Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage

We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real‐time PCR assay. The concentration of the bacterium was monitored at 5‐day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca. L. asiaticus‐infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus‐infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25‐, 360‐ and 130‐fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.     

利用单基因组扩增法对马传染性贫血病毒疫苗株异质性的分析

前期研究发现,马传染性贫血病毒（Equine infectious anemia virus EIAV）中国弱毒疫苗株并非单一病毒,而是由多种准种（quasispecies）组成的种群。阐明该疫苗株的具体构成,对于确定优势疫苗株和分析其在体内的进化具有重要意义。本研究比较了传统RNA病毒测序法（即bulk PCR）和单基因组扩增法（Single-genome Amplification, SGA）在扩增EIAV疫苗株囊膜表面蛋白gp90基因 V3~V5区序列上的差异。结果发现,利用SGA法和bulk PCR法获得的序列在组内差异率分别为1.84%和1.88%。进一步序列比较发现,SGA法扩增的序列中除了含有与bulk PCR法中同源性较高的序列外,还存在bulk PCR法未检出的含强毒株LN40特异性位点,以及单个氨基酸缺失的序列。上述序列的存在为该疫苗株“多克隆构成”假说提供了佐证。此外,在对抽样偏差分析中发现,由于疫苗株中各种病毒准种在量上的差异,使得传统bulk PCR法不能有效的扩增组成比例较低的病毒准种,而导致测得的序列组成不能完全代表实际情况。SGA法通过对单基因组分的扩增和测序,可避免bulk PCR法的以上缺陷,在分析以准种形式存在的RNA病毒序列方面具有独特的优势。     

Effects of different sources of nitrogen on performance,relative population of rumen microorganisms,ruminal fermentation and blood parameters in male feedlotting lambs

Slow-release urea (SRU) can substitute dietary protein sources in the diet of feedlotting ruminant species. However, different SRU structures show varying results of productive performance. This study was conducted to investigate the effect of different sources of nitrogen on performance, blood parameter, ruminal fermentation and relative population of rumen microorganisms in male Mehraban lambs. Thirty-five male lambs with an average initial BW of 34.7 ± 1.8 kg were assigned randomly to five treatments. Diets consisted of concentrate mixture and mineral and vitamin supplements plus (1) alfalfa and soybean meal, (2) wheat straw and soybean meal, (3) wheat straw and urea, (4) wheat straw and Optigen® (a commercial SRU supplement) and (5) wheat straw and SRU produced in the laboratory. No statistical difference was observed in animal performance and DM intake among treatments. The mean value of ruminal pH and ammonia was higher (P < 0.05) for the SRU diet compared with WU diet. The difference in pH is likely to be due to the higher ammonia level as VFAs concentrations were unchanged. The level of blood urea nitrogen (BUN) was different among treatments (P = 0.065). The highest concentration of BUN was recorded in Optigen diet (183.1 mg/l), whereas the lowest value was recorded in wheat straw-soybean meal diet (147 mg/l). The amount of albumin and total protein was not affected by the treatments. The relative population of total protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus in the SRU treatment was higher (P < 0.01) than that in urea treatment at 3 h post-feeding. During the period of lack of high-quality forage and in order to reduce dietary costs, low-quality forage with urea sources can be used in the diet. Results of microbial populations revealed that SRU can be used as a nitrogen source which can sustainably provide nitrogen for rumen microorganism without negative effects on the performance of feedlotting lambs.     

益生菌对幽门螺杆菌根除后肠道微生物群分布的影响

目的 检验益生菌对幽门螺杆菌根除后肠道微生物群分布的影响。方法 病人分为三联疗法根除组（A组）和三联疗法根除+双歧杆菌三联活菌胶囊联用组（B组）。治疗14天后对病人治疗效果进行多方面评估。收集治疗后患者的粪便样本进行qPCR分析,检测大肠埃希菌、肠球菌、肺炎克雷伯菌、乳杆菌、双歧杆菌及嗜热链球菌的数量。结果 120例患者中有112例按方案完成治疗,完成率为93.3%。A组患者中失访2例,不耐受5例,治疗完成率为88.3%;B组患者中失访3例,不耐受4例,治疗完成率为88.3%。双歧杆菌三联活菌胶囊降低了患者的不良反应并提高了三联疗法根除的治愈率。A组大肠杆菌及肺炎克雷伯菌相对表达水平明显高于B组;然而B组粪肠球菌、双歧杆菌、乳酸杆菌及嗜热链球菌表达高于A组。结论 益生菌的使用对提高三联疗法根除幽门螺杆菌有促进作用。     

Monitoring of Gene Expression Profiles and Isolation of Candidate Genes Involved in Pollination and Fertilization in Rice (Oryza Sativa L.) with a 10K cDNA Microarray

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.     

Expression of collagen type I and type II in consecutive stages of human osteoarthritis

In normal hyaline cartilage the predominant collagen type is collagen type II along with its associated collagens, for example, types IX and XI, produced by normal chondrocytes. In contrast, investigations have demonstrated that in vitro a switch from collagen type II to collagen type I occurs. Some authors have detected collagen type I in osteoarthritic cartilage also in vivo, especially in late stages of osteoarthritis, while others have not. In the light of these diverging results, we have attempted to elucidate which type of collagen, type I and/or type II, is synthesized in the consecutive stages of human osteoarthritis. We performed in situ hybridization and immunohistochemistry with cartilage tissue samples from patients suffering from various stages of osteoarthritis. Furthermore, we quantitated our results on the gene expression of collagen type I and type II with the help of real-time PCR. We found that with the progression of the disease not only collagen type II, but also increasing amounts of collagen type I mRNA were produced. This supports the conclusion that collagen type I gradually becomes one of the factors involved in the pathogenesis of osteoarthritis.     

High yield bacterial expression and purification of active recombinant PA28αβ complex

The PA28 complexes (also termed REG or 11S complexes) are described as activators of the 20S proteasome, a major intracellular protease in eukaryotic cells. They bind to the ends of the barrel-shaped 20S proteasome, and activate its peptidase activities. The interferon γ inducible PA28αβ, made of the two related subunits PA28α and β, is under sustained investigation as it plays important roles in the production by the proteasome of class I antigen peptides. However, in vitro studies of this complex have been impaired by the difficulty of producing large amount of this protein, mainly due to the poor solubility of its β subunit when expressed in Escherichia coli. Here we describe the construction of a bicistronic vector, allowing simultaneous production of functional human PA28α and β subunits in E. coli. Co-expression of the two proteins allows efficient formation of active PA28αβ complexes, that remain soluble and can be easily purified by regular chromatographic procedures.     

嗜盐菌Halobacterium species NRC-1不同盐浓度下基因表达差异分析

用RNA随机起始PCR（RAP—PCR）技术分析了盐杆菌NRC-1（Halobacterium NRC-1）不同NaCl盐度下基因表达的差异。81条引物用于比较17％、30％两种盐度下基因表达的差异,每条引物平均可以产生10条以上扩增条带,共得到15条在2种盐度下差异表达的条带,这些差异扩增条带的获得将有助从分子水平上了解Halobacterium NRC-1在高盐环境下的适应机制。     

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic β-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP’s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable β-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.