A near-infrared fluorescent probe for simultaneous detection of pH and viscosity.

In this work, we developed a ratiometric fluorescent probe (NT) based on ICT framework in near-infrared (NIR) which could detect pH and viscosity simultaneously. Long emission wavelength in NIR could protect the probe from interference of background fluorescence and improve the accuracy of the test. Due to the presence of thiazole-salt, the probe possessed good water solubility and could respond immediately to pH in water system. The pH values measured by NT in the actual samples were not much different from that measured by the pH meter, therefore, NT could give excellent accuracy. NT realized the reversible detection of pH by protonation and deprotonation. NT was used successfully to detect the pH of actual water samples, human serum and meat, as well as the viscosity variation caused by thickeners. Additionally, NT could monitor the changes of pH and viscosity in living cells. Therefore, the novel probe exhibited potential application in the fields of the environment, human health and food safety evaluation.

A near-infrared fluorescent probe based FRET for ratiometric sensing of H2O2 and viscosity in live cells.

Hydrogen peroxide (H2O2) and viscosity play vital roles in the cellular environment as signaling molecule and microenvironment parameter, respectively, and are associated with many physiological and pathological processes in biological systems. We developed a near-infrared fluorescent probe, CQ, which performed colorimetric and ratiometric detection of H2O2 and viscosity based on the FRET mechanism, and was capable of monitoring changes in viscosity and H2O2 levels simultaneously through two different channels. Based on the specific reaction of H2O2 with borate ester, CQ exhibited a significant ratiometric response to H2O2 with a large Stokes shift of 221 nm, a detection limit of 0.87 µM, a near-infrared emission wavelength of 671 nm, a response time of 1 h, a wide detection ranges of 0.87-800 µM and a high energy transfer efficiency of 99.9 %. CQ could also recognize viscosity by the TICT mechanism, and efficiently detect viscosity changes caused by food thickeners. More importantly, CQ could successfully detect endogenous/exogenous H2O2 and viscosity in live HeLa cells, which was expected to be a practical tool for detecting H2O2 and viscosity in live cells.

A highly sensitive ratiometric fluorescent probe for detecting HSO3-/SO32- and viscosity change based on FRET/TICT mechanism.

BACKGROUND: Sulfur dioxide (SO2) is a significant gas signaling molecule in organisms, and viscosity is a crucial parameter of the cellular microenvironment. They are both involved in regulating many physiological processes in the human body. However, abnormalities in SO2 and viscosity levels are associated with various diseases, such as cardiovascular disease, lung cancer, respiratory diseases, neurological disorders, diabetes and Alzheimer's disease. Hence, it is essential to explore novel and efficient fluorescent probes for simultaneously monitoring SO2 and viscosity in organisms. RESULTS: We selected quinolinium salt with good stability, high fluorescence intensity, good solubility and low cytotoxicity as the fluorophore and developed a highly sensitive ratiometric probe QQD to identify SO2 and viscosity changes based on Förster resonance energy transfer/twisted intramolecular charge transfer (FRET/TICT) mechanism. Excitingly, compared with other probes for SO2 detection, QQD not only identified HSO3-/SO32- with a large Stokes shift (218 nm), low detection limit (1.87 µM), good selectivity, high energy transfer efficiency (92 %) and wide recognition range (1.87-200 µM), but also identified viscosity with a 26-fold fluorescence enhancement and good linearity. Crucially, QQD was applied to detect HSO3-/SO32- and viscosity in actual water and food samples. In addition, QQD had low toxicity and good photostability for imaging HSO3-/SO32- and viscosity in cells. These results confirmed the feasibility and reliability of QQD for HSO3-/SO32- and viscosity imaging and environmental detection. SIGNIFICANCE: We reported a unique ratiometric probe QQD for detecting HSO3-/SO32- and viscosity based on the quinolinium skeleton. In addition to detecting HSO3-/SO32- and viscosity change in actual water and food samples, QQD could also monitor the variations of HSO3-/SO32- and viscosity in cells, which provided an experimental basis for further exploration of the role of SO2 derivatives and viscosity in biological systems.

A ratiometric fluorescent probe based on the FRET platform for the detection of sulfur dioxide derivatives and viscosity.

BACKGROUND: Sulfur dioxide (SO2) is a common gaseous pollutant that significantly threatens environmental pollution and human health. Meanwhile, viscosity is an essential parameter of the intracellular microenvironment, manipulating many physiological roles such as nutrient transport, metabolism, signaling regulation and apoptosis. Currently, most of the fluorescent probes used for detecting SO2 derivatives and viscosity are single-emission probes or probes based on the ICT mechanism, which suffer from short emission wavelengths, small Stokes shifts or susceptibility to environmental background. Therefore, the development of powerful high-performance probes for real-time monitoring of sulfur dioxide derivatives and viscosity is of great significance for human health. RESULTS: In this research, we designed the fluorescent probe QQC to detect SO2 derivatives and viscosity based on FRET platform with quinolinium salt as donor and quinolinium-carbazole as acceptor. QQC exhibited a ratiometric fluorescence response to SO2 with a low detection limit (0.09 µM), large Stokes shift (186 nm) and high energy transfer efficiency (95 %), indicating that probe QQC had good sensitivity and specificity. In addition, QQC was sensitive to viscosity, with an 9.10-folds enhancement of orange fluorescence and an excellent linear relationship (R2 = 0.98) between the logarithm of fluorescence intensity at 592 nm and viscosity. Importantly, QQC could not only recognize SO2 derivatives in real water samples and food, but also detect viscosity changes caused by food thickeners and thereby had broad market application prospects. SIGNIFICANCE: We have developed a ratiometric fluorescent probe based on the FRET platform for detecting sulfur dioxide derivatives and viscosity. QQC could not only successfully detect SO2 derivatives in food and water samples, but also be made into test strips for detecting HSO3-/SO32- solution. In addition, the probe was also used to detect viscosity changes caused by food thickeners. Therefore, this novel probe had significant value in food and environmental detection applications.

NBD-based colorimetric and ratiometric fluorescent probe in NIR for bisulfite.

This work presented a FRET-ICT based fluorescent probe (named NTC) composed of coumarin-benzothiazole as the acceptor and 4-nitrobenzo[c][1,2,5] oxadiazole (NBD) as the donor for the detection of SO2 derivatives in NIR. Probe NTC possessed superior performance including selectivity, quickly response toward SO32-/HSO3- and high energy transfer efficiency (94 %). The test strips provided a simple and effective tool in detecting the presence of bisulfite. Besides, NTC was applied to test the sulfur dioxide derivatives in food samples and cells.

A novel ratiometric fluorescence probe based on the FRET-ICT mechanism for detecting fluoride ions and viscosity.

Fluoride ion is not only important for dental health, but also a contributing factor in a variety of diseases. At the same time, fluoride ions and cell viscosity are both important to the physiological environment of mitochondria. We developed a dual-response ratiometric fluorescent probe BDF based on Förster resonance energy transfer (FRET) and intramolecular charge transfer (ICT) mechanism for the detection of F- and viscosity. BDF has an outstanding intramolecular energy transfer efficiency of 97.7% and shows excellent performance for fluorine ion detection. In addition, when the system viscosity increases, the fluorescence emission intensity of BDF is greatly heightened, indicating the possibility of viscosity detection. Finally, based on the fluorescence properties of BDF, we used the probe to detect F- in the toothpaste sample and image exogenous fluoride ions in HeLa cells.

Dual-response and lysosome-targeted fluorescent probe for viscosity and sulfur dioxide derivatives.

Viscosity and sulfur dioxide levels are important factors to evaluate the changes of cell micro-environment because a series of diseases usually occur when they are abnormal. At present, dual-response probes that can detect both viscosity and sulfur dioxide are rare. Therefore, we developed a novel fluorescent probe CBN for simultaneous detection of sulfur dioxide and viscosity. Besides, probe CBN could target lysosome of which normal function will be disrupted by the abnormality of viscosity. Therefore, probe CBN has the potential to be served as an effective biological tool to monitor the intracellular micro-environment.

FRET-based fluorescent probe with favorable water solubility for simultaneous detection of SO2 derivatives and viscosity.

The intracellular viscosity is an important parameter of the microenvironment and SO2 is a vital gas signal molecule. At present, some dual-response fluorescence probes for simultaneous measurements of viscosity and SO2 derivatives (HSO3-/SO32-) possessed poor water solubility. In this work, we developed a water-soluble fluorescence probe CIJ (0.0864 g/100 mL of water at 20 °C) for simultaneous measurements of viscosity and SO2 derivatives. CIJ exhibited a sensitive fluorescence enhancement to environmental viscosity from 0.97 to 28.04 cP based on a twisted intramolecular charge transfer mechanism and was applied to effective measurement of viscosity in vitro and in vivo. CIJ could also respond to SO2 derivatives with a low detection limit (44 nM) and a fast response time (5 min) based on the nucleophilic addition reaction. Furthermore, CIJ was applied to monitor SO2 derivatives in ratiometric response manner in living cells.

Esterase D interacts with metallothionein 2A and inhibits the migration of A549 lung cancer cells in vitro.

Esterase D (ESD) is a nonspecific esterase widely distributed in various organisms. ESD plays an important role in regulating cholesterol efflux, inhibiting viral replication and lung cancer growth. MT2A (metallothionein 2A) is the most important isoform of metallothionein (MTs) in human and high expression of MT2A in tumors represents poor prognosis and metastatic behavior. However, there are no reports about the molecular mechanism of ESD in the regulation of tumor metastasis. In this study, we found for the first time that activation ESD promoted its interaction with MT2A and decreased the protein level of MT2A, which resulting in the concentration of free zinc ions up-regulated, and inhibited the migration of A549 lung cancer cells in vitro.

Serum nicotinamide phosphoribosyltransferase as a novel biomarker for non-traumatic osteonecrosis of the femoral head.

OBJECTIVE: The aim of this study was to investigate the potential role of serum nicotinamide phosphoribosyltransferase (NAMPT) in non-traumatic osteonecrosis of femoral head (NONFH). METHODS: A total of 113 NONFH patients and 81 healthy individuals were included in this study. The NAMPT levels in serum were measured by a commercial enzyme-linked immunosorbent assay kit. Radiographic progression was determined using Association Research Circulation Osseous (ARCO) classification system. Clinical severity was assessed by Harris hip score (HHS) and visual analogue scale (VAS). Correlations between serum NAMPT and radiographic progression as well as clinical severity were evaluated statistically. Receiver operating characteristic (ROC) curves were performed to evaluate the diagnostic values of NAMPT in NONFH potential and disease severity. RESULTS: The serum NAMPT levels in NONFH patients were significantly lower than that in healthy controls. There were no significant differences among alcohol-induced group, steroids-induced group, and idiopathic group. NONFH patients with ARCO stage 4 had significant lower serum NAMPT levels in comparisons with ARCO stage 3 and 2, respectively. Lower serum NAMPT levels were also observed in bilateral NONFH cases compared with cases with unilateral NONFH. In addition, serum NAMPT was negatively correlated with ARCO stages and VAS scores, and positively correlated with HHS. ROC curve analysis indicated that serum NAMPT may serve as a novel biomarker for diagnosing early NONFH and for monitoring disease severity. CONCLUSIONS: Our results suggest that serum NAMPT may serve as a novel biomarker for NONFH potential and disease severity.

Activation of GRP78 ATPase suppresses A549 lung cancer cell migration by promoting ITGB4 degradation.

Hypochlorous acid (HOCl) is an essential signal molecule in cancer cells. Activated GRP78 ATPase by a HOCl probe named ZBM-H inhibits lung cancer cell growth. However, the role and underlying mechanism of GRP78 ATPase in lung cancer cell migration have not been established. Here, we reported that activation of GRP78 ATPase by ZBM-H suppressed A549 cell migration and inhibited EMT process. Notably, ZBM-H time-dependently decreased the protein level of integrin ß4 (ITGB4) in A549 cells. Combinatorial treatment of 3BDO (an autophagy inhibitor) and ZBM-H partially rescued the protein level of ITGB4. Consistently, 3BDO partially reversed ZBM-H-inhibited cell migration. Furthermore, ZBM-H promoted the interaction between ANXA7 and Hsc70, which participated in the regulation of selective autophagy and degradation of ITGB4.

Identification of a new way to induce differentiation of dermal fibroblasts into vascular endothelial cells.

BACKGROUND: Human dermal fibroblasts (HDFs) have the potential to differentiate into vascular endothelial cells (VECs), but their differentiation rate is low and the mechanism involved is not clear. The small molecule pathway controls the phenotype of fibroblasts by activating cellular signaling pathways, which is a more convenient method in the differentiation strategy of HDFs into VECs. METHODS: In this study, HDFs were treated with the different doses of CPP ((E)-4-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl) piperazin-1-yl) styryl)-1-methylpyridin-1-ium iodide), and the mRNA and protein levels of HDFs were detected by qPCR, Western blot, flow cytometry and immunofluorescent staining. The matrigel assays, acetylated-LDL uptake and angiogenesis assays of chick embryo chorioallantoic membrane (CAM) and hindlimb ischemia model of nude mice were performed to evaluate the functions of VECs derived from HDFs. RESULTS: Here, we report that the small chemical molecule, CPP, can effectively induce HDFs to differentiate into VECs. First, we observed the morphological changes of HDFS treated with CPP. Flow cytometry, Western blot and qRT-PCR analyses showed that CPP effectively decreased the level of the HDFs-marker Vimentin and increased levels of the VEC-markers CD31, CD133, TEK, ERG, vWF, KDR and CDH5. Detection of the percentage of CD31-positive cells by immunofluorescent staining confirmed that CPP can effectively induce HDFs to differentiate into VECs. The results of Matrigel assays, DiI-ac-LDL uptake, angiogenesis assays on CAM and hindlimb ischemia model of nude mice showed that CPP-induced HDFs have the functions of VECs in vitro and in vivo. Western blot and qRT-PCR analysis showed that CPP induces HDFs to differentiate into VECs by promoting the expression of pro-angiogenic factors (VEGF, FGF-2 and PDGF-BB). CONCLUSIONS: Our data suggest that the small chemical molecule CPP efficiently induces the differentiation of HDFs into VECs. Simultaneously, this new inducer provides a potential to develop new approaches to restore vascular function for the treatment of ischemic vascular diseases.

HOCI Probe CPP Induces the Differentiation of Human Dermal Fibroblasts into Vascular Endothelial Cells through PHD2/HIF-1α/HEY1 Signaling Pathway.

Human dermal fibroblasts (HDFs) have the potential to differentiate into endothelial cells (VECs). In our previous research, we reported that a hypochlorous acid (HOCl) probe CPP efficiently induced the differentiation of HDFs into VECs, however, the mechanism of differentiation was not clear. As an HOCI probe, CPP binds HOCI to modulate its effects. In this study, through Western blotting, qPCR, and PHD2 enzyme activity assay, we found that CPP inhibited the enzyme activity of prolyl-4-hydroxylase 2 (PHD2), thereby stabilizing HIF-1α. To further clarify the mechanism by which CPP inhibits PHD2 enzyme activity, we constructed plasmids, and found that CPP inhibited PHD2 activity to increase the HIF-1α level through the modulation of PHD2 at Cys302 by HOCl in HDFs. Furthermore, RNA-seq experiments showed that CPP could induce the expression of HEY1, which is not only a target gene regulated by HIF1α, but also a key transcription factor for VECs. We used siRNA transfection and in vivo experiments to confirm that CPP could induce HDFs to differentiate into VECs by HEY1. In summary, we identified a new inhibitor of PHD2, demonstrated the new role of HOCl in cell differentiation, and elucidated the mechanism by which HOCl probe CPP induced the differentiation of HDFs into VECs.

Grp94 Inhibitor HCP1 Inhibits Human Dermal Fibroblast Senescence.

Researchers are paying more and more attention to aging, especially skin aging. Therefore, it is urgent to find an effective way to inhibit aging. Here, we report a small chemical molecule, HCP1, that inhibited the senescence of human dermal fibroblasts (HDFs). First, we performed morphological experiment and found that HCP1-treated HDFs were no longer elongated and flat compared to DMSO-treated groups. Next, we found that the number of ß-gal positive cells decreased compared to DMSO-treated groups. Through flow cytometry, western blot, and immunofluorescence, we found that HCP1 could inhibit the senescence of HDFs. In the study of the mechanism, we found that HCP1 could regulate the AMPK/mTOR signal pathway through glucose-regulated protein 94 (Grp94). In addition, we found that HCP1 could promote the interaction between Grp94 and lysosomes, which led to an increase in the activity of lysosomes and inhibited the senescence of HDFs. At the same time, we found that HCP1 decreased the concentration of Ca2+ in mitochondria, inhibiting the senescence of HCP1. Therefore, we propose that HCP1 is a potential aging-inhibiting compound, and provide a new idea for the development of senescence-inhibiting drugs.

An imidazo[1,5-α]pyridines-based ratiometric fluorescent probe for sensing sulfur dioxide derivatives in real samples based on a FRET mechanism.

A novel fluorescence resonance energy transfer (FRET)-based ratiometric emission fluorescent probe AT was designed and developed in which the imidazo[1,5-α]pyridine was served as a FRET donor and tricyanofuran (TCF) as the FRET acceptor to detect SO32-/HSO3- based on the Michael addition reaction. Probe AT had a high energy transfer efficiency (95%) and a large pseudo-Stokes shift (259 nm) in EtOH/PBS buffer (5/5, v/v). It also possessed good selectivity and quick response to SO32-/HSO3-. There was good linearity between the ratio of fluorescence intensity (F499/F645) and the concentrations of SO32-/HSO3- in the ranges of 1.5-7.5 µM and 9-20 µM, with calculated detection limits (LOD) of 55 nM. In addition, the probe could also detect the concentrations of SO32-/HSO3- in real samples such as environmental water and sugar, allowing the probe to be used in a variety of applications.

A Simple but Effective Fluorescent Probe for the Detection of 4-Methylthiophenol.

In this paper, a fluorescent probe (QFR) for the detective work of 4-methylthiophenol was successfully synthesized with a simple but highly effective probe structure. In the buffer solution (V(ACN): V(PBS) = 3:7), by observing the response of the probe after the fluorescence was turned on, we concluded that the probe had good characteristics such as high selectivity, low detection limit (116 nM), and fast response speed (20 min). In addition, the probe was a rare fluorescent probe that detected 4-methylthiophenol but did not respond to thiophenol. Fluorescence intensity was linearly related to 4-methylthiophenol concentration in the range of 0 to 2 equivalents (0-10 µM). The probe demonstrated good results in the determination of the recovery rate (92.28% to 110.1%) of actual water samples, and has great potential in environmental monitoring.

Safrole oxide induced 5-HT neuron-like cell differentiation of bone marrow mesenchymal stem cells by elevating G9a.

In our previous study, we found that safrole oxide (SFO) could induce bone marrow mesenchymal stem cell differentiation into neuron-like cells. However, which kind of neuron cells was induced by SFO was unknown. Here, we found that SFO could induce BMSC differentiation into 5-hydroxytryptamine (5-HT) neuron-like cells. Microarray analysis of BMSCs treated with SFO for 6 h revealed a total of 35 genes changed more than twice. We selected G9a, a histone methyltransferase for further study. The upregulation of G9a was confirmed by RT-PCR and Western blot analysis. Small interfering RNA knockdown of G9a blocked SFO-induced BMSC differentiation. These results demonstrated that G9a was the pivotal factor in SFO-medicated 5-HT neuronal differentiation of BMSCs. Our findings provide a new clue for further investigating the gene control of BMSC differentiation into 5-HT neuron-like cells and provide a putative strategy for depression diseases therapies.

Activation of Esterase D by FPD5 Inhibits Growth of A549 Lung Cancer Cells via JAB1/p53 Pathway.

Esterase D (ESD) is widely distributed in mammals, and it plays an important role in drug metabolism, detoxification, and biomarkers and is closely related to the development of tumors. In our previous work, we found that a chemical small-molecule fluorescent pyrazoline derivative, FPD5, an ESD activator, could inhibit tumor growth by activating ESD, but its molecular mechanism is still unclear. Here, by using RNA interference (RNAi), andco-immunoprecipitation techniques, we found that ESD suppressed the nucleus exportation of p53 through reducing the interaction between p53 and JAB1. The protein level of p53 in the nucleus was upregulated and the downstream targets of p53 were found by Human Gene Expression Array. p53 inhibited the expression of CDCA8 and CDC20. Lastly, the cell cycle of A549 cells was arrested at the G0/G1 phase. Together, our data suggest that ESD inhibited the cancer cell growth by arresting the cell cycle of A549 cells via the JAB1/p53 signaling pathway. Our findings provide a new insight into how to inhibit the growth of lung cancer with the activation of ESD by FPD5.

A quinoline-coumarin near-infrared ratiometric fluorescent probe for detection of sulfur dioxide derivatives.

Sulfur dioxide derivatives (HSO3- and SO32-) play an important role in food preservative, antibacterial, antioxidant and other aspects, so it is urgent for us to develop more efficient detection methods to broaden their application in biochemical research and related disease diagnosis. Fluorescent probes are of particular interest because of their simplicity and high temporal and spatial resolution. Herein, we constructed a new near-infrared (NIR) fluorescence probe, CQC, composed of coumarin fluorophore and quinoline fluorophore, for detecting SO2 derivatives. The near-infrared emission probe CQC with a large Stokes shift (260 nm) not only kept the distance between the two emission peaks large enough (165 nm), but also had a particularly high energy transfer efficiency (99.5%), and was particularly sensitive to the detection of HSO3-/SO32- (LOD: 0.1 µM). The powerful probe CQC succeeded in real-time visualizing endogenous HSO3-/SO32- in living cells.

A spectroscopic probe with FRET-ICT feature for thiophenol monitoring in real water samples.

Thiophenol (PhSH) is widely used in industry, however, it is extremely harmful to the environment and human health due to its high toxicity. In this work, we developed a new FRET-ICT-based ratiometric fluorescent and colorimetric probe (DMNP) for detecting PhSH. DMNP had an ultrahigh energy transfer efficiency (99.7%) and clear spacing of two emission peaks (133 nm). DMNP achieved a fast response to PhSH and exhibited drastic enhancement (over 2100 folds) of the fluorescence intensity ratio upon addition of PhSH. DMNP showed good linear response in the PhSH concentration ranges of 0.5-13 µM and 17.0-22.0 µM. Meanwhile, DMNP could also be applied to monitor PhSH in a variety of real water samples.