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.     

Biflavonoids from Podocalyx loranthoides

Analysis of the ethyl acetate extract of the aerials parts of Podocalyx loranthoides led to the isolation of I 7, II 4'-dimethylamentoflavone (1) and II 4'-methylamentoflavone (2). Compound (1) gave a moderate effect against Leishmania mexicana promastigotes.     

Phenotypic differences may limit the potential for habitat restoration involving species translocation: a case study of shape ecophenotypes in different populations of Modiolus modiolus (Mollusca: Bivalvia)

相似文献   

Liquid chromatography-electrospray ionization-mass spectrometry method in multiple reaction monitoring mode to determine 17alpha-ethynylestradiol residues in cattle hair without previous digestion

A liquid chromatography-mass spectrometric (LC-MS/MS) method has been developed for determination of ethynylestradiol residues in cattle hair. Hair samples were pulverized with a cryogenic mill followed by a simple extraction with acetonitrile. A dansyl derivatization procedure to improve ethynylestradiol detection was used before the LC-MS/MS analysis in multiple reaction monitoring (MRM) mode using alpha-estradiol as an internal standard. The method was validated following the latest EU guidelines using blank hair samples spiked at 2 ng g(-1). The detection capability (CCbeta) was less than 2 ng g(-1), and the decision limit (CCalpha) was 1 ng g(-1). Incurred samples obtained 56 days after cow treatment with ethynylestradiol were analyzed, and the presence of ethynylestradiol in the hair was confirmed in all cases.     

Study of enrofloxacin depletion in the eggs of laying hens using diphasic dialysis extraction/purification and determinative HPLC-MS analysis

A study of the depletion of enrofloxacin residues in eggs was carried out using a diphasic dialysis procedure for the extraction of fluoroquinolone residues from the matrix. Enrofloxacin was administered to laying hens through the intramuscular route (15 mg/day) and orally (12 mg/day). After daily collection, the egg albumen and the egg yolk were separated, and the residue levels were determined using an HPLC-MS (API-ESI) method. The enrofloxacin and ciprofloxacin peaks gradually increased until the fifth day, because the drug was employed for 5 days. However, differences were observed in the depletion curves of enrofloxacin and its metabolite when both parts of the egg and the mode of administration were considered.     

Genetic characterization of 218 elite CIMMYT maize inbred lines using RFLP markers

Characterization of genetic diversity among maize inbred lines can facilitate organization of germplasm and improve efficiency of breeding programs. A set of 218 phenotypically diverse inbred maize lines developed at CIMMYT for hybrid production was characterized using 32 RFLP markers to: (1) analyze the genetic diversity present; (2) define potential heterotic groups based on clusters formed with marker data; and (3) identify the most representative testers for each potential heterotic group. Lines were clustered using five different genetic distance measurements to find consensus non-hierarchical clusters. Dendrograms were produced to study hierarchical classification within smaller groups of lines. A very high average allelic diversity was seen in this germplasm. Lines did not cluster based on phenotype, environmental adaptation, grain color or type, maturity, or heterotic response (as determined based on hybrid performance with testers), but lines related by pedigree usually did cluster together. Previously defined testers from opposite heterotic groups were not genetically differentiated, and did not represent well their heterotic group. Discrete clusters were difficult to find; thus, potential heterotic groups will be difficult to suggest using RFLP markers alone. However, suggestions on how to use molecular markers and cross performance information to refine heterotic groups and select representative testers are presented.     

Statistical methods for classifying genotypes

In genetic resource conservation and plant breeding, multivariate data on continuous and categorical traits are collected with the objective of selecting genotypes and accessions that best represent the entire population or gene collection with the minimum loss of genetic diversity. Therefore, the best numerical classification strategy is the one that produces the most compact and well-separated groups, that is, minimum variability within each group and maximum variability among groups. In this study, we review geometric classification techniques as well as statistical models based on mixed distribution models. The two-stage sequential clustering strategy uses all variables, continuous and categorical, and it tends to form more homogeneous groups of individuals than other clustering strategies. The sequential clustering strategy can be applied to three-way data comprising genotypes × environments × attributes. This approach groups genotypes with consistent responses for most of the continuous and categorical traits across environments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Soil organic carbon stock assessment for the different cropland land uses in Italy

The aim of this work was to quantify the soil organic C (SOC) stock in the top 30 cm of mineral soil for the whole Italian territory, according to the different land use types of the Intergovernmental Panel on Climate Change (IPCC) cropland category (arable land, agroforestry, vineyards, olive groves, orchards and rice fields), as a basis for future land use scenarios and to address mitigation policy at country level. A database for SOC stock was created with the data from the national project denominated SIAS and partly from regional map reports. All data were referred to the year 2000 since they were derived from surveys conducted from 1995 to 2005. The data were stratified according to the Italian climatic regions, the landscape position and the IPCC cropland subcategories. Taking into account the uncertainty in the estimate, the mean SOC stock values of the different subcategories show significant differences (p < 0.05) among climatic regions and landscapes, ranging from 41.9 ± 15.9 Mg C ha⁻¹ in the vineyards to 63.3 ± 27.9 Mg C ha⁻¹ in the rice fields. Generally, a small decrease of the SOC stock from the temperate regions toward the Mediterranean ones is observed. Taking into account the mean value of each subcategory and the country area they occupied in 2000, the total C stored in the upper 30 cm of soil was estimated at 490.0 ± 121.7 Tg C. The resulting estimate represents the 17% of the value reported by another study for the soil of the whole country down to 50 cm depth, suggesting the importance of preserving this large C pool. Considering the cropland category as a whole, the estimated mean SOC stock is 52.1 ± 17.4 Mg C ha⁻¹, similar to that reported for other European countries, 50–60 Mg C ha⁻¹. In conclusion, the assessment of the mean SOC stock of the different cropland land uses, landscape position and climate regions could notably help when assessing the impact of different agricultural practices and future stock change evaluation.