Microchip analytic system for multiplex analysis by real-time polymerase chain reaction with reagents immobilized in microreactors

A microchip analytic system that uses a silicon chip with immobilized in microreactor test-system for multiplex analysis of DNA by real-time polymerase chain reaction (real time PCR) was developed and optimized. We suggested the method of immobilization of PCR-components of a test-system, chose the stabilizer, and conducted the optimization of the composition of reaction mixture to achieve permanent stability of a microchip. We conducted optimization of preparation of samples using magnetic sorbent and indicated that, with 2.6 × 10⁴ copies/ml, 60 min are necessary to obtain positive identification including time for preparation of model samples. The abilities of the created system were demonstrated on the example of microchip analysis of samples with different content of DNA, low absolute limits of detection (20 DNA copies in microreactor), and high reproducibility of the analysis.     

A second generation universal microarray for subtyping influenza a virus

The microchip for influenza A subtyping working on the ??one spot-one subtype?? principle was developed. Each spot contains a set of oligonucleotide probes specific for particular subtypes of hemagglutinin, neuraminidase and matrix protein (influenza A marker). Reliability of the proposed chip is the same as for the full-size microchip for separate hemagglutinin and neuraminidase typing which was created in our group earlier. The image was visualized by labeling the analyzed nucleic acid by either fluorescent dye or biotin with the following fixation in streptavidin-gold nanoparticles and development by silver precipitation. In the second case, the image was analyzed using an ordinary scanner that essentially simplifies influenza A subtyping.     

Microarray for diagnostics of human pathogenic influenza A viruses subtypes

An oligonucleotide microchip was developed for diagnostics of human pathogenic Influenza A viruses subtypes. It contains discriminating probes for H1-, H2-, H3-, H5-, H7- and H9-subtypes of hemagglutinin and for N1-, N2-, and N7-subtypes of neuraminidase. The additional set of probes was used for M-gene of Influenza A viruses definition. Microchip was tested on samples pathogenic H5N1 avian influenza viruses, pandemic H1N1 swine influenza viruses and seasonal H1N1 and H3N2 influenza viruses. The microchip can be used for the analysis of both cultured strains and clinical specimens.     

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

In this study, we present a microarray approach for the typing of influenza A and B viruses, and the subtyping of H1 and H3 subtypes. We designed four pairs of specific multiplex RT-PCR primers and eight specific oligonucleotide probes and prepared microarrays to identify the specific subtype of influenza virus. Through amplification and fluorescent marking of the multiplex RT-PCR products on the M gene of influenza A and B viruses and the HA gene of subtypes H1 and H3, the PCR products were hybridized with the microarray, and the results were analyzed using a microarray scanner. The results demonstrate that the chip developed by our research institute can detect influenza A and B viruses specifically and identify the subtypes H1 and H3 at a minimum concentration of 1 × 10² copies/μL of viral RNA. We tested 35 clinical samples and our results were identical to other fluorescent methods. The microarray approach developed in this study provides a reliable method for the monitoring and testing of seasonal influenza.     

Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N‐linked oligosaccharides released from human serum without derivatization has been developed using on‐line nanoLC and high resolution TOF MS. The N‐linked oligosaccharides were analyzed with MALDI FT‐ICR MS and microchip LC MS (HPLC–Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43×0.075 mm² i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 mm² i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N‐linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.     

Evolutionary Rate and Genetic Heterogeneity of Human T-Cell Lymphotropic Virus Type II (HTLV-II) Using Isolates from European Injecting Drug Users

Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 × 10⁻⁴ and 2.7 × 10⁻⁵ nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10⁻² and 10⁻⁴.     

Types of Amylases in Rice Grains

The subunit structure of phenoloxidase from the larvae of housefly, Musca domestica vicina Maquart, was investigated by urea treatment, SDS-polyacrylamide gel electrophoresis and electron microscopy. Phenoloxidase was dissociated into a single type of subunit (MW 6.5 × 10⁴) by 5 m urea treatment. A similar subunit (MW 6.1 × 10⁴) was also detected by SDS-polyacrylamide gel electrophoresis. Furthermore, four additional protein bands with molecular weights of 4.8 × 10⁴, 3.5 × 10⁴, 2.4 × 10⁴ and 1.2 × 10⁴ were detected by SDS-polyacrylamide gel electrophoresis by extensive incubation of phenoloxidase with SDS. A cylindrical molecular structure was deduced from the electron microscopic analysis. The outside and inside diameter and the height of phenoloxidase molecule were evaluated to be 100, 50 and 70 Å, respectively.     

实时荧光定量RT-PCR在小鼠及人G蛋白mRNA检测中的应用

用自行设计的TaqMan双标探针和扩增引物建立检测鸟苷酸结合蛋白(G-protein)mRNA的实时荧光定量RT-PCR技术.用G蛋白纯品绘制定量标准曲线,实时荧光定量PCR仪检测ECV304细胞和小鼠G蛋白mRNA水平.10μmol/LGqαmRNA反义寡核苷酸(GqαODN)作用ECV304细胞24h后,Gqα的mRNA表达显著下降(3.18×108±1.75×108拷贝下降到1.44×106±4.82×105拷贝),48h和72h下降更明显;Gsα和Gi2α的mRNA表达变化不显著.10μmol/LGsαmRNA反义寡核苷酸(GsαODN)作用ECV304细胞24h后,Gsα的mRNA表达显著下降(2.97×108±2.68×107拷贝下降到4.16×106±2.00×106拷贝),48h和72h下降更明显;Gqα、Gi2α表达变化不显著.小白鼠油酸致伤后6h,GqαmRNA表达显著下降(1.16×108±8.73×106拷贝下降到3.30×106±1.68×106拷贝),24h下降更显著(9.32×107±1.47×107拷贝下降到4.14×106±1.67×106拷贝);Gsα和Gi2α表达变化的趋势同GqαmRNA.结果准确可靠,重现性好.说明建立的实时荧光定量RT-PCR方法成功地实现了对ECV304细胞和小白鼠肺组织G蛋白不同亚型不同丰度基因表达的检测.     

CUBN and NEBL common variants in the chromosome 10p13 linkage region are associated with multibacillary leprosy in Vietnam

Leprosy is caused by infection with Mycobacterium leprae and is classified clinically into paucibacillary (PB) or multibacillary (MB) subtypes based on the number of skin lesions and the bacillary index detected in skin smears. We previously identified a major PB susceptibility locus on chromosome region 10p13 in Vietnamese families by linkage analysis. In the current study, we conducted high-density association mapping of the 9.5 Mb linkage peak on chromosome region 10p13 covering 39 genes. Using leprosy per se and leprosy subtypes as phenotypes, we employed 294 nuclear families (303 leprosy cases, 63 % MB, 37 % PB) as a discovery sample and 192 nuclear families (192 cases, 55 % MB, 45 % PB) as a replication sample. Replicated significant association signals were revealed in the genes for cubilin (CUBN) and nebulette (NEBL). In the combined sample, the C allele (frequency 0.26) at CUBN SNP rs10904831 showed association [p = 1 × 10^?5; OR 0.52 (0.38–0.7)] with MB leprosy only. Likewise, allele T (frequency 0.42) at NEBL SNP rs11012461 showed association [p = 4.2 × 10^?5; OR 2.51 (1.6–4)] with MB leprosy only. These associations remained valid for the CUBN signal when taking into account the effective number of tests performed (type I error significance threshold = 2.4 × 10^?5). We used the results of our analyses to propose a new model for the genetic control of polarization of clinical leprosy.     

Universal oligonucleotide microarray for sub-typing of Influenza A virus

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1-H13, H15, H16) and neuraminidase (N1-N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.     

中国芸香科植物的花粉形态

本文用光学显微镜对我国21属52种2变种芸香科(Rutaceae)植物的花粉形态进行了详细地观察研究,其中对6属6种进行了扫描电镜观察。本科花粉具沟孔,是花粉单类型的科之一。根据萌发孔的数目和外壁纹饰特点,本文将芸香科的花粉分为具3孔沟和4-6孔沟两个花粉类型,并根据各属花粉形态特征,作出了花粉形态分属检索表。此外,本文还对花椒(Zanthoxylum L.)和Fagara两属的合并问题及花椒与吴茱萸(Evodia Scop.)两属之间的亲缘关系等问题进行了讨论。     

Investigation of expression and activity levels of primary rat hepatocyte detoxication genes under various flow rates and cell densities in microfluidic biochips

We investigated the behavior of primary rat hepatocytes in biochips using a microfluidic platform (the integrated dynamic cell culture microchip). We studied the effects of cell inoculation densities (0.2–0.5 × 10⁶ cells/biochip) and perfusion flow rates (10, 25, and 40 µL/min) during 72 h of perfusion. No effects were observed on hepatocyte morphology, but the levels of mRNA and CYP1A2 activity were found to be dependent on the initial cell densities and flow rates. The dataset made it possible to extract a best estimated range of parameters in which the rat hepatocytes appeared the most functional in the biochips. Namely, at 0.25 × 10⁶ inoculated cells cultivated at 25 µL/min for 72 h, we demonstrated better induction of the expression of all the genes analyzed in comparison with other cell densities and flow rates. More precisely, when primary rat hepatocytes were cultivated at these conditions, the time‐lapse analysis demonstrated an over expression of CYP3A1, CYP2B1, ABCC1b and ABCC2 in the biochips when compared to the postextraction levels. Furthermore, the AHR, CYP1A2, GSTA2, SULT1A1, and UGT1A6 levels remained higher than 50% of the postextraction values whereas values of HNF4α, CEBP, and PXR remained higher than 20% during the duration of the culture process. Nevertheless, an important reduction in mRNA levels was found for the xenosensors CAR and FXR, and the related CYP (CYP2E1, CYP7A1, CYP3A2, and CYP2D2). CYP1A2 functionality was illustrated by 700 ± 100 pmol/h/10⁶ cells resorufin production. This study highlighted the functionality in optimized conditions of primary rat hepatocytes in parallelized microfluidic cultures and their potential for drug screening applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:401–410, 2014     

Influence of the postantibiotic effect and postantibiotic Sub-MICs effect of netilmicin,tobramycin, ciprofloxacin and pefloxacin on alginate production byPseudomonas aeruginosa

The postantibiotic effect (PAE) (postantibiotic phase induced by 2× or 4×MIC) as well as the postantibiotic effect of subinhibitory concentrations (0.1×, 0.2× and 0.3× MIC) (PA SME) of netilmicin, tobramycin, ciprofloxacin and pefloxacin affected the production of the virulence factor alginate by aP. aeruginosa strain. Aminoglycosides and ciprofloxacin at a concentration of 4× MIC inhibited the alginate production more significantly than 2× MIC. Suprainhibitory concentrations of aminoglycosides were more effective than pefloxacin (2× or 4× MIC) and ciprofloxacin (2× MIC). PA SME demonstrated by the above antibiotics (with the exception of ciprofloxacin 2× MIC +00.1× MIC) suppressed alginate production more efficiently.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

肌醇磷脂激酶(PI4-K)单克隆抗体的制备及其对细胞生长的抑制

制备了抗肌醇磷脂激酶 ( PI4- K)单克隆抗体 ( A6D)并测定了抗原 -抗体反应基本特性及功能 .结果表明 ,单克隆抗体与固相及溶液中肌醇磷脂激酶的亲和常数分别为 7.5× 1 0 6和 6× 1 0 8( mol/L) -1.单抗 1 .9× 1 0 -7mol/L可以抑制从细胞提取液的 PI4- K酶活力 50 % .用 FITC标记单抗在蛋白微球引导下进入细胞内 ,主要富集在细胞质膜区 ,并对 He La细胞和小鼠小脑细胞生长有明显抑制作用 .     

Further studies on double-stranded RNA species from raspberry plants infected with aphid-borne viruses

Analysis by electrophoresis in polyacrylamide gels, followed by silver staining, of dsRNA extracted from many samples of raspberry leaves infected with raspberry leaf mottle virus (RLMV) and/or raspberry leaf spot virus (RLSV) failed to detect reliably any significant quantities of dsRNA species in excess of 1·0 × 10⁶mol. wt. This contrasts with results reported from Canada where three dsRNA species of estimated mol. wt 2·6 × 10⁶1·6 × 10⁶and 1·1 × 10⁶were consistently associated with infection with RLSV but none were associated with RLMV. However, in Scotland, four dsRNA species of estimated mol. wt 2·4 × 10⁶1·6 × 10⁶0·7 × 10⁶and 0·3 × 10⁶were detected in raspberry infected with apple mosaic ilarvirus. These results suggest that the dsRNA species reported from Canada are not those of RLSV but are probably those of a second virus, possibly an ilarvirus, which occurs together with RLSV and/or induces similar symptoms. A few samples from plants infected with RLMV and RLSV contained very small amounts of two dsRNA species of estimated mol. wt 4·7 × 10⁶and 4·5 × 10⁶. It is not known whether these species are those of RLMV and RLSV.     

Evaluating the effect of increased rates of rhizobial inoculation on grain legume productivity

Intrinsic promiscuity in cowpea and bean enables plants to nodulate with native rhizobia, though sometimes ineffective rhizobia may occupy nodules, resulting in poor response to inoculation. Field trials were conducted from 2014 to 2017 in Marondera, Natural Region II, Zimbabwe, to determine the effect of increasing inoculation rates on legume growth parameters, nitrogen uptake and grain productivity. Treatments included an un-inoculated control and inoculant rates of ×1 (standard), ×2, ×3, ×4, ×5, ×7 and ×10 for both cowpea (rhizobia - inoculant-strain-MAR 1510) and bean (rhizobia-inoculant-strain-CIAT 899). Biomass productivity ranged from 2.05 (×2) - 2.94 t ha^?1 (×4) and 1.10 (×10) – 1.95 t ha^?1 (×4) for cowpea and bean, respectively. Nitrogen uptake increased with increasing inoculation rates reaching up to 57.56 kg N ha^?1 for bean (×4) and 100.20 kg N ha^?1 for cowpea (×3). The uninoculated control was not significantly different from the standard, {(×1); 1 g inoculant 500 g seed^?1} treatment, for cowpea nodule weight and grain productivity. The highest cowpea and bean nodule weights were recorded from the ×3 and ×4 treatments, respectively, in the first season. Cowpea grain yield significantly varied across treatments, ranging between 0.63 and 1.55 t ha^?1 with the ×3 recording the highest yield. The “×4” treatment recorded the highest bean grain productivity reaching up to 0.88 t ha^?1. It can be concluded that, increasing rhizobia cells concentration per unit seed up to ×3 (cowpea) and ×4 (bean) improves response to inoculation and grain productivity suggesting a need to change product formulation or increase inoculation rate.     

Pharmacological study of the effects of leukotrienes C4, D4, E4 & F4 on guinea pig trachelis: Interaction with FPL-55712

Leukotrienes D₄ ? C₄ > E₄ ? F₄ produced qualitatively similar contractions of guinea-pig trachealis, which were antagonized by the SRS-antagonist FPL-55712. Schild analyses indicated that FPL-55712 when tested in a low concentration range (0.57–5.7 × 10^?6M) competitive antagonist of LTC₄, LTE₄ and LTF₄ (slope not significantly different from one). The interaction of FPL-55712 with LTD₄ may be noncompetitive (slope < 1). Comparison of the calculated dissociation constants (?log K_B) indicated that FPL-55712 was more effective at blocking LTE₄ and LTF₄ compared to LTC₄ and LTD₄. In the presence of higher concentrations of FPL-55712 (1.9 × 10^?5M) the antagonism of LTC₄ became noncompetitive. These findings indicate that important differences exist in the interaction of FPL-55712 with the various peptido leukotrienes in guinea pig trachealis. Discovery of more selective antagonists will be needed to determine if multiple receptor subtypes are present in this tissue.     

剪切应力对毛细血管内皮细胞代谢的影响

建立的平行平板流协腔装置适用于研究血管内皮细胞代谢对剪切流场的响应。将培养的人胚肾小球血管单层内皮细胞置于剪应力分别为５＊１０＾－５Ｎ／ｃｍ＾２,１＊１０＾－４Ｎ／ｃｍ＾２和１．５＊１０＾－４Ｎ／ｃｍ＾２的定常层流中剪切２５小时。