LNRLMI: Linear neighbour representation for predicting lncRNA‐miRNA interactions

LncRNA and miRNA are key molecules in mechanism of competing endogenous RNAs(ceRNA), and their interactions have been discovered with important roles in gene regulation. As supplementary to the identification of lncRNA‐miRNA interactions from CLIP‐seq experiments, in silico prediction can select the most potential candidates for experimental validation. Although developing computational tool for predicting lncRNA‐miRNA interaction is of great importance for deciphering the ceRNA mechanism, little effort has been made towards this direction. In this paper, we propose an approach based on linear neighbour representation to predict lncRNA‐miRNA interactions (LNRLMI). Specifically, we first constructed a bipartite network by combining the known interaction network and similarities based on expression profiles of lncRNAs and miRNAs. Based on such a data integration, linear neighbour representation method was introduced to construct a prediction model. To evaluate the prediction performance of the proposed model, k‐fold cross validations were implemented. As a result, LNRLMI yielded the average AUCs of 0.8475 ± 0.0032, 0.8960 ± 0.0015 and 0.9069 ± 0.0014 on 2‐fold, 5‐fold and 10‐fold cross validation, respectively. A series of comparison experiments with other methods were also conducted, and the results showed that our method was feasible and effective to predict lncRNA‐miRNA interactions via a combination of different types of useful side information. It is anticipated that LNRLMI could be a useful tool for predicting non‐coding RNA regulation network that lncRNA and miRNA are involved in.     

Metabolic profiles of fish nodavirus infection in vitro: RGNNV induced and exploited cellular fatty acid synthesis for virus infection

Red‐spotted grouper nervous necrosis virus (RGNNV), the causative agent of viral nervous necrosis disease, has caused high mortality and heavy economic losses in marine aquaculture worldwide. However, changes in host cell metabolism during RGNNV infection remain largely unknown. Here, the global metabolic profiling during RGNNV infection and the roles of cellular fatty acid synthesis in RGNNV infection were investigated. As the infection progressed, 71 intracellular metabolites were significantly altered in RGNNV‐infected cells compared with mock‐infected cells. The levels of metabolites involved in amino acid biosynthesis and metabolism were significantly decreased, whereas those that correlated with fatty acid synthesis were significantly up‐regulated during RGNNV infection. Among them, tryptophan and oleic acid were assessed as the most crucial biomarkers for RGNNV infection. In addition, RGNNV infection induced the formation of lipid droplets and re‐localization of fatty acid synthase (FASN), indicating that RGNNV induced and required lipogenesis for viral infection. The exogenous addition of palmitic acid (PA) enhanced RGNNV infection, and the inhibition of FASN and acetyl‐CoA carboxylase (ACC) significantly decreased RGNNV replication. Additionally, not only inhibition of palmitoylation and phospholipid synthesis, but also destruction of fatty acid β‐oxidation significantly decreased viral replication. These data suggest that cellular fatty acid synthesis and mitochondrial β‐oxidation are essential for RGNNV to complete the viral life cycle. Thus, it has been demonstrated for the first time that RGNNV infection in vitro overtook host cell metabolism and, in that process, cellular fatty acid synthesis was an essential component for RGNNV replication.     

A high‐throughput BAC end analysis protocol (BAC‐anchor) for profiling genome assembly and physical mapping

Traditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto‐ or allopolyploid species. Here, we developed a highly efficient and low‐cost BAC end analysis protocol, named BAC‐anchor, to identify paired‐end reads containing large internal gaps. Our approach mainly focused on the identification of high‐throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60–100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I‐ and 165 Mlu I‐derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high‐coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies.     

Conditioning adhesive contacts between the neutrophils and the endotheliocytes by Staphylococcus aureus

We have developed a model for evaluating the integral intercellular interactions in the “endotheliocyte‐neutrophil” system and have shown the high variability of adhesion contacts in different donors associated with different expression profiles of neutrophils. Two methods (forсe spectroscopy‐spectroscopy and scanning ion‐conductance microscopy) showed a decrease in the rigidity of the membrane‐cytoskeletal complex of neutrophils under the influence of Staphylococcus aureus 2879 M. Adding this strain to the “endotheliocyte‐neutrophil” system caused a statistically significant decrease in the adhesion force and adhesion work, which indicates a change in the expression profile and physicochemical properties of membranes of both types of interacting cells (neutrophils and endotheliocytes).     

mem-iLID,a fast and economic protein purification method

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein–protein interaction.     

双委夜蛾气味结合蛋白AdisOBP6的原核表达抗体制备及表达谱分析

【目的】本研究旨在对双委夜蛾Athetis dissimilis气味结合蛋白OBP6进行原核表达、抗体制备以及表达谱分析,便于今后对AdisOBP6功能展开研究。【方法】利用生物信息学软件分析AdisOBP6蛋白结构特征;利用通过原核表达获得的重组蛋白4次免疫新西兰大白兔,制备AdisOBP6抗体;采用荧光定量PCR和Western blot技术检测AdisOBP6在双委夜蛾雌雄成虫触角、不同时期精巢以及受精卵和未受精卵中的表达情况。【结果】同源建模预测显示,AdisOBP6具有6个保守半胱氨酸残基和7个α-螺旋结构,并 折叠成一个结合口袋。原核表达结果显示,在20℃下0.5 mmol/L IPTG诱导重组蛋白表达量最多、最稳定。4次免疫重复中,抗体效价分别超过1∶512 000, 1∶512 000, 1∶512 000和1∶64 000。Westernblot检测获得的抗体与AdisOBP6蛋白能够特异性结合。荧光定量PCR结果显示,AdisOBP6在双委夜蛾精巢中的表达量远高于在触角中的,在幼虫期精巢中AdisOBP6就已经开始表达,在蛹期精巢中表达量低于在幼虫期的, 成虫羽化后精巢中的表达量逐渐进入高峰,在未受精卵和受精卵中几乎不表达或表达量极低。Western blot分析发现,AdisOBP6蛋白也主要在精巢中表达,在雌雄成虫触角、受精卵和未受精卵中表达量很低。【结论】本研究实现了AdisOBP6的原核表达,成功地制备了抗体;并证实AdisOBP6在双委夜蛾精巢中大量表达,成虫羽化后表达量达到高峰,推测AdisOBP6可能参与了授精过程。本研究结果为今后进一步研究AdisOBP6的功能奠定了基础。     

Expression profiles and potential roles of transfer RNA-derived small RNAs in atherosclerosis

Knowledge regarding the relationship between the molecular mechanisms underlying atherosclerosis (AS) and transfer RNA-derived small RNAs (tsRNAs) is limited. This study illustrated the expression profile of tsRNAs, thus exploring its roles in AS pathogenesis. Small RNA sequencing was performed with four atherosclerotic arterial and four healthy subject samples. Using bioinformatics, the protein-protein interaction network and cellular experiments were constructed to predict the enriched signalling pathways and regulatory roles of tsRNAs in AS. Of the total 315 tsRNAs identified to be dysregulated in the AS group, 131 and 184 were up-regulated and down-regulated, respectively. Interestingly, the pathway of the differentiated expression of tsRNAs in cell adhesion molecules (CAMs) was implicated to be closely associated with AS. Particularly, tRF-Gly-GCC might participate in AS pathogenesis via regulating cell adhesion, proliferation, migration and phenotypic transformation in HUVECs and VSMCs. In conclusion, tsRNAs might help understand the molecular mechanisms of AS better. tRF-Gly-GCC may be a promising target for suppressing abnormal vessels functions, suggesting a novel strategy for preventing the progression of atherosclerosis.