Establishment of a multiplex polymerase chain reaction detection assay for three common harmful microalgae in the East China Sea.

It is urgent to develop techniques that can simultaneously detect multiple microalgae, due to the diversity of harmful algal blooms (HABs)-forming algal species. The target algae species in this study are Heterosigma akashiwo, Prorocentrum donghaiense and Karenia mikimotoi. These algae are the dominant species that cause HABs in the East China Sea, and the multiple detection technique focusing on these three algae is not common. Therefore, this study established a multiplex polymerase chain reaction(mPCR) to diagnose the three algae, which is simple and low cost. First, the corresponding specific primers were designed based on the D1-D2 region of the large subunit (LSU) ribosomal DNA sequence. Then, mPCR was established and the reaction conditions were optimized. And the specificity, sensitivity, and stability of mPCR were evaluated. The result of specificity test showed that the established mPCR had good specificity for the target microalgae and did not cross-react with eighteen non-target microalgae. The sensitivity of experiment was 3.3 × 10-1 ng µL-1, and the established mPCR was not affected by the interfering microalgae. Moreover, the practicability evaluation of mPCR by using the simulated natural water samples showed that the detection limit of target microalgae was 100 cells mL-1, which could meet the demand for early warning of HABs. In summary, the established mPCR is characterized by strong specificity, good stability, and multiple analysis to detect H. akashiwo, P. donghaiense, and K. mikimotoi.

The complete mitochondrial genome of Pethia padamya (Actinopteri, Cyprinidae).

Pethia padamya (Kullander and Britz, 2008) is a freshwater fish distributed in the Mekong River basin of Thailand. It has beautiful colors and can be used as an ornamental fish. The complete mitochondrial genome of P. padamya was determined using next-generation sequencing technology and its characteristics were analyzed. The mitochondrial genome is a closed circular molecule comprising 16,792 bp, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a major non-coding region. The overall base composition of the mitochondrial genome is 32.47% A, 25.39% C, 26.08% T, and 16.06% G, with a high A + T bias of 58.55%. Phylogenetic analysis revealed P. padamya as a sister group of Pethia conchonius+(Pethia ticto+Pethia cumingii) and Pethia gelius with maximal support, providing support for the monophyly of the genus Pethia based on concatenated nucleotide sequences. The results of this study proved the monophyly of the genus Pethia. These data for the first time provide information on the complete mitochondrial genome of P. padamya and can contribute to further studies on the biodiversity and management of P. padamya.

The Clinical Significance of Serum IL-33 and sST2 Alterations in the Post-Stroke Depression.

INTRODUCTION: This study was to test whether the serum levels of IL-33 and sST2 are correlated with the development of depression after acute ischemic stroke. METHODS: Patients diagnosed with acute ischemic stroke were selected. This study took the 24-item Hamilton Depression Rating Scale (HAMD) (score ≥20) as the diagnostic criteria for depression. On the 21st day after admission, patients who met the depression diagnostic criteria were included in the depression group, and patients who failed to meet the diagnostic criteria were included in the non-depression group. The serum levels of IL-33, sST2 and hsCRP were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: On 1st day after stroke, compared with the non-depression group, there was no significant difference in the serum IL-33, sST2 and hsCRP levels in the depression group; on 21st day after stroke, compared with the non-depression group, the serum IL-33 and hsCRP levels were significantly increased, while the sST2 level was significantly decreased in the depression group. Correlation analysis showed that IL-33 was positively correlated with the depression quantitative score and hsCRP, while sST2 was negatively correlated with the depression quantitative score and hsCRP. Regression analysis showed that IL-33 and sST2 were independent risk factors for the depression after acute ischemic stroke. DISCUSSION: The abnormal alterations of serum IL-33 and sST2 levels in the stroke patients may serve as one of the risk factors for the occurrence and exacerbation of the depression, and its mechanism may be related to the promotion of inflammatory factor production in vivo.

Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation.

N6-methyladenosine (m6A) RNA modification regulates multiple biological functions. Methyltransferase like 3 (METTL3), one of the major N6-methyltransferases, is highly expressed in gastric cancer, but its potential role in disease is unclear. The current study knocked out METTL3 (METTL3-KO) in human gastric cancer AGS cells using CRISPR/Cas9. METTL3-KO AGS cells exhibited decreased m6A methylation levels. A significant inhibition of cell proliferation was observed in METTL3-KO AGS cells. Silencing METTL3 in AGS cells altered the expression profile of many effector molecules that were previously demonstrated to serve key roles in AGS cell proliferation, including the suppressor of cytokine signaling (SOCS) family of proteins. The results further demonstrated that SOCS2 upregulation in METTL3-KO AGS cells was associated with a decreased RNA decay rate. Furthermore, SOCS2 KO or SOCS2 overexpression caused a significant increase and decrease in AGS cell proliferation, respectively. The current data suggested that METTL3-KO in gastric cancer cells resulted in the suppression of cell proliferation by inducing SOCS2, suggesting a potential role of elevated METTL3 expression in gastric cancer progression.

A heavy metal module coupled with the SWAT model and its preliminary application in a mine-impacted watershed in China.

Heavy metal behavior in soil and water requires modeling for a better understanding of the potential adverse impacts on the ecosystem as well as on humans. A heavy metal transport and transformation module is combined with the well-established SWAT (Soil and Water Assessment Tool) model for the purpose of simulating the fate and transport of metals at the watershed scale. The heavy metal module accounts for sorption, complexation and slow reactions among metal species; the heavy metals in the upland are allowed to transport vertically through percolation and evaporation-induced water rising as well as horizontally through soil erosion and surface/subsurface runoff; the heavy metals in the water body, in contrast, are modeled to undergo settling, resuspension, diffusion and burial processes. As a demonstration, the SWAT-laden heavy metal module (SWAT-HM) was calibrated to simulate zinc (Zn) and cadmium (Cd) dynamics in an upstream watershed of the Liuyang River in China, which has been impacted by mining activities for decades. The model simulations were found to agree reasonably well with the monitored results. In particular, the elevation of metal loads in channels with precipitation events was well represented, demonstrating that a considerable amount of Zn and Cd in the waste rock dumps and contaminated soil was released into rivers through rainfall. After a simulation of 6years (2009-2014), the simulated Zn and Cd concentrations were used as a surrogate for the Predicted Environmental Concentration (PEC), whereby an ecological risk assessment was conducted for the demonstrative mining area. This initiative toward developing a heavy metal module combined with SWAT has high potential for application in environmental risk analysis and pollution control.

The effect of L-thyroxine substitution on oxidative stress in early-stage diabetic nephropathy patients with subclinical hypothyroidism: a randomized double-blind and placebo-controlled study.

PURPOSE: To study oxidative stress status of early type 2 diabetic nephropathy (DN) patients with subclinical hypothyroidism (SCH) and to assess effect of L-thyroxine therapy on the oxidative stress in these patients. METHODS: It is a randomized double-blind and placebo-controlled trial. A total of 48 patients with early type 2 DN were included as Euthyroid group, and 92 early type 2 DN with SCH were selected and randomly assigned to L-thyroxine treatment group (LT4 group) and placebo group (SCH group). Changes in urinary albumin excretion rate (UAER), serum malondialdehyde (MDA), superoxide dismutase (SOD) activity, urine 8-hydroxyl deoxyguanosine (8-OHdG), serum creatinine, estimated glomerular filtration rate, and lipid profile before and after 24 weeks of follow-up were examined and compared. RESULTS: The levels of UAER, MDA, 8-OHdG were higher, while the SOD activity was lower in DN patients with SCH than in DN patients (p < 0.05 for all). In the LT4 group, the levels of UAER, MDA, 8-OHdG decreased significantly (p < 0.05) to levels no longer different from the Euthyroid group. The SOD activity increased significantly. But in SCH group, the levels of mAlb, MDA, 8-OHdG were greater after 24 weeks of follow-up and greater than patients in the Euthyroid group. SOD activity decreased significantly after 24 weeks in the SCH group (p < 0.05). CONCLUSION: Oxidative stress is greater in the DN patients with SCH, and SCH may exacerbate kidney injury in early DN patients. The LT4 treatment may decrease the oxidative stress and attenuate renal injury in DN patient with SCH.

Dune-scale distribution pattern of herbaceous plants and their relationship with environmental factors in a saline-alkali desert in Central Asia.

Sand dunes are the main landforms in modern deserts worldwide. As an important type of desert vegetation, herbaceous plants have long been acknowledged for their important ecological functions. We focus on the dune-scale distribution pattern of herbaceous plants and their relationship with the environment. During the early summer of 2010, the herbaceous plant density, coverage, aboveground biomass and eighteen environmental factors were surveyed from 214 plots (5m×5m) across two linear sand dunes (220m×110m) located in the Gurbantunggut Desert, China, a typical saline-alkali desert in Central Asia. Data were analyzed using univariate, ordination, and geostatistical techniques. Three vegetation variables represent a significant clumped distribution (P<0.01) across the entire study site. Obvious differences were observed among different slope positions/transects, with the lowest values observed at the summit. Geostatistics indicated that all of the variables showed a moderate spatial dependence and obvious zonal distribution along the sand dunes. The ranges (lag distance) of density (32.2m) and coverage (33.5m) were close to the average width (31.4m) of the transects. The biomass range (74.1m) was almost equal to the average width (75m) of the sand dunes. Pearson's correlation analysis and nonmetric multidimensional scaling analysis consistently demonstrated that the distributions of herbs on sand dunes were dominantly influenced by sand dune topography (slope position, convexity, and relative height), soil nutrients (total nitrogen and phosphorus) and deep-rooted shrubbery (Ephedra distachya). Compared to large, mobile and steep sand dunes, low, fixed and gentle sand dunes contribute to herbaceous plant abundance and distribution as well as the stability maintenance of the whole desert ecosystem.

Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while potently inhibiting estradiol-3-O-glucuronidation.

This in vitro study investigates the effects of diethylstilbestrol (DES), a widely used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O) glucuronidation, via culturing human liver microsomes (HLMs) or recombinant UDP-glucuronosyltransferases (UGTs) with DES and E2. DES can potently inhibit E2-3-O-glucuronidation in HLM, a probe reaction for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive inhibition mechanism, with the Ki value of 2.1±0.3µM, which is less than the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation, the acceleration is observed on E2-17-O-glucuronidation in HLM, in which cholestatic E2-17-O-glucuronide is generated. In the presence of DES (0-6.25µM), Km values for E2-17-O-glucuronidation are located in the range of 7.2-7.4µM, while Vmax values range from 0.38 to 1.54nmol/min/mg. The mechanism behind the activation in HLM is further demonstrated by the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing E2-17-O-glucuronidation. The presence of DES (2µM) can elevate Vmax from 0.016 to 0.81nmol/min/mg, while lifting Km in a much lesser extent from 4.4 to 11µM. Activation of E2-17-O-glucuronidation is well described by a two binding site model, with KA, α, and ß values of 0.077±0.18µM, 3.3±1.1 and 104±56, respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation are not observed in liver microsomes from several common experimental animals. In summary, this study issues new potential toxic mechanisms for DES: potently inhibiting the activity of UGT1A1 and powerfully accelerating the formation of cholestatic E2-17-O-glucuronide by UGT1A4.

[Analysis of volatile compounds of fresh tea leaves from yaoluoping nature preserve by SDE-GC-MS].

OBJECTIVE: To study the volatile compounds of fresh tea leaves from Yaoluoping Nature Preserve, and to provide scientific basis for the quality and medicinal value of tea from high mountainous area. METHODS: The volatile compounds were extracted from fresh tea leaves by simultaneous distillation and extraction, and analyzed by gas chromatography-mass spectrum (GC-MS). RESULTS: 41 volatile compounds were identified from two tea cultivars, Shifoxiang and Shifocui. Shifoxiang had 32 kinds of volatile compounds and Shifocui had 38 kinds of volatile compounds. The main volatile components of Shifoxiang and Shifocui were green leaf volatiles. The kind and relative content of terpenes of Shifocui were more than that of Shifoxiang. Also the kind and relative content of ketones of Shifocui were more than that of Shifoxiang. There was one kind of heterocyclic compound of Shifocui. CONCLUSION: The main volatile components of Shifoxiang and Shifocui had an important role for the formation of unique aroma of high mountain ous tea. The composition and proportion of volatile components between Shifoxiang and Shifocui had certain differences. Some trace components may be associated with defense in the process of tea plant growth. The functional components from the volatile components had good medicinal value, worthy of further research.

Benidipine protects kidney through inhibiting ROCK1 activity and reducing the epithelium-mesenchymal transdifferentiation in type 1 diabetic rats.

We investigated the protective effect of benidipine, by testing the changes of the activity of Rho kinase and transdifferentiation of renal tubular epithelium cells in vivo. Wistar rats were randomly divided into two groups: normal (N) and diabetes. STZ were used to make the rats type 1 diabetic and were randomly assigned as diabetes without treatment (D), diabetes treated with benidipine (B), and diabetes treated with fasudil (F) and treated for 3 months. Immunohistochemistry and western blotting were for protein expressions of ROCK1, α-SMA, and E-cadherin and real-time PCR for the mRNA quantification of ROCK1. Compared with N group, D group had significant proliferation of glomerular mesangial matrix, increased cell number, thickened basement membrane, widely infiltrated by inflammatory cells and fibrosis in the renal interstitial, and dilated tubular. Those presentations in F and B groups were milder. Compared with N group, D group showed elevated MYPT1 phosphorylation, increased expression of ROCK1, α-SMA protein, and ROCK1 mRNA and decreased expression of E-cadherin protein. B group showed attenuated MYPT1 phosphorylation, decreased ROCK1, α-SMA protein, and ROCK1 mRNA expression and increased expression of E-cadherin protein. In conclusion, benidipine reduces the epithelium-mesenchymal transdifferentiation and renal interstitial fibrosis in diabetic kidney by inhibiting ROCK1 activity.

The influence of fasudil on the epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells from diabetic rats.

To investigate the influence of fasudil on the epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells from diabetic rats and explore the mechanisms of this effect. Wistar rats were randomly divided into the following three groups: control, diabetes and fasudil-treatment. All rats were sacrificed after three months of feeding with or without fasudil treatment. Pathological changes to the glomeruli and renal interstitium were studied using Periodic acid-Schiff's staining and Masson staining, respectively. Expression of ROCK1, alpha-SMA, E-cadherin and the distribution of beta-catenin in rat renal cortex were revealed by immunohistochemistry. Changes in the MYPT1 phosphorylation profile and alpha-SMA, E-cadherin and membrane beta-catenin expression were revealed by western blot. Changes in the levels of ROCK1, E-cadherin and total beta-catenin mRNA expression were analyzed by real-time PCR. Fasudil treatment notably attenuates renal interstitial fibrosis in diabetic rats. Compared to the control rats, diabetic rats showed elevated phosphorylation of MYPT1, increased expression of ROCK1 and alpha-SMA, decreased expression of E-cadherin and membrane beta-catenin, and increased expression of ROCK1 and total beta-catenin mRNA, decreased expression of E-cadherin mRNA. Fasudil treatment of diabetic rats resulted in attenuated MYPT1 phosphorylation, decreased ROCK1 and alpha-SMA expression, increased E-cadherin and membrane beta-catenin expression, and reduced ROCK1 and total beta-catenin mRNA expression, increased expression of E-cadherin mRNA. In conclusion, fasudil may reduce the epithelial-mesenchymal transdifferentiation and renal interstitial fibrosis in diabetic rats through a mechanism by which ROCK activity is inhibited, which further facilitates the recovery of the cell-cell adhesions among renal tubular epithelial cells and adhesion complex formation.

A novel fluorescent distinguished probe for Cr (VI) in aqueous solution.

Salicylaldehyde rhodamine B hydrazone (SRBH) was developed as a new spectrofluorimetric probe for the selective and sensitive detection of CrO(4)(2-) in acidic conditions. The proposed method was based on the special oxidation reaction between non-fluorescent SRBH by potassium dichromate to produce a highly fluorescent rhodamine B, as a product. Under the optimum conditions described, the fluorescence enhancement at 591 nm was good linearly related to the concentration of CrO(4)(2-) from 1.0 x 10(-8) to 3.0 x 10(-7)M (0.42-12.6 ng mL(-1)) with a correlation coefficient of R(2)=0.9989 (n=10) and a detection limit of 1.5 x 10(-9)M (0.063 ng mL(-1)). The relative standard deviation (R.S.D.) was 2.0% (n=6). The proposed method was also successfully applied to the determination of chromium (VI) in drinking water, river water and synthetic samples.