a-simple-red-blood-cell-lysis-method-for-establishment-b

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

an-efficient-simple-method-to-establish-nk-t-cell-lines-from-patients

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Chinese Academy of Sciences

Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion.

Expression of Nanog gene promotes NIH3T3 cell proliferation.

The conserved sequences of the mouse histamine H3 receptor at the potential alternative splice junctions suggest that the splice isoforms found in guinea pig, rat, human, and hamster may also be present in the mouse. However, the trace amount isoforms are hard to be detected by the regular PCR approach. In this paper, we report a method in which the unspliced long isoform is cut by restriction endonuclease so that the short isoforms can be amplified to detectable levels to confirm the existence of the splice isoforms of H3 receptor mRNA in the mouse. This method is applicable to the detection of trace amounts of splice isoforms that coexist with the long, more abundant isoforms.

Combining restriction digestion and touchdown PCR permits detection of trace isoforms of histamine H3 receptor.

Bone morphogenetic protein 2 (BMP2) has great potential in therapeutic applications. We are working on generating transgenic plants as a bioreactor to produce BMP2. We have studied the effects of codon optimization on the expression of human BMP2 (hBMP2) in tobacco plants. Three modified hBMP2 genes were transformed into tobacco under the control of either cauliflower mosaic virus 35S (CaMV35S) promoter or double-CaMV35S promoter plus alfalfa mosaic virus (AMV) enhancer. The fused beta-glucuronidase (GUS) reporter gene was used to facilitate the assay of protein expression. The results indicated that codon optimization could increase the protein expression level obviously under CaMV35S promoter. However, under relatively stronger initiation condition (double-CaMV35S promoter plus AMV enhancer), only the gene with the lowest degree of codon optimization could increase the protein expression level. Our findings suggest that the action of codon optimization may be influenced by the factors of promoter strength and A+T content in tobacco plants.

Effects of codon modification on human BMP2 gene expression in tobacco plants.

Biomedical research in human beings is largely restricted to in vitro studies that lack complexity of a living organism. To overcome this limitation, humanized mouse models are developed based on immunodeficient characteristics of severe combined immunodeficiency (SCID) or recombination activating gene (Rag)(null) mice, which can accept xenografts. Peripheral constitution of human immunity in SCID or Rag(null) mice has been achieved by transplantation of mature human immune cells, foetal human thymus, bone marrow, liver tissues, lymph nodes or a combination of these, although efficiency needs to be improved. These mouse models with constituted human immunity (defined as humanized mice in the present text) have been widely used to investigate the basic principles of human immunobiology as well as complex pathomechanisms and potential therapies of human diseases. Here, elements of an ideal humanized mouse model are highlighted including genetic and non-genetic modification of recipient mice, transplantation strategies and proposals to improve engraftments. The applications of the humanized mice to study the development and response of human immune cells, human autoimmune diseases, virus infections, transplantation biology and tumour biology are reviewed as well.

Mouse models with human immunity and their application in biomedical research.

Comparative phylogeography of the Yellow River schizothoracine fishes (Cyprinidae): vicariance, expansion, and recent coalescence in response to the Quaternary environmental upheaval in the Tibetan Plateau.

Intron retention in histamine H(3) receptors has been found in many mammals, including rats and mice. The short transcript isoforms that exclude alternatively spliced introns are readily detected in very low abundance in rats and are undetectable in mice using the regular PCR approach. The detailed mechanism for the special alternative splicing remains poorly understood. The aim of this work was to investigate the effects of essential splice signals on intron retention and to identify sequence elements that determine the differences in splicing between rats and mice. We have constructed a minigene-splicing system to mimic natural alternative receptor splicing and analyzed the regulatory elements in HEK293 cells. Mutating suboptimal 5' and 3' splice sites toward the consensus sequences can enhance the splicing of corresponding alternative introns. The effect is much more significant for the 3' splice site of the longer intron than for the other two splice sites; it is also more significant in rats than in mice. The splicing differences between rats and mice are primarily determined by the six discrepant nucleotides within the alternative introns, which promote or suppress the intron splicing in different ways and cooperate to make splicing of the two introns more efficient in rats. From these results we conclude that (1) the weakness of the splice sites is an important determinant for very low efficiency during intron splicing and, (2) multiple sequence elements determine the splicing differences between rats and mice. The results provide insight into special alternative splicing regulation in H(3) receptors.

Multiple sequence elements determine the intron retention in histamine H3 receptors in rats and mice.

In the alternative splicing, intron retention, of histamine H(3) receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5' splice site and different 3' splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5' and 3' splice sites on intron retention in HeLa cells. Mutating weak 5' and 3' splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more significant for the 3' splice site of the longer alternative intron than for the 3' splice site of the shorter alternative intron and the common 5' splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3' splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.

Effects of splice sites on the intron retention in histamine H3 receptors from rats and mice.

Epithelial-derived thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that is mainly produced by epithelial cells. It has been shown to play a key role in the development of Th2-type allergic inflammation. Commercial antibodies available against TSLP can only be used in Western blot assay, which further limits investigation of its function. Here we efficiently generated a panel of anti-mouse TSLP monoclonal antibodies in rats with DNA priming-protein boosting strategy. Overall, four MAb strains (4B12, 4E11, 5D7, and 6H10) were obtained and their characterizations were identified. The MAbs can specifically bind to TSLP according to the ELISA and FACS assays. It was found that they recognized distinct epitopes. They are useful in detecting TSLP expression in the tissue by immunoblotting and in the cytoplasm by intracellular staining assay. Thus, these antibodies will be valuable tools for studying TSLP biological functions.

Development and characterization of monoclonal antibodies against mouse TSLP.

The schizothoracine Gymnocypris chilianensis is restricted to the Shiyang, Ruoshui and Shule Rivers, listed from east to west, along the northeast edge of the Tibetan Plateau. This distribution provides a valuable system to test hypotheses about postglacial colonization. We used mitochondrial DNA sequence data (a control region and the cytochrome b gene; 1894 bp) to assess the phylogeographic structure of this species based on 278 specimens sampled from throughout the species' entire geographical range. We found three lineages corresponding geographically to the three rivers, suggesting three independent glacial differentiation centers within the northeast edge of the Tibetan Plateau. The phylogenetic analysis suggested that the Shiyang River population forms a lineage that separated from the other populations of G. chilianensis at the basal phylogenetic split within this species. The molecular data further demonstrated a clear pattern of decreasing genetic diversity from the eastern Shiyang River towards the central Ruoshui River and western Shule River lineages, a pattern consistent with sequential western colonization. We therefore propose a phylogeographic scenario for G. chilianensis of a gradual westerly expansion from the Shiyang River population along the northeast edge of the Tibetan Plateau, with subsequent allopatric evolution at approximately 0.37 and 0.05 million years ago (Ma), through at least two glacial maxima. Together with the genetic evidence reported in other species, our findings suggest that this common biogeographic pattern emphasizes the importance of the northeastern edge region of the Tibetan Plateau as a hotspot of genetic diversity for some taxa.

Phylogeography of the endemic Gymnocypris chilianensis (Cyprinidae): sequential westward colonization followed by allopatric evolution in response to cyclical Pleistocene glaciations on the Tibetan Plateau.

A new strain of human H1N1 influenza A viruses was broken out in the April 2009 and caused worldwide pandemic emergency. The present study is trying to estimate a temporal reassortment history of 2009 H1N1 viruses by phylogenetic analysis based on a total 394 sequences of H1N1viruses isolated from swine, human and avian.

Evolutionary genomics of the pandemic 2009 H1N1 influenza viruses (pH1N 1v).

Human influenza infections display a strongly seasonal pattern. However, whether H7N9 and H5N1 infections correlate with climate factors has not been examined. Here, we analyzed 350 cases of H7N9 infection and 47 cases of H5N1 infection. The spatial characteristics of these cases revealed that H5N1 infections mainly occurred in the South, Middle, and Northwest of China, while the occurrence of H7N9 was concentrated in coastal areas of East and South of China. Aside from spatial-temporal characteristics, the most adaptive meteorological conditions for the occurrence of human infections by these two viral subtypes were different. We found that H7N9 infections correlate with climate factors, especially temperature (TEM) and relative humidity (RHU), while H5N1 infections correlate with TEM and atmospheric pressure (PRS). Hence, we propose a risky window (TEM 4-14 °C and RHU 65-95%) for H7N9 infection and (TEM 2-22 °C and PRS 980-1025 kPa) for H5N1 infection. Our results represent the first step in determining the effects of climate factors on two different virus infections in China and provide warning guidelines for the future when provinces fall into the risky windows. These findings revealed integrated predictive meteorological factors rooted in statistic data that enable the establishment of preventive actions and precautionary measures against future outbreaks.

Identification of climate factors related to human infection with avian influenza A H7N9 and H5N1 viruses in China.

The development of Chinese population genetics by Ruofu Du.

The influenza virus H1N1 has been prevalent all over the world for nearly a century. Many studies on its evolutionary history, substitution rate and antigenicity-associated sites have been done with small datasets. To have a complete view, we analysed 3171 full-length HA sequences from human H1N1 viruses sampled from 1918 to 2016, and discovered a new clade has formed with sequences isolated in Iran. Based on genetic distance calculations, we revealed an uneven evolutionary rate among sequences isolated in different years. We also found that the HA1 fragment of the new clade is like that of viruses that existed in the 1930s, while the HA2 fragment is closely associated with strains isolated after the 2009 pandemic. This new, "mixed" HA sequence indicates a cryptic antigenic shift event occurred, and it should draw more attention to the new clade identified from sequences from Iran.

Novel antigenic shift in HA sequences of H1N1 viruses detected by big data analysis.

RIG-I is a key cytosolic pattern recognition receptor that interacts with MAVS to induce type I interferons (IFNs) against RNA virus infection. In this study, we found that cyclophilin A (CypA), a peptidyl-prolylisomerase, functioned as a critical positive regulator of RIG-I-mediated antiviral immune responses. Deficiency of CypA impaired RIG-I-mediated type I IFN production and promoted viral replication in human cells and mice. Upon Sendai virus infection, CypA increased the interaction between RIG-I and its E3 ubiquitin ligase TRIM25, leading to enhanced TRIM25-mediated K63-linked ubiquitination of RIG-I that facilitated recruitment of RIG-I to MAVS. In addition, CypA and TRIM25 competitively interacted with MAVS, thereby inhibiting TRIM25-induced K48-linked ubiquitination of MAVS. Taken together, our findings reveal an essential role of CypA in boosting RIG-I-mediated antiviral immune responses by controlling the ubiquitination of RIG-I and MAVS.

Cyclophilin A-regulated ubiquitination is critical for RIG-I-mediated antiviral immune responses.

Porcine butyrophilin-like 5 (Btnl5) is a novel member of the butyrophilin family, which consists of immune regulators. The expression pattern and the function of this gene remain unclear. In this study, Btnl5 is identified as a negative regulator of the NF-κB pathway. Our study indicates that Btnl5 is mainly expressed in intestinal epithelial cells (IECs) and expressed in membrane systems. Btnl5 inhibits MyD88-mediated activation of the NF-κB pathway. Btnl5 interacts with TNF receptor-associated factor 2 (TRAF2) and transcription factor p65. Besides, Btnl5 inhibits p65-mediated activation of the NF-κB pathway and inhibits nuclear translocation of p65. These results suggest that Btnl5 may inhibit NF-κB pathway through binding and interfere nuclear translocation of p65.

ArticleTotal : 2	Year
Publication title Cited by 1	2017
Publication title Cited by 1	2018

Article	Year
Expression of Nanog gene promotes NIH3T3 cell proliferation. Biochemical and biophysical research communications\| PubMed ID: 16259959	2005
Combining restriction digestion and touchdown PCR permits detection of trace isoforms of histamine H3 receptor. BioTechniques\| PubMed ID: 16382901	2005
Effects of codon modification on human BMP2 gene expression in tobacco plants. Plant cell reports\| PubMed ID: 16491379	2006
Mouse models with human immunity and their application in biomedical research. Journal of cellular and molecular medicine\| PubMed ID: 18419795	2009
Comparative phylogeography of the Yellow River schizothoracine fishes (Cyprinidae): vicariance, expansion, and recent coalescence in response to the Quaternary environmental upheaval in the ... Molecular phylogenetics and evolution\| PubMed ID: 19364538	2009
Multiple sequence elements determine the intron retention in histamine H3 receptors in rats and mice. The international journal of biochemistry & cell biology\| PubMed ID: 19446035	2009
Effects of splice sites on the intron retention in histamine H3 receptors from rats and mice. Journal of genetics and genomics = Yi chuan xue bao\| PubMed ID: 19683670	2009
Development and characterization of monoclonal antibodies against mouse TSLP. Hybridoma (2005)\| PubMed ID: 21050044	2010
Phylogeography of the endemic Gymnocypris chilianensis (Cyprinidae): sequential westward colonization followed by allopatric evolution in response to cyclical Pleistocene glaciations on the ... Molecular phylogenetics and evolution\| PubMed ID: 21352931	2011
Evolutionary genomics of the pandemic 2009 H1N1 influenza viruses (pH1N 1v). Virology journal\| PubMed ID: 21600019	2011
Identification of climate factors related to human infection with avian influenza A H7N9 and H5N1 viruses in China. Scientific reports\| PubMed ID: 26656876	2015
The development of Chinese population genetics by Ruofu Du. Protein & cell\| PubMed ID: 27094622	2016
Novel antigenic shift in HA sequences of H1N1 viruses detected by big data analysis. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases\| PubMed ID: 28359834	2017
Cyclophilin A-regulated ubiquitination is critical for RIG-I-mediated antiviral immune responses. eLife\| PubMed ID: 28594325	2017
Porcine enterocyte protein Btnl5 negatively regulates NF-kappa B pathway by interfering p65 nuclear translocation. Gene\| PubMed ID: 29197592	2018

Ziyuan Duan

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