Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.     

Receptor-Galpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.     

Capillary electrophoresis as a verification tool for immunochemical drug screening

BACKGROUnd: The aim of this work was to develop a simple capillary electrophoretic method as the verification and confirmation tool in the screening analysis for amphetamines, opiates, benzodiazepines and cocaine and their metabolites for toxicological applications. METHODS: 50 mM phosphate Tris pH 2.0 with 30% (v/v) of methanol was used as a background electrolyte that enabled fast separation of drugs and their metabolites in saliva and urine. Verification of the data from the electrophoretic method was done by High Performance Thin Layer Chromatography (HPTLC) and the immunochemical screening test QuikScreen. RESULTS: The experimental conditions of the Capillary Electrophoresis (CE) were partially optimized (mainly the influence of concentration and types of additives, e.g. cyclodextrines, organic solvents) and validated; the method was used for analysing samples from drug abusers. CONCLUSIONS: The non-instrumental, immunoassay tests could only confirm qualitative addictions and are mainly employed when the emergency detection of drugs is needed. For quantitative analysis and verification of obtained results the confirmation step is strongly recommended. The simple screening capillary zone electrophoresis method allows recognition of the most abused drugs. The agreement of the results from CE, HPTLC and QuikScreen test was more than 95%.     

Electroantennographic bioassay as a screening tool for host plant volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant. When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control. Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.     

BESSICC, a COSMO-RS based tool for in silico solvent screening of biocatalyzed reactions

Many enzymatic reactions are near-equilibrium reactions. This often limits final yield and hence application of biocatalyzed processes in the industrial production. The most widely applied strategy to overcome this issue is solvent selection. It must be underlined that measuring the equilibrium position experimentally is a difficult and time-consuming procedure and any tool for predicting the solvent effect on the reaction equilibrium can be very valuable. The present work reports on the development of BESSICC, an algorithm to calculate the effect of medium composition on biocatalyzed reactions equilibrium. It is based on COSMO-RS calculation of activity coefficients of all the species in the reaction mixture and minimization of Gibbs free energy of the reaction. Starting from one single experimental measurement of the equilibrium position for a given biocatalyzed reaction it can predict the yield of the reaction in any other solvent or solvent mixture. Predictions are accurate, the errors of prediction are in average below 25% for the esterification of dodecanoic acid with menthol and below 65% for esterification of 1-dodecanoic acid with 1-dodecanol. The best predictions show an error well below 5%.     

Chlorophyll fluorescence as a tool for evaluation of drought stress in strawberry

The effect of water deficit on chlorophyll fluorescence, sugar content, and growth parameters of strawberry (Fragaria×ananassa Duch. cv. Elsanta) was studied. Drought stress caused significant reductions in leaf water potential, fresh and dry masses, leaf area, and leaf number. A gradual reduction of photochemical quenching (q_P) and quantum efficiency (Φ_PS2) was observed under drought stress while non-photochemical quenching (q_N) increased. Maximum efficiency of photosystem 2 (F_v/F_m) was not affected by drought stress.     

Capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection as a tool to monitor reactions of proteins

Capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) has the characteristics of high resolution, high speed and high sensitivity for separation of amphoteric biomolecules. These features enable a CIEF-LIF-WCID system to monitor the dynamic process of a protein reaction. The reaction can be a physical change or a chemical reaction, provided that the kinetics of the reaction is slower than the focusing speed or that the intermediates involved have long enough life-span compared to the analysis time. The processes of denaturation (a physical reaction), reduction and carbamylation (both chemical reactions) were dynamically monitored. The CIEF profiles at successive reaction times clearly displayed the formation of different products at different stages. At incomplete denaturation, intermediates with higher apparent pI values relative to the products at complete denaturation were detected. Carbamylation products of a protein were detected when the protein reacted with a urea solution that had prepared three months earlier, exhibiting gradually decreased pI values. Mechanisms involved in these reactions were rationalized. A combined mechanism of denaturation and reduction was suggested to explain the denaturing process under high concentrations of urea. Potential applications and critical factors to manipulate these reactions were also discussed.     

Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A

Botulinum neurotoxin serotype A (BoNT/A) is a proteolytic enzyme that induces muscle paralysis. It is a cause of food poisoning, a potential bioterrorist threat and, in low doses an emerging pharmaceutical product. No effective treatment is currently available for BoNT intoxication. Previously we developed a BoNT/A light chain enzyme assay using a peptide substrate based on the SNAP-25 protein target, with HPLC separation and UV detection of assay products, and applied the method to screen combinatorial peptide libraries for inhibitory activity to BoNT/A. We now report on development of a capillary electrophoresis laser-induced fluorescence (CE-LIF) method for measuring BoNT/A activity. The enzyme assay products were labeled with CBQCA dye followed by CE separation on a bare fused silica column in a HEPES-based buffer and LIF detection. All assay products were separated in CE within 8 min compared to incomplete separation of assay products within 1h by HPLC. The labeled products showed linear dependence of intensity versus concentration, and quantitative mole-fraction assignments. We used the CE-LIF method to screen combinatorial peptide libraries for potential modulating effects on BoNT/A peptidase activity. With some of the libraries, peptides co-migrated with assay products and interfered with quantitation. In such cases, interference was reduced by substituting sodium dodecyl sulfate (SDS) for Tween-20 in the running buffer. Separation in the capillaries then occurred by micellar electrokinetic chromatography (MEKC). The CE-LIF method is quick and lends itself to high-throughput or microfluidic formats.     

Caenorhabditis elegans as a chemical screening tool for the study o f neuromuscular disorders. Manual and semi-automated methods

We previously reported the use of the cheap and fast-growing nematode Caenorhabditis elegans to search for molecules, which reduce muscle degeneration in a model for Duchenne Muscular Dystrophy (DMD). We showed that Prednisone, a steroid that is generally prescribed as a palliative treatment to DMD patients, also reduced muscle degeneration in the C. elegans DMD model. We further showed that this strategy could lead to the discovery of new and unsuspected small molecules, which have been further validated in a mammalian model of DMD, i.e. the mdx mouse model. These proof-of-principles demonstrate that C. elegans can serve as a screening tool to search for drugs against neuromuscular disorders. Here, we report and discuss two methodologies used to screen chemical libraries for drugs against muscle disorders in C. elegans. We first describe a manual method used to find drugs against DMD. We further present a semi-automated method, which is currently in use for the search of drugs against the Schwartz-Jampel Syndrome (SJS). Both assays are simple to implement and can be readily transposed and/or adapted to screens against other muscle/neuromuscular diseases, which can be modeled in the worm. Finally we discuss, with respect to our experience and knowledge, the different parameters that have to be taken into account before choosing one or the other method.     

Chlorophyll fluorescence as a tool for nutrient status identification in rapeseed plants

In natural conditions, plants growth and development depends on environmental conditions, including the availability of micro- and macroelements in the soil. Nutrient status should thus be examined not by establishing the effects of single nutrient deficiencies on the physiological state of the plant but by combinations of them. Differences in the nutrient content significantly affect the photochemical process of photosynthesis therefore playing a crucial role in plants growth and development. In this work, an attempt was made to find a connection between element content in (i) different soils, (ii) plant leaves, grown on these soils and (iii) changes in selected chlorophyll a fluorescence parameters, in order to find a method for early detection of plant stress resulting from the combination of nutrient status in natural conditions. To achieve this goal, a mathematical procedure was used which combines principal component analysis (a tool for the reduction of data complexity), hierarchical k-means (a classification method) and a machine-learning method—super-organising maps. Differences in the mineral content of soil and plant leaves resulted in functional changes in the photosynthetic machinery that can be measured by chlorophyll a fluorescent signals. Five groups of patterns in the chlorophyll fluorescent parameters were established: the ‘no deficiency’, Fe-specific deficiency, slight, moderate and strong deficiency. Unfavourable development in groups with nutrient deficiency of any kind was reflected by a strong increase in F _o and ΔV/Δt ₀ and decline in φ _Po, φ _Eo δ _Ro and φ _Ro. The strong deficiency group showed the suboptimal development of the photosynthetic machinery, which affects both PSII and PSI. The nutrient-deficient groups also differed in antenna complex organisation. Thus, our work suggests that the chlorophyll fluorescent method combined with machine-learning methods can be highly informative and in some cases, it can replace much more expensive and time-consuming procedures such as chemometric analyses.     

His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohé L (1997) Biol Chem 378: 827–836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 1 l fermentation broth of A ₆₀₀ 2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b(+)/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k _cat of 1.7 s⁻¹ and limiting K _m values of 51.8 μM and 1.7 μM for t-butyl hydroperoxide and CfTXN2H6, respectively. Received: 3 September 1999 / Accepted: 1 November 1999     

Pyruvate as a fluorescence quencher: a new spectroscopic assay for pyruvate reactions

Optimal conditions for the extraction from brain tissue and the simultaneous quantification of catechol and indole derivatives were determined after a systematic degradation study in water and perchloric acid. The roles of three parameters, namely temperature, presence of antioxidant agents, and time, were considered. Adrenaline, noradrenaline, dopamine, homovanillic acid, 5-hydroxytryptophan, 5-hydroxyindole acetic acid, serotonin, and epinephrine were separated by HPLC and detected electrochemically. The results indicated a great instability of the indole derivatives at an ambient temperature, in an acid medium, and in the absence of a protective agent. Therefore, when perchloric acid has to be used for deproteinization, the lowest concentration (0.1 M) is preferable. The samples have to be kept on ice, in darkness, and protected by ascorbic acid and sodium ethylenediamine tetracetate.     

Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

Summary The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.     

Evaluation of chlorophyll fluorescence as a tool for salt stress detection in roses

The induction kinetic of the chlorophyll (Chl) fluorescence and the F_v/F_m ratio have been tested in order to find out the suitability of this technique to evaluate damage caused by salinity in plants of Rosa hybrida cv. Ilseta grafted on R. manetti growing in a greenhouse under non-saturating irradiance. Under these conditions salinity induced changes in plants morphology, nutrient and Chl contents and in the gas exchange parameters, but not in the F_v/F_m ratio. The Rfd index did not reveal more information. The F_v/F_m ratio as well as the fluorescence induction curves were more affected by salinity when an irradiation stress was added, therefore as an indicator of salt stress in roses, Chl fluorescence is of limited use when the plants are grown without additional stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chlorophyll fluorescence as a tool for evaluation of viability in freeze-stressed grapevine buds

This study examined the utility of the ratio of variable fluorescence to maximum fluorescence (F_v/F_m) to detect freezing injury on buds of two Vitis vinifera cultivars: Pinot noir and Pinot gris. Freezing treatments on buds caused a decrease both in F_v/F_m and percentage of budburst, more severely on Pinot gris than Pinot noir, specifically at the lower temperature (−20°C). F_v/F_m ratio showed a close correlation with percentage of budburst, and a threshold of the lethal F_v/F_m was proposed as an indicator of bud mortality.     

Trace-element analysis by charged-particle-induced reactions as a tool for physicians and chemists

The object of this article is to apprise physicians and chemists of nuclear analytical techniques and, in particular, of ion beam analysis (PIXE and PIGE) for the purpose of application to the clinical diagnostic method. The feasibility of the technique, sampling, and sample preparation for trace element analysis in biological and biomedical samples has been described previously (1–3). Analysis data from normal human blood samples and biomedical samples by ion beam reactions have been compared at the end. Emphasis will be placed on the use of the analytical technique on determination of the range of trace and toxic elements in human blood samples.     

Integrating DNA-encoded chemical libraries with virtual combinatorial library screening: Optimizing a PARP10 inhibitor

Two critical steps in drug development are 1) the discovery of molecules that have the desired effects on a target, and 2) the optimization of such molecules into lead compounds with the required potency and pharmacokinetic properties for translation. DNA-encoded chemical libraries (DECLs) can nowadays yield hits with unprecedented ease, and lead-optimization is becoming the limiting step. Here we integrate DECL screening with structure-based computational methods to streamline the development of lead compounds. The presented workflow consists of enumerating a virtual combinatorial library (VCL) derived from a DECL screening hit and using computational binding prediction to identify molecules with enhanced properties relative to the original DECL hit. As proof-of-concept demonstration, we applied this approach to identify an inhibitor of PARP10 that is more potent and druglike than the original DECL screening hit.