The Expert Consensus on the Diagnosis and Therapy of myelodysplastic syndrome (MDS) (2014) will be interpreted in this paper focusing on whether it is scientific and reasonable.Some advises and views will be put forward,hoping that it will be useful to improve the diagnostic and therapeutic ability on MDS for clinicians in our country.

Objective To study the influence of site-directed deglycosylation of the HIV-1 envelope (Env) on its immunogenicity and assembly of functional pseudovirus. Methods Site-directed deglycosylation were performed using cycling mutagenesis and selection of mutants with DpnⅠ. Single-cycle infection assay was employed to analyze the effect of the mutations on the ability of functional pseudovirus assembly. The influence of deglycosylations on the immunodeficiency of Env was evaluated using pseudovirusbased neutralization assay and ELISPOT assay. Results Mutant N197Q induced higher neutralization activities for both pseudoviruses, but lower Env-specific T-cell response. And N197Q rendered the Env to lose the ability of functional pseudovirus assembly. Mutant G2 induced higher neutralization activities for pseudovirus 74-2 but lower for pseudovirus Wt, and had almost no influence on Env-specific T-cell response and functional pseudovirus forming. Conclusion The site-directed deglycosylation of the HIV-1 Env affects the pseudovirus forming and its immunogenicity.

Objective To establish a pseudovirus-based neutralization assay. Methods The functional gp160 genes were amplified from plasmids containing HIV-1 gene. The products were ligased into pcDNA3.1 plasmid and positive clones were screened by digestion with restriction enzymes. The pseudoviruses were harvested by co-transfection of the positive clone and pSG3△env plasmid. The neutralizing activities of monoclonal antibodies and HIV-1 antibody positive plasma were measured by these pseudoviruses. Results The four strains of psedoviruses (CHB01, CHB02, CHBC03 and CHAE04) had been successfully obtained. Monoclonal antibody 4E10 could neutralize all of 4 pseudoviruses while 2G12 could not neutralize any pesudoviruses. Monoclonal antibody 2F5 could neutralize pseudovirus CHB01, CHB02 and CHAE04 but not CHBC03, while IgG1b12 could neutralize pseudovirus CHB01, CHB02 and CHBC03 but not CHAE04. The neutralizing activities of 43 of HIV-1 antibody positive plasma against different subtypes of pseudovirus were significant differences and the cross-neutralization effects for some samples exist. Conclusion The harvested pseudoviruses could be used in the neutralization assay. However, the neutralizing characteristics of different pseudoviruses may be different.

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around Nab epitopes and deletions of variable regions in env.The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γELISPOT.Overall,five mutants(dWt,M2,M5-2,M5-1 and dM7)induced highed neutralization activities for both pseudoviruses than plasmid Wt,while only two of the mutants(dWt and M5-2)showed significant differences(P<0.05).Two mutants(M2 and dM2)induced more Env-specific T cells than plasmid Wt.Statistically however,significance was only reached for mutant M2.Thus,properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.

Objective : To construct myeloid specific Spi1 gene knockout mice and analyze their genotypes , so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets . Methods : According to the principle of CRISPR/Cas9 technology and C re/LoxP system , sgRNA and Donor vectors were de signed and constructed . The transcript of Exon 2 ( Exon 2) was used as the knockout region , and Loxp elements were placed on both sides of Exon 2 . Cas9 protein , sgRNA and Donor vector were mixed and microinj ected into the fertilized eggs of C57BL/6J mice , the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice , and F0 generation was obtained after 19 ~ 20 days . Positive F0 mice were mated with C57BL/6J mice to ob tain stable F1 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/flox/Lyz2-cre - mice were used as wild type (WT) mice . DNA of WT and KO mice was extracted , and the genotypes were identified by agarose gel electro phoresis after PCR amplification . Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene , Spi - 1 /purine rich box - 1(PU . 1) in immune cells of WT and KO mice . Results: The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1 flox/flox homozygote , and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre + . Western blot showed that compared with WT group , the protein PU . 1 was not expressed in bone marrow derived macropha ges (BMDMs ) and peritoneal macrophages (PM) in KO group (P < 0.01) . There was no significant difference of statistics in the expression level of PU . 1 in T cells between KO mice and WT mice . The results of PCR and West ern blot showed that myeloid specific Spi1 KO mice were successfully constructed . Conclusion The myeloid spe cific Spi1 gene KO mice are successfully constructed and identified , which provides animal model basis for further revealing the potential mechanism of PU . 1 inimmune regulation .

Objective : To construct Csf1r-CreERT2 R26REYFP reporter gene mice and assess the efficacy of Csf1r- CreERT2 -mediated enhancement of CSF1R in CD45 + cells labeled with yellow fluorescein protein EYFP． Methods: Csf1r-CreERT2 mice were crossbred with R26REYFP homozygous mice，and Csf1r-CreERT2 R26REYFP mice were identified through PCR and Western Blot analyses．Flow cytometry was employed to evaluate CSF1R tag-efficiency in CD45 + cells across different mouse tissues following tamoxifen induction. Results : Csf1r-CreERT2 R26REYFP reporter gene mice were acquired．In addition，it was found that Csf1r-CreERT2 -mediated EYFP could effectively mark CSF1R in various tissues of mice and CD45 + cells in different locations．Compared to the R26REYFP group，the highest labeling efficiency was observed in the brain tissue (P＜0. 001) ，the lowest in the thymus tissue (P＜0. 05) ，and no sig- nificant difference was observed in the spleen tissue． Conclusion Adult Csf1r-CreERT2 mice and R26REYFP mice are effective ways to obtain Csf1r-CreERT2 R26REYFP induced conditional fluorescence mice．Csf1r-CreERT2 can mediate EYFP to effectively trace CSF1R in CD45 + cells in different parts of mice.

Objective : To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in- vestgation of the specific role of Spi1-encoded protein PU. 1 . Methods : The Lck-Cre mice were mated with Spi1 flox/flox mice to obtain Lck-Cre ×Spi1 flox/flox mice (T lymphocyte-specific Spi1 knockout mice) , and the genotype was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis . Magnetic beads were used to sort out the splenic T lymphocytes , and the knockdown efficiency of PU. 1 in T cells was detected by Western blot , quantitative real-time PCR ( qPCR) and flow cytometry. Results : The Lck-Cre ×Spi1 flox/flox mouse genotype was stably inherited . Compared with Spi1 flox/flox mice , the expression level of PU. 1 was significantly reduced in splenic T cells of Lck-Cre ×Spi1 flox/flox mice . Conclusion In this study , the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology , which provided a reliable an- imal model for the subsequent experiments of the specific role of PU. 1 in T cell-related diseases .