Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: Applications on detection, sequencing and virus isolation

Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93–99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10^5.55 TCID₅₀/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.     

Multivariate analysis for characteristics of heat tolerance in horses in Brazil

The environment in which the horse is reared affects its ability to maintain thermal balance which is in turn related to thermal characteristics and regulatory physiological mechanisms. In this study a multivariate analysis of physiological traits in relation to heat tolerance in horses was carried out in the Federal District, Brazil. The aim was to test the ability of these analyses to separate groups of animals and determine which physiological traits are most important in the adaptation to heat stress. Forty adult horses (4 to 13 years) were used, ten from each of four different genetic groups (English thoroughbred, Brazilian showjumper, crossbred and Breton). The traits examined included heart and breathing rate, rectal temperature as well as blood parameters. The data underwent multivariate statistical analysis including cluster, discriminate and canonical using Statistical Analysis System - SAS ® procedures CLUSTER, STEPDISC, CANCORR and DISCRIM. The tree diagram showed clear distances between groups studied and canonical analysis was able to separate individuals in groups. The discriminate analysis identified the variables which were most important in separating these groups. The multivariate analysis was able to separate the animals into groups with RR, HR and RT being important in this separation.     

Extraction of Antioxidants from Wheat Bran Using Conventional Solvent and Microwave-Assisted Methods

Total phenolic and tocopherol contents and free radical scavenging capability of wheat bran extracted using conventional and microwave-assisted solvent extraction methods were studied. Three different solvents (methanol, acetone, and hexane) were used in the conventional solvent extraction. Methanol was the most effective solvent, producing higher extraction yield (4.86%), total phenolic compound content (241.3 μg of catechin equivalent/g of wheat bran), and free radical scavenging capability (0.042 μmol of trolox equivalent/g of wheat bran) than either acetone or hexane. However, there was no significant difference in the total tocopherol contents (13.6–14.8 μg/g of wheat bran) among the three different solvent extraction methods. Microwave-assisted solvent extraction using methanol significantly increased the total phenolic compound content to 467.5 and 489.5 μg of catechin equivalent; total tocopherol content to 18.7 and 19.5 μg; and free radical scavenging capability to 0.064 and 0.072 μmol of trolox equivalent/g of wheat bran at extraction temperatures of 100 and 120°C, respectively. However, extraction yields of conventional methanol solvent and microwave-assisted methanol extractions at different temperatures were not significantly different.     

Analysis of soil microbial communities based on amplicon sequencing of marker genes

The use of cultivation independent methods has revolutionized soil biology in the last decades. Most popular approaches are based on directly extracted DNA from soil and subsequent analysis of PCR-amplified marker genes by next-generation sequencing. While these high-throughput methods offer novel possibilities over cultivation-based approaches, several key points need to be considered to minimize potential biases during library preparation and downstream bioinformatic analysis. This opinion paper highlights crucial steps that should be considered for accurate analysis and data interpretation.     

Foliar application of calcium and kinetin on soybean at reproductive stage

We characterized the pollination and fecundation times in soybean flowers and evaluated the effects of kinetin and calcium applications on physiological and productive traits of soybean plants during the reproductive stage. The anatomical study of flowers of eight soybean cultivars showed that fecundation occurred in closed flowers with visible petals, which presented embryo in the first cell divisions. These results indicate that fertilizers and growth regulation applications should be performed before flower opening, which is different from the current recommendation. Foliar applications of kinetin and calcium between the floral bud and full flowering stages did not affect carbon dioxide (CO₂) assimilation, yield components and final yield. The results obtained in this research showed the lack of viability of foliar application of calcium and kinetin in order to increase pod set and number of seeds per plant in soybean crop.     

Sensitivity of Soil P Availability Tests to Ca-P in Oxisols

We assessed the sensitivity of mixed exchange resin (MER) and Mehlich-3 (M3) to calcium-bound phosphate (Ca-P) in Oxisols. These methods were compared with Mehlich-1 (M1) and anion exchange resin (AER), which have high and low sensitivity, respectively, to this soil P form. We used maize (Zea mays L.) as a test plant. Three samples from two Oxisols were treated with five ratios of Triple Superphosphate—TS and Bayóvar Phosphate Rock—BPR (0 + 100; 25 + 75, 50 + 50, 75 + 25 and 100 + 0% TS + BPR). The MER extracted more P than AER and M3 at the highest BPR:TS ratios (high Ca-P) and similar quantities at the higher TS:BPR ratios (low Ca-P) for two soil samples. Unlike M1-extracted P, M3-P, AER-P, and MER-P positively correlated with maize dry matter production and P uptake. Mehlich-3 and MER were suitable to assess P availability in high Ca-P Oxisols.     

An explicit stem profile model for forked and un-forked teak (Tectona grandis) trees in West Africa

Studies on stem profile of teak (Tectona grandis) hardly exist. This can be possibly attributed to the occurrence of forks that is prevalent on teak trees. Stem profile model was therefore developed for teak in West Africa, which took into account the occurrence of forks. Trees were destructively sampled from Moist Evergreen Forest (MEF), Moist Semi-deciduous Forest (MSDF), Dry Semi-deciduous Forest (DSDF) and Savannah ecological zones in both Ghana and Côte d’Ivoire for the study. A single set of parameters could be used explicitly to predict stem profile of any teak tree, irrespective of country, eco-climatic zone, edaphic variables, site index, tree age, stand density and tree competition. The model efficiency and root mean square error (in relative diameter) were calculated to be 0.97 and 0.053, respectively. The relative position of the forks along the stem bole, with respect to total tree height, was highly variable and did not follow any particular trend, except higher value for the first fork in the Moist Evergreen Forest zone. However, average tree height to the first fork decreased along the ecological gradient from MEF, MSDF and DSDF to Savannah. The extent of diameter reduction due to the forks could be predicted. Relative tree form for zero-forked, one-forked and two-forked trees was calculated to be 0.39, 0.36 and 0.33, respectively. Relative loss of stem volume due to one fork and two forks was estimated to be 6.5 and 13.9%, respectively. The measured stem volume when related to the corresponding predicted value yielded coefficient and intercept not significantly different from unity and zero, respectively, with an r² value of 0.97.     

Validation of enzyme-linked immunosorbent assay for measurement of faecal cortisol in fish

Quantification of glucocorticoid (GC) levels in faeces has become an established method for the non-invasive assessment of adrenocortical activity. These hormones are frequently determined in plasma samples as parameters of adrenal activity and response to stress. Because GCs are metabolized and excreted with both intact hormone and their metabolites present in faeces, the concentration of GCs can be measured in excreta. Faecal samples present the advantages of easy collection, no stress to the animal and elimination of the issue of potentially misleading acute GC spikes. The aim of this study was to determine if an enzyme-linked immunosorbent assay (ELISA) for cortisol was appropriate for monitoring adrenocortical activity in faecal casts of fishes. Performance of the cortisol ELISA was validated by comparison to high-performance liquid chromatography, which is an established method for measuring free GCs and GC metabolites in faeces. Parallelism and sample extraction efficiency were compared for the two methods. Pearson's correlation across samples for these methods was 0.996. Results demonstrated that the ELISA was an efficient, sensitive and reliable method for cortisol measurement in faecal extracts, which should permit integration of non-invasive stress monitoring into studies of fish behaviour and physiology.     

以谷蛋白GluA-2信号肽增强外源蛋白在转基因水稻胚乳中的表达与积累

提高外源蛋白在特定目标组织器官中的表达量是转基因植物研究与开发的核心技术之一。谷蛋白是水稻种子中最主要的贮藏蛋白,其表达具有严格的时空特异性。为进一步研究谷蛋白信号肽序列在指导基因表达中的作用,本研究克隆了水稻谷蛋白Glu A-2基因的启动子及其信号肽编码序列,并与GUS报告基因编码区融合,构建了分别含有和不含有信号肽的表达载体p13GG和p13GSG;经农杆菌介导法分别转入同一水稻品种中,获得了20多个独立转化子,PCR证明外源基因都已整合进了水稻基因组中。Northern杂交结果表明,融合Glu A-2信号肽编码序列可显著提高GUS基因在水稻胚乳中的转录;利用GUS特异的抗体进行Western杂交分析,显示该信号肽序列可显著提高外源蛋白在转基因水稻胚乳中的积累,但是其所指导表达的GUS蛋白在水稻胚乳中并没有表现出相应的活性,其机制有待进一步深入解析。相关结果对于水稻品质改良基因工程研究以及以水稻种子作为生物反应器高效表达外源蛋白具有重要的指导作用。