In-situ reduction of Ag+ on black phosphorene and its NH2-MWCNT nanohybrid with high stability and dispersibility as nanozyme sensor for three ATP metabolites.

The environmental stability, water-processibility and life-span of black phosphorene (BP) severely limit the application of its electronic devices in aqueous system containing oxygen. We reported the controllable preparation of in-situ reduction and deposition of silver nanoparticles on the BP surface and its amino-functionalized multi-walled carbon nanotubes (NH2-MWCNT) nanocomposite. With the addition of both NH2-MWCNT and Ag+, the BP-based nanocomposite was prepared by ultrasonic-assisted liquid-phase exfoliation and was dispersed in carboxymethyl cellulose sodium (CMC) aqueous solution. The morphology, microstructure, and electrochemical properties of the nanohybrid were characterized. NH2-MWCNT-BP-AgNPs showed high environmental stability, good water-processibility, satisfactory life-spans, superior electrocatalytic capacity with enzyme-like kinetic characteristics. The nanohybrid was applied as electrochemical sensors for single/simultaneous analysis of uric acid (UA), xanthine (XT) and hypoxanthine (HX). Excellent voltammetric responses for simultaneous determination in linear ranges of 0.1-800 µM with a limit of detection (LOD) of 0.052 µM for UA, 0.5-680 µM with a LOD of 0.021 µM for XT, and 0.7-320 µM with a LOD of 0.025 µM for HX under optimal conditions. Besides, the developed nanozyme sensor was employed for simultaneous voltammetric analysis of UA, XT and HX in real samples with acceptable recoveries. This work will provide theoretical guidance and experimental support for the preparation and application of two-dimensional materials, nanozymes and sensing devices.

Simultaneous detection of ATP metabolites in human plasma and urine based on palladium nanoparticle and poly(bromocresol green) composite sensor.

A sensitive voltammetric sensor based on palladium nanoparticles (PdNPs) and poly-bromocresol green (pBG) composite layer immobilized on amide functionalized single-walled carbon nanotubes (AmSWCNTs) modified pyrolytic graphite (PdNPs:pBG/AmSWCNTs/PG) has been prepared for the simultaneous determination of adenosine triphosphate (ATP) catabolites, inosine (INO), hypoxanthine (HX), xanthine (XT), and uric acid (UA). The modified PdNPs:pBG/AmSWCNTs/PG was characterized by electrochemical experiments and surface analysis, which exhibited exceptional electrocatalytic effects towards the oxidation of INO, HX, XT, and UA with a significant enhanced peak current and well resolved peaks separation for all the analytes. The linear calibration curves were obtained in the concentration range of 0.001-175 µM, 0.001-200 µM, 0.001-150 µM, and 0.001-200 µM and limits of detection were found as 0.95 nM, 1.04 nM, 1.07 nM, and 0.43 nM corresponding to INO, HX, XT, and UA, respectively. The common metabolites present in the biological fluids did not interfere in the determination. The applicability of the proposed sensor was successfully demonstrated by determining INO, HX, XT, and UA in the human plasma and urine and the obtained results were validated by using HPLC.

Lysinibacillus fusiformis M5 Induces Increased Complexity in Bacillus subtilis 168 Colony Biofilms via Hypoxanthine.

In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, and systems biology, among others. The sociomicrobiology of multispecies biofilms, however, is still poorly understood. Here, we report a screening system that allowed us to identify soil bacteria which induce architectural changes in biofilm colonies when cocultured with Bacillus subtilis We identified the soil bacterium Lysinibacillus fusiformis M5 as an inducer of wrinkle formation in B. subtilis colonies mediated by a diffusible signaling molecule. This compound was isolated by bioassay-guided chromatographic fractionation. The elicitor was identified to be the purine hypoxanthine using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. We show that the induction of wrinkle formation by hypoxanthine is not dependent on signal recognition by the histidine kinases KinA, KinB, KinC, and KinD, which are generally involved in phosphorylation of the master regulator Spo0A. Likewise, we show that hypoxanthine signaling does not induce the expression of biofilm matrix-related operons epsABCDEFGHIJKLMNO and tasA-sipW-tapA Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation of B. subtilis biofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression but rather is caused by the excess of hypoxanthine within B. subtilis cells, which may lead to cell stress and death.IMPORTANCE Biofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be beneficial, for instance, in crop protection. In nature, biofilms are commonly found as multispecies communities displaying complex social behaviors and characteristics. The study of interspecies interactions will thus lead to a better understanding and use of biofilms as they occur outside laboratory conditions. Here, we present a screening method suitable for the identification of multispecies interactions and showcase L. fusiformis as a soil bacterium that is able to live alongside B. subtilis and modify the architecture of its biofilms.

[Constituents from the leaves of Aquilaria sinensis].

OBJECTIVE: To study the chemical constituents of the leaves of Aquilaria sinensis. METHOD: The compounds were isolated and purified by the methods of solvent extraction and chromatographic technique, and their structures were identified on the basis of the analyses of spectral data. RESULT: Thirty-three compounds were obtained. Among them, twelve compounds were identified as 5-hydroxyl-7,4'-dimethoxyflavone (1), acacetin (2), luteolin (3), genkwanin (4), yuankanin (genkwanin-5-O-beta-D-primeveroside, 5), adenosine (6), genkwanin-5-O-beta-D-glucopyranoside (7), hypolaetin-7-O-beta-D-glucopyranoside (8), hypoxanthine (9), uracil (10), 8-C-beta-D-galactopyranosylisovitexin (11), and 4-(1,2,3-trihydroxypropyl) -2,6-dimethoxyphenyl-1-O-beta-D-glucopyranoside (12), respectively. CONCLUSION: All compounds except for 1, 3 and 4 were isolated from the leaves of A. sinensis for the first time.

Overexpressed proteins may act as mops removing their ligands from the host cells: a case study of calf PNP.

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes--moping of ligands from the host cells--may take place in the case of other proteins with high overexpression yield.

Fabrication and characterization of open-tubular CEC modified with tentacle-type metal-chelating polymer chains.

A novel stationary phase with tentacle-type metal-chelating polymer chains was fabricated for open-tubular CEC. The preparation procedure of the stationary phase included the synthesis of monomer, silanization of capillary inner wall, in situ polymerization, and metal complexation. The effects of initiator concentration and reaction time on the column capacity were investigated. To compare with the tentacle-type metal-chelating capillary column, a monolayer ligand-modified capillary was also prepared. Immobilized copper(II) capacity of the tentacle-type polymer stationary phase was nearly 900 times higher than that of the monolayer one. The electroosmotic mobility was examined for its dependence on pH as well as phosphate and ACN concentrations. The tentacle-type metal-chelating capillary with high ligand capacity has proven to afford better retention and resolution for the separation of phenylalanine, tryptophan, and tyrosine mixtures and three purine derivatives. The separation was considered to be effected by a combination of ligand exchange and electrophoretic mobility.

[Identification of 5 constituents of the aqueous extract of Isatis indigotica by HPLC-MS2].

OBJECTIVE: To identify five constituents in the aqueous extract of Isatis indigotica Fort. METHODS: After separation of the aqueous extract of Isatis indigotica Fort. by HPLC, the eluates of five peaks were collected separatively and analysed by MS2. UV spectra and MS2 were compared with those of reference standards of cytidine, uridine, guanosine xanthine and hypoxanthine. RESULTS: Each HPLC elute of the aqueous extract had same retention time, UV spectra and fragment pattern in the MS2 spectrum as the corresponding standards. CONCLUSION: Five constituents of the aqueous extract of Isatis indigotica Fort. are identified as cytidine, hypoxanthine, uridine, xanthine and guanosine.

Screening for biologically active metabolites with endosymbiotic bacilli isolated from arthropods.

Endosymbiotic bacteria from the genus Bacillus were isolated from different compartments of the gut of various members of insects (Hexapoda) and millipedes (Diplopoda). They were grown in submerged culture and investigated by biological assays and HPLC-diode array analysis regarding their production of bioactive metabolites, which were isolated and determined in structure. Known compounds and yet unknown derivatives from the primary metabolism were detected, as well as antibacterially and antifungally acting peptide antibiotics.

Separation of inosine, hypoxanthine, and guanosine by high-performance liquid chromatography on silica.

The separation of inosine (Ino), hypoxanthine (Hyp), and guanosine (Guo) on silica has been studied. In adsorption normal-phase systems the peak shapes are unsatisfactory; a low selectivity has been observed for the Ino-Hyp and Guo-Hyp pairs. Chromatograms can be significantly improved if systems with a mixed partition-adsorption retention mechanism are applied.

[Separation and determination of purine bases and pyrimidine bases from nucleic acid hydrolysis by HPLC on BDS column].

The hydrolysates of nucleic acid, six purine bases and pyrimidine bases (cytosine, uracil, guanine, hypoxanthine, adenine and thymine) were separated and determined by using HPLC. It is discussed how the column and mobile phase affect the separation. The peaks of cytosine and adenine are tailed on ordinary C18 column, and they are very good on BDS-C18 column. The KH2PO4-H3PO4 buffer can be used in separation the hydrolysates of RNA and DNA, and the NaAc-HAc buffer is only used in DNA. In addition, pH value is a very important factor for separation. With pH value of mobile phase increasing, the retention times of guanine, hypoxanthine and thymine were first increased and then decreased, adenine was increased, and cytosine and uracil were almost constant. The chosen mobile phase was 0.1 mol/L KH2PO4-H3PO4 buffer, with a pH value of 4.05. It was detected at UV 260 nm. The determination was completed within 10 min. The RSDs were all less than 3% and the recoveries were in the range of 82%-114%. The method has been applied to the detection of yeast hydrolysates.

[Chemical constituents of rhizoma Pinelliae pedatisecta].

Five compounds were isolated from the alkaloid extract of Rhizoma Pinelliae Pedatisecta and their structures were determined as pedatisectine F(I), hypoxanthine (II), erythritol (III), uridine (IV) and pedatisectine G (V), of which I and V are new compounds, while II, III and IV were found in this plant for the first time.