An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.     

半乳糖凝集素‑3对骨关节炎成骨细胞矿化的作用研究

炎症反应以炎症因子为代表,是骨关节炎（osteoarthritis,OA）中软骨下骨发生病变的重要机制。炎症因子半乳糖凝集素?3（galectin?3,Gal?3）会引起软骨下骨OA样变,但机制尚不清楚。利用因膝关节OA行全膝关节置换患者的胫骨平台标本,培养成骨细胞。分别在正常氧和缺氧条件下用Gal?3处理成骨细胞后,检测骨钙素、ERRα和Sirtuin 1的表达情况。同时,给予矿化液培养的成骨细胞以Gal?3处理,28 d后使用茜素红染色检测成骨细胞矿化程度并进行定量分析。结果显示,Gal?3抑制成骨细胞骨钙素的表达,在缺氧条件下诱导ERRα和Sirtuin1的表达,在正常氧条件下Gal?3促进OA成骨细胞的矿化。正常氧条件下,Gal?3可以诱导OA成骨细胞的异常矿化,缺氧条件下Gal?3促进成骨,表明Gal?3在OA成骨细胞的矿化中扮演重要角色。     

Crystal structure of Drosophila melanogaster tryptophan 2,3-dioxygenase reveals insights into substrate recognition and catalytic mechanism

Tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidative cleavage of the indole ring of l-tryptophan to N-formylkynurenine in the kynurenine pathway, and is considered as a drug target for cancer immunotherapy. Here, we report the first crystal structure of a eukaryotic TDO from Drosophila melanogaster (DmTDO) in complex with heme at 2.7 Å resolution. DmTDO consists of an N-terminal segment, a large domain and a small domain, and assumes a tetrameric architecture. Compared with prokaryotic TDOs, DmTDO contains two major insertion sequences: one forms part of the heme-binding site and the other forms a large portion of the small domain. The small domain which is unique to eukaryotic TDOs, interacts with the active site of an adjacent monomer and plays a role in the catalysis. Molecular modeling and dynamics simulation of DmTDO-heme-Trp suggest that like prokaryotic TDOs, DmTDO adopts an induced-fit mechanism to bind l-Trp; in particular, two conserved but flexible loops undergo conformational changes, converting the active site from an open conformation to a closed conformation. The functional roles of the key residues involved in recognition and binding of the heme and the substrate are verified by mutagenesis and kinetic studies. In addition, a modeling study of DmTDO in complex with the competitive inhibitor LM10 provides useful information for further inhibitor design. These findings reveal insights into the substrate recognition and the catalysis of DmTDO and possibly other eukaryotic TDOs and shed lights on the development of effective anti-TDO inhibitors.     

Effects of dietary live yeast Hanseniaspora opuntiae C21 on the immune and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

A feeding experiment was conducted to determine effects of Hanseniaspora opuntiae C21 on immune response and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Sea cucumbers were fed with either diets containing C21 at 10⁴, 10⁵ and 10⁶ CFU g^?1 feed or a control diet for 30–50 days, respectively. After feeding for 30 days and 45 days, five sea cucumbers from each tank were sampled for immunological analyses. Results indicated that C21 significantly improved the phagocytic activity in coelomocytes of sea cucumbers (P < 0.05). Moreover, C21 administration significantly enhanced lysozyme (LSZ), phenoloxidase activity (PO), total nitric oxide synthase (T-NOS), superoxide dismutase (SOD), alkaline phosphatase (AKP) and acid phosphatase (ACP) activities in coelomic fluid, and LSZ, T-NOS, AKP and ACP activities in coelomocytes lysate supernatant (CLS) of sea cucumbers (P < 0.05). After feeding for 45 days, 10 sea cucumbers from each dose group were challenged with V. splendidus NB13. Cumulative incidence and mortality of sea cucumbers fed with C21 were found to be lower than those of control group. After feeding for 50 days, sea cucumbers in 10⁴ CFU g^?1 C21 treatment and control tanks were subjected to acute salinity changes (from 30 to 20) for 24 h in the laboratory, and the immunological parameters were measured to evaluate the immune capacities of the A. japonicus. Phagocytic, LAZ and T-NOS activities of C21-treated group were higher than those of control group, indicating that salinity stress tolerance of sea cucumber was enhanced by C21. The present results showed that a diet supplemented with C21 could stimulate the immune system of juvenile A. japonicus thus enhancing their resistance against V. splendidus.     

Identification and characterization of a pig FKBP5 gene with a novel expression pattern in lymphocytes and granulocytes

Abstract

The immunophilins are an important group of regulatory molecules in the immune system. FKBP5, expressed throughout mammals and in fish and birds, functions in both physiological and pathogenic pathways, including innate immunity and steroid-based diseases. In this study, we cloned the first porcine FKBP5 from Rongchang pig by the rapid amplification of cDNA ends technique. The full-length cDNA is 4097?bp, with an open reading frame of 1371?bp that codes for a 457-aa protein. Western blotting detected the porcine FKBP5 protein at highest levels in thymus, followed by spleen and lung. Immunohistochemistry detected the porcine FKBP5 protein in lymphocytes and granulocytes of the blood, and flow cytometry identified greater expression in unactivated (vs. activated) T lymphocytes. Finally, the expression level of porcine FKBP5 in the granulocytes was found to decline significantly from the time of birth to one-year-old. These collective data suggest that the newly identified porcine FKBP5 may function in activation of T cells in pig and in innate immunity in the newborn pig in particular.     

Molecular cloning,characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides): Serum amyloid A

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.     

快速STR分型在法医DNA分析中的应用研究进展

STR分型是目前世界通用的DNA指纹分析方法.传统的分型方法包括DNA提取、DNA定量、PCR扩增和对扩增后的STR片段进行检测和分析,耗时较长(8¹0 h).一些情况下很难满足案件的实际需求.法医学家们在加快STR分型的方法研究一直不懈努力.现从快速提取DNA、无提取直接PCR、快速PCR、微流控芯片技术在STR快速分型方面的应用四方面,对近年来出现的可进行快速STR分型的新技术、新方法进行综述.     

斑马鱼Fbxl5基因多克隆抗体的制备与检测

Fbxl5为F-box基因家族的一员,目前研究发现其与心脏的发育有关。为了在斑马鱼模型中进一步研究该基因的功能,有必要制备其多克隆抗体。通过桥式PCR扩增出亲水性和特异性均好的斑马鱼Fbxl5基因片段,将其克隆入表达载体pET-28a,转化至大肠杆菌Rosseta中。用IPTG诱导Fbxl5重组质粒得到His-Fbxl5的融合蛋白。这个融合蛋白用Ni-IDA凝胶柱亲和纯化,将纯化的His-Fbxl5融合蛋白免疫新西兰大白兔制备多克隆抗体。使用Western Blot检测,获得了Fbxl5原核表达重组融合蛋白及高效价的特异性兔抗Fbxl5多克隆抗体。随后检测了Fbxl5在斑马鱼胚胎和成体组织中蛋白的表达,通过基因芯片分析在Fbxl5-MO和Std胚胎样品的mRNA含量差异.所得结果显示获得了较高效价和特异性好的斑马鱼Fbxl5多克隆抗体,为Fbxl5功能的进一步研究奠定了基础。     

4种快速PCR检测试剂法医学应用的初步研究

采用快速PCR扩增,探索其法医学应用价值.将AmpFLSTRIdentifiler试剂盒分别与4种不同的快速PCR检测试剂联合构建快速PCR体系,以9947A为模板,采用各自优化的循环参数进行扩增,并将各分型结果与常规方法进行比较,结果表明:联合构建的4种快速PCR体系均可获得与常规方法一致的DNA分型结果,扩增用时最短可减少至22 min.可见应用快速PCR方法进行扩增,可获得与常规方法一致的STR分型,并且显著缩短扩增时间,提高DNA分型速率.     

野生和人工养殖鲤鱼口咽腔腭部结构与功能的初步研究

采用SONY DSC-F717数码相机记录了野生和人工养殖鲤鱼口咽腔腭部的形态学差异以及外源刺激对人工养殖鲤鱼口咽腔腭部的变化,并用KYKY-1000B扫描电镜和OLYMPUS DP70生物显微镜观察了鲤鱼口咽腔腭部的结构。结果表明:野生鲤鱼口咽腔的腭部密布栉状突起,而人工养殖鲤鱼口咽腔的腭部光滑平坦,机械刺激下口咽腔腭部的肌肉能强烈收缩,形成与野生鲤鱼栉状构造类似的圆锥状突起。与野生鲤鱼相比,人工养殖鲤鱼口咽腔腭部的味蕾和粘液细胞密度减小。这些变化可能与野生鲤鱼杂食性而人工养殖鲤鱼几乎完全吞食颗粒状配合饲料有关。