Distinct responses of chloroplasts to blue and green laser microbeam irradiations in the centric diatom Pleurosira laevis

The centric diatom Pleurosira laevis is a large unicellular alga, in which ca 200 chloroplasts migrate toward the nuclear cytoplasm through the transvacuolar cytoplasmic strands in response to blue-light irradiation and, on the contrary, toward the cortical cytoplasm in response to green-light irradiation. We analyzed these light-induced chloroplast migrations using a scanning laser microbeam provided by a confocal microscope for intracellular irradiation. Spot irradiation of a blue laser microbeam induced rapid assemblage of chroloplasts into the nuclear cytoplasm regardless of the spot position and spot number. On the other hand, one or two spots of green laser microbeam induced chloroplast accumulation at the spots, although increasing spot numbers suppressed chloroplast accumulation at each spot. In our experimental condition, ca 1 min of blue-light irradiation was sufficient to stimulate movement, whereas green-light irradiation required uninterrupted and longer irradiation time (ca 15 min). Chloroplast assemblage induced by blue-light required extracellular Ca2+, and was inhibited by Ca2+ channel antagonists. Furthermore, higher efficiencies of chloroplast migration were obtained when a single beam spot was fragmented and scattered over wider area of plasma membrane. These observations suggested that blue-light induced a response at the plasma membrane, which subsequently activated Ca2+ permeable channels. This sequence of physiological events is identical to what was previously observed with chloroplast movement in response to mechanical stimulation. Furthermore, experiments with the cytoskeleton-disrupting agents, colchicine and cytochalasin D, indicated that blue-light-induced chloroplast movement required microtubules whereas the green-light-induced response to beam spot required actin filaments.     

Multi-spot paper chromatograms of sodium orthophosphate

The formation of two zones on paper chromatograms was investigated by chromatographing orthophosphoric acid and the primary, secondary and tertiary sodium salts, using pyridine-ethyl acetate/water as mobile phase. Movement of the species from the origin depends on washing the paper with acid and suggests an exchange of protons for sodium as a prerequisite condition. Washing with EDTA does not alter this requirement. Zone movement and multi-spot formation require that the forming solvent contain at least 5% of water (v/v). Between this value and saturation, increasing water content enhances zone disengagement. A solute spot of a sodium salt exchanges with the protons to produce a fast-migrating protonated phosphate species. If the solute sample has a sodium content less than the amount of exchangeable protons, a single fast spot is produced ; if the concentration exceeds this capacity, a second slower spot results.     

A contribution to non-equilibrium chemical kinetics. II. Strongly non-equilibrium “hot” reactions in liquids and solids

The method proposed in part I for non-equilibrium chemical kinetics is applied to processes provoked by non-equilibrium assemblies of energetic particles in liquids and solids. The movement of such an energetic particle belonging to a certain energy group is considered as a stochastic process when the direction of the velocity is changed stochastically at each step. On the ground of this consideration a simplified model of such a process is introduced: the stochastic movement of a particle is replaced by the deterministic movement of the corresponding quasi-particle having parameters determined through corresponding averages of the stochastic process. By use of this model, group constants of kinetic equations of our abovementioned work were expressed through parameters of microscopic processes in solids and liquids, and systems of non-equilibrium chemical kinetics' equations were written for different case. The proposed approach also permits us to consider the non-equilibrium of the crystalline lattice created by energetic particles. “Hot spot” reactions were considered as an example and a method to distinguish between direct and “hot spot” reactions was indicated. The proposed approach and obtained kinetic equations can be applied to recoil atoms (ions), fission products, hot particles produced in radiation chemistry, photochemistry, by laser beams, flash-photolysis etc. The destruction of the crystalline lattice by laser beams can also be considered by use of these equations.     

Ionophoretic studies on mixed metal--nitrilotriacetate--penicillamine complexes

Paper ionophoresis is described for the study of equilibria in a mixed ligand complex system in solution. This method is based on the movement of a spot of metal ion in an electric field with the complexants added in the background electrolyte at pH 8.5. The concentration of the primary ligand (nitrilotriacetate) was kept constant, while that of the secondary ligand (penicillamine) was varied. The stability constants of the metal-nitrilotriacetate-penicillamine complexes have been found to be 6.26 +/- 0.09 and 6.68 +/- 0.13 (log K values) for Al3+ and Th4+ complexes, respectively, at 35 degrees C and ionic strength 0.1 M.     

The role of complex formation in partition paper chromatography

Further information of the possible effects of chemical reaction on differential ionic movement in partition paper chromatography was obtained by developing chromatograms of fifteen inorganic cations with alcoholic solvents containing varying concentrations of disodium ethylenediaminetetraacetate.An examination of these chromatograms indicated that variations in the concentrations of E.D.T.A. in the solvent or the presence of free acid or ammonia resulted in marked changes in R_F values and spot areas.It is therefore suggested that care should be taken in interpreting the results of any chromatograms developed with solvents capable of chemical interaction with the “spotted” solute.     

Relationship between Hot Spot Residues and Ligand Binding Hot Spots in Protein-Protein Interfaces

In the context of protein-protein interactions, the term "hot spot" refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research, a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening.     

Study on the paper substrate room temperature phosphorescence of theobromine,caffeine and theophylline and analytical application

Paper substrate room temperature phosphorescence (RTP) of theobromine (TB), caffeine (CF) and theophylline (TP) were investigated. The method is based on fast speed quantitative filter paper as substrate and KI-NaAc as heavy atom perturber. Various factors affecting their RTP were discussed in detail. Under the optimum experimental conditions, the linear dynamic range, limit of detection (LOD), and relative standard deviation (R.S.D.) were 14.41 approximately 576.54 ng per spot, 1.14 ng per spot, 4.8% for TB, 5.44 approximately 699.08 ng per spot, 0.78 ng per spot, 1.56% for CF, 7.21 approximately 360.34 ng per spot, 1.80 ng per spot, 3.80% for TP, respectively. The first analytical application for the determination of these compounds was developed. The recovery of standard samples added to commercial products chocolate, tea, coffee and aminophylline is in the range 92.80-106.08%. The proposed method was successfully applied to real sample analysis without separation.     

An alternative approach to deal with geometric uncertainties in computer analysis of two-dimensional electrophoresis gels

With the growing importance of proteomics in biomedical and pharmaceutical sciences a need has emerged for computing tools that are capable of digitally visualizing and analyzing protein spot patterns within two-dimensional electrophoresis (2-DE) gel. Matching programs need to meet requirements such as interlaboratory comparison and the comparison of samples from different origins. For such research purposes, we have developed the CAROL system that implements new algorithms for spot detection and matching, which enable researchers to take a different approach to protein spot identification and comparison. The present short communication discusses how the system deals with uncertain geometric spot information that arises from streaks and complex spot regions and how this can be amplified for the matching procedure.     

不同人群对正畸固定矫治中白垩斑的认知调查

目的：调查正畸患者、患者家长、正畸医生和全科牙医对固定正畸矫治中白垩斑的认知度,为正畸治疗中白垩斑的防治及医患沟通提供参考。方法对323名正畸患者、295名患者家长、63名正畸医生及117名全科牙医进行白垩斑认知度的问卷调查,对结果进行统计分析。结果4组人群均认为白垩斑使矫治后牙齿的美观度降低,患者要对白垩斑的发生负最主要责任,患者和家长与正畸医师和牙医对白垩斑的治疗看法迥异。结论不同人群均认为白垩斑降低了牙齿的美观度,其主要责任在于患者自身,医患双方应当加强沟通,消除医患矛盾隐患。     

Investigation of non-nucleophilic additives for the reduction of morphological anomalies in protein arrays

Uniform spot morphology is of critical importance in the fabrication and successful use of protein arrays, and solution additives are often needed to ensure good spot quality. Whereas hydroxyl-bearing molecules such as glycerol have found wide use, in our experience these reduce the efficiency of probe immobilization (particularly in the context of aldehyde-terminated surfaces). Here, we report a series of non-nucleophilic molecules that can be used as additives to improve spot homogeneity in protein arrays. Arrayed imaging reflectometry, a label-free optical biosensing technique, has been used along with spectroscopic ellipsometry to test the spot homogeneity, antibody immobilization efficiency, and activity of antihuman IgG arrays prepared with these non-nucleophilic additives on glutaraldehyde surfaces. It has been determined that 0.1% v/v 12-crown-4 performs optimally in MPBS buffer.     

Physical markers for landmarking fluorescently stained gels that facilitate automated spot-picking

The quantitative comparison of spot patterns relies heavily on protein stains that do provide an appropriate dynamic range. Unfortunately most spot picking robot devices are still limited to nonfluorescent protein stains and the appropriate equipment is still quite expensive. These problems are solved by the application of a newly developed "GelMarker" that combines a spot picking robot device and a UV scanner. The "GelMarkers" are detectable in both the visible and UV range of light and permit the comparison of gel pictures taken by such different devices. The application of these "GelMarkers" together with the transformation of spot coordinates by using a "spot matching" procedure allows the automated excision of selected protein spots. The obtained picking accuracies are as good as those obtainable from visible stained gels due to the shape stability of the gels even over a longer time period.     

A two-dimensional electrophoresis-related laboratory information processing system: spot matching.

An approach for the computer-assisted analysis of two-dimensional gels has been developed as a part of our laboratory information processing system (LIPS). This approach relies in part on an algorithm for the pairwise matching of protein spots. The matching process initially matches spots based on a cross-correlational measure of how well neighboring spots align. While this first pass correctly determines most spot correspondences and noncorrespondences, it can make errors. Higher accuracy is obtained by monitoring the consistency of spot match decisions in a second pass, which demands that neighboring spot pairs that align spatially must also have been found to match in the first pass. Pairwise comparisons of gels are combined into n-way comparisons by matching spot lists of gels to "master" gel spot lists, which in turn are matched to higher level masters, resulting in a hierarchy of matched spots. After each pairwise match the results are reviewed and corrected with the assistance of a graphical match-editor. Results are given for 19 single-cell-derived lymphoid clones in which the presence of a mutation had previously been established, each processed in duplicate. Only one of 46 spot changes failed to be detected, which demonstrates that the strategy is sensitive and efficient for detecting qualitative spot differences.     

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry

Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics. Targeted analyses of small molecules are the main application of dried blood spot samples and liquid chromatography-mass spectrometry, but emerging applications include untargeted metabolomics and proteomics. Applications are highly varied, including analyses related to newborn screening, diagnostics and monitoring of disease progression and treatment effects of virtually any disease, as well as studies into the physiology and effects of diet, exercise, xenobiotics, and doping. A range of dried blood spot products and methods are available, and applied liquid chromatography-mass spectrometry instrumentation is varied with regard to liquid chromatography column formats and selectivity. In addition, novel approaches such as on-paper sample preparation (e.g., selective trapping of analytes with paper-immobilized antibodies) are described. We focus on research papers published in the last 5 years.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis maps: relevance to proteomics

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.     

Electrophoretic studies on the mixed nitrilotriacetate-penicillamine complexes of Pb2+ and UO 2 2+

A technique involving the use of paper electrophoresis is described for the study of binary and mixed-complex systems in solution. This technique is based on the movement of a spot of metal ion in an electric field with the complexants added in the background electrolyte at pH 8.5. The concentration of primary ligand (nitrilotriacetate) was kept constant, while that secondary ligand (penicillamine) was varied. A graph of log[penicillamine] versus mobility was used to obtain information on the mixed complexes and to calculate stability constants. The stability constants of mixed-ligand complexes lead(II)-nitrilotriacetate-penicillamine and uranyl(II)-nitrilotriacetate-penicillamine have been found to be 5.68 ± 0.09 and 6.56 ± 0.05 (logarithm stability constant values), respectively, at ionic strength 0.1 M and a temperature of 35°C. The text was submitted by the author in English.     

Protein arrays on high-surface-area plasma-nanotextured poly(dimethylsiloxane)-coated glass slides

Treatment of poly(dimethylsiloxane) (PDMS) surfaces with SF(6) plasma results in the creation of high-surface-area nanotextured surfaces that considerably favour protein adsorption with respect to untreated ones. In order to employ such nanotextured surfaces as substrates for microarrays to be created and analysed using standard instrumentation, we fabricated thin PDMS films on top of standard low-cost microscope glass slides. The properties of both untreated and plasma-treated PDMS-coated slides towards spotting of protein solutions were evaluated in terms of spot signal intensity and homogeneity as well as of spot shape and size. It was found that the plasma-treated PDMS-coated glass slides provided highly homogeneous spots (mean intra-spot variation 7.6%) with spot signal intensity 6-times higher than that obtained using the untreated ones. In addition, comparison with commercially available polystyrene and aminosilanized-glass microarray slides showed that the proposed slides provided 3-times higher spot signal intensity and 2-times lower intra-spot signal variation. In addition, the implementation of long-aged-after-plasma-treatment nanotextured PDMS-coated glass slides provided spots whose shape and size matched those of the spotting tip. As a consequence, denser arrays of variable spot shape can be created using SF(6) plasma-treated PDMS-coated slides instead of standard microarray slides opening new potentials for bioanalytical applications.     

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two‐dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI‐MS, LC‐Q‐TOF MS and LC‐Orbitrap Velos MS for the identification of proteins within one spot. With LC‐Orbitrap Velos MS each Coomassie Blue‐stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large‐scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low‐abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE‐MS to separate at the protein species level. Therefore, 2DE coupled with high‐sensitivity LC‐MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom‐up LC‐MS investigations.     

Investigation of colloidal graphite as a matrix for matrix‐assisted laser desorption/ionisation mass spectrometry of low molecular weight analytes

The analysis of low molecular weight compounds by matrix‐assisted laser desorption/ionisation mass spectrometry is problematic due to the interference and suppression of analyte ionisation by the matrices typically employed – which are themselves low molecular weight compounds. The application of colloidal graphite is demonstrated here as an easy to use matrix that can promote the ionisation of a wide range of analytes including low molecular weight organic compounds, complex natural products and inorganic complexes. Analyte ionisation with colloidal graphite is compared with traditional organic matrices along with various other sources of graphite (e.g. graphite rods and charcoal pencils). Factors such as ease of application, spectra reproducibility, spot longevity, spot‐to‐spot reproducibility and spot homogeneity (through single spot imaging) are explored. For some analytes, considerable matrix suppression effects are observed resulting in spectra completely devoid of matrix ions. We also report the observation of radical molecular ions [M^–●] in the negative ion mode, particularly with some aromatic analytes. Copyright © 2016 John Wiley & Sons, Ltd.     

Dried plasma spot–based liquid chromatography–tandem mass spectrometry for the quantification of methotrexate in human plasma and its application in therapeutic drug monitoring

Methotrexate, a folic acid antitumor drug, is widely used to treat childhood acute lymphoblastic leukemia. Therapeutic drug monitoring is crucial for adjusting the dosage of methotrexate according to its plasma concentration and reducing adverse effects. Micro-sampling strategies, like dried plasma spot, is an attractive but underutilized method that has the desired features of easy collection, storage, and transport, and overcomes known hematocrit issues in dried blood spot analysis. This study describes a dried plasma spot–based liquid chromatography–tandem mass spectrometry method for quantification of methotrexate. The assay showed good linearity over 30–2000 ng/mL (R² ≥ 0.995) as well as excellent precision (0.6–9.3%) and accuracy (89.2–108.3%). Methotrexate was extracted from dried plasma spot and wet plasma samples with recoveries greater than 92.1%, and no significant matrix effect was observed. A comparison of dried plasma spot and wet plasma concentrations was assessed in 27 patients treated with methotrexate and Passing–Bablok regression coefficients showed that no significant difference between the two methods. The Bland–Altman plots showed similar agreement between the methods, indicating that the proposed dried plasma spot sampling method is an effective way to monitor the concentration of methotrexate in human plasma.     

Corrigendum to “Deposition of chemically reactive and repellent sites on biosensor chips for reduced non-specific binding” [Colloids Surf. B 79 (2010) 270–275]

Treatment of poly(dimethylsiloxane) (PDMS) surfaces with SF₆ plasma results in the creation of high-surface-area nanotextured surfaces that considerably favour protein adsorption with respect to untreated ones. In order to employ such nanotextured surfaces as substrates for microarrays to be created and analysed using standard instrumentation, we fabricated thin PDMS films on top of standard low-cost microscope glass slides. The properties of both untreated and plasma-treated PDMS-coated slides towards spotting of protein solutions were evaluated in terms of spot signal intensity and homogeneity as well as of spot shape and size. It was found that the plasma-treated PDMS-coated glass slides provided highly homogeneous spots (mean intra-spot variation 7.6%) with spot signal intensity 6-times higher than that obtained using the untreated ones. In addition, comparison with commercially available polystyrene and aminosilanized-glass microarray slides showed that the proposed slides provided 3-times higher spot signal intensity and 2-times lower intra-spot signal variation. In addition, the implementation of long-aged-after-plasma-treatment nanotextured PDMS-coated glass slides provided spots whose shape and size matched those of the spotting tip. As a consequence, denser arrays of variable spot shape can be created using SF₆ plasma-treated PDMS-coated slides instead of standard microarray slides opening new potentials for bioanalytical applications.