急性髓系白血病mRNA与非编码RNA差异表达谱及CeRNA调控网络分析

利用GEO数据库(gene expression omnibus database)通过生物信息学分析方法探讨急性髓系白血病(acute myelogenous leukemia,AML)的发病机制。检索GEO数据库中AML相关芯片数据集GSE142698、GSE142699和GSE96535。利用GEO2R分析得到差异mRNAs、miRNAs以及差异lncRNAs。利用在线生物信息学分析工具DAVID对差异mRNAs进行GO富集分析和KEGG通路分析。利用miRWalk数据库预测AML相关miRNAs的靶向mRNAs,利用Spongescan数据库预测AML相关miRNAs的靶向lncRNAs,构建lncRNA-miRNA-mRNA竞争性内源RNA （competing endogenous RNA,ceRNA）调控网络。共筛选出29个显著差异mRNAs、70个显著差异miRNAs和20 005个显著差异lncRNAs。GO富集分析和KEGG通路分析显示,差异表达基因主要涉及蛋白磷酸化、细胞分裂、细胞增殖的负调控、基因表达的正向调节、周期蛋白依赖的丝氨酸/苏氨酸激酶活性的调节等生物过程以及细胞周期、细胞衰老、癌症通路、PI3K-Akt通路等信号通路。将miRWalk数据库预测的靶向mRNAs与差异mRNAs取交集,Spongescan数据库预测的靶向lncRNAs与差异lncRNAs取交集,分别确定了25个mRNAs、6个lncRNAs参与AML相关ceRNA调控网络的构建。结果表明,lncRNAs可能作为关键的ceRNA,通过调控miRNA和相关靶基因参与AML的发生与发展,研究结果为AML诊断和治疗的分子生物学研究提供了新的依据。     

Molecular characterization of lung cancer: A two-miRNA prognostic signature based on cancer stem-like cells related genes

Lung cancer is one of the deadliest cancers worldwide. To increase the survival rate of lung cancer, it is necessary to explore specific prognosis markers. More and more evidence finds that noncoding RNA is closely associated with the survival of lung cancer, and cancer stem cells (CSCs) also play a significant role in the progress of lung cancer. The objective of this study is to find CSLCs genes that affect the prognosis of lung cancer. The differential expression of long noncoding RNAs (lncRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs) in the Cancer Genome Atlas (TCGA) database and differential expression data from microarray of CD326⁺ and CD326⁻ A549 cell are intersected to identify stable and consistent expression genes (2 lncRNAs, 15 miRNAs, and 134 mRNAs). The intersection of lncRNAs and miRNAs is analyzed by univariate and multivariate Cox regression to obtained prognostic genes. Two miRNAs (miR-30b-5p and miR-29c-3p) are significantly correlated with the overall survival rate. Then using these two miRNAs to construct a risk score model as a prognosis signature of lung cancer. Subsequently, we analyzed the association between two miRNAs and clinical information of lung cancer patients, of which T stage, Neoplasm cancer and risk score (P < .05) can be used as independent prognostic indicators of lung cancer. Finally, target genes of 2 miRNAs and 134 mRNAs were annotated with Gene Ontology and analyzed with Kyoto Encyclopedia of Genes and Genomes pathway, and verified with the GEO database. In summary, this study illustrates the role of miRNAs in the promotion of lung cancer by CSCs, which is important to find molecular biomarkers of lung cancer.     

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic ΔasaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The ΔasaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.     

Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis

Studies on the microbial ecology of gut microbiota in bats are limited and such information is necessary in determining the ecological significance of these hosts. Short-nosed fruit bats (Cynopterus brachyotis brachyotis) are good candidates for microbiota studies given their close association with humans in urban areas. Thus, this study explores the gut microbiota of this species from Peninsular Malaysia by means of biochemical tests and 16S rRNA gene sequences analysis. The estimation of viable bacteria present in the stomach and intestine of C. b. brachyotis ranged from 3.06 × 10¹⁰ to 1.36 × 10¹⁵ CFU/ml for stomach fluid and 1.92 × 10¹⁰ to 6.10 × 10¹⁵ CFU/ml for intestinal fluid. A total of 34 isolates from the stomach and intestine of seven C. b. brachyotis were retrieved. A total of 16 species of bacteria from eight genera (Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas and Serratia) were identified, Enterobacteriaceae being the most prevalent, contributing 12 out of 16 species isolated. Most isolates from the Family Enterobacteriaceae have been reported as pathogens to humans and wildlife. With the possibility of human wildlife transmission, the findings of this study focus on the importance of bats as reservoirs of potential bacterial pathogens.     

Comprehensive analysis of lncRNA–miRNA–mRNA during proliferative phase of rat liver regeneration

This study aims to reveal the regulatory mechanism of lncRNAs–miRNAs–mRNAs network during the proliferative phase of liver regeneration (LR). High-throughput sequencing technology was performed, and a total of 1,738 differentially expressed lncRNAs (DE lncRNAs), 167 known differentially expressed miRNAs (DE miRNAs), and 2,727 differentially expressed mRNAs were identified. Then, the target DE lncRNAs and DE mRNAs regulated by the same miRNAs were screened and a ceRNA regulatory network containing 32 miRNAs, 107 lncRNAs, and 270 mRNAs was constructed. Insulin signaling pathway, pyrimidine metabolism, axon guidance, carbohydrate digestion and absorption, and pyruvate metabolism were significantly enriched in the network. Through literature review and the regulatory relationship between lncRNAs and miRNAs, nine core lncRNAs were identified, which might play important roles during the proliferative phase of rat LR. This study analyzed lncRNA–miRNA–mRNA regulatory network for the first time during the proliferative phase of rat LR, providing clues for exploring the mechanism of LR and the treatment of liver diseases.     

Baltic salmon, Salmo salar, from Swedish river Lule älv is more resistant to furunculosis compared to rainbow trout

Background

Furunculosis, caused by Aeromonas salmonicida, continues to be a major health problem for the growing salmonid aquaculture. Despite effective vaccination programs regular outbreaks occur at the fish farms calling for repeated antibiotic treatment. We hypothesized that a difference in natural susceptibility to this disease might exist between Baltic salmon and the widely used rainbow trout.

Study Design

A cohabitation challenge model was applied to investigate the relative susceptibility to infection with A. salmonicida in rainbow trout and Baltic salmon. The course of infection was monitored daily over a 30-day period post challenge and the results were summarized in mortality curves.

Results

A. salmonicida was recovered from mortalities during the entire test period. At day 30 the survival was 6.2% and 34.0% for rainbow trout and Baltic salmon, respectively. Significant differences in susceptibility to A. salmonicida were demonstrated between the two salmonids and hazard ratio estimation between rainbow trout and Baltic salmon showed a 3.36 higher risk of dying from the infection in the former.

Conclusion

The finding that Baltic salmon carries a high level of natural resistance to furunculosis might raise new possibilities for salmonid aquaculture in terms of minimizing disease outbreaks and the use of antibiotics.     

In silico analysis of RNA interference components and miRNAs-like RNAs in the seed-borne necrotrophic fungus Alternaria brassicicola

RNA interference is a mechanism of suppressing gene expression in plants, animals and fungi. This regulation mechanism involves three main enzymes, Dicers (Dcr), Argonautes (Ago) and RNA Dependent RNA Polymerases (Rdrp) allowing to produce smallRNAs. RNA interference and smallRNAs have a role in the plant–microorganisms interaction, either in a pathogenic or in a symbiotic relationships. Alternaria brassicicola is a pathogenic fungus of the Brassicaceae plants. During plant infection, it is able to transmit itself vertically and horizontally, giving advantages for new infection and dissemination. To investigate RNA interference and the presence of smallRNAs in A. brassicicola, an in silico analysis was achieved. Two DCR, 4 AGO and 3 RDRP genes were identified comforting the presence of smallRNAs in A. brassicicola. SmallRNA sequencing from wild-type strain and DCR deleted mutants allowed the identifcation of 17 miRNAs in A. brassicicola. The synthesis of these miRNAs is only weakly influenced by the inactivation of DCR genes suggesting the possible existence of an alternative Dicer-independent miRNA synthesis pathway. Target's prediction of A. brassicicola miRNAs identified genes in the fungus and in the plant model Arabidopsis thaliana. Some miRNAs were predicted to target A. thaliana genes involved in the methylation of histone and in the disease resistance.     

Bisucaberin – A dihydroxamate siderophore isolated from Vibrio salmonicida, an important pathogen of farmed Atlantic salmon (Salmo salar)

A siderophore of the bacterial fish pathogen, Vibrio salmonicida, was isolated from low-iron culture supernatant and structurally characterized as bisucaberin by FTICR- and FAB-MS, NMR and GC-MS analysis of the hydrolysis products. Although the cyclic dihydroxamate bisucaberin has previously been isolated from a marine bacterium, Alteromonas haloplanktis, its involvement in cold-water vibriosis of Atlantic salmon (Salmon salar) is novel. Bisucaberin production in iron-limited media was highest at temperatures around and below 10 °C, correlating well with temperatures at which outbreaks of cold-water vibriosis occur. Due to the very high stability constant of K = 32.2, bisucaberin is a most efficient iron scavenger which may contribute to the virulence of V. salmonicida in Atlantic salmon.     

Leptin reduces Atlantic salmon growth through the central pro-opiomelanocortin pathway

Leptin (Lep) is a key factor for the energy homeostasis in mammals, but the available data of its role in teleosts are not conclusive. There are large sequence differences among mammalian and teleost Lep, both at the gene and protein level. Therefore, in order to characterize Lep function in fish, the use of species-specific Lep is crucial. In this study, the cDNA sequence of salmon leptin a1 (lepa1) was used to establish a production protocol for recombinant salmon LepA1 (rsLepA1) in Escherichia coli, that enabled a final yield of 1.7 mg pure protein L^?1 culture. The effects of 20-day administration of rsLepA1 on growth and brain neuroendocrine peptide gene expression [npy, cart, agrp (-1 and -2), pomc (-a1, -a2, -a2s, and -b)] were studied in juvenile, immature Atlantic salmon (96.5 ± 2.1 g) fed a commercial diet to satiation. Intraperitoneal osmotic pumps were used to deliver rsLepA1 at four different concentrations (calculated pumping rates were 0, 0.1, 1.0 and 10 ng g^?1 h^?1). In the highest dosage group (10 ng g^?1 h^?1), the growth rate was significantly reduced, and pomc-a1 gene expression was higher than in controls. The results support the lipostatic hypothesis and suggest that sLepA1 reduces growth in Atlantic salmon by affecting food intake through the central pro-opiomelanocortin pathway.     

ceRNA network construction and comparison of gastric cancer with or without Helicobacter pylori infection

Gastric cancer (GC) is a lethal disease, and among its variety of etiological factors, Helicobacter pylori (H. pylori) infection is the strongest risk factor. However, the genetic and molecular mechanisms underlying H. pylori-related GC need further elucidation. We investigated the competing endogenous RNA (ceRNA) network differences between H. pylori (+) and H. pylori (−) GC. The long noncoding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression data from 32 adjacent noncancerous samples and 18 H. pylori (+) and 141 H. pylori (−) stomach adenocarcinoma samples were downloaded from the TCGA database. After construction of lncRNA–miRNA–mRNA ceRNA networks of H. pylori (+) and H. pylori (−) GC, Panther and Kobas databases were used to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, survival analysis was used to discover the key genes. In H. pylori (+) GC, we identified a total of 1,419 lncRNAs, 82 miRNAs, and 2,501 mRNAs with differentially expressed profiles. In H. pylori (−) GC, 2,225 lncRNAs, 130 miRNAs, and 3,146 mRNAs were differentially expressed. Furthermore, three unique pathways (cytokine–cytokine receptor interaction, HIF-1 signaling pathway, and Wnt signaling pathway) were enriched in H. pylori (+) GC. According to the overall survival analysis, three lncRNAs (AP002478.1, LINC00111, and LINC00313) and two mRNAs (MYB and COL1A1) functioned as prognostic biomarkers for patients with H. pylori (+) GC. In conclusion, our study has identified the differences in ceRNA regulatory networks between H. pylori (+) and H. pylori (−) GC and provides a rich candidate reservoir for future studies.     

Antibodies to Aeromonas salmonicida Lipopolysaccharide and cell wall antigens in rabbit and salmon immune sera

Atlantic salmon (Salmo salar L.) immunised with A-layer positive or A-layer negative strains ofAeromonas salmonicida did not produce antibodies reactive with proteinase K-digested LPS in the low molecular weight area corresponding to the core-region of LPS. The salmon produced antibody titres as high as those produced by rabbit when assayed against whole bacteria or LPS in ELISA. The salmon antibodies against the A-layer positive strain of A. salmonicida lysed rabbit erythrocytes sensitised with LPS from the A-layer positive strain of A. salmonicida. This was in contrast to the non-haemolytic activity of the salmon antibodies against the A-layer negative strain, indicating differences in epitopes between the two strains.     

一株溶藻细菌对海洋原甲藻的溶藻效应

从深圳大鹏湾南澳赤潮爆发海域的表层海水中分离得到1株对海洋原甲藻(Prorocentrum micans)具有溶藻活性的海洋细菌,菌株编号为N10。利用液相感染法研究了该溶藻细菌的溶藻效果和溶藻作用方式。结果表明,菌株N10能使藻细胞失去运动活性,并膨胀变形,细胞膜内物质聚集于一端,藻细胞最终破裂死亡。菌悬液接种到藻液中的量越大,初始细菌密度越高,其溶藻效果越强。菌悬液以1∶10的体积比接种到藻液中时,藻细胞在24 h的死亡率为83%,至72 h全部溶解死亡;体积比为1∶20的藻细胞在24 h的死亡率为71%,之后藻细胞密度略有波动,120 h时死亡率达77%;而体积比为1∶100的藻细胞密度在前24 h有所下降,死亡率达39%,之后藻细胞密度又开始明显上升;对照组的藻细胞密度均呈明显上升趋势。菌悬液过滤液和高温加热处理后的菌悬液过滤液对海洋原甲藻均无溶藻活性,表明菌株N10的溶藻方式为直接溶藻。通过16S rRNA序列分析并与GenBank数据进行同源性检索,并结合细菌形态及生理生化特征,菌株N10隶属于黄杆菌科(Flavobacteriaceae)中的Muricauda sp.。     

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard^®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard^® stimulated CRP and complement responses to A. salmonicida infection in common carp.