Proteomic analyses of the Xiphophorus Gordon-Kosswig melanoma model

Interspecies hybridization between the platyfish X. maculatus Jp 163 A, and the swordtail X. helleri (Sarabia), generates F(1) hybrids with pronounced melanin pigmentation. Backcrossing of F(1) hybrids with the X. helleri parent results in 25% of progeny that will spontaneously develop melanoma. We have applied proteomic methods to this Gordon-Kosswig (G-K) melanoma model to identify candidate proteins that exhibit modulated expression in fin tissue due to interspecies hybridization and progression of hybrid tissues to spontaneous melanoma. Difference Gel Electrophoresis (DIGE) was used to minimize the variability commonly observed in quantitative analyses of comparative protein samples. Following identification of up- or down-regulated protein expression by DIGE, candidate protein spots were identified by mass spectrometric sequencing. Several protein expression differences displayed in interspecies hybrids were identified and compared to distinct differences that occur upon backcrossing and progression to melanoma. These studies are important for the identification of distinct biochemical pathways involved in the variety of Xiphophorus interspecies hybrid tumor models.     

多源空间数据融合的城市人居环境监测模型与应用研究

人居环境监测作为城市人居环境建设与管理实践提升的基本,是目前人居环境研究落地的重点。传统的城市人居环境监测在数据更新速度、精度等方面存在不足,难以满足精细化管理需求。提出利用遥感数据与互联网的兴趣点POI(Point of interest)数据结合,建立人居环境监测模型。模型主要有两个关键环节,一是构建自动化提取建筑物算法,该算法通过建立地物特征集,以POI点对应样本为种子,利用全局最优和区域生长算法,自动提取城市建筑物,再利用全局最优算法确定其他地类的阈值;二是人居环境指标计算,将建筑物、绿地、水体信息提取结果与POI数据结合,利用密度类与距离类空间分析算法,分别计算自然、社会经济类指标。基于上述模型,利用2018年4月的北京二号遥感影像和POI点数据在北京市回龙观社区进行实验验证,结果显示:信息提取结果中,总体精度超过95%,Kappa系数超过92%,提取效率提高2.3倍,表明信息提取精度高且可信,适合工程化应用。计算回龙观社区人居环境监测指标,分析结果认为,社区内自然类指标差异不大,但缺乏水体生态系统,生物多样性不够丰富,社区内的商业比较繁华,但是学校和医疗不充足,尤其是缺乏大型公立医院。综上,通过人居环境监测模型研究和应用分析,将遥感数据和互联网数据结合应用于人居环境质量监测有效提高了精度和速度,有利于业务化,服务政府管理。     

FMR1: A gene with three faces

The FMR1 gene is involved in three different syndromes, the fragile X syndrome (FXS), premature ovarian insufficiency (POI) and the fragile X-associated tremor/ataxia syndrome (FXTAS) at older age. Fragile X syndrome is caused by an expansion of a CGG repeat above 200 units in the FMR1 gene resulting in the absence of the FMR1 mRNA and protein. The FMR1 protein is proposed to act as a regulator of mRNA transport and of translation of target mRNAs at the synapse. FXS is seen as a loss of function disorder. POI and FXTAS are found in individuals with an expanded repeat between 50 and 200 CGGs and are associated with increased FMR1 mRNA levels. The presence of elevated FMR1 mRNA in FXTAS suggests that FXTAS may represent a toxic RNA gain-of-function effect. The molecular basis of POI is yet unknown. The role of the FMR1 gene in these disorders is discussed.     

A NANOS3 mutation linked to protein degradation causes premature ovarian insufficiency

Primary ovarian insufficiency (POI), or premature ovarian failure, is defined as the cessation of ovarian function before the age of 40. An insufficient ovarian follicle pool derived from primordial germ cells (PGCs) is an important cause of POI. Although the Nanos gene family is known to be required for PGC development and maintenance in diverse model organisms, the relevance of this information to human biology is not yet clear. In this study, we screened the coding regions of the NANOS1, NANOS2 and NANOS3 genes in 100 Chinese POI patients and identified four variants in the coding regions of these three genes, including one synonymous variant in NANOS3, one missense variant in each of NANOS1 and NANOS2 and one potentially relevant mutation (c.457C>T; p.Arg153Trp, heterozygous) in NANOS3. We demonstrated that the p.Arg153Trp substitution decreases the stability of NANOS3, potentially resulting in a hypomorph. Furthermore, an investigation of the relationship between the number of PGCs and the dosage of NANOS3 in mouse models showed that the population of PGCs is controlled by the level of NANOS3 protein. Taken together, our results provide new insight into the properties of the NANOS3 protein and establish that NANOS3 mutation is one possible cause of POI.     

Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display

There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10¹⁰ individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFα, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery

One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway. This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 β-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We show also that ISELATE enables focusing of an scFv library in soluble sequence space before functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. Our results reveal that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli.