Nanoparticle for siRNA delivery and its pancreatic cancer targeting ability

AIM: To synthesize a safe, efficient and targeted nanoparticulate carrier for siRNA delivery to pancreatic cancer cells. METHODS: Iron oxide nanocrystal with carboxylic acid group-polyethyleneimine (IONP-PEI) was synthesized and investigated as a nonviral carrier of siRNA to the pancreatic cells. The size, surface and charge using zeta potential were characterized. The perfect charge ratio between amino groups of IONP-PEI and phosphate groups of siRNA (N/P) was determined by the transfection efficiency detection, gel retardation assay and MTS assay. An antibody-directed nonviral vector, scFvCD44v6-IONP-PEI nanoparticle attaching to the cancer-associated CD44v6 single-chain variable fragment, was constructed as a cancer-targeting nanocarrier for siRNA delivery. Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFvCD44v6-IONP-PEI/siRNA complexes in the cells. The transfection efficiency, fluorescence intensity and the expression of KRAS at mRNA and protein levels in the cells transfected by IONP-PEI/siRNA and scFvCD44v6-IONP-PEI/siRNA were detected by flow cytometry, fluorescence microscopy, real-time PCR and Western blotting, respectively. RESULTS：The mass ratio of IONP to PEI was 0.75. The suitable ratio of N/P was 20. The averaged size and surface zeta potential of IONP-PEI/siRNA in deionized water were (51.3±2.2)nm (diameter) and (21.73±8.07)mV,respectively. Red fluorescence was seen in both targeting and nontargeting groups, which clearly revealed the intracellular distribution of siRNA and delivery agents. Transfection efficiencies in targeting and nontargeting groups were (89.75±1.81)% and (59.87±4.52)%, respectively. Down-regulation of the KRAS mRNA in Panc-1 cells transfected with siKRAS by scFvCD44v6-IONP-PEI and IONP-PEI was up to (34.02± 6.15)% and (51.09±6.70)%, respectively. The protein level of KRAS was lower in targeting group than that in nontargeting group. CONCLUSION：scFvCD44v6-IONP-PEI is a safe and efficient nanoparticulate carrier for gene delivery. It is more effective to transfer siRNA into the cells and mediate gene silencing effect in vitro than the nontargeting group.     

Transwell contact co-culture promotes growth and differentiation of single-dissociated iPSCs

AIM：To investigate the promoting role of Transwell contact co-culture system in the growth and differentiation of single-dissociated induced pluripotent stem cells (iPSCs). METHODS：Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃ and 5% CO2 for 8 h. Accutase digestion and 40 μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs, and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks. The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction (qPCR), immunofluorescence staining, live & dead cell staining and alkaline phosphatase (ALP) staining. The group of iPSCs cultured in conventional medium was used as control group 1. The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2. RESULTS：The bovine CECs showed typical hexagonal cobblestone shape. iPSCs showed colony-like growth, while became single-dissociated cells after Transwell contact co-culture with bovine CECs for 3 d. The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4. The mRNA expression levels of Nanog, Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05). The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01). After 14 d of induced differentiation co-culture, the single-dissociated iPSCs showed rather uniform polygonal morphology, increased dimension and no obvious colony existence. Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed. The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased, and had statistically significant difference compared with control group 1 (P<0.01). CONCLUSION：When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs. Transwell contact co-culture system not only enhances the growth of single-dissociated iPSCs, but also promotes their differentiation.     

北方蚕区第五批桑品种鉴定（山西点）试验报告

2011—2013年,山西省蚕业科学研究院对北方蚕区3个单位7个桑品种进行了鉴定。田间调查和实验室鉴定结果表明,7个参鉴桑品种生长势强、产叶量高,品质特性良好,在形态特征和生物学特性方面与对照存在不同程度的差异,而昌盛、9330两个品种的表现比较突出。     

稻瘟病菌MoYpt5蛋白互作网络预测

稻瘟病菌(Magnaporthe oryzae)共有11个假定的Rab蛋白家族成员,本文选取了MoYpt51(MGG_06241)和MoYpt52(MGG_01185)进行了生物信息学分析。通过搜索多个大型蛋白互作数据库和文献,共得到数百个与核心蛋白互作的蛋白和互作对。利用信息处理技术和高效制图平台将这些蛋白互作对构建成互作网络,得到若干个具有生物学意义的模块。结果表明,筛选得到的互作蛋白中,有的参与了蛋白降解的泛素途径(MGG_04053等)、囊泡介导的蛋白胞内运输(MGG_01238等),有的在蛋白、染色体的组装和修饰等过程(MGG_03677等)起重要作用。大部分假定互作蛋白定位于细胞质和质膜上,为其与目标蛋白互作提供了空间可能性。     

风沙黄土区排土场不同恢复类型植物群落与土壤种子库特征

风沙黄土区排土场作为一种人造生态系统,自然条件恶劣,土壤贫瘠,植被恢复困难。为了探明有效的人工促进植被恢复措施,采用植被调查与种子库萌发试验相结合的方法,通过研究不同植被类型地上植物群落与土壤种子库特征及二者的关系,探讨了其植被恢复效益及潜力。结果表明:研究区人工植被恢复下地上植物群落中共47种植物,分属16科40属,土壤种子库共14种植物,分属5科13属,其中均以禾本科、菊科、藜科、豆科占主导地位; 灌木植被的地上植被和土壤种子库的物种多样性均表现为最优; 地上植被与土壤种子库密度的变化范围为88.48～495.47株/m²,74.74～1422.91粒/m²,且均在草地类型下最大。土壤种子库和地上植被的相似性普遍较低,相似性系数仅为0.16～0.23。因此,风沙黄土区排土场的植被恢复与重建需要加强保护与管理,可以考虑构建以草灌配置为主的人工植被恢复模式,保障群落的恢复潜力,并提高群落多样性与稳定性,亦可考虑引入外源种子库提升群落恢复的潜力。     

In vitro Selection of DNAAptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

Aptamers are specific nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high affinity and specificity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment （SELEX） was used to select and PCR amplify DNA sequences （aptamers） capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simplified affinity separation approach was employed, in which L. monocytogenes in exponential （log） phase of growth was used as the separation target. A fluorescently-labeled aptamer assay scheme was devised for detecting L. monoeytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest affinity was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and purification of complex markers or targets.     

深松垄作对土壤物理性状及玉米产量的影响

为探讨深松耕作在半干旱地区气候条件下对玉米生长发育的影响,以玉米品种先玉335为试验材料,研究了春深松30cm,秋深松30cm垄作及常规表层旋耕15cm耕作模式对耕层土壤水分状况、物理性状和玉米生长发育及产量的影响。结果表明:深松30cm垄作有利于降低15~35cm耕层土壤紧实度与容重,常规表层旋耕有利于降低0~15cm耕层土壤紧实度与容重;深松垄作比常规表层旋耕有利于蓄积降水和降雨,秋深松30cm处理蓄水效果好于春深松30cm处理,秋深松30cm、春深松30cm垄作处理分别比表层旋耕15cm处理增产11.15%和7.26%。     

不同叶面肥对黄瓜产量和品质的影响

为了探索黑龙江省东部地区黄瓜叶面肥的施用效果,以黄瓜品种中农6号为试材,设4个叶面肥处理,分别施用利果美、金牌、凯丰和巨尔,研究了同种浓度不同功效的叶面肥对大田黄瓜产量和品质的影响。结果表明:4种叶面肥均有提高黄瓜产量、VC含量和可溶性蛋白含量的作用,其中1 000倍液巨尔叶面肥处理的黄瓜产量和品质与对照差异极显著。     

浙江桂幼龄材的生长特性及主要材性

以3株6年生浙江桂幼龄材为试材,研究了其生物量、材积、基本物理力学性能及生长轮的宽度、纤维长度、宽度、微纤丝角和结晶度的径向变异规律。结果表明,浙江桂幼龄材单株平均总生物量27.93 kg,平均材积0.0124 m3,生长轮平均宽度9.15 mm（3.12~10.57 mm）,基本密度为0.414 g/ cm3,除髓心外其余各生长轮宽度和密度无明显差异,反映浙江桂生长较为稳定;顺纹抗压强度、抗弯强度和弹性模量等物理力学综合性能中上等;纤维长度为830.6μm（581~1043μm）,纤维宽度为23.6μm（21~24μm）,结晶度为38.46％~58.43％,微纤丝角为13.6°~24.6°;纤维长度、宽度和结晶度自髓心向外逐渐增加,微纤丝角自髓心向外逐渐减小,结晶度与纤维长度、宽度间相关性极显著。     

露地栽培条件下大白菜氮肥投入阈值研究

通过田间试验,研究了不同施氮量对大白菜产量、氮肥利用率、硝态氮残留量的影响。结果表明,施氮对大白菜有显著的增产作用,但随着施氮量的增加产量变化不大,产量对施氮量的反应呈平台模式。氮肥利用率在3.3%-12.6%,并且随施氮量的增加呈先增加后降低的趋势,在施氮量为656.4 kg/hm^2（纯氮300 kg/hm^2＋有机肥12 000 kg/hm^2）时,氮肥利用率和作物产量最高。合理控制氮肥用量会促进作物对土壤氮素的吸收。施氮可明显提高0-180 cm剖面土壤NO3--N的累积量,残留的NO3--N的分布随施氮量的增加而显著增加,并且随施氮量的增加残留深度下移,表现出明显的底层累积。各处理土壤氮均集中在0-100 cm土层中,100 cm土层以下,各处理氮含量渐趋一致。综合不同施氮量对大白菜产量以及环境指标的影响,在本试验肥力水平下,大白菜氮肥投入阈值为656.4 kg/hm^2（纯氮300 kg/hm^2＋有机肥12 000 kg/hm^2）时,能够兼顾作物高产与环境安全。