人血清蛋白质组学方法的比较和优化

目的:对人未做处理的血清以及去除白蛋白和免疫球蛋白G(IgG)血清的蛋白质组学方法进行比较和优化。方法:应用双向电泳(2-DE)方法分离了未做处理的以及去除白蛋白和免疫球蛋白G(IgG)的血清,比较优化了高温变性、水化液成份组成及泡胀方式等影响血清2-DE分离效果的因素,并用质谱分析鉴定未做处理和已处理血清的2-DE谱图中部分差异蛋白点。结果:得到了分辨率和重复性较好的2-DE谱图,未做处理的血清、去除白蛋白及IgG血清的平均蛋白质点分别为(482±18)个和(523±29)个,质谱分析了9个差异蛋白点,鉴定为8种蛋白质,其中7种为功能蛋白质。仅出现在未做处理血清中的蛋白有4种,分别是维生素A结合蛋白、可溶性尿激酶血纤维蛋白溶酶原激活剂受体、蛋白激酶1抗原、血清白蛋白。4种蛋白仅出现在去除白蛋白和IgG的血清中,分别是NADH脱氢酶辅酶β亚基、肌动蛋白结合蛋白M1、T细胞活性受体β链、血小板生长因子C。结论:去除高丰度蛋白可增加一些低丰度蛋白质的检出,但非特异性吸附会导致部分功能蛋白质的丢失。     

Metagenomic insights into the environmental adaptation and metabolism of Candidatus Haloplasmatales,one archaeal order thriving in saline lakes

The KTK 4A-related Thermoplasmata thrives in the sediment of saline lakes; however, systematic research on its taxonomy, environmental adaptation and metabolism is lacking. Here, we detected this abundant lineage in the sediment of five artificially separated ponds (salinity 7.0%–33.0%) within a Chinese soda-saline lake using culture-independent metagenomics and archaeal 16S rRNA gene amplicons. The phylogenies based on the 16S rRNA gene, and 122 archaeal ubiquitous single-copy proteins and genome-level identity analyses among the metagenome-assembled genomes demonstrate this lineage forming a novel order, Candidatus Haloplasmatales, comprising four genera affiliated with the identical family. Isoelectric point profiles of predicted proteomes suggest that most members adopt the energetically favourable ‘salt-in’ strategy. Functional prediction indicates the lithoheterotrophic nature with the versatile metabolic potentials for carbohydrate and organic acids as well as carbon monoxide and hydrogen utilization. Additionally, hydrogenase genes hdrABC-mvhADG are linked with incomplete reductive citrate cycle genes in the genomes, suggesting their functional connection. Comparison with the coupling of HdrABC-MvhADG and methanogenesis pathway provides new insights into the compatibility of laterally acquired methanogenesis with energy metabolism in the related order Methanomassiliicoccales. Globally, our research sheds light on the taxonomy, environmental adaptative mechanisms, metabolic potentials and evolutional significance of Ca. Haloplasmatales.     

Development of a fluorescent probe hydrolysis-insulated isothermal PCR for rapid and sensitive on-site detection of African swine fever virus

Highlights
1. A probe-based insulated isothermal PCR (iiPCR) assay was developed for rapid and onsite detection of ASFV.
2. The developed iiPCR showed similar sensitivity and specificity with OIE recommended real-time PCR.
3. Blood samples could be directly applied as PCR template in iiPCR without DNA extraction.     

In vivo surveillance and elimination of teratoma-forming human embryonic stem cells with monoclonal antibody 2448 targeting annexin A2

This study describes the use of a previously reported chimerised monoclonal antibody (mAb), ch2448, to kill human embryonic stem cells (hESCs) in vivo and prevent or delay the formation of teratomas. ch2448 was raised against hESCs and was previously shown to effectively kill ovarian and breast cancer cells in vitro and in vivo. The antigen target was subsequently found to be Annexin A2, an oncofetal antigen expressed on both embryonic cells and cancer cells. Against cancer cells, ch2448 binds and kills via antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-drug conjugate (ADC) routes. Here, we investigate if the use of ch2448 can be extended to hESC. ch2448 was found to bind specifically to undifferentiated hESC but not differentiated progenitors. Similar to previous study using cancer cells, ch2448 kills hESC in vivo either indirectly by eliciting ADCC or directly as an ADC. The treatment with ch2448 post-transplantation eliminated the in vivo circulating undifferentiated cells and prevented or delayed the formation of teratomas. This surveillance role of ch2448 adds an additional layer of safeguard to enhance the safety and efficacious use of pluripotent stem cell-derived products in regenerative medicine. Thereby, translating the use of ch2448 in the treatment of cancers to a proof of concept study in hESC (or pluripotent stem cell [PSC]), we show that mAbs can also be used to eliminate teratoma forming cells in vivo during PSC-derived cell therapies. We propose to use this strategy to complement existing methods to eliminate teratoma-forming cells in vitro. Residual undifferentiated cells may escape in vitro removal methods and be introduced into patients together with the differentiated cells.     

Functional variants of hepatocyte growth factor identified in patients with adolescent idiopathic scoliosis

The genetic etiology of adolescent idiopathic scoliosis (AIS) remains obscure. Whole-genome sequencing was performed in four members of one family. Then, we performed a rigorous computational analysis to determine the deleterious effects of the identified variants. Furthermore, the structural differences between the native hepatocyte growth factor (HGF) protein and a protein encoded by an HGF variant containing one mutation (p.T596M) were analyzed using molecular dynamic stimulation. A novel heterozygous mutation (p.T596M) within the HGF gene was identified and found to cosegregate with scoliosis phenotypes in three affected family members. Subsequent modeling and structure-based analyses supported the theory that this mutation is functionally deleterious. Functional analyses demonstrated that the HGF p.T596 M mutation changed the ability of the HGF protein to be secreted and impaired migration and invasion in HEK293T cells. Furthermore, an HGF knockdown zebrafish model exhibited a curly tailed phenotype. Mutation in HGF is associated with an autosomal dominant pattern of inheritance of AIS. This finding increases our understanding of the genetic heterogeneity of AIS.     

Changing expression profiles of mRNA,lncRNA, circRNA,and miRNA in lung tissue reveal the pathophysiological of bronchopulmonary dysplasia (BPD) in mouse model

New perinatal care technologies have improved the survival rate of preterm neonates, but the prevalence of bronchopulmonary dysplasia (BPD), one of the most intractable problems in neonatal intensive care unit (NICU), remains unchanged. In present study, high-throughput sequencing (HTS) was performed to detect the expression profiles of long noncoding RNAs (lncRNAs), messenger RNAs (mRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs) in hyperoxia-induced BPD mouse model. Significant differentially expressed RNAs were selected and clustered between the BPD group and the control group. The results revealed that expressions of 1778 lncRNAs, 1240 mRNAs, 97 circRNAs, and 201 miRNAs were significantly altered in the BPD group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to predict the potential functions of differentially expressed RNAs. lncRNA-mRNA and circRNA-miRNA coexpression networks were constructed to detect their association with the pathogenesis of BPD. Our study provides a systematic perspective on the potential function of RNAs during BPD.     

拟双角斯氏线虫生物学特性的研究

本文对拟双角斯氏线虫Ｄ－４－３品系的生物学特性进行了研究。     

Male mouse meiotic chromosome cores deficient in structural proteins SYCP3 and SYCP2 align by homology but fail to synapse and have possible impaired specificity of chromatin loop attachment

The targeted deletion of the meiotic chromosome core component MmSYCP3 results in chromosome synaptic failure at male meiotic prophase, extended meiotic chromosomes, male sterility, oocyte aneuploidy and absence of the MmSYCP2 chromosome core component. To test the functions of SYCP2 and SYCP3 proteins in the cores, we determined the effect of their deletion on homology recognition by whole chromosome painting and the effect on chromatin loop attachment to the cores with endogenous and exogenous sequences. Because we observed that the alignment of cores is between homologs, it suggested that alignment is not a function of the chromosome core components but might be mediated by chromatin-chromatin interactions. The alignment function therefore appears to be separate from intimate synapsis function of homologous cores that is observed to be defective in the SYCP3-/- males. To examine the functions of the SYCP2 and 3 core proteins in chromatin loop attachment, we measured the loop sizes of the centromeric major satellite chromatin and the organization of an exogenous transgene in SYCP3+/+ and SYCP3-/- males. We observed that these satellite chromatin loops have a normal appearance in SYCP3-/- males, but the loop regulation of a 2-Mb exogenous lambda phage insert appears to be altered. Normally the insert fails to attach to the core except by flanking endogenous sequences, but in the absence of SYCP2 and SYCP3, there appears to be multiple attachments to the core. This suggests that the selective preference for the attachment of mouse sequences to the chromosome core in the wild-type male is impaired in the SYCP3-/- male. Apparently the SYCP2 and SYCP3 proteins function in the specificity of chromatin attachment to the chromosome core.