布鲁氏菌BP26基因标记疫苗株的构建及鉴别PCR方法的建立

[目的]由于现有的减毒活疫苗仍存在较强的毒力,因抗原与毒株的差异不大而很难区分疫苗免疫和自然感染等缺点,限制了现有布鲁氏菌减毒活疫苗的广泛应用.本文拟对布鲁氏菌的减毒活疫苗株M5进行遗传改造,克服这些缺点.[方法]本研究利用同源重组的方法,用卡那抗性基因替换了布鲁氏菌减毒疫苗株M5的BP26基因,得到了新的标记疫苗株M5△BP26.分别用标记疫苗株和野生株侵染巨噬细胞和感染小鼠,比较分析标记株在细胞内和小鼠体内的存活能力.根据种特异性保守基因dnaK和缺失的BP26基因设计引物,建立双重PCR,用于区分标记株与野生株.[结果]成功构建了.BP26基因标记疫苗株,细胞实验和动物实验结果表明,标记株仍能在胞内和小鼠内存活,具备作为减毒活疫苗的特性.小鼠实验结果显示,感染后两周野生株的细菌数为1022.9 ,而突变株为101.1 (P<0.01),至第3周野生株的细菌数为102.2 ,而突变株未能检出,表明与原疫苗株相比,标记株的感染力进一步减弱.根据DNA序列的差异,建立了能够区分标记疫苗株与野生株的双重PCR方法,标记株因只能扩增出一条带而能与野生株和毒株相区分,从而可以区分自然感染和疫苗免疫.[结论]基因标记疫苗株的构建及鉴别PCR方法的建立,为布鲁氏菌疫苗的进一步研发奠定了基础.     

抑制肿瘤生长活性的内皮生长抑制剂EDI-8t

对内源性的内皮生长抑制剂的序列来源分析后,用RT-PCR方法从人脐带中获得了417bp的cDNA,并克隆到毕赤酵母表达载体pPIC9,然后转化毕赤酵母GS115获得重组表达菌株。表达菌株通过甲醇诱导表达后,将发酵液通过分离纯化获得重组蛋白纯品,命名为EDI-8t。体外实验结果表明,这个胶原蛋白VIII来源的rhEDI-8t蛋白能特异性地抑制牛主动脉内皮细胞的增殖和迁移,并能引起内皮细胞的凋亡。动物体内实验结果表明,rhEDI-8t蛋白样品可有效抑制裸鼠皮下肝癌肿瘤的生长,抑制效果和参照品endostatin相同。但在小鼠黑色素瘤肺转移模型中,rhEDI-8t显示了高于参照品的抑癌效果。     

Length-weight relationships of eight fish species from the Hailang river,China

The present study reports the length-weight relationships (LWRs) for eight fish species sampled in Hailang River, a left-bank tributary of the Mudan River in Northeast China. The fishes were collected from April to October bimonthly 2017 by electrofishing (fishing 2 kilometers along the river and within 5 meters from the bank) and netting (drift gillnet: mesh size 2 cm × 3 cm; 200 m net length). The specimens were weighed (nearest 0.1 g) and measured (nearest 0.1 cm) in the laboratory. This study provides an update in maximum lengths for five species.     

Genome-wide association study identifies 7q11.22 and 7q36.3 associated with noise-induced hearing loss among Chinese population

Noise-induced hearing loss (NIHL) seriously affects the life quality of humans and causes huge economic losses to society. To identify novel genetic loci involved in NIHL, we conducted a genome-wide association study (GWAS) for this symptom in Chinese populations. GWAS scan was performed in 89 NIHL subjects (cases) and 209 subjects with normal hearing who have been exposed to a similar noise environment (controls), followed by a replication study consisting of 53 cases and 360 controls. We identified that four candidate pathways were nominally significantly associated with NIHL, including the Erbb, Wnt, hedgehog and intraflagellar transport pathways. In addition, two novel index single-nucleotide polymorphisms, rs35075890 in the intron of AUTS2 gene at 7q11.22 (combined P = 1.3 × 10⁻⁶) and rs10081191 in the intron of PTPRN2 gene at 7q36.3 (combined P = 2.1 × 10⁻⁶), were significantly associated with NIHL. Furthermore, the expression quantitative trait loci analyses revealed that in brain tissues, the genotypes of rs35075890 are significantly associated with the expression levels of AUTS2, and the genotypes of rs10081191 are significantly associated with the expressions of PTPRN2 and WDR60. In conclusion, our findings highlight two novel loci at 7q11.22 and 7q36.3 conferring susceptibility to NIHL.     

Anticancer effects of melatonin via regulating lncRNA JPX-Wnt/β-catenin signalling pathway in human osteosarcoma cells

Osteosarcoma (OS) is a type of malignant primary bone cancer, which is highly aggressive and occurs more commonly in children and adolescents. Thus, novel potential drugs and therapeutic methods are urgently needed. In the present study, we aimed to elucidate the effects and mechanism of melatonin on OS cells to provide a potential treatment strategy for OS. The cell survival rate, cell viability, proliferation, migration, invasion and metastasis were examined by trypan blue assay, MTT, colony formation, wound healing, transwell invasion and attachment/detachment assay, respectively. The expression of relevant lncRNAs in OS cells was determined by real-time qPCR analysis. The functional roles of lncRNA JPX in OS cells were further examined by gain and loss of function assays. The protein expression was measured by western blot assay. Melatonin inhibited the cell viability, proliferation, migration, invasion and metastasis of OS cells (Saos-2, MG63 and U2OS) in a dose-dependent manner. Melatonin treatment significantly downregulated the expression of lncRNA JPX in Saos-2, MG63 and U2OS cells. Overexpression of lncRNA JPX into OS cell lines elevated the cell viability and proliferation, which was accompanied by the increased metastasis. We also found that melatonin inhibited the OS progression by suppressing the expression of lncRNA JPX via regulating the Wnt/β-catenin pathway. Our results suggested that melatonin inhibited the biological functions of OS cells by repressing the expression of lncRNA JPX through regulating the Wnt/β-catenin signalling pathway, which indicated that melatonin might be applied as a potentially useful and effective natural agent in the treatment of OS.     

分离自苏云金芽胞杆菌的首例细菌乌头酸异构酶TbrA的酶学性质研究(英文)

【目的】乌头酸异构酶(aconitate isomerase,AI)可介导具有多重生物学活性及应用潜力的小分子物质反式乌头酸(trans-aconiticacid,TAA)的合成。本文通过表征来自苏云金芽胞杆菌的生物体首条AI基因(tbrA)的产物——TbrA蛋白的催化性质,填补人们对于AI酶学特性的认识。【方法】我们利用大肠杆菌Rosetta菌株和Ni²⁺柱亲和纯化获得了His6-TbrA蛋白,并在体外通过HPLC检测了产物生成及对应酶活。【结果】TbrA蛋白的最适pH、温度与离子强度分别为8.0,37°C和25 mmol/L。TbrA在10°C时仍保留约60%的活性,展现了较好的耐低温特性。金属离子Mg²⁺、Ca²⁺与还原剂DTT可显著增强TbrA活性,而Fe²⁺、Cu²⁺、Zn²⁺、Mn²⁺则强烈抑制TbrA活性。TbrA将顺式乌头酸(cis-aconitic acid,CAA)异构为TAA的正反应K_m、V...     

Variation in floral sex allocation, female success, and seed predation within racemiform synflorescence in the gynomonoecious Ligularia virgaurea (Asteraceae)

Studies of diclinous species have showed that floral sex allocation and female reproductive success were quite variable within inflorescences. However, little attention has been paid to gynomonoecious species, in which individuals produce both female and bisexual flowers. The purpose of this study was to investigate the variations in reproductive patterns at different capitulum positions within racemiform synflorescence in Ligularia virgaurea, and to determine selective mechanisms of variations in reproductive patterns. We conducted observational and experimental studies in natural populations of the gynomonoecious composite L. virgaurea. Floral sex allocation, seed production and pre-dispersal seed predation were quantified in the field. The results showed several patterns of variation from top to bottom capitula, including an increase in bisexual flowers and flower number per capitulum, but a decrease in seed set and size. Removing earlier capitula during bud stage did not change floral sex allocation in later capitula. And no effect was found on seed set under supplemental pollination. Thus, although it has been reported many times in previous studies, the variation of floral sex allocation in L. virgaurea may not result from architectural effect or mating environment, and the variation of seed production could not be fully explained by pollination success. Additionally, our results showed that L. virgaurea was susceptible to high levels of bisexual biased predation, which was greater for top capitula. We therefore suggest that these variations may help to enhance reproductive success of L. virgaurea in the face of bisexual-biased seed predation.     

Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: A comparative proteomic analysis

Background

Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study.

Methods

We utilized proteomic study to indentify the protein expression changes of Sertoli TM4 cells treated with 10^− 8 M and 10^− 5 M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis.

Results

Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10^− 8 M and 10^− 5 M BPA, respectively, for 24 h. Exposure to 10^− 5 M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10^− 8 M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages.

Conclusions

Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism.

General significance

Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.     

Association analysis of ERBB2 amplicon genetic polymorphisms and STARD3 expression with risk of gastric cancer in the Chinese population

The purpose of this study was to investigate whether risk of gastric cancer (GC) was associated with single nucleotide polymorphisms (SNPs) in a gene cluster on the chromosome 17q12-q21 (ERBB2 amplicon) in the Chinese Han population. We detected twenty-six SNPs in this gene cluster containing steroidogenic acute regulatory-related lipid transfer domain containing 3 (STARD3), protein phosphatase 1 regulatory subunit 1B (PPP1R1B/DARPP32), titin-cap (TCAP), per1-like domain containing 1(PERLD1/CAB2), human epidermal growth factor receptor-2 (ERBB2/HER2), zinc-finger protein subfamily 1A 3 (ZNFN1A3/IKZF3) and DNA topoisomerase 2-alpha (TOP2A) genes in 311 patients with GC and in 425 controls by Sequenom. We found no associations between genetic variations and GC risk. However, haplotype analysis implied that the haplotype CCCT of STARD3 (rs9972882, rs881844, rs11869286 and rs1877031) conferred a protective effect on the susceptibility to GC (P = 0.043, odds ratio [OR] = 0.805, 95% confidence intervals [95% CI] = 0.643–0.992). The STARD3 rs1877031 TC genotype endued histogenesis of gastric mucinous adenocarcinoma and signet-ring cell carcinoma (P = 0.021, OR = 2.882, 95% CI = 1.173–7.084). We examined the expression of STARD3 in 243 tumor tissues out of the 311 GC patients and 20 adjacent normal gastric tissues using immumohistochemical (IHC) analysis and tissue microarrays (TMA). The expression of STARD3 was observed in the gastric parietal cells and in gastric tumor tissues and significantly correlated with gender (P = 0.004), alcohol drinking (P < 0.001), tumor location (P = 0.007), histological type (P = 0.005) and differentiation (P = 0.023) in GC. We concluded that the combined effect of haplotype CCCT of STARD3 might affect GC susceptibility. STARD3 expression might be related to the tumorigenesis of GC in the Chinese population.     

Occurrence and development of Eucryptorrhynchus brandti and E. chinensis (Coleoptera: Curculionidae) on Ailanthus altissima trees subjected to different levels of mechanical damage

Eucryptorrhynchus brandti (Harold) and E. chinensis (Oliver) are herbivores of Ailanthus altissima (Mill.) Swingle (tree of heaven) in China. Eucryptorrhynchus brandti has been recommended as a potential biological control agent of A. altissima in North America. In China, the majority of adults of both species were found on tree trunks 0–5 m above the ground, from May to September. In October and November, most adults were found at the base of A. altissima trees. Trees were subjected to different levels of mechanical injury: very low mechanical injury, one to two 0.015 m² bark sections were removed; low, two to four bark sections were removed; moderate, 8 to 22 bark sections removed; high, tree was girdled; and extreme, tree was felled and the remaining stump was sampled. Very low and low mechanical injury trees had very few adult emergence holes with few larvae developing beyond the first or second instar after 29 months. The health of these trees remained good. An increase in adult emergence holes occurred in moderately injured trees that appeared as healthy as very low to low mechanically injured trees. This may indicate that only a small increase in tree stress can increase the adult emergence rate and thus hasten a decline in tree health. Moderate, high and extreme mechanically injured trees had significantly more emergence holes/m than very low and low injured trees and late larval stages were present. The development of Eucryptorrhynchus species was more successful the greater the degree of mechanical injury. Based on this study both Eucryptorrhynchus species appear to be secondary pests of A. altissima in China.