A novel label-free live-cell biosensor for G-protein-coupled receptor functional assay with enhanced sensitivity

This study developed a surface plasmon resonance (SPR)-based live-cell biosensor with enhanced sensitivity for label-free ligand binding assay of G-protein-coupled receptors (GPCRs). The β2-adrenoceptor was heterologously expressed in human embryonic kidney-293 cells. The specific ligand binding function of expressed β2-adrenoceptor was monitored by SPR via refractive index measurement. The results indicate the expressed β2-adrenoceptor can respond to isoprenaline with high specificity. The SPR signals can be enhanced more than three times by the use of LY294002. This biosensor can be applied in the functional assay of GPCRs by detecting the specific interactions between GPCRs and their target ligands.     

Surface processes of olfactory receptors

Summary The free surface morphology of olfactory receptor cells from the nasal mucosa of Cynomolgus monkeys was studied electron microscopically. The receptor cell, in addition to showing characteristic cilia, possesses several branched or unbranched shorter elevations or spiny processes covered by numerous delicate lace-like filaments not previously described. These filaments diminish in length and number toward the base of the microvillous protrusions.     

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.     

A surface acoustic wave sensor functionalized with a polypyrrole molecularly imprinted polymer for selective dopamine detection

A surface acoustic wave sensor operating at 104 MHz and functionalized with a polypyrrole molecularly imprinted polymer has been designed for selective detection of dopamine (DA). Optimization of pyrrole/DA ratio, polymerization and immersion times permitted to obtain a highly selective sensor, which has a sensitivity of 0.55°/mM (≈550 Hz/mM) and a detection limit of ≈ 10 nM. Morphology and related roughness parameters of molecularly imprinted polymer surfaces, before and after extraction of DA, as well as that of the non imprinted polymer were characterized by atomic force microscopy. The developed chemosensor selectively recognized dopamine over the structurally similar compound 4‐hydroxyphenethylamine (referred as tyramine), or ascorbic acid,which co‐exists with DA in body fluids at a much higher concentration. Selectivity tests were also carried out with dihydroxybenzene, for which an unexpected phase variation of order of 75% of the DA one was observed. Quantum chemical calculations, based on the density functional theory, were carried out to determine the nature of interactions between each analyte and the PPy matrix and the DA imprinted PPy polypyrrole sensing layer in order to account for the important phase variation observed during dihydroxybenzene injection. Copyright © 2015 John Wiley & Sons, Ltd.     

A comparison of protocols for the optimisation of detection of bacteria using a surface acoustic wave (SAW) biosensor

A dual channel surface acoustic wave (SAW) device has been used as a biosensor to detect two different microorganisms, Legionella and Escherichia coli, simultaneously. A series of experiments was conducted to optimise the use of the SAW for bacterial detection using a novel protocol of coating bacteria on the sensor surface prior to addition of the antibody. Results were compared with an experiment in which a conventional protocol was utilised, where antibody was coated on the sensor surface prior to exposure to bacteria. The concentration of bacteria that attached to the surface of the SAW device was related to the antibody that specifically bound to it and therefore to frequency in a dose dependent fashion. Unlike conventional microbiological techniques quantitative results can be obtained for Legionella and E. coli down to 10(6) cells per ml within 3 h. In addition E. coli was detected down to 10(5) cells per ml in a modified protocol using sheep IgG as a blocking agent.     

Viscoelastic properties' characterization of corneal stromal models using non-contact surface acoustic wave optical coherence elastography (SAW-OCE)

Viscoelastic characterization of the tissue-engineered corneal stromal model is important for our understanding of the cell behaviors in the pathophysiologic altered corneal extracellular matrix (ECM). The effects of the interactions between stromal cells and different ECM characteristics on the viscoelastic properties during an 11-day culture period were explored. Collagen-based hydrogels seeded with keratocytes were used to replicate human corneal stroma. Keratocytes were seeded at 8 × 10³ cells per hydrogel and with collagen concentrations of 3, 5 and 7 mg/ml. Air-pulse-based surface acoustic wave optical coherence elastography (SAW-OCE) was employed to monitor the changes in the hydrogels' dimensions and viscoelasticity over the culture period. The results showed the elastic modulus increased by 111%, 56% and 6%, and viscosity increased by 357%, 210% and 25% in the 3, 5 and 7 mg/ml hydrogels, respectively. To explain the SAW-OCE results, scanning electron microscope was also performed. The results confirmed the increase in elastic modulus and viscosity of the hydrogels, respectively, arose from increased fiber density and force-dependent unbinding of bonds between collagen fibers. This study reveals the influence of cell-matrix interactions on the viscoelastic properties of corneal stromal models and can provide quantitative guidance for mechanobiological investigations which require collagen ECM with tuneable viscoelastic properties.     

Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatography assay for the novel antitumour agent CT2584 in human plasma

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the novel anticancer agent CT2584, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, which has recently completed a phase I trial at the Christie Hospital, Manchester under the auspices of the CRC phase I/II committee. Following addition of CT2576, 1-(11-octylamino-10-hydroxylundecyl)-3,7-dimethylxanthine, as internal standard, a solid-phase extraction cartridge (100 mg cyanopropyl) was used to isolate the drug CT2584 from human plasma. Analysis was performed by reversed-phase chromatography. CT2576 was used as internal standard at a concentration of 4 μg ml⁻¹ for the quantification of CT2584 from plasma for the duration of this work. The lower limit of quantification for the drug CT2584 in buffer using this assay was found to be 0.0122 μM (0.008 μg ml⁻¹) and 0.048 μM (0.027 μg ml⁻¹) when extracted from human plasma.     

A rapid and sensitive assay for quantification of siRNA efficiency and specificity

RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin β4 (Tβ4); determined their effectiveness at silencing Tβ4 and have both excluded off-target silencing of the Tβ4 homologue Thymosin β10 (Tβ10) and demonstrated partial knockdown of Tβ10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling.     

Assay and analysis for anti- and pro-oxidative effects of ascorbic acid on DNA with the bulk acoustic wave impedance technique

A bulk acoustic wave (BAW) impedance sensor has been applied for in situ monitoring of the whole process of DNA oxidative damage induced by the vitamin C (Vc)-Fe (III) system, based on its real-time responses to the density-viscosity change of the tested solution due to the damages occurring on the DNA molecules. The results showed that Vc exhibited two conflicting effects, i.e., pro-oxidation and anti-oxidation on the DNA at different Vc concentrations in the damage system, and the "threshold" concentration of Vc for these two effects was estimated to be about 100 micromol/L. The end-point frequency change of the sensor (Deltaf(m)) was found to be linearly related to the initial concentration of the soybean DNA (C(DNA)) in the range of 40-1000 microg/mL, and the exponential relationship between the frequency change (Deltaf(0)) vs damaging time suggested that the Fe (III)-mediated DNA damage by Vc could be described as a first-order kinetics reaction. The effects of variations in concentrations of Vc and Fe3+ on the DNA oxidative damage were discussed, and based on investigations for the enhancing influence of H2O2 and inhibiting influence of HO* scavengers on the DNA damage, the nature and physiological toxicity of the damage in biological system were also examined. In addition, UV-vis spectra and electrophoresis analysis were also used, and the experimental observations were in good agreement with the above results.     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.     

Taurine-induced attenuation of MPP+ neurotoxicity in vitro: a possible role for the GABA(A) subclass of GABA receptors

Taurine is a sulphur-containing beta-amino acid found in high (millimolar) concentrations in excitable tissues such as brain and heart. Its suggested roles include osmoregulator, thermoregulator, neuromodulator, and potential neurotransmitter. This amino acid has also been shown to be released in large concentrations during ischaemia and excitotoxin-induced neuronal damage. Here we report a protective effect of taurine against MPP(+)-induced neurotoxicity in coronal slices from rat brain. Significant protective effects were observed at taurine concentrations of 20 and 1 mM, suggesting a potential role for taurine in cases of neuronal insult. Studies with the synthetic taurine analogues taurine phosphonate, guanidinoethane sulphonate, and trimethyltaurine suggested the observed effect to be mediated via an extracellular mechanism. The use of GABA receptor ligands muscimol and bicuculline indicated the effect to be mediated through activation of GABA(A) receptors.     

Novel application of surface plasmon resonance biosensor chips for measurement of advanced glycation end products in serum of Zucker diabetic fatty rats

Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.     

Challenging Theophrastus: A common core list of plant traits for functional ecology

Abstract. Ecologists need a common language of plant traits in order to make comparisons across regions and scales, pool data, and maximize the utility of the data. To develop such a set of traits we began with the primary challenges faced by plants: dispersal, establishment, and persistence in order to identify fundamental traits. Most of these traits are hard to measure, but advances in comparative ecology have suggested a number of easy to measure analogs. Unfortunately, some of the fundamental traits have no simple analog. The common core list includes: seed mass, seed shape, dispersal mode, clonality, specific leaf area, leaf water content, height, above-ground biomass, life history, onset of flowering, stem density, and resprouting ability. Most of the traits can be measured quantitatively, but several traits on the list must still be measured qualitatively due to logistical problems or lack of an easy analog. Key problem areas include: dispersal ability, capacity for vegetative spread, germination, palatability, plasticity, and all the various below-ground traits. Comparative studies need to address these problem areas. The common core list is suggested as a common starting point for studies of functional ecology. The idiosyncrasies of regional floras and specific research agendas will dictate which traits can be ignored and those that need to be added.     

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

Aims

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

Main methods

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4 weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4 mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10 mg/kg b.w.), GP 1a (2.5 mg/kg b.w.) or ZnPP IX (1 μM). Isolated KC were treated with Zymosan (0.5 mg/ml) in addition to JWH-133 (5 μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western blot or confocal microscopy.

Key findings

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

Significance

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.