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关键词： 胚胎干细胞   邻苯二甲酸二丁酯   胚胎毒性  

摘要： 目的应用胚胎干细胞实验模型初步评价塑化剂邻苯二甲酸二丁酯的胚胎毒性。方法体外培养小鼠胚胎干细胞E14TG2a和小鼠成纤维细胞3T3,MTT法检测阳性对照5-氟尿嘧啶、阴性对照青霉素G和受试物邻苯二甲酸二丁酯对E14TG2a细胞和3T3细胞的细胞毒性,计算3种化合物对E14TG2a细胞和3T3细胞的半数生长抑制浓度值IC503T3、IC50E14TG2a;采用悬滴-悬浮-贴壁法体外诱导胚胎干细胞向心肌细胞分化,根据浓度-反应曲线计算3种化合物对E14TG2a细胞的半数分化抑制浓度值ID50E14TG2a。利用胚胎毒性预测模型预测邻苯二甲酸二丁酯的胚胎毒性。结果邻苯二甲酸二丁酯对E14TG2a细胞和3T3细胞的增殖均有抑制作用,其IC503T3和IC50E14TG2a分别为(321.20±8.60)μg/ml和(180.60±4.84)μg/ml。邻苯二甲酸二丁酯对E14TG2a细胞的分化亦有抑制作用,其ID50E14TG2a为(34.40±3.91)μg/ml。根据EST预测模型计算得出邻苯二甲酸二丁酯为弱胚胎毒性。结论邻苯二甲酸二丁酯为弱胚胎毒性。

关键词： 扩增子测序   晚期乳腺癌   循环游离DNA   循环肿瘤DNA   基因突变  

摘要： 目的:应用扩增子测序方法检测乳腺癌循环游离DNA肿瘤相关突变。方法:军事医学科学院附属医院的10例HER2阳性晚期乳腺癌患者血样入组,应用扩增子测序技术检测血细胞和血浆中的循环游离DNA(cfDNA),筛选出肿瘤相关基因突变,即循环肿瘤DNA(ctDNA)。结果:检测10例晚期乳腺癌患者外周循环血50个基因,共检出11个突变基因,其中PIK3CA、ERBB4的阳性检出率均达到100%,AKT、TP53的阳性检出率为90%。结论:检测覆盖度及深度均良好,结果提示11个基因的热点突变区域出现单核苷酸的多态性基因突变,突变基因与PI3K-AKT-m TOR通路、Ras-Raf-MEK-ERK通路明显相关。扩增子测序技术和筛选方法可以用于乳腺癌ctDNA的无创性检测。

关键词： TRANSCRIPTION FACTOR GATA-1   GENE-EXPRESSION   IN-VIVO   DEFINITIVE HEMATOPOIESIS   RETICULATED PLATELETS   FUNCTIONAL PLATELETS   MEGAKARYOPOIESIS   MATURATION   GENERATION   MUTATION  

摘要： Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10(13)-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.

关键词： ENZYMATIC AMPLIFICATION   OLIGONUCLEOTIDE PROBES   FORENSIC CASEWORK   GENOME   QUANTIFICATION   HETEROPLASMY   NUCLEAR   SAMPLES   ALLELE  

摘要： Aim To apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples. Methods A duplex polymerase chain reaction (PCR) assay targeting the mitochondrial DNA (mtDNA) hypervariable regions I/II (HVI/HVII) was developed for NGS analysis on the Roche 454 GS Junior instrument. Eight sets of multiplex identifier-tagged 454 fusion primers were used in a combinatorial approach for amplification and deep sequencing of up to 64 samples in parallel. Results This assay was shown to be highly sensitive for sequencing limited DNA amounts (similar to 100 mtDNA copies) and analyzing contrived and biological mixtures with low level variants (similar to 1%) as well as "complex" mixtures (>= 3 contributors). PCR artifact "hybrid" sequences generated by jumping PCR or template switching were observed at a low level (< 2%) in the analysis of mixed samples but could be eliminated by reducing the PCR cycle number. Conclusion This study demonstrates the power of NGS technologies targeting the mtDNA HVI/HVII regions for analysis of challenging forensic samples, such as mixtures and specimens with limited DNA.

关键词： Human embryonic stem cells   NKX2.1   Respiratory endoderm   Lung   HRV   Differentiation   Rhinovirus   GFP   TRANSCRIPTION FACTOR-I   PDGF RECEPTOR-ALPHA   DIRECTED DIFFERENTIATION   LUNG MORPHOGENESIS   PRIMITIVE STREAK   AIRWAY EPITHELIA   VITRO MODEL   MOUSE   GENERATION   EXPRESSION  

摘要： Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions.

关键词： MEGAKARYOCYTES   CHALLENGES  

摘要： The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megalcaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications.

关键词： retrotransposons   repetitive elements   primate evolution   human ESC   pluripotent state regulators   NANOG   POU5F1 (OCT4)   CTCF   methyl-cytosine deamination   LTR7 RNAs   L1 retrotransposition   LINE   LTR   LTR7/HERVH   evolution of Modern Humans   TRANSCRIPTIONAL NETWORK   BINDING   DIFFERENTIATION   RETROTRANSPOSITION   PLURIPOTENCY   ORGANIZATION   DEAMINATION   EVOLUTION   MUTATION   ORIGIN  

摘要： Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators(NANOG[Nanoghomeobox], POU5F1[POU class 5 homeobox 1], CCCT C-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals' genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions such as

关键词： 酶工程   基因工程   应用  

摘要： 酶工程是指对酶分子进行研究和人为改造,以便了解、生产和利用酶。文章介绍了酶的分子生物学研究方法,以及酶工程在医药、环保和食品工业中的应用。并展望了酶工程的发展前景。

关键词： Correction  

摘要： A correction to the article 'RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells,' that was published in the 9(11) issue.

关键词： NONCODING RNA   CHROMATIN INTERACTOME   SELF-RENEWAL   TRANSCRIPTION   EXPRESSION   EVOLUTION   ENHANCERS   LINCRNAS   IDENTITY   PLURIPOTENCY  

摘要： Long noncoding RNAs (lncRNAs) have been implicated in controlling various aspects of embryonic stem cell (ESC) biology, although the functions of specific lncRNAs, and the molecular mechanisms through which they act, remain unclear. Here, we demonstrate discrete and opposing roles for the lncRNA transcript Haunt and its genomic locus in regulating the HOXA gene cluster during ESC differentiation. Reducing or enhancing Haunt expression, with minimal disruption of the Haunt locus, led to upregulation or downregulation of HOXA genes, respectively. In contrast, increasingly large genomic deletions within the Haunt locus attenuated HOXA activation. The Haunt DNA locus contains potential enhancers of HOXA activation, whereas Haunt RNA acts to prevent aberrant HOXA expression. This work reveals a multifaceted model of lncRNA-mediated transcriptional regulation of the HOXA cluster, with distinct roles for a lncRNA transcript and its genomic locus, while illustrating the power of rapid CRISPR/Cas9-based genome editing for assigning lncRNA functions.