Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5 °C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12 °C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5 °C/min from 20 to −50 °C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5 °C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.     

猪环状RNA IGF1R促进脂肪细胞分化

环状RNA(circular RNA, circRNA)作为竞争性内源RNA(competitive endogenous RNA, ceRNA)在细胞分化调控中发挥着重要作用。本研究旨在对猪环状RNA IGF1R(circular RNA insulin-like growth factor 1 receptor, circIGF1R)进行鉴定及分析,探明其表达规律,构建猪circIGF1R相关的ceRNA调控网络,并探究其异位表达对小鼠间充质干细胞(C3H10T1/2)成脂分化的调控作用。通过正反向引物PCR、Sanger测序、RNase R酶消化检测和qRT-PCR验证circIGF1R是胰岛素样生长因子1受体(insulin-like growth factor 1 receptor, IGF1R)第二外显子形成的circRNA,它在猪各组织中均有表达,且其表达量在脂肪组织中随日龄增加呈上升趋势;使用miRDB、TargetScan和miRWalk在线软件预测circIGF1R靶基因,运用RNAhybrid软件进行结合位点预测,使用DAVID生物信息功能分析软件对候选靶基因进行GO和KEGG富集分析,运用Cytoscape软件构建ceRNA网络,基于基因表达相关性和预测的靶标关系,绘制了GO和KEGG富集分析及构建了ceRNA网络;双荧光素酶报告基因分析证明circIGF1R及FABP4可与ssc (Sus scrofa chromosome) -miR-133a-5p结合;成功构建circIGF1R过表达载体,在间充质干细胞C3H10T1/2中异位表达,过表达circIGF1R后关键成脂调控因子CEBPα、CEBPβ、FABP4和PPARγ极显著升高(P＜0.01),脂滴数量显著增加。本研究结果证明,circIGF1R在猪脂肪组织中存在,并且可能通过ceRNA机制正调控C3H10T1/2细胞成脂分化,为进一步研究circIGF1R调控猪前体肌内脂肪细胞成脂分化奠定理论基础。     

Epstein-Barr Virus_Encoded LMP1 Upregulates MicroRNA-21 to Promote the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin-Induced Apoptosis by Suppressing PDCD4 and Fas-L

Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC₅₀ value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.     

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco, sorghum and rice

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.     

Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed

Background

Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.

Methodology/Principal Findings

Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0–8.0, which is in agreement with the normal environment of digestive tract.

Conclusion/Significance

Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.     

Immunostimulating Effects of Extract of Acanthopanax sessiliflorus

As malfunction/absence of immune cells causes a variety of immunosuppressive disorders and chemical synthetic drugs for curing these diseases have many adverse effects, vigorous studies are being conducted. The Acanthopanax family has been used as traditional medicines for gastric ulcer, diabetes, etc. and culinary materials in East-South Asia. In this study, the immunostimulating properties of A. sessiliflorus were evaluated. A. sessiliflorus increased not only the splenocyte number but also immune-related cytokines such as TNF-α. However, it could not upregulate the expressions of IFN-γ and IL-2. A. sessiliflorus increased the swimming time, and comparison of organ weights relative to body weights for immune-related organs such as the spleen and thymus after a forced swim test showed that it could recover the spleen and thymus weights. It also increased the expression of TNF-α and slightly increased the concentration of IFN-γ but not IL-2. From the results, we concluded that as A. sessiliflorus has not only a host defense effect but also a stress-ameliorating property, further study it will be a promising material of immunostimulating material.     

Ⅲ型纤连蛋白组件包含蛋白5通过抑制ERK1/2磷酸化抑制C3H10T1/2细胞成脂分化

旨在探究Ⅲ型纤连蛋白组件包含蛋白5(type Ⅲ domain-containing protein5,FNDC5)对C3H10T1/2细胞成脂分化的调控作用.利用qRT-PCR和Western印迹检测FNDC5在C3H10T1/2细胞成脂分化过程中的时序性表达规律;构建慢病毒包被的过表达/干扰FNDC5载体,转染C3...     

1989—2019年西北地区干燥度指数时空变化及其对气候因子的响应

基于西北地区143个气象站点的气象数据,采用FAO-56 Penman-Monteith公式计算潜在蒸发量,并结合降水量计算西北地区1989—2019年干燥度指数(AI),采用Mann-Kendall趋势分析、小波分析、偏微分方程等方法来揭示其变化趋势、变化周期和气候因子对AI的贡献率。结果表明: 1989—2019年,西北地区AI整体呈不显著的减小趋势,其中,青海呈显著减小趋势,新疆呈不显著的上升趋势;研究区AI在2010年发生了突变,AI变化存在1个17年的主周期。西北地区AI呈现出由东南部向中部、西北部向中部增加的空间格局。西北地区AI变化的倾向率为-1.267·(10 a)^-1,其中,甘肃、宁夏、陕西、青海和新疆AI变化的倾向率分别为-1.17、-0.41、-0.49、-1.77和-2.73·(10 a)^-1。青海小灶火、新疆库尔勒、阿克苏和吐鲁番地区干旱风险发生的可能性较高。降水量和实际水汽压是影响甘肃、宁夏、青海、陕西AI变化的主要气侯因子,影响新疆AI变化的主要气侯因子为潜在蒸散、太阳辐射和平均气温。     

动物对孢子植物的传播模式及进化意义

孢子植物物种多样性丰富, 是自然生态系统的重要组成部分。孢子植物的传播通常被认为主要依靠风、水、弹力等非生物媒介, 而动物的作用往往被忽略。本文主要概述了: (1)孢子植物对动物传播的适应: 一方面孢子植物可为动物提供食物、庇护所、繁殖场所等, 另一方面孢子植物也可产生视觉、嗅觉等方面的线索来吸引动物, 从而促进动物传播其繁殖体。(2)动物对孢子植物的传播模式: 包括体内传播(消化道和组织寄生)和体外传播两种, 这些模式都能对孢子植物繁殖体进行有效传播。由于动物间形态或生活习性的不同, 以致传播距离存在差异, 最短距离为0.1 cm, 最长距离可从北半球至南半球。(3)动物对孢子植物传播的生态与进化意义; 由于某些孢子植物繁殖体的结构特点或萌发的需求, 以致其繁殖体只能通过动物的传播才能得以定殖, 因此动物与孢子植物之间存在密不可分的关系。目前, 动物对孢子植物的传播研究主要是描述性的内容以及研究单方面的传播途径, 建议在今后的研究中考虑动物对孢子植物传播的有效性以及多途径同时传播对孢子植物定殖的影响, 同时应更加关注孢子植物和动物互惠关系的形成、维持机制及将来的进化趋势。