[Determination of glutathione in cells by capillary electrophoresis-laser induced fluorescence].

Glutathione (GSH) is vital for oxidative stress resistance and heavy metals detoxification. It is significant to develop a sensitive and accurate quantitative GSH approach for the toxicity mechanism for studying heavy metals in cells. A high-sensitive capillary electrophoresis-laser induced fluorescence (CE-LIF) detection approach was proposed in this study to detect GSH content in cells. The approach employed HepG2 cells as an object and 2,3-naphthalenedicarboxaldehyde (NDA) with the active group of aromatic o-dialdehyde as a labeling reagent. The effects of buffer solution types, pH, additives on the GSH reaction rate with NDA, and the sensitivity of NDA-GSH were systematically investigated. The sensitivity of NDA-GSH and the reaction rate of GSH with NDA were compared in tris(hydroxymethyl)aminomethane (Tris) buffer solution at pH 7.4 or 9.2 and borate-Tris buffer solution at pH 9.2. The results revealed that the NDA-GSH sensitivity was the highest and the reaction rate of GSH and NDA was the fastest in borate buffer solution at pH 9.2. The effects of the four additives on the sensitivity of NDA-GSH were further compared. The best additive was revealed to be ß-cyclodextrin (ß-CD). GSH reacted with NDA to reach equilibrium within 5 min under the optimal experimental conditions, and the electrophoretic signal of NDA-GSH could be seen in 3 min. Quantitative analysis of GSH in HepG2 cells was performed using an external standard approach by determining a series of GSH standard solutions. The results revealed that the approach had a good linear relationship with the peak area vs. concentration (0.01-20.00 mmol/L) of GSH. The limit of detection (LOD) and limit of quantification (LOQ) of GSH were determined using signal-to-noise ratios of 3 (S/N=3) and 10 (S/N=10), which were 0.006 µmol/L and 0.020 µmol/L, respectively. The approach's spiked recoveries were 95.7%-112.6%, with relative standard deviations of the approach being 3.8%-5.0% (n=3). This approach offers high sensitivity, good stability, accuracy, and reliability. To study the relationship between the toxicity of arsenic and chromium on HepG2 cells and the content of GSH in HepG2 cells, the effects of arsenic and chromium with different valences on cell viability were analyzed. The results illustrated that the cytotoxicity of potassium dichromate (Cr(Ⅵ)) was the strongest. The variations of GSH content in HepG2 cells stimulated with arsenite (As(Ⅲ)), arsenate (As(Ⅴ)), chromium chloride (Cr(Ⅲ)), and Cr(Ⅵ) were analyzed by the proposed approach and analysis of intracellular GSH imaging. The results revealed that the stimulation group i. e. analyzed doses (low-dose 2 mg/L, high-dose 5 mg/L) of As(Ⅲ), As(Ⅴ), and Cr(Ⅲ) had no obvious effect on GSH content in HepG2 cells compared with the control group, whereas high-dose Cr(Ⅵ) can significantly reduce GSH content in HepG2 cells. Considering the analysis of cytotoxicity of As(Ⅲ), As(Ⅴ), Cr(Ⅲ), and Cr(Ⅵ), it shows that the content of GSH in HepG2 cells is related to cytotoxicity, and the content of GSH will decrease with the increase in cytotoxicity.

Investigation on selenium and mercury interactions and the distribution patterns in mice organs with LA-ICP-MS imaging.

It is the first time to investigate local distribution patterns of mercury (Hg) in mice organs after Hg and Se exposure with detection of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Two batch of adult mice were employed to be exposed to inorganic mercury (iHg) and methylmercury (MeHg) with or without Se at the dose of 55 µmol kg-1. Tissue sections of brain, kidney, liver, and spleen from one batch mice were prepared to get local imaging of Hg by LA-ICP-MS. Tissues from another batch mice were used to quantify Hg and Se in tissues with ICP-MS after acid digestion. The results indicated that, for mice exposed to iHg, Hg mainly distributed in kidney, a little in liver, and hardly in brain and spleen; for mice exposed to MeHg, lower amount of Hg was found in kidney, liver and spleen, and almost no Hg was found in brain. It was interesting that for Hg and Se co-administration groups, higher level of Hg was observed in kidney, liver, spleen and even in brain than single Hg administration groups. In addition, Se level in organ tissues increased obviously not only in Se exposure group but also in MeHg exposure group, while the phenomenon was not observed in iHg exposure group. HepG2 cells were employed to investigate Se and Hg interactions in single cell level, similar bioaccumulation behavior of Hg was found between cells and mice organs. Higher level of Hg was observed in cells cultured with Se and Hg medium than cells cultured with single Hg medium. The results are expected to provide new insight to investigate Hg and Se interactions in animal bodies and in-vitro cells.

Transcriptomic study of the mechanism by which the Kai Yu Zhong Yu recipe improves oocyte quality in a stressed mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The Kai Yu Zhong Yu recipe (KYZY) is a classic herbal formula in traditional Chinese medicine (TCM) that has been used to treat infertility associated with psychological stress for more than three hundred years. AIM OF THE STUDY: Psychological stress has major impacts on fertility, with variable outcomes depending on the nature, strength, and duration of the stress. Stress can directly disturb ovulation, oocyte quality, maturation, and embryo development. The aim of this study is to investigate the molecular mechanism by which KYZY improves oocyte developmental potential under psychological stress. MATERIALS AND METHODS: ICR female mice aged 4-5 weeks were randomly divided into five groups: control, stressed in the chronic unpredictable stress model (CUSM), and stressed plus KYZY treatment at 38.2 g/kg (KYZYH), 19.1 g/kg (KYZYM), or 9.6 g/kg (KYZYL). Ovary function was assessed by measuring serum levels of estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH). Oocyte quality was evaluated in terms of reactive oxygen species (ROS) levels, apoptotic DNA fragmentation, and mitochondria distribution. We used RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) between groups and then further analyzed the DEGs for gene ontology (GO) term enrichment and protein-protein interactions. RESULTS: Mice in the stressed group had reduced serum E2, LH, and FSH as well as increased ROS levels, increased apoptosis, and disturbed mitochondria distribution in oocytes. Treatment with KYZY at all three doses reversed or ameliorated these negative effects of stress. DEG analysis identified 187 common genes between the two comparisons (stressed vs. control and KYZYM vs. stressed), 33 of which were annotated with six gene ontology (GO)'s biological process (BP) terms: cell differentiation, apoptosis, ATP synthesis, protein homo-oligomerization, neuron migration, and negative regulation of peptidase activity. Protein-protein interaction network analysis of DEGs identified key hub genes. Notably, the genes Atp5o and Cyc1 were both involved in the ATP synthesis and among the top three hub genes, suggesting that regulation of oocyte mitochondrial electron transport and ATP synthesis is important in the response to stress and also is a possible mechanism of action for KYZY. CONCLUSIONS: KYZY was effective in ameliorating the adverse effects of stress on oocyte competence, possibly by targeting the mitochondrial respiratory chain and ATP synthase.

DMSA-Functionalized Mesoporous Alumina with a High Capacity for Selective Isolation of Immunoglobulin G.

A novel dimercaptosuccinic acid-functionalized mesoporous alumina (DMSA-MA) is synthesized by the dicarboxylic acid groups of dimercaptosuccinic acid molecules coordinating to the Al3+ ions located in the mesostructure. The as-prepared DMSA-MA composites possess a large surface area of 91.17 m2/g as well as a uniform pore size and a high pore volume of 17.22 nm and 0.23 cm3/g, respectively. DMSA coating of mesostructures significantly enhanced their selectivity for glycoprotein adsorption through a powerful hydrophilic binding force, and the maximum adsorption capacity of immunoglobulin G (IgG) can reach 2298.6 mg g-1. The captured IgG could be lightly stripped from the DMSA-MA composites with an elution rate of 98.3% by using 0.5 wt % CTAB solution as the elution reagent. DMSA-MA is further employed as a sorbent for the enrichment of IgG heavy chain and light chain from human serum sample. SDS-PAGE assay results showed the obtained IgG with high purity compared to that of the standard solution of IgG.

Thermo/pH dual-stimuli-responsive drug delivery for chemo-/photothermal therapy monitored by cell imaging.

A thermo/pH dual-stimuli-responsive drug delivery system (DDS) based on polymer coated mesoporous silica nanostructures (MSNs) is developed for facilitating chemotherapy and photothermal therapy. Thermo/pH-responsive polymer, poly((N-isopropylacrylamide, NIPAM)-co-methacrylic acid, MA), is grafted onto MSNs by in situ polymerization, followed by loading a chemotherapeutic drug (doxorubicin hydrochloride, DOX) and a near-infrared-absorbing phototherapeutic agent (indocyanine green, ICG) to construct the intelligent drug delivery system, shortly as DOX-ICG-MSN@p(NIPAM-co-MA). At NIR irradiation, the photothermal conversion capability of ICG raises the temperature of the DDS and opens the gatekeeper by shrinkage of the copolymer p(NIPAM-co-MA), which triggers controlled release of DOX at an elevated temperature. On the other hand, drug release is also realized at pH 5.3, a characteristic pH value in cancer cell microenvironment, at which it not only causes the shrinkage of the pH-sensitive polymeric moiety of methacrylic acid in MSN@p(NIPAM-co-MA) but also deteriorates electrostatic interaction of DOX molecules in the mesoporous channel by protonation of silanols. In addition, ICG further ensures photothermal therapy (PTT) and photodynamic therapy (PDT). The cytotoxicity assay of HeLa cells shows obvious synergistic effect by demonstrating that the combined use of DOX and ICG is more effective in killing HeLa cells than free DOX and ICG. The endocytosis of the drug is monitored by cell imaging.

[Selection and purification potential evaluation of woody plant in vertical flow constructed wetlands in the subtropical area].

In order to solve the problem that wetland herbaceous plants tend to die during winter in subtropics areas, selection and purification potential evaluation experiments were carried out by introducing into the constructed wetlands 16 species of woody wetland plants. Cluster analysis was performed by including the morphological characteristics, physiological characteristics, as well as nitrogen and phosphorus accumulation of the woody wetland plants. The results indicated that there were significant differences among the tested woody plants in their survival rate, height increase, root length increase and vigor, Chlorophyll content, Superoxide dismutase, Malonaldehyde, Proline, Peroxidase, biomass, average concentration and accumulation of nitrogen and phosphorus. Based on the established evaluation system, the tested plants were clustered into 3 groups. The plants in the 1st group possessing high purification potentials are Nerium oleander and Hibiscus syriacus. Those in the 2nd group possessing moderate purification potentials are Trachycarpus fortune, Llex latifolia Thunb., Gardenia jasminoides, Serissa foetida and Ilex crenatacv Convexa. And those in the 3rd group with low purification potentials are Jasminum udiflorum, Hedera helix, Ligustrum vicaryi, Ligustrum lucidum, Buxus sempervives, Murraya paniculata, Osmanthus fragrans, Mahoniafortune and Photinia serrulata.

Selenium adsorption and speciation with Mg-FeCO3 layered double hydroxides loaded cellulose fibre.

A novel adsorbent was developed by coating Mg-FeCO(3) layered double hydroxides (LDHs) on cellulose fibre. The LDHs take up significant amount of selenite and selenate in a wide pH range with similar sorption capacities (pH 3.8-8.0 for selenite and pH 5.8-7.0 for selenate). A mini-column packed with Mg-FeCO(3) LDHs layer coated cellulose fibre particles was incorporated into a sequential injection system for uptake of selenite at pH 6.0. The retained selenite was afterwards collected with 70 µ L of 0.8%(m/v) NaOH as eluent, followed by hydride generation and atomic fluorescence spectrometric detection. Total inorganic selenium was adsorbed at pH 6.0 by the LDHs-cellulose fibre mini-column after selenate was pre-reduced to selenite by 2.0 mol L(-1) HCl at 80°C, and selenium speciation was performed by difference. With a sample volume of 1.0 mL, an enrichment factor of 13.3 was derived with a detection limit of 11 ng L(-1) within a linear range of 0.04-4.0 µg L(-1). A relative standard deviation (RSD) of 3.3% (0.5 µg L(-1), n=11) was achieved. The procedure was validated by analyzing selenium in a certified reference material GBW 10010 (rice), and speciation of inorganic selenium in natural water samples.

Thiolated eggshell membranes sorb and speciate inorganic selenium.

Eggshell membranes (ESMs) provide a unique, disulfide bond-rich surface. Thioglycolate reduction was used to generate thiol (-SH) groups on the ESM surface by S-S bond cleavage. The thiol-bearing ESMs (TESMs) were characterized by scanning electron microscopy and Raman spectroscopy. The fibrous network structure of the ESM is retained in the TESMs. TESMs adsorb both Se(IV) and Se(VI) but by different mechanisms: Se(VI) is retained reversibly, possibly via ionic interactions, while Se(IV) is reduced to Se(0) and deposited. We thus demonstrate speciation of selenium species, by using samples (a) as such and after prior oxidation to Se(VI), (b) preconcentration on a TESM microcolumn, (c) elution by 0.5 M HNO(3) that only elutes Se(VI) and (d) detection by graphite furnace atomic absorption spectrometry (GFAAS). The Se(IV) amount is determined by difference. For a 1.0 mL sample, the enrichment factor was 17.2, the S/N = 3 detection limit was 0.06 µg L(-1) and the precision was 3.3% at 0.50 µg L(-1). The linear range was 0.25-2.50 µg L(-1). The procedure was validated by analyzing selenium in certified reference materials of human hair (GBW 09101) and rice (GBW 10010). We further demonstrate utility by speciation of inorganic selenium in a series of water samples.

[Selection of winter plant species for wetlands constructed as sewage treatment systems and evaluation of their wastewater purification potentials].

In order to establish an evaluation system for selection of winter wetland plants possessing high wastewater purification potentials in subtropics areas, designed sewage treatment experiments were carried out by introducing into the constructed wetlands 25 species of winter wetland plants. Cluster analysis was performed by including harmful environment-resistant enzyme and substrate enzyme activities into the commonly applied plant screening and assessment indexes system. The obtained results indicated that there were significant differences among the tested winter plants in their root length and vigor, leaf malonaldehyde (MDA), biomass, average nitrogen and phosphorus concentration and uptake, and urease and phosphoric acid enzyme activities in the root areas. Based on the established evaluation system, the tested plants were clustered into 3 groups. The plants in the 1st group possessing high purification potentials are Oenanthe javanica, Brassicacapestris, Juncus effusu, Saxifragaceae, Iris pseudoacorus, Osmanthus fragrans and Iris ensata; those in the 2nd group possessing moderate purification potentials are Brassica oleracea var acephala, Calendula officinalis, Aucuba japonica, Ligustrum lucidu, Beta vulgaris, Rhododendron simsii and Ilex latifolia; and those in the 3rd group with low purification potentials are Brassica oleracea var acephala, Calistephus chinensis, Rosa chinensis, Antirrhinums, Liriope palatyphylla, Zephyranthes candida, Fatshedera lizei, Petunia hybrida, Ilex quihoui, Dianthus caryophyllus and Loropetalum chinensis.

Precipitate coating on cellulose fibre as sorption medium for selenium preconcentration and speciation with hydride generation atomic fluorescence spectrometry.

Lanthanum hydroxide precipitate is for the first time coated onto cellulose fibre and serves as a novel sorption medium for separation and speciation of inorganic selenium. A micro-column packed with precipitate-layer-coated cellulose fibre is incorporated into a sequential injection system for selenite retention from a neutral aqueous solution, which is afterwards stripped with a NaBH(4)-NaOH solution as eluent. The hydride generation is actuated by merging the eluate and hydrochloric acid downstream, followed by the detection with atomic fluorescence spectrometry. Total inorganic selenium is derived by pre-reduction of selenate and speciation is estimated by difference. The coated precipitate layer can be used for 150 runs for selenium sorption, offering a clear advantage over the conventional precipitation protocols where a large amount of precipitate is dissolved into a small volume of eluent which might interfere with the detection. With a sample volume of 1.0 mL, an enrichment factor of 9.7 and a detection limit of 9 ng L(-1) are obtained in a linear range of 0.05-2.5 microg L(-1). A sampling frequency of 24 h(-1) is achieved along with a R.S.D. of 1.7% at 0.5 microg L(-1) Se(IV). The procedure is validated by analyzing selenium in a reference material GBW 10010 (rice) and a human hair sample. It is further demonstrated by speciation of inorganic selenium in surface water samples by pre-reduction of selenate.