几家HCV抗体诊断试剂盒检测系列血清的比较

应用Abbott、UBI及国内几家的HCV第二代抗体诊断试剂盒A、B、c及D检测25名HCV感染者的210份感染后不同时期的系列血清,首次采用ELISA及RIBA分片段检测抗体,并与敏感的PCR法检测血清中HCV RNA结果相比较。虽然各试剂盒的总体检测符合率无显著性差异,但个别国产试剂盒的早期检测能力尚有待提高。用任何一家抗体检测试剂盒均会漏检小部分标本,抗体检测阴性的血清在100000倍稀释后仍可检出HCV RNA,因此,有必要发展包括更多片段包被的第三代HCV抗体诊断试剂盒。     

Detection of human norovirus in cherry tomatoes, blueberries and vegetable salad by using a receptor-binding capture and magnetic sequestration (RBCMS) method

In this study, we developed a sensitive receptor-binding capture and magnetic sequestration (RBCMS) method capable of concentrating human norovirus (HuNoV) from various food samples within few hours. We found that distilled water was suitable for the elution of HuNoV from inoculated tomatoes and blueberries, and glycine buffer improved the elution of HuNoV from inoculated salad. A significant improvement in post-extraction RNA yield was achieved by sequentially heat-releasing and column-extracting over either technique alone. The viral recovery of the RBCMS method was significantly higher than both the same-day PEG method (90 min PEG precipitation) and the two-day PEG method (overnight PEG precipitation) with a recovery rate of 8.75%, 1.03% and 5.40%, respectively. The detection limit of HuNoV by RBCMS method was significantly improved to 0.056 RTU. The estimated minimal concentration powers (MCPs) were 6.11, 30.48, and 63.60-fold for the same-day PEG, two-day PEG, and RBCMS methods, respectively. RNase protection assay suggests that the viral genome was protected from RNase attack by remaining within the viral capsid. The signal detected by the RBCMS method might be more biologically relevant, as it requires both intact viral capsid to bind to HBGA receptors and the presence of viral genome to be amplified. Overall, the RBCMS method takes significantly less time than current PEG precipitation methods, recovers a higher yield of HuNoV from various food samples, and hence exhibits higher sensitivity.     

Application of electrospray ionization ion trap/time-of-flight mass spectrometry for chemically-synthesized small RNAs

In this study, we have demonstrated an accurate and rapid small RNA analytical method with both sequence determination and detailed modification analysis by electrospray ionization-ion trap/time-of-flight mass spectrometry (ESI-IT/TOFMS). To develop this ideal method, we have examined the performance of ESI-IT/TOFMS using various chemically-synthesized model sequences of modified or unmodified microRNAs (miRNAs). The deconvoluted mass of a 22-nucleotide (nt) miRNA was obtained from a multiply charged precursor ion (MS(1)). The ion exhibited high mass accuracy (< 7 ppm) and high mass resolution (a value of m/Δm=10,000) and was therefore very useful in RNA composition assignment. The optimized MS(2) method using ion trap collision-induced dissociation, as well as automatic annotation analysis of product ions based on the accurate mass information, enabled the precise sequencing determination of intact miRNAs. Further, the detailed structural analysis of 3'-terminal modified nucleic acid in intact methylated miRNA was carried out using the MS(3) capability of the hybrid IT/TOFMS. The direct infusion method also provided a high throughput and good sensitivity because the analytical time and sample concentration needed in a series of experiments with reliable data were only 3 min and 100 nM, respectively. This study provides a novel approach for characterizing the intact chemically-synthesized small RNA without chemical and enzymatic digestions and would be widely applicable for the structural analysis of complicated modified small RNAs.     

Characterization of microbial communities in a fluidized-pellet-bed bioreactor for wastewater treatment

In order to make clear the microbiological characteristics of the fluidized-pellet-bed bioreactor (FPB) which is a newly developed wastewater treatment device to perform coagulation, particle pelletization, biological degradation and solid-liquid separation in a single unit, the method of denaturing gradient gel electrophoresis (DGGE) was applied in this study paying attention to the microbial diversity of the granular sludge. Spread plate method was also used for enumeration of aerobic bacteria in unit weight of granular sludge. As a result, slight difference was found between the total aerobic bacteria at the bottom, middle, and top sections though the dissolved oxygen (DO) concentration decreased from about 3.5 mg/L at the bottom inlet to 0.23 mg/L at the top of the FPB bioreactor. From the DGGE finger printing, 17 common species were identified from all these sections, and certain specific species were also identified from each section. The comparability of the microbial communities in the three sections was 83.1%, indicating a very stable structure of the microbial communities. The 16 S rRNA sequence analysis results revealed that the 18 operational taxonomic units (OTUs) obtained all belong to Eubacteria. Among them 11 are Proteobacteria, 3 are Actinobacteria, 2 are low G + C gram-positive bacteria and the remaining 2 belong to other bacteria branches. The dominant microbial communities are typical aerobes or facultative anaerobes commonly encountered in conventional activated sludge.     

RNA Relics and Origin of Life

A number of small RNA sequences, located in different non-coding sequences and highly preserved across the tree of life, have been suggested to be molecular fossils, of ancient (and possibly primordial) origin. On the other hand, recent years have revealed the existence of ubiquitous roles for small RNA sequences in modern organisms, in functions ranging from cell regulation to antiviral activity. We propose that a single thread can be followed from the beginning of life in RNA structures selected only for stability reasons through the RNA relics and up to the current coevolution of RNA sequences; such an understanding would shed light both on the history and on the present development of the RNA machinery and interactions. After presenting the evidence (by comparing their sequences) that points toward a common thread, we discuss a scenario of genome coevolution (with emphasis on viral infectious processes) and finally propose a plan for the reevaluation of the stereochemical theory of the genetic code; we claim that it may still be relevant, and not only for understanding the origin of life, but also for a comprehensive picture of regulation in present-day cells.     

Transmission electron microscopy and scanning force microscopy of poly r(A-U) and poly r(A-U)-ethidium bromide

Transmission electron microscopy and scanning force microscopy of negative-stained, carbon-coated replica and mica-adsorbed preparations of 200 μM poly r(A-U) and 50 μM ethidium bromide/200 μM poly r(A-U) have been employed to evaluate ethidium-induced changes in poly r(A-U) topology. Poly r(A-U) alone exhibits elongated conformations 85–115 nm in length that possess a number of hairpin loops as well as single-stranded domains. While the double-stranded domains are found predominately at the base of the hairpin loops (diameter = 5–30 nm), other rod-like (presumably double-stranded) regions ranging from 25–80 nm in length are present in other portions of the poly r(A-U). In contrast with the poly r(A-U) alone, the EB/poly r(A-U) combination appears as a heterogeneous population of condensed structures whose lengths and widths vary from 12–88 nm and 15–45 nm, respectively. These conformational changes are due to a number of factors, including the displacement of ordered water surrounding the poly r(A-U) and charge shielding of the phosphate groups of the poly r(A-U) upon the binding of the ethidium.     

Minor Intron Splicing from Basic Science to Disease

Pre-mRNA splicing is an essential step in gene expression and is catalyzed by two machineries in eukaryotes: the major (U2 type) and minor (U12 type) spliceosomes. While the majority of introns in humans are U2 type, less than 0.4% are U12 type, also known as minor introns (mi-INTs), and require a specialized spliceosome composed of U11, U12, U4atac, U5, and U6atac snRNPs. The high evolutionary conservation and apparent splicing inefficiency of U12 introns have set them apart from their major counterparts and led to speculations on the purpose for their existence. However, recent studies challenged the simple concept of mi-INTs splicing inefficiency due to low abundance of their spliceosome and confirmed their regulatory role in alternative splicing, significantly impacting the expression of their host genes. Additionally, a growing list of minor spliceosome-associated diseases with tissue-specific pathologies affirmed the importance of minor splicing as a key regulatory pathway, which when deregulated could lead to tissue-specific pathologies due to specific alterations in the expression of some minor-intron-containing genes. Consequently, uncovering how mi-INTs splicing is regulated in a tissue-specific manner would allow for better understanding of disease pathogenesis and pave the way for novel therapies, which we highlight in this review.     

Effects of siRNAs targeting the spacer sequence of plant RNAi vectors on the specificity and efficiency of RNAi

Small interfering RNAs (siRNAs) generated from the spacer region of hairpin RNAs were not detected in the RNA interference (RNAi) plants targeting the fatty acid desaturase gene. The expression of the desaturase gene was stably suppressed even when siRNAs targeting the spacer sequences were introduced into this plant.