Risk assessment of broflanilide for human and non-target terrestrial organisms in cauliflower production.

Broflanilide is a newly-developed meta-diamide insecticide, proposed for the control of a wide variety of chewing pests on many crops. In view of the proposed use of broflanilide and its environmental fate, it may be exposed to consumers and non-target organisms, which adversely affect human and the environment. In this paper, a rapid, sensitive and valid UPLC-MS/MS method was established for simultaneous analysis of broflanilide and its two major metabolites, DM-8007 and S (PFP-OH)-8007, in cauliflower. Then, the dissipation behaviors and final residues of broflanilide and its two major metabolites in cauliflower from eight sites with different climatic conditions in China were studied via the described analytical method. In addition, the acute toxicity test of 9.5 % suspension concentrate of broflanilide, broflanilide standard, DM-8007 and S (PFP-OH)-8007 were conducted to non-target terrestrial organisms. Risk assessment for human and non-target terrestrial organisms in cauliflower production was evaluated based on the maximum annual application rates and intervals. The results showed that the highest residue of broflanilide detected in cauliflower samples was all lower than the corresponding MRLs (2 mg/kg) in Japan. Chronic food dietary risk estimates for broflanilide do not exceed 50 % for all the Chinese population groups. Moreover, broflanilide is of low acute toxicity to birds and earthworm, while broflanilide and its metabolites is classified as highly toxic to adult honeybees. Acute risks of broflanilide to birds and earthworms were deemed to be acceptable in a realistic worst-case scenario, while its risk to adult honeybees and ladybug was unacceptable. A protection statement for honeybees and ladybug is required to recognize the high toxicity of broflanilide on related product labels. The study will be conducive to provide guidance for the rational application of broflanilide in cauliflower production.

Dissipation and dietary exposure risk assessment of spinosad, thiocyclam, and its metabolite nereistoxin in cucumber and groundwater for different population groups.

A QuEChERS (quick, easy, cheap, effective, rugged, and safe) technique using ultrahigh-performance liquid chromatography with tandem mass spectrometry for the analysis of spinosad (spinosyn A + spinosyn D), thiocyclam, and nereistoxin in cucumber was developed with mean recoveries of 93-104%, relative standard deviations of ≤9%, and limits of quantification of 0.01 mg/kg. Field trials of spinosad and thiocyclam were performed in 12 representative cultivating areas in China. Field trial results indicate that spinosyn A and spinosyn D easily dissipated in cucumber with half-lives of 2.48-6.24 and <3 days, respectively. Nereistoxin was produced after thiocyclam application and was more persistent than its parent. The terminal residues of spinosad were all below the maximum residue limits (0.2 mg/kg) in China, whereas the terminal concentration of nereistoxin (calculated as the stoichiometric equivalent of thiocyclam), which was much higher than that of thiocyclam, was far beyond the maximum residue limits of thiocyclam in cucumber (0.01 mg/kg) established by the European Union. The predicted no-effect concentrations of spinosyn A, spinosyn D, thiocyclam, and nereistoxin leaching into groundwater were estimated using China-PEARL (Pesticide Emission Assessment at Regional and Local scales) models after application. However, the dietary (food and water) exposure risk quotient for different populations was below 1 with a preharvest interval set at 5 days after the last application, indicating that the application of spinosad and thiocyclam in cucumber was unlikely to pose unacceptable risk for human health. This study provides data for the safe use of spinosad and thiocyclam in cucumber ecosystem.

Autotoxicity of Endogenous Organic Acid Stress in Two Ganoderma lucidum Cultivars.

Ganoderma lucidum has been used as a rare medical mushroom for centuries in China, due to its health-promoting properties. Successive cropping obstacles are common in the cultivation of G. lucidum, although the remaining nutrients in the germ substrate are sufficient for a second fruiting. Here, we aimed to study the metabolite profile of G. lucidum via nontargeted metabonomic technology. Metabonomic data revealed that organic acids played an important role in the cropping obstacles of G. lucidum, which is accordance with the pH decrease in the germ substrate. A Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis indicated that most differential acids participated in the metabolic pathways. Five acids were all significantly upregulated by two MS with high energy (MSE) modes in two cultivars, among which 5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylic acid is also involved in purine metabolism regulation and microbial metabolism in diverse environments. Taken together, this work illustrated the organic acid stress generated by G. lucidum, which formed the autotoxicity feedback, and resulted in cropping obstacles. Determining the cause of the cropping obstacles in G. lucidum will promote the utilization rate of fungus substrate to realize the sustainable use of this resource.

Residue behavior and dietary risk assessment of spinetoram (XDE-175-J/L) and its two metabolites in cauliflower using QuEChERS method coupled with UPLC-MS/MS.

Spinetoram (XDE-175-J/L), a new spinosyn-based insecticide, is one of the most widely used bio-pesticide worldwide and its registration for direct application on cauliflower to control Plutella xylostella is currently under review in China. In this study, an accredited method for simultaneous determination of spinetoram and its two metabolites in cauliflower was established and validated using QuEChERS (quick, easy, cheap, effective, rugged, and safe) preparation coupled with ultra-liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The average recoveries using this method were ranged from 74 to 99% with relative standard deviations (RSDs) of 2.4-10.5%. The dissipation kinetics and terminal residues of spinetoram and its two metabolites in cauliflower were studied in Tianjin and Guizhou over two years under open field conditions. The dissipation experiments revealed that spinetoram was swiftly degraded in cauliflower, with the half-lives less than or equal to 4.85 days. The terminal residues of total spinetoram (sum of spinetoram and its two metabolites) detected in cauliflower samples were in the range of 0.009 mg/kg-0.337 mg/kg. Dietary risk assessment study was implemented based on the scientific data of field trials, food consumption and acceptable daily intake (ADI). The estimated long-term dietary risk probability (RQ) of total spinetoram from cauliflower was between 5.79% and 5.91%, indicating that spinetoram was associated with acceptable risk for dietary cauliflower consumption. The results would provide scientific guidance for proper usage of spinetoram in cauliflower field ecosystem.

Sorafenib induces autophagic cell death and apoptosis in hepatic stellate cell through the JNK and Akt signaling pathways.

Increasing hepatic stellate cell (HSC) death is an attractive approach for limiting liver fibrosis. We investigated the molecular mechanisms underlying the effects of sorafenib on HSCs. LX2 cells were incubated with sorafenib and a variety of inhibitors of apoptosis, autophagy, and necrosis. Electron microscopy was used to observe autophagosomes. Inhibitors and siRNA were used to examine the role of the Akt/mTOR/p70S6K and JNK pathways. Ultrastructural analysis revealed that rat HSCs treated with 5 µmol/l sorafenib accumulated residual digested material and empty or autophagic vacuoles. Incubating LX2 cells with lysosomal protease inhibitors increased the accumulation of LC3-II, indicating that sorafenib enhances autophagic flux in HSCs. Autophagy may precede apoptosis. Lower concentrations of sorafenib and a shorter treatment time resulted in the dominance of autophagic cell death over apoptosis. Further analysis showed that Beclin 1 is inactivated by the caspases induced by sorafenib during apoptosis. Inhibition of autophagy in LX2 cells using 3-methyladenine treatment or siRNA-mediated knockdown of Atg5 resulted in a marked increase in apoptosis. Finally, sorafenib induced programmed cell death by attenuation and activation of Akt/mTOR/p70S6K and JNK signaling. Sorafenib-induced cell death is mediated by both autophagy and apoptosis. Elucidation of the signaling pathways activated by sorafenib could potentially lead to novel antifibrosis therapies for chronic liver diseases.

Knockdown of Astrocyte Elevated Gene-1 Inhibits Activation of Hepatic Stellate Cells.

BACKGROUND: Astrocyte elevated gene-1 (AEG-1) is a positive regulator of tumorigenesis and a valuable prognostic marker of a diverse array of cancers, including liver cancer; however, the relationship between AEG-1 and hepatic fibrogenesis is not known. OBJECTIVE: The objective of this study was to explore the expression of AEG-1 during hepatic fibrogenesis and determine how AEG-1 regulates the profibrogenic phenotype of hepatic stellate cells (HSCs). METHODS: The levels of AEG-1 were monitored in the fibrotic livers and transforming growth factor-ß (TGF-ß)- or lipopolysaccharide (LPS)-stimulated HSCs. The expression of AEG-1 was knocked down by lentivirus-mediated short hairpin RNA in HSCs, and collagen expression, proliferation assays, apoptosis induction studies, and migration assays were simultaneously conducted in vitro. RESULTS: AEG-1 expression was increased in the fibrotic livers. At the cellular level, TGF-ß or LPS stimulation, which caused HSC activation, induced AEG-1 expression in HSC-T6 and primary rat HSCs (P < 0.05). Knockdown of AEG-1 inhibited collagen I and α-smooth muscle actin expression (P < 0.05), reduced cell proliferation (P < 0.05) and motility (P < 0.05), and induced cell apoptosis (P < 0.05) in HSCs. This antifibrotic effect caused by lack of AEG-1 was associated with the inactivation of PI3K/Akt and the mitogen-activated protein kinase pathway. CONCLUSIONS: Knockdown of AEG-1 suppressed the activation of HSCs by modulating the phenotype and inducing apoptosis. AEG-1 might be a potential target in treatment of hepatic fibrosis.

The uncertainty of boundary can improve the classification accuracy of BI-RADS 4A ultrasound image.

PURPOSE: The Breast Imaging-Reporting and Data System (BI-RADS) for ultrasound imaging provides a widely used reporting schema for breast imaging. Previous studies have shown that in ultrasound imaging, 90% of BI-RADS 4A tumors are benign lesions after biopsies. Unnecessary biopsy procedures can be avoided by accurate classification of BI-RADS 4A tumors. However, the classification task is challenging and has not been fully investigated by existing studies. For benign and malignant tumors of BI-RADS 4A, the appearances of intra-class tumors are highly variable, the characteristics of inter-class tumors is overall-similar. Discriminative features need to be found to improve classification accuracy of BI-RADS 4A tumors. METHODS: In this study, we designed the network using the clinical features of BI-RADS 4A tumors to improve the discrimination ability of network. The boundary information is embedded into the input of the network using the uncertainty. A fine-grained data augmentation method is used to find discriminative features in tumor information embedded with boundary information. Two mathematical methods, voting-based and variance-based, are used to define the uncertainty of boundary, and the differences of these two definitions are compared in a classification network. RESULTS: The dataset we used to evaluate our method had 1155 2D grayscale images. Each image represented a unique BI-RADS 4A tumor. Among them, 248 tumors were proven to be malignant by biopsy, and the remaining 907 were benign. A weakly supervised data augmentation network (WS-DAN) was used as the backbone classification network, which showed competitive performance in finding discriminative features. Using the auxiliary input of the uncertain boundaries defined by the voting method, the area under the curve (AUC) value of our method was 0.8347 (sensitivity = 0.7774, specificity = 0.7459). The AUC value of the variance-based uncertainty was 0.7789. The voting-based uncertainty was higher than the baseline (AUC = 0.803), which only inputs the original image. Compared with the classic classification network, our method had a significant effect improvement (p < 0.01). CONCLUSIONS: Using the uncertain boundaries defined by the voting methods as auxiliary information, we obtained a better performance in the classification of BI-RADS 4A ultrasound images, while variance-based uncertain boundaries had no effect on improving classification performance. Additionally, fine-grained network helped find discriminative features comparing with the commonly used classification networks.

Risk Factors for Brain Metastasis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy with high mortality, especially in HCC patients with brain metastases (BMS). However, few studies have investigated the risk factors for BMS among HCC patients based on large-scale population. The study involved clinical data of 36,091 patients who met the inclusion criteria from the SEER database, from 2004 to 2016. Univariate analysis and multifactor logistics regression analysis was used to analyze risk factors affecting BMS among HCC patients. This study revealed that BMS occurred in 108 of 36,091 patients, with an incidence of 0.33%. Median survival was 7 months for patients with BMS, but 12 months for patients without BMS. Univariate analysis showed that pathological low differentiation and undifferentiation, lymph node metastasis, no surgical treatment, and no chemotherapy and radiotherapy increased risk of BMS (P < 0.05). Multivariate analysis suggested that no surgical treatment and no chemotherapy or radiotherapy were independent risk factors for BMS (P < 0.001). Our findings highlighted that the independent risk factors for BMS were no surgical treatment, no chemotherapy, and no radiotherapy.

Automatic segmentation of the pectoral muscle based on boundary identification and shape prediction.

The purpose of this work is to identify the pectoral muscle region in mediolateral oblique (MLO) view mammograms even when the boundary is blurred or obscured. The problem is decoupled into two subproblems in our study: identifying parts of boundaries with high confidence and predicting the overall shape of the pectoral muscle. Due to the similarity in intensity and texture between pectoral muscle and gland tissue, we trained a deep neural network to distinguish them in the first subproblem. The boundary with high confidence can be obtained according to the consistency of predictions from multiple converged models. For the shape prediction problem, a generative adversarial network (GAN) is used to learn mapping from a given identified region and the breast shape to the overall pectoral muscle shape. Our method is evaluated on a mammogram dataset including 633 MLO view mammograms collected from three different datacenters. We take U-Net as our baseline model and the dataset is divided into three groups according to the performance of U-Net for evaluation. In all three groups, U-Net achieves 80.1%, 92.9%, and 98.3% in the Dice similarity coefficient, respectively, and our method achieves 85.2%, 94.8%, and 98.1% in the Dice similarity coefficient, respectively. The experiment shows that our method effectively estimates the pectoral muscle boundary, even parts of boundaries that are difficult to detect, and greatly improves the performance of segmentation in this case.

Development of immunoaffinity solid phase microextraction rods for analysis of three estrogens in environmental water samples.

In this study, immunoaffinity solid phase microextraction (SPME) rods were developed for the analysis of diethylstilbestrol (DES), hexestrol (HES) and dienestrol (DIS) followed by ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This immunoaffinity SPME device was built with three stainless steel rods bundled together as one and modified with porous silicate particles. As an extraction sorbent, antibody immobilization approach was employed based on the covalent attachment of the anti-diethylstilbestrol monoclonal antibody (mAb) onto the rods. The immunoaffinity SPME rod was characterized and confirmed by scanning electron microscopy and X-ray photoelectron spectroscopy analysis. The porous network showed a very large active surface area and significantly increased the adsorption capacity which can reach 49.6pmol/cm2. Moreover, the immunoaffinity sorbent showed good sturdiness at least 10 times with stable extraction performance. Once the main experimental parameters were optimized, the method was used to detect DES, HES and DIS in environmental water samples. The limits of detection for the three estrogens were 0.05-0.15ng/mL, and the limits of quantification was 0.5ng/mL. The average recoveries ranged from 34.2 to 62.7% were achieved with good intra-day and inter-day precision ranging from 7.8 to 12.7% and from 8.2 to 13.5% respectively. The newly developed immunoaffinity SPME showed high adsorption capacity, good sensitivity and reproducibility and was successfully applied to the analysis of DES, HES and DIS in environmental water samples.

Overexpression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor Mediates Liver Fibrosis in Transgenic Mice.

BACKGROUND: The role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver fibrosis is not clear and is sometimes even contradictory. To clarify this role, a HB-EGF transgenic (Tg) mouse model was, for the first time, used to evaluate the functions of HB-EGF in liver fibrosis. MATERIALS AND METHODS: For the in vivo study, carbon tetrachloride injection and bile duct ligation treatment were used to induce liver fibrosis in HB-EGF Tg mice and wild-type (WT) mice, respectively. Primary hepatic satellite cells (HSCs) were isolated from HB-EGF Tg and WT mice for the in vitro study. RESULTS: Compared with the WT mice, HB-EGF Tg mice were shown to develop more severe liver fibrosis when treated with carbon tetrachloride or bile duct ligation, with increased matrix metalloproteinases 13 activity and enhanced expression of fibrogenic genes including α-smooth muscle actin and collagen I. HB-EGF gene transfer led to an increase in proliferation and a decrease in apoptosis in primary HSCs. The ERK signaling pathway was more highly activated in primary HSCs from HB-EGF Tg mice than in those from WT mice. CONCLUSIONS: Our investigation confirmed the profibrotic effect of HB-EGF on the liver using a Tg mouse model. This result may contribute to the elucidation of HB-EGF as a therapeutic target in liver fibrosis.

Iodine and freeze-drying enhanced high-resolution MicroCT imaging for reconstructing 3D intraneural topography of human peripheral nerve fascicles.

BACKGROUND: The precise annotation and accurate identification of the topography of fascicles to the end organs are prerequisites for studying human peripheral nerves. NEW METHOD: In this study, we present a feasible imaging method that acquires 3D high-resolution (HR) topography of peripheral nerve fascicles using an iodine and freeze-drying (IFD) micro-computed tomography (microCT) method to greatly increase the contrast of fascicle images. RESULTS: The enhanced microCT imaging method can facilitate the reconstruction of high-contrast HR fascicle images, fascicle segmentation and extraction, feature analysis, and the tracing of fascicle topography to end organs, which define fascicle functions. COMPARISON WITH EXISTING METHODS: The complex intraneural aggregation and distribution of fascicles is typically assessed using histological techniques or MR imaging to acquire coarse axial three-dimensional (3D) maps. However, the disadvantages of histological techniques (static, axial manual registration, and data instability) and MR imaging (low-resolution) limit these applications in reconstructing the topography of nerve fascicles. CONCLUSIONS: Thus, enhanced microCT is a new technique for acquiring 3D intraneural topography of the human peripheral nerve fascicles both to improve our understanding of neurobiological principles and to guide accurate repair in the clinic. Additionally, 3D microstructure data can be used as a biofabrication model, which in turn can be used to fabricate scaffolds to repair long nerve gaps.

Simultaneous enantioselective determination of phenylpyrazole insecticide flufiprole and its chiral metabolite in paddy field ecosystem by ultra-high performance liquid chromatography/tandem mass spectrometry.

A novel and sensitive ultra-high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous enantioselective determination of flufiprole and its hydrolysis metabolite in paddy field ecosystem. The separation and determination were performed using reversed-phase chromatography on a novel cellulose chiral stationary phase, a Lux Cellulose-4 (150 mm × 2.0 mm) column, under isocratic conditions at 0.25 mL/min flow rate. The effects of other four different polysaccharide-based chiral stationary phases (CSPs) on the separation and simultaneous enantioseparation of the two target compounds were also evaluated. The elution orders of the eluting enantiomers were identified by an optical rotation detector. Modified QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) method and solid-phase extraction (SPE) were used for the enrichment and cleanup of paddy water, rice straw, brown rice and paddy soil samples, respectively. Parameters including the matrix effect, linearity, precision, accuracy and stability were evaluated. Under the optimal conditions, the mean recoveries for all enantiomers from the above four sample matrix were ranged from 83.6% to 107%, with relative standard deviations (RSD) in the range of 1.0-5.8%. Coefficients of determination R(2)≥0.998 were achieved for each enantiomer in paddy water, rice straw, brown rice and paddy soil matrix calibration curves within the range of 5-500 µg/kg. The limits of quantification (LOQ) for all stereoisomers in the above four matrices were all below 2.0 µg/kg. The methodology was successfully applied for simultaneously enantioselective analysis of flufiprole enantiomers and their chiral metabolite in the real samples, indicating its efficacy in investigating the environmental stereochemistry of flufiprole in paddy field ecosystem.

A Comparison of Novel Entomopathogenic Nematode Application Methods for Control of the Chive Gnat, Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga Yang & Zhang (Diptera: Sciaridae) is the most serious pest of Chinese chive, Allium tuberosum Chemical pesticide application has become a necessary step to control B. odoriphaga after each of the four to six harvests during the growing season. We compared the effects of application type (nematode-infected cadaver, aqueous nematode suspension, and mixture of cadaver and aqueous suspension) and nematode species (Heterorhabditis bacteriophora vs. Steinernema carpocapsae) on B. odoriphaga control. Nematode species combinations and the use of the cadaver method has only been tested in a relatively few studies, and has not been tested for this target pest, B. odoriphaga. Furthermore, this is the first report of combining application methods (aqueous and cadaver). Results indicated that the cadaver treatment produced higher mortality of B. odoriphaga than the aqueous treatment, and H. bacteriophora caused higher mortality of B. odoriphaga than S. carpocapsae. The mortality of B. odoriphaga was 96.7% in H. bacteriophora cadaver treatment and only 27.5% in control treatment without nematode addition. Cadaver treatments caused higher biomass of A. tuberosum than that of aqueous treatment. The plant biomass in H. bacteriophora cadaver treatment was 10 folds as much as that in the control treatment. The mixed aqueous suspension of H. bacteriophora and S. carpocapsae did not increase the mortality in pot trial. Our findings indicate that entomopathogenic nematodes applied as cadavers might be a potential alternative biological agent in the integrated management of B. odoriphaga for Chinese chive production.

[Simultaneous determination of 4 kinds of 29 banned and restricted veterinary drugs in animal-derived foods by ultra performance liquid chromatography-tandem mass spectrometry and modified QuEChERS for sample preparation].

An ultra performance liquid chromatography-tandem mass spectrometric method, with modified QuEChERS method for sample preparation, was developed for the determination of the 4 kinds of 29 banned and restricted veterinary drugs in chicken muscle, chicken liver, porcine muscle and porcine liver samples. The drugs were extracted with McIlvaine buffer solution and acetonitrile, then cleaned-up by modified QuEChERS method, and finally analyzed by ultra performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode via positive electrospray ionization. The method was validated at three fortification levels (LOQ level, 20 µg/kg and 50 µg/kg) in chicken muscle, chicken liver, porcine muscle and porcine liver samples. The validation results were as follows. The correlation coefficients of the standard calibration curves for the 29 veterinary drugs were all between 0. 993 and 0. 999 over their own concentration ranges. The average recoveries of the 29 drugs were between 71. 5% and 93. 2%, with the relative standard deviations (RSDs) between 0. 8% and 7. 7%. The limits of determination (S/N=3) of the drugs were between 0. 3 and 3. 0 µg/kg. The limits of quantification (S/N≥10) were 1. 0 µg/kg for amantadine, 10.0 µg/kg for the tetracycline drugs, and 5. 0 µg/kg for all the other drugs. It was indicated that the method developed is easy, sensitive, and has a good purification effect. The sensitivity, accuracy and precision of the method were all acceptable, and it can meet the requirements of the multiple residue analysis. So, this method can be further applied to investigate the veterinary drug residues in animal derived foods.

[Simultaneous determination of sulfonylurea herbicides in ginseng by ultra performance liquid chromatography-tandem mass spectrometry].

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was developed for the determination of the fifteen sulfonylurea herbicides in ginseng. The pesticides were extracted with acetonitrile, cleaned-up by an ENVI-Carbon solid phase extraction cartridge, eluted with 1% formic acid in methanol-dichlormethane (20:80, v/v), separated with UPLC and detected with MS/MS in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI+). The method was validated at three fortification levels in ginseng. The validation results were as follows. The standard calibration curves for the fifteen sulfonylurea herbicides all showed linear over the range of 2-100 microg/L with the correlation coefficients between 0.996 and 0.999. The average recoveries of the fifteen sulfonylurea herbicides at the three fortification levels of 5, 25 and 50 microg/kg were between 84.9% and 104.3% with the relative standard deviations (RSDs) between 2.4% and 11.9%. The limit of quantification (LOQ) was defined as the lowest concentration that could be measured with acceptable precision and accuracy. The LOQs of all the fifteen sulfonylurea herbicides in ginseng were 5 microg/kg. It was indicated that this method is easier, more sensitive and has a better purification effect. The sensitivity, accuracy and precision of the method were all acceptable. So, this method can be further applied to investigating the contamination status of traditional Chinese medicine by the sulfonylurea herbicides