Alkaline transfer of DNA to plastic membrane

DNA forms a stable complex with Gene Screen Plus membrane at alkaline pH. Based on this, a method of alkaline transfer of DNA from agarose gel to Gene Screen Plus membrane was elaborated. The procedure entails the use of 0.4 M NaOH for both, the DNA denaturation and DNA transfer steps. The alkaline transfer offers a higher hybridization efficiency and simplifies the transfer procedure as compared with the standard method of DNA transfer at neutral pH. In addition, it can be used to remove RNA contamination from the transferred DNA.     

山羊不同组织器官的内参基因筛选

本研究通过比较9个内参基因在山羊不同组织中的表达水平进而确定最适合研究山羊组织表达的内参基因。本试验以简州大耳羊为试验材料,利用实时荧光定量PCR技术分析9个内参基因(GAPDH,PPIA,18S rRNA,PPIB,UXT,RPLP0,ACTB,EIF3K和TBP)在心脏、肝脏、脾脏、肺脏、肾脏、大肠、瘤胃、背最长肌和皮下脂肪等组织中的表达差异情况,并利用geNorm、NormFinder和BestKeeper等程序分析了它们的表达稳定性。geNorm和NormFinder程序一致显示TBP表达最稳定,其次是UXT和RPLP0;BestKeeper分析显示18S rRNA表达最为稳定,其次为TBP和ACTB;3个程序一致认为GAPDH表达稳定性最差。综合3个程序分析得出TBP最适合作为山羊组织中的内参基因,其次为UXT和RPLP0,GAPDH表达稳定性最差,不适合作为山羊组织内参,这为后续研究其他目的基因在山羊组织器官中的表达模式提供数据保障。     

Assessment of the total effective xenoestrogen burden in extracts of human placentas

We have standardized a method to assess the total effective xenoestrogen burden (TEXB) in human placentas by the extraction and separation by high-performance liquid chromatography of two fractions containing lipophilic xenoestrogens (alpha) and endogenous hormones (beta), followed by assessing their estrogenicity in MCF-7 breast cancer cell-based E-Screen and Yeast Estrogen Screen (YES) bioassays. The means of TEXB alpha concentrations (in estradiol equivalent (Eeq) units) were 1.32 and 0.77 Eeq pM g^?1 placenta in the E-Screen and YES, respectively; TEXB beta concentrations were 6.97 and 11.56 Eeq pM g^?1 placenta, respectively. The interclass correlation coefficient was low and a fair level of agreement was observed after kappa test correction. According to the E-Screen and YES, TEXB alpha was ≥LOD in 70.0 and 55.0% of the placentas and 92.5 and 82.5% in beta, respectively. Although both bioassays can be recommended for assessing TEXB, there is greater experience with the use of the E-Screen for estrogenic assessment after extensive extraction of complex human matrices.     

Design,synthesis and algicides activities of thiourea derivatives as the novel scaffold aldolase inhibitors

By using a new Fragment-Based Virtual Screen strategy, two series of novel FBA-II inhibitors (thiourea derivatives) were de novo discovered based on the active site of fructose-1, 6-bisphosphate aldolase from Cyanobacterial (CyFBA). In comparison, most of the N-(2-benzoylhydrazine-1-carbonothioyl) benzamide derivatives (L14～L22) exhibit higher CyFBA-II inhibitory activities compared to N-(phenylcarbamothioyl) benzamide derivatives (L1～L13). Especially, compound L14 not only shows higher CyFBA-II activity (K_i?=?0.65?μM), but also exhibits most potent in vivo activity against Synechocystis sp. PCC 6803 (EC₅₀?=?0.09?ppm), higher (7-fold) than that of our previous inhibitor (EC₅₀?=?0.6?ppm). The binding modes of compound L14 and CyFBA-II were further elucidated by jointly using DOX computational protocol, MM-PBSA and site-directed mutagenesis assays. The positive results suggest that strategy adopted in this study was promising to rapidly discovery the potent inhibitors with novel scaffolds. The satisfactory algicide activities suggest that the thiourea derivatives is very likely to be a promising lead for the development of novel specific algicides to solve Cyanobacterial harmful algal blooms (CHABs).     

微藻抗菌物质及筛选模型

由于海洋环境和微藻本身的特殊性,从微藻中筛选抗菌物质已成为当今研究的热点之一,并且具有广阔的应用前景。对近年来从微藻中筛选抗菌物质的研究现状和进展进行了综述,重点总结了抗菌活性物质筛选过程中的模型选用,并介绍了新筛选模型的发展,指出了难点问题及可行的解决方法。     

Reversible AChE inhibitors in C. elegans vs. rats, mice

We are investigating whether Caenorhabditis elegans could be used as a screen for vertebrates by comparing the responses of components of its cholinergic system to well-characterized toxicants. We assessed whether C. elegans displays similar toxicity as rats and mice to reversible acetylcholinesterase (AChE) inhibitors, and sought to corroborate that the toxicity mechanism is the same. To determine relative potencies, movement-concentration curves were generated, 50th percentiles for movement were located, ranked and compared statistically to rat and mouse oral acute LD50s. The ranking was significantly correlated to rat and mouse rankings (alpha=0.05). We measured a concentration-dependent decrease in AChE activity correlating to a decrease in movement for each carbamate, suggesting that the mechanism of toxicity is the same. Finally, as seen in mammals, inhibition of AChE activity occurred before a movement decrease. The response of C. elegans to carbamate exposure shows significant correlation to rat and mouse data.     

Pathogenicity of Sclerotinia sclerotiorum on Ranunculus acris in Dairy Pasture

Fifty-four isolates of Sclerotinia sclerotiorum from Ranunculus acris and other natural hosts were applied as mycelial infested kibbled wheat onto 6 month-old R. acris plants in two glasshouse screening experiments. Most isolates (90%) did not differ in their pathogenicity towards R. acris. One isolate, S. sclerotiorum G45, was selected based on its ability to cause severe disease and suppress regeneration of R. acris. A field experiment was conducted to determine the efficacy of S. sclerotiorum (G45) against R. acris in infested dairy pastures in the Takaka Valley, Golden Bay, New Zealand. Isolate G45 was formulated as a wettable powder and was applied as a slurry at 20 and 40 ml/plant in December 1995. After 10 weeks, regeneration from the crown of treated plants was apparent and a second application of S. sclerotiorum was made in February 1996. Best control of R. acris was obtained when the plants were inoculated in full flower in December. At the first time of treatment, the 40 ml application of S. sclerotiorum slurry reduced the total dry weight of R. acris by an average of 57%. The second application had no effect on total dry weight, possibly because moisture levels were not sufficient for S. sclerotiorum infection. This study confirmed S. sclerotiorum to be an aggressive pathogen of R. acris under both glasshouse and field conditions. As a result, this pathogen has potential as a mycoherbicide for R. acris. Further experiments are required to explore ways of enhancing the efficacy of S. sclerotiorum against R. acris by manipulation of the host, pathogen and environment.     

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.