Intrinsic Fluorescence Signal Research Articles

Molecular Engineering of a Doubly Quenched Fluorescent Probe Enables Ultrasensitive Detection of Biothiols in Highly Diluted Plasma and High-Fidelity Imaging of Dihydroartemisinin-Induced Ferroptosis.

The occurrence and development of diseases are accompanied by abnormal activity or concentration of biomarkers in cells, tissues, and blood. However, the insufficient sensitivity and accuracy of the available fluorescence probes hinder the precise monitoring of associated indexes in biological systems, which is generally due to the high probe intrinsic fluorescence and false-negative signal caused by the reactive oxygen species (ROS)-induced probe decomposition. To resolve these problems, we have engineered a ROS-stable, meso-carboxylate boron dipyrromethene (BODIPY)-based fluorescent probe, which displays quite a low background fluorescence due to the doubly quenched intrinsic fluorescence by a combined strategy of the photoinduced electron transfer (PET) effect and "ester-to-carboxylate" conversion. The probe achieved a high S/N ratio with ultrasensitivity and good selectivity toward biothiols, endowing its fast detection capability toward the biothiol level in 200×-diluted plasma samples. Using this probe, we achieved remarkable distinguishing of liver injury plasma from normal plasma even at 80× dilution. Moreover, owing to its good stability toward ROS, the probe was successfully employed for high-fidelity imaging of the negative fluctuation of the biothiol level in nonsmall-cell lung cancer (NSCLC) during dihydroartemisinin-induced ferroptosis. This delicate design of suppressing intrinsic fluorescence reveals insights into enhancing the sensitivity and accuracy of fluorescent probes toward the detection and imaging of biomarkers in the occurrence and development of diseases.

High-Speed Clearing and High-Resolution Staining for Analysis of Various Markers for Neurons and Vessels.

Tissue clearing enables deep imaging in various tissues by increasing the transparency of tissues, but there were limitations of immunostaining of the large-volume tissues such as the whole brain. Here, we cleared and immune-stained whole mouse brain tissues using a novel clearing technique termed high-speed clearing and high-resolution staining (HCHS). We observed neural structures within the cleared brains using both a confocal microscope and a light-sheet fluorescence microscope (LSFM). The reconstructed 3D images were analyzed using a computational reconstruction algorithm. Various neural structures were well observed in three-dimensional (3D) images of the cleared brains from Gad-green fluorescent protein (GFP) mice and Thy 1-yellow fluorescent protein (YFP) mice. The intrinsic fluorescence signals of both transgenic mice were preserved after HCHS. In addition, large-scale 3D imaging of brains, immune-stained by the HCHS method using a mild detergent-based solution, allowed for the global topological analysis of several neuronal markers such as c-Fos, neuronal nuclear protein (NeuN), Microtubule-associated protein 2 (Map2), Tuj1, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) in various anatomical regions in the whole mouse brain tissues. Finally, through comparisons with various existing tissue clearing methodologies such as CUBIC, Visikol, and 3DISCO, it was confirmed that the HCHS methodology results in relatively less tissue deformation and higher fluorescence retention. In conclusion, the development of 3D imaging based on novel tissue-clearing techniques (HCHS) will enable detailed spatial analysis of neural and vascular networks present within the brain.

Reductive carbon dots for reduction, ratiometric fluorescence determination, and intracellular imaging of Au3+

Many studies show that ortho-phenylenediamine (OPD) produces an oxidized fluorescent product when exposed to an oxidizing agent that enables the direct or indirect fluorescence detection of a range chemical and biochemical analytes. However, there is no report on this unique optical behavior for other two isomers of phenylenediamine. This study demonstrates that a simple hydrothermal treatment of para-phenylenediamine (PPD) in the presence of sulfuric acid results in the formation of fluorescent N, S-doped carbon dots (CDs) with triple functionalities including the reduction of Au3+ into gold nanoparticles (AuNPs), the stabilization of the produced AuNPs, and the determination of Au3+ concentration through an intrinsic ratiometric fluorescence signal. In the presence of Au3+, the blue emission of CDs at 437 nm quenched, and a green emission at 540 nm emerged. The linear concentration range for the determination of Au3+ was 20 nM–16 µM with a detection limit of 16 nM. Additionally, the dual emissive CDs-AuNPs hybrid probe showed potential for the indirect fluorescence ratiometric determination of cysteine and sulfide ions. The linear concentration range for cysteine and sulfide ions were 0.25–8 μM and 0.1–6 μΜ, with detection limits of 0.095 μM and 0.041 μM, respectively. Accordingly, CDs were applied to detect Au3+ and S2− in real water samples. Moreover, the synthesized CDs showed no cytotoxicity for HeLa cells up to 300 µg mL−1, as determined by the MTT assay. Therefore, their potential for intracellular imaging of Au3+ in living cells was also investigated.

Can We See SEI Directly byNaked Eyes?

Stable solid electrolyte interface (SEI) is the key to improve the electrochemical performance of lithium metal batteries (LMBs). However, there are still many puzzles about SEI film that have not been well explained, due to the complexity of electrochemical reactions involving in SEI formation and the absence of direct observation methods for SEI. Here, this work realizes the direct observation of SEI by skillfully designed fluorescent tracers acting as an SEI film-forming additive for electrolytes. These fluorescent tracers have three important moieties: an olefin group for polymerization on anode surface so as to participate in SEI film formation during charge/discharge cycles, a polar group for Li-ion conduction, and an AIEgen for fluorescent tracing. Therefore, the tracers participate in SEI film-forming and result in a shining SEI film. This shining SEI film with intrinsic fluorescence signal allows direct observation and quantification on the distribution, relative abundance, and macro morphology of SEI. These fluorescent tracers can also reveal the SEI formation growth destruction regularity during charge/discharge cycles. Several summarized typical macro morphologies and evolution stages of SEI will enrich knowledge and understanding of SEI and help to gain insight into the interaction between electrolyte and anode, electrochemical performance, and cycle life of batteries.

Towards a better understanding of flow-related processes in the vertically distributed compartments of karst aquifers by combining natural tracers and stable isotopes

Understanding infiltration pathways throughout the different compartments of karst systems (soil-epikarst-unsaturated zone-saturated zone) is vital to assess recharge processes, vulnerability to pollution, and the general hydrogeological functioning of these kind of aquifers. To gain deeper insight into the soil-water-rock hydrogeochemical processes occurring throughout the vertically hierarchized compartments of the aquifer and to evaluate the applicability of different soil natural parameters as groundwater flow tracers, the present work analyzes the spatial and temporal evolution of total organic carbon -TOC-, intrinsic fluorescence related to organic matter, nitrates, δ13CTDIC, and the values of stable isotopes of δ2H and δ18O from water molecules and at different sampling points in a geologically complex karst aquifer in south Spain (Jarastepar carbonate massif). Over two years, rain and soil water (30 and 60 cm deep) samples were collected simultaneously at an experimental plot along with groundwater from the top of the saturated zone and a base-level spring. Results show that the variation in the concentration of natural soil tracers and the signal of water stable isotopes (δ2H and δ18O) smooth throughout the different compartments of the aquifer, with rapid variations in the unsaturated zone after recharge events and delayed effects in the saturated zone. At the same time, δ13CTDIC and nitrate values rise along the subsurface flow. While the carbon isotope evidences a shorter transit time between recharge areas and the top of the saturated zone than the subsequent path toward the permanent base springs, nitrate evolution is affected by pollution sources near discharge areas, so its applicability is questionable. Finally, organic matter (TOC and intrinsic fluorescence) mineralizes (or degrades) up to 90% from the first centimeters of soil to the permanent basal springs. An exponential decreasing trend defines the relationship between TOC and δ13CTDIC caused by organic matter attenuation along the groundwater flow path, while δ13CTDIC increases because of a higher residence time of water in contact with the host rock. These results, together with previous research based on other natural tracers (chemical, isotopes, and dating tools) and dye tracing experiences, permit to link TOC and δ13CTDIC values with a range of transit times, from hours-days to years-decades. Meanwhile, the attenuation of natural intrinsic fluorescence and TOC signal evidence the filter function of the aquifer from the soil to the saturated zone. All these findings highlight the usefulness of natural fluorescence and, particularly, TOC and δ13CTDIC, as tracers of groundwater flow and hydrogeochemical processes within the different compartments of complex karst aquifers.

Epitaxial self-assembly of Bimetallic MOF heterostructure for fluorescent and colorimetric detection of tetracyclines

The irrational use of tetracycline antibiotics (TCs) leads to the pollution of environmental resources and finally threatens human health, which makes it urgent to develop a sensor for sensitively and selectively detecting the antibiotics. In this work, a Zn-based metal-organic framework (Zn-MOF) anchored on an amino-functionalized aluminum-based metal-organic framework (NH2-MIL-53(Al)) was constructed as a fluorescence sensor for TCs determination. The abundant –NH2 made NH2-MIL-53(Al) a self-assembly core, which facilitated the formation of MOF-on-MOF heterojunction by amide reaction with the exposed –COOH of Zn-MOF. The single characteristic emission peak of Zn-MOF-on–NH2–MIL-53(Al) at 402 nm could identify TCs by internal filtration effect. Upon introduction of TCs, Zn-MOF-on–NH2–MIL-53 (Al) enhanced the intrinsic fluorescence signal of TCs at 517 nm through the coordination via the metal active center (Zn (II)), forming a ratiometric fluorescent sensor with a reverse response. Zn-MOF-on–NH2–MIL-53(Al) sensor displayed excellent anti-interference capability with a broader linear range (0.1–80 μM), and a lower limit of detection (LOD) of 8.24 nM for tetracycline (TC), 2.77 nM for oxytetracycline (OTC), and 8.85 nM for doxycycline (DC), respectively. By merit of Zn-MOF-on–NH2–MIL-53(Al), a portable smartphone-integrated test hydrogel colorimetric platform was fabricated for visual colorimetric detecting TCs by incorporating the sensor with a smartphone. Additionally, the bimetal-organic framework smartphone sensing platform exhibited satisfactory recovery (95.66–107.65%) and reliability in beef, pork, and chicken. Accordingly, the tactics presented a new strategy for developing an integrated system for environmental pollutant detection.

Nanoscopy on drug-encapsulating nanosystems by phasor-based fluorescence lifetime analysis.

Understanding the supramolecular organization of active compounds (drugs) encapsulated within standard formulations is becoming increasingly important in nanomedicine. In fact, the synthetic identity of a drug-encapsulating nanosystem is supposed to change throughout its journey, from administration to final fate within the target tissue/cell. Here we addressed this issue by exploiting phasor-based fluorescence lifetime imaging (FLIM) of a drug intrinsic fluorescence signal. As case-study, we selected Irinotecan (CPT-11), a chemotherapy medication used to treat colon cancer, small lung cancer and pancreatic cancer, and focused on its liposomal formulation Onivyde®, launched in 2015 as an orphan drug for the treatment of metastatic ductal adenocarcinoma (mPDAC). After measuring the FLIM signature of the drug's individual physical states, we extracted the fractional intensities of each coexisting physical state of the active principle within the nanoparticle. Our analysis was boosted by means of dedicated custom algorithms based on preliminary data processing and multi-dimensional histogram inference to identify regions of interest in the phasor plot. Whereas, in the case of biological samples, we achieved autonomous cell segmentation by means of computer vision algorithms. Onivyde® in commercial vials was found to coexist in multiple physical states (i.e. gelated/precipitated, free and membrane-associated) and undergo extensive leakage in media mimicking complex biological environments. Moreover, we identified multiple mechanism of cellular uptake and were able to investigate subcellular localization and supramolecular organization of irinotecan in a model of pancreatic cells. In conclusion, we believe that Phasor-FLIM represents a useful platform to quantitatively address the synthetic identity of encapsulated fluorescent drugs, both in cuvette and cellular environments. In this context, we envision many potential applications ranging from batch-to-batch analysis, at the production level, to drug development optimization.

Surfactant Semiconductors as Trojan Horses in Cell-Membranes for On-Demand and Spatial Regulation of Oxidative Stress.

Oxidative stress is a cause for numerous diseases and aging processes. Thus, researchers are keen to tune the level of intracellular stress and to learn from that. An unusual approach is presented here. The methodology involves multifunctional surfactants. Although their molecular design is nonbiological-a fullerenol head group attached covalently to pi-conjugated dyes-the surfactants possess superior biocompatibility. Using an intrinsic fluorescence signal as a probe,it is shown that the amphiphiles become incorporated into the Caco-2 cells. There, they are able to exhibit additional functions. The compound reduces cellular stress in dark reaction pathways. The antagonistic property is activated under irradiation, the photocatalytic production of reactive oxygen species (ROS), resulting in cell damage. The feature is activated even by near-infrared light (NIR-light) via a two-photon process. The properties as molecular semiconductors lead to a trojan horse situation and allows the programming of the spatial distribution of cytotoxicity.

Study of Aerosol Classification Technique Based on Normalized Intrinsic Fluorescence Signal

Study of Aerosol Classification Technique Based on Normalized Intrinsic Fluorescence Signal

Tunable ultraviolet laser induces high signal-to-noise ratio in intrinsic fluorescence titration for protein- ligand interaction studies

Protein intrinsic fluorescence emitted by tryptophan residues is widely used for macromolecular mechanism studies, such as the protein binding by ligands and enzyme kinetics. However, modifications in fluorescence intensity can be weak using the intrinsic fluorescence in many proteins containing few tryptophan residues, or with their weak involvement in the binding. A low signal to noise ratio is a main drawback, due to the weak intensity of Xenon lamp illumination in the spectrofluorometer at the ultraviolet (UV) range, in addition to the small intrinsic fluorescence modifications with ligand binding, in many cases. We report herein the montage and the utilization of a laser light source in a spectrofluorometer in the UV range for the titration of protein intrinsic fluorescence. A significantly stronger modification of the protein intrinsic fluorescence was obtained with the titrations represented by E. coli Arginyl tRNA synthetase and human 17β-hydroxysteroid dehydrogenase type 7 with their natural ligands using a wavelength-tunable UV laser source. Also, a higher signal to noise ratio was obtained with the laser source. Finally, a similar KD was obtained with the titration using the two light sources. These results are supported by the direct binding study of equilibrium dialysis, showing that the laser source yields a reliable binding analysis. Thus, the utilization of a laser source in the intrinsic fluorescence titration and related usage can give a significant opening for the wider applications of the spectroscopy and photo technology to many new proteins with limited intrinsic fluorescence signals, while their mechanisms are critical to understand biochemistry and will facilitate medical applications. This study includes fundamental, applied, and interdisciplinary areas of physics.

PolyQ aggregation studied by model peptides with intrinsic tryptophan fluorophores

Polyglutamine (polyQ) model peptides are ideally suited to analyze the involvement of glutamines in the disease-related aggregation onset. Here we use a template-assisted design of polyQ-rich hairpin peptides (Trpzip-Qn) to monitor structural stability with fluorescence spectroscopy. The hairpin model imitates the monomeric motif of a polyQ fibril and is stabilized by hydrophobic interactions of two cross-strand pairs of tryptophans (Trps) which are used as fluorophores to report on structural changes. The Trps also frame the polyQ repeats located on each hairpin strand with a different number of glutamines (Qn). Single-stranded sequences mimic the unfolded state and were used as references to differentiate the intrinsic fluorescence signal from the spectral effect caused by structural changes. Temperature-induced hairpin unfolding was monitored by the spectral shift of the Trp fluorescence signal and transition temperatures were determined. The magnitude of the spectral shift indicates the degree of structural disorder. We observed that a longer polyQ repeat is more disordered and weakens the cross-strand Trp-Trp interactions resulting in a decrease of the spectral shift. Aggregation to a fibrillar and more ordered structure shows an increase of the spectral shift. In addition, a band at 280 nm occurs in the spectrum which clearly correlates with the turbidity of the sample and is attributed to scattering of larger aggregated structures. Our study reveals that the number of glutamines, pH and temperature affect structural stability and aggregation of polyQ repeats.

DIFFERENTIAL STABILIZING EFFECTS OF BUFFERS ON STRUCTURAL STABILITY OF BOVINE SERUM ALBUMIN AGAINST UREA DENATURATION

The conformational stability of bovine serum albumin (BSA) against urea denaturation was investigated in aqueous solutions both in the absence and presence of buffers. Various buffers differing in polar and nonpolar characters such as sodium phosphate, Tris-HCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES and [3-(N-morpholino)propanesulfonic acid] MOPS buffers were used in this study. Urea-induced structural changes were analyzed using different probes, i.e., intrinsic fluorescence, ANS fluorescence and UV-difference spectral signal. Presence of different buffers in the incubation medium offered different degrees of resistance to the protein against urea-induced structural changes compared to those obtained in water (in the absence of buffers). A similar trend of buffer-induced structural resistance was noticed with three different probes. The stabilizing effect of these buffers followed the order: MOPS > HEPES > sodium phosphate > Tris-HCl > water. As found in MOPS and HEPES buffers, the highest stability of BSA can be attributed to the presence of morpholine and piperazine rings, respectively, in their structures. These groups might have produced a hydrophobic environment around the protein surface, thus stabilizing protein conformation against urea denaturation.

Empirical method for rapid quantification of intrinsic fluorescence signals of key metabolic probes from optical spectra measured on tissue-mimicking turbid medium.

.Significance: Optical fluorescence spectroscopy technique has been explored extensively to quantify both glucose uptake and mitochondrial metabolism with proper fluorescent probes in small tumor models in vivo. However, it remains a great challenge to rapidly quantify the intrinsic metabolic fluorophores from the optically measured fluorescence spectra that contain significant distortions due to tissue absorption and scattering.Aim: To enable rapid spectral data processing and quantify the in vivo metabolic parameters in real-time, we present an empirical ratio-metric method for rapid fluorescence spectra attenuation correction with high accuracy.Approach: A first-order approximation of intrinsic fluorescence spectra can be obtained by dividing the fluorescence spectra by diffuse reflectance spectra with some variable powers. We further developed this approximation for rapid extraction of intrinsic key metabolic probes (2-NBDG for glucose uptake and TMRE for mitochondrial function) by dividing the distorted fluorescence spectra by diffuse reflectance intensities recorded at excitation and emission peak with a pair of system-dependent powers. Tissue-mimicking phantom studies were conducted to evaluate the method.Results: The tissue-mimicking phantom studies demonstrated that our empirical method could quantify the key intrinsic metabolic probes in near real-time with an average percent error of .Conclusions: An empirical method was demonstrated for rapid quantification of key metabolic probes from fluorescence spectra measured on a tissue-mimicking turbid medium. The proposed method will potentially facilitate real-time monitoring of key metabolic parameters of tumor models in vivo using optical spectroscopy, which will significantly advance translational cancer research.

A gel-forming clusteroluminogenic polymer with tunable emission behavior as a sustained-release carrier enabling real-time tracking during bioactive agent delivery

Sustained-release carriers with intrinsic luminescence exhibit the potential to combine imaging with bioactive agent delivery in practice. This study reports the synthesis and properties of CT, which is a gel-forming and highly tunable non-conjugated polymer with intrinsic luminescence. Owing to the excitation wavelength-dependent emission tunability of CT, optical imaging can be performed in a wide range of excitation wavelengths. This study is the first proof-of-concept study successfully integrating the property of clusterization-triggered emission into carrier design for combining imaging with bioactive agent delivery. By using cancer therapy as a practical example, our results demonstrate that CT is a depot system that cannot only enhance in vivo antitumor efficacy but can also allow its location and gelation process to be tracked in real time by giving an intrinsic blue-colored fluorescence signal. Together with its high biocompatibility, CT shows high practical potential for executing self-illuminating therapy to tackle cancer.

Structure Dynamics Guides the Enhancement of Ligand Affinity for MdmX

Mdm2 and MdmX are highly homologous and Mdm2 inhibitors exhibit weak affinity for MdmX. The structural mechanism underlying how Mdm2/MdmX distinguish ligand binding affinity remains elusive. Guided by NMR study, we have identified three flexible regions on N-MdmX that affected inhibitor-binding affinity. Furthermore, using disulfide staple, we found that the Phe19-binding site on MdmX was one of the most flexible regions weakening nutlin-3a binding. On the other hand, we continued to pursue high-resolution structure of Mdm2 in complex with ntutin-3a. An X-ray crystallographic structure of Mdm2/nutlin-3a at 1.10 Å identified that His72 had two conformations, a bound state and an unbound state. Molecular dynamics simulation also revealed that His72 in MdmX existed two states. Thus, our work started to search fragments that could rigidify Phe19-binding site. We designed a fusion protein model that p5315-29 was fused with MdmX through a flexible linker, i.e., p5315-29-MdmX, for fragment screening. By use of the intrinsic fluorescence signal of p5315-29 to monitor high-throughput screening, we found that the imidazoindole was a bona fide scaffold for template-based design of MdmX inhibitors. Next the p5315-29-MdmX fusion protein was modified to leave Phe19-binding site unoccupied, i.e., p5323-29-MdmX. By screening a fragment library, a small fragment, CPD-01, could bind to Phe19-binding site in an allosteric mode that enhanced the binding of p5323-29 to MdmX. Furthermore, CPD-01 was conjugated with p5323-29 to synthesize p53 peptide analogues with different linkers. These peptide analogues exhibited enhanced binding affinity for MdmX, compared with p5323-29. Then, CPD-01 was integrated into nutlin-3a to replace Phe19-binding-site-pharmacophore. The new nutlin analogue exhibited high-affinity binding MdmX and Mdm2 as well. Taken together, our work provided a promising strategy to design inhibitors of MdmX/Mdm2. This strategy should be applicable for lead optimization in drug discovery.

Optimization of frequency-doubled Er-doped fiber laser for miniature multiphoton endoscopy.

Frequency-doubled femtosecond Er-doped fiber laser is a low-cost and portable excitation source suitable for multiphoton endoscopy. The frequency-doubled wavelength at 780nm is used to excite the intrinsic fluorescence signal. The frequency-doubling with a periodically poled MgO : LiNbO3 (PPLN) is integrated in the distal end of the imaging head to achieve fiber connection. The imaging speed is further improved by optimizing the excitation laser source. A 0.3-mm length of PPLN crystal is selected and the Er-doped fiber laser is manipulated to match its bandwidth with the acceptance bandwidth of the PPLN. Through this optimization, a reduced pulsewidth of 80fs of the frequency-doubled pulse is achieved. All-fiber dispersion compensation and pulse compression by single mode fiber is conducted, which makes the fiber laser directly fiber-coupled to the imaging head. An imaging speed of 4 frames / s is demonstrated on ex vivo imaging of unstained biological tissues, which is 10 times faster than our previous study using a 1-mm-long PPLN. The results show that miniature multiphoton endoscopy using frequency-doubled Er-doped fiber laser has great potential for clinical applications.

Effect of temperature on FAD and NADH-derived signals and neurometabolic coupling in the mouse auditory and motor cortex.

Tight coupling of neuronal metabolism to synaptic activity is critical to ensure that the supply of metabolic substrates meets the demands of neuronal signaling. Given the impact of temperature on metabolism, and the wide fluctuations of brain temperature observed during clinical hypothermia, we examined the effect of temperature on neurometabolic coupling. Intrinsic fluorescence signals of the oxidized form of flavin adenine dinucleotide (FAD) and the reduced form of nicotinamide adenine dinucleotide (NADH), and their ratios, were measured to assess neural metabolic state and local field potentials were recorded to measure synaptic activity in the mouse brain. Brain slice preparations were used to remove the potential impacts of blood flow. Tight coupling between metabolic signals and local field potential amplitudes was observed at a range of temperatures below 29°C. However, above 29°C, the metabolic and synaptic signatures diverged such that FAD signals were diminished, but local field potentials retained their amplitude. It was also observed that the declines in the FAD signals seen at high temperatures (and hence the decoupling between synaptic and metabolic events) are driven by low FAD availability at high temperatures. These data suggest that neurometabolic coupling, thought to be critical for ensuring the metabolic health of the brain, may show temperature dependence, and is related to temperature-dependent changes in FAD supplies.

Pyrrolo-dC modified duplex DNA as a novel probe for the sensitive assay of base excision repair enzyme activity

We develop a novel approach to determine formamidopyrimidine DNA glycosylase (Fpg) activity by taking advantage of the unique fluorescence property of pyrrolo-dC (PdC) positioned opposite to 8-oxoguanine (8-oxoG) in duplex DNA. In its initial state, PdC in duplex DNA undergoes the efficient stacking and collisional quenching interactions, showing the low fluorescence signal. In contrast, the presence of Fpg, which specifically removes 8-oxoG and incises resulting apurinic (AP) site, transforms duplex DNA into single-stranded (ss) DNAs. As a result, the intrinsic fluorescence signal of PdC in ssDNA is recovered to exhibit the significantly enhanced fluorescence signal. Based on this Fpg-dependent fluorescence response of PdC, we could reliably determine Fpg activity down to 1.25U/ml with a linear response from 0 to 50U/ml. In addition, the diagnostic capability of this strategy was successfully demonstrated by reliably assaying Fpg activity in human blood serum, showing its great potential in the practical applications.

A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors.

In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 μM. The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library, including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

Using Intrinsic Flavoprotein and NAD(P)H Imaging to Map Functional Circuitry in the Main Olfactory Bulb.

Neurons exhibit strong coupling of electrochemical and metabolic activity. Increases in intrinsic fluorescence from either oxidized flavoproteins or reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the mitochondria have been used as an indicator of neuronal activity for the functional mapping of neural circuits. However, this technique has not been used to investigate the flow of olfactory information within the circuitry of the main olfactory bulb (MOB). We found that intrinsic flavoprotein fluorescence signals induced by electrical stimulation of single glomeruli displayed biphasic responses within both the glomerular (GL) and external plexiform layers (EPL) of the MOB. Pharmacological blockers of mitochondrial activity, voltage-gated Na+ channels, or ionotropic glutamate receptors abolished stimulus-dependent flavoprotein responses. Blockade of GABAA receptors enhanced the amplitude and spatiotemporal spread of the flavoprotein signals, indicating an important role for inhibitory neurotransmission in shaping the spread of neural activity in the MOB. Stimulus-dependent spread of fluorescence across the GL and EPL displayed a spatial distribution consistent with that of individual glomerular microcircuits mapped by neuroanatomic tract tracing. These findings demonstrated the feasibility of intrinsic fluorescence imaging in the olfactory systems and provided a new tool to examine the functional circuitry of the MOB.