled ligand binding assay was involved to evaluate the ability of CGP binding to thromboxane prostanoid (TP) and P2Y12 receptors. Results CGP inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid (AA), significantly. Furthermore, it is also found that LGP exhibited obvious inhibitory effects on αIIbβ3 integrin activation, serotonin (5-HT) secretion from granule and thromboxane A2 (TXA2) synthesis. Next, we found that CGP suppressed RhoA and PI3K/Akt/GSK3β signal transduction. Data from radiolabelled ligand binding assay showed that CGP displayed apparent competing effects on TP and P2Y12 receptors. Conclusion Collectively, the data presented here demonstrated that CGP, a natural compound from Callicarpa nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual-receptor inhibition on P2Y12 and TP receptors.

Aiming at the problem of fitting error in accelerated degradation data processing, a model optimization method was proposed. Firstly, the best fitting model was selected according to the minimum Residual Sum of Squares, and pseudo-failure lifetime was calculated for each degradation data group. Secondly, the optimization method of the lifetime distribution model was proposed and the best lifetime distribution was obtained according to the minimum fitting error. Finally, a case example was given, which indicated that the deviation may occurred without the model optimization.

In order to meet the requirements of alignment capability, gap tolerance and welding track control, dual-beam laser welding has been put forward to weld tailored blanks with different thicknesses. The arrangement mode and power ratio of dual-beam laser determine the heat transfer and temperature field distribution, and then influence the weld profile and weld quality. In this study, based on consideration of the temperature dependence of material physical properties, convection and radiation heat transfer, and material latent heat, the finite element model was developed. The model validated was conducted to compare with experimental results and it showed good agreement. The arrangement mode and power ratio of dual-beam laser welding were compared and optimized based on the consideration of the weld width on top surface and gap tolerance. Finally, the temperature field characteristics with the preferable process were analyzed in detail according to the numerical simulation. The calculated results indicated the weld width is mainly controlled by the laser irradiation on the thin plate, and dual-beam laser welding of tailored blanks has great advantages on improving heat transfer, weld profile and gap tolerance, which helps to reduce welding defects and enhance welding quality.

Undertaking varying working conditions is common for a machine tool. One single reliability model for determining a single effect is not suitable for the reliability evaluation of machine tools due to the multi-effect of working conditions on the reliability of machine tools. This paper proposes a generalized model using hazard functions and Weibull distribution to assess the reliability of machine tools under different effects of working conditions. This model considers three effects, including the proportional hazard, time, and additive hazard effects. The distribution of time effect is discussed. Furthermore, a secure parameter estimation method is presented to calculate the proposed reliability model. A case study based on field data is presented. The proposed model is compared with classical reliability models. The result shows that the proposed model has better goodness of fit for the realistic cumulative distribution of machine tools.

An on-line and continuous approach was demonstrated for in vivo measurement of bisulfide in rat's brain. A modified droplet-based microfluidic system was constructed, which allowed on-line qualification of the fluorescence responses of the gold nanoparticle-glutathione-fluorescein isothiocyanate probe to the variation of bisulfide in the presence of the cerebral microdialysate background. The on-line method achieved a dynamic working range from 5.0 μM to 40 μM and a detection limit of 2.5 μM. The in vivo bisulfide concentration in the hippocampus of rat's brain was measured under different physiological conditions. The on-line method may facilitate the study of H2S biology by providing a previously unattainable continuous record of H2S variation in living animals. It also provides a practical platform for in vivo and continuous monitoring of other important species in cerebral systems.

Under an electric field, the complexes formed by DNA and polylysine exhibit novel features, such as selective merging of particles, ejecting of daughter vehicles, and differentiation of particles of varying mobility. The mobility of the complex could be three times faster than that of free DNA.

Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca

To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa. QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes. Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

Platelet activation and subsequent accumulation at sites of vascular injury perform a central role in thrombus formation, which is believed to be the trigger of several cardiovascular diseases, such as atherosclerosis, myocardial infarction and strokes. In this sense, the search for agents that are capable of blocking platelets aggregation has important implications for these diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicine herb for eliminating stasis to subdue swelling and hemostasis. Our previous study found several compounds extracted from this herb, including 1, 6-di-O-caffeoyl-β-D-glucopyranoside (CGP), showed inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation.The aim of current study is confirmation of the anti-platelet effects and elucidation of the probable mechanisms.The experiments were performed on platelet rich plasma freshly isolated from SD rat. ADP, U46619 or arachidonic acid (AA) induced platelet aggregation assay were performed to evaluate the anti-platelet properties of CGP. Activated αCGP inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid (AA), significantly. Furthermore, it is also found that LGP exhibited obvious inhibitory effects on αCollectively, the data presented here demonstrated that CGP, a natural compound from Callicarpa nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual-receptor inhibition on P2Y

Elevating cytosolic Ca2+stimulates glucose uptake in skeletal muscle, but how Ca2+affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca2+leading to contraction preclude analysis of the impact of individual, Ca2+-derived signals. In L6 muscle cells stably expressing GLUT4 myc, the Ca2+ionophore ionomycin raised cytosolic Ca2+and caused a gain in cell surface GLUT4 myc. Extra- and intracellular Ca2+chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4 myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4 myc. Ionomycin stimulated GLUT4 myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4 myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4 myc endocytosis triggered by ionomycin. In summary, rapid Ca2+influx into muscle cells accelerates GLUT4 myc exocytosis while slowing GLUT4 myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4 myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca2+-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.

In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes.

Real-time fluorescence imaging of the activity of nucleases in living cells has been a difficult issue because of unintended degradation of the natural oligonucleotides by nontarget nucleases or interactions with other proteins. In this work, we demonstrate two types of highly selective, sensitive, and robust oligonucleotide probes for simultaneous imaging of the activities of two different nucleases in living cells. The probes consist of the desired substrate structure of the target nuclease and partially phosphorothioate modified backbone labeled with fluorophore and quencher for protection from undesired degradation by other nucleases and signal transduction. Upon reaction with the target nuclease, the initially fluorescence quenched probe was cleaved and the fluorophore was separated from the quencher, giving out strong fluorescence signals. Two nucleases, DNase I and Exonuclease III, were employed as model enzymes to demonstrate the concept. In vitro studies proved that the two probes could discriminate their respective target nucleases in serum with high resistance to other coexisting enzymes. The lower limits of detection for DNase I and Exonuclease III were observed to be 40 U/L and 2.0 U/L, respectively. By labeling the two probes with different fluorophores and quenchers, simultaneous visualization of the activities of DNases and 3' exonucleases was achieved in both HeLa cells and the suspension cells of Arabidopsis thaliana. The developed approaches may greatly facilitate the studies on the intracellular functions of the two nucleases and other related biological processes. The probe design concept may also be further adapted to the detection of many other nucleases.

Surface imprinted superparamagnetic nanoparticles (Fe3O4@MIP) for bisphenol A (BPA) have been prepared by a rapid and simple UV irradiation initiated suspension polymerization method. The obtained nanoparticles show high binding affinity for BPA and enable rapid and efficient extraction of BPA from water samples. By combination with liquid chromatography–mass spectrometry, the method allows sensitive and reliable quantification of BPA in tap water with a detection limit as low as 0.1 ng/L for a sample volume of 100 mL. The polymerization method may be further extended to the preparation of surface imprinted magnetic nanoparticles for other analytes of interest.

Simultaneous determination of indole-3-acetic acid and methyl indole-3-acetic acid ester in small amounts of plant tissue is essential for elucidating their mutual transformation mechanism and the in vivo function of methyl indole-3-acetic acid ester. Rapid quantification of flavonoids in the same sample is important for clarifying their roles in the transport of auxins and other phytohormones. Herein, we describe a simple method for the simultaneous determination of indole-3-acetic acid and its methyl ester in the roots of the Arabidopsis thaliana seedlings and a protocol for the rapid extraction and quantification of quercetin and kaempferol in these seedlings. High-performance liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry was used for the detection of all the compounds. Negative data for indole-3-acetic acid and positive data for methyl indole-3-acetic acid ester were collected in two successive files with a single injection of the extracted sample. Under optimized conditions, the limit of detection for the four compounds was 2 ng/mL for indole-3-acetic acid, 0.5 ng/mL for methyl indole-3-acetic acid ester, 5 ng/mL for quercetin, and 1 ng/mL for kaempferol, respectively. Because of the high sensitivity of the assay, only 2-10 mg of the plant material was required to obtain quantitative results.

Quick & easy: A simple and positive-readout fluorescent immunosensor was developed based on a quencher composed of a 3 nm gold nanoparticle (GNP) covalently linked to a Fab fragment, which proved to be stable and specific. The method allows for direct and sensitive detection of target antigens in homogeneous solutions in one step without any separation steps. It has been successfully applied to rapidly quantify the amount of secretory immunoglobulin A (sIgA) in human saliva samples.

In this work, we developed a novel class-specific immunoaffinity column for the natural isoprenoid cytokinins (CTKs) by using trans-zeatin riboside as the hapten to generate a complete antigen. By combination with a mixed-mode solid phase extraction step for pre-cleanup and a high performance liquid chromatography-quadrupole-time of flight mass spectrometry for the quantification, an efficient analytical protocol was established which allowed simultaneous quantification of eight endogenous isoprenoid CTKs in Arabidopsis thaliana leaves with a wide linear range from 25 to 500pg/g fresh weight and a detection limit of 12.5pg/g fresh weight. The method will be very useful for comprehensive research on the networks of signaling interactions of the active phytohormones and their regulation of the plant functions.

The sharp increase in the cost of lithium resource has driven the research on sodium-ion batteries (SIBs) as sodium shares a similar electrochemical property as lithium. Carbonaceous materials are important anodes for rechargeable batteries, but the prevailing graphite only shows a limited activity towards sodium storage. Herein, we demonstrate that carbon nanoflakes serve as an efficient anode material for SIBs, exhibiting a stable capacity of 148[Formula: see text]mAh[Formula: see text]g[Formula: see text] over 600 continuous cycles at 150[Formula: see text]mA[Formula: see text]g[Formula: see text] and an excellent rate capability of 120[Formula: see text]mAh[Formula: see text]g[Formula: see text] at 1500[Formula: see text]mA[Formula: see text]g[Formula: see text]. More importantly, sodium storage in carbon nanoflakes exhibits a pseudocapacitive behavior, possibly due to their larger interlayer spacing and less-ordered structure vs. crystallized carbon.

In this work, glycyrrhetinic acid-modified chitosan (mGA-suc-CTS) used as liver targeted carrier for drug delivery, was prepared via hemisuccinate as a bridged group. The structure of the product was confirmed by IR and NMR methods and the degree of substitution (DS) of glycyrrhetinic acid groups was estimated via elemental analysis. Nanoparticles were formed by ionic gelation methold. The drug-loading and release behavior of the nanoparticles were investigated using BSA as the model drug. The results indicated that the carrier with a highest DS of 5.19% could be got and the DS was controlled by changing reaction temperature or feed ratio. BSA could be entrapped into the nanoparticles with the drug-loading ratio of 26.3% and the encapsulation efficiency of 81.5%. A sustained release over an 11-day period was observed in pH 7.4 in vitro.

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.

Although numerous strategies are now available to generate rudimentary forms of synthetic cell-like entities, minimal progress has been made in the sustained excitation of artificial protocells under non-equilibrium conditions. Here we demonstrate that the electric field energization of coacervate microdroplets comprising polylysine and short single strands of DNA generates membrane-free protocells with complex, dynamical behaviours. By confining the droplets within a microfluidic channel and applying a range of electric field strengths, we produce protocells that exhibit repetitive cycles of vacuolarization, dynamical fluctuations in size and shape, chaotic growth and fusion, spontaneous ejection and sequestration of matter, directional capture of solute molecules, and pulsed enhancement of enzyme cascade reactions. Our results highlight new opportunities for the study of non-equilibrium phenomena in synthetic protocells, provide a strategy for inducing complex behaviour in electrostatically assembled soft matter microsystems and illustrate how dynamical properties can be activated and sustained in microcompartmentalized media., Synthetic cells are designed to mimic basic life processes, but it is challenging to prepare cells that can persist and operate at non-equilibrium conditions. Here, Yin et al. show complex dynamical behaviors of protocells made by exposing polylysine and single-stranded DNA droplets to an electric field.

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-γ, 2-methyl-thio-ATP (2MA), PGE2, and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited

We examined the major patterns of changes in gene expression in mouse splenic B cells in response to stimulation with 33 single ligands for 0.5, 1, 2, and 4 h. We found that ligands known to directly induce or costimulate proliferation, namely, anti-IgM (anti-Ig), anti-CD40 (CD40L), LPS, and, to a lesser extent, IL-4 and CpG-oligodeoxynucleotide (CpG), induced significant expression changes in a large number of genes. The remaining 28 single ligands produced changes in relatively few genes, even though they elicited measurable elevations in intracellular Ca2+ and cAMP concentration and/or protein phosphorylation, including cytokines, chemokines, and other ligands that interact with G protein-coupled receptors. A detailed comparison of gene expression responses to anti-Ig, CD40L, LPS, IL-4, and CpG indicates that while many genes had similar temporal patterns of change in expression in response to these ligands, subsets of genes showed unique expression patterns in response to IL-4, anti-Ig, and CD40L.

Transcription factors of the Forkhead Box, class O (FOXO) family promote cell-cycle arrest and/or apoptosis in a variety of cell types. Mitogenic stimuli inactivate FOXO function by way of an evolutionarily conserved pathway involving the activation of phosphoinositide 3-kinase (PI3K) and its downstream effector, Akt. Although PI3K activation is required for B-lymphocyte proliferation, it is not known whether PI3K-dependent inactivation of FOXO proteins is important for cell-cycle progression and survival of these cells. Here, we show that B-cell receptor (BCR) engagement triggers PI3K-dependent phosphorylation and nuclear export of FOXO1. Furthermore, forced expression of PI3K-independent variants of FOXO1 or FOXO3a in activated B cells induces partial arrest in G1 phase of the cell cycle and increases apoptosis. These findings establish that FOXO inactivation is a functionally important consequence of PI3K signaling in primary B cells.

Indole-3-acetyl-myo-inositol (IAInos) is one of the most important auxin conjugates for storage and transportation of auxin. The information of its composition, distribution, and metabolism is particularly desired for elucidating the related signal transduction pathways of the plant hormones. However, separation and quantification of the four individual IAInos isomers in plant tissues have not been reported so far. In this work, we first synthesized and isolated four IAInos isomers using semi-preparative high-performance liquid chromatography (HPLC). The IAInos isomer structures were characterized using liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC-QTOF/MS) and nuclear magnetic resonance spectroscopy (NMR). Using these pure compounds as internal or external standards, an efficient LC-MS method was developed for simultaneous detection of indole-3-acetic acid, methyl indole-3-acetic acid ester, and the four IAInos isomers in plant tissue samples. The linear working range and lower limit of detection for the four IAInos isomers are 10-2,000 ng mL(-1) and 5.0 ng mL(-1), respectively. The stabilities and interconversion pathways of IAInos isomers were studied using our synthetic isomers. It was found that two IAInos isomers existed in Zea mays kernels, while all of the four IAInos isomers were present in the roots of Arabidopsis thaliana. The content of IAInos in A. thaliana roots was much lower than in the Z. mays kernels. The methodology in this article provides useful techniques and methods for systematic study on the phytophysiology and phytochemistry of IAA conjugates and other related plant hormones.

Young Alicia rabbits use VHa-negative genes, VHx and VHy, in most VDJ genes, and their serum Ig is VHa negative. However, as Alicia rabbits age, VHa2 allotype Ig is produced at high levels. We investigated which VH gene segments are used in the VDJ genes of a2 Ig-secreting hybridomas and of a2 Ig+ B cells from adult Alicia rabbits. We found that 21 of the 25 VDJ genes used the a2-encoding genes, VH4 or VH7; the other four VDJ genes used four unknown VH gene segments. Because VH4 and VH7 are rarely found in VDJ genes of normal or young Alicia rabbits, we investigated the timing of rearrangement of these genes in Alicia rabbits. During fetal development, VH4 was used in 60–80% of nonproductively rearranged VDJ genes, and VHx and VHy together were used in 10–26%. These data indicate that during B lymphopoiesis VH4 is preferentially rearranged. However, the percentage of productive VHx- and VHy-utilizing VDJ genes increased from 38% at day 21 of gestation to 89% at birth (gestation day 31), whereas the percentage of VH4-utilizing VDJ genes remained at 15%. These data suggest that during fetal development, either VH4-utilizing B-lineage cells are selectively eliminated, or B cells with VHx- and VHy-utilizing VDJ genes are selectively expanded, or both. The accumulation of peripheral VH4-utilizing a2 B cells with age indicates that these B cells might be selectively expanded in the periphery. We discuss the possible selection mechanisms that regulate VH gene segment usage in rabbit B cells during lymphopoiesis and in the periphery.

We demonstrate a novel fluorescent sensor for real-time and continuous monitoring of the variation of bisulfide in microdialysis effluents by using a nanoparticle-glutathione-fluorescein isothiocyanate (AuNP-GSH-FITC) probe coupled with on-line droplet-based microfluidic chip. The AuNP-GSH-FITC fluorescent probe was firstly developed and used for bisulfide detection in bulk solution by quantitative real-time PCR, which achieved a linear working range from 0.1 μM to 5.0 μM and a limit of detection of ~50 nM. The response time was less than 2 min. With the aid of co-immobilized thiol-polyethylene glycol, the probe exhibited excellent stability and reproducibility in high salinity solutions, including artificial cerebrospinal fluids (aCSF). By adding 0.1% glyoxal to the probe solution, the assay allowed quantification of bisulfide in the presence of cysteine at the micro-molarity level. Using the AuNP-GSH-FITC probe, a droplet-based microfluidic fluorescent sensor was further constructed for online monitoring of bisulfide variation in the effluent of microdialysis. By using fluorescence microscope-charge-coupled device camera as the detector, the integrated microdialysis/microfluidic chip device achieved a detection limit of 2.0 μM and a linear response from 5.0 μM to 50 μM for bisulfide in the tested sample. The method was successfully applied for the on-line measurement of bisulfide variation in aCSF and serum samples. It will be a very useful tool for tracking the variation of bisulfide or hydrogen sulfide in extracellular fluids.

Enzymes containing 3'-5' exonuclease activities play vital roles in maintaining genome stability. Though a wide variety of methods have been developed for detection of these enzymes, few of them can be directly applied for in situ and real-time monitoring of the secretion of these active substances by living cells. Taking advantages of the free 3'-end of stacked guanine-quenched photoinduced electron transfer fluorescent probes, here we demonstrate a novel assay capable of in situ and real-time monitoring of the 3'-5' exonucleases secreted by living cells. The detection limit of the new method achieved as low as 0.04 U/mL, allowing direct monitoring of the target enzymes in an extracellular environment without preconcentration steps. False positive signals caused by other nonspecific enzymes were easily ruled out by the use of a control probe with the 3'-end modified with exonuclease-resistant phosphorothioate guanines. Using Alexa Fluor 488 as the fluorophore, the probe is adaptable to a wide range of pH conditions. The approach was successfully applied for in situ, real-time monitoring of the 3'-5' exonucleases secreted by suspension cells of Arabidopsis thaliana. It also holds great potential for in situ and real-time detection of many other DNA end-processing enzymes produced by other types of cells.

In this chapter, recent achievements in the development of luminescent optical sensors based on nanoscale molecularly imprinted polymers were reviewed. The first part focused on introduction of the commonly used ways to convert the specific interactions between imprinted cavities and corresponding template into measurable luminescent signals. In the following parts, the MIP-based luminescent optical sensors are classified as fluorescent sensors, chemiluminescent sensors, colorimetric sensors, and other types of luminescent optical sensors. The principal analytical features and applications of these different types of MIP sensors that have been developed recently are all summarized and discussed. In the last part, some remaining challenges are briefly discussed for further development of the field.

Molecular beacons (MBs) have shown great potential in measurement of enzyme activities. However, currently available methods for monitoring of phosphatases only use MBs as a signal reporter. Extra substrates for the phosphatases are needed to hybridize to the MB either as a primer or as a template. Moreover, few MB-based methods have been used to detect enzyme activities in real biological samples due to insufficient sensitivity or false positive interference signals caused by nonspecific nucleases. In this work, a novel type of fluorescent probe was designed and synthesized for monitoring of phosphatases by integrating the DNA substrate and the signaling structures into a single molecule. Such a new design not only significantly simplified the probing system and greatly enhanced the sensitivity, but also offered a practical way to guard against the false-positive signal problems in the application to real samples. The unique design of the assay format should be widely applicable to many other enzymatic assays using oligonucleotide fluorescent probes.

The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E(2), that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-alpha, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor kappaB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates.

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.

In order to improve the properties of laser overlap welded T-joint, laser-additional current hybrid welding process is put forward. In this paper, the welding characteristics of laser-additional current hybrid welded aluminum and titanium alloy T-joint were conducted and compared. The weld width at faying surface increase, which results in tensile shear load increasing compared with those of laser welding for both aluminum and titanium alloy, but the effect of current on aluminum alloy is more obvious. The porosity defect within the laser-additional current hybrid welded joint sharply reduces compared with that within laser welding. The tensile shear load of aluminum alloy and titanium alloy hybrid welded joints respectively increase 21% and 15%. The effects of additional current on welding characteristics of aluminum alloy and titanium alloy are compared and analyzed.