TRAP-rc,Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.     

Continuous removal of endocrine disruptors by versatile peroxidase using a two‐stage system

The oxidant Mn³⁺‐malonate, generated by the ligninolytic enzyme versatile peroxidase in a two‐stage system, was used for the continuous removal of endocrine disrupting compounds (EDCs) from synthetic and real wastewaters. One plasticizer (bisphenol‐A), one bactericide (triclosan) and three estrogenic compounds (estrone, 17β‐estradiol, and 17α‐ethinylestradiol) were removed from wastewater at degradation rates in the range of 28–58 µg/L·min, with low enzyme inactivation. First, the optimization of three main parameters affecting the generation of Mn³⁺‐malonate (hydraulic retention time as well as Na‐malonate and H₂O₂ feeding rates) was conducted following a response surface methodology (RSM). Under optimal conditions, the degradation of the EDCs was proven at high (1.3–8.8 mg/L) and environmental (1.2–6.1 µg/L) concentrations. Finally, when the two‐stage system was compared with a conventional enzymatic membrane reactor (EMR) using the same enzyme, a 14‐fold increase of the removal efficiency was observed. At the same time, operational problems found during EDCs removal in the EMR system (e.g., clogging of the membrane and enzyme inactivation) were avoided by physically separating the stages of complex formation and pollutant oxidation, allowing the system to be operated for a longer period (～8 h). This study demonstrates the feasibility of the two‐stage enzymatic system for removing EDCs both at high and environmental concentrations. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:908–916, 2015     

不同低温胁迫下粳稻耐冷种质的孕穗期耐冷性比较

为粳稻孕穗期耐冷标准品种的建立和耐冷遗传育种研究提供优异基因资源,利用人工低温气候室,采用15℃、17℃、19℃3种胁迫温度,4d和6d两种胁迫时间,3×2交互式设计的方法,对来自黑龙江、吉林、辽宁和云南的12份粳稻种质进行了孕穗期耐冷性强度的研究。结果表明,随着低温胁迫的增强,各水稻品种的平均空壳率都随之增加;在15℃/6d胁迫下,供试品种平均空壳率的方差和变异系数达到最大,该胁迫强度可被选用于孕穗期强耐冷种质的筛选。依据15℃/6d胁迫下供试品种平均空壳率的方差分析及多重比较结果,空育131和龙稻3号具有极强的孕穗期耐冷性。不同地区可根据参试品种在本试验中的耐冷表现,并结合当地水稻种植区的光温条件选择相应的耐冷标准品种。     

北京夏植黄瓜内生真菌区系研究

为了解黄瓜植株内生真菌的区系组成及其变化,从而为进一步研究黄瓜内生真菌的生态和功能奠定基础,对采自北京延庆的不同品种和不同生育期的40株黄瓜进行了内生真菌的分离培养。经形态学鉴定和18S rDNA序列分析,分离到的1,024株内生真菌属于18属,其中Exserohilum和Neocosmospora尚未见内生真菌的报道。Alternaria、Aspergillus、Chaetomium、Cladosporium和Fusarium在各生育期和各器官普遍存在。其中,Alternaria在叶中的定殖率达47.0%,远高于在其他器官中的定殖率;Fusarium在根中的定殖率达32.5%,远高于在其他器官中的定殖率。多数真菌类群表现出不同程度的器官偏好性,有些真菌类群则只出现在特定器官中。叶和根的内生真菌类群数量和总定殖率均高于茎和果实。随着黄瓜的生长,各器官内生真菌的类群数在增加,部分真菌的定殖率也呈上升趋势,但Neocosmospora和Chaetomium在各器官中的定殖率则随植株生长呈下降趋势。     

Relationship between survival rates of associative nitrogen-fixers on roots and yield response of plants to inoculation

Abstract: The population dynamics of associative nitrogen-fixers Azospirillum lipoferum 137, Arthrobacter mysorens 7, Flavobacterium sp. L30 and phosphate-solubilizing strain Agrobacterium radiobacter 10 in soil and the rhizoplane of inoculated plants was studied in pot and field experiments. All of the present strains were able to actively colonize the rhizoplane of barley, wheat, oat, tomatoes, rape, and alfalfa. For the most part the population size and dynamics of introduced bacteria depended only slightly on the plant genotype and soil conditions. The overall pictures of survival of the strains in soil and on plant roots were similar. The reliable effect of inoculation on plants was observed only in individual cases. No correlation was established between survival of introduced bacteria and their effect on plant development. It was concluded that the influence of plants on survival of bacteria was not specific. In contrast, the plant response to inoculation was conditioned to a greater extent by the plant genotype.     

A cell-detaching reactor for inoculation of anchorage-dependent CHD and Vero cells between stepwise-expanded bioreactors

A cell-detaching reactor was developed to collect cells growing on microcarriers for inoculation between stepwise-expanded bioreactors. It consisted of a trypsinization zone and a separation zone, which were separated by a 200-mesh stainless steel screen. The screen allowed the cells only to pass through to the next bioreactor, after the cells have been trypsinized and detached from microcarriers. The operating feasibility of the cell-detaching reactor was tested with anchorage-dependent recombinant Chinese hamster ovary (rCHO) and African green monkey kidney (Vero) cells. rCHO and Vero cells were first cultured in a small microcarrier bioreactor, and then inoculated via the cell-detaching reactor into either a packed-bed bioreactor (for rCHO cells) or a larger microcarrier bioreactor (for Vero cells). For rCHO cells, the cell density reached 1.3 × 10⁷ cells/ml in the perfusion culture, and Vero cells reached 1.3 × 10⁶ cells/ml in the batch culture.