A rhodamine-based fluorescent probe used to determine nitroxyl (HNO) in lysosomes.

The reactive nitrogen species (RNS) in lysosomes play a major role during the regulation of lysosomal microenvironment. Nitroxyl (HNO) belongs to active nitrogen species (RNS) and is becoming a potential diagnostic and therapeutic biomarker. However, the complex synthesis routes of HNO in biosystem always hinder the exact determination of HNO in living cells. Here, a rhodamine-based fluorescent probe used to determine nitroxyl (HNO) in lysosomes was constructed and synthesized. 2-(Diphenylphosphino)benzoate was utilized as the sensing unit for HNO and morpholine was chose as the targeting group for lysosome. Before the addition of HNO, the probe displayed a spirolactone structure and almost no fluorescence was found. After the addition of HNO, the probe existed as a conjugated xanthene form and an intense green fluorescence was observed. The fluorescent probe possessed fast response (3 min) and high selectivity for HNO. Furthermore, fluorescence intensity of the probe linearly related with the HNO concentration in the range of 6.0 × 10-8 to 6.0 × 10-5 mol L-1. The detection limit was found to be 1.87 × 10-8 mol L-1 for HNO. Moreover, the probe could selectively targeted lysosome with excellent biocompatibility and had been effectually utilized to recognize exogenous HNO in A549 cells.

A new lysosome-targeted fluorescent probe for hydrogen peroxide based on a benzothiazole derivative.

As an important member of reactive oxygen species, hydrogen peroxide (H2O2) plays a key role in oxidative stress and cell signaling. Abnormal levels of H2O2 in lysosomes can induce damage or even loss of lysosomal function, leading to certain diseases. Therefore, real-time monitoring of H2O2 in lysosomes is very important. In this work, we designed and synthesized a novel lysosome-targeted fluorescent probe for H2O2-specific detection based on a benzothiazole derivative. A morpholine group was used as a lysosome-targeted unit and a boric acid ester was chosen as the reaction site. In the absence of H2O2, the probe exhibited very weak fluorescence. In the presence of H2O2, the probe showed an increased fluorescence emission. The fluorescence intensity of the probe for H2O2 displayed a good linear relationship in the concentration range of H2O2 from 8.0 × 10-7 to 2.0 × 10-4 mol·L-1. The detection limit was estimated to be 4.6 × 10-7 mol·L-1 for H2O2. The probe possessed high selectivity, good sensitivity and short response time for the detection of H2O2. Moreover, the probe had almost no cytotoxicity and had been successfully applied to confocal imaging of H2O2 in lysosomes of A549 cells. These results illustrated that the developed fluorescent probe in this study could provide a good tool for the determination of H2O2 in lysosomes.

[Metabolite identification of isochlorogenic acid A in rats by HPLC-Q-Exactive Orbitrap-MS].

Isochlorogenic acid A(ICA) is the main active component of several TCMs, such as Artemisiae Scopariae Herba. This study aims to identify the metabolites of orally administered ICA in rat plasma, urine, and feces, and to speculate on its potential metabolic pathways. Rats were administered ICA orally, and samples of plasma, urine, and feces were collected at different time points. High-performance liquid chromatography-quadrupole Exactive Orbitrap-mass spectrometry(HPLC-Q-Exactive Orbitrap-MS) was used in combination with reference standards, retention time comparison, fragmentation pattern analysis, and literature data to identify the metabolites in the biological samples. A total of 39 metabolites(M1-M39) of ICA were preliminarily identified from rat samples, including 31 from plasma(M1-M10, M12-M24, M26-M28, M30, M34-M35, M38-M39), 34 from urine(M1-M11, M13-M15, M19-M25, M27-M39), and 11 from feces(M2-M3, M6, M15, M21-M23, M32, M34, M36-M37). The main metabolic pathways included hydrolysis, glucuronidation, methylation, and sulfonation reactions. This study revealed the metabolic profile of ICA in rat plasma, urine, and feces, providing references for the in-depth elucidation of its pharmacologically active components.

USP39 mediates p21-dependent proliferation and neoplasia of colon cancer cells by regulating the p53/p21/CDC2/cyclin B1 axis.

Ubiquitin-specific protease 39 (USP39) is frequently overexpressed in a variety of cancers, and involved in the regulation of various biological processes, such as cell proliferation, cell cycle progression, apoptosis and pre-messenger RNA splicing. Nevertheless, the biological roles and mechanisms of USP39 in colon cancer remain largely unknown. In this study, we analyzed whether USP39 can be a molecular target for the treatment of colon cancer. Whilst overexpression of USP39 was detected in human colon cancer tissues and cell lines, USP39 knockdown was observed to inhibit the growth and subcutaneous tumor formation of colon cancer cells. Further analysis showed that USP39 knockdown can stabilize p21 by prolonging the half-life of p21 and by upregulating the promoter activity of p21. The RS domain and USP domain of USP39 were found to play an essential role. Additionally, our findings revealed that USP39 plays a regulatory role in the proliferation of colon cancer cells by the p53/p21/CDC2/cyclin B1 axis in a p21-dependent manner. Taken together, this study provided the theoretical basis that may facilitate the development of USP39 as a novel potential target of colon cancer therapy.

Knocking down USP39 Inhibits the Growth and Metastasis of Non-Small-Cell Lung Cancer Cells through Activating the p53 Pathway.

Ubiquitin-specific protease 39 (USP39), a member of the deubiquitinating enzyme family, has been reported to participate in cytokinesis and metastasis. Previous studies determined that USP39 functions as an oncogenic factor in various types of cancer. Here, we reported that USP39 is frequently overexpressed in human lung cancer tissues and non-small-cell lung cancer (NSCLC) cell lines. USP39 knockdown inhibited the proliferation and colony formation of A549 and HCC827 cells and decreased tumorigenic potential in nude mice. Specifically, knocking down USP39 resulted in cell cycle arrest at G2/M and subsequent apoptosis through the activation of the p53 pathway, including upregulation of p21, cleaved-cas3, cleaved-cas9 and downregulation of CDC2 and CycinB1. Moreover, USP39 knockdown significantly inhibited migration and invasion of A549 and HCC827 cells, also via activation of the p53 pathway, and downregulation of MMP2 and MMP9. Importantly, we verified these results in metastasis models in vivo. Collectively, these results not only establish that USP39 functions as an oncogene in lung cancer, but reveal that USP39 has an essential role in regulating cell proliferation and metastasis via activation of the p53 pathway.

A coumarin-naphthalimide-based ratiometric fluorescent probe for nitroxyl (HNO) based on an ICT-FRET mechanism.

Nitroxyl (HNO) is an important reactive nitrogen that is associated with various states in physiology and pathology and plays a unique function in living systems. So, it is important to exploit fluorescent probes with high sensitivity and selectivity for sensing HNO. In this paper, a novel ratiometric fluorescent probe for HNO was developed utilizing intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) mechanisms. The probe selected coumarin as energy donor, naphthalimide as energy receptor and 2-(diphenylphosphino)benzoate as the sensing site for detecting HNO. When HNO was not present, the 2-(diphenylphosphino)benzoate unit of the probe restricted electron transfer and the ICT process could not occur, leading to the inhibition of FRET process as well. Thus, in the absence of HNO the probe displayed the intrinsic blue fluorescence of coumarin. When HNO was added, the HNO reacted with the 2-(diphenylphosphino)benzoate unit of the probe to yield a hydroxyl group which resulting in the opening of ICT process and the occurring of FRET process. Thus, after providing HNO the probe displayed yellow fluorescence. In addition, the probe showed good linearity in the ratio of fluorescence intensity at 545 nm and 472 nm (I545 nm/I472 nm) with a concentration of HNO (0.1-20 µM). The probe processed a detection limit of 0.014 µM and a response time of 4 min. The probe also specifically identified HNO over a wide pH scope (pH = 4.00-10.00), including physiological conditions. Cellular experiments had shown that this fluorescent probe was virtually non-cytotoxic and could be applied for ratiometric sensing of HNO in A549 cells.

A lysosome-located and rhodamine-based fluorescence probe for recognizing hydrogen polysulfide.

Hydrogen polysulfide (H2Sn, n≥2), as a kind of active sulfur species (RSS), has become a hot topic in RSS. It can regulate the biological activity of many proteins through S-sulfhydrylation of cysteine residues (protein Cys-SSH), and has a protective effect on cells. Although there have been some studies on hydrogen polysulfide, its production, degradation pathway and regulation mechanism still need further be researched. In presented study, an original lysosome-localized fluorescent probe for determining H2Sn was developed utilizing rhodamine as the fluorogen. The probe used morpholine as the locating unit of lysosomes and chose 2-fluoro-5-nitrobenzoate as the recognizing group. Before adding H2Sn, the proposed probe displayed a spironolactone structure and emitted very weak fluorescence. After adding H2Sn, a conjugated xanthene was formed and the probe demonstrated green fluorescence. When the H2Sn concentration was varied from 6.0×10-7 mol·L-1 to 10.0×10-5 mol·L-1, the fluorescence intensity of the probe was linearly dependent on the H2Sn concentration. And the detection limit was 1.5×10-7 mol·L-1. The presented probe owned a fast response speed, good selectivity, excellent sensitivity and broad pH work scope. In addition, the probe had been well utilized to sense endogenic and exogenic H2Sn in lysosomes.

Green synthesis of yellow-green emissive silicon nanoparticles and their application for the sensitive fluorescence detection of bilirubin.

Bilirubin, a tetrapyrrole compound metabolized by heme, is an important biomarker for diagnosis and prognosis of patients with liver diseases. Highly sensitive detection of bilirubin is essential for disease prevention and treatment. In recent years, silicon nanoparticles (SiNPs) have received intense attention due to their excellent optical properties and environmental friendliness. In this paper, water-soluble yellow-green fluorescent SiNPs were synthesized by a mild water bath method using 2-aminophenylboronic acid hydrochloride as the reducing agent and 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEEA) as the silicon source. The preparation process does not require high temperature, high pressure and complex modifications. The SiNPs possessed excellent photostability and good water dispersibility. It was found that the fluorescence of SiNPs at 536 nm could be significantly quenched by bilirubin. By using SiNPs as a fluorescent probe, a novel fluorescence method for sensitive detection of bilirubin was established with a wide linear range of 0.05-75 µM and a limit of detection (LOD) of 16.67 nM. The detection mechanism was mainly due to the internal filtration effect (IFE). More significantly, the established method could successfully determine the contents of bilirubin in biological samples with good recoveries.

An ICT-FRET-based ratiometric fluorescent probe for hydrogen polysulfide based on a coumarin-naphthalimide derivative.

Hydrogen polysulfide (H2Sn, n > 1), as one of the important members of reactive sulfur species (RSS), plays a vital part in the processes of both their physiology and pathology. In this work, a ratiometric fluorescent probe for H2Sn had been designed and prepared based on the combination mechanism of intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET). The probe chose a coumarin derivative as the energy donor, a naphthalimide derivative as the energy acceptor and 2-fluoro-5-nitrobenzoate as the H2Sn recognition group. When H2Sn was not present in the system, the ICT process of the naphthalimide acceptor was inhibited and the FRET process from the coumarin donor to the naphthalimide acceptor was turned off. When H2Sn was added, both ICT and FRET occurred due to the nucleophilic substitution-cyclization reactions between the probe and hydrogen polysulfide. In addition, the ratio value of the emission intensities at 550 nm and 473 nm (I550 nm/I473 nm) of this probe had a good linear relationship with H2Sn concentration in the range of 6.0 × 10-7-5.0 × 10-5 mol·L-1, and a detection limit of 1.8 × 10-7 mol·L-1 was obtained. The developed probe had high selectivity and sensitivity, as well as good biocompatibility. Additionally, the probe had been used to successfully image both indigenous and exogenous hydrogen polysulfide in A549 cells using confocal microscope.

A naphthalimide-based and Golgi-targetable fluorescence probe for quantifying hypochlorous acid.

The Golgi apparatus (GA) is a vital organelle in biological systems and excess reactive oxygen species (ROS) is produced during stress in the Golgi apparatus. Hypochlorous acid (HOCl) is a significant reactive oxygen species and has strong oxidative and antibacterial activity, but excessive secretion of hypochlorous acid can affect Golgi structure or function abnormally, it will lead to a series of diseases including Alzheimer's disease, neurodegenerative diseases, autoimmune diseases, and Parkinson's disease. In present work, a novel fluorescent probe for Golgi localization utilizing naphthalimide derivatives was constructed to detect hypochlorous acid. The fluorescent probe used a derivatived 1,8-naphthalimide as the emitting fluorescence group, phenylsulfonamide as the localization group and dimethylthiocarbamate as the sensing unit. When HOCl was absent, the intramolecular charge transfer (ICT) process of the developed probe was hindered and the probe exhibited a weak fluorescence. When HOCl was present, the ICT process occurred and the probe showed strong green fluorescence. When the HOCl concentration was altered from 5.0 × 10-7 to 1.0 × 10-5 mol·L-1, the fluorescence intensity of the probe well linearly correlated with the HOCl concentration. The detection limit of 5.7 × 10-8 mol·L-1 was obtained for HOCl. The HOCl fluorescent probe possessed a rapid reaction time, a high selectivity and a broad working pH scope. In addition, the probe possessed good biocompatibility and had been magnificently employed to image Golgi HOCl in Hela cells. These characteristics of the probe demonstrated its ability to be used for sensing endogenous and exogenous hypochlorous acids within the Golgi apparatus of living cells.

Highly sensitive and selective detection of triphosgene with a 2-(2'-hydroxyphenyl)benzimidazole derived fluorescent probe.

In this work, a 2-(2'-hydroxyphenyl)benzimidazole derived fluorescent probe, 2-(2'-hydroxy-4'-aminophenyl)benzimidazole (4-AHBI), was synthesized and its fluorescent behavior toward triphosgene were evaluated. The results showed that 4-AHBI exhibited high sensitivity (limit of detection, 0.08 nM) and excellent selectivity for triphosgene over other acyl chlorides including phosgene in CH2Cl2 solution. Moreover, 4-AHBI loaded test strips were prepared for the practical sensing of triphosgene.

Methyl 2-(4-meth-oxy-3-nitro-benzamido)-acetate.

The title compound, C(11)H(12)N(2)O(6), crystallizes with two independent mol-ecules in the asymmetric unit, which differ slightly in conformation. The dihedral angle between the amide O=C-N plane and the attached benzene ring is 19.5 (3)° in one mol-ecule and 23.4 (3)° in the other. In the crystal, the two independent mol-ecules are connected alternately by N-H⋯O hydrogen bonds, forming a chain along the a axis.

Water Soluble Silicon Nanoparticles as a Fluorescent Probe for Highly Sensitive Detection of Rutin.

In this work, water-soluble fluorescent silicon nanoparticles (SiNPs) were prepared by one-pot hydrothermal method using 3-(2-aminoethylamino)propyldimethoxymethylsilane (AEAPDMMS) as a silicon source and amidol as a reducing agent. The prepared SiNPs showed bright green fluorescence, excellent stability against photobleaching, salt tolerance, temperature stability, and good water solubility. Due to the internal filtration effect (IFE), rutin could selectively quench the fluorescence of the SiNPs. Based on such phenomena, a highly sensitive fluorescence method was established for rutin detection. The linear range and limit of detection (LOD) were 0.05-400 µM and 15.2 nM, respectively. This method was successfully applied to detect rutin in the samples of rutin tablets, Sophora japonica, fry Sophora japonica, and S. japonica carbon with satisfactory recovery.

A novel lysosome-located fluorescent probe for highly selective determination of hydrogen polysulfides based on a naphthalimide derivative.

Hydrogen polysulfides (H2Sn, n > 1) belongs to sulfane sulfur in the reactive sulfur species (RSS) family and plays a significant regulatory role in organisms. Highly selective and lysosome-located probes for detecting hydrogen polysulfides are rare. Thus, it is important to develop a technique to detect the changes of H2Sn level in lysosomes. In this work, a lysosome-targeting fluorescent probe for H2Sn was designed and developed based on a naphthalimide derivative. 4-Hydroxynaphthalimide was selected as the fluorescent group and 2-chloro-5-nitrobenzoate group was used as a specific recognition unit for H2Sn. A morpholine unit was chosen as a lysosome-located group. In the absence of H2Sn, the fluorescent probe exhibited almost no fluorescence. In the presence of H2Sn, the fluorescent probe showed strong fluorescence owing to H2Sn-mediated aromatic substitution-cyclization reactions. The fluorescence emission intensity at 548 nm of the probe showed a good linear relationship toward H2Sn in the range of 2.0 × 10-7 - 9.0 × 10-5 mol·L-1, and the detection limit was found to be 1.5 × 10-7 mol·L-1. The probe possessed a wide work range of pH, including the pH of physiological environment, and high selectivity for H2Sn. There are almost no cytotoxicity and the ability of detecting endogenous and exogenous H2Sn in lysosomes. These results indicate that the fluorescent probe can provide a good tool for intracellular and extracellular detection of H2Sn.

Effect of Different Temperatures on the Hydration Kinetics of Urea-Doped Cement Pastes.

Urea can solve the problem of concrete cracking due to temperature stress. However, its effect is affected by temperature. The influencing mechanism of temperature on urea-doped cement pastes is still unclear. This paper explores the effect of different temperatures on the hydration kinetics of urea-doped cement pastes. The isothermal calorimeter (TAM Air) was used to test hydration at three constant temperatures (20 °C, 40 °C, and 60 °C). The effects of the urea admixture and temperature on the hydration process and hydration kinetics parameters were investigated. The hydration mechanism was analyzed, and the changes in macroscopic mechanical compressive strength and porosity were tested. The results show that, as the urea content (UC) increases, the rate of hydration gradually decreases, and the increase in temperature promotes the inhibitory effect of urea. At 60 °C, UC of 8% can be reduced by 23.5% compared with the pure cement (PC) group's hydration rate. As the temperature increases from 20 °C to 60 °C, the Krstulovic-Dabic model changes from the NG-I-D process to the NG-D process. The effect of urea on the compressive strength of the cement is mainly shown in the early stage, and its effect on later strength is not obvious. In addition, urea will increase its early porosity. The porosity will gradually decrease in the later stage. The results of the study clarify the effect of temperature on urea-doped cement pastes. The optimal content of urea in cement is about 8%, which will provide theoretical guidance for solving the cracking problem of large-volume concrete due to temperature stress.

Rhodamine-Based Fluorescent Probe for Highly Selective Determination of Hg2.

The determination of mercuric ions (Hg2+) in environmental and biological samples has attracted the attention of researchers lately. In the present work, a novel turn-on Hg2+ fluorescent probe utilizing a rhodamine derivative had been constructed and prepared. The probe could highly sensitively and selectively sense Hg2+. In the presence of excessive Hg2+, the probe displayed about 52-fold fluorescence enhancement in 50% H2O/CH3CH2OH (pH, 7.24). In the meantime, the colorless solution of the probe turned pink upon adding Hg2+. Upon adding mercuric ions, the probe interacted with Hg2+ and formed a 1:1 coordination complex, which had been the basis for recognizing Hg2+. The probe displayed reversible dual colorimetric and fluorescence sensing of Hg2+ because rhodamine's spirolactam ring opened upon adding Hg2+. The analytical performances of the probe for sensing Hg2+ were also studied. When the Hg2+ concentration was altered in the range of 8.0 × 10-8 to 1.0 × 10-5 mol L-1, the fluorescence intensity showed an excellent linear correlation with Hg2+ concentration. A detection limit of 3.0 × 10-8 mol L-1 had been achieved. Moreover, Hg2+ in the water environment and A549 cells could be successfully sensed by the proposed probe.

A Highly Selective Fluorescent Probe for Hypochlorous Acid in Living Cells Based on a Naphthalene Derivative.

Hypochlorous acid (HOCl) was crucial for maintaining the homeostasis in cells and plays vital roles in many physiological and pathological processes. In this work, a highly selective fluorescent probe for hypochlorous acid in living cells was constructed and prepared based on a naphthalene derivative. A naphthalene derivative was utilized as the fluorescent group, and N,N-dimethylthiocarbamate was applied as the selective recognition site for HOCl. Before adding HOCl, the fluorescent probe exhibited weak fluorescence. Upon adding HOCl, the fluorescent probe displayed remarkable fluorescence enhancement. The fluorescence intensity at 502 nm showed a linear response to the concentration of HOCl from 3.0 × 10-7 to 1.0 × 10-5 mol·L-1. The detection limit was estimated to be 1.5 × 10-7 mol·L-1 for HOCl. The fluorescent probe showed fast response and outstanding selectivity toward HOCl. It owned good biocompatibility and had also been successfully applied in the confocal imaging of exogenous and endogenous HOCl in living cells.

Ratiometric Determination of Nitroxyl Utilizing a Novel Fluorescence Resonance Energy Transfer-Based Fluorescent Probe Based on a Coumarin-Rhodol Derivative.

Nitroxyl (HNO) is a member of the reactive nitrogen species, and how to detect it quickly and accurately is a challenging task. In this work, we designed and prepared a fluorescent ratiometric probe based on the fluorescence resonance energy transfer (FRET) mechanism, which can detect HNO with high selectivity. The coumarin derivative was used as an energy donor, the rhodol derivative was applied as an energy receptor, and 2-(diphenylphosphine)benzoate was utilized as the recognition group to detect nitroxyl. In the absence of HNO, the rhodol derivative exists in a non-fluorescent spironolactone state, and the FRET process is inhibited. Upon adding HNO, the closed spironolactone form is transformed into a conjugated xanthene structure and the FRET process occurs. This probe could specifically recognize nitroxyl, showing high sensitivity and selectivity. When the HNO concentration was changed from 3.0 × 10-7 to 2.0 × 10-5 mol·L-1, I 543nm/I 470nm exhibited a satisfactory linear correlation with the concentration of HNO. A detection limit of 7.0 × 10-8 mol·L-1 was obtained. In addition, almost no cell toxicity had been verified for the probe. The probe had been successfully applied to the ratiometric fluorescence imaging of HNO in HepG2 cells.

Protective effect and mechanism of loganin and morroniside on acute lung injury and pulmonary fibrosis.

BACKGROUND: Loganin and morroniside are two iridoid glycosides with anti-inflammatory, antioxidant and anti-tumor effects. Whether they have effect on acute lung injury and pulmonary fibrosis are still unknown. PURPOSE: To explore the potential effects of loganin and morroniside against acute lung cancer and pulmonary fibrosis, and the underlying molecular mechanism. STUDY DESIGN AND METHODS: Cell and animal models of acute lung injury were established by the induction of LPS. After intervention with loganin and morroniside, the pathological symptom of lung tissue was assessed, pro-inflammatory factors in cells and lung tissues were detected, NF- κB/STAT3 signaling pathway related proteins were detected by western blotting. Mice pulmonary fibrosis model was induced by bleomycin, pathological symptom was assessed by HE and Masson staining. Fibrosis related indicators were detected by qPCR or western blot. CD4+/CD8+ was detected by flow cytometry. RESULTS: Loganin and morroniside relieved the pathological symptom of lung tissue in acute lung injury, pro-inflammatory factors such as IL-6, IL-1ß, TNF-α mRNA were inhibited. Expression of p-p65 and STAT3 in lung tissues were also downregulated. In addition, loganin and morroniside downregulated the expression of collagen fiber, hydroxyproline and TGF-ß1, collagen I and α-SMA mRNA in lung tissues of pulmonary fibrosis model. This study proved that loganin and morroniside have protective effect on acute lung injury and pulmonary fibrosis, and may provide theoretical basis for the development of new clinical drugs.

(2-Bromo-acet-yl)ferrocene.

In the title mol-ecule, [Fe(C(5)H(5))(C(7)H(6)BrO)], the C atoms of the substituted ring have disparate Fe-C bond lengths compared with the unsubstituted ring. In the bromo-acetyl residue, the Br and O atoms are co-planar [the O-C-C-Br torsion angle is 5.7 (4)°] and are syn to each other. Helical supra-molecular chains along the b axis are formed in the crystal structure mediated by C-H⋯O contacts; the carbonyl-O atom is bifurcated. The chains are linked into layers by C-H⋯π(unsubstituted ring) inter-actions that stack along the a-axis direction.