Xiu-qi Wang

The relationship between gene expression of cationic and neutral amino acid transporters in the small intestine of chick embryos and chick breed, development, sex, and egg amino acid concentration

This study was conducted to investigate the gene expression of cationic and neutral amino acid (AA) transporters in the small intestine of chick embryos with different genetic backgrounds [Wenshi Yellow-Feathered chick (WYFC) and White Recessive Rock chick (WRRC)]. The study also investigated the correlation between the abundance of AA transporter mRNA and the AA content of fertilized eggs. Intestinal samples were collected on embryonic d 9, 12, 14, 17, and 19 and the day of hatch. The results showed that, before incubation, the AA content of WRRC eggs was lower (P < 0.05) than the AA content of WYFC eggs. In WYFC, the mRNA abundance of CAT-1 [solute carrier (SLC) family 7 member 1], CAT-4 (SLC family 7 member 4), rBAT (SLC family 3 member 1), y(+)LAT-1 (SLC family 7 member 7), y(+)LAT-2 (SLC family 7 member 6), LAT-4 (SLC family 43 member 2), and SNAT-2 (SLC family 38 member 2), as detected by real-time reverse transcriptase PCR, was greater (P < 0.05) than the mRNA abundance detected in the WRRC samples. The mRNA abundance of all measured AA transporters was affected (P < 0.05) by embryonic age. Sex had the largest effect (P < 0.05) on the mRNA expression of CAT-1, CAT-4, y(+)LAT-2, and LAT-4 in WYFC and on CAT-4 and B(0)AT-1 (SLC family 6 member 19) mRNA expression in WRRC. In WYFC, only CAT-1 mRNA expression was negatively correlated (r = -0.68 to -0.84, P < 0.05) with all AA content. However, few correlations were detected between AA content and the mRNA expression of multiple transporters in WRRC. These findings provide a comprehensive profile of the temporal and spatial mRNA expression of AA transporters in the small intestine of chick embryos. Few correlations were detected between the AA content of the eggs and mRNA expression of specific AA transporters in the small intestine.

The Differential Proliferative Ability of Satellite Cells in Lantang and Landrace Pigs

Here, for the first time, we evaluate the hypothesis that the proliferative abilities of satellite cells (SCs) isolated from Lantang (indigenous Chinese pigs) and Landrace pigs, which differ in muscle characteristics, are different. SCs were isolated from the longissimus dorsi muscle of neonatal Lantang and Landrace pigs. Proliferative ability was estimated by the count and proliferative activity of viable cells using a hemocytometer and MTT assay at different time points after seeding, respectively. Cell cycle information was detected by flow cytometry. Results showed that there was a greater (P<0.05) number of SCs in Lantang pigs compared with Landrace pigs after 72 h of culture. The percentage of cell population in S phase and G2/M phases in Lantang pigs were higher (P<0.05), while in G0/G1 phase was lower (P<0.05) in comparison with the Landrace pigs. The mRNA abundances of MyoD, Myf5, myogenin and Pax7 in SCs from Lantang pigs were higher (P<0.05), while those of myostatin, Smad3 and genes in the mammalian target of rapamycin (mTOR) pathway (with the exception of 4EBP1) were lower (P<0.05) than the Landrace pigs. Protein levels of MyoD, myogenin, myostatin, S6K, phosphorylated mTOR and phosphorylated eIF4E were consistent with the corresponding mRNA abundance. Collectively, these findings suggested that SCs in the two breeds present different proliferative abilities, and the proliferative potential of SCs in Lantang pigs is higher than in Landrace pigs.

Effects of dietary lysine levels on apparent nutrient digestibility and cationic amino acid transporter mRNA abundance in the small intestine of finishing pigs, Sus scrofa.

One hundred and twenty pigs were used to evaluate the effects of different dietary lysine levels on the growth performance, apparent nutrient digestibility, and abundance of cationic amino acid transporter messenger RNA (mRNA) in the small intestine of finishing pigs. Pigs received a low lysine diet (LL, 0.60% lysine), moderate lysine diet (ML, 0.80% lysine) or a high lysine diet (HL, 1.00% lysine) for 28 days. A digestion test was carried out during the third week. Although the apparent nutrient digestibility in pigs fed experimental diets were different (P < 0.05) and the highest when pigs were fed ML diet, diets did not change the growth performance. In the duodenum, mRNA abundance of PepT-1, as detected by real-time RT-PCR, was reduced in the LL diet (P < 0.05). A greater abundance of b(0,+) AT and PepT-1 mRNA was associated with the ML diet (P < 0.05) in the jejunum and ileum, respectively. In the ileum, the HL diet had a lower abundance of CAT-1 mRNA compared with other diets. These results showed that the finishing pigs would gain better nutrient digestibility when the dietary lysine content was 0.80%, and dietary lysine levels influenced the expression of cationic amino acid transporter mRNA in the small intestine of finishing pigs.

Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia)

The objective of this study was to investigate the relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth in domestic pigeons (Columba livia). One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions (temperature of 38.1 °C and relative humidity of 55%). Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day (E) 9, 11, 13, 15 and day of hatch (DOH). The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax) were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The mRNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The mRNA abundances of b0,+AT, EAAT3, y+LAT2, PepT1, LAT4, NHE2, and NHE3 increased linearly with age, whereas mRNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b0,+AT, EAAT3, PepT1, LAT4, NHE2, NHE3, and y+LAT2 had positive correlations with body weight (0.71<correlation coefficient (CC)<0.82, P<0.0001), while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight (−0.86<CC<−0.64, P<0.0001). The gene expressions of b0,+AT, EAAT3, LAT4, PepT1, NHE2, NHE3, and y+LAT2 showed positive correlations with intestinal weight (0.80<CC<0.91, P<0.0001),while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight (−0.84<CC<−0.67, P<0.0001). It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter mRNA in the small intestine during embryonic growth of pigeons.概要目的探索家鸽胚胎期器官生长发育及其小肠营养素转运载体基因表达规律, 为家鸽胚胎期的营养调控提供理论基础。创新点第一次较全面地研究了家鸽胚胎期小肠氨基酸转运载体基因表达规律。方法利用实时荧光定量逆转录聚合酶链反应 (real-timeRT-PCR) 技术, 同时结合了多内标校正方法对小肠营养素转运载体基因表达模式进行分析。结论在家鸽胚胎期发育的过程中, 不同的器官生长模式不完全一致。营养素转运载体b0,+AT、EAAT3、LAT4、PepT1、NHE2、NHE3 和y+LAT2 基因的表达与小肠重量呈正相关关系, CAT1、CAT2 和EAAT2 基因的表达与小肠重量呈负相关关系。

Heat stress inhibits proliferation, promotes growth, and induces apoptosis in cultured Lantang swine skeletal muscle satellite cells

Proliferation suppression and apoptosis are the prominent characteristics induced by heat stress (HS) in cells, whereas the effects of HS on cell growth (mass accumulation) are unknown. In this study, Lantang swine (an indigenous breed of China) skeletal muscle satellite cells (SCs) were pre-cultured at 37 °C for 24 h. The HS group was subjected to HS at 41 °C, while the control group was maintained at 37 °C. Heat shock protein 70 (HSP70) expression and SC size are significantly increased (P<0.05) by HS, but cell proliferation is suppressed (P<0.05) and apoptosis is induced (P<0.05). HS led to a lower percentage of SCs in the G0/G1 phase (P<0.05) together with a higher percentage of SCs in the S phase (P<0.05). However, the percentage of SCs in the G2/M phase was decreased (P<0.05) at 48 h but then increased (P<0.05) at 72 h with HS. In addition, the phosphorylation ratios of protein kinase b (Akt), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 were increased (P<0.05) by HS. Nevertheless, the phosphorylation ratios of the 4E binding protein 1 and the eukaryotic initiation factor-4E were indistinguishable (P>0.05) from those of the control group. The phosphorylation ratio of the mammalian target of rapamycin (mTOR) (Ser2448) increased(P<0.05) within 48 h, and apparent differences were abrogated at 72 h (P>0.05). Moreover, cleaved caspase-3 expression was increased at 72 h (P<0.05). These findings indicate that HS induces apoptosis and disrupts cell cycle distribution to decrease the number of cells. Additionally, HS can promote SC growth via an activated Akt/mTOR/S6K signaling pathway.概要目的探究热应激对蓝塘猪骨骼肌卫星细胞增殖与生长的影响, 分析其作用机制。创新点通过体外细胞模型研究了热应激对猪骨骼肌卫星细胞增殖与生长的影响,发现热应激通过 Akt/mTOR/S6K 途径调控细胞的生长。方法本试验选用1 日龄蓝塘猪背最长肌的骨骼肌卫星细胞, 对照组为37 °C 正常培养, 热应激组培养温度为41 °C, 其它培养条件相同。采用细胞计数、MTT 法、流式细胞术等手段分析比较热应激对蓝塘猪骨骼肌卫星细胞增殖与生长的影响; 利用实时逆转录聚合酶链式反应 (RT-PCR) 和蛋白质印迹法 (Western blot) 等方法, 检测了热休克蛋白HSP70、半胱氨酸蛋白酶-3 (caspase-3) 及哺乳动物雷帕霉素靶蛋白 (mTOR) 信号通路关键基因的表达。结论(1) 41 °C 热处理可引起蓝塘猪骨骼肌卫星细胞产生热应激反应; (2) 热应激72 h 会通过改变细胞周期、抑制细胞增殖并诱导细胞凋亡等共同作用减少细胞数目; (3) 热应激通过Akt/mTOR/S6K途径调控细胞的生长。

Enantioselective analysis and dissipation of triazole fungicide penconazole in vegetables by liquid chromatography-tandem mass spectrometry.

Penconazole is a typical triazole fungicide, which is commonly used to control powdery mildew in vineyard and vegetable field. In this study, the enantioselective dissipation of penconazole in cucumber, tomato, head cabbage, and pakchoi was investigated by field experiments. A sensitive method for enantiomeric analysis of penconazole was established on the basis of the buffered QuEChERS sample preparation technique followed by reverse-liquid chromatography equipped with a TSQ Discovery triple quadrupole mass spectrometer and a Lux Cellulose-2 chiral column. Methanol and 2 mM ammonium acetate buffer solution containing 0.1% formic acid (70:30, v/v) were used as mobile phase at a 0.2 mL L(-1) flow rate isocratic elution. The linearity, recovery, and precision of this method were also evaluated. Finally, the results of this study demonstrated that enantioselective dissipation occurred in head cabbage and pakchoi, with the preferential degradation of (-)-penconazole, and resulting in an enrichment of the (+)-penconazole residue in the two vegetables. However, the enantioselective behavior was not observed in cucumber and tomato. More importantly, this is the first report of enantioselective behavior of penconazole, and the result may provide useful information for the risk evaluation of penconazole in food and environmental safety.

GSK1904529A, a Potent IGF-IR Inhibitor, Reverses MRP1-Mediated Multidrug Resistance

Overexpression of multidrug‐resistant efflux transporters is one of the major causes of chemotherapy failure. MRP1, a 190 kDa efflux transporter, confers resistance to a wide of range of chemotherapeutic drugs. Here we study the cellular effects of GSK1904529A in reversing MRP1‐mediated drug resistance. Cytotoxicity of GSK1904529A was determined by MTT assay. Reversal effects of GSK1904529A in combination with MRP1 substrates were determined. The intracellular accumulation and efflux of MRP1 substrate was measured by scintillation counter and protein expression was determined by Western blotting analysis. Cell cycle effects of GSK1904529A in combination with MRP1 substrates were determined by flow cytometric analysis. GSK1904529A, at non‐toxic concentrations, enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 cells. Furthermore, GSK1904529A increased the intracellular accumulation of [3H]‐vinblastine by inhibiting the efflux function of MRP1. GSK1904529A did not alter the expression level of MRP1, induced a G0/G1 phase cell cycle arrest. Our results indicated that GSK1904529A significantly increased the sensitivity of MRP1 overexpressing cells to chemotherapeutic agents. Furthermore, GSK1904529A enhanced the efficacy of chemotherapeutic drugs that are substrates of MRP1. J. Cell. Biochem. 118: 3260–3267, 2017.

L-Glutamate deficiency can trigger proliferation inhibition via down regulation of the mTOR/S6K1 pathway in pig intestinal epithelial cells

The objective of this study was to investigate the effects of L-glutamate (Glu) deficiency or L-trans pyrrolidine-2,4-dicarboxylic acid (PDC) supplementation on the proliferation of pig intestinal epithelial cells (IPEC-1). First, IPEC-1 cells were cultured in normal growing medium supplemented with 0 (Control), 50, 100, or 200 µmol/L PDC to determine an appropriate concentration of PDC supplementation. Second, IPEC-1 cells were cultured in Glu-deficient medium supplemented with 0 µmol/L Glu (Glu deficiency), 50 µmol/L Glu (Control), or 50 µmol/L Glu plus 100 µmol/L PDC (PDC supplementation). Cell proliferation ( = 24), cell cycle distribution ( = 6), cell apoptosis ( = 6), and expression levels of proteins of interest ( = 4) were determined by MTT assay, flow cytometry, or western blot. The results showed that cell proliferation was inhibited ( < 0.05) by 50, 100, and 200 µmol/L PDC supplementation at 24 and 48 h after treatment. Variance analysis was performed using the GLM procedure, and the results demonstrated that Glu deficiency or PDC supplementation led to the inhibition ( < 0.05) of cell proliferation, a greater ( < 0.05) percentage of cells in the G1 phase, and a lower ( < 0.05) percentage of cells in the S phase. Moreover, Glu deficiency or PDC supplementation reduced ( < 0.05) the expression levels of excitatory AA transporter 3 (EAAT3), phosphor-mammalian target of rapamycin (p-mTOR; Ser2448), p-ribosomal protein S6 kinase 1 (S6K1; Thr389), and p-S6 (Ser235/236). This study demonstrates that Glu deficiency or PDC supplementation inhibits proliferation of IPEC-1 cells via downregulation of the mTOR/S6K1 pathway and EAAT3 expression indicating that Glu deficiency may lead to the disturbances of intestinal epithelial renewal in pigs, particularly in neonates.

The effect of dietary supplementation with phytase transgenic maize and different concentrations of non-phytate phosphorus on the performance of laying hens

Abstract 1. The objective of this study was to evaluate the effects of dietary supplementation with phytase transgenic corn (maize) (PTC) which has a phytase activity of 21 000 units (U) phytase per kg of maize on productive performance, egg quality, tibia bone quality and phosphorus (P) excretion in laying hens. 2. In the experiment, 1800 44-week-old Hy-line brown laying hens were divided into 5 groups with 6 replicates per group and 60 hens per replicate. The experiment lasted for 12 weeks. The layers in the control group (control) were given a basal diet with 0.36% non-phytate P (NPP), while the treatment groups received diets containing 360 U of exogenous phytase/kg with 0.26% NPP (EP) or 360 phytase U of PTC/kg diet with 0.26% (PTC1), 0.21% (PTC2) or 0.16% (PTC3) NPP. 3. The results showed that there was no significant difference in egg production, average daily feed intake, feed efficiency, rate of broken or soft-shell egg production or egg mass among the treatments. There was no significant difference in eggshell thickness or eggshell strength. On the other hand, no differences in any of the bone variables were found between treatments. The faecal P percentage content in EP, PTC1, PTC2 and PTC3 groups was significantly lower than the control group. 4. In summary, the PTC could be used in the feed of laying hens instead of EP to reduce P excretion without effecting production and bone mineralisation.

CDX2 Stimulates the Proliferation of Porcine Intestinal Epithelial Cells by Activating the mTORC1 and Wnt/β-Catenin Signaling Pathways

Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/β-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/β-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/β-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/β-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H –thiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/β-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/β-catenin signaling pathways.