秋茄KcRD22基因的克隆与功能分析

本文以用200 mmol/L NaCl处理24 h后的秋茄幼苗为材料提取秋茄叶片总RNA,利用RT-PCR方法克隆获得KcRD22基因的全长cDNA,通过构建pCAMBIA-2300-KcRD22过表达载体,利用农杆菌侵染的方式获得过表达KcRD22的烟草转基因株系,并对转基因株系的耐盐性做出初步分析.实验结果显示:KcRD22基因的ORF长1 131 bp,编码1个等电点为9.07、分子量为39.8 kD、由375个氨基酸组成的蛋白.PCR及RT-PCR鉴定结果表明,KcRD22基因已经分别整合到8株烟草的染色体中,并在两个株系中获得表达.对转基因烟草进行光合作用测定,结果显示100 mmol/L NaCl处理显著降低了野生型烟草的净光合速率,而转基因植株叶片的光合作用受到的影响较小.盐浓度达到200 mmol/L时,转基因植株及野生型烟草净光合速率都明显降低,但盐胁迫解除后,转基因烟草光合作用的恢复情况明显好于野生型烟草,说明KcRD22的过表达提高了烟草的抗盐性.本文初步确定了KcRD22基因对植物耐盐性的贡献,这为进一步深入研究该基因在耐盐机制中的功能奠定了良好基础.     

Reproducibility and quantitation of amplicon sequencing-based detection

To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the β-diversity of microbial communities.     

A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer

Cancer secretomes are a promising source for biomarker discovery. The analysis of cancer secretomes still faces some difficulties mainly related to the intracellular contamination, which hinders the qualification and follow-up validations. This study aimed to establish a high-quality secretome of A549 cells by using the cellular proteome as a reference and to test the merits of this refined secretome for biomarker discovery for non-small cell lung cancer (NSCLC). Using one-dimensional gel electrophoresis followed by liquid-chromatography tandem mass spectrometry, we comprehensively investigated the secretome and the concurrent cellular proteome of A549 cells. A high-quality secretome consisting of 382 proteins was refined from 889 initial secretory proteins. More than 85.3% of proteins were annotated as secreted and 76.8% as extracellular or membrane-bound. The discriminative power of the lung-cancer associated secretome was confirmed by gene expression and serum proteomic data. The elevated level of C4b-binding Protein (C4BP) in NSCLC blood was verified by enzyme-linked immunosorbent assays (ELISA, p = 6.07e-6). Moreover, the serum C4BP level in 89 patients showed a strong association with the clinical staging of NSCLC. Our reference-experiment-driven strategy is simple and widely applicable, and may facilitate the identification of novel promising biomarkers of lung cancer.     

Molecular phylogenetic analysis of the Amiota apodemata and Amiota sinuata species groups (Diptera: Drosophilidae), with descriptions of four new species

The phylogenetic relationships among the East Asian species of the apodemata and sinuata species groups of the genus Amiota were investigated based on DNA sequences of the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. A total of 23 samples of 12 species were employed as in‐group taxa, and one sample for each of four other Amiota species were used as out‐groups. The results suggested with strong confidence the monophyly of both the apodemata and the sinuata groups, whereas the monophyly of the ‘apodemata group + sinuata group’ cluster was less supported. Based on its geographical distribution, the origin of the sinuata group is supposed to be southern China. Four new species were described from Guangxi and Yunnan, China: Amiota reikae Xu & Chen sp. nov. , Amiota guiensis Xu & Chen sp. nov. , Amiota hesongensis Xu & Chen sp. nov. , and Amiota polytreta Xu & Chen sp. nov. © 2013 The Linnean Society of London     

宁夏石砚子墓地颅骨缺损个体的古病理学

本文对出土于宁夏东汉时期的石砚子墓地中一例罕见颅骨病理性损伤个体进行了古病理学诊断分析,该男性个体颅骨上可见不规则形大面积骨质破坏,累及额骨、左侧顶骨、左侧颞骨以及右侧顶骨,缺损周缘可见成骨性、溶骨性反应混合特征,呈向内、外翻卷状,边缘伴有骨膜反应,颅内壁可见较深血管压迹。通过CT影像分析骨骼损伤形态、骨骼损伤区域特征,并结合临床医学资料进行鉴别诊断,排除了朗格汉斯细胞组织细胞增生症、骨血管瘤、Gorham-Stout综合征、骨髓瘤和非特异性感染,推测该病例的病理诊断为甲状腺癌导致的继发性颅骨转移癌。     

The CXCR4 Antagonist AMD3465 Regulates Oncogenic Signaling and Invasiveness In Vitro and Prevents Breast Cancer Growth and Metastasis In Vivo

CXCR4, the receptor for stromal-derived factor-1, is reportedly involved in breast carcinogenesis. However, the mechanisms through which CXCR4 contributes to breast cancer cell growth and metastases are poorly understood. In this study, we examined the putative in vitro and in vivo anti-cancer effects of the specific CXCR4 inhibitor AMD3465. Here, we report that AMD3465 triggers a reduction in breast cancer cell invasiveness in vitro, and promotes marked changes in oncogenic signaling proteins including a reduction in STAT3, JAK2, AKT, and CXCR4 phosphorylation and the reduced expression of GSK3 and cMYC. Using three breast cancer cell lines as murine syngeneic immunocompetent breast cancer models, we found that AMD3465 inhibited breast tumor formation and reduced tumor cell metastases to the lung and liver. Furthermore, treatment with AMD3465 significantly reduced the infiltration of myeloid CD11b positive cells at the aforementioned metastatic sites as well as the spleen implying this agent could regulate the formation of the tumor microenvironment and conceivably the premetastatic niche. In conclusion, our studies suggest that AMD3465 inhibits breast cancer growth and metastases by acting on tumor cells as well as immune cells that constitute the tumor microenvironment. This process appears to be regulated, at least in part, through the modulation of oncogenic signaling that includes the STAT3 pathway. Thus, CXCR4 could be a novel target for breast cancer therapy.     

Production of gene-edited pigs harboring orthologous human mutations via double cutting by CRISPR/Cas9 with long single-stranded DNAs as homology-directed repair templates by zygote injection

Transgenic Research - Precise gene editing of model organisms is required for accurately modeling human diseases and deciphering gene functions. In this study, we used a pair of guide RNAs...     

CXCL12/CXCR4/CXCR7轴在子宫内膜异位症中的研究进展

子宫内膜异位症(endometriosis, EMT)是常见的妇科疾病,发病率高,且有年轻化的趋势。因其治疗困难且复发率高,严重影响了女性的生活质量和生育能力。研究发现趋化因子CXCL12与其受体CXCR4和CXCR7在恶性肿瘤中起重要作用。虽然EMT为良性疾病,但有恶性肿瘤的生物学特征,近来发现CXCL12/CXCR4/CXCR7轴可以影响子宫内膜异位症的定植、侵袭和转移。本文就当前国内外研究CXCL12/CXCR4/CXCR7轴在EMT发生发展过程中的作用进行了综述,旨在为EMT的治疗找到新靶点。     

Isolation of brain mitochondria from neonatal mice

Mitochondria are key contributors to many forms of cell death including those resulting from neonatal hypoxic-ischemic brain injury. Mice have become increasingly popular in studies of brain injury, but there are few reports evaluating mitochondrial isolation procedures for the neonatal mouse brain. Using evaluation of respiratory activity, marker enzymes, western blotting and electron microscopy, we have compared a previously published procedure for isolating mitochondria from neonatal mouse brain (method A) with procedures adapted from those for adult rats (method B) and neonatal rats (method C). All three procedures use Percoll density gradient centrifugation as a key step in the isolation but differ in many aspects of the fractionation procedure and the solutions used during fractionation. Methods A and B both produced highly enriched fractions of well-coupled mitochondria with high rates of respiratory activity. The fraction from method C exhibited less preservation of respiratory properties and was more contaminated with other subcellular components. Method A offers the advantage of being more rapid and producing larger mitochondrial yields making it useful for routine applications. However, method B produced mitochondria that were less contaminated with synaptosomes and associated cytosolic components that suits studies that have a requirement for higher mitochondrial purification.     

Atomic force microscopy study of the rabbit skeletal muscle ryanodine receptors in different functional states

Atomic force microscope was applied to investigate the effect of extrinsic phospholipid on the structure of rabbit skeletal muscle ryanodine receptor/calcium release channel (RyR1). In addition, in the presence of extrinsic phospholipid, the height and elasticity of the RyR1s in different functional states were also measured. The results indicate: (i) most of the RyR1s showed a normal structure only in the presence of extrinsic phospholipid; (ii) treatment of the RyR1s with AMP and Ca²⁺ together could increase their Young’s Modulus but not change their apparent height; (iii) no detectable change in either height or Young’s Modulus of the RyR1s appeared, if the RyR1s were treated with other activators or inhibitors.