Effective methods for isolation and purification of extracellular vesicles from plants

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.     

Targeted Delivery of GDNF through the Blood–Brain Barrier by MRI-Guided Focused Ultrasound

Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), are promising therapeutic agents for neurodegenerative diseases. However, the application of GDNF to treat these diseases effectively is limited because the blood–brain barrier (BBB) prevents the local delivery of macromolecular therapeutic agents from entering the central nervous system (CNS). Focused ultrasound combined with microbubbles (MBs) using appropriate parameters has been previously demonstrated to be able to open the BBB locally and noninvasively. This study investigated the targeted delivery of GDNF MBs through the BBB by magnetic resonance imaging (MRI)-guided focused ultrasound. Evans Blue extravasation and histological examination were used to determine the optimum focused ultrasound parameters. Enzyme-linked immunosorbent assay was performed to verify the effects of GDNF bound on MBs using a biotin–avidin bridging chemistry method to promote GDNF delivery into the brain. The results showed that GDNF can be delivered locally and noninvasively into the CNS through the BBB using MRI-guided focused ultrasound combined with MBs under optimum parameters. MBs that bind GDNF combined with MRI-guided focused ultrasound may be an effective way of delivering neurotrophic factors directly into the CNS. The method described herein provides a potential means of treating patients with CNS diseases.     

Reductive soil disinfestation (RSD) alters gross N transformation rates and reduces NO and N2O emissions in degraded vegetable soils

Background and aims

Continuous vegetable cultivation in greenhouses can easily induce soil degradation, which considerably affects the development of sustainable vegetable production. Recently, the reductive soil disinfestation (RSD) is widely used as an alternative to chemical soil disinfestations to improve degraded greenhouse vegetable soils. Considering the importance of nitrogen (N) for plant growth and environment effect, the internal N transformation processes and rates should be well investigated in degraded vegetable soils treated by RSD, but few works have been undertaken.

Methods

Three RSD-treated and three untreated degraded vegetable soils were chosen and a ¹⁵?N tracing incubation experiment differentially labeled with ¹⁵NH₄NO₃ or NH₄ ¹⁵NO₃ was conducted at 25 °C under 50 % water holding capacity (WHC) for 96 h. Soil gross N transformation rates were calculated using a ¹⁵?N tracing model combined with Markov Chain Monte Carlo Metropolis algorithm (Müller et al. 2007), while the emissions of N₂O and NO were also measured.

Results

RSD could significantly enhance the soil microbial NH₄ ⁺ immobilization rate, the heterotrophic and autotrophic nitrification rates, and the NO₃ ^? turnover time. The ratio of heterotrophic nitrification to total inorganic N supply rate (mineralization + heterotrophic nitrification) increased greatly from 5.4 % in untreated vegetable soil to 56.1 % in treated vegetable soil. In addition, low release potential of NO and N₂O was observed in RSD-treated vegetable soil, due to the decrease in the NO and N₂O product ratios from heterotrophic and autotrophic nitrifications. These significant differences in gross N transformation rates, the supply processes and capacity of inorganic N, and the NO and N₂O emissions between untreated and treated vegetable soils could be explained by the elimination of accumulated NO₃ ^?, increased pH, and decreased electrical conductivity (EC) caused by RSD. Noticeably, the NO₃ ^? consumption rates were still significantly lower than the NO₃ ^? production rates in RSD-treated vegetable soil.

Conclusions

Except for improving soil chemical properties, RSD could significantly alter the supply processes of inorganic N and reduce the release potential of N₂O and NO in RSD-treated degraded vegetable soil. In order to retard the re-occurrence of NO₃ ^? accumulation, acidification and salinization and to promote the long-term productivity of greenhouse vegetable fields, the rational use of N fertilizer should be paid great attention to farmers in vegetable cultivation.     

Bifidobacterium lactis Meile et al. 1997 is a subjective synonym of Bifidobacterium animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974

Bifidobacterium lactis JCM 10602T (T = type strain) and Bifidobacterium animalis JCM 1190T were found to be phenotypically similar. These strains were subjected to investigation of their genetic relationships. The 16S rRNA sequence of B. animalis JCM 1190T was aligned with that of other Bifidobacterium species. B. animalis and B. lactis were the most closely related species in the phylogenetic tree and showed a high similarity in sequences (98.8%). The levels of DNA-DNA hybridization between the type strains of B. lactis and B. animalis ranged from 85.5 to 92.3%, showing that they represent a single species. It is proposed that B. lactis should be considered as a junior subjective synonym of B. animalis.     

不同性状的棉花及其杂交F1代对棉铃虫的抗性

通过连续两年的田间试验,系统、全面地研究了棉花对棉铃虫(Helicoverpa armigera)的形态抗虫作用。研究结果表明：在小区试验条件下,以百株累积卵量、百株平均幼虫量及百株平均蕾铃受害量为考察指标,红叶可使危害减轻28.1%、31.45%和36.O%;鸡脚叶可使其下降19.0%、25.4%和28.4%;窄卷苞叶使其降低25.8%、32.9%和34.0%;光叶使各指标分别降低32.7%、35.1%和37.3%。高棉酚(油腺)抗性主要表现为对幼虫的抗生作用,使幼虫量下降了45.1%,使蕾铃受害量下降了51.4%。此外,研究还表明,两种抗虫性状的抗性效果有累加作用,鸡脚叶与红叶联合作用使三项指标分别下降达48.3%、55.2%和57.9%;红叶与窄卷苞叶则使危害减轻了52.7%、58.3%和60.6%;具有光叶与较密油腺两个抗虫性状的品种受害可减轻29.9%、54.9%和58.3%。将一些具有相对性状的品种进行杂交,其F₁有不同的抗虫表现。红叶、鸡脚叶和光叶品种与其对照杂交后的F1代因在不同程度上保持了抗虫性状故能表现出抗性,而窄卷苞叶品种与其对照杂交后F1代苞叶正常,则未能表现出抗性。因小区条件限制了无蜜腺抗性的表达,无蜜腺小区未表现出明显差异。     

Activation of gelatinases by fibrin is PA/plasmin system-dependent in human glomerular endothelial cells

Evidence suggests that fibrin deposit is related to severity of glomerulonephropathy. Fibrin is considered to play an active role beyond a haemostatic plug or temporary matrix in response to injury. We have reported that fibrin induced specific morphological changes and up-regulated intercellular adhesion molecule-1 expression of glomerular endothelial cells (GECs). Changes of gelatinases activity have been implicated playing a prominent role in glomerular diseases involving matrix turnover. This study examined whether overlying fibrin influences the expression of gelatinase A and B in cultured human GECs and mechanism underlying the activation. No gelatinase activity was detectable in supernatant of cultured GECs; however, physiological concentration of fibrin (0.5–2.0 mg/ml) induced a dramatic expression of activated MMP-2 and MMP-9 at both mRNA and protein level in a dose and time dependent manner. Increased mRNA level of membrane-type 1 matrix metalloproteinases (MT1-MMPs) was also found. Interestingly, we observed that fibrin also induced the expression of tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 by casein zymographic and reverse zymographic analysis. Fibrin plate assay revealed the net activity was PA predominant. Serine protease inhibitor aprotinin blocked the conversion of pro-gelatinase A and B to their active forms. The results demonstrate that overlying fibrin increased the secretion of gelatinase A and B from GECs. PA/plasmin proteolytic pathways contributed to the activation of gelatinases.     

小麦-中间偃麦草双体异附加系的选育和鉴定

在小麦-中间偃麦草59个杂交后代种质系中,筛选出6个小麦-中间偃麦草双体异附加系(line0605,line0607,line0609,line0610,line0611,line0625),并对其进行了形态学、白粉病抗性、细胞学和RAPD鉴定。形态学结果表明:6个双体异附加系农艺性状较好地结合了双亲的优良特点;细胞学结果表明:6个双体异附加系具有高度的细胞学稳定性,花粉母细胞减数分裂中期Ⅰ(PMCMI)的染色体构型为2n=22Ⅱ;RAPD分析表明:在供试的209个随机引物中有5个引物分别能在6个异附加系中稳定地扩增出不同的特异带型,可以作为各个异附加系所附加染色体的特异分子标记;白粉病抗性鉴定结果表明:line0605表现免疫,line0610和line0625表现高抗,line0607表现中抗,line0609和line0611表现中感。     

Ad-sTNFRI-IgGFc腺病毒载体的构建及其转染人气道平滑肌细胞

TNF-a是一种具有广泛生物学活性的前炎症细胞因子, 参与哮喘整个病理生理过程。可溶性肿瘤坏死因子受体(sTNFR)可以拮抗肿瘤坏死因子活性, 已被用来治疗与TNF相关的炎性疾病。将sTNFRI-IgGFc基因插入腺病毒穿梭质粒pDC316, 与辅助质粒pBHGloxΔE1, 3Cre共转染HEK293细胞, 重组产生Ad-sTNFRI-IgGFc, PCR鉴定毒种正确后, 进行扩增、纯化和滴度测定, 转染人气道平滑肌细胞, 利用RT-PCR、免疫组化方法, 流式细胞仪, ELISA检测转染后细胞中sTNFRI-IgGFc的转录和表达。实验结果证明成功构建了Ad-sTNFRI-IgGFc腺病毒载体, 感染性滴度达3×1010 TCID50/mL, 200 moi转染气道平滑肌细胞阳性率达99.32%, 转染后气道平滑肌细胞在mRNA和蛋白水平均有sTNFRI-IgGFc表达。转染上清稀释64倍后仍对TNF有拮抗活性。为将表达sTNFRI-IgGFc腺病毒基因治疗哮喘的实验研究提供了基础。     

中国蜘蛛抱蛋属一新记录种——博格纳蜘蛛抱蛋

报道了产自中国云南喀斯特地区的蜘蛛抱蛋属一新记录种——博格纳蜘蛛抱蛋（Aspidistra bogneri H.-J.Tillich）。该种以前报道仅产于越南宁平省菊芳国家公园（Ninh Binh,Cuc Phuong National Park）,本次是中国首次记录。对该种的特征进行了详细描述并提供了彩色图片,凭证标本存放于中国科学院昆明植物研究所标本馆（KUN）。     

寇氏隐甲藻（Crypthecodinium cohnii）细胞核骨架与中间纤维...

Nuclear matrix (NM) and intermediate filament (IF) scaffold in primitive eukaryote Crypthecodinium cohnii were shown using selective extraction together with embedment-free electron microscopy, whole mount cell preparation and immunoblot techniques. There exists a delicate NM-IF network spreading over cytoplasm and nucleus in dinoflagellate cells, however, nuclear lamina is undeveloped. The diameter of NM fiber is about 3-5 nm and IF is 10 nm. Chromosomes are connected with NM filament network. Immunoblot analysis showed that dinoflagellate contained keratin-like polypeptides (63 kD and 67 kD) while mammalian lamin antibodies did not crossreact with dinoflagellate total protein. Our experiment results demonstrated that a framework similar to NM-IF scaffold in mammalian cell appeared in primitive eukaryote. We propose that: (1) NM-IF scaffold is not restrict to vertebrate cell, and it may be originated from early stages of eukaryote evolution; (2) Keratin is probably very conservative; (3) Compared with IF, lamina might appear late in evolution, and some of primitive characteristics of dinoflagellate nucleus may be related to the lack of lamina.