Probes In Experiments Research Articles

Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria.

Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [2H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC–MS. Feeding experiments with [2H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [2H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [2H3]methionine and 34SO42−, synthesized from elemental 34S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.

Development of Insulated Conductive AFM Probes for Experiments in Electrochemical Environment

In this work, we developed a process to make insulated conductive AFM probes. The process is based on a previously reported PtxSiy CP-AFM. Following this fabrication, a feedback controlled electrochemical deposition of Au in a HAuCl4 based electrolyte is applied to coat a homogeneous gold cluster layer exclusively on the conductive tip apex. This modified device has the potential for connecting molecules and to measure their properties, which include both the mechanical force and the charge transport between the tip and a conducting substrate.

Polarization and Symmetry of Electronic Transitions in Long Fluorescence Lifetime Triangulenium Dyes

To fully exploit the capabilities of fluorescence probes in modern experiments, where advanced instrumentation is used to probe complex environments, other photophysical properties than emission color and emission intensity are monitored. Each dye property can be addressed individually as well as collectively to provide in-depth information unavailable from the standard intensity measurements. Dyes with long emission lifetimes and strongly polarized transitions enable the monitoring of lifetime changes as well as emission polarization (anisotropy). Thus experiments can be designed to follow slow dynamics. The UV and visible electronic transitions of a series of red-emitting dyes based on the triangulenium motif are investigated. We resolve overlapping features in the spectra and assign the orientation of the transition moments to the molecular axes. The result is the complete Jablonski diagram for the UV and visible spectral region. The symmetries of the studied dyes are shown to have a large influence on the optical response, and they are clearly separated into two groups of symmetry by their photophysical properties. The C(2v) symmetric dyes, azadioxatriangulenium (ADOTA(+)) and diazaoxatriangulenium (DAOTA(+)), have high emission anisotropies, fluorescence lifetimes around 20 ns, and fluorescence quantum yields of ∼50%. The trioxatriangulenium (TOTA(+)) and triazatriangulenium (TATA(+)) dyes-nominally of D(3h) symmetry-have fluorescence lifetimes around 10 ns lifetimes and fluorescence quantum yields of 10-15%. However, the D(3h) symmetry is shown to be lowered to a point group, where the axes transform uniquely such that the degeneracy of the E' states is lifted.

Application of Polythiophene–Sawdust Nano-Biocomposite for Basic Dye Removal Using a Continuous System

A novel dye adsorbent was developed by a poly (3-methyl thiophene) nano layer coated onto sawdust obtained from Narra wood. The polymer was synthesized chemically under in-situ conditions on the surface of the sawdust. Methylene blue, a typical cationic textile dye, was employed as a test probe for adsorption experiments. The experiments were conducted in both batch and fixed-bed column systems. The effects of variables such as pH, initial dye concentration, flow rate, and bed height were investigated. The Thomas dynamic model was applied to calculate the sorption capacity of adsorbents. Based on the breakthrough curves analysis, the highest column capacity of 191 mg/g was obtained. The introduced nano-biocomposite was found to be more than seven times more efficient than unmodified sawdust for methylene blue removal. The regeneration of the modified sawdust is quite possible using 0.10 M HCl in ethanol as a washing solution with high efficiency.

Probing the Influence of the Particle in Single Particle Tracking Measurements on Lipids

We present a systematic study of the influence of the nanoparticle used as a probe in Single Particle Tracking experiments with a supported lipid bilayer as a model system. Quantum Dots, 40 nm and 200 nm diameter fluorescent latex spheres, and 40 nm diameter gold colloids were attached to headgroup-labeled lipid molecules in the bilayer by means of an antibody. The percentage of the particle's surface covered with antibody was also varied. We argue that the best estimate of the diffusion coefficient is derived from the first two points of the particle's mean square displacement. The accuracy of this estimate is equivalent to a more rigorous analysis based on the cumulated probability function. We show that, under optimal conditions, Quantum Dots and also 40 nm latex beads have a negligible influence on the lipid diffusion. The diffusion coefficient of gold colloids and 200 nm diameter latex particles was systematically reduced 2- to 3-fold compared to the reference value obtained by Fluorescence Recovery After Photobleaching. Unexpectedly, the diffusion coefficient of Quantum Dots and 40 nm latex beads depended on the percentage of the nanoparticle's surface covered with antibody: at very low percentage the perturbation to the lipid diffusion due to the particle increased. We speculate that in this regime subtle details of antibody binding to the nanoparticle become important.In an alternative system using particles tethered with 30 or 90 bp DNA strands, particles attached with the longer tether showed the highest diffusion coefficient. We propose that the proximity of the nanoparticle to the membrane may modify lipid dynamics.ReferenceP. Mascalchi et al. 2012. Probing the influence of the particle in Single Particle Tracking measurements of lipid diffusion. Soft Matter, 2012,8, 4462-4470.

Evaluation of quantitative probes for weaker Cu(i) binding sites completes a set of four capable of detecting Cu(i) affinities from nanomolar to attomolar

Copper plays essential roles in biology, but abnormal interactions are damaging. Reliable quantification of copper-protein interactions will underpin the molecular understanding of copper nutrition and toxicity. We have previously established two high affinity probes, Bathocuproine disulfonate (Bcs) and Bicinchoninate (Bca) anions, that are capable of in vitro quantification of Cu(i) binding with affinities from pico- to atto-molar concentrations. Quantitative probes are required for Cu(i) binding of lower affinity for proteins and peptides typically associated with neurodegenerative diseases. The present work evaluates two classic Fe(ii) ligands Ferene S (Fs) and Ferrozine (Fz) as quantitative probes for Cu(i). Both react with Cu(i) quantitatively to yield well-defined complex anions [Cu(I)(Fs)2](3-) (λmax = 484 nm, ε = 6700 cm(-1) M(-1)) and [Cu(I)(Fz)2](3-) (λmax = 470 nm, ε = 4320 cm(-1) M(-1)). These complexes are sensitive to aerial oxidation (E1/2∼ +0.36 V vs. SHE) and to substitution by other ligands (e.g., Cl(-), MeCN). However, they can be protected effectively under anaerobic conditions by suitable reductants and an excess of the free probe ligands. Formation constants β2 were determined by two approaches: direct metal ion titration and ligand competition. They provided estimates which differed by ∼3 orders of magnitude. The sources of these differences were examined carefully to consolidate the affinities of the two probes to a unified standard (10(15.1) M(-2) for Fz and 10(13.7) M(-2) for Fs). It is apparent that application of direct metal ion titrations to quantification of Cu(i) binding affinities is problematical and should be avoided. The four ligands Bcs, Bca, Fz and Fs in combination form a set of versatile probes for ligand competition experiments and are capable of detecting and differentiating an extended spectrum of Cu(i) binding affinities from nano- to atto-molar concentrations. Selected examples of quantification of weaker Cu(i) binding in proteins and peptides are provided, including that of an amyloid-β peptide.

Structural Characterization of the Interaction of Human Lactoferrin with Calmodulin

Lactoferrin (Lf) is an 80 kDa, iron (Fe3+)-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf’s interaction with calmodulin (CaM), a ubiquitous, 17 kDa regulatory calcium (Ca2+)-binding protein localized in the cytoplasm and nucleus of activated cells. Due to the size of this intermolecular complex (∼100 kDa), TROSY-based NMR techniques were employed to structurally characterize Ca2+-CaM when bound to intact apo-Lf. Both CaM’s backbone amides and the ε-methyl group of key methionine residues were used as probes in chemical shift perturbation and cross-saturation experiments to define the binding interface of apo-Lf on Ca2+-CaM. Unlike the collapsed conformation through which Ca2+-CaM binds the CaM-binding domains of its classical targets, Ca2+-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca2+-CaM, enabling the N-terminal lobe to potentially bind another target. Our use of intact apo-Lf has made possible the identification of a secondary interaction interface, removed from CaM’s primary binding domain. Secondary interfaces play a key role in the target’s response to CaM binding, highlighting the importance of studying intact complexes. This solution-based approach can be applied to study other regulatory calcium-binding EF-hand proteins in intact intermolecular complexes.

Revealing Rotational Modes of Functionalized Gold Nanorods on Live Cell Membranes

A full understanding of cell mechanics requires knowledge of both translational and rotational dynamics. The single particle orientation and rotational tracking (SPORT) technique is combined here with correlation analysis to identify the fundamental rotational modes: in-plane rotation and out-of-plane tilting, as well as other more complex rotational patterns, from the vast image data captured at a temporal resolution of 5 ms for single gold nanorod probes in live cell imaging experiments. The unique capabilities of visualizing and understanding rotational motions of functional nanoparticles on live cell membranes allow correlation of the rotational and translational dynamics in unprecedented detail and provide new insights into complex membrane processes. Particles with functionalized surfaces, which interact with the membrane in fundamentally different ways, can exhibit distinct rotational modes and are, for the first time, directly visualized, and these show the early events for membrane approach and attachment.

Contrasting evolution of a satellite DNA and its ancestral IGS rDNA inPhaseolus(Fabaceae)

CC4 is a satellite DNA from common bean (Phaseolus vulgaris L.) that is similar to its intergenic spacer (IGS) rDNA. CC4 was originally hypothesized to be an old, fast evolving satellite family that has invaded common bean rDNA. To test this hypothesis and contribute to the understanding of IGS-like satellite DNA evolution, we have investigated its distribution in the genus Phaseolus and related species. CC4 was cloned and used as probe for Southern blot and FISH experiments. CC4 was observed as an independent satellite in common bean, forming two to three major and a few minor pericentromeric clusters. In Phaseolus coccineus, CC4 was present in four major clusters, also not co-localized with the 45S rDNA sites. Remarkably, in the less related species of the genus, signals were detected co-localized with the 45S rDNA sites, but co-localization was not observed in the species where CC4 is present as an independent satellite. No signal was detected in species from related genera. Altogether, the data suggest that CC4 has originated from the IGS rDNA in the P. vulgaris-P. coccineus lineage and has evolved slower than the IGS rDNA from this lineage.

Ultra-high energy probes of classicalization

Classicalizing theories are characterized by a rapid growth of the scattering cross section. This growth converts these sort of theories in interesting probes for ultra-high energy experiments even at relatively low luminosity, such as cosmic rays or Plasma Wakefield accelerators. The microscopic reason behind this growth is the production of N-particle states, classicalons, that represent self-sustained lumps of soft Bosons. For spin-2 theories this is the quantum portrait of what in the classical limit are known as black holes. We emphasize the importance of this quantum picture which liberates us from the artifacts of the classical geometric limit and allows to scan a much wider landscape of experimentally-interesting quantum theories. We identify a phenomenologically-viable class of spin-2 theories for which the growth of classicalon production cross section can be as efficient as to compete with QCD cross section already at 100TeV energy, signaling production of quantum black holes with graviton occupation number N ∼ 104.

Integration of Co-expression Networks for Gene Clustering

AbstractSimultaneous overexpression or underexpression of multiple genes, used in various forms as probes in the high-throughput microarray experiments, facilitates the identification of their underlying functional proximity. This kind of functional associativity (or conversely the separability) between the genes can be represented proficiently using co-expression networks. The extensive repository of diversified microarray data encounters a recent problem of multi-experimental data integration for the aforesaid purpose. This paper highlights a novel integration method of gene co-expression networks, based on the search for their consensus network, derived from diverse microarray experimental data for the purpose of clustering. The proposed methodology avoids the bias arising from missing value estimation. The method has been applied on microarray datasets arising from different category of experiments to integrate them. The consensus network, thus produced, reflects robustness based on biological validation.

Integration of Co-expression Networks for Gene Clustering

Simultaneous overexpression or underexpression of multiple genes, used in various forms as probes in the high-throughput microarray experiments, facilitates the identification of their underlying functional proximity. This kind of functional associativity (or conversely the separability) between the genes can be represented proficiently using co-expression networks. The extensive repository of diversified microarray data encounters a recent problem of multi-experimental data integration for the aforesaid purpose. This paper highlights a novel integration method of gene co-expression networks, based on the search for their consensus network, derived from diverse microarray experimental data for the purpose of clustering. The proposed methodology avoids the bias arising from missing value estimation. The method has been applied on microarray datasets arising from different category of experiments to integrate them. The consensus network, thus produced, reflects robustness based on biological validation.

Perturbed Angular Correlation of the stretched cascade in the decay of 180mHf using a digital spectrometer

We report on the measurement of the nuclear quadrupole interaction (NQI) at Hf sites using the nuclear probe 180mHf in HfF4·HF·2H2O at 300 K by exploiting all possible start quanta in the stretched cascade with a digital Time Differential Perturbed Angular Correlation (TDPAC) spectrometer. With conventional spectrometers, multiple prompt start signals would paralyze the router. The gain in coincidence rate is about a factor of 5 compared to a conventional spectrometer using a single start only. With multiple starts 180mHf is a promising new isomeric nuclear probe in TDPAC experiments. As an additional feature we implemented the possibility to measure up to four cascades simultaneously in order to save data collection time or to measure isobaric contaminations like 111mCd and 111In.

Probing the influence of the particle in Single Particle Tracking measurements of lipid diffusion

We present a systematic study of the influence of the nanoparticle used as a probe in Single Particle Tracking experiments with a supported lipid bilayer as a model system. Quantum Dots, 40 nm and 200 nm diameter fluorescent latex spheres, and 40 nm diameter gold colloids were attached to headgroup-labeled lipid molecules in the bilayer by means of an antibody. The percentage of the particle's surface covered with antibody was also varied. We made the choice to extract the diffusion coefficient from the first two points of the mean square displacement of the particle after demonstrating that the accuracy of this method is equivalent to a more rigorous but less prevalent analysis based on the cumulated probability function. We show that, under optimal conditions, Quantum Dots and also 40 nm latex beads have a negligible influence on the lipid diffusion, whereas the diffusion coefficient of gold colloids and 200 nm diameter latex particles was systematically reduced 2- to 3-fold compared to the reference value obtained by Fluorescence Recovery After Photobleaching. Interestingly, the diffusion coefficient of Quantum Dots and 40 nm latex beads depended on the percentage of the surface of the nanoparticle covered with antibody in an unexpected manner: at very low percentage the perturbation to the lipid diffusion due to the particle increased. We speculate that in this regime subtle details of antibody binding to the nanoparticle become important.

A FRET-based probe with a chemically deactivatable quencher

A new concept of a chemically deactivatable quencher is proposed for a FRET-based probe that turns-on its fluorescence by either an enzymatic cleavage or a chemical reagent (sodium dithionite). This concept allowed us to quantify the caspase-3 cleavage activity in solution and to reveal unreacted probes in cell experiments.

Advances in the chemistry of small molecule fluorescent probes

Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes.

Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody–ligand interaction thermodynamics and kinetics

For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA>108M−1; KD<10–8M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand–antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.

Thermal product estimation method for aerodynamics experiments

This paper presents an experimental technique to estimate the appropriate thermal product values of rugged and fast response temperature probes (TPs) for hypersonic aerodynamic experiments. Two types of scratches were used, mainly abrasive papers with different grit sizes and scalpel blades with different thicknesses, to form the probe junction. The effect of the scratch technique on the probe’s thermal product is investigated. The probes are tested and calibrated in the test section and in the end wall of shock tube facility. It is observed that the thermal product of a particular TP depends on the Mach number, junction scratch technique, and junction location, as well as on the enthalpy conditions. It is noted that, depending on the scratch technique, some of the TPs do not require individual calibration; however, calibration for others is likely to be needed.

High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli

Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown. In order to initiate ligand identification, structural studies, and epitope mapping experiments, we developed a simple and efficient expression and purification protocol allowing to produce large amounts of pure soluble monomeric mouse NKR-P1C(B6). Our protein encompassed approximately half of the stalk region and the entire C-terminal globular ligand binding domain. The identity of protein that was devoid of N-terminal initiation methionine and had all three expected disulfides closed was confirmed using high resolution ion cyclotron resonance mass spectrometry. Protein produced into inclusion bodies in Escherichia coli was efficiently refolded into a unique three dimensional structure as confirmed by NMR using 1H– 15N-HSQC spectra of uniformly labeled protein. The exceptional purity of the protein should allow its crystallization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand identification and antibody epitope mapping experiments.

Microarray estimation of genomic inter-strain variability in the genus Ectocarpus (Phaeophyceae).

BackgroundBrown algae of the genus Ectocarpus exhibit high levels of genetic diversity and variability in morphological and physiological characteristics. With the establishment of E. siliculosus as a model and the availability of a complete genome sequence, it is now of interest to analyze variability among different species, ecotypes, and strains of the genus Ectocarpus both at the genome and the transcriptome level.ResultsWe used an E. siliculosus gene expression microarray based on EST sequences from the genome-sequenced strain (reference strain) to carry out comparative genome hybridizations for five Ectocarpus strains: four E. siliculosus isolates (the male genome strain, a female strain used for outcrosses with the genome strain, a strain isolated from freshwater, and a highly copper-tolerant strain), as well as one strain of the sister species E. fasciculatus. Our results revealed significant genomic differences between ecotypes of the same species, and enable the selection of conserved probes for future microarray experiments with these strains. In the two closely related strains (a male and a female strain used for crosses), genomic differences were also detected, but concentrated in two smaller genomic regions, one of which corresponds to a viral insertion site.ConclusionThe high variability between strains supports the concept of E. siliculosus as a complex of cryptic species. Moreover, our data suggest that several parts of the Ectocarpus genome may have evolved at different rates: high variability was detected particularly in transposable elements and fucoxanthin chlorophyll a/c binding proteins.