An automatic flow-based system as a front end to liquid chromatography (LC) for on-line dynamic leaching of microplastic materials (polyethylene of medium density and poly(vinyl chloride)) with incurred phthalates and bisphenol A is herein presented. The microplastic particles were packed in a metal column holder, through which seawater was pumped continuously by resorting to advanced flow methodology. Each milliliter of the leachable (bioaccessible) fraction of chemical additives was preconcentrated on-line using a 10 mm-long octadecyl monolithic silica column placed in the sampling loop of the injection valve of a HPLC system that served concomitantly for analyte uptake and removal of the seawater matrix. After loading of the leachate fraction, the LC valve was switched to the inject position and the analytes were eluted and separated by a monolithic column (Onyx C18HD 100 × 4.6 mm) using an optimized acetonitrile/water gradient with UV detection at 240 nm. The automatic flow method including dynamic flow-through extraction, on-line sorptive preconcentration, and matrix clean-up was synchronized with the HPLC separation, which lasted ca. 9 min. The only two currently available multi-component certified reference materials (CRM) of microplastics (CRM-PE002 and CRM-PVC001) were used for method development and validation. Out of the eight regulated phthalates contained in the two CRMs, only the 2 most polar species, namely, dimethyl phthalate and diethyl phthalate as well as bisphenol A, were leached significantly by the seawater in less than 2 h, with bioaccessibility percentages of 51-100%. The leaching profiles were monitored and modeled with a first-order kinetic equation so as to determine the rate constants for desorption in a risk assessment scenario. Intermediate precision values of bioaccessibility data for three batches of CRMs were for the suite of targeted compounds ≤22%. This work for the first time reports a fully automatic flow method with infinite sink capacity (i.e., using a surplus of extracting solution) for the target species able to mimic the leaching of additives from plastic debris across the water body in marine settings under worst-case extraction conditions.
Vernix caseosa, the waxy substance that coats the skin of newborn babies, has an extremely complex lipid composition. We have explored these lipids and identified nonhydroxylated 1-O-acylceramides (1-O-ENSs) as a new class of lipids in vernix caseosa. These ceramides mostly contain saturated C11-C38 ester-linked (1-O) acyls, saturated C12-C39 amide-linked acyls, and C16-C24 sphingoid bases. Because their fatty acyl chains are frequently branched, numerous molecular species were separable and detectable by HPLC/MS: we found more than 2,300 molecular species, 972 of which were structurally characterized. The most abundant 1-O-ENSs contained straight-chain and branched fatty acyls with 20, 22, 24, or 26 carbons in the 1-O position, 24 or 26 carbons in the N position, and sphingosine. The 1-O-ENSs were isolated using multistep TLC and HPLC and they accounted for 1% of the total lipid extract. The molecular species of 1-O-ENSs were separated on a C18 HPLC column using an acetonitrile/propan-2-ol gradient and detected by APCI-MS, and the structures were elucidated by high-resolution and tandem MS. Medium-polarity 1-O-ENSs likely contribute to the cohesiveness and to the waterproofing and moisturizing properties of vernix caseosa.
- MeSH
- Ceramides metabolism MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Mass Spectrometry MeSH
- Skin metabolism MeSH
- Humans MeSH
- Lipids blood MeSH
- Magnetic Resonance Spectroscopy MeSH
- Infant, Newborn MeSH
- Gas Chromatography-Mass Spectrometry MeSH
- Vernix Caseosa metabolism MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Humans MeSH
- Infant, Newborn MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutations introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude, and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method's application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as α/β-hydrolases containing > 100 000 protein sequences based on the Pfam database.
- MeSH
- Alkanes chemistry metabolism MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Biocatalysis MeSH
- Databases, Protein MeSH
- Escherichia coli genetics metabolism MeSH
- Gene Expression MeSH
- Fluorescence Polarization MeSH
- Hydrocarbons, Halogenated chemistry metabolism MeSH
- Hydrolases chemistry genetics metabolism MeSH
- Catalytic Domain MeSH
- Kinetics MeSH
- Cloning, Molecular MeSH
- Ligands MeSH
- Mutagenesis, Site-Directed MeSH
- Protein Engineering * MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Solvents chemistry MeSH
- High-Throughput Screening Assays MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
Currently, the interest in microalgae as a source of biologically active components exploitable as supplementary ingredients to food/feed or in cosmetics continues to increase. Existing research mainly aims to focus on revealing and recovering the rare, cost competitive components of the algae metabolom. Because these components could be of very different physicochemical character, a universal approach for their isolation and characterization should be developed. This study demonstrates the systematic development of the extraction strategy that represents one of the key challenges in effective algae bioprospecting, which predefines their further industrial application. By using of Trachydiscus minutus as a model microalgae biomass, following procedures were tested and critically evaluated in order to develop the generic procedure for microalgae bioprospecting: (i) various ways of mechanical disintegration of algae cells enabling maximum extraction efficiency, (ii) the use of a wide range of extraction solvents/solvent mixtures suitable for optimal extraction yields of polar, medium-polar, and non-polar compounds, (iii) the use of consecutive extractions as a fractionation approach. Within the study, targeted screening of selected compounds representing broad range of polarities was realized by ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometric detection (UHPLC-HRMS/MS), to assess the effectiveness of undertaken isolation steps. As a result, simple and high-throughput extraction-fractionation strategy based on consecutive extraction with water-aqueous methanol-hexane/isopropanol was developed. Moreover, to demonstrate the potential of the UHPLC-HRMS/MS for the retrospective non-target screening and compounds identification, the collected mass spectra have been evaluated to characterize the pattern of extracted metabolites. Attention was focused on medium-/non-polar extracts and characterization of lipid species present in the T. minutus algae. Such detailed information on the composition of native (non-hydrolyzed) lipids of this microalga has not been published yet.
The aim of the work was to synthesize a molecularly imprinted material for the selective solid-phase extraction (SPE) of β-N-methylamino-L-alanine (L-2-amino-3-methylpropionic acid; BMAA) from cyanobacterial extracts. BMAA and its structural analogs that can be used as template are small, polar and hydrophilic molecules. These molecules are poorly soluble in organic solvents that are commonly used for the synthesis of acrylic-based polymers. Therefore, a sol gel approach was chosen to carry out the synthesis and the resulting sorbents were evaluated with different extraction procedures in order to determine their ability to selectively retain BMAA. The presence of imprinted cavities in the sorbent was demonstrated by comparing elution profiles obtained by using molecularly imprinted silica (MIS) and non-imprinted silica (NIS) as a control. The molecularly imprinted solid-phase extraction (MISPE) procedure was first developed in a pure medium (acetonitrile) and further optimized for the treatment of cyanobacterial samples. It was characterized by high elution recoveries (89% and 77% respectively in pure and in real media).The repeatability of the extraction procedure in pure medium, in real medium and the reproducibility of MIS synthesis all expressed as RSD values of extraction recovery of BMAA were equal to 3%, 12% and 5%, respectively. A MIS capacity of 0.34 µmol/g was measured. The matrix effects, which affected the quantification of BMAA when employing a mixed mode sorbent, were completely removed by adding a clean-up step of the mixed-mode sorbent extract on the MIS.
Ultra high performance liquid chromatography with quadrupole/time-of-flight mass spectrometry was applied to evaluate the potential of nontarget metabolomic fingerprinting in order to distinguish Fusarium-infected and control barley samples. First, the sample extraction and instrumental conditions were optimized to obtain the broadest possible representation of polar/medium-polar compounds occurring in extracts obtained from barley grain samples. Next, metabolomic fingerprints of extracts obtained from nine barley varieties were acquired under ESI conditions in both positive and negative mode. Each variety of barley was tested in two variants: artificially infected by Fusarium culmorum at the beginning of heading and a control group (no infection). In addition, the dynamics of barley infection development was monitored using this approach. The experimental data were statistically evaluated by principal component analysis, hierarchical clustering analysis, and orthogonal partial least-squares discriminant analysis. The differentiation of barley in response to F. culmorum infection was feasible using this metabolomics-based method. Analysis in positive mode provided a higher number of molecular features as compared to that performed under negative mode setting. However, the analysis in negative mode permitted the detection of deoxynivalenol and deoxynivalenol-3-glucoside considered as resistance-indicator metabolites in barley.
- MeSH
- Principal Component Analysis MeSH
- Fusarium physiology MeSH
- Mass Spectrometry MeSH
- Hordeum metabolism microbiology MeSH
- Metabolomics * MeSH
- Least-Squares Analysis MeSH
- Plant Diseases microbiology MeSH
- Cluster Analysis MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.
- MeSH
- Actins analysis MeSH
- Bridged Bicyclo Compounds, Heterocyclic pharmacology MeSH
- Cell Nucleus ultrastructure MeSH
- Cryptococcus neoformans drug effects physiology ultrastructure MeSH
- Dimethyl Sulfoxide pharmacology MeSH
- Microscopy, Electron MeSH
- Phalloidine analogs & derivatives MeSH
- Microscopy, Fluorescence MeSH
- Actin Cytoskeleton ultrastructure MeSH
- Microtubules drug effects MeSH
- Tubulin Modulators pharmacology MeSH
- Marine Toxins pharmacology MeSH
- Nocodazole pharmacology MeSH
- Rhodamines MeSH
- Free Radical Scavengers pharmacology MeSH
- Thiazolidines pharmacology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Ambient mass spectrometry employing a direct analysis in real time (DART) ion source coupled to a medium high-resolution/accurate mass time-of-flight mass spectrometer (TOFMS) was used as a rapid tool for metabolomic fingerprinting to study the effects of supplemental feeding with cereals (triticale) on the composition of muscle metabolites of common carp (Cyprinus carpio L.). First, the sample extraction and DART-TOFMS instrumental conditions were optimized to obtain the broadest possible representation of ionizable compounds occurring in the extracts obtained from common carp muscle. To this end, a simultaneous (all-in-one) extraction procedure was developed employing water and cyclohexane mixture as the extraction solvents. Under these conditions both polar as well as non-polar metabolites were isolated within a single extraction step. Next, the metabolomic fingerprints (mass spectra) of a large set of common carp muscle extracts were acquired. Finally, the experimental data were statistically evaluated using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Using this approach, differentiation of common carp muscle in response to dietary supplementation (feeding with and without cereals) was feasible. Correct classification was obtained based on the assessment of polar and as well as non-polar extracts fingerprints. The current study showed that DART-TOFMS metabolomic fingerprinting represents a rapid and powerful analytical strategy enabling differentiation of common carp muscles according to feeding history by recording metabolomic fingerprints of ionizable components under the conditions of ambient MS.
- MeSH
- Principal Component Analysis MeSH
- Cyclohexanes MeSH
- Diet MeSH
- Liquid-Liquid Extraction MeSH
- Edible Grain * MeSH
- Carps growth & development metabolism MeSH
- Muscle, Skeletal chemistry metabolism MeSH
- Metabolome * MeSH
- Least-Squares Analysis MeSH
- Dietary Supplements * MeSH
- Solvents MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Water MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
... Culture 398 -- Hybrid Cells Called Hybridomas Produce Abundant -- Monoclonal Antibodies 400 -- HAT Medium ... ... trans-Golgi 603 -- Several Pathways Sort Membrane Proteins to the Apical or Basolateral Region of Polarized ... ... Monomers Assemble into Long, Helical -- F-Actin Polymers 717 -- F-Actin Has Structural and Functional Polarity ... ... Levels and Kinase Activity of Mitosis-Promoting Factor (MPF) Change Together in Cycling Xenopus Egg Extracts ... ... Most Somatic Cells Are Complete 961 -- Gastrulation Creates Multiple Tissue Layers, Which -- Become Polarized ...
6th ed. xxxvii, 1150 s. : il., tab. ; 29 cm
- MeSH
- Cell Biology MeSH
- Molecular Biology MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- biologie
- cytologie, klinická cytologie
... -- L-tense, 77-8 lambda-extraction, 221-2 language change, see diachrony language-games, 227, 280-1; ... ... 16, 49, 101, 113,131,238, 241 meaningfulness, 170-1, 227 measures : of time, 73ff of space, 79ff medium ... ... presupposition, see under presupposition test, for presuppositions, see constancy under negative polarity ...
Cambridge textbooks of linguistics
15th ed. xvi, 420 s.
