Rapid and sensitive quantification of capsaicinoids for edible oil adulteration by immunomagnetic solid-phase extraction coupled with time-resolved fluorescent immunochromatographic assay.

Immunochromatographic methods are acknowledged analytic assay to analyze capsaicinoids. Immunomagnetic solid-phase extraction (IMSPE) coupled with time-resolved fluorescence immunochromatographic assay (TRFICA) was proposed to quantify capsaicinoids in oil samples. Monoclonal antibodies (mAb) were synthesized with CNBr-Magnetic Crystarose 4B particles (CNBr-MCPs) under mild condition. The resultant CNBr-MCPs@mAb were conjugated high affinity mAbs on its surface, which was utilized to extract capsaicinoids from lipid matrices via antibodies-antigens capture. Under the optimized conditions, the whole IMSPE procedure was achieved within 15 min, and quantified by TRFICA strips. The results showed coefficients up to 0.9975 and the visual detection limit as low as 0.6 µg kg-1. The recoveries were ranging from 88.3 % to 112.4 % with the intra-day and inter-day precision lower than 11.6 %. Finally, the proposed IMSPE-TRFICA method was successfully used to detect capsaicinoids in lipid matrices, which has great utility to quantify capsaicinoids and adulteration detect vegetable oils.

Reducing False Positives through the Application of Fluorescence Lifetime Technology: A Comparative Study Using TYK2 Kinase as a Model System.

The predominant assay detection methodologies used for enzyme inhibitor identification during early-stage drug discovery are fluorescence-based. Each fluorophore has a characteristic fluorescence decay, known as the fluorescence lifetime, that occurs throughout a nanosecond-to-millisecond timescale. The measurement of fluorescence lifetime as a reporter for biological activity is less common than fluorescence intensity, even though the latter has numerous issues that can lead to false-positive readouts. The confirmation of hit compounds as true inhibitors requires additional assays, cost, and time to progress from hit identification to lead drug-candidate optimization. To explore whether the use of fluorescence lifetime technology (FLT) can offer comparable benefits to label-free-based approaches such as RapidFire mass spectroscopy (RF-MS) and a superior readout compared to time-resolved fluorescence resonance energy transfer (TR-FRET), three equivalent assays were developed against the clinically validated tyrosine kinase 2 (TYK2) and screened against annotated compound sets. FLT provided a marked decrease in the number of false-positive hits when compared to TR-FRET. Further cellular screening confirmed that a number of potential inhibitors directly interacted with TYK2 and inhibited the downstream phosphorylation of the signal transducer and activator of transcription 4 protein (STAT4).

Time-resolved fluorescence and chemometrics-assisted excitation-emission fluorescence for qualitative and quantitative analysis of scopoletin and scopolin in Erycibe obtusifolia Benth.

This paper presents a new strategy for qualitative identification of scopoletin and scopolin in Erycibe obtusifolia Benth using time-resolved (lifetimes) fluorescence and quantitative analysis with chemometrics-assisted excitation-emission matrix (EEM) fluorescence. Due to the significant spectral overlapping among analytes and interference, the use of the more selective time-resolved fluorescence is proposed for qualitative identification in quality control of traditional Chinese medicine (TCM) for the first time. Using the strategy of combining EEM fluorescence with second-order calibration method, i.e. parallel factor analysis (PARAFAC), the simultaneous quantification of scopoletin and scopolin in the complex system of Erycibe obtusifolia Benth was achieved successfully. The predicted concentrations were compared with the values obtained using high performance liquid chromatography-coupled to fluorimetric detector (HPLC-FLD), and no significant differences between them were observed. Therefore, the proposed methods using time-resolved fluorescence for qualitative analysis and EEMs coupled with second-order calibration for quantitative analysis in TCM are comparable and provide a suitable alternative to the chromatography-based method.

Elucidating the interaction of Spathodea campanulata leaf extracts mediated potential bactericidal gold nanoparticles with human serum albumin: spectroscopic analysis.

Nanomaterials in different form have been thoroughly used in the area of pharmaceutics and medicine for drug delivery. The large scale of nanoparticles (NPs) synthesis from plant extract is much safe, cheap and eco-friendly. Here, we demonstrated a new, one-step, ultra-fast biosynthesis of gold nanoparticles (sc-AuNPs, 19.54 nm) by using aqueous Spathodea campanulata leaf extracts as a reducing and capping agent. And also, we presented the synthesis of citrate capped gold nanoparticles (cit-AuNPs) of approximately same size (19.66 nm). These two NPs were characterized by UV-Visible, dynamic light scattering, transmission electron microscope and energy dispersive X-ray spectroscopy. Fourier transform infrared spectroscopy confirmed that the functional groups like OH, NH, OH of COOH and CO were contributed in the sc-AuNPs formation. The negative zeta potential (-20.5, -22.8 mV) established the stability and dispersion of the sc- and cit-AuNPs. The anti-bacterial activity of the sc- and cit-AuNPs were checked against Escherichia coli (DH5-Alpha). Minimum inhibitory concentration was 2.4 and 3.0 nM, respectively for sc- and cit-AuNPs. The interaction study of the sc-AuNPs/cit-AuNPs-human serum albumin (HSA) system was done by UV-Visible absorbance, fluorescence, circular dichroism, time resolved fluorescence spectroscopy and the measurement of zeta potential. Absorbance, three dimensional fluorescence, synchronous fluorescence and circular dichroism spectroscopy showed a minor conformational change of HSA upon interaction with the sc-AuNPs compared to cit-AuNPs. The present comparative study will advance our knowledge about the binding mode, mechanism and conformational change of the protein upon interaction with green synthesized sc-AuNPs and cit-AuNPs. Communicated by Ramaswamy H. Sarma.

Pentacyclic Triterpenes from Cecropia telenitida Can Function as Inhibitors of 11ß-Hydroxysteroid Dehydrogenase Type 1.

Plant extracts from the genus Cecropia have been used by Latin-American traditional medicine to treat metabolic disorders and diabetes. Previous results have shown that roots of Cecropia telenitida contain pentacyclic triterpenes and these molecules display a hypoglycemic effect in an insulin-resistant murine model. The pharmacological target of these molecules, however, remains unknown. Several lines of evidence indicate that pentacyclic triterpenes inhibit the 11ß-hydroxysteroid dehydrogenase type 1 enzyme, which highlights the potential use of this type of natural product as phytotherapeutic or botanical dietary supplements. The main goal of the study was the evaluation of the inhibitory effect of Cecropia telenitida molecules on 11ß-hydroxysteroid dehydrogenase type 1 enzyme activity. A pre-fractionated chemical library was obtained from the roots of Cecropia telenitida using several automated chromatography separation steps and a homogeneous time resolved fluorescence assay was used for the bio-guided isolation of inhibiting molecules. The screening of a chemical library consisting of 125 chemical purified fractions obtained from Cecropia telenitida roots identified one fraction displaying 82% inhibition of the formation of cortisol by the 11ß-hydroxysteroid dehydrogenase type 1 enzyme. Furthermore, a molecule displaying IC50 of 0.95 ± 0.09 µM was isolated from this purified fraction and structurally characterized, which confirms that a pentacyclic triterpene scaffold was responsible for the observed inhibition. Our results support the hypothesis that pentacyclic triterpene molecules from Cecropia telenitida can inhibit 11ß-hydroxysteroid dehydrogenase type 1 enzyme activity. These findings highlight the potential ethnopharmacological use of plants from the genus Cecropia for the treatment of metabolic disorders and diabetes.

A novel bioassay based on aptamer-functionalized magnetic nanoparticle for the detection of zearalenone using time resolved-fluorescence NaYF4: Ce/Tb nanoparticles as signal probe.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by fungi on stored grains. The earlier detection methods used for ZEN rely on expensive equipment, time-consuming sample preparation and temperature sensitive antibodies. The current work, proposed a novel strategy based on ZEN aptamer labeled with amine-functionalized magnetic nanoparticle (MNPs) as a capture probe and time-resolved fluorescence (TRFL) nanoparticles labeled with complementary DNA (cDNA) as a signal probe. Under the optimized conditions, TRFL intensity at 544 nm was used to measure ZEN (R2 = 0.9920) in the range of 0.001-10 ng mL-1 and limits of detection (LOD) for proposed method was 0.21 pg mL-1. The specificity of bioassay was also determined by using other mycotoxins (OTA, AFB2, DON and Patulin) and results showed that the aptamer are specific to recognize only ZEN. The analytical applications of the present bioassay in maize and wheat samples were also examined and results were compared with existing methods. Based on these findings, it is suggested to use current rapid and simple bioassay for the determination of ZEN in food and agricultural products.

Discovery and characterization of an iminocoumarin scaffold as an inhibitor of MEKK2 (MAP3K2).

The kinase MEKK2 (MAP3K2) activates the MEK5/ERK5 cell signaling pathway and may play an important role in tumor growth and metastasis. Thus, MEKK2 may represent a novel kinase target for cancer. In order to identify inhibitors of MEKK2, we screened a library of compounds using a high throughput MEKK2 intrinsic ATPase enzyme assay. We identified two hits with validated structures and confirmed activity in the primary assay (IC50 values = 322 nM and 7.7 µM) and two orthogonal MEKK2 biochemical assays. Compound 1, the more potent hit, was the subject of further investigation. Limited structure-activity relationship (SAR) studies were performed on this iminocoumarin hit which resulted in ≥20-fold more potent analogs (e.g. 8 and 16 nM IC50). Two analogs had improved selectivity in a 50-member kinase profiling panel compared to the hit. These studies suggested that substitutions around the phenoxy ring of this scaffold can impart improved potency and selectivity for MEKK2. Analog Compound 1s (16 nM IC50) was further verified by external testing to inhibit MEKK2 and MEKK3 with similar potencies. Compound 1s displayed activity in cell-based assays in which it inhibited ERK5 pathway activation in cells and inhibited cell migration in a scratch assay. Thus, we have identified a scaffold that has promising potential to be developed into a highly selective and potent inhibitor of MEKK2. Information from these SAR studies provides specific guidance for the future design of MEKK2 inhibitor probes.

Time-Resolved Fluorescence Assays.

Fluorescence-based detection techniques are popular in high throughput screening due to sensitivity and cost-effectiveness. Four commonly used techniques exist, each with distinct characteristics. Fluorescence intensity assays are the simplest to run, but suffer the most from signal interference. Fluorescence polarization assays show less interference from the compounds or the instrument, but require a design that results in change of fluorophore-containing moiety size and usually have narrow assay signal window. Fluorescence resonance energy transfer (FRET) is commonly used for detecting protein-protein interactions and is constrained not by the sizes of binding partners, but rather by the distance between fluorophores. Time-resolved fluorescence resonance energy transfer (TR-FRET), an advanced modification of FRET approach utilizes special fluorophores with long-lived fluorescence and earns its place near the top of fluorescent techniques list by its performance and robustness, characterized by larger assay window and minimized compound spectral interference. TR-FRET technology can be applied in biochemical or cell-based in vitro assays with ease. It is commonly used to detect modulation of protein-protein interactions and in detection of products of biochemical reactions and cellular activities.

Monitoring the Collapse of pH-Sensitive Liposomal Nanocarriers and Environmental pH Simultaneously: A Fluorescence-Based Approach.

Nowadays, the encapsulation of therapeutic compounds in so-called carrier systems is a very smart method to achieve protection as well as an improvement of their temporal and spatial distribution. After the successful transport to the point of care, the delivery has to be released under controlled conditions. To monitor the triggered release from the carrier, we investigated different fluorescent probes regarding their response to the pH-induced collapse of pH-sensitive liposomes (pHSLip), which occurs when the environmental pH falls below a critical value. Depending on the probe, the fluorescence decay time as well as fluorescence anisotropy can be used equally as key parameters for monitoring the collapse. Especially the application of a fluorescein labeled fatty acid (fPA) enabled the monitoring of the pHSLips collapse and the pH of its microenvironment simultaneously without interference. Varying the pH in the range of 3 < pH < 9, anisotropy data revealed the critical pH value at which the collapse of the pHSLips occurs. Complementary methods, e.g., fluorescence correlation spectroscopy and dynamic light scattering, supported the analysis based on the decay time and anisotropy. Additional experiments with varying incubation times yielded information on the kinetics of the liposomal collapse.

Development of a CERT START Domain-Ceramide HTRF Binding Assay and Application to Pharmacological Studies and Screening.

Sphingomyelin (SM) metabolism deregulation was recently associated with cell metastasis and chemoresistance, and several pharmacological strategies targeting SM metabolism have emerged. The ceramide (Cer) generated in the endoplasmic reticulum (ER) is transferred to the Golgi apparatus to be transformed into SM. CERamide Transfer (CERT) protein is responsible for the nonvesicular trafficking of Cer to Golgi. Blocking the CERT-mediated ER-to-Golgi Cer transfer is an interesting antioncogenic therapeutic approach. Here, we developed a protein-lipid interaction assay for the identification of new CERT-Cer interaction inhibitors. Frequently used for protein-protein interaction by enzymatic and analyte dosage assays, homogeneous time-resolved fluorescence technology was adapted for the first time to a lipid-protein binding assay. This test was developed for high-throughput screening, and a library of 672 molecules was screened. Seven hits were identified, and their inhibitory effect quantified by EC50 measurements showed binding inhibition three orders of magnitude more potent than that of HPA12, the unique known CERT antagonist to date. Each compound was tested on an independent test, confirming its high affinity and pharmacological potential.

Cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching for time-resolved fluorescence detection of biothiols in serum.

We report here an efficient pyrene excimer signaling-based time-resolved fluorescent sensor for the measurement of biothiols (cysteine (Cys), homocysteine (Hcy), glutathione (GSH)) in human serum based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and the inclusion interaction of cyclodextrin. The sensing mechanism of the approach is based on the competitive ligation of Hg(2+) ions by Hcy/Cys/GSH and T-T mismatches in a bis-pyrene-labeled DNA strand with the self-complementary 5' and 3' ends. The introduction of γ-cyclodextrin can provide cooperation for the molecular level space proximity of the two labeled pyrene molecules, moreover the hydrophobic cavity of γ-cyclodextrin can also offer protection for the pyrene dimer's emission from the quenching effect of environmental conditions and enhance the fluorescence intensity of the pyrene excimer. When the biothiols are not presented, the sensing ensemble is in the "off" state due to the long distance between the two labeled pyrene molecules resulted from the formation of a more stable T-Hg(2+)-T structure. While in the presence of biothiols, Hg(2+) interacts very strongly with thiol groups and the T-Hg(2+)-T structure is dehybridized, and then the pyrene excimer will be formed due to the self-complementary 5' and 3' ends of the DNA probe and the cooperation interaction of γ-cyclodextrin to pyrene dimer, thus resulting in switching the sensing ensemble to the "on" state. In the optimum conditions described, the linear concentration range of 1.0-100 µM with the limit of detection (LOD) of 0.36 µM for GSH was obtained. Moreover, due to the much longer lifetime of the pyrene excimer fluorescence than those of the ubiquitous endogenous fluorescent components, the time-resolved fluorescence technique has been successfully used for application in complicated biological samples.

Influence of cooking on the levels of bioactive compounds in Purple Majesty potato observed via chemical and spectroscopic means.

Tubers rich in phytochemicals can exhibit a potential health benefit. This work aims at studying the relative effect of different domestic cooking techniques by monitoring the level of total phenolic compounds (TP), total anthocyanins (TA) and anti-oxidant activity (AOA) on a variety of pigmented potatoes. Raw purple potatoes are a good source of anthocyanins (219 mg/kg FW) and the level of these compounds increased using different cooking techniques, with the exception of baking. However, the levels of phenolic compounds (originally 209 mg GAE/100 g FW) decreased in the cooked potatoes. Although potatoes contain different antioxidants in this work the antioxidant activity seems to be related to the levels of phenolic compounds present in the pigmented potato. The fact that some of the compounds present fluoresce enabled both steady state and time-resolved fluorescence techniques to be assessed as a non destructive means of monitoring. This elucidated the presence of different components (via spectral deconvolution and time-resolved emission spectra). Their relative contribution to the fluorescence emission was found to be affected by the different cooking process, with a longer wavelength emission appearing to relate to reflect the presence of anthocyanins.

Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2.

Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z' factor = 0.58) emerged as a superior screening strategy compared with FP (Z' factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions.

Tyrosine kinase inhibitory activity of dehydroabietylamine derivatives tested by homogeneous time-resolved fluorescence based high throughput screening model.

Protein tyrosine kinases (PTKs) are attractive targets in searching for therapeutic agents against many diseases. In this study, a series of dehydroabietylamine derivatives were first determined to show PTK inhibitory activity using a high-throughput screening (HTS) method based on homogeneous time-resolved fluorescence (HTRF) technology. The structure-activity relationships of the dehydroabietylamine derivatives were established, and it was found that the compounds with a nitrogen-containing side chain had better inhibitory activity. Further studies showed that the compounds substituted with halogen in the phenyl ring resulted in higher inhibitory activity on the epidermal growth factor receptor (EGFR), and can be a guide to modify the structure of dehydroabietylamine derivatives. Dehydroabietylamine derivatives might be a new class of multi-targeted and effective PTK inhibitors with structure modifications.

Honokiol: a non-adipogenic PPARγ agonist from nature.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators. METHODS: We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists. RESULTS: The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain. CONCLUSION: We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo. GENERAL SIGNIFICANCE: This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.

A new method for the detection of adenosine based on time-resolved fluorescence sensor.

In this work, we report a thrombin binding aptamer complex based time-resolved fluorescence sensor for small molecule detection. The sensor employs two strands (DNA1 and DNA2) of oligonucleotides. This two strands of oligonucleotides contain two aptamer (α-aptamer and ß-aptamer) respectively. DNA1 and DNA2 were labeled with biotin and DIG at the 3'-end, respectively. Binding of the α-aptamer and ß-aptamer to the thrombin promotes the hybridization between the complementary stem sequences attached to the two oligonucleotide sequences. The hybridization then brings biotin to be hidden in the shield part on DNA1, shielding biotin from being approached by the streptavidin modified on the microplate due to the steric hindrance effect of the shield part of DNA1. Result in the thrombin-aptamer complex cannot be modified on the surface of microplate which further leads to no signal reported. The strategy integrates the distinguishing features of aptamer and fluorescent techniques. As a proof-of-principle, adenosine in serum was detected with a detection limit of 0.5 nM. A nice detection limit and linear relationship were obtained.

Tyrosine kinase inhibitory activity of dehydroabietylamine derivatives tested by homogeneous time-resolved fluorescence based high throughput screening model / 中国天然药物

Protein tyrosine kinases (PTKs) are attractive targets in searching for therapeutic agents against many diseases. In this study, a series of dehydroabietylamine derivatives were first determined to show PTK inhibitory activity using a high-throughput screening (HTS) method based on homogeneous time-resolved fluorescence (HTRF) technology. The structure-activity relationships of the dehydroabietylamine derivatives were established, and it was found that the compounds with a nitrogen-containing side chain had better inhibitory activity. Further studies showed that the compounds substituted with halogen in the phenyl ring resulted in higher inhibitory activity on the epidermal growth factor receptor (EGFR), and can be a guide to modify the structure of dehydroabietylamine derivatives. Dehydroabietylamine derivatives might be a new class of multi-targeted and effective PTK inhibitors with structure modifications.