Development of a rapid visual detection technology for BmNPV based on CRISPR/Cas13a system.

Pathogenic microorganism of silkworm are important factors that threaten the high-quality development of sericulture. Among them, Bombyx mori nucleopolyhedrovirus (BmNPV) caused diseases often lead to frequent outbreaks and high mortality, resulting in huge losses to sericultural industry. Current molecular detection methods for BmNPV require expensive equipment and sikilled technical personnel. As a result, the most commonly detection method for silkworm egg production enterprises involves observing the presence of polyhedra under a microscope. However, this method has low accuracy and sensitivity. There is an urgent need to develop a new detection technology with high sensitivity, high specificity, and applicability for silkworm farms, silkworm egg production enterprises and quarantine departments. In this study, we successfully established the CRISPR/Cas13a BmNPV visualized detection technology by combining Recombinase Polymerase Amplification (RPA) technology and CRISPR/Cas13a system. This technology is based on microplate lateral, flow test strips and portable fluorescence detector. The detection sensitivity can reach up to 1 copies/µL for positive standard plasmid and 1 fg/µL for BmNPV genome in 30-45 min, demonstrating high sensitivity. By detecting silkworm tissues infected with different pathogens, we determined that CRISPR/Cas13a detection technology has good specificity. In summary, the newly established nucleic acid detection technology for BmNPV is characterized by high sensitivity, high specificity, low cost and convenience for visualization. It can be applied in field detection and silkworm egg quality monitory system.

Synthesis, structure and antiproliferative and optical activities of two new biphenyl-derived Schiff bases.

Two novel Schiff bases derived from indole and biphenyl have been designed and synthesized, namely 3-((E)-{(E)-[1-(biphenyl-4-yl)ethylidene]hydrazinylidene}methyl)-1-methyl-1H-indole (3-BEHMI) acetonitrile monosolvate, C24H21N3·CH3CN, and 3-((E)-{(E)-[1-(biphenyl-4-yl)ethylidene]hydrazinylidene}methyl)-1-methyl-1H-indole (3-BEHEI) acetonitrile monosolvate, C24H21N3·CH3CN. Their structures were characterized by elemental analysis, quadrupole time-of-flight MS, NMR and UV-Vis spectroscopy. The single-crystal packing structure of 3-BEHMI is largely dominated by C-H...π interactions and weak van der Waals interactions. The in vitro cytotoxicity of the two title compounds have been evaluated against two tumour cell lines (A549 human lung cancer and 4T1 mouse breast cancer) and two normal cell lines (MRC-5 normal lung cells and NIH 3T3 fibroblasts) by MTT assay. The results indicate that 3-BEHEI exhibits a slightly weaker antiproliferative capability (IC50 = ∼50 µM) than the previously reported similar Schiff base 3-BEHI (IC50 = ∼20 µM). This is in line with docking results. 3-BEHMI demonstrates a weak cytotoxic activity, with IC50 values around 110 µM, which disagrees with its docking results. Overall, the tested compounds manifest relevant cytotoxicities on the selected cancer cell lines and normal cell lines. The UV-Vis and fluorescence spectra were recorded and reproduced through the TD-DFT method with four types of hybrid density functionals, including B3LYP, M062X, PBE1PBE and WB97XD.

Preparation of imprinted polymers at surface of magnetic nanoparticles for the selective extraction of tadalafil from medicines.

In this paper, highly selective core-shell molecularly imprinted polymers (MIPs) of tadalafil on the surface of magnetic nanoparticles (MNPs) were prepared. Three widely used functional monomers 2-(trifluoromethyl) acrylic acid (TFMAA), acrylic acid (AA), and methacrylic acid (MAA) were compared theoretically as the candidates for MIP preparation. MIP-coated magnetic nanoparticles (MIP-coated MNPs) showed large adsorption capacity, high recognition ability, and fast binding kinetics for tadalafil. Furthermore, because of the good magnetic properties, MIP-coated MNPs can achieve rapid and efficient separation with an external magnetic field simply. The resulting MIP-coated MNPs were used as dispersive solid-phase extraction (DSPE) materials coupled with HPLC-UV for the selective extraction and detection of tadalafil from medicines (herbal sexual health products). Encouraging results were obtained. The amounts of tadalafil that were detected from the herbal sexual health product was 43.46 nmol g(-1), and the recoveries were in the range of 87.36-90.93% with the RSD < 6.55%.

Quantification of pantoprazole in human plasma using LC-MS/MS for pharmacokinetics and bioequivalence study.

A highly sensitive and rapid method for the analysis of pantoprazole in human plasma using liquid chromatography coupled to tandem electrospray ionization mass spectrometry was developed. The procedure involves a simple protein precipitation method with methyl alcohol and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 384.1→200.0 and m/z 346.1→198.0, for quantification of pantoprazole and IS, respectively. The standard calibration curves showed good linearity within the range of 5-5,000 ng mL(-1). The lower limit of quantitation (LLOQ) was about 5 ng mL(-1). The extractive recovery of pantoprazole from the biological matrix was more than 77.58% and the matrix effect was complied with relevant provision. The intra-day accuracy of the drug containing serum samples was more than 92.19% with a precision of 0.79-5.36%. The inter-day accuracy was 85.49% or more, with a precision of 0.91-12.67%. Intra and inter-day accuracy of the assay at four concentrations were 97.9-98.2% with a precision of 4.2-13.9%. This method offered good precision and accuracy and was successfully applied to the pharmacokinetic and bioequivalence studies of 40 mg of enteric-coated pantoprazole in 20 healthy Chinese volunteers.

Determination of itopride hydrochloride in human plasma by RP-HPLC with fluorescence detection and its use in bioequivalence study.

A sensitive, selective and simple method using a precipitation of protein with 10% perchloric acid, followed by high-performance liquid chromatography (HPLC) with fluorescence detection was developed for the determination of itopride hydrochloride in human plasma, using levofloxacin as the internal standard (IS). Chromatographic separation was obtained within 7.0 min using a reverse phase Hypersil BDS C(18) (250 mm x 4.6 mm, 5 microm) column and an isocratic mobile phase, constituting of a mixture of 0.1 mol/l ammonium acetate-methanol (30:70, v/v) flowing at 1.1 ml/min. The excitation and emission wavelengths were set at 304 and 344 nm, respectively. The method was validated over the concentration range of 5 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 5 ng/ml. The extractive recovery of itopride hydrochloride from the biological matrix was more than 80.77%. The intra-day accuracy of the drug containing serum samples was more than 82.94% with a precision of 2.81-4.37%. The inter-day accuracy was 82.91% or more, with a precision of 6.89-9.54%. The limit we have used (70-143%) is based on the local regulatory authority (SFDA). The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.