A loop-mediated isothermal amplification procedure targeting the sodA gene for rapid and specific identification of Gallibacterium anatis.

This paper reports on the development and validation of a real-time loop-mediated isothermal amplification assay (LAMP) for rapid and specific identification of Gallibacterium anatis. To design a set of 6 primers using the LAMP technique, the conserved region of the G. anatis sodA gene was selected as a target. To evaluate primer specificity we used 120 field strains, the reference strain G. anatis ATCC 43329, and 9 non-G. anatis bacteria. The results confirmed positive reactions for all G. anatis strains tested by LAMP at 63°C for 60 min, with no cross-reactivity observed for the negative control bacteria, i.e., Haemophilus parainfluenzae (ATCC 51505 and ATCC 33392), Aggregatibacter aphrophilus ATCC 7901, Avibacterium endocarditis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Avibacterium paragallinarum, Ornithobacterium rhinotracheale, and Escherichia coli. The lowest detectable amount of DNA for the LAMP reaction was 0.2561 pg, which was detected in about 34 min, while the highest available concentration of the G. anatis reference strain was detected in about 10 min. The lowest detectable amount of DNA for the real-time PCR reaction was 21.24 pg, which was detected in about 20 min, while the highest available concentration of the G. anatis reference strain was detected in about 7 min. Moreover, using the real-time LAMP assay the reaction could be effectively carried out in a volume of just 13 µL, about half the officially recommended reaction volume (25 µL). The aim of this study was to develop a highly sensitive and specific G. anatis real-time LAMP assay that is less time-consuming and less costly than quantitative PCR.

Arm bone loading index predicts DXA musculoskeletal outcomes in two samples of post-menarcheal girls.

OBJECTIVE: A site-specific bone loading index was developed to predict post-menarcheal arm bone mass, geometry, areal density and non-bone lean mass using organized activity records. METHODS: Two cohorts of post-menarcheal girls (A= 55, B= 48) met analysis inclusion criteria: (1) Whole body and non-dominant radius DXA scans +1.0 to +2.6 years post-menarche; (2) detailed, organized activity records available for 36 months prior to the focal DXA scan; (3) accompanying anthropometric data. DXA non-dominant arm and radius regions of interest (1/3, Ultradistal (UD)) were evaluated. An arm bone loading index (arm totBLI) was developed and refined to describe >50 activities. Separate regression analyses for Cohorts A&B tested explanatory value of arm totBLI for DXA outcomes, accounting for gynecological age, height and whole body non-bone lean mass. RESULTS: In both cohorts, arm totBLI reflecting 3 years of peri-menarcheal activity exposure exhibited strong explanatory value for post-menarcheal radius and arm outcomes (squared semi-partial r =0.07-0.34, p<0.05), except Arm Area. For both cohorts and most outcomes, arm totBLI explained significant variance, even after adjusting for local muscle mass. CONCLUSIONS: In two independent cohorts, arm totBLI may consistently indicate osteogenic and sarcogenic properties of represented activities; additional research is necessary for further refinement and validation.

Glacial history of the European marine mussels Mytilus, inferred from distribution of mitochondrial DNA lineages.

Mussels of the genus Mytilus have been used to assess the circumglacial phylogeography of the intertidal zone. These mussels are representative components of the intertidal zone and have rapidly evolving mitochondrial DNA, suitable for high resolution phylogeographic analyses. In Europe, the three Mytilus species currently share mitochondrial haplotypes, owing to the cases of extensive genetic introgression. Genetic diversity of Mytilus edulis, Mytilus trossulus and Mytilus galloprovincialis was studied using a 900-bp long part of the most variable fragment of the control region from one of their two mitochondrial genomes. To this end, 985 specimens were sampled along the European coasts, at sites ranging from the Black Sea to the White Sea. The relevant DNA fragments were amplified, sequenced and analyzed. Contrary to the earlier findings, our coalescence and nested cladistics results show that only a single M. edulis glacial refugium existed in the Atlantic. Despite that, the species survived the glaciation retaining much of its diversity. Unsurprisingly, M. galloprovincialis survived in the Mediterranean Sea. In a relatively short time period, around the climatic optimum at 10 ky ago, the species underwent rapid expansion coupled with population differentiation. Following the expansion, further contemporary gene flow between populations was limited.

Complete sequences of maternally inherited mitochondrial genomes in mussels Unio pictorum (Bivalvia, Unionidae).

Mitochondrial genomes are frequently used to infer phylogenetic relationships. Some taxa are, however, poorly represented. To facilitate better understanding of the potential of mitochondrial genome data in freshwater mussels, we present here, for the first time, the mitochondrial sequences of 4 complete F-type mitochondrial genomes from the European freshwater bivalve Unio pictorum (Unionidae). These genomes are very compact (15,761 bp) but have a typical gene complement for bilaterian mitochondrial genomes and a very similar organization to other unionid genomes available in databases. Very low nucleotide diversity within the species suggests a small effective population size of Polish U. pictorum, a phenomenon of potential importance for environmental management policies.

[Integration of DNA oscillatory vector pSV neo to genome of NIH3T3: importance in investigations of transformation with c-Ha-ras oncogene]. / Integracja DNA wektora wahadlowego pSVneo do genomu komórek NIH3T3: znaczenie w badaniach transformacji onkogenem c-Ha-ras.

Main aim of this study was evaluation of application of stable clones of transforming cells, containing DNA of plasmid vector for the investigation of oncogenes. Plasmid vector was constructed basing on pSV2neo vector, containing activated oncogene c-Ha-ras-1, derived from pT 24-C3 which was followed by evaluation of phenotypic and genetic changes in standard line of NIH3T3 mouse fibroblast line after transfection with constructed plasmid. After two weeks of culture in selective conditions, transformants resistant to geneticin were obtained and analysis of clones was performed after transfection with constructed vector containing ras oncogene and after transfection pSV2neo. Analysis of efficiency of cloning and transformation basing on growth independent from placement and morphology and investigation of karyotypes demonstrated similar irregularities in both investigated groups and subchromosomal aberrations of NIH3T3 cells were even more frequent than in initial lines of NIH3T3 cells. Southern hybridization with pSV2neo-ras probe demonstrated that only restrictive DNA fragments, obtained by Pst1 enzyme contain copies of neo in cell genomes. Integration of gene cf geneticin-resistance increases thus normally unstable genetically NIH3T3 cells.

Genetic changes in Morris hepatoma 7777 cells.

Analysis of genetic changes in Morris hepatoma 7777 is described. Three different approaches were applied: DNA and chromosome transfection, karyotype analysis, and Southern hybridization modified by use of PFGE. Changes in the genome of tested cells were identified by PFGE and chromosome transfection. This supports the statement that PFGE is a useful method in transformed cell genome analysis.

Delayed hypersensitivity to polyoma and "self" antigens in syngeneic mice.

Using the tail swelling test, delayed-type hypersensitivity (DTH) to tumor and self antigens accompanying the development of syngeneic polyoma tumor in CBA mice was observed. The soluble polyoma cell-surface antigen contained both polyoma and H-2k specificities and induced weak proliferation response of spleen cells in the presence of Il-2 in vitro. Appearance of a measurable tail swelling reaction at the side of the midtail, subcutaneous injection of the eliciting antigen not earlier than at 16th hour, maximum of swelling after 24 hours and the results of histological examination proved that the swelling was caused by a typical DTH to the antigens. Maximum DTH to both antigens occurred in the mice 6 days after polyoma cell transplantation (about 3 days before the appearance of palpable tumor) and weakened as the tumor progressed. DTH activity was transferred by spleen T lymphocytes to naive recipients. Five-day restimulation of splenocytes from polyoma transplanted donors with antigen in the presence of Il-2 led to an increase of antigen-lymphocytes affinity but resulted in a decrease of DTH activity of these cells. The mechanisms of these processes are still discussed.