Selection of surfactants for cell lysis in chemical cytometry to study protein-DNA interactions

Protein-DNA interactions play a defining role in many cellular processes. Studying such interactions at the single-cell level is important and challenging. Here we make the first step toward achieving this goal with chemical cytometry. Chemical cytometry utilizes capillary separation for detailed chemical analyses of single cells. The cell is injected into a capillary, lysed, and its components are analyzed by CE or capillary chromatography with highly sensitive detection. In order to apply chemical cytometry to studies of protein-DNA interactions, cell lysis must not destroy protein-DNA complexes. Surfactants represent the most practical means of cell lysis inside the capillary. This work aimed at finding surfactants and lysis conditions that do not destroy protein-DNA complexes. We studied three groups of surfactants--ionic, zwitterionic, and nonionic--with respect to their ability to lyse the cell membrane without significantly influencing the stability of protein-DNA complexes. Nonequilibrium CE of equilibrium mixtures with surfactants in the equilibrium mixtures and in the run buffer was used to measure the equilibrium constant, K(d), and rate constant, k(off), of protein-DNA complex dissociation. We found that nonionic surfactants worked best: they lyse the plasma membrane without significantly influencing K(d), k(off), or the EOF. This work creates the foundation for studies of protein-DNA interactions in single cells by chemical cytometry.     

Ligandless temperature-controlled ionic liquid-phase microextraction of lead(II) ion prior to its determination by FAAS

We describe the application of temperature-controlled ionic liquid based microextraction (TC-IL-ME) of lead(II) ion. The method does not require the use of an organic solvent or a ligand. Rather, the IL is directly added to the aqueous sample containing Pb(II) in a centrifuge tube, and the mixture is heated to 80 °C for 4 min. After cooling at 0 °C, the solution turns cludy due to the formation of fine droplets of the IL containing Pb(II). The IL is separated by centrifugation, acidified, and directly submitted to FAAS by microinjection. The effects of pH value, volume of IL, extraction time, temperature, sample volume and matrix were optimized to result in a preconcentration factor of 30, a detection limit of 5.8 μg L^?1, and a limit of quantification of 19.3 μg L^?1. The method was validated by analyzing a certified reference material (NCSZC81002B; hair). A recovery test performed with spiked samples gave values between 102 % and 105 %. The method was also used to determine Pb(II) in hair samples.

Anti-Alzheimer and Anti-COX-2 Activities of the Newly Synthesized 2,3′-Bipyridine Derivatives (II)

3-Amino-4-aryl-6-pyridin-3-ylthieno[2,3-b]pyridine-2-carbohydrazides 3a,b were used as the starting materials in the present study. Our targets here were represented by the synthesis of several 3-amino-N′-4-arylmethylenes 5a,b, N-formamides 8a,b, ethyl imidoformate 10b, N,N-dimethyl-N′-imidoformamides 12a,b, N-acetyl-N-acetamides 14a,b, 1,3,4-oxadiazole-2-thiol 16a,b, pyrazolothienopyridines 20a,b, 2-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]-4-aryl-6-pyridin-3-ylthieno[2,3-b]pyridin-3-amines 22a,b, 2-carbonyl-5-methyl-2,4-dihydro-3H-pyrazol-3-ones 24a,b, and 2-carbonyl-pyrazolidine-3,5-diones 26a,b. The newly synthesized heterocyclic compounds were tested as anti-Alzheimer and anti-COX-2 agents, and their structures were elucidated by considering the data of IR, ¹H NMR, mass spectra, and elemental analyses.

Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.

     

Synthesis of Nano-Cadmium Sulfide by Pulsed Laser Ablation in Liquid Environment

A pulsed laser-assisted in liquid environment method has been developed successfully to synthesize size-tunable (5–12 nm) and different shapes (sphere, rod, rope) of nano II–VI semiconductor (cadmium sulfide). This method can be carried out in two ways; the first one is the top-down technique, which has been discussed in publications in the last few decades, and the other one is the bottom-up technique, which appears for the first time in this paper. X-ray diffraction, ultraviolet-visible spectroscopy, and transmission electron microscopy confirm that the nanoparticles are crystalline. The methods lead to the production of nanomaterials, which are important for photonics and biosensing applications. Both synthesized methods can be applied in all materials because of their ability to ablate almost all kinds of materials due to the ultrahigh energy density and control over the growth process by manipulating the process parameters such as intensity, wavelength, and so on.     

Synthesis of chromenone,pyrimidinone, thiazoline,and quinolone derivatives as prospective antitumor agents

Hydrazide-hydrazone namely, 2-cyano-N′-((1-phenyl-3-[thiophen-2-yl]-1H-pyrazol-4-yl)methylene)acetohydrazide (3) underwent a series of reactions with some chemical reagents to construct new biologically active N-heterocycles, for example, chromenone, benzochromenone, thiazoline, and quinolone derivatives. Treating the nitrile derivative 3 with 2,4-dichlorobenzaldehyde and pyrazole aldehyde 1 afforded the corresponding condensed products. Some of the synthesized compounds were screened for their in vitro antitumor activities against two different human tumor cell lines including hepatocellular liver carcinoma (HepG2) and breast adenocarcinoma (MCF7) activities. Compound 3 was the most potent against the two tumors.     

Effect of Gamma Radiation on the IR-Spectra of Polymethylmethacrylate

The IR-spectra of polymethylmethacryate (PMMA) have been measured before and after irradiation in air, in the region of wave number 700–4000 cm^?1. The results showed that the rate of decrease in the absorbance of the IR absorption bands correlated with the type of bond, and the type of vibration whether being stretching, bending or rocking one.

Es wurden die IB-Spektren von Polymethylmethacrylat vor und nach Bestrahlung mit Gammastrahlen in Luft im Wellenzahlbereich von 700 — 4000 cm?1 untersucht. Die Ergebnisse zeifgten, daβ die Geschwindigkeit der Abnahme der IR-Absorptionsbanden mil der Art der Bindung korreliert werden konnten und mit der Art der Schwingung, ob Streck-, Deformations- oder Schaukelschwingung.     

Highly sensitive UHPLC–DAD method for simultaneous determination of two synergistically acting antiepileptic drugs; levetiracetam and lacosamide: Application to pharmaceutical tablets and human urine

A simple and highly sensitive ultra‐high‐performance liquid chromatographic–diode array (UHPLC‐DAD) detection method was developed and validated for the simultaneous estimation of levetiracetam (LEV) and lacosamide (LAC). It was clinically proven that the combination of LEV and LAC exhibits a synergistic effect against refractory seizures in mice, which was the motivation for the analysis of this binary mixture both in bulk and in human urine samples. The binary mixture was resolved on a Hypersil BDS C₁₈ analytical column, utilizing a mobile phase of 0.050 mol L^?1 phosphate buffer (pH 5.60), methanol and acetonitrile in the ratio (80:10:10 v/v/v) using catechol as an internal standard. The mobile phase was pumped at a flow rate of 1.2 mL min^?1 with diode array detection at 205 nm for both drugs and 270 nm for IS. Calibration curves were linear with correlation coefficient >0.9990 over the studied concentration range of 0.1–70.0 μg mL^?1 for both drugs. The developed method was reproducible with low relative standard deviation values for intra‐ and inter‐day precision (<2.0%). Both drugs were determined in bulk, pharmaceutical formulations and human urine samples without any interference from complex matrices.     

Pencil Graphite Electrode Decorated with Xylenol Orange Flakes for Studying Possible Pharmacokinetic Interaction Between Vardenafil and Daclatasvir

The main aim of the current work is to investigate possible pharmacokinetic interactions between vardenafil hydrochloride (VAR), which is used for the treatment of erectile dysfunction and daclatasvir dihydrochloride (DAC), which is used for the treatment of chronic hepatitis C viral infection when they are concomitantly administered. Therefore, a sensitive and selective square‐wave voltammetric method was developed and validated for simultaneous determination of VAR and DAC using disposable pencil graphite electrode (PGE) modified with xylenol orange (X.O.) flakes as an electrochemical sensor. A full investigation of the experimental parameters for obtaining the highest electroanalytical signal with sufficient resolution between the oxidation peaks of two compounds was performed. It was found that VAR and DAC were resolved on X.O./PGE with different potentials at 1.4 V and 0.9 V, respectively using Britton‐Robinson buffer (pH 2.2) and 0.1 mol L^?1 KCl as a supporting electrolyte. In addition, with the aid of cyclic voltammetry, a mechanistic scheme for the oxidation behaviour of both VAR and DAC was suggested. The proposed square wave voltammetric method was successfully applied for trace quantification of VAR and DAC in male rabbits. The suggested approach shows detection and quantification limits in rabbit plasma samples of 0.06 and 0.17 μmol L^?1, respectively for VAR and 0.13 and 0.39 μmol L^?1, respectively for DAC. The pharmacokinetic parameters of VAR alone and in combination with DAC after oral administration to rabbits were successfully estimated. The obtained results confirm that when DAC is co‐administered with VAR, plasma concentration of VAR increases, which necessitates dose adjustment for VAR to prevent toxicological consequences in patients.     

Chemical Constitution and Antimicrobial Activity of Kombucha Fermented Beverage

Kombucha is a traditional beverage of sweetened black tea fermented with a symbiotic association of acetic acid bacteria and yeasts. In this study, kombucha fermented beverage (KFB) appeared to include nine chemical groups (alcohols, acids, lactones, condensed heterocyclic compounds, antibiotics, esters, aldehydes, fatty acids, and alkaloids) of many bioactive metabolites, as elucidated by gas chromatography–mass spectrometry (GC-MS) and IR spectra. The fermented metabolic components of KFB seem collectively to act in a synergistic action giving rise to the antimicrobial activity. Four types of kombucha preparations (fermented, neutralized, heat-treated and unfermented) were demonstrated with respect to their antimicrobial activity against some pathogenic bacterial and fungal strains using agar well diffusion assay. KFB exerted the strongest antimicrobial activities when compared with neutralized and heat-treated kombucha beverages (NKB and HKB). Staphylococcus aureus ATCC6538 (S. aureus) and Escherichia coli ATCC11229 (E. coli) were the organisms most susceptible to the antimicrobial activity of kombucha beverage preparations. Finally, the KFB preparation showed remarkable inhibitory activity against S. aureus and E. coli bacteria in a brain heart infusion broth and in some Egyptian fruit juices (apple, guava, strawberry, and tomato). These data reveal that kombucha is not only a prophylactic agent, but also appears to be promising as a safe alternative biopreservative, offering protection against pathogenic bacteria and fungi.