The significance of hosting the 2008 Olympic Games for elite sport and sport for all in China

This thesis explores the significance of hosting the Beijing 2008 Olympic Games (OGs) on elite sport and sport for all development in China. The impacts of the OGs have received significant attention from both academics and practitioners worldwide in the last 20 years and attention has been predominantly paid to political, cultural, economic, and environmental impacts of hosting them, especially as these emerge after the event. However, little concern was given to changes in the host country s sport development that are due to games related preparations. This study identifies the characteristics of the sport system, the policy actors, and how such actors were involved in preparations for the 2008 OGs, and it also outlines the development of policy concerning elite sport and sport for all. A case study approach was adopted focusing on the 2008 OGs. Adopting a qualitative methodology, the study utilised document analysis and semi-structured interviews to elicit data regarding the significance of preparations for the 2008 OGs on elite sport and sport for all. Globalisation, governance and policy making were found to be useful lenses through which to explore the processes of the emergence of such impacts. This thesis found that central government and the General Administration of Sport (GAOS) were the two most powerful policy actors in both elite sport and sport for all development in China, and made decisions as regards how to develop China s sport taking the opportunities of hosting the 2008 OGs. The research reveals that preparations for the 2008 OGs have various impacts on the elite sport and sport for all sectors. On one hand, the impacts can be witnessed in increased funding, more attention received from central government and GAOS, more sport policies, increased number of sport venues, new and updated facilities and equipment, technological, scientific and medical support, and increased sport participation; on the other hand, through providing such support, GAOS exerted its control over non-governmental organisations and individuals, such as via the restrictions by GAOS on athletes commercial activities, and national competitions. The research found evidence that globalisation had influenced China s general governance (including sport governance) process since the 1970s, with governance becoming more privatised and decentralised. However, sport governance took a different path after China won the bid in 2001. Against the backdrop of decentralisation having been previously officially adopted for Chinese sport governance, the research revealed that in pursuit of the aim of winning more medals in the 2008 OGs temporarily recentralisation occurred as required by central government and GAOS. The research also revealed that increased numbers of policies were produced to develop both elite sport and sport for all, however the interests of the public had not always been satisfied because of China's closed policy making process. Therefore, some impacts had not turned out as expected for the public

CCR5-/CR2 cells confer resistant to R5-tropic viruses and exhibits selective advantage during R5-tropic HIV-1 infection.

<p>A. HIV-1 replication in sorted CCR5-/CR2 and CCR5+/GF1 cells infected with X4-tropic HIV-1 Bru3. B. HIV-1 replication in sorted CCR5-/CR2 and CCR5+/GF1 cells infected with R5-tropic HIV-1 Bru-Yu2. C. FACS analysis of cell surface expression of CD4 and CCR5 in mixtures of CCR5-/CR2 and parental CEMss-CCR5 cells at 6 and 18 days post-infection with X4-tropic HIV-1 Bru3 (middle panel), R5-tropic HIV-1 Bru-Yu2 (right panel), or no virus control (left panel). D. <i>CCR5</i> gene analysis on uncleaved versus cleaved bands in parental CEMss-CCR5 cells (line 1) or CCR5-/CR2 and parental CEMss-CCR5 mixture at 0, 6 and 18 days post infection with X4 tropic HIV-1 Bru3 (lines 2 to 4) or R5-tropic HIV-1 Bru-Yu2 (lines 5 to 7) by T7EI assay. DPI: days post infection.</p

<i>CCR5</i> gene disruption by RNA-guided Cas9 endonuclease in transduced TZM.bl cells.

<p>A. Schematic diagrams of lentiviral transfer vectors containing Cas9 endonuclease or sgRNAs CR1, CR2, Cr3 and GF1. HA: HA epitope tag; NLS: nuclear localization signal; PGK: promoter sequence derived from phosphoglycerate kinase-1; U6: promoter sequence derived from polymerase III U6. B. Cell surface expression of CCR5 on TZM.bl cells co-transduced with lentiviral vectors expressing Cas9-HA-NLS and sgRNAs CR1, CR2, CR3 or GF1. Transduced (open curves) and mock-transduced (shaded curves) cells were stained with anti-CCR5 antibody followed by FACS analysis. C. FSC and SSC analysis mock-transduced TZM.bl cells and TZM.bl cells transduced with lentiviral vectors expressing Cas9-HA-NLS fusion protein and sgRNAs CR1, CR2, CR3 or GF1, respectively. Percentages of gated (live) cells are shown at the lower and left corner of the figures. D. <i>CCR5</i> gene disruption analysis by T7EI assay using prime pairs CR1/F990-CR1/R1750 and CR2/3F593-CR2/3R1254 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s003" target="_blank">S1 Table</a>). E. <i>CCR5</i> gene disruption analysis by T7EI assay using prime pair CR1/2/3F2559-CR1/2/3R3893 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s003" target="_blank">S1 Table</a>).</p

<i>CCR5</i> Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

<div><p>CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced <i>CCR5</i> via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4⁺ cells yields high frequencies of <i>CCR5</i> gene disruption. <i>CCR5</i> gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over <i>CCR5</i> gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of <i>CCR5</i> via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.</p></div

Distribution of BMI in 930 IgAN patients.

<p>(A) BMI goups were separated by age categories in all patients with IgAN, and then divided into males (B) and females(C).</p

<i>CCR5</i> gene disruption by RNA-guided Cas9 endonuclease in transduced CEM ss-CCR5 cells.

<p>A. Cell surface expression of CCR5 on CEMss-CCR5 cells co-transduced with lentiviral vectors expressing Cas9-HA-NLS and one of sgRNA (CR2 or GF1) as compared to mock-transduced CEMss-CCR5 cells. B. FSC and SSC analysis mock-transduced CEMss-CCR5 cells and CEMss-CCR5 cells transduced with lentiviral vectors expressing Cas9-HA-NLS fusion protein and sgRNAs CR2 or GF1, respectively. Percentages of gated (live) cells are shown at the lower and right corner of the figures. C. <i>CCR5</i> gene disruption analysis in mock-transduced CEMss-CCR5 cells or CEMss-CCR5 cells co-transduced with lentiviral vectors expressing Cas9-HA-NLS and one of sgRNAs (CR2 or GF1) by T7EI assay. D. Mean and median values of fluorescence intensity of cell surface expression of CD4, CXCR4 and CCR5 in sorted CCR5-/CR2 and CCR5+/GF1 cells as compared to parental CEMss-CCR5 cells. E. Relative fluorescent intensity of DDAO-labeled CCR5-/CR2 and CCR5+/GF1 cells as compared to parental CEMss-CCR5 cells at 0, 12, 24 and 36 hours post culture.</p

<i>CCR5</i> gene disruption by RNA-guided Cas9 endonuclease in sorted CCR5-/CR1, CCR5-/CR2 and CCR5-/CR3 cells by Sanger sequencing analysis.

<p>A-C. Cell surface expression of CCR5 on sorted CCR5-/CR1 (A), CCR5-/CR2 (B) and CCR5-/CR3 (C) TZM.bl cells as compared to mock-transduced TZM.bl cells. D-F. Representative Sanger sequencing of CCR5 target sites by CR1 (D), CR2 (E) or CR3 (F) in co-transduced TZM.bl cells. The full list of Sanger sequencing of CCR5 target sites by CR1, CR2 or CR3 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s002" target="_blank">S2 Fig</a>. For each <i>CCR5</i> site targeted by CR1 (D), CR2 (E) or CR3 (F), the upper panel shows representative Sanger sequencing of targeted CCR5 sequences amplified by a prime pair that covers both the endogeous <i>CCR5</i> gene and transgene. The lower panel shows representative Sanger sequencing of targeted <i>CCR5</i> sequences by a prime pair that only covers the endogenous <i>CCR5</i> gene.</p

<i>CCR5</i> gene disruption in CEMss-CCR5 cells transduced with a single lentiviral vector expressing both CR2 sgRNA and Cas9.

<p>A. Schematic diagrams of a lentiviral transfer vector pRRL-CR2 sgRNA-Cas9/EGFP. Cas9: Cas9 endonuclease; CR2 sgRNA: single guided RNA CR2; HA: HA epitope tag; NLS: nuclear localization signal; U6: polymerase III U6 promoter sequence; EF1α: promoter sequence derived from elongation factor 1α; 2A: 2A self cleaving peptide sequence; and EGFP: sequence encoding enhanced green fluorescent protein. B. FSC and SSC analysis in mock-transduced CEMss-CCR5 cells and CEMss-CCR5 cells transduced with a single lentiviral vector pRRL-CR2 sgRNA-Cas9/EGFP. Percentages of gated (live) cells are shown at the lower and right corner of the top-right and top-left panels. CCR5 and EGFP analysis in mock-transduced CEMss-CCR5 cells and CEMss-CCR5 cells transduced with a single lentiviral vector pRRL-CR2 sgRNA-Cas9/EGFP. C. <i>CCR5</i> gene disruption analysis in mock-transduced CEMss-CCR5 cells or CEMss-CCR5 cells transduced with a single lentiviral vector pRRL-CR2 sgRNA-Cas9/EGFP by T7EI assay.</p

T7 endonuclease I (T7EI) analysis of potential off-target loci.

<p>A. T7EI analysis of three potential off-target loci (AKAP9, ULK1 and MED16) to CR2 as well as on-target locus CCR5. “–”: genomic DNA isolated from mock-transduced TZM.bl; “+”: genomic DNA isolated from sorted CCR5-/CR2 cells. D21 and D84: genomic DNA samples isolated from sorted CCR5-/CR2 cells at 21 and 84 days post transduction. B. T7EI analysis of nine potential off-target loci (SH2D5, ASB9P1, PRRT1, LINC00265, ENDOV, NR2F1, ASB9, CLPP and SOBP) to CR3 as well as on-target locus CCR5 control. “–”: genomic DNA isolated from parental TZM.bl; “+”: genomic DNA isolated from sorted CCR5-/CR3 cells. D21 and D84: genomic DNA samples isolated from sorted CCR5-/CR3 cells at 21 and 84 days post transduction.</p

Compare baseline data among different BMI groups categorized by WHO Asian standard.

<p>Compare baseline data among different BMI groups categorized by WHO Asian standard.</p