Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

Long non‐coding RNA LINC01225 promotes proliferation,invasion and migration of gastric cancer via Wnt/β‐catenin signalling pathway

Emerging evidence has classified the aberrant expression of long non‐coding RNAs (lncRNAs) as a basic signature of various malignancies including gastric cancer (GC). LINC01225 has been shown to act as a hepatocellular carcinoma‐related gene, with its expression pattern and biological function not clarified in GC. Here, we verified that LINC01225 was up‐regulated in tumour tissues and plasma of GC. Analysis with clinicopathological information suggested that up‐regulation of LINC01225 was associated with advanced disease and poorer overall survival. Receiver operating characteristic (ROC) analysis showed that plasma LINC01225 had a moderate accuracy for diagnosis of GC. In addition, knockdown of LINC01225 led to retardation of cell proliferation, invasion and migration, and overexpression of LINC01225 showed the opposite effects. Mechanistic investigations showed that LINC01225 silencing inhibited epithelial‐mesenchymal transition (EMT) process and attenuated Wnt/β‐catenin signalling of GC. Furthermore, ectopic expression of Wnt1 or suppression of GSK‐3β abolished the si‐LINC01225‐mediated suppression against EMT, thereby promoting cell proliferation, invasion and migration of GC. In conclusion, LINC01225 promotes the progression of GC through Wnt/β‐catenin signalling pathway, and it may serve as a potential target or strategy for diagnosis or treatment of GC.     

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.     

IgA1 isolated from Henoch‐Schönlein purpura children promotes proliferation of human mesangial cells in vitro

Previous studies show that the proliferation of human mesangial cells (HMCs) played a significant part in the pathogenesis of Henoch‐Schönlein purpura nephritis (HSPN). The aim of this study was to explore the proliferation of HMCs induced by IgA1 isolated from the sera of HSP patients. HMCs were cultured in three different types of media, including IgA1 from patients with HSP (HSP IgA1 group), healthy children (healthy IgA1 group) and medium (control group). The proliferation of HMCs incubated with IgA1 was determined by cell counting kit‐8 assay and bromodeoxyuridine incorporation. The expression of ERK1/2 and phosphatidylinositol 3 kinase/protein kinase B/mammalian targets of the rapamycin (PI3K/AKt/mTOR) signals and transferrin receptor (TfR/CD71) was detected with the methods of immunoblotting. The results indicated that the proliferation of HMCs significantly increased in the HSP IgA1 group compared with that in the control group or the healthy IgA1 group (P < 0.001). Moreover, we found that IgA1 isolated from HSP patients activated ERK and PI3K/AKt/mTOR signals, and markedly increased TfR/CD71 expression in HMCs. These effects induced by IgA1 isolated from patients with HSP were inhibited by human TfR polyclonal antibody (hTfR pAb) and soluble human transferrin receptor (sTfR), indicating that IgA1‐induced HMC proliferation and ERK1/2 and PI3K/AKt/mTOR activation were dependent on TfR/CD71 engagement. Altogether, these data suggested that TfR/CD71 overexpression and ERK1/2 and PI3K/AKt/mTOR activation were engaged in HMC proliferation induced by IgA1 from HSP patients, which might be related to the mesangial injury of HSPN.     

The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2‘,7‘-dichlorodihydrofluorescein diacetate, to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry, with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide(PI), indicated that As2O3 (2μM) could induce apoptosis in NB4 cells prevailingly from G2/M phase, and this efficacy was enhanced upon co-administration of 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) (2.5μM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells‘ susceptibility to apoptosis by As2O3.     

垂体腺苷环化酶激活多肽抑制β淀粉样蛋白对Neuro-2a细胞神经毒性作用的机制

通过MTT方法检测细胞活性 ,同时采用激光共聚焦显微镜技术检测细胞内游离钙离子的瞬时运动 ,研究了垂体腺苷环化酶激活多肽 (pituitaryadenylatecyclaseactivatingpolypeptide 2 7,PACAP2 7)通过调节细胞内钙对抗淀粉样蛋白Aβ2 5 35引起Neuro 2a细胞神经毒性作用的可能机制。结果表明 ,PACAP在一定浓度范围内 (<0 1μmol/L)可提高Neuro 2a细胞增殖能力并对抗Aβ引起的神经毒性 ,此作用可以被PACAP受体竞争性拮抗剂PACAP6 2 7所抑制。 2 5 μmol/LAβ使细胞内钙离子缓慢上升 ,并有一个较长时间的平台期。 0 1μmol/L的PACAP使细胞内钙离子迅速升高后下降 ,10min后回到接近基线水平 ,伴有较长时间的不应期。用PACAP预处理细胞 10min后Aβ引起细胞内钙的慢上升不再出现。推测 ,PACAP受体激活引起瞬时内向钙离子运动 ,而后伴随一个较长时间的不应期 ,可能是一个消除凋亡或阻止凋亡启动的保护机制。     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.