A colorimetric detection of dopamine in urine and serum based on the CeO2 @ZIF-8/Cu-CDs laccase-like nanozyme activity.

This study reports a sensitive and selective colorimetric approach for the analysis of dopamine (DA) based on CeO2 @ZIF-8/Cu-CDs laccase-like nanozymes activity. The CeO2 @ZIF-8/Cu-CDs was synthesized using cerium oxide (CeO2 ) and copper-doped carbon dots (Cu-CDs) with 2-methylimidazole by a facilely hydrothermal approach. The CeO2 @ZIF-8/Cu-CDs exhibited excellent laccase-like nanozymes activity and can oxidize the colorless substrate (DA) to red product with 4-aminoantipyrine as the chromogenic agent. The Michaelis-Menten constant (Km ) and the maximal velocity (Vmax ) of CeO2 @ZIF-8/Cu-CDs are 0.20 mM and 1.48 µM/min, respectively. The detection method has a linear range of 0.05-7.5 µg/mL and a detection limit as low as 8.5 ng/mL with good reproducibility. The developed colorimetric sensor was applied to rapid and precise quantitative evaluation of DA levels in serum and urine samples. This study presents a new approach for detecting biological molecules by utilizing the controlled regulation of nanozymes' laccase-like activity.

Nucleoporin Seh1 controls murine neocortical development via transcriptional repression of p21 in neural stem cells.

Mutations or dysregulation of nucleoporins (Nups) are strongly associated with neural developmental diseases, yet the underlying mechanisms remain poorly understood. Here, we show that depletion of Nup Seh1 in radial glial progenitors results in defective neural progenitor proliferation and differentiation that ultimately manifests in impaired neurogenesis and microcephaly. This loss of stem cell proliferation is not associated with defects in the nucleocytoplasmic transport. Rather, transcriptome analysis showed that ablation of Seh1 in neural stem cells derepresses the expression of p21, and knockdown of p21 partially restored self-renewal capacity. Mechanistically, Seh1 cooperates with the NuRD transcription repressor complex at the nuclear periphery to regulate p21 expression. Together, these findings identified that Nups regulate brain development by exerting a chromatin-associated role and affecting neural stem cell proliferation.

Nuclear import carrier Hikeshi cooperates with HSP70 to promote murine oligodendrocyte differentiation and CNS myelination.

Dysregulation of factors in nucleocytoplasmic transport is closely linked to neural developmental diseases. Mutation in Hikeshi, encoding a nonconventional nuclear import carrier of heat shock protein 70 family (HSP70s), leads to inherited leukodystrophy; however, the pathological mechanisms remain elusive. Here, we showed that Hikeshi is essential for central nervous system (CNS) myelination. Deficiency of Hikeshi, which is observed in inherited leukodystrophy patients, resulted in murine oligodendrocyte maturation arrest. Hikeshi is required for nuclear translocation of HSP70s upon differentiation. Nuclear-localized HSP70 promotes murine oligodendrocyte differentiation and remyelination after white matter injury. Mechanistically, HSP70s interacted with SOX10 in the nucleus and protected it from E3 ligase FBXW7-mediated ubiquitination degradation. Importantly, we discovered that Hikeshi-dependent hyperthermia therapy, which induces nuclear import of HSP70s, promoted oligodendrocyte differentiation and remyelination following in vivo demyelinating injury. Overall, these findings demonstrate that Hikeshi-mediated nuclear translocation of HSP70s is essential for myelinogenesis and provide insights into pathological mechanisms of Hikeshi-related leukodystrophy.

Cu, I-doped carbon dots as simulated nanozymes for the colorimetric detection of morphine in biological samples.

As newly developed synthetic enzymes with exceptional catalytic capabilities and outstanding stability, nanozymes have drawn considerable interest in the realm of sensing. Using a simple hydrothermal process, iodine and copper-doped carbon dots (Cu,I-CDs) with simulated enzymes were fabricated in the current investigation. Cu,I-CDs demonstrate peroxidase-mimicking function together with high catalytic effectiveness due to aforementioned features. This led to generation of a colorimetric sensor for quick and accurate quantitative assessment of morphine (MOR). The outcomes showed the method's usefulness for the colorimetric detection of MOR. The linear range for MOR detection is 0.25-25 µg/mL having a reduced detection limit of 64 ng/mL. This sensor's successful use in the analysis of MOR in biological material is more noteworthy.

Nucleoporin Seh1 maintains Schwann cell homeostasis by regulating genome stability and necroptosis.

Schwann cells play critical roles in peripheral neuropathies; however, the regulatory mechanisms of their homeostasis remain largely unknown. Here, we show that nucleoporin Seh1, a component of nuclear pore complex, is important for Schwann cell homeostasis. Expression of Seh1 decreases as mice age. Loss of Seh1 leads to activated immune responses and cell necroptosis. Mice with depletion of Seh1 in Schwann cell lineage develop progressive reduction of Schwann cells in sciatic nerves, predominantly non-myelinating Schwann cells, followed by neural fiber degeneration and malfunction of the sensory and motor system. Mechanistically, Seh1 safeguards genome stability by mediating the interaction between SETDB1 and KAP1. The disrupted interaction after ablation of Seh1 derepresses endogenous retroviruses, which triggers ZBP1-dependent necroptosis in Schwann cells. Collectively, our results demonstrate that Seh1 is required for Schwann cell homeostasis by maintaining genome integrity and suggest that decrease of nucleoporins may participate in the pathogenesis of periphery neuropathies.

Dual-Emission Carbon-Dot Ratiometric Fluorescence Sensor for Morphine Recognition in Biological Samples.

Herein, a novel nitr[ogen-doped carbon dot (N-CD) fluorescence sensor with a dual emission ratio is developed using the microwave-assisted synthesis of m-phenylenediamine and spermidine. As a result of the fluorescence inner filtration effect (IFE) effect between morphine (MOR) and N-CD, the blue fluorescence of N-CDs at 350 nm was reduced in the presence of MOR, whereas the fluorescence of N-CDs at 456 nm increased substantially. The results demonstrated that the approach has a tremendous potential and that the linear range of MOR detection is 0.25-25 µg/mL, with a 71.8 ng/mL detection limit. Under UV light, the blue fluorescent system is easily visible to the naked eye. More significantly, the sensor proved successful in providing satisfactory results for the speciation measurement of MOR in a variety of biological samples.

Novel N,Cl-doped deep eutectic solvents-based carbon dots as a selective fluorescent probe for determination of morphine in food.

In the present study, new N,Cl co-doped carbon dots (N,Cl-CDs) based on deep eutectic solvent (DES) were fabricated by a facile hydrothermal process. This fluorescent probe exhibited a good quantum yield of 14% and was applied for the sensitive and selective quantification of morphine in foods. In addition, the influence of solution pH, interaction time, system temperature, interfering substances and analogues on the determination was also investigated. Under the optimized conditions, the luminescence intensity of carbon dots increased linearly with the addition of morphine in the concentration range of (0.15-280.25) µg mL-1 (R 2 > 0.9969) and the limit of detection (LOD) of 46.5 ng mL-1. Based on these results, it is suggested that N,Cl-CDs is a promising fluorescent probe for sensitive and selective quantification of morphine in foods.

Comprehensive Investigation of Moringa oleifera from Different Regions by Simultaneous Determination of 11 Polyphenols Using UPLC-ESI-MS/MS.

In this study, we develop and validate a simultaneous quantification of polyphenols method based on an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) to adequately understand how different habitats influence the quality and profile of Moringa oleifera polyphenol. Furthermore, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to compare and discriminate 25 samples collected from different areas. A significant correlation was found between the polyphenol profile and the collection area. Significant differences in the polyphenol content of Moringa oleifera from different regions indicate that the genetic diversity of Moringa oleifera was relatively rich, possibly due to differences in cultivation conditions, climate, or soil environment resulting in the accumulation of different polyphenols. These observations provide a theoretical basis for subsequent Moringa oleifera germplasm selection and development research. Furthermore, the quantitative analysis methodology used to characterize the polyphenols may be used toward developing quality assessment and future pharmacodynamic investigations of Moringa oleifera.

Screening of multiclass pesticide residues in maca and Moringa oleifera by a modified QuEChERS sample preparation procedure and UPLC-ESI-MS/MS analysis.

In the present study, a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was proposed for the simultaneous analysis of 75 pesticides in maca and Moringa oleifera with ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). The developed method was validated in accordance with linearity, linear range, limit of detection, limit of quantification, accuracy, precision, and matrix effect. Each analyte had good linearity (R 2 > 0.99) in the corresponding concentration range. The method LOD and LOQ values of all the analytes ranged from 0.01 µg kg-1 to 303.35 µg kg-1 and 0.03 µg kg-1 to 1011.15 µg kg-1, respectively. The recoveries (n = 6) of the analyzed pesticides were in the range of 75.92-113.43%. The RSDs of precision were between 0.60% and 7.36%. All matrix effect values ranged from 81.79% to 118.71% and 80.36% to 119.64% in maca and Moringa oleifera, respectively. The analysis of 103 samples showed the presence of isofenphos-methyl in some of them. The method had a good application prospect and could be used as a general approach for the quantitative determination of pesticide residues in food.

Mechanism of Oxymatrine-induced Human Esophageal Cancer Cell Apoptosis by the Endoplasmic Reticulum Stress Pathway.

Endoplasmic reticulum stress is one of the mechanisms of cell apoptosis. In this study, the mechanism of oxymatrine-induced human esophageal cancer Eca-109 cell apoptosis by the endoplasmic reticulum stress pathway was investigated. Eca-109 cells were cultured in vitro with different doses of oxymatrine (0.5, 1, 2 µg/mL) for 48 h. The cell viability and proliferation inhibition rate were examined by MTT assay and cell cycle assay. The apoptosis rate was examined by Annexin V-FITC/propidium iodide assay. The expression of endoplasmic reticulum stress markers, including binding immunoglobulin protein and CCAAT-enhancer-binding protein homologous protein, were determined by real-time quantitative polymerase chain reaction and western blotting, respectively. MTT data showed that oxymatrine significantly inhibited the proliferation of Eca-109 cells. The cell apoptosis rate was quantified by flow cytometry. The expression of binding immunoglobulin protein was markedly downregulated in oxymatrine-treated Eca-109 cells while that of CCAAT-enhancer-binding protein homologous protein was upregulated. Oxymatrine inhibited proliferation and induced apoptosis of human esophageal carcinoma Eca-109 cells. Thus, oxymatrine may be a potential agent for treating human esophageal cancer.

Influence of Tanshinone IIA on apoptosis of human esophageal carcinoma Eca-109 cells and its molecular mechanism.

BACKGROUND: Previous studies have shown that Tanshinone (Tan) IIA exerts obvious antitumor efficacy; however, its molecular mechanism remains unclear. This study was conducted to identify the influence of Tan IIA on Eca-109 cell apoptosis, explore its molecular mechanism, and provide a theoretical basis for clinical application. METHODS: Eca-109 cells were cultured in vitro and treated with different concentrations of Tan IIA. Morphologic changes were viewed under inverted fluorescence microscope with dual acridine orange/ethidium bromide staining assay. Methyl-thiazolyl-tetrazolium and Annexin V propidium iodide assays were respectively used to measure cell viability and apoptosis rate. The protein and messenger (m)RNA expression of binding immunoglobulin protein (BIP), mitochondrial cytochrome c (CytC), and caspase-9 were detected by Western blot and quantitative real-time PCR, respectively. RESULTS: Cell viability decreased and the apoptosis rate significantly increased with increasing concentrations of Tan IIA (0, 20, 40, 60 µg/mL), which indicated that Tan IIA inhibited the proliferation and induced the apoptosis of Eca-109 cells in a dose-dependent manner. Eca-109 cells treated with 60 µg/mLTan IIA showed typical morphological changes of apoptosis under the inverted microscope. Moreover, compared with the negative control group, protein and mRNA expression of BIP decreased significantly (P < 0.05), whereas protein and mRNA expression of CytC and caspase-9 increased significantly (P < 0.05). CONCLUSION: Tan IIA can induce apoptosis in human esophageal carcinoma Eca-109 cells by regulating BIP, CytC, and caspase-9 expression. Endoplasmic reticulum stress and mitochondrial-dependent may be involved in Tan IIA-induced Eca-109 cell apoptosis.

A Computational Investigation of the Equilibrium Constants for the Fluorescent and Chemiluminescent States of Coelenteramide.

In spite of recent advances in understanding the mechanism of coelenterate bioluminescence, there is no consensus about which coelenteramide specie and/or state are the light emitter. In this study, a systematic investigation of the geometries and spectra of all possible light emitters has been performed at the TD ωB97XD/6-31+G(d) level of theory, including various fluorescent and chemiluminescent states in vacuum, in a hydrophobic environment and in aqueous solution. To deduce the most probable form of the fluorescent and chemiluminescent coelenteramide emitter, the equilibrium constants for the fluorescent and chemiluminescent states connecting the various species have been calculated. ωB97XD gives a qualitatively good description of fluorescent and chemiluminescent structures. Coelenteramide is formed in a "dark" chemiluminescent state and must evolve to a bright fluorescent state. Moreover, the photoacidity of the phenol group is significantly higher in the fluorescent state than in the chemiluminescent state, which allows the formation of phenolate coelenteramide and clarifies its role as the bioluminescent emitter.

Effects of Leptin on Na+/Ca2+ Exchanger in PC12 Cells.

BACKGROUND/AIMS: Alzheimer's disease (AD) is known to be related to alterations in neuronal intracellular calcium activity ([Ca2+]i). The present study revealed the distinct role of leptin in Na+/Ca2+-exchanger activity. METHODS: [Ca2+]i was determined utilizing Fura-2 fluorescence. The activity of NCX was measured by removal of extracellular Na+ in the presence of external Ca2+. Na+/Ca2+-exchanger activity was further quantified from whole cell currents following removal of extracellular Na+. Na+/Ca2+-exchanger isoform NCX1 transcript levels and protein abundance were quantified by RT-PCR and Western blotting, respectively. RESULTS: Exposure of PC12 cells to 30 µM amyloid (Aß42) increased [Ca2+]i, an effect significantly blunted by 6 hours incubation with leptin before Aß42 treatment. Moreover, leptin treatment significantly increased Na+/Ca2+-exchanger mediated Ca+ transport and current, NCX1 transcript level as well as NCX1 membrane protein abundance. CONCLUSION: We show that leptin blunts Aß42-evoked [Ca2+]i increase by increasing expression and activity of Na+/Ca2+-exchanger NCX1.

Influence of Tanshinone IIA on the Apoptosis of Human Esophageal Ec-109 Cells.

The induced-apoptosis effect and mechanism of human esophageal cancer Ec-109 cells via tanshinone IIA was investigated. The Ec-109 cells were cultured in vitro with different concentrations of tanshinone IIA (2 µg/mL, 4 µg/mL, or 8 µg/mL) for 12, 24, 36, and 48 hours. MTT assay was used to evaluate the proliferative inhibition rate of tanshinone IIA on esophageal Ec-109 cells. After 24 hours of culturing in vitro, a control group was assigned. The apoptosis rate was detected by the AO/EB and annexin V-FITC/propidium iodide assay, and the protein levels of Caspase-4 and CHOP were determined by the Western blot technique. MTT data showed that tanshinone IIA could significantly inhibit the proliferation of Ec-109 cells with a dose- and time-dependent mode. Compared with the control group, tanshinone IIA could apparently induce apoptosis of Ec-109 cells, and the level of Caspase-4 and CHOP (p < 0.01) obviously increased. Tanshinone IIA can significantly induce the apoptosis of Ec-109 cells, which may take effect by the stress pathway of the endoplasmic reticulum.

Oxymatrine inhibited cell proliferation by inducing apoptosis in human lung cancer A549 cells.

To investigate the inhibition effect of oxymatrine induces human lung cancer A549 cells apoptosis. The A549 cells were cultured for 24 h, than the various concentration of oxymatrine (2 mmol/L, 4 mmol/L, 8 mmol/L, 15 mmol/L) were added into different experimental group cells, and 5-fluorouracil were added into the positive control group cells for 12 h, 24 h, 36 h, 48 h respectively. The A549 cells inhibition rate, apoptosis, and the expression of Bcl-2 and Bax were examined by MTT method, Annexin V/PI double staining method, real-time quantitative PCR and western blot, respectively. At same time, the morphological changes of A549 cells were observed with an inverted microscope. In the range of 2 mmol/L~15 mmol/L, oxymatrine had obvious inhibition effects on the proliferation of A549 cells. Compared with the negative control group, it has significantly different (P<0.01). There was remarkably the time- and dose-dependent correlation. After A549 cells were treated with 8 mmol/L oxymatrine for 24 h, the morphological change of cell apoptosis was observed and the extent of apoptosis was quantified by flow cytometry. Furthermore, the expression of Bcl-2 was reduced and the expression of Bax was increased remarkably (P<0.05). Oxymatrine has significant inhibition effects on the cells proliferation and the effects showed time-dependent and dose-dependent. Oxymatrine can induce apoptosis of the A549 cells by regulating the expression of Bcl-2 and Bax.

Reactive oxygen species (ROS) accumulation induced by mononaphthalimide-spermidine leads to intrinsic and AIF-mediated apoptosis in HeLa cells.

Developing polyamine conjugates having the potential of transporting naphthalimide selectively into tumor cells is attractive. However, the evaluation of their cytotoxic mechanism has not been comprehensive. This study focused on the effects of mononaphthalimide spermidine (MNISpd) conjugate on apoptosis induction and the relationship between MNISpd-induced apoptosis and reactive oxygen species (ROS) in HeLa cells. Our findings indicated that 9 µM MNISpd induced apoptosis in HeLa cells during a 48-h period. MNISpd induced apoptosis in HeLa cells following cytochrome c release, elevation of caspase 3/9 activity, apoptosis-inducing factor (AIF) translocation and up-/down-regulation of Bax/Bcl-2 protein expression, respectively, and these effects were completely antagonized by pre-incubation with 10 mM NAC for 2 h. MNISpd induced significant ROS accumulation following up-regulation of polyamine oxidase (PAO) activity and complex variations in glutathione levels. It is concluded that MNISpd-induced apoptosis is related to intrinsic caspase-dependent and AIF-mediated caspase-independent apoptosis pathways in HeLa cells. MNISpd-induced apoptosis correlates to MNISpd-induced ROS production resulting from GSH (reduced form of glutathione) pool depletion, and PAO is likely to be the source of ROS.

Synthesis and biological evaluations of novel bendazac lysine analogues as potent anticataract agents.

Novel bendazac analogues and their salts have been designed and prepared. The resulting compounds (13c-d, 15c, 17c) showed very good aqueous solubility (>100 mg/mL). An in vitro assay showed that most of the resulting compounds had potent protective activity against the oxidative damage. Particularly, compound 13d was also able to enhance the WSP and T-AOC level in the H(2)O(2)/FeCl(3)-induced oxidative damage model, indicating the resulting compound may protect the lens through an antioxidant pathway.

[Molecular cloning of an amidase gene from Nocardia sp. and its expressionin Escherichia coli].

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.