脂肪组织源性干细胞研究进展

增加具有完整功能的种子细胞数目是细胞移植的首要环节。近来研究发现,成体动物脂肪组织中含有大量的具有多向分化潜能的间充质干细胞,在特定条件下可分化为多种组织细胞,如脂肪细胞、成骨细胞、软骨细胞、肌细胞及神经星状细胞等,且具有极强的自我复制能力,有望成为组织工程理想的种子细胞。本文综述了脂肪组织源性干细胞（ADSCs）的发现、生物学特性、多向分化潜能、应用前景及存在的问题。     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

shRNA‑mediated knockdown of KNTC1 inhibits non-small-cell lung cancer through regulating PSMB8

In view of the important roles played by Kinetochore proteins in mitosis, we believed that they may contribute to the development and progression of human cancers, which has been reported recently elsewhere. Kinetochore-associated 1 (KNTC1) participates in the segregation of sister chromatids during mitosis, the effects of which on non-small-cell lung cancer (NSCLC) remain unclear. Here, we sought to identify the biological significance of KNTC1 in NSCLC. KNTC1 protein expression in NSCLC tissues was investigated by immunohistochemistry. Lentivirus delivered short hairpin RNA (shRNA) was utilized to establish KNTC1 silence NSCLC cell lines. The effects of KNTC1 depletion on NSCLC cell proliferation, migration, apoptosis, and tumor formation were analyzed by MTT assay, wound-healing assay, transwell assay, flow cytometry assay, and in nude mouse models in vivo. After KNTC1 reduction, NSCLC cell viability, proliferation, migration, and invasion were restrained. A xenograft tumor model was also provided to demonstrate the inhibited tumorigenesis in NSCLC. In addition, the downstream mechanism analysis indicated that KNTC1 depletion was positively associated with PSMB8. The findings of the present study suggested that KNTC1 may have a pivotal role in mediating NSCLC progression and may act as a novel therapeutic target for NSCLC.Subject terms: Non-small-cell lung cancer, Cell migration     

人胰岛素A、B链基因的合成、克隆及其在大肠杆菌中的表达

根据人胰岛素A、B链的多肽顺序,设计并用一种简便快速的多肽基因合成了A、B链基因。合成的A 链和B 链基因分别克隆到pWR590质粒上,构建了表达型质粒pWR590-HIA和pWR 590-HIB,它们能够表达由β-半乳糖苷酶N-端约590个氨基酸残基与A 链或B 链组成的融合蛋白(两者之间由Met 连接)。A 链或B 链融合蛋白经BrCN 降解,磺化及分离纯化等步骤,得到了磺化A、B 链。磺化A、B链体外重组得到人胰岛素。     

Floral scent emission is the highest at the second night of anthesis in Lonicera japonica (Caprifoliaceae)

Floral color change in diverse plants has been thought to be a visual signal reflecting changes in floral rewards, promoting pollinator foraging efficiency as well as plant reproductive success. It remains unclear whether olfactory signals co-vary with floral color change. We investigated the production rhythms of floral scent and nectar associated with floral color change in Lonicera japonica. The flowers generally last 2–3 days. They are white on opening at night (N1) and become light yellow the following day (D1), yellow on the second night (N2), and golden on the second day of flowering (D2). Our measurements in the four stages indicated that nectar production decreased significantly from N1 and D1 to N2 and D2, tracking the floral color change. A total of 34 compounds were detected in floral scent and total scent emission was significantly higher in N2 than in the other three stages. The scent emission of three major compounds, Linalool, cis-3-Hexenyl tiglate, and Germacrene D was also significantly higher in N2, but the relative content of Linalool decreased gradually, cis-3-Hexenyl tiglate increased gradually, and the relative content of Germacrene D did not differ among the four measured stages. Greater scent emission by night than by day suggested a strong olfactory signal to attract nocturnal hawkmoths, the effective pollinators. However, floral scent rhythms in the four stages did not match the color change and nectar secretion, suggesting that floral color (visual) and scent (olfactory) in this species may play different roles in attracting or filtering various visitors.     

The protein conformation and a zinc-binding domain of an autoantigen from mouse seminal vesicle.

The protein conformation of a mouse seminal vesicle autoantigen was studied by circular dichroism spectroscopy. At pH 7.4, the spectrum in the UV region appears as one negative band at 217 nm and one positive band at 200 nm. This together with the predicted secondary structures indicates no helices but a mixture of beta form, beta turn, and unordered form in the protein molecule. The conformation is stable even at pH 10.5 or 3.0. The spectrum in the near-UV region consists of fine structures that are disturbed in acidic or alkaline solution. The environments around Trp2 and Trp82 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 345 nm, and about 87% of them are accessible to quenching by acrylamide. Correlating the quenching effect of CsCl and Kl on the protein fluorescence to the charged groups along the polypeptide chain suggests the difference in the "local charge" around the two tryptophan residues. The presence of ZnCl2 in the protein solution effects no change in the circular dichroism but perturbs the fluorescence due to Trp82. Analysis of the fluorescence data suggests a Zn(2+)-binding site on the protein, which cannot coordinate with both Ca2+ and Mg2+. The association constant for the complex formation is 1.35 x 10(5) +/- 0.04 x 10(5) M-1 at pH 7.4.     

Design and selection of trap color for capture of the tea leafhopper,Empoasca vitis,by orthogonal optimization

The tea leafhopper, Empoasca vitis (Göthe) (Hemiptera: Cicadellidae), is a major pest of the tea plant, Camellia sinensis (L.) O. Kuntze (Theaceae). In this study, the RGB color model was used to describe the colors of sticky traps. The most effective color for attraction of E. vitis was investigated by orthogonal optimization. The selected color was verified in tea gardens and the most effective height for positioning of color sticky traps for capturing tea leafhoppers was investigated. After the determination of the effect of the three color parameters and their interactions by orthogonal optimization, the color gold (RGB: 255, 215, 0) was selected as the most effective color to trap tea leafhoppers. In tea gardens, more leafhoppers were captured using gold sticky traps (RGB: 226, 204, 4) than using commercially available yellow sticky traps. The most effective height of gold sticky traps for trapping leafhoppers was 40–60 cm above the tea canopy. Few lady beetles were captured at this height. We conclude that the orthogonal optimization method is a convenient and efficient method to screen digitally generated colors for attracting and trapping of pests.     

基于微滴式数字PCR检测肠癌病人游离环状DNA KRAS突变的新方法

基于微滴式数字聚合酶链式反应(Droplet digital polymerase chain reaction,dd PCR)设计一种检测肠癌游离循环DNA(Circulating cell free DNA,cf DNA)中KRAS(V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog)基因突变的新方法并评估其灵敏度和准确性。根据肠癌病人KRAS基因的突变类型设计并合成,采用dd PCR扩增并评估其灵敏度和准确性;根据AMRS-PCR引物设计原理设计KRAS基因的实时定量PCR扩增引物并评估其准确性,进而比较dd PCR和q PCR二者之间的优缺点;最后针对52例肠癌病人的cf DNA采用dd PCR进行检测,研究dd PCR在cf DNA KRAS基因突变检测的应用。成功使用dd PCR和q PCR两种方法对KRAS野生型及7种突变型建立检测方法,使用质粒标准品及实际样品验证该两种方法可行并对其假阳性率、线性范围及检测下限等性能进行了评价,最后成功对52例临床患者和20例正常人的血浆cf DNA样本进行检测,临床灵敏度为97.64%,临床特异性为81.43%。dd PCR的检测性能优于q PCR,LOD达到个位数DNA拷贝,最低可确认突变浓度达到0.01%–0.04%。样本提取效率在方法学建立中也十分重要,直接影响到灵敏度和Cut Off值的判定。临床患者检测结果显示其KRAS突变率接近报道水平。     

Study of Double-Side Ultrasonic Embossing for Fabrication of Microstructures on Thermoplastic Polymer Substrates

Double-side replication of polymer substrates is beneficial to the design and the fabrication of 3-demensional devices. The ultrasonic embossing method is a promising, high efficiency and low cost replication method for thermoplastic substrates. It is convenient to apply silicon molds in ultrasonic embossing, because microstructures can be easily fabricated on silicon wafers with etching techniques. To reduce the risk of damaging to silicon molds and to improve the replication uniformity on both sides of the polymer substrates, thermal assisted ultrasonic embossing method was proposed and tested. The processing parameters for the replication of polymethyl methacrylate (PMMA), including ultrasonic amplitude, ultrasonic force, ultrasonic time, and thermal assisted temperature were studied using orthogonal array experiments. The influences of the substrate thickness, pattern style and density were also investigated. The experiment results show that the principal parameters for the upper and lower surface replication are ultrasonic amplitude and thermal assisted temperature, respectively. As to the replication uniformity on both sides, the ultrasonic force has the maximal influence. Using the optimized parameters, the replication rate reached 97.5% on both sides of the PMMA substrate, and the cycle time was less than 50 s.