The infection of chicken tracheal epithelial cells with a H6N1 avian influenza virus

Sialic acids (SAs) linked to galactose (Gal) in α2,3- and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. We demonstrate that chicken tracheal ciliated cells express α2,3-linked SA, while goblet cells mainly express α2,6-linked SA. In addition, the plant lectin MAL-II, but not MAA/MAL-I, is bound to the surface of goblet cells, suggesting that SA2,3-linked oligosaccharides with Galβ1-3GalNAc subterminal residues are specifically present on the goblet cells. Moreover, both α2,3- and α2,6-linked SAs are detected on single tracheal basal cells. At a low multiplicity of infection (MOI) avian influenza virus H6N1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the H6N1 virus. At a MOI of 1, ciliated, goblet and basal cells are all permissive to the AIV infection. This result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry.     

Identification of a set of miRNAs differentially expressed in transiently TIA-depleted HeLa cells by genome-wide profiling

Background

In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established.

Results

Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof ¹ /+; mnk ^p6 /+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks.

Conclusion

mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway.     

Identification and characterization of differentially expressed miRNAs between bamboo shoot and rhizome shoot

As one of the largest members of Poaceae family, bamboo is a very important agricultural plant in the world. The development of bamboo shoot is very special and particularly significant to bamboo production. Understanding the developmental differences between bamboo shoot and rhizome shoot is extremely valuable for us to further elucidate the mechanism of bamboo shoot formation since both bamboo shoot and rhizome shoot develop directly from rhizome bud underground. In this paper, miRNA chips with 413 miRNA probes were used to compare miRNA expressions between bamboo shoot and rhizome shoot. The experiment revealed 64 bamboo shoot upregulated and 56 rhizome shoot up-regulated miRNAs which were classified into four major categories according to deep sequencing based target prediction. Meristem and morphological development related miRNAs were most important in bamboo shoot, especially miR171 and miR156 members. While in rhizome shoot the mainstream of miRNA expressions was metabolism and nutrition related ones, especially miR395 members. The meristem and morphological development related miRNAs in bamboo shoot showed some embryonic characteristics and suggested the participation of several phytohormones like gibberellin, cytokinin and auxin, which were absent in those miRNAs of rhizome shoot. Further qRT-PCR detections of 21 up-regulated miRNAs in bamboo seedlings indicated that 12 ones were regulated to varying degrees by some environmental factors. Among them, rhizome shoot upregulated osa-miR395b was the most environment-sensitive miRNA, particularly to dehydration. And the bamboo shoot up-regulated osa-miR399j proved uniquely and strongly induced by phosphor. The existence of multiple regulation sites from same miRNA suggested the probability of crosstalks among meristem development, metabolism and stress response during bamboo shoot and rhizome shoot development.     

重组禽腺联病毒介导的miRNA抑制传染性法氏囊病病毒在鸡胚内的复制

【目的】在鸡胚水平上探索VP1和VP2基因特异miRNA抑制传染性法氏囊病病毒(infectious bursaldisease virus,IBDV)复制的可行性。【方法与结果】将表达VP1基因特异miRNA重组载体pAITR-RFPmiVP1或VP2基因特异miRNA重组载体pAITR-RFPmiVP2E与禽腺联病毒(avian adeno-associated virus,AAAV)包装载体pcDNA-ARC和腺病毒辅助载体pHelper共转染AAV-293细胞,获得重组病毒rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E,用同样方法获得不表达miRNA的rAAAV-RFP和表达对照miRNA的rAAAV-RFPmiVP2con。电镜观察显示重组病毒具有典型的AAAV颗粒形态;PCR检测结果表明其基因组中含miRNA表达盒;经poly(A)加尾RT-PCR检测证明重组病毒感染细胞能表达基因特异的miRNA。分别将重组病毒经卵黄囊途径接种8日龄SPF鸡胚,然后经绒毛尿囊膜途径用Lukert株IBDV攻毒,收获鸡胚进行IBDV组织细胞半数感染剂量(TCID50)测定。结果在攻毒后第3天,rAAAV-RFP和rAAAV-RFPmiVP2con接种组的IBDV TCID50为8.0 log10,rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E接种组的IBDV TCID50分别下降到1.0和1.5 log10;在攻毒后第6天,rAAAV-RFP和rAAAV-RFPmiVP2con接种组的IBDV TCID50仍为8.0 log10,rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E接种组的TCID50分别下降到0.8和2.0 log10。【结论】rAAAV是有效的miRNA鸡胚导入载体,表达的VP1和VP2基因特异miRNA能有效阻断IBDV复制。     

NHE1 gene associated with avian leukosis virus subgroup J infection in chicken

As a kind of binding protein, the type 1 Na⁺/H⁺ exchanger (NHE1) is a receptor for the highly pathogenic Avian leukosis viruses-J subgroup (ALV-J) in chicken. In order to investigate the potential effect of chicken NHE1 gene on leukosis, we compared its expression between ALV-J-affected and -unaffected chicken, screened variations across the whole gene, and then performed association analysis with ALV-J affected/unaffected trait in three un-related chicken populations. We found that the NHE1 gene expressed in four immune tissues including spleen, bursa fabricius, liver, and thymus, and its expression was significantly up-regulated in liver and thymus of ALV-J-affected chickens (with leukosis phenotype) compared to -unaffected ones (ALV-J-negative controls). Thirty-six single nucleotide polymorphisms (SNP) were identified in a 6,105 bp region of the chicken NHE1 gene, giving rise to every 170 bp per SNP. Two SNP of g.4405A>G and g.5886C>G were genotyped with PCR-RFLP method. Results showed that g.4405A>G was significantly associated (P < 0.05) with ALV-J infection in all of the three chicken populations, including White Recessive Rock (WRR), Dwarf Yellow (DY) and Shiki Yellow (SY), while g.5886C>G was significantly associated (P < 0.05) with ALV-J infection in SY. These results indicated that the NHE1 gene was related to ALV-J infection in chicken.     

Identification of soybean microRNAs involved in soybean cyst nematode infection by deep sequencing

Soybean cyst nematode (SCN), Heterodera glycines, is the most devastating pathogen of soybean worldwide. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are known to play important role in plant stress response. However, there are few reports profiling the miRNA expression patterns during pathogen stress. We sequenced four small RNA libraries from two soybean cultivar (Hairbin xiaoheidou, SCN race 3 resistant, Liaodou 10, SCN race 3 susceptible) that grown under un-inoculated and SCN-inoculated soil. Small RNAs were mapped to soybean genome sequence, 364 known soybean miRNA genes were identified in total. In addition, 21 potential miRNA candidates were identified. Comparative analysis of miRNA profiling indicated 101 miRNAs belong to 40 families were SCN-responsive. We also found 20 miRNAs with different express pattern even between two cultivars of the same species. These findings suggest that miRNA paly important role in soybean response to SCN and have important implications for further identification of miRNAs under pathogen stress.     

Identification of differentially expressed genes in American cockroach ovaries and testes by suppression subtractive hybridization and the prediction of its miRNAs

Studies on the cockroach have contributed to our understanding of several important developmental processes, especially those that can be easily studied in the embryo. However, our knowledge on late events such as gonad differentiation in the cockroach is still limited. The major aim of the present study was to identify sex-specific genes between adult female and male Periplaneta americana. Two cDNA libraries were constructed using the suppression subtractive hybridization method; a total of 433 and 599 unique sequences were obtained from the forward library and the reverse library, respectively, by cluster assembly, and sequence alignment of 1,032 expressed sequence tags. The analysis of the differentially expressed gene functions allowed these genes to be categorized into three groups: biological process, molecular function, and cellular component. The differentially expressed genes were suggested to be related to the development of the gonads of P. americana. Twelve differentially expressed genes were randomly selected and verified using relative quantitative real-time polymerase chain reaction (qRT-PCR). Meanwhile, by adopting a range of filtering criteria, we predicted two potential microRNA sequences for P. americana, pam-miR100-3p and pam-miR7. To confirm the expression of potential microRNAs (miRNAs) in American cockroach, a qRT-PCR approach was also employed. The data presented here offer the insights into the molecular foundation of sex differences in American cockroach, and the first report for the miRNAs in this species. In addition, the results can be used as a reference for unraveling candidate genes associated with the sex and reproduction of cockroaches.