MiR-126 Suppresses the Glucose-Stimulated Proliferation via IRS-2 in INS-1 β Cells

Background

Increasing evidence suggests that miR-126 participates in the glucose homeostasis through its target molecules. Although bioinformatics analysis predicts that miR-126 can bind with the insulin receptor substrate-2(IRS-2) mRNA at the “seed sequence”, but there are still no definitely reports to support it. In this study, we provided evidences that IRS-2 was one of the target genes of miR-126. And miR-126 has a proliferation inhibiting effects in INS-1 β cells, mainly through the suppression of IRS-2.

Methods

The 3’-UTR of IRS-2 regulated by miR-126 was analyzed by the luciferase assay and western blot. Furthermore, proliferation of INS-1 β cells stimulated by glucose was tested, and the association between IRS-2 and miR-126 were analyzed.

Results

We found that mutation of only three of the 6 “seed sequences” can eliminate the inhibition effect of miR-126. In INS-1 β cells, administration of miR-126 suppresses the proliferation, together with the unbalanced down-regulation of IRS-2 and IRS-1. Over-expression of IRS-2 can reverse the proliferation effect of miR-126, while not of IRS-1. These results suggested that miR-126 inhibited the β-cell proliferation via the inhibition of IRS-2 instead of IRS-1.Additionally, we also found that high glucose and insulin could stimulate the rapid production of endogenous miR-126 within 6 hours, together with the short term suppression of IRS-1 and IRS-2 expression, and intensify the unbalanced expression of IRS-1 and IRS-2.

Conclusions

IRS-2 was one of the targets of miR-126. MiR-126 inhibited the β-cell proliferation through IRS-2 instead of IRS-1. MiR-126 may take part in the glucose homeostasis both through its target IRS-2 and IRS-1. The unbalance between IRS-1 and IRS-2 caused by miR-126 may play an important role in type 2 diabetes.     

Probing the polarity and water environment at the protein-peptide binding interface using tryptophan analogues

7-Azatryptophan and 2,7-diazatryptophan are sensitive to polarity changes and water content, respectively, and should be ideal for studying protein-protein and protein-peptide interactions. In this study, we replaced the tryptophan in peptide Baa (LKWKKLLKLLKKLLKLG-NH₂) with 7-azatryptophan or 2,7-diazatryptophan, forming (7-aza)Trp-Baa and (2,7-aza)Trp-Baa, to study the calmodulin (CaM)-peptide interaction. Dramatic differences in the (7-aza)Trp-Baa and (2,7-aza)Trp-Baa fluorescence properties between free peptide in water and calmodulin-bound peptide were observed, showing a less polar and water scant environment at the binding interface of the peptide upon calmodulin binding. The affinity of the peptides for binding CaM followed the trend Baa (210±10 pM)<(7-aza)Trp-Baa (109±5 pM)<(2,7-aza)Trp-Baa (45±2 pM), showing moderate increase in binding affinity upon increasing the number of nitrogen atoms in the Trp analogue. The increased binding affinity may be due to the formation of more hydrogen bonds upon binding CaM for the Trp analogue with more nitrogen atoms. Importantly, the results demonstrate that (7-aza)Trp and (2,7-aza)Trp are excellent probes for exploring the environment at the interface of protein-peptide interactions.     

包埋法固定化对硫氧化微生物菌群结构和功能的影响

【目的】为探讨包埋法固定化过程对硫氧化菌群硫化物去除能力及菌群微生物群落结构的影响,【方法】以聚乙烯醇-海藻酸钠-活性炭为载体,对硫氧化菌群进行了固定化,并采用富含硫化物的无机盐培养基,对比固定化与非固定化硫氧化菌群对硫化物的氧化去除能力。同时,利用PCR-DGGE技术,探讨硫氧化菌群在固定化前后以及在硫化物氧化去除过程中微生物群落结构变化。【结果】在对硫氧化菌群进行固定化之后,12 h之内对硫化物的最大去除能力从1000 mg/L下降为600 mg/L。硫氧化菌群的微生物群落结构发生了明显变化,但菌群中的硫氧化菌Catenococcus thiocycli未受影响,硫氧化菌Thioclava pacifica在菌群中的地位反而得到了强化。【结论】受制于底物在载体材料中的扩散迁移效率,硫氧化菌群对硫化物的氧化去除能力在固定化之后有所下降。由于不同微生物对固定化形成的微环境的适应能力以及对载体附着能力的不同,固定化对硫氧化菌群的微生物群落结构产生较大影响。     

表面展示表达果胶酯酶的重组酿酒酵母构建及乙醇发酵

【目的】以淀粉为原料的乙醇发酵工艺仍然是当前燃料乙醇的主要生产方式。然而,一些原料中含有的果胶物质不仅降低了乙醇产率,而且会导致醪液粘度增大,从而会进一步影响传质和传热、增加设备负担等。构建能够自主降解果胶质的重组酿酒酵母并应用于燃料乙醇生产是值得探索的领域。【方法】论文将来源于黑曲霉的果胶酯酶基因克隆于α因子信号肽下游并通过酵母α-凝集素C-端蛋白的介导构建了在细胞表面锚定表达果胶酯酶的重组酿酒酵母PE。【结果】重组酵母的果胶酯酶表达水平达到2.6 U/g(菌体湿重),并进一步鉴定了重组果胶酯酶性质。以甘薯粉为原料的同步糖化发酵实验中,重组酵母PE的乙醇浓度和乙醇转化率分别达到95 g/L和88.1%,与出发菌株相比提高了2.2%。更重要的是,表面展示果胶酯酶能够显著降低发酵过程中的发酵液粘度。【结论】通过在工业酿酒酵母表面展示表达果胶酯酶不仅能够提高糖化酶等的作用效果和酿酒酵母的代谢能力,而且能够显著降低乙醇生产过程中发酵液的粘度,将对工业规模乙醇生产在降低设备负担、节约能耗方面具有一定的潜在价值。     

Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension

Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.     

Development of a loop‐mediated isothermal amplification assay for rapid and sensitive detection of Sporisorium scitamineum in sugarcane

Smut disease caused by Sporisorium scitamineum is one of the most destructive sugarcane diseases worldwide. The pathogen spreads primarily through infected sugarcane setts, and hence, the use of disease‐free planting materials is essential for preventing disease development in the field. In this study, a species‐specific loop‐mediated isothermal amplification (LAMP) assay was developed for rapid and accurate detection of S. scitamineum. Based on the differences in internal transcribed spacer (ITS) sequences of S. scitamineum, a set of four species‐specific primers, F3, B3, FIP and BIP, were designed by using a panel of fungal and bacterial species as controls. After optimization of the reaction conditions, the detection limit of LAMP assay was about 2 fg of the S. scitamineum genomic DNA in 25 µL reaction solution, 100‐fold lower than that of conventional polymerase chain reaction. The assay showed high specificity to discriminate all S. scitamineum isolates from nine other fungal and bacterial pathogens. The LAMP assay also detected smut infection from young sugarcane leaves with no visible smut‐disease symptoms. The findings from this study provide a simple, highly sensitive, rapid and reliable technique for early detection of S. scitamineum, which may be useful for sugarcane quarantine and production of smut‐free seedcanes. This is the first report of LAMP‐based assay for the detection of S. scitamineum in sugarcane.     

Chlorophylls <Emphasis Type="Italic">d</Emphasis> and <Emphasis Type="Italic">f</Emphasis> and their role in primary photosynthetic processes of cyanobacteria

The finding of unique Chl d- and Chl f-containing cyanobacteria in the last decade was a discovery in the area of biology of oxygenic photosynthetic organisms. Chl b, Chl c, and Chl f are considered to be accessory pigments found in antennae systems of photosynthetic organisms. They absorb energy and transfer it to the photosynthetic reaction center (RC), but do not participate in electron transport by the photosynthetic electron transport chain. However, Chl d as well as Chl a can operate not only in the light-harvesting complex, but also in the photosynthetic RC. The long-wavelength (Q_y) Chl d and Chl f absorption band is shifted to longer wavelength (to 750 nm) compared to Chl a, which suggests the possibility for oxygenic photosynthesis in this spectral range. Such expansion of the photosynthetically active light range is important for the survival of cyanobacteria when the intensity of light not exceeding 700 nm is attenuated due to absorption by Chl a and other pigments. At the same time, energy storage efficiency in photosystem 2 for cyanobacteria containing Chl d and Chl f is not lower than that of cyanobacteria containing Chl a. Despite great interest in these unique chlorophylls, many questions related to functioning of such pigments in primary photosynthetic processes are still not elucidated. This review describes the latest advances in the field of Chl d and Chl f research and their role in primary photosynthetic processes of cyanobacteria.