Development and characterization of marker‐free and transgene insertion site‐defined transgenic wheat with improved grain storability and fatty acid content

Development of marker‐free and transgene insertion site‐defined (MFTID) transgenic plants is essential for safe application of transgenic crops. However, MFTID plants have not been reported for wheat (Triticum aestivum). Here, we prepared a RNAi cassette for suppressing lipoxygenase (LOX) gene expression in wheat grains using a double right border T‐DNA vector. The resultant construct was introduced into wheat genome via Agrobacterium‐mediated transformation, with four homozygous marker‐free transgenic lines (namely GLRW‐1, ‐3, ‐5 and ‐8) developed. Aided by the newly published wheat genome sequence, the T‐DNA insertion sites in GLRW‐3 and GLRW‐8 were elucidated at base‐pair resolution. While the T‐DNA in GLRW‐3 inserted in an intergenic region, that of GLRW‐8 inactivated an endogenous gene, which was thus excluded from further analysis. Compared to wild ‐type (WT) control, GLRW‐1, ‐3 and ‐5 showed decreased LOX gene expression, lower LOX activity and less lipid peroxidation in the grains; they also exhibited significantly higher germination rates and better seedling growth after artificial ageing treatment. Interestingly, the three GLRW lines also had substantially increased contents of several fatty acids (e.g., linoleic acid and linolenic acid) in their grain and flour samples than WT control. Collectively, our data suggest that suppression of grain LOX activity can be employed to improve the storability and fatty acid content of wheat seeds and that the MFTID line GLRW‐3 is likely of commercial value. Our approach may also be useful for developing the MFTID transgenic lines of other crops with enhanced grain storability and fatty acid content.     

Resveratrol inhibits cell differentiation in 3T3-L1 adipocytes via activation of AMPK

Resveratrol (Res) is a natural polyphenolic compound with anti-inflammatory and antioxidant properties. Also, Res can inhibit lipogenesis and adipocyte differentiation. However, the underlying mechanisms of Res's functions remain largely unknown. AMP-activated protein kinase (AMPK) is a key player in adipocyte differentiation. Therefore, the purpose of our study was to determine the role played by AMPK in the Res-mediated regulation of adipocyte differentiation. Incubation of 3T3-L1 cells with Res confirmed that Res inhibited adipocyte differentiation. The phosphorylation of AMPKα was increased by Res in a dose-dependent manner, while total AMPKα levels were unchanged, and peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) levels were decreased. Interestingly, pretreatment with AMPKα siRNA and Res promoted adipocyte differentiation, while the decrease of p-AMPKα increased PPARγ, C/EBPα, and SREBP-1c protein expression. Our study shows that Res is capable of inhibiting lipogenesis and differentiation of 3T3-L1 adipocytes via activation of AMPK, suggesting its potential therapeutic application in the treatment or prevention of obesity.     

A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

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Highlights? Splice-isoform switching favors prosurvival BCL2 family expression in human BC ? BC LSCs are quiescent and TKI-resistant in the marrow niche ? Sabutoclax, a pan-BCL2 inhibitor, enhances TKI sensitivity of bone marrow BC LSCs     

Bradykinin-bradykinin receptor (B1R) signalling is involved in the blood–brain barrier disruption in moyamoya disease

Moyamoya disease (MMD) is a rare disorder of the cerebrovascular system. It is a steno-occlusive disease that involves angiogenesis and blood–brain barrier (BBB) disruption. Bradykinin (BK), its metabolite des-Arg9-BK, and receptor (B1R) affect angiogenesis and BBB integrity. In this study, we aimed to investigate the changes in BK, B1R and des-Arg9-BK levels in the serum and brain tissues of patients with MMD and explore the underlying mechanism of these markers in MMD. We obtained the serum samples and superficial temporal artery (STA) tissue of patients with MMD from the Department of Neurosurgery of the Jining First People's Hospital. First, we measured BK, des-Arg9-BK and B1R levels in the serum of patients by means of ELISA. Next, we performed immunofluorescence to determine B1R expression in STA tissues. Finally, we determined the underlying mechanism through Western blot, angiogenesis assay, immunofluorescence, transendothelial electrical resistance and transcytosis assays. Our results demonstrated a significant increase in the BK, des-Arg9-BK and B1R levels in the serum of patients with MMD compared to healthy controls. Furthermore, an increase in the B1R expression level was observed in the STA tissues of patients with MMD. BK and des-Arg9-BK could promote the migratory and proliferative abilities of bEnd.3 cells and inhibited the formation of bEnd.3 cell tubes. In vitro BBB model showed that BK and des-Arg9-BK could reduce claudin-5, ZO-1 and occluding expression and BBB disruption. To the best of our knowledge, our results show an increase in BK and B1R levels in the serum and STA tissues of patients with MMD. BK and Des-Arg9-BK could inhibit angiogenesis, promote migratory and proliferative capacities of cells, and disrupt BBB integrity. Therefore, regulating BK, des-Arg9-BK and B1R levels in the serum and the brain could be potential strategies for treating patients with MMD.     

Soil bacterial community composition and diversity respond to cultivation in Karst ecosystems

Soil microorganisms play vital roles in recovering and maintaining the health of ecosystems, particularly in fragile Karst ecosystems that are easily degraded after cultivation. We investigated the composition and diversity of soil bacterial communities, based on RFLP and 16S rDNA sequencing, in a cropland, a naturally revegetated land with former cultivation disturbance and a primeval forest in the subtropical Karst of southwest China. Our results illustrated that Proteobacteria accounted for 44.8% of the 600 tested clones, making it the most dominant phylum observed. This phylum was followed by Acidobacteria and Planctomycetes for the three Karst soils analyzed. Compared with the primeval forest soil, the proportions of Proteobacteria were decreased by 30.2 and 37.9%, while Acidobacteria increased by 93.9 and 87.9%, and the Shannon-Wiener diversity indices and the physicochemical parameters declined in the cropland and the revegetated land, respectively. Among the three soils, the proportion of dominant bacterial phyla and the diversity indices in the revegetated land were similar to the cropland, implying the bacterial community in the cropland was relatively stable, and the after-effects of cultivation were difficult to eliminate. However, similar distributions of the four Proteobacteria subphyla were observed between the revegetated land and the primeval forest soil. Furthermore, the proportion of Rhizobiales belonging to α-Proteobacteria was sharply decreased with cultivation compared to the primeval forest soil, while a small cluster of Rhizobiales recurred with vegetation recovery. These results indicated that although the subphyla of the dominant bacterial phylum had some positive responses to 20 years of vegetation recovery, it is a slow process. Our results suggest that priority should be given to conserve the primeval forest and inoculation of functional microorganisms on the basis of vegetation recovery may be more effective for the restoration of Karst ecosystems after cultivation.