A high‐throughput BAC end analysis protocol (BAC‐anchor) for profiling genome assembly and physical mapping

Traditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto‐ or allopolyploid species. Here, we developed a highly efficient and low‐cost BAC end analysis protocol, named BAC‐anchor, to identify paired‐end reads containing large internal gaps. Our approach mainly focused on the identification of high‐throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60–100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I‐ and 165 Mlu I‐derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high‐coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies.     

Tle1 attenuates hepatic ischemia/reperfusion injury by suppressing NOD2/NF-κB signaling

ABSTRACT

Liver damage induced by ischemia/reperfusion (I/R) remains a primary issue in multiple hepatic surgeries. Innate immune-mediated inflammatory responses during the reperfusion stage aggravate the injury. Nevertheless, the detailed mechanism of hepatic I/R has not been fully clarified yet. Our research focuses on the role of Transducin-like enhancer of split-1 (Tle1) in the liver I/R injury and the relation between Tle1 and Nucleotide-binding oligomerization domain 2 (NOD2). To answer these questions, we constructed mouse models of I/R and cell models of hypoxia/reoxygenation (H/R). We found decreased Tle1 accompanied by increased NOD2 during reperfusion. Mice pro-injected with Tle1-siRNA emerged aggravated liver dysfunction. Repression of Tle1 had a significant impact on NOD2 and downstream NF-κB signaling in vitro. However, alteration of NOD2 failed to affect the expression of Tle1. To conclude, our study demonstrates that Tle1 shelters the liver from I/R injury through suppression of NOD2-dependent NF-κB activation and subsequent inflammatory responses.     

TGF-β induces GBM mesenchymal transition through upregulation of CLDN4 and nuclear translocation to activate TNF-α/NF-κB signal pathway

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor. The unregulated expression of Claudin-4 (CLDN4) plays an important role in tumor progression. However, the biological role of CLDN4 in GBM is still unknown. This study aimed to determine whether CLDN4 mediates glioma malignant progression, if so, it would further explore the molecular mechanisms of carcinogenesis. Our results revealed that CLDN4 was significantly upregulated in glioma specimens and cells. The inhibition of CLND4 expression could inhibit mesenchymal transformation, cell invasion, cell migration and tumor growth in vitro and in vivo. Moreover, combined with in vitro analysis, we found that CLDN4 can modulate tumor necrosis factor-α (TNF-α) signal pathway. Meanwhile, we also validated that the transforming growth factor-β (TGF-β) signal pathway can upregulate the expression of CLDN4, and promote the invasion ability of GBM cells. Conversely, TGF-β signal pathway inhibitor ITD-1 can downregulate the expression of CLDN4, and inhibit the invasion ability of GBM cells. Furthermore, we found that TGF-β can promote the nuclear translocation of CLDN4. In summary, our findings indicated that the TGF-β/CLDN4/TNF-α/NF-κB signal axis plays a key role in the biological progression of glioma. Disrupting the function of this signal axis may represent a new treatment strategy for patients with GBM.Subject terms: CNS cancer, Epithelial-mesenchymal transition     

Potato virus Y HC-Pro Reduces the ATPase Activity of NtMinD,Which Results in Enlarged Chloroplasts in HC-Pro Transgenic Tobacco

Potato virus Y (PVY) is an important plant virus and causes great losses every year. Viral infection often leads to abnormal chloroplasts. The first step of chloroplast division is the formation of FtsZ ring (Z-ring), and the placement of Z-ring is coordinated by the Min system in both bacteria and plants. In our lab, the helper-component proteinase (HC-Pro) of PVY was previously found to interact with the chloroplast division protein NtMinD through a yeast two-hybrid screening assay and a bimolecular fluorescence complementation (BiFC) assay in vivo. Here, we further investigated the biological significance of the NtMinD/HC-Pro interaction. We purified the NtMinD and HC-Pro proteins using a prokaryotic protein purification system and tested the effect of HC-Pro on the ATPase activity of NtMinD in vitro. We found that the ATPase activity of NtMinD was reduced in the presence of HC-Pro. In addition, another important chloroplast division related protein, NtMinE, was cloned from the cDNA of Nicotiana tabacum. And the NtMinD/NtMinE interaction site was mapped to the C-terminus of NtMinD, which overlaps the NtMinD/HC-Pro interaction site. Yeast three-hybrid assay demonstrated that HC-Pro competes with NtMinE for binding to NtMinD. HC-Pro was previously reported to accumulate in the chloroplasts of PVY-infected tobacco and we confirmed this result in our present work. The NtMinD/NtMinE interaction is very important in the regulation of chloroplast division. To demonstrate the influence of HC-Pro on chloroplast division, we generated HC-Pro transgenic tobacco with a transit peptide to retarget HC-Pro to the chloroplasts. The HC-Pro transgenic plants showed enlarged chloroplasts. Our present study demonstrated that the interaction between HC-Pro and NtMinD interfered with the function of NtMinD in chloroplast division, which results in enlarged chloroplasts in HC-Pro transgenic tobacco. The HC-Pro/NtMinD interaction may cause the formation of abnormal chloroplasts in PVY-infected plants.     

沙门氏菌CRISPR位点的结构特征比较

成簇规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR),是存在于多数细菌和古菌中的遗传结构,能够有效防御外源DNA的入侵(质粒、噬菌体等),进而防御外源基因的水平转移。【目的】本研究以沙门氏菌属中常见的鸡伤寒沙门氏菌(Salmonella gallinarum)、鼠伤寒沙门氏菌(Salmonella typhimurium)、猪霍乱沙门氏菌(Salmonella choleraesuis)以及肠炎沙门氏菌(salmonella enteritidis)等30个菌株为研究对象。探索CRISPR位点在不同沙门氏菌种中的结构差异。【方法】通过生物信息学的方法比较间隔序列与插入序列的同源性以及CRISPR位点与质粒数量关系。【结果】30株沙门氏菌中均存在CRISPR结构,包括CRISPR位点61个以及可疑位点12个。重复序列和cas1基因均不能作为这4类细菌的分类依据。【结论】虽然我们发现CRISPR位点数量与间隔区数量和质粒数量之间均不存在统计学关系,但间隔序列整合子、耐药基因等移动遗传原件具有一定的同源性,说明沙门氏菌在进化过程中不断受外源基因的侵袭。     

A protein-bound polysaccharide from the stem bark of Eucommia ulmoides and its anti-complementary effect

Bioactivity-guided fractionation of a hot-water extract from the stem bark of Eucommia ulmoides led to the isolation of a homogeneous polysaccharide EWDS-2, which was identified as a highly branched protein-bound polysaccharide with average molecular weight between 1000 and 2000 kDa, composed of Glc, Gal, Ara, and Rha in the ratio of 2.2:1.0:0.4:0.2, along with traces of Man and 6.55% of protein. The main linkages of the residues of EWDS-2 include terminal, 1,3-linked, 1,4-linked, 1,2,6-linked, 1,3,6-linked Glc; 1,6-linked, 1,2,6-linked, 1,3,4-linked, 1,4,6-linked Gal; 1,5-linked, 1,3,5-linked Ara; terminal and 1,2,5-linked Rha. The bioassay revealed that EWDS-2 inhibits complement activation on both the classic and alternative pathways with CH₅₀ and AP₅₀ values of 282 ± 11 μg/mL and 144 ± 17 μg/mL, respectively. Preliminary mechanism studies indicate that EWDS-2 inhibits the activation of the complement system by interacting with C1q, C1r, C1s, C2, C3, C4, C5, and C9. The results suggested that EWDS-2 could be valuable for the treatment of diseases associated with the excessive activation of the complement system.     

Identification and characterization of MtMTP1, a Zn transporter of CDF family,in the Medicago truncatula

Zn is an essential micronutrient in plants, and the mechanisms of Zn homeostasis are under intensive study. In this report, we have identified MtMTP1, a Zn transporter of the CDF family in the legume model plant Medicago truncatula. The ORF of the MtMTP1 cDNA encodes a protein consisting of 407 amino acid residues with a predicted molecular mass of 45 kDa. Like other metal tolerance proteins (MTPs) in plants, heterologous expression of MtMTP1 can complement the Zn-susceptible zrc1 cot1 yeast double mutant. The expression pattern was studied by quantitative fluorescent PCR. The expression of MtMTP1 was detected in all vegetative organs with the highest level of expression observed in leaves. With Zn supplementation its expression in roots was reduced while its expression in stems was increased in the first 2 days. No obvious changes were detected in leaves. Inoculation with Rhizobium meliloti down-regulated its expression in roots.     

香溪河河流连续统特征研究

以香溪河附石藻类密度、藻类叶绿素a浓度、Shannon-Wiener多样性指数及生态系统初级生产力随河流级别的变化为例探索该河流的连续统特征。结果表明,附石藻类密度、藻类叶绿素a浓度和Shannon-Wiener多样性指数总体上都有随着河流级别增加而增加的趋势,但在1～3级河段之间这种趋势并不明显,甚至出现了逆转。河流初级生产力也有随河流级别增加而增加的趋势,并在第4级河流达到最大,随后有所下降。在全水系范围内河流初级生产力(P)与群落呼吸(R)比值P／R均大于1,说明香溪河是一条以自养生产为主的河流。所研究的特征并不完全与连续统概念的预测相一致,表明河流已经受到了一定程度的人为干扰。     

黄淮海平原封丘试区水体理化性状的多元分析

在封丘试区内两个最具代表性水体(曹岗湖,潘店芦苇荡)水质调查的基础上,对主要水质指标及其集合进行方差分析和聚类分析,结果表明:封丘试区水体具有典型的北方水体特征,其水质的变化主要受季节影响,水体间无甚大差别。各水体pH值偏高(年平均值为8.0—9.0),属弱碱性;硬度较大(17.54—20.63德国度),均属硬水且有逐年上升趋势;离子总量较大(860—1448mg/L);碱度(4.57—5.20me/L)和主要营养物含量(总氮1.31—2.08mg/L,总磷0.100—0.135mg/L)较高,属富营养型水体。文中指出:该试区水体水质状况较好,渔业开发潜力较大,但磷是水体生物生产力的主要限制性营养物。     

Divergence between general public's risk perception and environmental risk assessment: A case study of p-xylene projects in China

The growing public opposition to p-xylene (PX) project proposals in China requires understanding the divergence between the general public's risk perception and environmental risk assessment (ERA) results and its implications on its decision-making. In a case study in Fujian Province of China, a questionnaire was designed to investigate the public perception of risks of a proposed PX project, interviews were further conducted in Zhangzhou City and Xiamen City, and the survey results were compared with the ERA results of the proposed PX project. Results indicate that the environmental risk is acceptable according to current technical ERA guidelines and standards, while the public acceptance of the PX project is very low. The underlying factors causing the difference between the general public's risk perception and ERA results were identified from the ERA technical perspective. A better understanding of the risk perception divergence from different disciplinary perspectives is helpful for the public acceptance of these kinds of projects, which are on high demand in China. This study not only highlights the necessity to improve the risk communication but also provides insights on future research of ERA for this kind of large-scale project proposal.