Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish

BACKGROUND: The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters. RESULTS: The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus. CONCLUSIONS: Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile

Antimicrobial peptides: A natural alternative to chemical antibiotics and a potential for applied biotechnology

A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, cationic peptides are the most widespread. Interestingly, the variety and diversity of these peptides seem to be much wider than suspected. In fact, novel classes of peptides with varying chemical propertiescontinue to be isolated from different vertebrate and invertebrate species, as well as from bacteria. To the early characterized peptides, mostly cationic in nature, anionic peptides, aromatic dipeptides, processed forms of oxygen-binding proteins and processed forms of natural structural and functional proteins can now be added, just to name a few. In spite of the astonishing diversity in structure and chemical nature displayed by these molecules, all of them present antimicrobial activity, a condition that has led researchers to consider them as "natural antibiotics" and as such a new and innovative alternative to chemical antibiotics with a promising future as biotechnological tools. A resulting new generation of anti microbial peptides (AMPs) with higher specific activity and wider microbe-range of action could be constructed, and hopefully endogenously expressed in genetically-modified organisms

The computer as a tool for the writer: the quest for the "right" word

This symposium paper discusses the role of dictionaries in the process of writing and the effects of computerization. The authors include examples of several different hypothetical lexical searches and their outcomes, based on a writer's needs, and argue that these relationships differ from what is to be found in lexicons. A key point is that writers often want to break habits of thought, predigested thinking and prejudice, in which case dictionaries will not directly serve their writing requirements. The dictionary could be seen as a decision support system for writing, with a computerized system that tracks what has been written and suggests ideas based on the evolving text or on dictionary searches carried out or attempted by the writer

Structural Properties of Planar Graphs of Urban Street Patterns

Recent theoretical and empirical studies have focused on the structural properties of complex relational networks in social, biological and technological systems. Here we study the basic properties of twenty 1-square-mile samples of street patterns of different world cities. Samples are represented by spatial (planar) graphs, i.e. valued graphs defined by metric rather than topologic distance and where street intersections are turned into nodes and streets into edges. We study the distribution of nodes in the 2-dimensional plane. We then evaluate the local properties of the graphs by measuring the meshedness coefficient and counting short cycles (of three, four and five edges), and the global properties by measuring global efficiency and cost. As normalization graphs, we consider both minimal spanning trees (MST) and greedy triangulations (GT) induced by the same spatial distribution of nodes. The results indicate that most of the cities have evolved into networks as efficienct as GT, although their cost is closer to the one of a tree. An analysis based on relative efficiency and cost is able to characterize different classes of cities.Comment: 7 pages, 3 figures, 3 table

Gill tissues of the mussel Mytilus edulis chilensis : A new source for antimicrobial peptides

Antimicrobial peptides are small-sized, cationic and amphipathic molecules able to neutralize pathogenic microorganisms. Their antimicrobial effects tie them to mechanisms of immune defense, which is why they have been normally purified from immune cells. We describe an apparently new group of antimicrobial peptides from gill tissues of the mussel Mytilus edulis chilensis . 20 specimens yielded 40 g of gills which produced 16 mg of an enriched fraction with antimicrobial activity as low as 0.045 \u3bcg/\u3bcl over reference strains. Considering the chemical nature of these molecules we used an acid extraction procedure followed by consecutive cationic exchange and hydrophobic interaction chromatography steps for peptide enrichment. The resulting post Sep-pak C-18\uae 20% acetonitrile (ACN) eluate was fractionated by reverse phase HPLC and all resulting fractions were the source for in vitro antimicrobial activity evaluation. Active fractions were characterized by SDS-containing protein gel electrophoresis. All fractions were particularly enriched with low molecular weight peptides displaying neutralizing growth activity against Gram positive and Gram negative bacteria and 10 times more efficient over fungal pathogens. Active fractions resulted to be thermostable and non cytotoxic to eukaryotic cells. Considering these results, industrial waste gills of bivalves arise as a new source for antimicrobial molecules

Lipopolyamine-mediated transfection of reporter plasmids into a fish cell line

Conditions have been optimised to transfect the fish cell line CHSE-214 to measure expression, maintenance and putative chromosomal integration of the reporter gene LUC, spliced into two versions of an expression vector. The first is pCMVL, and the second p103, a novel pCMVL-derived plasmid to which a highly conserved tandem repeat from the salmon genome was added in an inverted configuration flanking the LUC gene to promote its chromosomal integration. A minimal ratio of one to one, lipopolyamine carrier to plasmid DNA, was enough to efficiently transfect the cell line to follow the fate of target DNAs up to five cell passages. In this time-span we demonstrated the maintenance of the foreign DNA in the cells, the concomitant expression of the reporter gene, and a higher stability of p103 over the control plasmid which might suggest a higher potential for integration. Thus, we define an efficient model system for future in vitro evaluation of potential target genes of commercial interest for fish transgenesis