Droplet-based microfluidics for dose–response assay of enzyme inhibitors by electrochemical method

A simple but robust droplet-based microfluidic system was developed for dose–response enzyme inhibition assay by combining concentration gradient generation method with electrochemical detection method. A slotted-vials array and a tapered tip capillary were used for reagents introduction and concentration gradient generation, and a polydimethylsiloxane (PDMS) microfluidic chip integrated with microelectrodes was used for droplet generation and electrochemical detection. Effects of oil flow rate and surfactant on electrochemical sensing were investigated. This system was validated by measuring dose–response curves of three types of acetylcholinesterase (AChE) inhibitors, including carbamate pesticide, organophosphorus pesticide, and therapeutic drugs regulating Alzheimer's disease. Carbaryl, chlorpyrifos, and tacrine were used as model analytes, respectively, and their IC₅₀ (half maximal inhibitory concentration) values were determined. A whole enzyme inhibition assay was completed in 6 min, and the total consumption of reagents was less than 5 μL. This microfluidic system is applicable to many biochemical reactions, such as drug screening and kinetic studies, as long as one of the reactants or products is electrochemically active.     

N'-(2-羟基-5-甲氧基苯甲基)-4-二甲氨基苯甲酰肼及其氧钒(V)配合物:合成、晶体结构和脲酶抑制活性

制备了一个苯甲酰腙化合物N'-(2-羟基-5-甲氧基苯甲基)-4-二甲氨基苯甲酰肼(H₂L).利用H₂L、乙酰氧肟酸(HAHA)和VO(acac)₂在甲醇中反应得到了配合物[VOL(AHA)].通过元素分析、红外和紫外光谱,以及单晶X-射线衍射对H₂L和其配合物进行了表征。苯甲酰腙配体作为二价阴离子,利用其酚羟基氧原子、亚胺基氮原子、以及烯醇氧原子与V原子进行配位.乙酰氧肟酸配体利用其羰基氧原子和去质子化的羟基氧原子进行配位.配合物中的V原子为八面体配位构型。测试了H₂L、HAHA和钒配合物的脲酶抑制活性.在浓度为100 μmol·L^-1时,钒配合物对幽门螺旋杆菌脲酶的抑制率为63%,其IC₅₀值为45μmol·L^-1.还利用分子对接技术研究了配合物分子与脲酶的作用方式.     

Composition and in vitro Antifungal Activity of Essential Oils of Erigeron canadensis and Myrtus communis from France

Essential oils of Erigeron canadensis L. and Myrtus communis L. were tested in vitro as growth inhibitors against phytopathogenic fungi Rhizoctonia solani Kuhn, Fusarium solani (Mart.) Sacc. and Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. Both showed weak fungicidal acitivity, except the essential oil of M. communis that exerted a 60% growth inhibition against R. Solani at a dose of 1600 ppm. Microscopic observation revealed that the essential oil of M. Communis caused morphological alterations of hyphae of all fungi at 1600 ppm, while, at the same dose, only the hyphal morphology of C. Lindemuthianum was affected by the essential oil of Er. Canadensis.     

细管电泳-激光诱导荧光检测用于多个胞内蛋白激酶的抑制剂筛选和选择性评价

发展了一种基于毛细管电泳(CE)-激光诱导荧光(LIF)检测的多个细胞内源激酶的抑制剂平行筛选及选择性评价方法。CE高效的分离能力和LIF检测器的高选择性,使得同时测试多个胞内激酶的活性成为可能。共4种细胞系、3种特异性蛋白激酶底物肽、2种选择性蛋白激酶抑制剂和1种非选择性蛋白激酶抑制剂用于方法的建立。特异性底物肽与细胞裂解液混合后孵育,被其相应的激酶选择性地磷酸化,利用CE-LIF分离检测磷酸化产物和底物肽。同时测定一个抑制剂对几种蛋白激酶的抑制活性,用于评价抑制剂的选择性。与传统的单靶标筛选模式相比,这种基于细胞裂解液的多靶标筛选方法能提供更多的信息,更加高效,且细胞裂解液作为一种廉价的激酶来源大大降低了筛选成本。     

Pharmacophore modelling,molecular docking and virtual screening for EGFR (HER 1) tyrosine kinase inhibitors

A pharmacophore model has been developed using diverse classes of epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitors useful in the treatment of human tumours. Among the top 10 generated hypotheses, the second hypothesis, with one hydrogen bond acceptor, one ring aromatic and three hydrophobic features, was found to be the best on the basis of Cat Scramble validation as well as test set prediction (r _training?=?0.89, r _test?=?0.82). The model also maps well to the external test set molecules as well as clinically active molecules and corroborates the docking studies. Finally, 10 hits were identified as potential leads after virtual screening of ZINC database for EGFR TK inhibition. The study may facilitate the designing and discovery of novel EGFR TK inhibitors.     

Computer modeling of the dynamic properties of the cAMP-dependent protein kinase catalytic subunit

The structural dynamics of the cAMP-dependent protein kinase catalytic subunit were modeled using molecular dynamics computational methods. It was shown that the structure of this protein as well as its complexes with ATP and peptide ligand PKI(5-24) consisted of a large number of rapidly inter-converting conformations which could be grouped into subsets proceeding from their similarity. This cluster analysis revealed that conformations which correspond to the “opened” and “closed” structures of the protein were already present in the free enzyme, and most surprisingly co-existed in enzyme–ATP and enzyme–PKI(5-24) complexes as well as in the ternary complex, which included both of these ligands. The results also demonstrated that the most mobile structure segments of the protein were located in the regions of substrate binding sites and that their dynamics were most significantly affected by the binding of the ATP and peptide ligand.     

Generation of anti-trenbolone monoclonal antibody and establishment of an indirect competitive enzyme-linked immunosorbent assay for detection of trenbolone in animal tissues, feed and urine

Trenbolone (TRE) is a steroid used by veterinarians on livestock to increase appetite and body weight. The use of TRE has been restricted because of its harmful side effect for consumers. To effectively control TRE residue in food and food product, a rapid and convenient immunoassay was developed by preparing an anti-TRE monoclonal antibody. The immunogen and coating antigen were prepared by coupling TRE hapten with carrier proteins via 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) method. The optimized method gave an average IC₅₀ value of 0.323 ng mL⁻¹ towards TRE and an average detection limit (LOD) of 0.06 ng mL⁻¹, which is much lower than the maximum residue levels (2.0 ng g⁻¹) accepted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The specificity of the antibody was evaluated by measuring cross-reactivity of six structurally related compounds, including 19-nortestosterone (9.7%), testosterone (0.13%), methyltestosterone (<0.01%), methandrostenolone (<0.01%), (+)-dehydroisoandrosterone (<0.001%) and β-estradiol (<0.001%). The recovery rates of the test in detection of TRE-fortified animal tissue, urine and animal feed samples were in the range of 81.3-89.4%, while the intra- and inter-assay coefficients of variation were less than 12.0%.     

Polyphenolic profiling of Ipomoea carnea Jacq. by HPLC-DAD and its implications in oxidative stress and cancer

Ipomoea carnea Jacq. is an important folklore medicinal plant, assessed for its underexplored biological potential. Antioxidant, cytotoxic, antiproliferative and polyphenolic profile of whole plant was evaluated using various techniques. Maximum extract recovery (29% w/w), phenolic [13.54 ± 0.27 μg GAE/mg dry weight (DW)] and flavonoid (2.11 ± 0.10 μg QE /mg DW) content were recorded in methanol-distilled water (1:1) flower extract. HPLC-DAD analysis quantified substantial amount of six different polyphenols ranging from 0.081 to 37.95 μg/mg extract. Maximum total antioxidant and reducing potential were documented in methanol-distilled water and acetone-distilled water flower extracts (42.62 ± 0.47 and 24.38 ± 0.39 μg AAE/mg DW) respectively. Ethanol-chloroform root extract manifested highest free radical scavenging (IC₅₀ of 61.22 μg/mL) while 94.64% of the extracts showed cytotoxicity against brine shrimps. Ethanol leaf extract exhibited remarkable activity against THP-1 cell line (IC₅₀ = 8 ± 0.05 μg/mL) and protein kinases (31 mm phenotype bald zone).     

Polyepoxysuccinic acid with hyper-branched structure as an environmentally friendly scale inhibitor and its scale inhibition mechanism

In order to improve the scale inhibition efficiency of existing polyepoxysuccinic acid (PESA) and to study the impact of their molecular structure on scale inhibition efficiency, a series of PESA with linear and hyper-branched structure have been designed and synthesized through co-polymerization reaction with glycidyl and epoxy succinate. The scale inhibition behavior of PESA with linear and hyper-branched structure against CaCO₃ and CaSO₄ scales was evaluated using static scale inhibition method, and their ability to retard deposition of CaCO₃ was also examined. The experimental results showed that, for CaCO₃ and CaSO₄, the PESA with hyper-branched structure provides a scale inhibiting efficiency as high as 95.9% and 94.3%, respectively, at an inhibitor concentration of 15?mg/L. In addition, the processes of crystal nucleation, growth and crystal morphology have been analyzed. The experimental results show that the PESA with hyper-branched structure not only prolongs the induction period of CaCO₃ crystal nucleation, but also reduces the number of crystal nuclei and changes the size and morphology of the CaCO₃ crystal. Moreover, the FTIR, SEM and XRD analyses showed that the PESA with hyper-branched structure can induce the irregularity of growing CaCO₃ crystal, destroy the formation of crystals and change the polymorphs of calcium scale crystal. This conclusion indicates that the prepared PESA with hyper-branched structure has great potential for applying in the treatment of industrial water.     

Dinuclear cobalt and nickel complexes of a mercaptoacetic acid substituted 1,2,4‐triazole ligand: syntheses,structures and urease inhibitory studies

Because of its versatile coordination modes and strong coordination ability, the mercaptoacetic acid substituted 1,2,4‐triazole 2‐{[5‐(pyridin‐2‐yl)‐4H‐1,2,4‐triazol‐3‐yl]sulfanyl}acetic acid ( H₂L ) was synthesized and characterized. Treatment of H₂L with cobalt and nickel acetate afforded the dinuclear complexes {μ‐3‐[(carboxylatomethyl)sulfanyl]‐5‐(pyridin‐2‐yl)‐4H‐1,2,4‐triazol‐4‐ido‐κ²N¹,N⁵:N²,O}bis[aqua(methanol‐κO)cobalt(II)] methanol disolvate, [Co₂(C₉H₆N₄O₂S)₂(CH₃OH)₂(H₂O)₂]·2CH₃OH ( 1 ), and {μ‐3‐[(carboxylatomethyl)sulfanyl]‐5‐(pyridin‐2‐yl)‐4H‐1,2,4‐triazol‐4‐ido‐κ²N¹,N⁵:N²,O}bis[diaquanickel(II)] methanol disolvate dihydrate, [Ni₂(C₉H₆N₄O₂S)₂(H₂O)₄]·2CH₃OH·2H₂O ( 2 ), respectively. Complex 1 crystallized in the monoclinic space group P2₁/c, while 2 crystallized in the tetragonal space group I4₁/a. Single‐crystal X‐ray diffraction studies revealed that H₂L is doubly deprotonated and acts as a tetradentate bridging ligand in complexes 1 and 2 . For both of the obtained complexes, extensive hydrogen‐bond interactions contribute to the formation of their three‐dimensional supermolecular structures. Hirshfeld surface analysis was used to illustrate the intermolecular interactions. Additionally, the urease inhibitory activities of 1 , 2 and H₂L were investigated against jack bean urease, where the two complexes revealed strong urease inhibition activities.