Assessment of macro, trace and toxic element intake from rice: differences between cultivars, pigmented and non-pigmented rice.

Pigmented and non-pigmented rice varieties (grown in different areas) were collected in China, Yunnan, to investigate the content of macro-, trace elements and potentially toxic elements (PTEs), and to assess the health risk associated with dietary intake. The order of elemental concentrations in rice was Mn > Zn > Fe > Cu > Se for trace elements, P > K > Mg > Ca > Na for macro elements, and Cr > As > Cd for PTEs. Rice with a high concentration of essential elements also associated with a high content of PTEs. In addition, higher content of Cr, Mn and Na were found in pigmented rice. The health risk assessment showed that the daily intake of all elements was below the tolerable limit (UL). Moreover the intake of Fe, Zn and Se was far from sufficient for the nutrient requirement. The PTEs in rice dominated the health risk. Of concern is that this rice consumption is likely to contribute to carcinogenic risks in the long term and that adults are at higher health risk from pigmented rice compared to non-pigmented rice. This study confirms that the lack of essential micronutrients in rice and the health risk associated with rice diets should remain a concern.

Multiple spinal gas in an 86-year-old diabetic man.

Although spinal gas is common and can be found in various sites and lesions, it should prompt a search for the underlying cause, given that the clinical significance of ectopic gas varies from benign to scary. Spinal gas can occur in the traumatic, iatrogenic, degenerative, osteoporotic, infectious, or neoplastic lesions. The imaging similarity may cause the misdiagnosis or delayed diagnosis which sometimes requires immediate attention. The pattern of gas distribution, detailed appearance, clinical history, and findings on examination can provide clues to diagnosis. Computed tomography is the best method for sensitive detection of gas.

Smoothened mediates medaka spermatogonia proliferation via Gli1-Rgcc-Cdk1 axis†.

The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.

Gata2a Mutation Causes Progressive Microphthalmia and Blindness in Nile Tilapia.

The normal development of lens fiber cells plays a critical role in lens morphogenesis and maintaining transparency. Factors involved in the development of lens fiber cells are largely unknown in vertebrates. In this study, we reported that GATA2 is essential for lens morphogenesis in Nile tilapia (Oreochromis niloticus). In this study, Gata2a was detected in the primary and secondary lens fiber cells, with the highest expression in primary fiber cells. gata2a homozygous mutants of tilapia were obtained using CRISPR/Cas9. Different from fetal lethality caused by Gata2/gata2a mutation in mice and zebrafish, some gata2a homozygous mutants of tilapia are viable, which provides a good model for studying the role of gata2 in non-hematopoietic organs. Our data showed that gata2a mutation caused extensive degeneration and apoptosis of primary lens fiber cells. The mutants exhibited progressive microphthalmia and blindness in adulthood. Transcriptome analysis of the eyes showed that the expression levels of almost all genes encoding crystallin were significantly down-regulated, while the expression levels of genes involved in visual perception and metal ion binding were significantly up-regulated after gata2a mutation. Altogether, our findings indicate that gata2a is required for the survival of lens fiber cells and provide insights into transcriptional regulation underlying lens morphogenesis in teleost fish.

Homozygous Mutation of gsdf Causes Infertility in Female Nile Tilapia (Oreochromis niloticus).

Gonadal somatic cell-derived factor (Gsdf) is a member of the TGF-ß superfamily, which exists mainly in fishes. Homozygous gsdf mutations in Japanese medaka and zebrafish resulted in infertile females, and the reasons for their infertility remain unknown. This study presents functional studies of Gsdf in ovary development using CRISPR/Cas9 in Nile tilapia (Oreochromis niloticus). The XX wild type (WT) female fish regularly reproduced from 12 months after hatching (mah), while the XX gsdf-/- female fish never reproduced and were infertile. Histological observation showed that at 24 mah, number of phase IV oocyte in the XX gsdf-/- female fish was significantly lower than that of the WT fish, although their gonadosomatic index (GSI) was similar. However, the GSI of the XX gsdf-/- female at 6 mah was higher than that of the WT. The mutated ovaries were hyperplastic with more phase I oocytes. Transcriptome analysis identified 344 and 51 up- and down-regulated genes in mutants compared with the WT ovaries at 6 mah. Some TGF-ß signaling genes that are critical for ovary development in fish were differentially expressed. Genes such as amh and amhr2 were up-regulated, while inhbb and acvr2a were down-regulated in mutant ovaries. The cyp19a1a, the key gene for estrogen synthesis, was not differentially expressed. Moreover, the serum 17ß-estradiol (E2) concentrations between XX gsdf-/- and WT were similar at 6 and 24 mah. Results from real-time PCR and immunofluorescence experiments were similar and validated the transcriptome data. Furthermore, Yeast-two-hybrid assays showed that Gsdf interacts with TGF-ß type II receptors (Amhr2 and Bmpr2a). Altogether, these results suggest that Gsdf functions together with TGF-ß signaling pathway to control ovary development and fertility. This study contributes to knowledge on the function of Gsdf in fish oogenesis.

Computational simulation studies on the binding selectivity of Wee1 and Checkpoint kinase 1 by molecular dynamics simulation combined with free energy calculations.

Wee1 kinase and Checkpoint kinase 1 (Chk1) kinase, which are well known to be involved in cancer, are promising targets for cancer therapy. Most of developed Wee1 inhibitors can inhibit activity of Chk1 kinase to different degrees as well. The poor selectivity brought side effects and selective inhibitor is needed. However, the selective mechanisms of Wee1 versus Chk1 are not clear. Therefore, the design of selective Wee1 and Chk1 inhibitors would provide a meaningful starting for the development of anticancer drugs with optimal efficacy. In this study, Wee1 inhibitors with different selectivity over Chk1 were chosen to analyze the selectivity mechanism by means of molecular docking, molecular dynamics simulations and binding free energy calculations. Two key residues of Wee1 kinase and two critical residues of Chk1 were mutated to detect their effect on ligand binding into protein. The results indicated that these residues play a pivotal role in the binding interactions of ligands to receptors through hydrogen bond and hydrophobic interaction with inhibitors. This may provide a better understanding of the selective mechanism of Wee1 and Chk1. It would be beneficial to the discovery and optimization of selective Wee1 and Chk1 inhibitors.Communicated by Ramaswamy H. Sarma.

Regulation of Female Folliculogenesis by Tsp1a in Nile Tilapia (Oreochromis niloticus).

TSP1 was reported to be involved in multiple biological processes including the activation of TGF-ß signaling pathways and the regulation of angiogenesis during wound repair and tumor growth, while its role in ovarian folliculogenesis remains to be elucidated. In the present study, Tsp1a was found to be expressed in the oogonia and granulosa cells of phase I to phase IV follicles in the ovaries of Nile tilapia by immunofluorescence. tsp1a homozygous mutants were generated by CRISPR/Cas9. Mutation of tsp1a resulted in increased oogonia, reduced secondary growth follicles and delayed ovary development. Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries. Furthermore, transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated, while cAMP and MAPK signaling pathway genes which inhibit cell proliferation and promote cell differentiation were significantly down-regulated. In addition, aromatase (Cyp19a1a) expression and serum 17ß-estradiol (E2) concentration were significantly decreased in the mutants. These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia, which in turn, resulted in increased oogonia, reduced secondary growth follicles and decreased E2. Taken together, our results indicated that tsp1a was essential for ovarian folliculogenesis in Nile tilapia.

[Rapid determination of multiple prohibited and restricted veterinary drug residues in fish and shrimp by ultra high performance liquid chromatography-tandem mass spectrometry].

A method was established for the rapid determination of 28 prohibited and restricted veterinary drugs of five classes (sulfonamides, quinolones, chloramphenicols, nitrofurans metabolites and triphenylmethane) in fish and shrimp by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) coupled with simultaneous extraction and purification. After optimizing the sample pretreatment methods and chromatographic conditions, the samples were hydrolyzed with hydrochloric acid. The five kinds of residual drugs were simultaneously extracted with acetonitrile after nitrofuran metabolites derivatized with 2-nitrobenzaldehyde. The extraction solutions were dehydrated with sodium chloride and salted out. The acetonitrile layer was purified with n-hexane and concentrated to near dryness. The residues were dissolved in 1.0 mL methanol. Then, the solutions were purified by dispersive solid phase extraction with C18, N-(n-propyl)ethylenediamine (PSA) and amino bonded silica gel (NH2) sorbents. The residues were separated on a ZORBAX C18 chromatographic column (50 mm×2.1 mm, 1.8 µm). The MS data were acquired in positive and negative multiple reaction monitoring (MRM) modes, and quantitated by the isotope internal standard method. With this approach, the linear relationships of the 28 veterinary drugs were good. The linear correlation coefficients were greater than 0.99. The limits of detection and limits of quantification were in the range of 0.1-0.8 µg/kg and 0.3-2.7 µg/kg, respectively. The average recoveries at three spiked levels (1, 5, and 20 fold of the LOQs) were 70.0%-120% with relative standard deviations of 0.9%-10.0%. The method is simple, rapid, accurate and sensitive, and it is suitable for the rapid determination of various prohibited and restricted drugs in fish and shrimp.

[Simultaneous determination of antibiotics and triphenylmethanes veterinary drug residues in fish and shrimp by dispersive solid phase extraction purification-ultra high performance liquid chromatography-tandem mass spectrometry].

A method was established to rapidly determine 20 kinds of veterinary drug residues including three catagories of antibiotics (sulfonamides, quinolones, and chloramphenicols) and two kinds of triphenylmethanes (malachite green (MG) and leucomalachite green (LMG)) in fish and shrimp, based on dispersive solid phase extraction purification-ultra high performance liquid chromatography-tandem mass spectrometry. The samples were first hydrolyzed using a dipotassium hydrogen phosphate solution, and then extracted using acetonitrile. Afterward, the extraction solution was dehydrated and salted out with sodium chloride and condensed to nearly dry using a rotating evaporator. This residue was dissolved in 1.0 mL methanol. The resulting solution was purified by dispersive solid phase extraction method with C18 and PSA sorbents, and filtered through a filter. The target compounds were separated employing a ZORBAX C18 column. The mass spectrometer datas were acquired by multiple reaction monitoring (MRM) of positive and negative modes and quantitated applying the isotope internal standard method. The 20 veterinary drugs showed a good linear relationship in the range of 0.2-300 µg/L. The limits of detection and the limits of quantification were 0.1-0.6 and 0.3-1.8 µg/kg, respectively, while the correlation coefficients were greater than 0.99. The average recoveries at the three spiked levels (1, 5, and 20 times of quantitative limits) ranged between 72.5%-118%, with the relative standard deviations of 1.9%-9.8%. The advantages of method include a simple pretreatment, a high detection efficiency, and a low cost. Moreover, it is suitable for the simultaneous determination of multiple veterinary drug residues in fish and shrimp.

Design and synthesis of novel artemisinin derivatives with potent activities against colorectal cancer in vitro and in vivo.

A series of novel derivatives of artemisinin-4-(arylamino)quinazoline have been designed and synthesized, and most of them showing potent in vitro cytotoxic activity against HCT116 and WM-266-4 cell lines. Compound 32 was the most active derivative against HCT116 cell line with an IC50 of 110 nM, and significantly improved the antitumor activity of the parent compounds dihydroartemisinin (DHA) (IC50 = 2.85 µM) and Gefitinib (IC50 = 19.82 µM). In vivo HCT116 xenografts assay showed that compound 32 exhibited potent antitumor activity with obvious tumor growth delay and tumor shrunken after 18 days treatment on xenografted mice, and especially without loss of body weight. Our results indicate that compounds 32 may represent a safe, novel structural lead for developing new chemotherapy of colorectal cancer.

Establishment of three estrogen receptors (esr1, esr2a, esr2b) knockout lines for functional study in Nile tilapia.

Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptors (ESRs) in all vertebrates. To date, distinct roles of estrogen receptors have been characterized only in human and model organisms, including mouse, rat, zebrafish and medaka. Physiological role of estrogen/receptor signaling in reproduction remains poorly defined in non-model organisms. In the present study, we successfully generated esr1, esr2a and esr2b mutant lines in tilapia by CRISPR/Cas9 and examined their phenotypes. Surprisingly, the esr1 mutants showed no phenotypes of reproductive development and function in both females and males. The esr2a mutant females showed significantly delayed ovarian development and follicle growth at 90 and 180 dah, and the development caught up later at 360 dah. The esr2a mutant males showed no phenotypes at 90 dah, and displayed smaller gonads and efferent ducts, less spermatogonia and more abnormal sperms at 180 dah. In contrast, the esr2b mutants displayed abnormal development of ovarian ducts and efferent ducts which failed to connect to the genital orifice, and which in turn, resulted in infertility in female and male, respectively, although they produced gametes in their gonads. Taken together, our study provides evidence for differential functions of esr1, esr2a and esr2b in fish reproduction.

Identification of potential AMPK activator by pharmacophore modeling, molecular docking and QSAR study.

AMP-activated protein kinase (AMPK) plays a major role in maintaining cellular energy homeostasis by sensing and responding to AMP/ADP concentrations relative to ATP. AMPK has attracted widespread attention as a potential therapeutic target for metabolic diseases such as cancer and cardiovascular diseases. The structure-based 3D pharmacophore model was developed based on the training set. The best pharmacophore model Hypo5 was proposed and validated using a decoy set, an external test set. Hypo5, with the correlation coefficient value of 0.936, cost difference value of 112.08 and low RMS value of 1.63, includes a ionizable positive, a hydrogen bond donor, a hydrogen bond acceptor and two hydrophobic features, which showed a high goodness of fit and enrichment factor. Thus it was used as a 3D query to find potential activator from the SPECS Database. Then the ADMET descriptors were used to filter all of 158 screening molecules. The 41 filtering compounds were subsequently subjected to molecular docking and Quantitative structure-activity relationship (QSAR) analysis. Finally, the compound H2 was picked out from those filtering compounds based on the receptor-ligand interaction analysis and the prediction of the QSAR models. And then it was submitted for molecular dynamics (MD) simulations to explore the stability of complex. The result indicates that the candidate could be considered a potential AMPK activator.

[Determination of bisultap residue in tropical fruits by ultra performance liquid chromatography-tandem mass spectrometry].

A method for the determination of bisultap in tropical fruits was developed by modified QuEChERS method and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The tropical fruit samples were twice extracted with acetonitrile containing 1% (v/v) acetic acid, and purified by 200 mg of primary secondary amine (PSA), 100 mg of C18, 300 mg of MgSO4 and 500 mg of disodium hydrogen citrate. The analyte was separated on a CAPCELL CORE PC hydrophilic column, detected in the negative ion multiple reaction monitoring (MRM) mode, and quantified by external standard method. A good linear relationship in the range of 0.1-10.0 µg/L was obtained with the correlation coefficient (r2) of 0.9993. The limit of detection and limit of quantification were 0.015 µg/kg and 0.05 µg/kg, respectively. The spiked recoveries were in the range of 81.0%-88.3% with the relative standard deviations from 3.9% to 6.2%. The method has good purification effect and high sensitivity, and is suitable for the rapid analysis of the bisultap residues in tropical fruits.

Discovery of novel wee1 inhibitors via structure-based virtual screening and biological evaluation.

Wee1 plays a critical role in the arrest of G2/M cell cycle for DNA repair before entering mitosis. Many cancer cells have been identified as overexpression of Wee1. In this research, pharmacophore modeling, molecular docking and molecular dynamics simulation approaches were constructed to identify novel potential Wee1 inhibitors. A compound 8 was found to have a novel skeleton against Wee1 with an IC50 value of 22.32 µM and a Ki value of 13.11 µM. Kinetic assays were employed to evaluate the compound 8 as a competitive inhibitor. Compound 8 was tested against A-549 tumor cell lines with IC50 value of 17.8 µM. To investigate the intermolecular interaction of Wee1 and compound 8, further molecular dynamics simulations were performed. It indicates that the binding mode of compound 8 and reference ligand is similar. The active core scaffold of compound 8 could represent a promising lead compound for studying Wee1 and be used for further structural optimization to design more potent Wee1 inhibitors.

Pharmacophore modeling, molecular docking and molecular dynamics simulations toward identifying lead compounds for Chk1.

Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase, plays an important role in G2/M checkpoint, which is a key regulator in response to DNA damage. In this study, the structure-based drug design approach and molecular dynamics (MD) simulations were used to explore potent Chk1 inhibitors. A series of the best fitting candidates were picked out from the Specs database. Out of these, five candidates were submitted for MD simulations to explore the stability of complex. The result indicates that these five candidates could be considered potential Chk1 inhibitors and represents a promising starting point for developing potent inhibitors of Chk1 for the treatment of tumor.

Computational Analysis of Artimisinin Derivatives on the Antitumor Activities.

The study on antitumor activities of artemisinin and its derivatives has been closely focused on in recent years. Herein, 2D and 3D QSAR analysis was performed on the basis of a series of artemisinin derivatives with known bioactivities against the non-small-cell lung adenocarcinoma A549 cells. Four QSAR models were successfully established by CoMSIA, CoMFA, topomer CoMFA and HQSAR approaches with respective characteristic values q2 = 0.567, R2 = 0.968, ONC = 5; q2 = 0.547, R2 = 0.980, ONC = 7; q2 = 0.559, R2 = 0.921, ONC = 7 and q2 = 0.527, R2 = 0.921, ONC = 6. The predictive ability of CoMSIA with r2 = 0.991 is the best one compared with the other three approaches, such as CoMFA (r2 = 0.787), topomer CoMFA (r2 = 0.819) and HQSAR (r2 = 0.743). The final QSAR models can provide guidance in structural modification of artemisinin derivatives to improve their anticancer activities.

Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia.

It is well accepted that Forkhead box protein L2 (Foxl2) and aromatase (Cyp19a1; the enzyme responsible for estrogen synthesis) are critical for ovarian development in vertebrates. Knockouts of Foxl2 and Cyp19a1 in goat, mouse, and zebrafish have revealed similar but not identical functions across species. Functional analyses of these two genes in other animals are needed to elucidate their conserved roles in vertebrate sexual development. In this study, we established foxl2 and cyp19a1a mutant lines in Nile tilapia. Both foxl2-/- and cyp19a1a-/- XX fish displayed female-to-male sex reversal. Sf1, Dmrt1, and Gsdf were upregulated in the foxl2-/- and the cyp19a1a-/- XX gonads. Downregulation of Cyp19a1a and serum estradiol-17ß level, and upregulation of Cyp11b2 and serum 11-ketotestosterone level were observed in foxl2-/- XX fish. The mutant phenotype of foxl2-/- XX individuals could be rescued by 17ß-estradiol treatment from 5 to 30 days after hatching (dah). Upregulation of Star1, the enzyme involved in androgen production in tilapia, was also observed in the foxl2-/- XX gonad at 30 and 90 dah. In vitro promoter analyses consistently demonstrated that Foxl2 could suppress the transcription of star1 in a dose-dependent manner. In addition, compared with the control XX gonad, fewer germ cells were detected in the foxl2-/- XX, cyp19a1a-/- XX, and control XY gonads 10 dah. These results demonstrate that Foxl2 promotes ovarian development by upregulating Cyp19a1a expression and repressing male pathway gene expression. These results extend the study of Foxl2 and Cyp19a1a loss of function to a commercially important fish species.

Does intraoperative navigation improve the accuracy of pedicle screw placement in the apical region of dystrophic scoliosis secondary to neurofibromatosis type I: comparison between O-arm navigation and free-hand technique.

PURPOSE: To assess the accuracy of O-arm-navigation-based pedicle screw insertion in dystrophic scoliosis secondary to NF-1 and compare it with free-hand pedicle screw insertion technique. METHODS: 32 patients with dystrophic NF-1-associated scoliosis were divided into two groups. A total of 92 pedicle screws were implanted in apical region (two vertebrae above and below the apex each) in 13 patients using O-arm-based navigation (O-arm group), and 121 screws were implanted in 19 patients using free-hand technique (free-hand group). The postoperative CT images were reviewed and analyzed for pedicle violation. The screw penetration was divided into four grades: grade 0 (ideal placement), grade 1 (penetration <2 mm), grade 2 (penetration between 2 and 4 mm), and grade 3 (penetration >4 mm). RESULTS: The accuracy rate of pedicle screw placement (grade 0, 1) was significantly higher in the O-arm group (79 %, 73/92) compared to 67 % (81/121) of the free-hand group (P = 0.045). Meanwhile, a significantly lower prevalence of grade 2-3 perforation was observed in the O-arm group (21 vs. 33 %, P < 0.05), and the incidence of medial perforation was significantly minimized by using O-arm navigation compared to free-hand technique (2 vs. 15 %, P < 0.01). Moreover, the implant density in apical region was significantly elevated by using O-arm navigation (58 vs. 42 %, P < 0.001). CONCLUSION: We reported 79 % accuracy of O-arm-based pedicle screw placement in dystrophic NF-1-associated scoliosis. O-arm navigation system does facilitate pedicle screw insertion in dystrophic NF-1-associated scoliosis, demonstrating superiorities in the safety and accuracy of pedicle screw placement in comparison with free-hand technique.