Resonance Energy Transfer Method Research Articles

Fluorescence lifetime imaging of metMyoglobin formation due to nitric oxide stress.

Myoglobin is a protein that is expressed quite unevenly among different cell types. Nevertheless, it has been widely acknowledged that the Fe3+ state of myoglobin, metmyoglobin (metMb) has a broad functional role in metabolism, oxidative/nitrative regulation and gene networks. Accordingly, real-time monitoring of oxygenated, deoxygenated and metMb proportions- or, more broadly, of the mechanisms by which metMb is formed, presents a promising line of research. We had previously introduced a Förster resonance energy transfer (FRET) method to read out the deoxygenation/oxygenation states of myoglobin, by creating the targetable oxygen (O2) sensor Myoglobin-mCherry. In this sensor, changes in myoglobin absorbance features that occur with lost O2 occupancy -or upon metMb production- control the FRET rate from the fluorescent protein to myoglobin. When O2 is bound, mCherry fluorescence is only slightly quenched, but if either O2 is released or met is produced, FRET will increase- and this rate competing with emission reduces both emission yield and lifetime. Nitric oxide (NO) is an important signal (but also a toxic molecule) that can oxidize myoglobin to metMb with absorbance increases in the red visible range. mCherry thus senses both met and deoxygenated myoglobin, which cannot be easily separated at hypoxia. In order to dissect this, we treat cells with NO and investigate how the Myoglobin-mCherry lifetime is affected by generating metMb. More discriminatory power is then achieved when the fluorescent protein EYFP is added to Myoglobin-mCherry, creating a sandwich probe whose lifetime can selectively respond to metMb while being indifferent to O2 occupancy.

Synthesis, crystal structure, BSA binding and antibacterial studies of Ni(II) complexes derived from dithiocarbazate based ligands

A series of nickel(II) bis-complexes of the type [NiL2] C1-C4, where L is a tridentate (NNS) ligand have been isolated and characterized by conductivity studies, elemental analyses, IR and UV–Vis spectral measurements. The single crystal X-ray diffraction studies on [Ni(L1)2] C1 and [Ni(L3)2] C3 reveal that the deprotonated tridentate (NNS) ligands are meridionally coordinated to ascertain a distorted octahedral coordination geometry around nickel(II). The [Ni(L1)2] complex crystallizes in the monoclinic space group P21/c with a = 9.5009(4) Å, b = 13.3354(4) Å, c = 16.7988(7) Å, α = 90 °, β = 98.946(4)°, γ = 90° and Z = 4 while [Ni(L3)2] crystallizes in the monoclinic space group P21/c with a = 16.1362(19), b = 20.2502(11), c = 8.5462(5), α = 90 °, β = 102.778(8)°, γ = 90 ° and Z = 4. The antibacterial potencies of the compounds C1-C4 were estimated on the growth of P. aeruginosa, a Gram-negative bacterium, and S. mutans, a Gram-positive bacterium by broth culture and biofilm formation assay. The interaction between bovine serum albumin (BSA) was investigated using fluorescence quenching techniques. The results revealed that the fluorescence quenching of BSA is through a static quenching process. The binding constants and the number of binding sites were determined using fluorescence quenching data. The binding distance between BSA and Ni-complexes has been determined by using the fluorescence resonance energy transfer method.

Conformational Dynamics of DNA Polymerases Revealed at the Single-Molecule Level.

DNA polymerases are intrinsically dynamic macromolecular machines. The purpose of this review is to describe the single-molecule Förster resonance energy transfer (smFRET) methods that are used to probe the conformational dynamics of DNA polymerases, focusing on E. coli DNA polymerase I. The studies reviewed here reveal the conformational dynamics underpinning the nucleotide selection, proofreading and 5′ nuclease activities of Pol I. Moreover, the mechanisms revealed for Pol I are likely employed across the DNA polymerase family. smFRET methods have also been used to examine other aspects of DNA polymerase activity.

P133 Point-of-care measurement of infliximab and adalimumab trough levels by the fluorescence resonance energy transfer (FRET) assay

Abstract Background Therapeutic drug monitoring (TDM) is the clinical practice of measuring serum drug concentrations to guide clinical decision-making. In IBD patients, it is applied mainly in infliximab (IFX) and adalimumab (ADL) therapy. TDM is either performed reactively in response to disease activity, or proactively in the absence of symptoms. The aim of the present study was to compare two analytical methods for the detection of trough infliximab and adalimumab levels (TL): point-of-care (POCT) fluorescence resonance energy transfer (FRET) method, and the enzyme- linked immunosorbent immunoassay (ELISA). Methods TL IFX were measured in n=24 and TL ADL in n= 21 blood samples. For ELISA, standard peripheral venous samples were used, and for POCT FRET measurements, capillary finger puncture was performed. Two assay results were compared evaluating the regression relationship using Spearman’s correlation coefficient and the Bland-Altman plots. Cohen’s kappa analysis was used to compare the degree of qualitative agreement between the two data sets. Repeatability was assessed by the difference of the repeated measurements, their means and standard deviations. Results The high degree of correlation was found between the measured values, both qualitative and quantitative (p < 0.0001 for both IFX and ADL), either expressed by the Spearman’s correlations, or by Bland-Altman analysis. Cohen’s weighted kappa 0.903 for IFX and 0.834 for ADL showed substantial/almost perfect agreement of qualitative results. Closeness of the agreement of successive measurements was very acceptable for both TLs of both drugs. Conclusion Point-of-care detection of IFX and ADL trough levels by FRET is accurate, comparable to the reference method, analysis is sufficiently consistent. For clinical sites with a proactive approach to TDM, FRET may be a useful tool for decision-making regarding individualization of biologic therapy. Acknowledgements: We thank ProciseDX company for the ProciseDX analyzer rental, and for providing the laboratory diagnostics.

Exploration of SARS-CoV-2 3CLpro Inhibitors by Virtual Screening Methods, FRET Detection, and CPE Assay.

COVID-19 caused by a novel coronavirus (SARS-CoV-2) has been spreading all over the world since the end of 2019, and no specific drug has been developed yet. 3C-like protease (3CLpro) acts as an important part of the replication of novel coronavirus and is a promising target for the development of anticoronavirus drugs. In this paper, eight machine learning models were constructed using naïve Bayesian (NB) and recursive partitioning (RP) algorithms for 3CLpro on the basis of optimized two-dimensional (2D) molecular descriptors (MDs) combined with ECFP_4, ECFP_6, and MACCS molecular fingerprints. The optimal models were selected according to the results of 5-fold cross verification, test set verification, and external test set verification. A total of 5766 natural compounds from the internal natural product database were predicted, among which 369 chemical components were predicted to be active compounds by the optimal models and the EstPGood values were more than 0.6, as predicted by the NB (MD + ECFP_6) model. Through ADMET analysis, 31 compounds were selected for further biological activity determination by the fluorescence resonance energy transfer (FRET) method and cytopathic effect (CPE) detection. The results indicated that (+)-shikonin, shikonin, scutellarein, and 5,3′,4′-trihydroxyflavone showed certain activity in inhibiting SARS-CoV-2 3CLpro with the half-maximal inhibitory concentration (IC50) values ranging from 4.38 to 87.76 μM. In the CPE assay, 5,3′,4′-trihydroxyflavone showed a certain antiviral effect with an IC50 value of 8.22 μM. The binding mechanism of 5,3′,4′-trihydroxyflavone with SARS-CoV-2 3CLpro was further revealed through CDOCKER analysis. In this study, 3CLpro prediction models were constructed based on machine learning algorithms for the prediction of active compounds, and the activity of potential inhibitors was determined by the FRET method and CPE assay, which provide important information for further discovery and development of antinovel coronavirus drugs.

Simply amplificated signal in electrochemiluminescence sensor using nano-gold film as a bridge

Normally, analytical applications of metal oxide semiconductors (MOS) in electrochemiluminescence (ECL) sensors need to be coupled with electrochemical catalysts, some special coreactants or resonance energy transfer method, since MOS exhibited relatively low ECL intensity. In this research, we developed a simple ECL intensity enhancement method for MOS by covering a nanogold-film (NAuF) on the surface of fluorine-doped tin oxide-coated (FTO) conductive glass electrode. Aminated MnO2 nanoparticles (NH-MnO2) was chosen as exemplary MOS emitter to discuss the role of NAuF in ECL system. The results indicated that the NAuF bridged ECL sensor using NH-MnO2 as emitter (NH-MnO2-NAuF/FTO) exhibited a stronger ECL intensity and extremely better stability. In furazolidone (FZD) determination test, the quenched ECL intensity showed a good linear relationship to FZD concentrations in the range 1.0 × 10−8 M ∼ 1.0 × 10−5 M, with a detection limit of 6.6 × 10−9 M, in optimized conditions. Given these attributes, we developed a novel platform for ECL system that could simply enhance the intensity and stability. This work also efficiently offered insights into application potential of ECL analysis.

Driving co-precipitation of hydrophobic drugs in water by conjugating alkyl chains

Co-precipitated nanoaggregates (NAs) of small-molecule hydrophobic drugs have been widely used as carrier-free drug co-delivery systems, but how to construct stable NAs for potential co-delivery of combination drugs has been remaining unclear. Herein, we performed such investigation using our previously reported fluorescence resonance energy transfer (FRET) method, which was based on the fluorescent hydrophobic drugs of camptothecin (CPT), curcumin (CUR), and their lipophilic derivatives containing different alkyl chains. It is shown that unmodified CPT and CUR failed to form stable co-assembling NAs. Increasing hydrophobicity of one drug by conjugating an alkyl chain could promote the co-assembling ability of CPT and CUR, but the resulting NAs were very unstable and undergone a burst disassembly in the plasma. Only simultaneously enhancing the hydrophobicity of CPT and CUR could obtain stable co-assembling NAs. These results highlighted the co-assembly and disassembly of NAs formed by various hydrophobic drugs, which can effectively guide the design of co-assembling small-molecule drug co-delivery systems with high stability to resist the dilution of blood after intravenous injection.

Elucidation of Interactions Between Integration Host Factor and A DNA Four‐Way Junction

Integration host factor (IHF) is an E. coli nucleoid-associated protein that was initially discovered in the integration of bacterial phage DNA. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence and induces a bend of ~160° upon binding. Our previous results have shown that IHF binds to DNA 4WJ that do not contain the consensus sequence with high affinity. DNA Four-Way Junctions (4WJ) are important intermediates in double-strand break repair and homologous recombination. These junctions can adopt either an open or stacked conformation, in which the open conformation facilitates the process of branch migration and strand exchange. In this study, we investigated the IHF-induced changes in the population distribution of junction conformations, using ensemble and single molecule Förster resonance energy transfer (FRET) methods. Our steady state and time-resolved FRET data indicated that under conditions stabilizing the stacked junction conformation, upon binding, IHF induces the open conformation of junction. Single-molecule FRET (smFRET) experiments, performed with total internal reflection fluorescence microscopy, measured population distributions of the junction and showed clear opening of the junctions upon IHF binding. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Cross correlation analyses reveal that the protein slows down the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+, consistent with stabilization of the open conformation. These findings which show that IHF binds to 4WJ with high affinity, induces the open conformation and alters conformational dynamics, suggest that IHF may play multiple roles in the processes of integration and recombination.

Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods.

Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.

Measuring calibration factors by imaging a dish of cells expressing different tandem constructs plasmids.

Three-cube Förster resonance energy transfer (FRET) method is the most extensively applied approach for live-cell FRET quantification. Reliable measurements of calibration factors are crucial for quantitative FRET measurement. We here proposed a modified TA-G method (termed as mTA-G) to simultaneously obtain the FRET-sensitized quenching transition factor (G) and extinction coefficients ratio (γ) between donor and acceptor. mTA-G method includes four steps: (1) predetermining the ratio ranges of the sensitized emission of acceptor (FC ) to the donor excitation and donor channel image (IDD [(DA])) for all FRET plasmids; (2) culturing the cells which express every FRET plasmid in one dish respectively; (3) distinguishing and marking the cells expressing different FRET plasmids by detecting their FC /IDD (DA) values; (4) linearly fitting FC /IAA (DA) (acceptor excitation and acceptor channel image) to IDD (DA)/IAA (DA) for different kinds of cells. We implemented mTA-G method by imaging tandem constructs cells with different FRET efficiency cultured in one dish on different days, and obtained consistent G and γ values. mTA-G method not only circumvents switchover of different culture dishes but also keep the constant imaging conditions, exhibiting excellent robustness, and thus will expands the biological applications of quantitative FRET analysis in living cells.

Evaluation of siRNA Stability and Interaction with Serum Components Using an Agarose Gel-Based Single-Molecule FRET Labeling Method.

Small interfering RNAs (siRNAs) are RNA molecules with promising therapeutic potential as a result of their selective mRNA cleavage. However, despite recent progress, low stability in the bloodstream is an impediment to successful administration in vivo. Thus, the availability of flexible and rapid methods for studying siRNA stability and vehicles is crucial for future novel siRNA-based therapeutics. Herein, we report a fast Förster resonance energy transfer (FRET) method based on agarose gel electrophoresis to evaluate the stability of siRNA in serum as well as siRNA interaction with serum proteins and enzymes.

A Molecularly Imprinted Electrochemical Luminescence Sensor for Detection of Gibberellin Based on Energy Transfer

A molecularly imprinted sensor was fabricated, and a method of enhancing the electrochemiluminescence (ECL) signal of Ru(bpy) 3 Cl 2 using resonance energy transfer (RET) method was established for ultra-sensitive determination of gibberellin (GA3). Au-doped g-C 3 N 4 (g-C 3 N 4 /Au) material was prepared on the surface of glassy carbon electrode (GCE), and then o -phenylenediamine was electropolymerized to achieve a molecularly imprinted polymer (MIP) film. Elution was performed with a methanol-acetic acid eluent to obtain a cavity that specifically recognized GA3. In the experiment, Ru(bpy) 3 2+ was used as the probe and g-C 3 N 4 /Au was used as the energy donor. The RET between Ru(bpy) 3 2+ and g-C 3 N 4 /Au was used to enhance the ECL intensity of Ru(bpy) 3 2+ . In addition, ECL-RET was combined with molecular imprinting technology to improve the selectivity of the sensor. With the increase of the concentration of GA3 in the sample, the molecularly imprinted cavities recombined with GA3 which made the ECL intensity gradually decrease. Under the optimized conditions, it was found that the there was a linear relationship between ECL intensities and the GA3 concentrations in the range of 4.00 × 10 −14 − 7.00 × 10 −11 mol/L, and the detection limit was 1.64 × 10 –14 mol/L (DL = 3 S / K ). The sensor was successfully applied to the detection of GA3 in beer, and the recoveries were 95.7%–103.7%. A molecularly imprinted sensor was fabricated for gibberellin (GA3) determination by electrochemiluminescence resonance energy transfer (ECL-RET) method. g-C 3 N 4 /Au was modified on a glassy carbon electrode, and then a MIP film was synthesized on its surface. The ECL signal of Ru(bpy) 3 Cl 2 probe was enhanced significantly by ECL-RET system, by which the g-C 3 N 4 /Au donor transferred energy to the Ru(bpy) 3 Cl 2 acceptor. High sensitivity was achieved for GA3 detection with a detection limit of 1.64 × 10 -14 mol/L.

Fluorescence Detection of Glutathione Using N-Doped Graphene Quantum Dots–MnO2 Nanoarchitecture

We build a novel fluorescence resonance energy transfer (FRET) method based on N-doped graphene quantum dots (NGQDs)–MnO2 nanocomposite for rapid, sensitive detection of glutathione (G SH) levels in human serum. In this strategy, MnO2 nanosheets on the NGQDs surface serve as a quencher. NGQDs fluorescence can make a recovery by the addition of GSH, which can reduce MnO2 to Mn2+, and thus the GSH can be monitored. The MnO2 platform affords minimal background and high sensitivity for detecting GSH in this proposed scheme. Meanwhile, relevant fluorescence on–off–on processes were monitored, and the sensing mechanism was explored.

Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

Conformational Dynamics of the Periplasmic Chaperone SurA.

The periplasmic protein SurA is the primary chaperone involved in the biogenesis of bacterial outer membrane proteins and is a potential antibacterial drug target. The three-dimensional structure of SurA can be divided into three parts, a core module formed by the N- and C-terminal regions and two peptidyl-prolyl isomerase (PPIase) domains, P1 and P2. Despite the determination of the structures of several SurA-peptide complexes, the functional mechanism of this chaperone remains elusive and the roles of the two PPIase domains are yet unclear. Herein, we characterize the conformational dynamics of SurA by using solution nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer methods. We demonstrate a "closed-to-open" structural transition of the P1 domain that is correlated with both chaperone activity and peptide binding and show that the flexible P2 domain can also occupy conformations that closely contact the NC core module. Our results offer a structural basis for the counteracting roles of the two PPIase domains in regulating the SurA chaperone activity.

Alteration of the Conformational Dynamics of a DNA Hairpin by α-Synuclein in the Presence of Aqueous Two-Phase Systems.

The effect of an amyloidogenic intrinsically disordered protein, α‐synuclein, which is associated with Parkinson's disease (PD), on the conformational dynamics of a DNA hairpin (DNA‐HP) was studied by employing the single‐molecule Förster resonance energy transfer method. The open‐to‐closed conformational equilibrium of the DNA‐HP is drastically affected by binding of monomeric α‐synuclein to the loop region of the DNA‐HP. Formation of a protein‐bound intermediate conformation is fostered in the presence of an aqueous two‐phase system mimicking intracellular liquid‐liquid phase separation. Using pressure modulation, additional mechanistic information about the binding complex could be retrieved. Hence, in addition to toxic amyloid formation, α‐synuclein may alter expression profiles of disease‐modifying genes in PD. Furthermore, these findings might also have significant bearings on the understanding of the physiology of organisms thriving at high pressures in the deep sea.

Serum Activity Against G Protein-Coupled Receptors and Severity of Orthostatic Symptoms in Postural Orthostatic Tachycardia Syndrome.

BackgroundPostural orthostatic tachycardia syndrome (POTS) is characterized by excessive heart rate increase on standing and orthostatic intolerance. Previous data indicate autoimmune involvement. We studied serum activity against G protein–coupled receptors in relation to symptoms in patients with POTS and controls using a commercial cell‐based assay.Methods and ResultsForty‐eight patients with POTS (aged 28.6±10.5 years; 44 women) and 25 healthy individuals (aged 30.7±8.6 years; 21 women) were included. The 10‐item Orthostatic Hypotension Questionnaire (OHQ) was completed by 33 patients with POTS and all controls. Human embryonic kidney 293 cells overexpressing one G protein–coupled receptor: adrenergic α1 receptor, adrenergic β2 receptor, cholinergic muscarinic type 2 receptor, and opioid receptor‐like 1 were treated with sera from all patients. Receptor response was analyzed using a β‐arrestin–linked transcription factor driving transgenic β‐lactamase transcription by fluorescence resonance energy transfer method. Receiver operating characteristic curves were constructed. G protein–coupled receptor activation was related to OHQ indices in linear regression models. Sera from patients with POTS activated all 4 receptors to a higher degree compared with controls (P<0.01 for all). The area under the curve was 0.88 (0.80–0.97, P<0.001) combining all 4 receptors. Adrenergic α1 receptor activation associated with OHQ composite score (β=0.77 OHQ points per SD of activity, P=0.009) and with reduced tolerability for prolonged standing (P=0.037) and walking for short (P=0.042) or long (P=0.001) periods. All 4 receptors were associated with vision problems (P<0.05 for all).ConclusionsOur results indicate the presence of circulating proteins activating adrenergic, muscarinic, and nociceptin receptors in patients with POTS. Serum‐mediated activation of these receptors has high predictive value for POTS. Activation of adrenergic α1 receptor is associated with orthostatic symptoms severity in patients with POTS.

Utilization of KASP technology for wheat improvement

Hundreds of experiments involving genotyping and phenotyping of the plants are planned daily by plant researchers worldwide for wheat improvement. There are a plethora of molecular markers available that are being widely used in wheat genetic studies but have their own limitations. Developments in the areas of next-generation sequencing and bioinformatics applications have aided in the development of SNP-based markers. SNP-based assays have revolutionized the method of genotyping as these consume less time and have high accuracy and effectiveness. Among the SNP genotyping platforms, kompetitive allele-specific PCR (KASP) assay is a promising technology for high throughput SNP genotyping which is based on the fluorescence resonance energy transfer (FRET) method. KASP genotyping mainly involves two allele-specific forward primers, one common reverse primer and FRET cassettes which results in the identification of respective alleles having a particular SNP or InDel. It is playing a significant role in performing various genetic studies such as genetic diversity analysis, genome-wide association studies and marker assisted selection in wheat. In this review, for the first time, we attempt to give an overview of the KASP assays developed for various biotic and abiotic stresses, agronomic, physiological and quality traits for wheat improvement and the assays developed for genetic diversity analysis and genotyping by sequencing.

Rapid and sensitive detection of diazinon in food based on the FRET between rare-earth doped upconversion nanoparticles and graphene oxide.

Diazinon is a typical phosphorothionate, which is widely used to prevent and control harmful organisms that endanger the agriculture productions. However, it is among the most toxic substances and can cause damage to the environment, food and human health even in very low concentrations. Hence, ultra-sensitive screening methods are urgently required for the detection of this extensively used pesticide. In this study, a rapid and sensitive fluorescence resonance energy transfer (FRET) method was developed for low concentration detection of diazinon in food. The aptamer-modified upconversion nanoparticles (Apt-UCNPs) were synthesized and conjugated with graphene oxide (GO) through π-π interaction. Due to the FRET between UCNPs and GO, the fluorescence was quenched. When diazinon was added, the aptamer preferentially bound with it, caused the separation of GO, and resulted in the enhancement of fluorescent signal. Under the optimal conditions, a wide linear detection range from 0.05 to 500ng/mL was achieved, with a limit of detection (LOD) of 0.023ng/mL. The proposed method was successfully applied to measure diazinon in real samples. Results showed that the proposed nanosensor offers an efficient, specific and simple approach for the detection of diazinon in food and has a high potential for food safety and quality control.

Molecular Regulation of the RhoGAP GRAF3 and Its Capacity to Limit Blood Pressure In Vivo.

Anti-hypertensive therapies are usually prescribed empirically and are often ineffective. Given the prevalence and deleterious outcomes of hypertension (HTN), improved strategies are needed. We reported that the Rho-GAP GRAF3 is selectively expressed in smooth muscle cells (SMC) and controls blood pressure (BP) by limiting the RhoA-dependent contractility of resistance arterioles. Importantly, genetic variants at the GRAF3 locus controls BP in patients. The goal of this study was to validate GRAF3 as a druggable candidate for future anti-HTN therapies. Importantly, using a novel mouse model, we found that modest induction of GRAF3 in SMC significantly decreased basal and vasoconstrictor-induced BP. Moreover, we found that GRAF3 protein toggles between inactive and active states by processes controlled by the mechano-sensing kinase, focal adhesion kinase (FAK). Using resonance energy transfer methods, we showed that agonist-induced FAK-dependent phosphorylation at Y376GRAF3 reverses an auto-inhibitory interaction between the GAP and BAR-PH domains. Y376 is located in a linker between the PH and GAP domains and is invariant in GRAF3 homologues and a phosphomimetic E376GRAF3 variant exhibited elevated GAP activity. Collectively, these data provide strong support for the future identification of allosteric activators of GRAF3 for targeted anti-hypertensive therapies.