Abstract:This year an outbreak of influenza in Mexico comes from a new epidemic of influenza viruses: A/H1N1 influenza virus. This virus is a kind of virus mixture, including human, avian and swine influenza virus gene fragments. The pathogenic mechanism of avian, swine and human influenza virus in its natural host were compared. The main purpose is to assess the risk of which pigs and poultry has the possibility to become one of zoonotic disease and to assess the possible role in process of avian flu transmission from pigs to people. As zoonotic disease, avian influenza and swine influenza virus have the key role in the process of human infection. But the influenza virus spread of crossing species barrier is not sufficient to cause a large outbreak of human influenza. Animal influenza viruses must have a significant genetic variation before they could live for a long term in the crowd.

Abstract:The mechanism of hepatitis B virus entry is an interesting area in HBV research but still enigmatic. The difficulties in HBV entry research were primarily caused by the lack of easily accessible in vitro infection models. Recent years, primary hepatocytes from Tupaia belangeri has been substituted for primary human hepatocytes and upon induction of differentiation in vitro. A human hepatoma cell line named HepaRG has been found to be susceptible for HBV infection too. The two cell models enabled researchers to obtain a number of important discoveries for HBV entry. This article are focusing on these discoveries, including the domains of HBV surface proteins involved in HBV entry, potential HBV receptor candidates and the questions to be resolved in future years.

Abstract:Dual coding is a phenomenon which refers to a mature mRNA that contains two open reading frames (ORFs) in the same direction to code two different proteins. This phenomenon is quite common in prokaryote, bacteriophages and viruses as an economical and effective way to present the genome information. However, recent reports suggest that the dual coding phenomenon does also occur in eukaryote. Dual coding will help us to further understand the complicated regulation of eukaryotic genes or even the law of molecular evolution.

Abstract:Besides its function as a pathogenicity determinant, the Tombusvirus P19 also serves as a suppressor of RNA interference (RNAi) by sequestering intracellular small RNAs such as the small interfering RNAs (siRNAs) and microRNAs (miRNAs). However, the effect of P19 on mammalian cells has not been evaluated before. A human embryonic kidney 293 cell line that stably expressed p19 (HEK293-p19) was generated. Flow cytometric analysis revealed that over-expression of P19 caused a significant accumulation of G2/M phase cells. Cell proliferation assays demonstrated a reduced DNA replication and cell growth in HEK293-p19 cells. Moreover, p19 altered the expression profiles of a number of cell cycle regulators in HEK293 cells, such as upregulation of cyclin A1, CDK2, CDK4, CDK6, p18, cyclin D2, p19INK4d and E2F1, and downregulation of p15, cyclin A2, cyclin B1 and cyclin E1. Thus, the data strongly indicate that p19 might influence multiple G2/M regulators to cause G2/M arrest.

Abstract:To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells. CT358 gene from the Chlamydia trachomatis(C. trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedC1 vectors. The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins. The GST-CT358 fusion protein was used to immunize mice to raise the antibodies, which specifically recognized CT358 without cross-reacting with other unrelated proteins. The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay (IFA). Meanwhile, pDSRedC1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection. The hypothetical protein CT358 was identified in the inclusion membrane of C. trachomatis-infected cells for the first time,and it was detected as early as 12 h after C. trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of CT358 via a transgene failed to affect the subsequent chlamydial infection. These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein, giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.

Abstract:Agrobacterium tumefaciens possesses many advantages as a model bacterium for the study of a wide variety of biological processes. Gene disruption or inactivation is a powerful and direct tool for investigation of in vivo gene functions. The intensive study of A. tumefaciens has increased the need for simple and highly efficient procedures to manipulate its genome. The sacB gene was used as a counterselectable marker to develop a gene replacement procedure that allows precise insertion, deletion, and allele substitution of any gene sequence in A. tumefaciens without altering the genome in any other way. A kanamycin resistance (Km^R) cassette was constructed to the suicide vector as the positive selection marker. The suicide plasmid containing DNA fragments homologous to the flanking sequences of the target gene was integrated into the recipient cell genome at the target gene locus by intermolecular homologous recombination, generating the Km^R-single cross-over colonies. The effect of homologous sequence length on the intermolecular homologous recombination was analyzed. The second cross-over colonies generated by intramolecular homologous recombination occurring between two tandem repeats were simply screened out by counter-selection of sacB. Data showed that the intervening sequence length between two repeats significantly affected the intramolecular homologous recombination frequency in A. tumefaciens, indicating that A. tumefaciens adopted the homologous recombination mechanism similar to that in E. coli. All these results demonstrated that investigators could minimize the numbers of colonies to be analyzed and reduce the overall workload by optimizing the relative length of two homologous fragments and using the specific type of single cross-over transformants for screening the second cross-over event. This mutagenesis strategy had successfully been used to generate the double unmarked Δvbp2Δvbp3 mutant in two A. tumefaciens strains.

Abstract:Insulin-like growth factorⅠ(IGF-Ⅰ) is essential for the growth and differentiation of hair follicles which is an important part of wool and cashmere. But there is no report on polymorphisms of the IGF-Ⅰ gene in cashmere goat, and also few candidate genes for cashmere production traits have been reported in cashmere goats. The objectives of this work were to detect the single nucleotide polymorphism (SNP) in the 5′ flanking region of IGF-Ⅰ gene and to determine their association with fibre traits in Liaoning cashmere goats. The fibre traits data investigated in the experiment were combed cashmere weight, cashmere fibre length and cashmere fibre diameter. A few individuals of the Liaoning cashmere goats, selected according to phenotypic character, were used for SNPs detection in the 5′flanking region of IGF-Ⅰ gene, and four point mutation G→C (388 bp), A→G (668 bp), A→ C (719 bp), G→A (752 bp) were identified, which result in a CdxA bonding-site lack, and score increase of C/EBP site in the 5′ flanking region compared with the wild type. Then different genotypes were detected in 520 Liaoning cashmere goats using create restriction site (CRS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), three genotypes, AA, AB and BB were observed coded for by two different alleles A and B for each SNP, and 13 diplotypes were identified in the groups of the four SNPs. The relationships between the genotypes and combed cashmere weight, cashmere fibre length and cashmere fibre diameter were analyzed. The statistical analysis showed that animals with the genotype AA of SNP2 in Liaoning cashmere goats had the thinnest cashmere fiber diameter compared with those with the AB and BB genotype (P < 0.01), and individuals with genotype AA of the SNP4 had the highest cashmere weight compared with those with the other genotypes (P < 0.05). The relationship between diplotype and cashmere fiber traits was also analyzed, the results showed that both cashmere fiber diameter and cashmere weight were significantly correlated with the diplotype H7H7 (P < 0.05). These results suggested that IGF-Ⅰ gene would be useful candidate gene in selection program on cashmere fiber traits in cashmere goats.

Abstract:Treatment of chronic osteomyelitis has become a difficult problem in clinical medicine due to its extended pathological process, high occurrence of complication and recurrence of disease. The major pathogenesis mechanism is Gram-negative bacteria infection, such as staphylococci. LPS is an important substance found in the cell wall of Gram-negative bacteria and administration of LPS to skeleton relevant cells in vitro could simulate the pathological characteristics of osteomyelitis patients. The results of quantitative real-time PCR and Western blot showed that CHI3L1 was up-regulated obviously in the infected bone tissues of osteomyelitis patients and LPS-stimulated osteoblasts. Analysis of the luciferase activity of NF-κB reporter gene vector revealed that LPS could activate NF-κB. Bay11-7082, an inhibitor of NF-κB activation, suppressed the elevation of CHI3L1 expression induced by LPS. Pre-incubation of osteoblasts with anti-TNF-α antibody or silencing TNF-α receptor expression by siRNA inhibited the induction effect of LPS on CHI3L1. Inhibition of NF-κB activation also prevented up-regulation of TNF-α induced by LPS. In conclusion, LPS stimulated TNF-α expression through activating NF-κB, then TNF-α induced CHI3L1 expression. It was demonstrated for the first time that CHI3L1 expression is promoted in osteomyelitis and LPS-treated osteoblasts and investigates the molecular mechanism of LPS-induced CHI3L1 expression in osteoblasts.

Abstract:To investigate transduction mechanism on carboxyl terminal activating region 3 of Epstein - Barr virus encoded latent membrane protein 1(LMP1) in nasopharyngeal epithelial cells NP69, the NP69 cell lines were respectively infected, applied the approach of retroviruses infection, by RV-LMP1 and RV-LMP1^Δ252～351 retroviruses. Therefore, the NP69-LMP1 and NP69-LMP1^Δ252～351 cell lines of stable expression LMP1 were established. Sequentially, cellular proliferation of the NP69-LMP1 and NP69-LMP1^Δ252～351 cell lines was compared to draw growth curve, experiment plate clone formation and test soft agar colony forming. Meanwhile, adopted proteomic methods, the differential expression proteins were identified between NP69-LMP1and NP69-LMP1^Δ252～351 cell lines, and the expression levels of partial identified proteins were verified by Western blotting and fluorescent real-time quantitative RT-PCR. The results showed: 1) the ability of LMP1^Δ252～351 promoting NP69 cell proliferation was obviously decreased to compare with wild type LMP1(n=3，P < 0.05). 2) 16 proteins, 8 up- and 8 down-regulated ones, of LMP1-CTAR₃ mediated regulation were identified from infected NP69 cell lines. 3) The differential expression of partial identified proteins was similar with 2-DE separated ones and confirmed by Western blotting and fluorescent real-time quantitative RT-PCR. These results suggested that LMP1-CTAR₃ is the important activating domain of inducing cellular proliferation and the domain probably plays the role of mediating to regulate the expression of G-protein, isocitric enzyme, and so on.

Abstract:The transcription factor E2F1 may form a complex with RB and which may play a major role in promoting growth control. Small interfering RNAs are short, double-stranded RNA molecules that can target mRNAs with complementary sequence for degradation via a cellular process to induce gene silence through degradation of homologous transcripts. The experiments were designed to inhibit proliferation of human retinoblastoma cells by knockdown human transcription factor E2F1 expression level using RNA interference technology. Three different hairpin shRNA inserts were designed targeting human transcription factor E2F1. The three inserts were synthesized and subcloned into a shRNA espression vector pSilencer H1puro which contains the H1 RNA polymerase Ⅲ promoter. The three plasmids encoding E2F1 short hairpin were transfected into human retinoblastoma cells line HXO-Rb-44 and human liver cancer cell line SMMC-7721 in vitro. Real-time RT-PCR was used to measure E2F1 mRNA level. For function analysis, propidium iodide (PI)-stained DNA content method was used to identify cell cycle by flow cytometer. The selected shE2F1 were cloned into an adenovirus system and packaged to adenovirus. Retinoblastoma HXO-Rb-44-gfp cell line which has been marked with green fluorescent protein (GFP) were infected by Ad-shE2F1 for 48h, and Ad-shNegative was used as control. The infected cells were injected into anterior chamber of nude mice eyes. The eyes were investigated and the consecutive and dynamic photos of the condition of RB tumor′s growth were taken by the special fluorescent microscope. One in three RNAi expression plasmids delivery resulted in 83.5% suppression of E2F1 mRNA transcription level. The same shRNA showed 89% inhibition in entering S phase. In nude mice eye anterior chamber, Ad-shE2F1 infected tumor cells grew more slowly than the negative control. The results showed that efficacy of Ad-shE2F1 were helpful in the process of limiting the growth of retinoblastoma. RNAi gene therapy directed against the human transcription factor E2F1 is a new therapeutic approach to suppress retinoblastoma cells proliferation.

Abstract:The drug resistant mutations in human immunodeficiency virus type 1 (HIV-1) are a major impediment to successful highly active antiretroviral therapy (HAART) and new drug design. In order to understand the drug resistance mechanism of HIV-1 integrase (IN) mutually existed for multiple drug-resistant strains to the most potent IN inhibitors diketo acids (DKAs), three S-1360-resistant HIV-1 strains were selected and molecular docking and molecular dynamics (MD) simulations were performed to obtain the inhibitor binding modes. Based on the binding modes, compelling differences between the wild-type and the 3 mutants for IN have been observed. The results showed that: 1) In the mutants, the inhibitor is close to the functional loop 3 region but far away from the DNA binding site. Different binding sites lead to the decrease in susceptibility to S-1360 in mutants compared to the wild-type IN. 2) The fluctuations in the region of residues 138~166 are important to the biological function of IN. 2 hydrogen-bonds between S-1360 with residues N155 and K159 restrict the flexibility of the region. Drug resistant mutations result in a lack of the interaction, consequently, the less susceptible to S-1360. 3) In the 3 mutant IN complexes, the benzyl ring of S-1360 is far from the viral DNA binding site, thus, S-1360 can not prevent the end of the viral DNA from exposure to human DNA. 4) After T66I mutation, the long side chain of I occupied the active pocket in the 3 mutants，consequently， the inhibitor could not move into the same binding site or have the same orientation. All the above contribute to drug resistance. These results will be useful for the rational inhibitor modify and design.

Abstract:Tissue factor (TF) is a cell surface glycoprotein playing an important role in the initiation of the blood coagulation cascade. The functions of TF in other physiological or pathological activities, such as tumor cell migration, are also acknowledged gradually in recent years. A recombinant protein of mouse tissue factor (mTF) extracellular part fused with His tag was constructed, and it was expressed and purified successfully in high-level soluble form. This recombinant mTF can effectively initiate plasma clotting in vitro and enhance blood coagulation in vivo, which prove its biological activity. Using this recombinant mTF as immunogen, a murine monoclonal function-blocking antibody to mTF was also generated, which can inhibit mTF-initiated plasma clotting in vitro. And by treating with this function-blocking antibody, the thrombus formation in a murine deep vein thrombosis model was attenuated successfully, which suggests the important role of tissue factor in deep vein thrombosis. In all, with the active mTF recombinant protein and the mTF function-blocking antibody, the functional investigations of TF in murine models of various research areas become more convenient and feasible.

Abstract:MicroRNAs (miRNAs) act by binding to complementary sites on target messenger RNA (mRNA) to induce mRNA degradation and/or translational repression. To investigate the influence of miRNAs at transcript levels, two human miRNAs (miR-1 and miR-124) were transfected into HeLa cells and microarrays used to examine changes in the mRNA profile showed that many genes were downregulated and that the fold decreases in levels of these target mRNAs differed remarkably. Features depicting interactions between miRNAs and their respective target mRNAs, such as the number of putative binding sites, the strength of complementary matches and the degree of stabilization of the binding duplex, were extracted and analyzed. It was found that, for a given target mRNA, both the quality and quantity of miRNA binding sites significantly affected its degree of destabilization. To delineate these types of interactions, a simple statistical model was proposed, which considers the combined effects of both the quality and quantity of miRNA binding sites on the degradation levels of target mRNAs. The analysis provides insights into how any animal miRNA might interact with its target mRNA. It will help us in designing more accurate methods for predicting miRNA targets and should improve understanding of the origins of miRNAs.

Abstract:To investigate the role of abnormal protein expression of the tumor metastasis related-genes in invasion and metastasis of nasopharyngeal cancer (NPC) and to seek the metastasis-related markers for NPC, high-throughput tissue microarray and immunohistochemistry were used to detect protein expression of membrane type 1-matrix metalloproteinase (MT1-MMP), membrane type 2-matrix metalloproteinase (MT2-MMP), membrane-cytoskeletal crosslinker Ezrin (Ezrin), metastasis suppressor gene nm23-H1, the epithelial cell adhesion molecule E-cadherin (E-cad)and tissue inhibitor of metalloproteinase-2 (TIMP-2) in NPC. Results showed that significant high expression of MT1-MMP and Ezrin proteins and very low expression of nm23-H1 and TIMP-2 proteins were found in NPC compared with non-cancerous nasopharyngeal epithelium (P < 0.05, P < 0.01, respectively). Expression of MT1-MMP, MT2-MMP and Ezrin proteins in the stageⅡ, stageⅢ and Ⅳ NPC and NPC with lymph node metastasis is significantly higher than that of in the stageⅠ NPC and NPC without lymph node metastasis (P < 0.05, P < 0.01 respectively), but expression of nm23-H1 protein in the stageⅡ, stageⅢ and Ⅳ NPC and NPC with lymph node metastasis is significantly lower than that of in the stageⅠ NPC and NPC without lymph node metastasis (P < 0.05). There were significantly positive correlation between MT1-MMP and MT2-MMP (r = 0.308, P < 0.001), nm23-H1 and E-cad(r = 0.167, P < 0.05), nm23-H1 and TIMP-2(r = 0.279, P = 0.001); E-cad and TIMP-2(r = 0.279, P = 0.001) in the NPC. Also, there were obviously negative correlation between MT1-MMP and E-cad(r = -0.188, P < 0.05), MT1-MM and TIMP-2(r = -0.233, P < 0.05), Ezrin and E-cad (r = -0.204, P < 0.05) in NPC. Based on the cluster analysis, there was common higher expression of MT1-MMP, MT2-MMP and Ezrin proteins in the NPC than that of in the chronic inflammatory nasopharyngeal epithelium (P < 0.05). However, common higher loss expression of nm23-H1, E-cad and TIMP-2 proteins were found in NPC compared with the peri-cancer nasopharyngeal epithelium and chronic inflammatory nasopharyngeal epithelium (P < 0.05, P < 0.01). Logistic regression analysis and validity estimation indicated that MT1-MMP gene might be better independent prognostic value for metastasis and clinical progress of NPC. The results suggested that high protein expression of multiple metastasis related-genes, low expression of the metastasis suppressor genes and loss balance between metastasis genes and metastasis suppressor genes might play important role in the metastasis and clinical progression of NPC. MT1-MMP protein can be used as the better independent prognostic molecular marker for metastasis of NPC.

Abstract:Microenvironment plays a critical role in directing the progression of stem cells into differentiated cells. It is necessary to investigate the role of cardiac microenvironment in directing the differentiation of adipose tissue-derived stem cells (ADSCs). Human ADSCs were cocultured with rat cardiomyocytes directly or indirectly by cell culture inserts. For direct coculture, hADSCs were labeled with carboxyfluorescein succinimidyl ester (CFSE), then mixed with cardiomyocytes at a 1∶5 ratio in complete media and seeded at a cell density of 50 000 cells/ml. Fluorescence-activated cell sorting was used to extract and examine the directly cocultured differentiated ADSCs. For indirect coculture, differentiated ADSCs were collected directly. The assays used include scanning electron microscope(SEM) and transmission electron microscope(TEM) for the ultrastructure of differentiated cells, immunostaining against myosin heavy chain, troponinⅠ and connecxin43, Western blotting and RT-PCR for the expression of cardiac specific proteins and genes respectively. Results showed that the differentiated ADSCs experienced the coculture presented cardiac ultrastructure and expressed cardiac specific genes and proteins, and the fractions of ADSCs expressing these markers by direct coculture were higher than those of indirect coculture. These data indicate that in addition to soluble signaling molecules, the direct cell-to-cell contact is obligatory in relaying the external cues of the microenvironment controlling the differentiation of ADSCs to cardiomyocytes.

Abstract:LCRG1(laryngeal carcinoma related gene1, LCRG1), a new candidate tumor suppressor gene of laryngeal carcinoma. However, it is known little about the possible regulatory mechanisms of LCRG1 gene expression. Restriction endonuclease digestion was used to obtain a set of the 5′, or 3′deletion mutants from the region(-169～+127) of the LCRG1 gene. It has been found that the minimal promoter of the LCRG1 gene is mapped at the region from -169～-57. Linker scanning mutational analysis in the region(-169～+127) of the LCRG1 gene was used to identify the crucial cis-elements within the promoter region, The key cis-elements are within the region from -137～-122. SP1, E2F1/DP1, EKLF and ZF9 transcription factor binding site sites were predicted in the region by bioinformatics analysis. Co-transfection with each of a panel of the expression plasmids of the known transcription factors with the relevant reporter construct indicates Sp1 is potent transcription factor for enhancement of the promoter activity, SP1 can also up-regulate the endogenous expression of LCRG1 gene. Electrophoretic mobility shift assay (EMSA) was applied to verify that the key cis-elements of LCRG1 gene exist sequence of Sp1 binding sites. The findings, which showed that the key cis-elements within the region from -137～-122 play an important role in expression of the LCRG1 gene, provide a novel evidence for further study of the function of LCRG1 gene.

Abstract:The detection and classification of extracellular action potentials (i.e. spike) of various single neurons from extracellular recordings are crucial for extracting neuronal spike sequences and thereby for investigating the mechanisms of neural information processing in the central nervous system. In order to increase the correctness of spike detecting and sorting, a new analysis algorithm for processing multi-channel spike signals recorded from rat hippocampi with silicon microelectrode arrays is presented. Four recording contacts on the electrode array are arranged close enough to simultaneously record spikes emitted from same neurons. Firstly, the algorithm extracts all spikes in the four channel recordings by using a multi-channel threshold detection method. Secondly, the algorithm classifies the spikes based on a principle component analysis for a specifically designed type of compound spike waveforms. The compound spike waveform is formed by linking four spike waveforms of a same neuronal firing in the four recording channels one by one in series. The test results with both synthetic datasets and experimental recordings reveal that compared with corresponding traditional single-channel algorithm, the multi-channel algorithm can significantly enhance both the number of extracted spikes and the correctness of spike classifications. The algorithm can also increase the number of isolated neurons from a single experimental preparation. These results indicate that the novel method is efficient for the automatic detection and classification of neuronal spikes.

Abstract:Caenorhabditis elegans is one kind of the most important modal animals for studying heredity, development and behaviors, and for investigating the mechanisms underlying from molecular to systematic levels. Gene microinjection and integration are the most important ways to make transgenic animals in C. elegans. They are widely used in the studies of expression, function and interaction of genes. Briefly, a DNA construct (plasmid or cosmid) or PCR product which carried gene(s) of interest is mixed with a co-injection marker and injected into the distal gonad (syncytium) through a micropipette. The DNA injected is taken up into the mature oocytes and exists as an unintegrated extrachromosomal array, which segregates randomly and can be lost. The extrachromosomal arrays are integrated optionally into chromosomes by irradiation of γ rays, X rays, or by TMP/UV integration method. Here, the detail processes and key points of microinjection and gene integration in C. elegans was introduced and the know-how in these techniques was offered.