Detection of deoxynivalenol using biolayer interferometry

Biolayer interferometry allows for the real time monitoring of the interactions between molecules without the need for reagents with enzymatic, fluorescent, or radioactive labels. The technology is based upon the changes in interference pattern of light reflected from the surface of an optical fiber when materials bind to the tip of the fiber. The technique represents an alternative to technologies such as surface plasmon resonance, with an advantage in that the flow of extracts through small capillaries is not required. In this report, a deoxynivalenol-bovine serum albumin (DON-BSA) conjugate was non-covalently immobilized to the surface of aminopropylsilane sensors and the change in interference pattern resulting from the binding of DON-specific antibodies was measured. The basis for the assay was the competition between DON and the immobilized DON-BSA for binding to limited amounts of antibody. The technique was used to measure DON in extracts of spiked whole wheat flour, with a limit of detection of 0.10 mg DON/kg. Matrix interferences were an issue, and adequate quantification required using matrix-matched standards. When samples were tested with sensors that had not been conditioned to remove loosely attached DON-BSA, the recoveries at five spiking levels over the range from 0.2 to 5 mg/kg averaged 108.8% [relative standard deviation (RSD) 16.0%]. Using sensors that had been conditioned lowered the average recovery (101.4%) and improved the RSD (13.2%). This suggests that conditioning the sensors helped reduce a bias in the assay towards overestimation. These results, and the ease with which assays can be conducted, suggest further exploration of this technology for detection of mycotoxins is warranted.     

Detection of Karenia mikimotoi by spectral absorption signatures

This study investigated the performance of a spectral similarityindex and a multivariate partial least-squares regression techniquefor detecting the presence of the gyroxanthin-diester-carryingtoxic dinoflagellate Karenia mikimotoi from measurements ofspectral light absorption. The methods were applied to fourth-derivativeabsorption spectra of K. mikimotoi mixed with the toxic dinoflagellateProrocentrum minimum, which does not contain gyroxanthin-diester.Acclimating the cultures to different light and nutrient conditionsallowed us to evaluate the sensitivity of the methods to changesin pigmentation and intracellular light absorption (pigmentpackaging) of the algae. Both methods were able to determinethe fraction of K. mikimotoi and the gyroxanthin-diester concentration.However, whereas the partial least-squares predictions werealmost insensitive to the induced variability in optical propertiesof the algae, predictions based on the similarity index differedsignificantly depending on the acclimation of the algae. Furthermore,discriminating K. mikimotoi from natural phytoplankton assemblagesindicated a significant influence of cell-size composition,through pigment packaging, on the accuracy of the similarityindex. Our results suggest that optical discrimination of phytoplanktonspecies from spectral absorption signatures will improve significantlyby applying the partial least-squares regression technique.     

Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy

Abnormal aggregation of misfolded proteins and their deposition as inclusion bodies in the brain have been implicated as a common molecular pathogenesis of neurodegenerative diseases including Alzheimer, Parkinson, and the polyglutamine (poly(Q)) diseases, which are collectively called the conformational diseases. The poly(Q) diseases, including Huntington disease and various types of spinocerebellar ataxia, are caused by abnormal expansions of the poly(Q) stretch within disease-causing proteins, which triggers the disease-causing proteins to aggregate into insoluble beta-sheet-rich amyloid fibrils. Although oligomeric structures formed in vitro are believed to be more toxic than mature amyloid fibrils in these diseases, the existence of oligomers in vivo has remained controversial. To explore oligomer formation in cells, we employed fluorescence correlation spectroscopy (FCS), which is a highly sensitive technique for investigating the dynamics of fluorescent molecules in solution. Here we demonstrate direct evidence for oligomer formation of poly(Q)-green fluorescent protein (GFP) fusion proteins expressed in cultured cells, by showing a time-dependent increase in their diffusion time and particle size by FCS. We show that the poly(Q)-binding peptide QBP1 inhibits poly(Q)-GFP oligomer formation, whereas Congo red only inhibits the growth of oligomers, but not the initial formation of the poly(Q)-GFP oligomers, suggesting that FCS is capable of identifying poly(Q) oligomer inhibitors. We therefore conclude that FCS is a useful technique to monitor the oligomerization of disease-causing proteins in cells as well as its inhibition in the conformational diseases.     

Detection method for quantifying global DNA methylation by fluorescence correlation spectroscopy

A method for quantifying global DNA methylation using fluorescence correlation spectroscopy (FCS) has been established. The single-molecule methylation assay (SMMA) is based on two methodologies. One methodology, FCS, estimates the translational diffusion coefficient of molecules in solution, whereas the other methodology uses the high affinity of methyl-CpG-binding domain protein 2 (MBD2) to bind specifically to methylated DNA. We studied the specific binding rates of fluorescence-labeled MBD2 and methylated DNA from biological samples using the automated FCS system. Using a standard curve with methylated control DNA, we developed the SMMA index to assess the global DNA methylation level of the biological samples. A marked decrease in the SMMA index was observed when human leukemia cell lines (U937 and K562) were cultured with DNA demethylating agents. Our findings clearly indicate the applicability of SMMA as a simple and rapid tool for quantifying global DNA methylation. SMMA may prove useful for genome-wide comparative methylation analyses of malignancies and as an indicator of the demethylation effects of epigenetic drugs.     

Modification of nerve membrane sodium channels by the insecticide pyrethroids

1. The synthetic pyrethroids exert potent and selective actions on nerve membrane sodium channels. (+)-trans tetramethrin and (+)-trans allethrin cause repetitive discharges to be produced in the isolated crayfish and squid giant axons in response to a single stimulus as a result of an increase in depolarizing after-potential. 2. The latter effect is due to slowing of the sodium channel kinetics which causes a prolonged sodium current following the normal peak sodium current. 3. A kinetic model is proposed to account for the action of the pyrethroids in which the pyrethroid molecule binds to the sodium channels at both closed and open states to produce a modified open state. 4. (-)-trans and (-)-cis isomers of tetramethrin are ineffective in causing the effects, but prevent the active (+)-trans and (+)-cis isomers from exerting the effects. This stereospecificity provides us with an excellent opportunity for the study of binding sites of pyrethroids and other sodium channel modulators.     

基于多指标的中国淡水拟除虫菊酯水质基准

在US EPA水质基准制定指南框架基础上,利用从ECOTOX和CNKI数据库获取的3种拟除虫菊酯农药对中国淡水水生生物的急性毒性数据和生物标记物毒性数据,得出了基于多指标的中国淡水水体中溴氰菊酯、氯氰菊酯和氰戊菊酯的基准最大浓度(criteria maximum concentration,CMC)和基准连续浓度(criteria continuous concentration,CCC),分别为:7.5和0.9,110和19,21和3.5 ng·L-1。引入SOD、CAT、Ach E、MDA、G-SH、DNA/RNA含量等生物标记物指标,可以从多角度、多层次、多水平反映污染物的生态毒性效应,提出的基准值可以更加精准地预测污染物的生态风险。本研究可为水质基准推导方法的完善及拟除虫菊酯农药的水生生态风险控制提供参考。     

Use of spectral correlation method in a study of human emotional states

Application of a new approach to EEG analysis, i.e. of the method of spectral correlation, permitted identification of significant differences in spatial distribution of spectral power correlation for emotions of different quality, namely: anger and fear. It was shown that against the background of fear the topographic distribution of intracortical connections in the delta band was more extensive and included frontal, central, temporal, parietal and occipital regions. The emotion of anger evoked changes in the picture of spatial distribution of intracortical connections in the alpha band which resulted in appearance of a powerful focus of connections in the frontal regions. The greatest number of connections was found for the emotion of anger in the beta band--22 statistically significant correlations. Thus, a generalization of connections in the EEG high frequency bands is noted for the emotion of anger which is not characteristic of the emotion of fear.     

Study of protein aggregation using two-dimensional correlation infrared spectroscopy and spectral simulations

Two-dimensional (2D) correlation spectroscopy establishes correlations between intensity variations in a series of spectra obtained by the application of an external perturbation. However, spectral effects (wavenumber shift or bandwidth change) are known to generate apparent asynchronisms in 2D maps. Surprisingly, spectral effects are often neglected in the literature when interpreting experimental maps, which can lead to erroneous conclusions. In an attempt to evaluate the contribution of these effects and that of true asynchronisms on 2D maps, the heat-induced aggregation of glutamyl-tRNA synthetase (GluRS) was studied as a typical example of the application of Fourier transform infrared (FTIR) spectroscopy in the amide I region. The data were compared with those obtained from a mutant protein that differs by one amino acid. To determine whether the aggregation mechanisms are identical for both proteins, the experimental 2D maps were compared to simulations based on curve fitting of the initial and final spectra of the series, which allows change in position and bandwidth of the components to be taken into account. Intermediate spectra were generated using a convenient function that mimics the spectral evolution. The speed and the delay of each component were controlled. Apart from the appearance of turns that occur for the mutant and not for GluRS, the aggregation mechanisms of both proteins seems to be essentially identical. In particular, the loss of alpha-helices seems to be concomitant with the formation of intermolecular beta-sheets, whereas the loss of intramolecular beta-sheets is delayed. Since the experimental maps are satisfactorily simulated when almost all the components are in phase, it appears that many of the asynchronous features are mainly due to spectral effects. Thus, one has to be aware that true asynchronisms are not necessarily at the origin of peaks observed in asynchronous maps.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 × 10^− 9 cm² s^− 1 independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.     

High throughput detection of antibody self-interaction by bio-layer interferometry

Self-interaction of an antibody may lead to aggregation, low solubility or high viscosity. Rapid identification of highly developable leads remains challenging, even though progress has been made with the introduction of techniques such as self-interaction chromatography (SIC) and cross-interaction chromatography (CIC). Here, we report a high throughput method to detect antibody clone self-interaction (CSI) using bio-layer interferometry (BLI) technology. Antibodies with strong self-interaction responses in the CSI-BLI assay also show delayed retention times in SIC and CIC. This method allows hundreds of candidates to be screened in a matter of hours with minimal material consumption.     

Supercritical-fluid extraction of synthetic pyrethroids from wool

A stepwise approach was used to develop a supercritical fluid extraction (SFE) method for analysis of synthetic pyrethroids (SPs) on a wool matrix, commencing with a simple inert matrix to examine the solubility of the pyrethroids in the extraction fluid CO(2) and then extended to the real wool matrix. Chemometric approaches were used to determine the SFE optimum conditions. It was found that pyrethroids were readily extractable from an inert matrix over a wide range of pressure (170-350 atm) and at low temperature (<90 degrees C). Subambient hexane efficiently trapped the compounds from the depressurised fluid. Excessively high pressure and temperature resulted in poor trapping, isomerisation and possibly degradation of some components. With spiked wool samples method modifications focused on reducing the coextraction of grease, a bulk matrix component of raw wool. By using alumina (containing 8% moisture) and operating the extraction at 50 degrees C, 200 atm for 60 min, sufficiently clean extracts of pyrethroids suitable for gas chromatography-electron-capture detection analysis were obtained. The recoveries of all SPs were satisfactory (78-101%) over the range of 0.5-5 microg/g levels of these compounds. The precision of the entire analysis procedure was comparable to the conventional Soxhlet extraction method. Detection limits of some commonly used SPs for sheep treatment were also evaluated. Comparable results relative to those achieved by solvent extraction for incurred wool samples were obtained with a recovery of 81-85%. The results, however, suffered high uncertainties (R.S.D. approximately 19-24%) due to the small amount of wool sample taken in each extraction and the suspected inhomogeneity of the wool. Different persistences of cypermethrin isomers in wool were observed.     

Resurgences of spider mites (Acari: Tetranychidae) induced by synthetic pyrethroids

Causes of spider mite (Acari: Tetranychidae) population resurgences consequent upon exposure to synthetic pyrethroid (SP) treatments are reviewed. Resurgences may be seen as soon as 1 week, or even as late as a whole season, post-treatment. Synthetic pyrethroids vary in their adverse effects on spider mites, and also differ in their ability to invoke resurgences of different spidermite species on diverse plants. These pesticides are lethal as well as repellent to phytoseiids and other predators that prey on spider mites, may inhibit fungi which attack the latter, and affect phytophagous competitors. Spider mites are likewise repelled by SPs, thus becoming more evenlydistributed and less web-restricted, with a resultant increase in fecundity. Spider-mite development is shortened due to SPs and the sex ratio becomes more female-biased; onset of winter diapause also seems to be delayed. Synthetic pyrethroids appear to sensitize to spider-mite infestation plants which have not hitherto been attacked. Some SP effects (whether on spider mites, natural enemies or competitors) appear to be direct, whereas others may be mediated through the host plants. The effect of SPs on the other Acari is variable within the Prostigmata and Astigmata. Most Mesostigmata and Metastigmata (ticks) are very sensitive, whilst the Cryptostigmata (Oribatei) appear to be insensitive. Synthetic pyrethroids-induced resurgences of Homoptera are comparatively reviewed, with the conclusion that some of the phenomena may be similar to those observed in spider mites. Various resurgence models are discussed, as well as the three main causes of variation (SPs, spider-mite species, host plants) in the observed phenomena. The need for more rigorous and carefully controlled experimentation is emphasized.     

Infestation by pyrethroids resistant bed bugs in the suburb of Paris,France

Bed bugs are hematophagous insects responsible for a re-emerging and challenging indoor pest in many countries. Bed bugs infestations may have health consequences including nuisance biting, cutaneous and systemic reactions. This resurgence can probably be attributed to factors such as increased international travel and development of resistance against insecticides. Resistance against pyrethroids has been reported several times from the USA and rarely in Europe. In France, very few data on bed bugs are available. The present study aimed to assess the infestation by bed bugs of a complex of two high-rise apartment buildings in the suburb of Paris and to evaluate their susceptibility to pyrethroid insecticides. We inspected for bed bugs 192 out of 198 apartments units (97%) and interviewed their residents. 76 (39.6%) apartments were infested. Among the 97 residents living in infested apartments, 53 (54.6%) reported bed bug bites. A total of 564 bed bugs were collected in the infested units. Bioassays showed that 54 out of 143 bed bugs were resistant to pyrethroids (37.8%; 95% confidence interval: 29.9-45.7%). DNA sequencing showed that all bed bugs tested (n = 124) had homozygous L925I kdr-like gene mutation. The level of pyrethroid resistance found indicates that this phenomenon was already established in the site and prompts the need to reevaluate the wide use of pyrethroids to control bed bugs.     

Interactions of pyrethroids with phosphatidylcholine liposomal membranes

Interactions of several pyrethroids with membrane lipids in the form of dipalmitoylphosphatidylcholine (DPPC) liposomes have been studied using fluorescent membrane probes. Fluorescence anisotropy values and lifetimes (determined by phase-shift and demodulation techniques) of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, were decreased in gel phase liposomes by pyrethroids at concentrations on the order of 10 microM. The pyrethroids containing a cyano substituent were also observed to cause collisional quenching of diphenylhexatriene fluorescence. Pyrethroids differed in their effectiveness at lowering the phase transition temperature of DPPC, and in their ability to broaden the temperature range of this transition. The fluorescence intensity of DPPC-incorporated chlorophyll a was used to monitor the pretransition of DPPC and the lateral diffusion of a membrane component located in the polar headgroup region. Permethrin did not affect chlorophyll a fluorescence intensity at any temperature. It may be concluded from these results that pyrethroids are preferentially located in the interior hydrophobic regions of the lipid bilayer, and that these compounds can disorder hydrocarbon packing in the bilayer core. However, polar headgroups were not disordered, and diffusion of membrane components in the polar headgroup region was not altered.     

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381-410; (1985) Biochim. Biophys. Acta 812, 361-368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.