Speeding up a genetic algorithm for EPR-based spin label characterization of biosystem complexity

Complexity of biological systems is one of the toughest problems for any experimental technique. Complex biochemical composition and a variety of biophysical interactions governing the evolution of a state of a biological system imply that the experimental response of the system would be superimposed of many different responses. To obtain a reliable characterization of such a system based on spin-label Electron Paramagnetic Resonance (EPR) spectroscopy, multiple Hybrid Evolutionary Optimization (HEO) combined with spectral simulation can be applied. Implemented as the GHOST algorithm this approach is capable of handling the huge solution space and provides an insight into the "quasicontinuous" distribution of parameters that describe the biophysical properties of an experimental system. However, the analysis procedure requires several hundreds of runs of the evolutionary optimization routine making this algorithm extremely computationally demanding. As only the best parameter sets from each run are assumed to contribute into the final solution, this algorithm appears far from being optimized. The goal of this study is to modify the optimization routine in a way that 20-40 runs would be enough to obtain qualitatively the same characterization. However, to keep the solution diversity throughout the HEO run, fitness sharing and newly developed shaking mechanisms are applied and tested on various test EPR spectra. In addition, other evolutionary optimization parameters such as population size and probability of genetic operators were also varied to tune the algorithm. According to the testing examples a speed-up factor of 5-7 was achieved.     

Development and Verification of a Precolumn Derivatization LC-MS/MS Method for the Pharmacokinetic Study of Houttuynine of Houttuynia Essential Oil

Houttuynia essential oil (HEO) has excellent antiviral, anti-inflammatory, and other pharmacological effects, but the lack of effective analytical methods to quantify HEO in plasma has hindered its better clinical monitoring. Houttuynine (Hou) is one of the main active ingredients and quality control substances of HEO, so the pharmacokinetic study of HEO could be conducted by determining Hou blood concentration. Hou is active and not stable in plasma, which makes its blood concentration difficult to measure. In this work, a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method for Hou determination in rat blood was established that involves Hou being derivatized with 2, 4-dinitrophenylhydrazine to form a stable compound to prevent degradation. Herein, p-Tolualdehyde-2,4-dinitrophenylphenylhydrazone was selected as an internal standard substance and the LC-MS/MS method was evaluated for selectivity, precision, accuracy, calibration limit, matrix effect, recovery, and stability. Good linearity (r² = 0.998) was reached in the range of 2–2000 ng/mL, and the lower limit of quantification of Hou was determined to be 2 ng/mL. The mean intra-assay accuracy ranged from 77.7% to 115.6%, whereas the intra-assay precision (relative standard deviation, RSD) was below 11.42%. The matrix effect value for Hou in rat plasma was greater than 75%, and for the internal standard (IS) it was 104.56% ± 3.62%. The extraction recovery of Hou were no less than 90%, and for the IS it was 96.50% ± 4.68%. Our method is sensitive and reliable and has been successfully applied to the pharmacokinetic analysis of Hou in rats given HEO via gavage and injection.     

Polymeric materials as biomaterials under particular consideration of biodegradable polymers

Biomaterials may be defined as artificial materials which fulfill the mechanical requirements and interact with the biosystem they are in contact with in same way as a natural material would react in the same place. While the requirements of mechanical properties can be reached by suitable organo-polymeric and inorganic materials the interfacial biocompatiblity is neither understood in all its complexity nor can be fulfilled by any of the applied materials. Surface modification and characterization with greatest scrutiny and the observation of the answer of selected parameters of the biosystem are a subject of utmost interest. A few examples will be presented. In the long range, however, it has to be considered that any material is degraded and hence should present continuously a renewable biocompatible surface. On the other hand, materials are desired which deliberately are biodegradable. Presently available materials are polylactides and copolymers. An alternative could be presented by polydepsipeptides because of two reasons, (i) the local concentration of acid formed upon degradation would be reduced as compared to polylactides which in certain cases might be advantageous and (ii) the aminoacid units could carry side groups with bioactive molecules attached. Therefore, a new method of acylation of an aminoacid with a hydroxyacid is presented as well as the cyclisation to result in the cyclic depsipeptide and the polymerisation to yield the polydepsipeptide. The microstructure of the polymers, the thermal properties and the degradation behaviour is presented.     

基于超高效液相色谱-八端口转子阀的微升级预分级系统的建立

复杂生物系统蛋白质组的深度覆盖鉴定得益于近年来快速发展的高效色谱分离和串联质谱分析技术。二维高效液相色谱（2-D HPLC）是实现复杂肽混合物正交分离的有效手段，但其缺点是运行时间长，通常要求肽上样量在毫克级别，且收集时的组分体积大，后续合并流程复杂，而有望替代其的StageTip技术，则由于有限的色谱分离梯度，难以达到充分的正交分离。该文探索了超高效液相色谱（UPLC）和八端口转子阀联用，作为高效、便捷肽段预分离和收集系统的可行性。研究结果显示，将UPLC的高pH反相色谱分馏与在线LC-MS/MS相结合，可以实现高pH和低pH条件下基于肽段不同色谱保留行为的正交互补分离，在蛋白质鉴定方面表现出优越的性能。应用该方法对人肝癌细胞系HepG2进行3次技术重复试验，重复试验间具有非常高的定量重现性（决定系数R²>0.95）。与传统StageTip方法相比，肽段鉴定数提高了23.52%。该分级方法灵活、稳定，能够针对较少的样品开展蛋白质组深度覆盖分析。     

Exploring the local conformational space of a membrane protein by site-directed spin labeling

Molecular modeling based on a hybrid evolutionary optimization and an information condensation algorithm, called GHOST, of spin label ESR spectra was applied to study the structure and dynamics of membrane proteins. The new method is capable of providing detailed molecular information about the conformational space of the spin-labeled segment of the protein in a membrane system. The method is applied to spin-labeled bacteriophage M13 major coat protein, which is used as a model membrane protein. Single cysteine mutants of the coat protein were labeled with nitroxide spin labels and incorporated in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. The new computational method allows us to monitor distributions of local spatial constraints and molecular mobility, in addition to information about the location of the protein in a membrane. Furthermore, the results suggest that different local conformations may coexist in the membrane protein. The knowledge of different local conformations may help us to better understand the function-structure relationship of membrane proteins.     

A novel spectrophotometric method for the simultaneous kinetic analysis of ternary mixtures by mean centering of ratio kinetic profiles

In this paper, a novel and very simple method was developed for the simultaneous spectrophotometric determination of ternary mixtures, without prior separation steps. The method is based on the mean centering of ratio kinetic profiles. The mathematical explanation of the procedure is illustrated. In order to investigate the applicability of the proposed method, it was applied to the simultaneous spectrophotometric determination of hydrazine, phenylhydrazine and acetylhydrazine based on their condensation reactions with p-(dimethylamino)benzaldehyde (DAB) and p-nitrobenzaldehyde (NB) in micellar sodium dodecyl sulfate (SDS) media as a typical ternary mixture. The analytical characteristics of the method such as accuracy, precision, relative standard deviation (R.S.D.) and relative standard error (R.S.E.) were calculated.     

Versatile method for electroosmotic flow measurements in microchip electrophoresis

A novel versatile method for the determination of low or high electroosmotic mobility values in microdevices of variable microchannel design is presented. The electroosmotic flow (EOF) calculation is based on the difference between the apparent and effective mobilities of a reference compound. The proposed method uses microchip frontal electrophoresis for the determination of these mobilities. This requires simple monochannel microchip design and demonstrates versatile and time-saving procedure when compared to conventional current monitoring method when measuring low EOF. It has been applied successfully to the characterization of different coating procedure in glass and poly(dimethylsiloxane) microchips.     

金属-氧共价性增强的缺陷态高熵岩盐型氧化物用于电催化析氧

高熵材料可以在单一晶相中引入五种或五种以上元素以优化电子结构和配位环境,可作为一类新兴的电催化剂.本文制备了一种岩盐型高熵氧化物Mg0.2Co0.2Ni0.2Cu0.2Zn0.2O(HEO)用于催化氧析出反应(OER).由于相邻的不同金属离子晶格失配,所制备的HEO具有丰富的缺陷.此外,电负性更小的Mg和Zn元素的存在...     

Crystal Structure and Properties of a New 2, 2'‐Ortho(metaxylene) E‐Bridged Stilbenophane

The synthesis and characterization of novel E and Z‐2, 2'‐ortho(metaxylene)‐bridged stilbenophane 2 by reductive McMurry condensation are described and the X‐ray structure determination of the E‐isomer is reported. The structure analysis of 2 ‐E shows weak hydrogen bonding. The complexation of Na⁺ ions in 2, 2'‐ortho(metaxylene)‐Z‐bridged stilbenophane using sodium chloride and measurements of conductivity were performed. The formation constant logK was determined to 1.45.     

Flow-injection chemiluminescence determination of quinine using on-line electrogenerated cobalt(III) as oxidant

A novel chemiluminescence (CL) flow system for the determination of quinine is described. It is based on the direct chemiluminescence reaction of quinine and cobalt(III) in sulfuric acid medium. The unstable Co(III) was on-line electrogenerated by constant-current electrolysis. The chemiluminescence intensity was linear with a quinine concentration in the range of 0.1-100 mug ml(-1). The determination limit was 3.3x10(-8) g ml(-1). The whole process could be completed in 1 min. The proposed method is suitable for automatic and continuous analysis, and has been applied successfully to the analysis of quinine in pharmaceutical preparation.     

Electrochemical Characterization of In Situ Functionalized Gold Cysteamine Self‐Assembled Monolayer with 4‐Formylphenylboronic Acid for Detection of Dopamine

Functionalization of gold cysteamine (Au? CA) self‐assembled monolayer with 4‐formylphenylboronic acid (BA) via Schiff's base formation, through in situ method to fabricate Au‐CA‐BA electrode is presented and described. The fabricated electrode was used as a novel sensor for accumulation and determination of dopamine (DA). The accumulation of DA as a diol on the topside of Au‐CA‐BA as a Lewis acid, was performed via esterification (Au? CA? BA? DA), and followed for determination of DA. Functionalization, characterization, and determination steps were probed by electrochemical methods like cyclic voltammetry and electrochemical impedance spectroscopy. The data will be presented and discussed from which a new sensor for DA is introduced.     

荧光桃红分光光度法测定微量盐酸异丙嗪

在pH 1.85的HCl-NaAc缓冲溶液中,荧光桃红与盐酸异丙嗪依靠静电引力和疏水作用力相互作用形成离子缔合物,其组成比为1∶2,最大褪色波长537 nm,表观摩尔吸光系数ε=4.07×104L.mol-1.cm-1,研究了体系的光谱特征,适宜的反应条件和影响因素,发现体系的吸光度与盐酸异丙嗪的质量浓度在0.04~3.6μg/mL范围内呈良好的线性关系,线性回归方程A=-0.04991-0.1269ρ(μg/mL),线性相关系数r=0.9998,检出限和RSD分别为0.0177μg/mL和2.2%。本法可用于药物制剂中盐酸异丙嗪含量的快速测定。     

Simultaneous determination of polysorbate 20 and unbound polyethylene-glycol in protein solutions using new core–shell reversed phase column and condensation nucleation light scattering detection

A novel fast and sensitive method has been developed for the specific simultaneous determination of polysorbate 20 (Tween 20) and unbound polyethylene-glycol (PEG) from liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and condensation nucleation light scattering detection (CNLSD or NQAD™). The method uses a Kinetex core–shell column (100 mm × 3 mm, 2.6 μm) and methanol–water–trifluoroacetic acid mobile phase. The rapid HPLC-CNLSD method presented here is suitable for quantifying polysorbate 20 in the range of 10–60 μg/ml and unbound PEG in the range of 2–40 μg/ml in protein solutions within good manufacturing practices (GMP) of the pharmaceutical industry.     

Formation of a surface covalent organic framework based on polyester condensation

The reaction between 1,3,5-tris(4-hydroxyphenyl)benzene and benzene-1,3,5-tricarbonyl trichloride leads to polyester condensation and formation of a novel COF on an Au(111) surface. The characterization performed in situ by means of variable temperature STM and XPS reveals the formation of an array of hexagonal cavities with ca. 2 nm size.     

Improvement of the prediction ability of multivariate calibration by a method based on the combination of data fusion and least squares support vector machines

This paper suggests a novel method named DF-LS-SVM, which is based on least squares support vector machines (LS-SVM) regression combined with data fusion (DF) to enhance the ability to extract characteristic information and improve the quality of the regression. Simultaneous multicomponent determination of Fe(III), Co(II) and Cu(II) was conducted for the first time by using the proposed method. Data fusion is a technique that integrates information from disparate sources to produce a single model or decision. The LS-SVM technique allows for learning a high-dimensional feature with fewer training data, and reduces the computational complexity by only requiring the solution of a set of linear equations instead of a quadratic programming problem. Experimental results showed that the DF-LS-SVM method was successful for simultaneous multicomponent determination even when severe overlap of spectra existed. The DF-LS-SVM method is an attractive and promising hybrid approach that combines the best properties of the two techniques. The results obtained from an additional test case, simultaneous differential pulse voltammetric determination of o-nitrophenol, m-nitrophenol and p-nitrophenol, also demonstrated that the DF-LS-SVM method performed somewhat better than LS-SVM and PLS methods.     

Autoadaptative sequential injection system for nitrite determination in wastewaters

A novel autoadaptative sequential injection system for the analysis of nitrite is described. The automatic determination uses a direct spectrophotometric method, based on the Griess-Ilosvay reaction. In this method the absorbance of the purple azo dye formed is measured at 555 nm. In the sequential injection operation, the sample and the reagent are aspirated and mixed by reverse flow. The sequencing and overlapping of stacked (reagent) zones as well as selection of volumes have been studied in detail. The proposed analytical system is intelligent, simple and robust, allowing for nitrite determination in a double concentration range, by a simple and automatic programmable operation change. These two ranges are 0.0-3.0 and 0.0-20.0 ppm with detection limits being 0.048 and 0.4 ppm, respectively. Next surroundings have been developed allowing autocalibration and independent monitoring of nitrite concentration. The experimental set-up has been evaluated applying it to real samples analysis of very diverse concentration samples coming from a WWTP. The throughput of the method was 12 samples per hour.     

Membrane switch hypothesis. 1. Cell density influences lateral domain structure of tumor cell membranes

The domain structure of human cancer cells membranes was investigated by electron paramagnetic resonance (EPR) in different phases of cell growth, and the results were compared to those obtained for nonmalignant cells. On the basis of computer simulation of the EPR spectra using a newly developed GHOST condensation routine it was suggested that plasma membranes of cancer cells have less lateral lipid domain types at confluent conditions than in the exponential growing phase, while in nonmalignant cells the domain structure does not change significantly during cell growth. In accordance to our experimental data we propose a membrane switch hypothesis: disappearance of certain membrane domain types might act as a switch promoting the clustering of membrane constituents into the active units in a common lipid membrane domain and thus influencing the physiology of cells.     

New experiments for the quantification of counterion condensation

The condensation of counterions is an important aspect of charged macromolecules. Therefore an experimental characterization of the condensation of counterions is desirable. In this contribution two experimental techniques for the characterization of counterion condensation are introduced and compared: Anomalous Small Angle X-Ray Scattering (ASAXS) is able to probe the spatial distribution of counterions and electrophoresis nuclear magnetic resonance (NMR) measures counterion condensation via the effective charge obtained from the dynamic behaviour of molecules and complexes in an electric field.     

Flow Injection Chemiluminescence Determination of Gentamycin Using On-Line Electrogenerated Co(III) As the Oxidant

ABSTRACT

A novel flow-injection Chemiluminescence (CL) system for the determination of gentamycin is described, based on the direct chemiluminescence reaction of gentamycin and Co(III) in sulfuric acid media. The unstable strongly oxidative Co(III) was electrogenerated on-line with constant current electrolysis. The chemiluminescence intensity was linear with gentamycin concentrations of 0.01～80μg/ml, the detection limit being 0.005μg/ml. The whole process could be completed in 1 min with a relative standard deviation of less than 3.2%. The proposed method is rapid and simple and is suitable for automatic and continuous analysis. This method has been applied satisfactorily to the analysis of gentamycin in pharmaceutical preparations.     

Synthesis and characterization of a novel carbazole cyclic oligomer and main-chain polymer

The efficient synthesis of a novel cyclic carbazole tetramer and carbazole main-chain polymer via the Knoevenagel condensation has been developed. The carbazole cyclic tetramer could be obtained in a high yield by a one-stage Knoevenagel condensation of 3,6-diformyl-9-heptylcarbazole and 3,6-bis(cyanoacetoxymethyl)-9-heptylcarbazole in tetrahydrofuran (THF) without the use of the high-dilution principle. The corresponding carbazole main-chain polymer could also be obtained as a main product by a two-stage Knoevenagel polycondensation. Detailed structural characterization of this novel oligomer by spectroscopy and elemental analysis confirmed the cyclic structure. The corresponding main-chain polymer with large molecular weight was found to be amorphous by differential scanning calorimetry. Studies on the nonlinear optical and photorefractive properties of these materials are in progress. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35 : 2041–2047, 1997