G145R rHBsAg抗原衰减对抗体亲和层析的影响

目的：探讨G145R rHBsAg抗原衰减对抗体亲和纯化的影响与意义。方法：采用抗野生重组HBs G6-McAb制备层析载体,对含rG145R HBsAg的2A8细胞上清做亲和层析。以SDS-PAGE、Western Blot及ELISA等对产物纯度、含量及回收率进行评价,并与同法纯化之野生HBsAg进行比较。结果：梯度洗脱层析显示G145R rHBsAg、自然表达野生HBsAg及其r-wHBsAg三者纯化产物的纯度分别为90.3%、95.2%及93.1%;回收率为43.3%、72.0%及66.4%,其亲和洗脱峰型前者较后两者略宽,主峰前部出现明显顿挫;pH线性梯度洗脱显示,G145R rHBsAg洗脱曲线主峰较前梯度洗脱进一步增宽,顿挫更为明显,并在主峰前出现一低平的蛋白峰。ELISA检测显示HBsAg活性贯穿主峰始终,SDS-PAGE与Weistern blot显示两法洗脱产物纯度（92.5%与89.3%）大致相似,分子大小与野生HBsAg相同。结论：洗脱峰加宽与顿挫作为G145RrHBsAg抗原性渐进性衰减的特征性表现,在同类生物材料实验性制备与产品考评中有一定参考价值。     

Species diversity of the genus Osmundea (Ceramiales,Rhodophyta) in the Macaronesian region

Species diversity within the genus Osmundea in the Macaronesian region was explored by conducting a comprehensive sampling in the Azores, the Canary, and the Madeira archipelagos. Toward identification, all specimens were first observed alive to verify the absence of corps en cerise, a diagnostic character for the genus and morphometric data were measured (thallus length and width, first‐order branches length and width, branchlets length and width, cortical cell length and width in surface view, cortical cell length and width in transverse section). Specimens were sequenced for COI‐5P (39 specimens) and three species delimitation methods (Generalized Mixed Yule Coalescent, Automatic Barcode Gap Discovery method, and Poisson Tree Processes) were used to assess the threshold between infra‐ and interspecific relationships. Subsequently, one or several sequences of plastid‐encoded large subunit of RuBisCO (21 specimens) per delimited species were generated to assess the phylogenetic relationships among Macaronesian Osmundea. Moreover, for each delineated species, vegetative and reproductive anatomy was thoroughly documented and, when possible, specimens were either assigned to existing taxa or described as novel species. This integrative approach has provided data for (i) the presence of O. oederi, O. pinnatifida, and O. truncata in Macaronesia; (ii) the proposal of two novel species, O. prudhommevanreinei sp. nov. and O. silvae sp. nov.; and (iii) evidence of an additional species referred as “Osmundea sp.1,” which is a sister taxon of O. hybrida.     

Quantitative evaluation of siRNA delivery in vivo

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.     

QF-PCR在染色体异常男性不育症诊断中的应用

为评估定量荧光PCR(QF-PCR)方法在男性不育遗传学诊断中的应用价值,文章对78例非梗阻性男性不育患者,采用精液常规检测精子情况,并检测患者性激素水平;采用QF-PCR方法对患者性染色体多态性STR位点及特异性位点进行检测;采用常规染色体G显带方法进行核型分析;PCR检测AZF微缺失。结果显示78例非梗阻性男性不育患者中发现无精子症患者18例,少精子症患者20例,总检出率为48.72%。采用QF-PCR方法检出3例47,XXY患者,2例46,XX(SRY+)性反转患者,1例AZFc区微缺失患者,与细胞培养染色体分析和AZF微缺失PCR检测结果相符。与传统方法相比,QF-PCR技术能更迅速、直接、可靠地检测到男性不育患者的染色体异常区域,及早发现染色体细微结构异常,有助于染色体异常造成的男性不育症的鉴别诊断。     

Neuroprotective and nootropic drug noopept rescues α-synuclein amyloid cytotoxicity

Parkinson's disease is a common neurodegenerative disorder characterized by α-synuclein (α-Syn)-containing Lewy body formation and selective loss of dopaminergic neurons in the substantia nigra. We have demonstrated the modulating effect of noopept, a novel proline-containing dipeptide drug with nootropic and neuroprotective properties, on α-Syn oligomerization and fibrillation by using thioflavin T fluorescence, far-UV CD, and atomic force microscopy techniques. Noopept does not bind to a sterically specific site in the α-Syn molecule as revealed by heteronuclear two-dimensional NMR analysis, but due to hydrophobic interactions with toxic amyloid oligomers, it prompts their rapid sequestration into larger fibrillar amyloid aggregates. Consequently, this process rescues the cytotoxic effect of amyloid oligomers on neuroblastoma SH-SY5Y cells as demonstrated by using cell viability assays and fluorescent staining of apoptotic and necrotic cells and by assessing the level of intracellular oxidative stress. The mitigating effect of noopept against amyloid oligomeric cytotoxicity may offer additional benefits to the already well-established therapeutic functions of this new pharmaceutical.     

流行性感冒季节性的基本原因和模型理论

流行性感冒（流感）在世界范围内每年造成（300～500）万例严重疾病和（25～50）万例死亡。流感可造成严重的公共卫生和经济问题。流感流行和爆发都是以流感季节为特征。因为与甲型流感病毒相关疾病的发病率和死亡率水平较高,因此本研究从病毒学、流行病学、生物学和数学等方面重点讨论甲型流感季节性的基本原因和模型理论。     

香蕉MaASR1基因的抗干旱作用

ASR(ABA, stress, ripening induced protein)是一类响应植物干旱胁迫的关键转录因子, 在许多植物中已有报道, 然而尚未见香蕉(Musa acuminata)中ASR与抗旱作用的相关研究。该实验从香蕉果实cDNA文库中筛选出1个ASR基因, 即MaASR1(登录号为AY628102)。干旱胁迫下, 该基因在叶片中的表达量高于根部。将MaASR1转入拟南芥(Arabidopsis thaliana), Southern检测确定了两株独立表达的转基因株系(命名为L14和L38)。表型观察发现, 此两转基因株系的叶片变小且变厚; Northern和Western检测结果表明, MaASR1在L14和L38中表达。控水处理后, L14和L38的存活率及脯氨酸含量均高于野生型。经干旱胁迫和外源ABA处理后, 对MaASR1转基因株系中ABA/胁迫响应基因的表达分析, 发现MaASR1可增强转基因株系对ABA信号的敏感度, 但不能增强植株依赖于ABA途径的抗旱性。     

Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.     

Detection and validation of EST‐derived SNPs for poplar leaf rust Melampsora medusae f. sp. deltoidae

We report the discovery, characterization and validation of 118 single nucleotide polymorphisms (SNPs) for poplar leaf rust Melampsora medusae f. sp. deltoidae identified using a gene‐targeted approach in an expressed sequence tag (EST) library. We developed a genotyping assay using the iPLEX? primer extension method for two multiplex assays of 28 and 22 SNPs.