Surface imprinted bio-nanocomposites for affinity separation of a cellular DNA repair protein

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and “interactomes” of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio-nanocomposite with antibody-like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin-modified surface of silica-coated magnetic nanoparticles, we first added 3-aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2-acrylamido-2-methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG-NH₂). The resulting molecularly imprinted polymer-based bio-nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio-nanocomposite with high activity. The bio-nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.     

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.     

Circumscription of Fulbrightiella gen. nov. and Sherwoodiella gen. nov., Two Novel Genera in the Calotrichaceae (Nostocales,Cyanobacteria)

Three novel strains in Calotrichaceae from tropical habitats were isolated and characterized with regard to their morphology, phylogenetic placement, and secondary structures of conserved domains in the 16S-23S internal transcribed spacer (ITS). The strains fell into two clades formerly identified as Calothrix from freshwater and brackish habitats. Based on both morphology and ecology, they differed from the type species of Calothrix, C. confervicola, which is marine, has wide trichomes with short cells, and narrows abruptly to a hyaline hair. The first clade grouped species with heteropolar filaments widened at the base and narrowed gradually toward the apex but not ending in a hair, with basal heterocytes that are formed in series as the apically placed heterocytes senesce; this clade is being named Fulbrightiella gen. nov., with two named species, F. bharadwajae sp. nov. and F. oahuensis sp. nov. The second clade was comprised of a single species with isopolar trichomes that are untapering as hormogonia, but which widen midfilament and taper toward both ends following growth. These trichomes develop pairs of heterocyte mid-filament, causing fragmentation into heteropolar trichomes with basal heterocytes and ends that taper, but not to a hair. This clade consists of a single species at present, Sherwoodiella mauiensis. With this action, four clades in the Calotrichaceae have been named: Macrochaete, Dulcicalothrix, Fulbrightiella, and Sherwoodiella. Calothrix sensu stricto is truly marine, morphologically distinct, and unsequenced; finding and sequencing the generitype for Calothrix remains as the most important and unfinished task in the revision of the Calotrichaceae.     

βVI Turns in peptides and proteins: A model peptide mimicry

To investigate the role of proline in defining β turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L - and D -proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L -proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D -proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala-), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo(-Pro-Ala-Ala-Ala-Ala--), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon βVI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated. © 1993 Wiley-Liss, Inc.     

A Rapid Determination of 30 Prohibited Pesticide Residues in Lycii Fructus by UPLC-MS/MS

Prohibited pesticide residues have become one of the main factors affecting the quality and safety of Lycii Fructus, However, rarely studies focus on the rapid determination of these residues. Here, a total of 30 kinds of prohibited pesticide residues were determined by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-MS/MS) in five different process ways. Pretreatment methods, chromatographic separation and detection conditions in mass spectrometry were all optimized accordingly. Among the five different pretreatment methods, the first and third solid phase extraction failed to provide high recoveries of sulfosulfuron compounds (both lower than 60%). Recovery of chlorphenamidine by the Quick Easy Cheap Effective Rugged and Safe multiresidue method (QuEChERS) was lower than 60%, which did not meet the requirements of trace determination. The concentrations of 30 prohibited pesticides residues treated by straightforward and solid phase extraction showed good linearity in their corresponding ranges, with correlation coefficients over 0.99. The average recoveries of straightforward ranged from 78.13% to 110.9%, while RSD ranged from 1.3% to 16.9%, albeit poor purification was observed. The recovery yield from solid phase extraction was between 67.75% and 103.08% with RSD value from 0.8% to 14.0%, which met the requirements of trace determination, this method has good precision and stability. These results could be employed to other Traditional Chinese Medicines (TCMs) in detecting prohibited pesticide residues.     

Extraction,Antioxidant Activity and Identification of Flavonoids from Root Tubers of <i>Kosteletzkya virginica</i>

Kosteletzkya virginica (K. virginica) is used for revegetation of salt-affected coastal tidal flats and as a raw material of biodiesel. K. virginica root tuber, a biowaste with low economic value, is rich in bioactive compounds. This study aimed to extract and identify flavonoids from K. virginica root tubers. The optimal extraction conditions were 1/25 (w/v) solid/liquid ratio, 40% ethanol concentration at 40°C for 60 min. Under these conditions, 65.2 ± 3.7 mg/g total flavonoid content was extracted from the roots, which were collected from salinized soil in late autumn of the third year. Antioxidant activity was evaluated through 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radical, and superoxide anion scavenging assays. The extracted flavonoids exhibited antioxidant activity in a dose-dependent manner. Five flavonoids, glucoliquiritin apioside, licoisoflavone B, 5-methoxy-7,8-diprenyl- flavone, 7,2′-dihydroxy-6,8-dimethyl-4′,5′-methylenedioxyflavan, and 5,7,4′-trihydroxy-3′-methoxy-6,8-di-Cmethylflavanone, were identified by ultra-performance liquid chromatography–tandem mass spectrometry. Our results suggest that the flavonoids of K. virginica root tubers might be potent antioxidants and can be effectively applied as an ingredient in food and natural medicine.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.