GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.     

樟芝多糖通过降低NLRP3 Caspase1炎性小体表达改善帕金森小鼠神经行为学的机制研究

目的:研究樟芝多糖通过降低NLRP3-Caspase1炎性小体表达改善6-OHDA构建的帕金森小鼠模型的行为学机制。方法:利用6-OHDA脑内注射构建帕金森小鼠模型，通过酪氨酸羟化酶（TH）免疫组化染色和行为学判定小鼠模型的构建成功。利用樟芝多糖进行干预，分别在干预前、干预后的第1、3、7天4个时间点进行神经行为学实验，分别采用转棒实验、爬杆实验检测小鼠自主行为能力以及协调能力，4个时间点取小鼠尾静脉外周血采用ELISA法检测外周血中Caspase1和IL-1β的表达，樟芝多糖干预第7天时待进行完行为学实验后小鼠断颈处死，取小鼠脑组织-纹状体，Western blot法检测纹状体中Caspase1、proCaspase1、NLRP3的表达，高效液相色谱检测纹状体中单胺类神经递质的表达，RT-QPCR检测Caspase1、NLRP3、IL-1β、IL-4、IL-6的mRNA表达。NISSl染色检测小鼠脑组织神经细胞凋亡情况。 结果:6-OHDA脑内注射可以造成小鼠帕金森样病变，且TH蛋白表达显著下调，樟芝多糖干预后小鼠的行为学得到显著改善（P<0.05），纹状体中Caspase1、proCaspase1、NLRP3的表达显著下调，与模型小鼠相比具有统计学差异（P<0.05），且相关炎症因子Caspase1、NLRP3、IL-1β、IL-4、IL-6的mRNA表达下调（P<0.05），纹状体中单胺类神经递质表达上升（P<0.05）。结论:樟芝多糖可以通过下调NLRP3-Caspase1炎性小体表达来改善6-OHDA构建的帕金森小鼠模型行为改善，这可能是樟芝多糖治疗帕金森的机制之一。     

Milk from cows fed a diet with a high forage:concentrate ratio improves inflammatory state,oxidative stress,and mitochondrial function in rats

Excessive energy intake may evoke complex biochemical processes characterized by inflammation, oxidative stress, and impairment of mitochondrial function that represent the main factors underlying noncommunicable diseases. Because cow milk is widely used for human nutrition and in food industry processing, the nutritional quality of milk is of special interest with respect to human health. In our study, we analyzed milk produced by dairy cows fed a diet characterized by a high forage:concentrate ratio (high forage milk, HFM). In view of the low n-6:n-3 ratio and high content of conjugated linoleic acid of HFM, we studied the effects of this milk on lipid metabolism, inflammation, mitochondrial function, and oxidative stress in a rat model. To this end, we supplemented for 4 wk the diet of male Wistar rats with HFM and with an isocaloric amount (82 kJ, 22 mL/d) of milk obtained from cows fed a diet with low forage:concentrate ratio, and analyzed the metabolic parameters of the animals. Our results indicate that HFM may positively affect lipid metabolism, leptin:adiponectin ratio, inflammation, mitochondrial function, and oxidative stress, providing the first evidence of the beneficial effects of HFM on rat metabolism.     

Stabilizing temperature-capacitance dependence of (Sr,Pb, Bi)TiO3-Bi4Ti3O12 solutions for energy storage

(1-x)Sr_0.7Pb_0.15Bi_0.1TiO₃-xBi₄Ti₃O₁₂ ((1-x)SPBT-xBIT, x = 0-0.125) bulk ceramics were developed and calcined via the solid-state method, aimed at the application of pulsed power capacitors. The phase structures, temperature stability, hysteresis loop, and discharge properties were systematically investigated. Considering both the temperature stability and dielectric properties, 0.925SPBT-0.075BIT bulk ceramics with a capacitance variation satisfying the X7R specification were developed for pulsed power capacitors. The energy storage density was 0.252 J/cm³, and the ceramics showed high temperature stability at 80 kV/cm. The discharge current waveforms of the 0.925SPBT-0.075BIT ceramics were recorded. A high discharge power density of approximately 1.01 × 10⁸ W/kg with an 8 Ω load resistor and short discharge period of 84 ns were achieved at 50 kV/cm. The good temperature stability properties and high power density show that the 0.925SPBT-0.075BIT ceramics are well suited for pulsed power capacitors with a wide temperature range.     

Evaluation of the effect of processing on cocoa polyphenols: antiradical activity,anthocyanins and procyanidins profiling from raw beans to chocolate

The content and composition of anthocyanins and procyanidins in fermented cocoa beans (from different geographic origins: Ecuador, Cameroon, Ivory Coast, Ghana and Nigeria), roasted nibs, cocoa mass and chocolate were determined, beside the determination of the total antiradical capacity. Concerning geographic origin, cocoa beans and processed products from Ecuador showed the highest levels of anthocyanins, followed by Nigeria and Cameroon. Generally, as cocoa beans were further processed, the levels of anthocyanins and flavan‐3‐ols decreased. The largest observed losses of phenolics occurred during roasting. A progressive decreasing trend in polyphenol concentration was observed in the other processed samples as well. Despite the original content of polyphenols in raw cocoa beans, technological processes imply a significant impact on cocoa quality, confirming the need of specific optimisation to obtain high value chocolate.     

Properties and applications of β‐glycosidase from Bacteroides thetaiotaomicron that specifically hydrolyses isoflavone glycosides

To modify the glycan part of glycosides, the gene encoding β‐glycosidase was cloned from Bacteroides thetaiotaomicron VPI‐5482. The cloned gene, bt_1780, was expressed in Escherichia coli MC1061 and the expressed enzyme was purified using Ni‐NTA affinity chromatography. The purified enzyme, BTBG, showed optimal activity at 50 °C and pH 5.5. Interestingly, this enzyme did not have any hydrolysing activity on ordinary β‐linkage–containing substrates such as xylobiose, lactose and cello‐oligosaccharide, but specifically hydrolysed isoflavone glycosides such as daidzin, genistin and glycitin. Compared to a commercial beta glucosidase, BTBG selectively hydrolysed isoflavone glycosides in soybean extract mixture solution. These results suggest that BTBG may be a specialized enzyme for the hydrolysis of glycosides and that the substrate specificity of BTBG is applicable for the bioconversion of isoflavone glycosides in the food industry.     

Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.     

Catalytic Hydrocracking of a Bitumen‐Derived Asphaltene over NiMo/γ‐Al2O3 at Various Temperatures

Hydrocracking of a bitumen‐derived asphaltene over NiMo/γ‐Al₂O₃ was investigated in a microbatch reactor at varying temperatures. The molar kinetics of asphaltene cracking reaction was examined by fitting the experimental data. Below a defined temperature, the molar reaction showed the first‐order kinetic feature while at higher temperatures secondary reactions such as coke formation became significant, causing deviation of the reaction behavior from the proposed first‐order kinetic model. Selectivity analysis proved that dominant products varied from gases to liquids to gases with increasing temperature, shifting the dominant reaction from C–S bonds cleavage to C–C bonds cleavage.     

Elucidation of colour development and microstructural characteristics of Allium sativum fumigated with acetic acid

It has been well known that the greening of Allium sativum cloves could be formed after immersed in acetic acid solution. Nonetheless, no investigation was reported on colour development of A. sativum in response to acetic acid vapour until now. In this study, the brief exposure of A. sativum to acetic acid vapour (200–400 ppm) was combined with controlled atmosphere (5%, 20% and 80% CO₂) packaging storage to break cell membrane and green garlic. The garlic bulbs were fumigated with acetic acid before controlled CO₂ atmosphere packaging for 25 days at 4 °C. Fumigation with 200 ppm acetic acid followed by high CO₂ atmosphere packaging (80% CO₂) facilitated the garlic greening. It was also verified that γ‐glutamyl transpeptidase was involved in garlic greening in present study, and the compromise of glacial acetic acid vapour fumigation and CO₂ gas atmosphere in package stored at low temperature could result in garlic greening as well.