A novelα-ketoamide reactivity-based two-photon fluorogenic probe for visualizing peroxynitrite in Parkinson's disease models

Peroxynitrite(ONOO^(-))contributes to oxidative stress and neurodegeneration in Parkinson's disease(PD).Developing a peroxynitrite probe would enable in situ visualization of the overwhelming ONOO^(-)flux and understanding of the ONOO^(-)stress-induced neuropathology of PD.Herein,a novelα-ketoamide-based fluorogenic probe(DFlu)was designed for ONOO^(-)
monitoring in multiple PD models.The results demonstrated that DFlu exhibits a fluorescence turn-on response to ONOO^(-)with high specificity and sensitivity.The efficacy of DFlu for intracellular ONOO^(-)
imaging was demonstrated systematically.The results showed that DFlu can successfully visualize endogenous and exogenous ONOO^(-)in cells derived from chemical and biochemical routes.More importantly,the two-photon excitation ability of DFlu has been well demonstrated by monitoring exogenous/endogenous ONOO^(-)production and scavenging in live zebraflsh PD models.This work provides a reliable and promising
α-ketoamide-based optical tool for identifying variations of ONOO^(-)in PD models.

THE DETECTION OF MDR1 GENE EXPRESSION USING FLUOROGENIC PROBE QUANTITATIVE RT-PCR METHOD

Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107 copies/μg RNA and (8.49±0.67)×105 copies/μg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.

Fluorogenic Detection of Duck Tembusu Virus( DTMUV ) by Loop-mediated Isothermal Amplification(LAMP)

This study was to develop an efficient and simple method for the detection of duck Tembusu virus( DTMUV) by loop-mediated isothermal amplification( LAMP). Six pairs of LAMP primers were designed according to the conserved region of the DTMUV E gene sequence in Gen Bank,which were then used for the optimization of various reaction components and reaction system of specific LAMP for DTMUV. Further the fluorescent reagent SYBR Green I and a certain proportion of calcium and manganese ion were used to determin the color development of products for visible analysis instead of agarose gel electrophoresis. The results showed that the sensitivity SYBR Green I as the fluorescent reagent was 10 copies viruses per μL,which is 100 times higher than normal PCR method,while the detection limit of combined use of calcium and manganese ion was 1 000 copies viruses per μL. Although the sensitivity of mixture of calcium and manganese ion is lower than SYBR Green I,it can avoid the aerosol contamination. The fluorogenic analysis-based LAMP system established in our study has a high sensitivity and avoid the cross contamination,which is of huge potential in research institutions,grass-roots laboratories and field testing and can provide effective means to completely curb the occurrence and spreading of DTMUV.

A fluorogenic probe for SNAP-tag protein based on ESPT ratiometric signals

Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells.In this paper,we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins.In this probe,the O6-benzylguanine derivative of 3-hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein.The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity.The excited state proton transfer occurred after illumination,to obtain the ratio fluorescence signal from blue emission to red emission,realizing the wash-free fluorescence imaging of the target proteins.

Modulation of dynamic aggregation in fluorogenic SNAP-tag probes for long-term super-resolution imaging

The combination of super-resolution microscopy and synthetic fluorescence probes has emerged as a universal tool to monitor dynamic biological events at the nanometer scale.However,the limited site-specificity and fluorogenicity of synthetic fluorescent probes make it still difficult to realize long-term super-resolution imaging.Herein,we introduce a dynamic aggregation mediated SNAP-tag fluorogenic probe,BGAN-Aze,which can specifically bind to various SNAP-tag fusion proteins with 41-fold fluorescence enhancement.The equilibrium between the non-fluorescent aggregate/dimer(A–D)and the fluorescent monomer(M)of BGAN-Aze acts as an effective method to reduce the fluorescence background and endow BGAN-Aze with the capability of conducting washing-free super-resolution imaging of various intracellular and extracellular proteins.Using this probe,we monitored multiple dynamic biological events,such as MMC,mitophagy,the fusion of nucleolus,and the growth and contact of filopodia.We expect that BGAN-Aze will become a widely used SNAP-tag for super-resolution imaging of dynamic biological events and the A-D-M equilibrium can be a general strategy for designing fluorogenic probes.

A self-assembly/disassembly two-photo ratiometric fluorogenic probe for bacteria imaging

Fluorescence imaging has facilitated fluorescent probes to analyze the subcellular localization and dynamics of biological targets. In this paper, we reported a fluorogenic probe for bacteria imaging. The probe was an imidazolium-derived pyrene compound, which self-assembled to form nano-particles and the pyrene fluorescence was quenched by the aggregation effects. When the self-assembly nanoparticles interacted with anionic bacteria surfaces, synergistic effects of electrostatic interaction and hydrophobic force caused competing binding between bacteria surfaces and imidazoliums. This binding resulted in the disassembly of the aggregates to give fluorescence turn-on signal. Meanwhile, the probe bound bacteria surfaces and displayed both pyrene-excimer and pyrene-monomer fluorescence, which gave ratiometric signal. Then, fluorescent labeling by the probe enabled the two-photo ratiometric imaging of bacteria.

Two-photon dual-channel fluorogenic probe for in situ imaging the mitochondrial H_(2)S/viscosity in the brain of drosophila Parkinson’s disease model

H2S is an essential gas signal molecule in cells,and viscosity is a key internal environmental parameter.Recent studies have shown that H_(2)S acts as a cytoarchitecture agent and gas transmitter in many tissues,e.g.,as a regulator of neuroendocrine in the brain for mediating vascular tone in blood vessels.Mitochondrial viscosity is an important parameter for judging whether mitochondrial function is normal.It has been reported that oxidative stress and mitochondrial dysfunction are connected with Parkinson’s disease(PD),and the protective role of H_(2)S in PD models has been extensively demonstrated.Herein,Mito-HS,a new two-photon fluorescent probe was demonstrated to detect cross-talk between the two channels of mitochondrial viscosity and H_(2)S content.Moreover,this probe could detect the relative amount of and changes in mitochondrial H2S in situ due to the reduced mitochondrial targeting ability after reaction with H_(2)S.The results show that H2S in mitochondria is inversely related to viscosity.The PD model has a lower H2S in mitochondria and a higher mitochondrial viscosity than did the normal.This result is important for our deep understanding of PD and its causes.

A novel chromogenic and fluorogenic scaffold for detection of oxidative radicals

Radical detection has attracted significant attention recently. Here we have developed a scaffold through covalent assembly principle（OR570）, which could facile applications in detection of oxidative radicals.The primary advantage of the assembly type probe lies at the turn-on fluorescence signal from a zero background and hence high fluorescence turn-on ratio for sensitive detection of weak signal.

High-contrast and real-time visualization of membrane proteins in live cells with malachite green-based fluorogenic probes

Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670 nm by restricting flexibility of dye. Integrating a phosphate-caged quinone methide precursor with MG yielded a covalent labeling fluorogenic probe, allowing real-time imaging of membrane alkaline phosphatase(ALP,a model catalytic protein) activity in live cells with over 100-fold enhancement of fluorescence intensity.Moreover, MG is also applicable to image non-catalytic protein by conjugation with protein-specific ligand. A fluorogenic probe consisted of c-RGDf K peptide and MG proved to be compatible with wash-free and real-time visualization of non-catalytic integrin α_(v)β_(3) in live cells with high contrast.

Fluorescence imaging mitochondrial copper(Ⅱ) via photocontrollable fluorogenic probe in live cells

Monitoring mitochondrial derived copper（Ⅱ） in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper（Ⅱ）. Herein, we have reported the design,synthesis and characterization of photocontrollable fluorogenic probe, MCu-3, which is equipped with a photo-labile group（nitrobenzyl group） and mitochondria targeting unit（triphenylphosphonium salt）.This novel probe showed an intense fluorescence enhancement in response to copper（Ⅱ） without interference from other metal cations in the biological condition（p H 6–9）. The detection limit is 1.7 ×10^（-7） mol/L in HEPES buffer. The confocal fluorescence imaging results demonstrated MCu-3 can visualize mitochondrial copper（Ⅱ） in live mammalian cells. The clear advantage of our photocontrollable method is successful to avoid the influence of cytoplasmic copper（Ⅱ） during mitochondria specific detection.

SNAP-tag fluorogenic probes for wash free protein labeling

Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus,real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this review,we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.

Spectrofluorimetric Determination of Cu^2＋ Using New Fluorogenic Reagent

A new synthesized fluorogenic reagent, 8-[(2-pyridine)methylideneamino] quinoline (PMAQ), was utilized for spectrofluorimetric determination of Cu(II) at trace levels. PMAQ, a good fluorogenic reagent, though insoluble in water, but is soluble in ethanol and 20% ethanol-water. The excitation and the fluorescence wavelengths of PMAQ were 310 and 434 nm respectively. When the reagent was complexed with Cu(II), the fluorescence intensity decreased proportionally with the concentration of Cu(II) at pH 4.5 by a static quenching effect. The highest sensitivity to Cu2+ determination was shown to be at PMAQ concentration of 1.0 X 10(-5) mol.L-1. In order to enhance the quenching effect, the Cu(II)-PMAQ complex solution was kept at 22 degreesC for 20 min. Though the interferences by Co(II) and Fe(III) were very serious, they were however, completely eliminated by being masked with oxalate and ascorbate ions respectively. The linear dynamic range for Cu(II) determination was between 25-441 mug.L-1 with the detection limit of 18 mug.L-1 (RSD=3.7%, n=6). The proposed method was successfully applied to the determination of Cu(II) in real samples including human blood serum, commercial tea and wheat flour.

Biphasic Nano-Domains of Planar Lipid Bilayer Complexed with Fluorogenic Polymer Reporter Tailored for Antimicrobial Detection

We have fabricated an unexpected type of supported planar bilayer composed of receptor phospholipids and single-chained diacetylenes as fluorogenic reporters using protruded anchor moieties with a positive terminal charge.Nanoscale topographical and surface thermodynamic analyses,as well as molecular dynamics simulations,revealed the coexistence of well-dispersed liquid-condensed(L_(c))domains forming nano-islands and liquid-expanded(L_(e))region in the planar bilayer,enhancing sensitivity against a prototype of ubiquitous membrane-associated antimicrobial peptides,melittin.The L_(e)regions,acting as target receptors,enabled sensitive detection as the melittin adsorbed and inserted into these regions due to strong hydrophobic interactions between phospholipids and melittin.The L_(c)domains,serving as signal reporters,enabled diacetylenes to assemble,polymerize,and fluoresce in response to the insertion of melittin into the L_(e)regions.Thus,biphasic nanodomains of the planar lipid bilayer finally endowed this sensor system with a detection range of 100μMto 50 nM and a limit of detection(LOD)of∼37 nM for melittin.This exceeded the operational performance of the colorimetric polydiacetylene vesicle solution 45 times,which reportedly ranged from 100 to 4μM with an LOD of∼1.7μM.

Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.

Synthesis of New Near-infrared Fluorescent Dyes

A new near-infrared fluorescent dye, 9-N-(2-hydroxyethyl)-N-methylamino-6-carbethoxy-5H-benzo[a]phenoxazin-5-one 1, was prepared from the reaction of N-(2-hydroxyethyl)-N-methyl-4-nitrosoaniline hydrochloride and ethyl 1,3-dihydroxynaphthoate. Five more fluorescent compounds were synthesized by the reaction of the resulting dye 1 with appropriate amino acid or carboxylic acids.

Evaluation of the effect of compound aqueous solubility in cytochrome P450 inhibition assays

It is a common practice in drug discovery organizations to screen new chemical entities in order to predict future drug-drug interactions. For this purpose, there are two main assay strategies, one based on recombinant cytochrome P450 (rCYP) enzymes and fluorescent detection, and other on human liver microsomes (HLM) and liquid chromatography coupled to mass spectrometry. Many authors have reported a poor correlation between both technologies, giving rise to concerns about the usefulness of fluorometric methods for predicting drug-drug interactions. In this study, we investigated the role that compound aqueous kinetic solubility may play in this lack of correlation. We found that drug discovery compounds with unacceptable kinetic solubility, measured by a turbidimetric solutibility assay, tended to yield higher IC50 values in in vitro models based on human liver microsomes, whereas compounds with kinetic solubility values higher than 50 μM showed very similar IC50 values in both in vitro models. Our results show that the turbidimetric solubility assay is a useful tool to identify those discovery compounds that may require further investigation in order to avoid overlooking future drug-drug interactions.

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics

A titrant for the SARS-CoV-2 main protease(M^(pro))was developed that enables,for the first time,the exact determination of the concentration of the enzymatically active M^(pro) by active-site titration.The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme–titrant complex.Four fluorogenic substrates of M^(pro),including a prototypical,internally quenched Dabcyl-EDANS peptide,were compared in terms of solubility under typical assay conditions.By exploiting the new titrant,key kinetic parameters for the M^(pro)-catalyzed cleavage of these substrates were determined.

RNA Imaging in Live Cells

Traditional molecular biology tools have elucidated the identities and functions of RNA molecules,which are essential to the understanding of gene transcription and protein translation.Deepening this research field would further require the direct visualization of RNA dynamics such as the DNA-RNA interactions and RNA-protein interactions.Towards this goal,the rise of RNA imaging tools over the past 15 years has reformed how we looked at these processes.In this Emerging Topic,we first highlighted recent advances on three main RNA imaging tools based on the species of interacting molecules:RNA-RNA pairing,RNA-protein binding,and small molecule-RNA complex.We introduced the advantages of these tools from a technical viewpoint,including binding affinity,fluorescent turn-on ratio,stability,and impacts on targeted RNA.Next,we discussed new rising opportunities and future directions,echoing the state-of-the-art imaging tools in the fields of fluorescent proteins and small fluorescent molecules.Together,we believe this emerging field will bring new insights on how we study RNA biology in living systems.

Aniline as a TICT rotor to derive methine fluorogens for biomolecules:A curcuminoid-BF2 compound for lighting up HSA/BSA

The unique structure of fluorescent proteins in which the fluorophore is encapsulated by the protein shell to restrict rotation and emit light inspired the screening of chromophores that selectively bind to biomolecules to generate fluorescence. In this paper, we report a curcuminoid-BF2-like fluorescent dye NBF2containing 4-dimethylaniline as an electron-donating group. When this dye is combined with HSA or BSA, the fluorescence is enhanced 90/112-fold, and the fluorescence quantum yield increases from <0.001to 0.16/0.19. Such a large change in fluorescence enhancement is due to the encapsulation of N-BF2in the protein cavity by HSA/BSA, which inhibits the intramolecular rotation of the aniline moiety caused by charge transfer after the fluorophore is excited by light. N-BF2has fast and strong binding to HSA or BSA and was found to be reversible in solution and intracellularly. Since N-BF2also has the ability to target lipid droplets, the complex of N-BF2/HSA realizes the regulation of reversible lipid droplet staining in cells.