Methylglyoxal activates transient receptor potential A1/V1 via reactive oxygen species in the spinal dorsal horn.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.

Bioluminescent imaging systems boosting near-infrared signals in mammalian cells.

Bioluminescence (BL) is broadly used as an optical readout in bioassays and molecular imaging. In this study, the near-infrared (NIR) BL imaging systems were developed. The system was harnessed by prototype copepod luciferases, artificial luciferase 30 (ALuc30) and its miniaturized version picALuc, and were characterized with 17 kinds of coelenterazine (CTZ) analogues carrying bulky functional groups or cyanine 5 (Cy5). They were analyzed of BL spectral peaks and enzymatic kinetics, and explained with computational modeling. The results showed that (1) the picALuc-based system surprisingly boosts the BL intensities predominantly in the red and NIR region with its specific CTZ analogues; (2) both ALuc30- and picALuc-based systems develop unique through-bond energy transfer (TBET)-driven spectral bands in the NIR region with a Cy5-conjugated CTZ analogue (Cy5-CTZ); and (3) according to the computational modeling, the miniaturized version, picALuc, has a large binding pocket, which can accommodate CTZ analogues containing bulky functional groups and thus allowing NIR BL. This study is an important addition to the BL imaging toolbox with respect to the development of orthogonal NIR reporter systems applicable to physiological samples, together with the understanding of the BL-emitting chemistry of marine luciferases.

Spinopelvic fusion surgery from lower thoracic spine to pelvis increased hip joint moment-motion analysis.

PURPOSE: Spinal fusion surgery is often performed with pelvic fixation to prevent distal junctional kyphosis. The inclusion of spinopelvic fixation has been reported to induce progression of hip joint arthropathy in a radiographic follow-up study. However, its biomechanical mechanism has not yet been elucidated. This study aimed to compare the changes in hip joint moment before and after spinal fusion surgery. METHODS: This study was an observational study and included nine patients (eight women and one man) who were scheduled to undergo spinopelvic fusion surgery. We calculated the three-dimensional external joint moments of the hip during gait, standing, and climbing stairs before and 1 year after surgery. RESULTS: During gait, the maximum extension moment was 0.51 ± 0.29 and 0.63 ± 0.40 before and after spinopelvic fusion surgery (p = 0.011), and maximum abduction moment was 0.60 ± 0.33 and 0.83 ± 0.34 before and after surgery (p = 0.004), respectively. During standing, maximum extension moment was 0.76 ± 0.32 and 1.04 ± 0.21 before and after spinopelvic fusion surgery (p = 0.0026), and maximum abduction moment was 0.12 ± 0.20 and 0.36 ± 0.22 before and after surgery (p = 0.0005), respectively. During climbing stairs, maximum extension moment was - 0.31 ± 0.30 and - 0.48 ± 0.15 before and after spinopelvic fusion surgery (p = 0.040), and maximum abduction moment was 0.023 ± 0.18 and - 0.02 ± 0.13 before and after surgery (p = 0.038), respectively. CONCLUSION: This study revealed that hip joint flexion-extension and abduction-adduction moments increased after spinopelvic fixation surgery in the postures of standing, walking, and climbing stairs. The mechanism was considered to be adjacent joint disease after spinopelvic fusion surgery including sacroiliac joint fixation.

BRET Q-Body: A Ratiometric Quench-based Bioluminescent Immunosensor Made of Luciferase-Dye-Antibody Fusion with Enhanced Response.

A quenchbody (Q-body) is an immunosensor comprising an antibody fragment containing an antigen-binding site that is site-specifically labeled with a fluorescent dye. The fluorescent dye of a Q-body is quenched in the absence of an antigen; however, its fluorescence recovers in the presence of an antigen, offering simple and rapid systems for antigen detection. In this study, we fused luciferase NanoLuc to a Q-body to construct a new immunosensor termed the "BRET Q-body" that can detect antigens based on the bioluminescence resonance energy transfer (BRET) principle. The resulting BRET Q-bodies for an osteocalcin peptide that emit three different emission colors could detect an antigen without the requirement of an external light source, based on ratiometric detection and color change with two wavelengths for the luciferase and fluorophore. Furthermore, the BRET Q-body produced unexpectedly higher responses up to 12-fold because of the increased BRET efficiency, probably associated with antigen-dependent dye movement. Thus, the BRET Q-body is a useful biosensor as a core of point-of-care tests.

Detection and destruction of HER2-positive cancer cells by Ultra Quenchbody-siRNA complex.

Ultra Quenchbody (UQ-body) is a biosensor that utilizes the quenching behavior of the fluorescent dye linked to the antibody V region. When the corresponding antigen is bound to the UQ-body, the fluorescence is restored and allows the detection of target molecules easily and sensitively. In this paper, we constructed UQ-bodies to sensitively detect the human epidermal growth factor receptor 2 (HER2) cancer marker in solution or on cancer cells, which was further used to kill the cancer cells. A synthetic Fab fragment of anti-HER2 antibody Fab37 with many Trp residues at hypervariable region was prepared and labeled with fluorescent dyes to obtain the UQ-bodies. The UQ-body could detect HER2 in solution at concentrations as low as 20 pM with an EC50 of 0.3 nM with a fourfold response. Fluorescence imaging of HER2-positive cells was successfully performed without any washing steps. To deliver small interfering RNA (siRNA) to cancer cells, a modified UQ-body with C-terminal 9R sequence was also prepared. HER2-positive cancer cells were effectively killed by polo-like kinase 1 siRNA intracellularly delivered by the UQ-body-9R. The novel approach employing siRNA-empowered UQ-body could detect and image the HER2 antigen easily and sensitively, and effectively kill the HER2-positive cancer cells.

Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration with Analysis of Firefly Luciferase Mutants.

Firefly luciferase has been widely used in biotechnology and biophotonics due to photon emission during enzymatic activity. In the past, the effect of amino acid substitutions (mutants) on the enzymatic activity of firefly luciferase has been characterized by the Michaelis constant, KM. The KM is obtained by plotting the maximum relative luminescence units (RLU) detected for several concentrations of the substrate (luciferin or luciferyl-adenylate). The maximum RLU is used because the assay begins to violate the quasi-steady state approximation when RLU decays as a function of time. However, mutations also affect the time to reach and decay from the maximum RLU. These effects are not captured when calculating the KM. To understand changes in the RLU kinetics of firefly luciferase mutants, we used a Michaelis-Menten model with the non-steady state approximation. In this model, we do not assume that the amount of enzyme-substrate complex is at equilibrium throughout the course of the experiment. We found that one of the two mutants analyzed in this study decreases not only the dissociation rate ( koff) but also the association rate ( kon) of luciferyl-adenylate, suggesting the narrowing of the structural pocket containing the catalytic amino acids. Furthermore, comparative analysis of the nearly complete oxidation of luciferyl-adenylate with wild-type and mutant firefly luciferase reveals that the total amount of photons emitted with the mutant is 50-fold larger than that with the wild type, on average. These two results together indicate that the slow supply of luciferyl-adenylate to the enzyme increases the total number of photons emitted from the substrate, luciferyl-adenylate. Analysis with the non-steady state approximation model is generally applicable when enzymatic production kinetics are monitored in real time.

Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation.

The homogeneous immunological detection of small molecules at high sensitivity is still a daunting task. Here, we tried sensitive noncompetitive detection of small peptides based on the open-sandwich immunoassay principle, which was combined with a bioluminescent protein-fragment complementation assay (PCA) in vitro. Since the detection of antigen-induced approximation of the two antibody variable region fragments VH and VL by the standard Nanoluc-based PCA utilizing larger (LgBiT) and shorter (SmBiT) fragments was not successful, we decided to further split LgBiT into two, yielding smaller N-terminal derivative (LnBiT) and two C-terminal, 11 residue peptides (LcBiT and SmBiT) corresponding to consecutive beta strands, to which VH and VL were each fused and expressed in Escherichia coli cells. Through the optimization of reaction conditions and peptide sequence, the antigen osteocalcin peptide can be noncompetitively detected with a low background signal and limit of detection, yielding a high light emission of 88% compared to that of the wild-type enzyme. Since the luminescence of this open sandwich bioluminescent immunoassay (OS-BLIA) can be observed with the naked eye, it could become the foundation of many point-of-care detection systems.

Evaluation of protein-ligand interactions using the luminescent interaction assay FlimPIA with streptavidin-biotin linkage.

Post-translational modifications, such as phosphorylation, are crucial in the regulation of protein-protein interactions and protein function in cell signaling. Here, we studied the interaction between the transactivation domain peptide of cancer suppressor protein p53 and its negative regulator Mdm2 using a novel protein-protein interaction assay, based on the modified FlimPIA using the streptavidin-biotin interaction to link the p53 peptide and the probe enzyme. We succeeded in detecting an attenuation in the affinity of p53 towards Mdm2 caused by the phosphorylation at Thr18. It showed that the targets, which are not easy to fuse with the FlimPIA probes, such as phosphorylated peptides can be used in this system. Also, the use of streptavidin nanobeads was found effective to get clearer signal, probably due to concentration of the detection system onto the bead surface. The system was further applied to the detection of FKBP-FRB interaction using biotinylated FKBP domain, which suggested another potential merit of this system that allows to avoid misfolding and steric hindrance often observed for the fusion protein approach.

Correction: Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation.

Correction for 'Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation' by Jiulong Su et al., Analyst, 2018, 143, 2096-2101.

Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation.

In this study, a novel noncompetitive homogeneous immunoassay for antigen detection was developed. We utilized ß-glucuronidase (GUS), a homotetrameric enzyme, the assembly of all of whose subunits is necessary to attain its activity. By using a mutant GUS (GUSm), wherein the dimerization of dimers, which is a rate-limiting step, can be effectively inhibited by a set of interface mutations, we attempted to create a biosensor for detecting various molecules. Usually, the affinity between the two variable region domains (VH and VL) of an antibody, especially for a small molecule, is relatively low. However, in the presence of an antigen, the affinity increases so that they bind tighter to each other. A pair of fusion proteins, comprising the VH and VL regions of the antibody as the detector tethered to a GUSm subunit as the reporter, was constructed to detect antigen 4-hydroxy-3-nitrophenylacetyl (NP) and bone Gla protein (BGP) through GUS activity measurement. Colorimetric and fluorescence assays could detect NP, 5-iodo-NP, and BGP within 1 h without separation steps and with a higher signal/background ratio than conventional ELISA. The instantaneous response after simple mixing of the components makes this system convenient and high-throughput. The system could be effective for the analyses of various small molecules in environmental and clinical settings.

Improved sensitivity of firefly luminescent intermediate-based protein interaction assay using Ser 440 mutant with lower adenylation activity.

Protein-protein interaction assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel protein-protein interaction assay, the firefly luminescent intermediate-based protein interaction assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half-reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity ('Acceptor' Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin-induced FKBP12-FRB interactions.

Development of a Quenchbody for the Detection and Imaging of the Cancer-Related Tight-Junction-Associated Membrane Protein Claudin.

Claudins (CLs) are membrane proteins found in tight junctions and play a major role in establishing the intercellular barrier. However, some CLs are abnormally overexpressed on tumor cells and are valid clinical biomarkers for cancer diagnosis. Here, we constructed antibody Fab fragment-based Quenchbodies (Q-bodies) as effective and reliable fluorescent sensors for detecting and visualizing CLs on live tumor cells. The variable region genes for anti-CL1 and anti-CL4 antibodies were used to express recombinant Fab fragments, and clones recognizing CL4 with high affinity were selected for making Q-bodies. When two fluorescent dyes were conjugated to the N-terminal tags attached to the Fab, the fluorescent signal was significantly increased after adding nanomolar-levels of purified CL4. Moreover, addition of the Q-body to CL4-expressing cells including CL4-positive cancer cells led to a clear fluorescence signal with low background, even without washing steps. Our findings suggested that such Q-bodies would serve as a potent tool for specifically illuminating membrane targets expressed on cancer cells, both in vitro and in vivo.

Improved protein-protein interaction assay FlimPIA by the entrapment of luciferase conformation.

Recently we reported a novel protein-protein interaction assay FlimPIA (firefly luminescent intermediate-based protein-protein interaction assay) based on the functional complementation of two mutant firefly luciferases (Fluc), each defective in its one of two half reactions. The assay detects approximation of two mutant Flucs, namely, a "Donor" that catalyzes ATP-driven luciferin adenylation to produce a luciferyl-adenylate intermediate, and an "Acceptor" that mainly catalyzes subsequent oxidative luminescent reaction. However, there was a problem in FlimPIA that the remaining adenylation activity of the Acceptor constituted its background signal and hampered its wider use. In this study, we aimed at reducing the background signal by trapping the Acceptor to the "oxidation" conformation, either chemically or by disulfide bonding. The results showed higher sensitivity and detection over the longer distance of the developed assay compared to conventional FlimPIA, Fluc-based protein-fragment complementation assay, and fluorescent protein-based FRET assay.

A protein-protein interaction assay based on the functional complementation of mutant firefly luciferases.

We report a novel bioluminescent protein-protein interaction (PPI) assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named "Donor", which virtually lacks oxidative activity, while the other, named "Acceptor", is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure proteins. When the interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The assay was found to work both in vitro and in cultured cells with strong signals. This would be the first example of reconstituting two divided reactions of one enzyme to detect PPI, which will not only be utilized as a robust PPI assay, but also open a way to control the activity of similar enzymes in acyl/adenylate-forming enzyme superfamily.

Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments.

BACKGROUND: Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics. RESULTS: Split Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner's size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degree C up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system. CONCLUSION: Fluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro, which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu.

Glow-type conversion and characterization of a minimal luciferase via mutational analyses.

Luciferases are widely used as reporter proteins in various fields. Recently, we developed a minimal bright luciferase, picALuc, via partial deletion of the artificial luciferase (ALuc) derived from copepods luciferases. However, the structures of copepod luciferases in the substrate-bound state remain unknown. Moreover, as suggested by structural modeling, picALuc has a larger active site cavity, unlike that in other copepod luciferases. Here, to explore the bioluminescence mechanism of picALuc and its luminescence properties, we conducted multiple mutational analyses, and identified residues and regions important for catalysis and bioluminescence. Mutations of residues likely involved in catalysis (S33, H34, and D55) markedly reduced bioluminescence, whereas that of residue (E50) (near the substrate in the structural model) enhanced luminescence intensity. Furthermore, deletion mutants (Δ70-Δ78) in the loop region (around I73) exhibited longer luminescence lifetimes (~ 30 min) and were reactivated multiple times upon re-addition of the substrate. Due to the high thermostability of picALuc, one of its representative mutant (Δ74), was able to be reused, that is, luminescence recycling, for day-scale time at room temperature. These findings provide important insights into picALuc bioluminescence mechanism and copepod luciferases and may help with sustained observations in a variety of applications.

Improving bioluminescence of a minimal luciferase by adding a charged oligopeptide: picALuc2.0.

Luciferases are widely used as reporter proteins in diverse fields from basic biology to medical and environmental researches. Development of luciferase applications for reporter proteins requires small size without target inhibition, appropriate genomic insertion for high expression level, and bright emission for detection sensitivity. We previously developed the minimal luciferase picALuc, but its luminescence was still dim compared to other bright luciferases in terms of expression in Escherichia coli. In this study, diverse additions of oligopeptides with charged residues (eight amino acids in total) to the C-terminus of picALuc enhanced luminescence by up to approximately 50-fold, that is, enhanced enzymatic activity. Moreover, these high luminescence activities were achieved in bacterial and mammalian expression, suggesting their further applicability in many expression systems. The finding in this study that the simple addition of oligopeptides with charged residues (or charge engineering of this kind) enhances enzymatic activity may be applied to a wide variety of enzymatic reactions and protein functions.

Miniaturization of Bright Light-Emitting Luciferase ALuc: picALuc.

Luciferases are widely used as sensitive reporters in various fields ranging from basic biology to medical diagnosis, public health, and food inspection. Scientists have isolated novel luciferases from bioluminescent organisms and concentrated on improving their brightness and thermostability. Recently, small bright luciferases such as artificial luciferase (ALuc) (21 kDa), NanoLuc (19 kDa), GLuc (18 kDa), and TurboLuc (16 kDa) have been reported. However, smaller, brighter, and more stable luciferases are desired for further applications. Here, we constructed the smallest and bright mutant of ALuc, named "picALuc" (13 kDa). picALuc retained the luminescence activity of the full-length ALuc; moreover, its brightness and thermostability were at the same levels as NanoLuc. Furthermore, we showed the advantage of picALuc for the bioluminescence resonance energy transfer-based assay due to its smallness. Our development has opened the door for wider and more practical applications of luciferases.

Simple Fluorogenic Cellular Assay for Histone Deacetylase Inhibitors Based on Split-Yellow Fluorescent Protein and Intrabodies.

Histone deacetylase (HDAC) inhibitors that regulate the posttranslational modifications of histone tails are therapeutic drugs for many diseases such as cancers, neurodegenerative diseases, and asthma; however, convenient and sensitive methods to measure the effect of HDAC inhibitors in cultured mammalian cells remain limited. In this study, a fluorogenic assay was developed to detect the acetylation of lysine 9 on histone H3 (H3K9ac), which is involved in several cancers, Alzheimer's disease, and autism spectrum disorder. To monitor the changes in H3K9ac levels, an H3K9ac-specific intrabody fused with a small fragment FP11 of the split-yellow fluorescent protein (YFP) (scFv-FP11) was expressed in mammalian cells, together with a larger YFP fragment FP1-10 fused with a nuclear localization signal. When the intranuclear level of H3K9ac is increased, the scFv-FP11 is more enriched in the nucleus via passive diffusion through the nuclear pores from the cytoplasm, which increases the chance of forming a fluorescent complex with the nuclear YFP1-10. The results showed that the YFP fluorescence increased when the cells were treated with HDAC inhibitors. Moreover, the sensitivity of the split YFP reporter system to three HDAC inhibitors was higher than that of a conventional cell viability test. The assay system will be a simple and sensitive detection method to evaluate HDAC inhibitor activities at the levels of both single cells and cell populations.