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Odd-electron bonds, i.e., the two-center, three-electron (2c/3e), or one-electron (2c/1e) bonds, have attracted tremendous interest owing to their novel bonding nature and radical properties. Herein, complex [K(THF)6][LSn:···Sn:L] (1), featuring the first and unsupported 2c/1e Sn···Sn σ-bond with a long distance (3.2155(9) Å), was synthesized by reduction of stannylene [LSn:] (L = N,N-dpp-o-phenylene diamide) with KC8. The one-electron Sn–Sn bond in 1was confirmed by the crystal structure, DFT calculations, EPR spectroscopy, and reactivity studies. This compound can be viewed as a stabilized radical by delocalizing to two metal centers and can readily mediate radical reactions such as C–C coupling of benzaldehyde.

Accomplishments of enhanced activity and durability are a major concern in the design of catalysts for acidic water oxidation. To date, most studied supported metal catalysts undergo fast degradation in strongly acidic and oxidative environments due to improper controlling of the interface stability caused by their lattice mismatches. Here, we evaluate the activity–stability trends of in situ crystallized antimony-doped tin oxide (Sb-SnO2)@RuOx(Sb-SnO2@RuOx) heterostructure nanosheets (NSs) for acidic water oxidation. The catalyst prepared by atomic layer deposition of a conformal Ru film on antimony-doped tin sulfide (Sb-SnS2) NSs followed by heat treatment highlights comparable activity but longer stability than that of the ex situ catalyst (where Ru was deposited on Sb-SnO2followed by heating). Air calcination for in situ crystallization allows the formation of hierarchical mesoporous Sb-SnO2NSs from as-prepared Sb-SnS2NSs and parallel in situ transformation from Ru to RuOx, resulting in a compact heterostructure. The significance of this approach significantly resists corrosive dissolution, which is justified by the enhanced oxygen evolution reaction (OER) stability of the catalyst compared to most of the state-of-the-art ruthenium-based catalysts including Carbon@RuOx(which shows ∼10 times higher dissolution) as well as Sb-SnO2@Com. RuOxand Com. RuO2. This study demonstrates the controlled interface stability of heterostructure catalysts toward enhancing OER activity and stability.

Objective:We explore the pharmacodynamic targets of kaempferol in treating osteoporosis using transcriptomics and animal experiment. Methods:Firstly, we constructed an ovariectomized rat model (OVX). The anti-osteoporosis effect of kaempferol was evaluated by bone mineral density (BMD) and hematoxylin-eosin staining comprehensively. Moreover, differential genes between groups were screened by RNA sequencing technology (RNA-seq) for transcriptomics and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways enrichment analysis were performed. Finally, partial results of transcriptomics were verified to detect the expression of the relevant gene expression by immunohistochemistry. Results:The pharmacodynamic findings indicated that the administration of kaempferol resulted in an elevation in BMD (P< .01) and a notable enhancement in tibia microstructural indices in the experimental rats (P< .01). The transcriptomic analysis revealed that NTN1, LTBP4, GSN, and EBF1 were identified as the principal targets for therapeutic intervention in osteoporosis. The results of animal experiments showed that kaempferol promoted osteogenesis and inhibited bone resorption by downregulating the protein expression of NTN1, LTBP4, GSN, and EBF1 (P< .01). Conclusion:Kaempferol enhances BMD levels, retards tibial bone loss with structural deterioration in OVX model rats, and promotes bone formation while curbing bone resorption through NTN1, LTBP4, GSN, and EBF1 protein down-regulation.

Neutral N-pyrazolyl radicals [3,5-R2pz]•as reactive intermediates were generated by one-electron oxidization of the corresponding 3,5-disubstituted pyrazolato anions [3,5-R2pz]−(R = tBu, Ph) with BiCl3and trapped by the use of 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap, which was confirmed by electron paramagnetic resonance spectral analysis. With dimerization of the postulated pyrazolato low-valent BiIIradical species, two novel paddlewheel pyrazolatodibismuthanes [L2(Bi–Bi)L2] [L = η1,η1-3,5-R2pz; R = tBu (5α, 5β, and 5γ), Ph (6)] were isolated and structurally characterized.

The influences of biological, chemical, and flow processes on soil structure through microbial-induced carbonate precipitation (MICP) are not yet fully understood. In this study, we use a multi-level thresholding segmentation algorithm, genetic algorithm (GA) enhanced Kapur entropy (KE) (GAE-KE), to accomplish quantitative characterization of sandy soil structure altered by MICP cementation. A sandy soil sample was treated using MICP method and scanned by the synchrotron radiation (SR) micro-CT with a resolution of 6.5 μm. After validation, tri-level thresholding segmentation using GAE-KE successfully separated the precipitated calcium carbonate crystals from sand particles and pores. The spatial distributions of porosity, pore structure parameters, and flow characteristics were calculated for quantitative characterization. The results offer pore-scale insights into the MICP treatment effect, and the quantitative understanding confirms the feasibility of the GAE-KE multi-level thresholding segmentation algorithm.

A novel 2,6-naphthyridine was identified by high throughput screen (HTS) as a dual protein kinase C/D (PKC/PKD) inhibitor. PKD inhibition in the heart was proposed as a potential antihypertrophic mechanism with application as a heart failure therapy. As PKC was previously identified as the immediate upstream activator of PKD, PKD vs PKC selectivity was essential to understand the effect of PKD inhibition in models of cardiac hypertrophy and heart failure. The present study describes the modification of the HTS hit to a series of prototype pan-PKD inhibitors with routine 1000-fold PKD vs PKC selectivity. Example compounds inhibited PKD activity in vitro, in cells, and in vivo following oral administration. Their effects on heart morphology and function are discussed herein.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein–ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivoantitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

The multifunctionalization of unactivated cyclic ketones was developed via an electrochemically intermolecular α-amination under metal-free conditions. The reaction can be carried out smoothly with a broad scope of the aromatic amines substrates under mild conditions, affording a variety of α-enaminones with good to excellent yields in one step.

The life cycle environmental impacts of the new model radial passenger vehicle tire product was evaluated and compared. the silicon dioxide was added to replace part of the carbon black as filler, with its previous model tire product. The study covers all relevant life cycle stages: from the acquisition of raw materials to the production, use, and end-of-life. The data collected onsite in 2014 by one of the largest Chinese tire companies were used in the assessment. The evaluation is presented in terms of individual impact category according to the CML model. Five impact categories (i.e., global warming potential (GWP), acidification potential (AP), photochemical oxidant creation potential (POCP), eutrophication potential (EP), and human toxicity potential (HTP)) were considered. The research was conducted in accordance with the ISO 14040/14044 standards. The results showed that In all the five impact categories, new model tire product is lower than the previous model in the whole life cycle. It is mostly due to the reduction of the fuel consumption during the use stage, which resulted in replacing part of the carbon black with silicon dioxide. Fuel (gasoline) consumption represents an important contribution to most impact categories, including the GWP, AP, POCP, and EP, during the use stage. Vehicle fuel consumption and its proportion consumed by the tires during the use stage are key factors that contribute to environmental impact during tire life time. The largest contributor to the HTP category is raw material acquisition, mainly because of the impact of the production of organic chemicals. During the raw material acquisition stage, natural rubber, synthetic rubber, carbon black, and organic chemicals represent the largest contribution to the environmental impact categories, which could be mitigated by reducing the use of raw materials, replacing the aromatic oil with environmental oil in the new model tire product.

The catalytic transformation of N2to NH3by transition metal complexes is of great interest and importance but has remained a challenge to date. Despite the essential role of vanadium in biological N2fixation, well-defined vanadium complexes that can catalyze the conversion of N2to NH3are scarce. In particular, a V(NxHy) intermediate derived from proton/electron transfer reactions of coordinated N2remains unknown. Here, we report a dinitrogen-bridged divanadium complex bearing POCOP (2,6-(tBu2PO)2-C6H3) pincer and aryloxy ligands, which can serve as a catalyst for the reduction of N2to NH3and N2H4. Low-temperature protonation and reduction of the dinitrogen complex afforded the first structurally characterized neutral metal hydrazido(2−) species ([V]═NNH2), which mediated 15N2conversion to 15NH3, indicating that it is a plausible intermediate of the catalysis. DFT calculations showed that the vanadium hydrazido complex [V]═NNH2possessed a N–H bond dissociation free energy (BDFEN–H) of as high as 59.1 kcal/mol. The protonation of a vanadium amide complex ([V]–NH2) with [Ph2NH2][OTf] resulted in the release of NH3and the formation of a vanadium triflate complex, which upon reduction under N2afforded the vanadium dinitrogen complex. These transformations model the final steps of a vanadium-catalyzed N2reduction cycle. Both experimental and theoretical studies suggest that the catalytic reaction may proceed via a distal pathway to liberate NH3. These findings provide unprecedented insights into the mechanism of N2reduction related to FeV nitrogenase.

A metal-free electrophotochemical C(sp3)-H arylation was developed under mild conditions. This method enables a switchable synthesis of diaryl alcohols and diaryl alkanes from inactive benzylic carbons. More importantly, a cheap and safe mediator N-chlorosuccinimide (NCS) was developed, which was employed for the hydrogen atom transfer (HAT) process of the benzylic C–H bond. In addition, this active radical was captured and identified by electron paramagnetic resonance (EPR).

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1–4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1–4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4S× 4Imatrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC50-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC50-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.

SHP099, a selective inhibitor of signalling meditator SHP2 with drug-like properties, has an allosteric mechanism of action whereby it stabilizes SHP2 in an auto-inhibited conformation, and suppresses RAS–ERK signalling and proliferation in receptor-tyrosine-kinase-driven cancer cell lines and mouse tumour xenograft models.

Silicon powder is a kind of important nonmetallic mineral. It not only reduce the cost of polymer materials, but also can improve the performance of materials. In order to make silicon powder play a better role, this paper shows that surface modification of silicon powder by using the silane coupling agents KH550 and KH560. Through hydrophobic property, infiltrating performance, activation index, infrared spectroscopy(FT-IR) and scanning electron microscopy (SEM).It can be find that the evaluation and characterization of silicon powder modification effect. Comprehensive analysis shows that the modification effect is best,when KH550 and KH560 is the ratio of 1:2.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

Firstly, the importance and necessity of automotive eco-design were introduced. And then, the limit standards, testing methods and weight coefficient of basic indexes in assessment index system were pointed out. A linear-type dimensionless automotive eco-design assessment calculation method of individual index was proposed. Finally, the automotive eco-design assessment results calculation method was illustrated.

Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment.

Degradation of polymer electrolyte membrane fuel cell (PEMFC) systems has emerged as a critical issue. A thin metal oxide layer coated with Pt/carbon via ALD (atomic layer deposition) is one of the potential approaches for preserving electrochemical activity; however, the exact interfacial effects of metal oxide on enhancing PEMFC durability are unclear. Herein, interfacial engineering of the TiO2layers within the in situ-synthesized Pt/carbon catalysts (Pt/TiO2/C and TiO2/Pt/C) was studied using fluidized bed reactor (FBR) ALD to investigate the exact effects of the catalysts. For the Pt/TiO2/C catalyst, the TiO2layer was first conformally coated on the carbon surfaces, whereas for TiO2/Pt/C, the TiO2layer was selectively formed on the Pt NP surface via the ALD mechanism. The Pt/TiO2/C catalyst has a higher Pt loading with suppressed micropores due to the introduction of the TiO2layer on the carbon support, whereas the TiO2/Pt/C catalyst remained in the 2–3 nm mesopores. The electrochemical durability of both ALD catalysts is superior to that of the commercial Pt catalyst. Encapsulating the TiO2layer on the Pt surface specializing in blocking Pt dissolution resulted in better long-term stability of the electrochemical characteristics compared to the stability of those of Pt/TiO2/C, which especially showed the better initial performance of the electrochemically active surface area, oxygen reduction reaction, and PEMFC single-cell performance. This study provides the direction and steps toward an efficient nanostructure design of metal oxide by ALD in most catalyst fields.

A dimer acid was synthesized by Diels-Alder action using single oleic acid as raw material. Effects of different reaction conditions on composition and mechanism for Diels-Alder action of oleic acid were discussed. The result showed that the conversion of dimer acid was 71.7% under using the amount of organic montmorillonite as catalyst 9% (in term of mass of system), reaction temperature 260°C, reaction time 8h. It is to be a certain significance to improve the quality of dimer acid with oleic acid as raw material.

Firstly, the process of establishing automotive ecology related systems and the necessity of automotive eco-design assessment to carry out were introduced. The overall objective of automotive eco-design assessment about China was described. And then, automotive eco-design assessment system of China was established by screening the most vulnerable index of energy-saving, environmental protection and health impact in all automotive life cycle stages by the approaches of desk information research, business investigation visit and industry expert consultation. Finally, a wide range of automotive eco-design assessment strategy model was proposed.

The spongy dressing was widely used in the pharmacy and medicine field. In this paper the classification, preparation and defects of the spongy composite wound dressings based on the literature at domestic and overseas were introduced. The freeze-drying method was commonly used to prepare spongy composites.

In this paper, we studied the influence of the amount of alkyl glycosides emulsifier, reaction temperature and the amount of initiator on emulsion polymerization kinetics. The results show that when we apply the new alkyl glycosides emulsifier in acrylic emulsion polymerization, the relationship between polymerization rate Rp and emulsifier concentration [S] is Rp∝[S]0.70 and the correlation coefficient is 0.93; the relationship between polymerization rate Rp and the initiator concentration [I] is Rp∝[I]1.00 and the correlation coefficient is 0.86; the apparent activation energy of polymerization is 32.34KJ/mol and the correlation coefficient is 0.93. We got good results after applying the new type emulsifier in acrylic emulsion polymerization.

In this paper，lower polyethylene(LDPE) was melt to graft maleie anhydride (MAH) in reactive extruder by using dicumyl peroxide(DCP) and benzoyl peroxide(BPO) as the initiator. It can be found that MAH has been grafted to LDPE large molecular chain using by infrared spectrum and graft rate measurement. Orthogonal experiments were employed to confirm the influences of initiator, MAH and LDPE. The results showed that the reaction graft ratio firstly increased，then began to decrease with the increasing of the contents of MAH and initiator. The maximum shear strength was at 2.12MPa when graft polymer was used to adhesive the board of aluminum and polyethylene. The graft rate of graft polymer was at 15.68%.

Linear Motors have been used more and more widely in high precision and high-speed linear motion for CNC machine tools, but uniform testing standards and methods for linear motor performance have not been established in China. Based on the virtual instrument software platform by means of computer technology, research and development of the test platform for synthetic performance parameters of linear motors can realize the rapid and accurate measurement, automatic storage and processing of linear-motor output, currents, positions, temperatures and other performance parameters. , Meanwhile, the platform can also provide the technical direction for design or improvement of linear motors, and technically support the formulation of related standards. Experiments showed that the test platform for synthetic performance parameters of linear motors can achieve high precision and automatic measurement to meet the testing requirements for synthetic performance of linear motors.

Aimed at the longitudinal vibration of rope hoisting system with time-varying length, the governing equations are developed employing the Hamilton’s principle considering mutual influence of the rigid motion and deformation of rope. The Galerkin’s method is used to discretize the governing equations. In the mean time, The motions of elevator hoisting system were illustrated to evaluate the proposed mathematical models. The results of simulation show that the modeling methods can represent the longitudinal vibration of rope hoisting system with time-varying length in the paper. The governing equations derived can provide guidelines for further study on flexible hoisting system.

New initiator of FFM6 is used to initiate the acrylic emulsion polymerization. The influences of concentration of FFM6 (c[I]) and polymerization temperature (T) on polymerization reaction rate (Rp) were discussed. Rp is proportional to (c[I])1.4 which is different with classical emulsion polymerization whose Rp is proportion to (c[I])0.4, that indicate polymerization mechanism of the reaction in the study is different with classical mechanism. The value of Ea, 56.4 kJ/mol, is lower than the value of general radical polymerization’s Ea (80.0-96.0 kJ/mol), which indicates the FFM6 can initiate acrylic emulsion polymerization at a lower temperature compared with the other kinds of initiator.

The properties of UHMW-PE film produced by hot compression molding and films modified by acid and anti-static agent were characterized. The results showed that: a) the thickness distributing of the film which processing by calendar rolling with conventional design roller is inhomogeneity; the tensile strength was 37.22 MPa, the elongation at break was 368.00%; the transmittance increases with wavelength redshift shows short-wave absorption and long-wave transmission; initial temperature of dissociation is 320˚C; the surface resistance 1010 Ω and maximum is higher than 1016 Ω. b) Acid modification the surface of UHMW-PE film moderates the surface antistatic performance. C) After modified the film with external anti-static agent, the surface resistance was around 107Ω, but anti-static properties loss rapidly after friction. If modified the film with internal anti-static agent, the surface resistance was around 1010 Ω and have a long-lasting anti-static effect.

In this paper, flame retardant Polyamide 6 (PA6) composites were prepared by nano-magnesium hydroxide (NMH) or its composites with melamine cyanurate(MCA) and ammonium polyphosphate(APP). Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were used to analyze the structure of nano-magnesium hydroxide. The properties including tensile properties, molten index (MFI), rockwell hardness and density of flame retardant PA6 were analyzed. Orthogonal experiments were used to study flame retardancy of PA6 with NMH, MCA and APP. The results showed NMH had hexagonal orthorhombic crystal structure with size of 300×200×100nm. Density of polyamide 6 showed an upward trend when the content of NMH was increasing, the mechanical properties and hardness changed little while processing performance serious declined. The flame retardance of nitrogen-phosphorus -inorganic flame retardants was not desirable.

Modified barium sulfate (MBS) /nature rubber(NR) composite materials were prepared by direct blending methods. Barium sulfate was modified by coupling agent Si-69, titanium esters and stearic acid. Tensile property, hardness and wear resistance of composite materials were studied by various methods. The results showed that the modified barium sulfate fillers had great effect on the reinforcement of the composites.

The styrene-acrylic emulsions were prepared by copolymerization of styrene, acrylic acid and n-butyl acrylate; coatings were achieved by adding calcium sulfate whiskers and titanium dioxides at the presence of dispersing agents and rheological additives. The physical and mechanical properties as well as contact angles of coating films formed on tinplate were characterized in detail. The pencil hardness was slightly decreased; the contact angle was greatly decreased from 69.8o to 12oby adding the calcium sulfate to indicate the hydrophilic coating films may have self-cleaning properties.

Attapulgite (AT)/natural rubber (NR)/ styrene-butadiene rubber (SBR) nanocomposites have been prepared after attapulgite was modified by different coupling agent. The treatment of AT caused the adhesion between AT nanorods and the nature rubber/styrene-butadiene rubber was improved, which enhanced the tensile properties of the matrix. The tensile strength of composites attained 15.6 MPa after AT was modified by 3%wt Si-69 coupling with addition of 20 phr.

Spherical C@PSt microspheres with average size around 2 μm were successfully prepared using high-speed homogenization-assisted suspension polymerization of styrene in the presence of carbon black modified by oleic acid. The effects of initiator varieties and initiators concentration on the fractional conversion were investigated. Compared with BPO, AIBN has higher efficiency to initiate the polymerization. LPSA, SEM and OM results showed the prepared microspheres had spherical morphology with the carbon black in the core and PSt in the shell. About 10 weight percentages of carbon black were encapsulated in the complex microspheres proved by TGA results.

The core-shell acrylic emulsion was synthesized，with P(MMA/BA) as core，and with P(2-EHA/MBA/HPA) as shell. The influence of the mass ratio of core/shell on mechanical properties was investigated in detail. The high elongation at break proved that the emulsion may have a potential application in coating industry, afforded by HPA as functional monomer. However, when the amount of HPA exceeds 1.5 wt. % to the total monomers, the emulsion will become unstable and precipitation occurs.

Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.

In this paper, attapulgite(AT)/nature rubber (NR) composites have been prepared through melt blending process. First, different types of attapulgite were prepared.Secend,Fourier infrared spectroscopic (FTIR) was used to analyze attapulgite.Then,different types of attapulgite were filled into natural rubber according to different proportion.Finally, The mechanisms of composites were analyzed. The results showed that siliane coupling agent Si-69 can improve the dispersion of calcined attapulgite and enhance the chemical interfacial adhesion.

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.

Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen™ and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

Abstract The proton motive force (pmf) across the thylakoid membrane is composed of the proton gradient and the membrane potential, which promotes millisecond-delayed light emission (ms-DLE). In this study, the time courses of LHC II phosphorylation and ms-DLE were investigated in spinach chloroplast during State-2 transition. Red light illumination resulted in an exponential rise in LHC II phosphorylation and a biphasic time course of ms-DLE. The phospho-LHC II appeared upon ∼ 1 min illumination. The phosphorylation level increased exponentially when illumination was elongated to 20 min. The t½ of saturated LHC II phosphorylation was estimated 4–5 min under present illumination. During this process, the amplitudes of ms-DLE increased transiently to a maximal amplitude within 0.5 min illumination, and the reached maximum of the fast phase of ms-DLE was ∼ 140% of the dark control. Then, ms-DLE decreased from the maximum. After ≥3 min illumination, ms-DLE decreased to a lower level than the dark control. In the presence of uncouplers and inhibitors, the transient increase in the biphasic time course of ms-DLE was removed by nigericin and DCMU, and the sequential decrease was delayed by DCCD. The time course was not affected significantly by valinomycin and DBMIB. Moreover, the level of LHC II phosphorylation was enhanced by nigericin, valinomycin and DCCD, and was inhibited completely by DCMU and partially by DBMIB. Taken together, we proposed that the PS II photochemical activity remained unaffected even with a higher level of LHC II phosphorylation, which was reflected by the effect of DCCD on the time course of ms-DLE. Probably, the evidence of LHC II phosphorylation is the rearrangement of LHC II–PS II complex and the thylakoid, a feedback to light-exposure, rather than the redistribution of excitation energy from PS II to PS I.

This article will review both the binding and the functional cellular assays that can be used to characterize nuclear receptor ligands in vitro. The discussion will be broken into two sections, concentrating on cell-free and cell-based systems. Each section will provide an overview of available assays moving from generic screening assays to more specific technologies that can be used to discriminate the pharmacology of receptor / ligand interactions.

Brain-derived neurotrophic factor (BDNF) may play an important role in the modulation of N-methyl-d-asparate (NMDA) receptor function. To elucidate the underlying mechanisms, whole-cell patch-clamp recording was used to assess the effect of BDNF on the responses of cultured hippocampal neurons to the glutamate receptor agonist NMDA. We found that peak amplitude of NMDA-evoked currents in cultured hippocampal pyramidal neurons at Day 18 in vitro decreased significantly compared to that of NMDA currents at Day 10 or 14. Interestingly, NMDA-evoked currents were greatly enhanced by BDNF (50 ng/ml) in cultured neurons at Day 18, but not at Day 10 or 14. Treatment with Rp-cAMP abolished the potentiating effects of BDNF on NMDA current. Elevating the amount of cytosolic cAMP by preincubation with forskolin or Sp-cAMP also enhanced NMDA currents as effectively as BDNF in 18-day-old hippocampal neurons. Measurement of the cellular content of cAMP by RIA indicated that cultured hippocampal neurons showed decreased basal cAMP levels at the time NMDA currents were decreased and BDNF increased the decreased cAMP levels. Taken together, these results suggest that BDNF may restore decreased NMDA receptor activity in cultured hippocampal neurons by the cAMP pathway.