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We developed pISA-tree, a straightforward and flexible data management solution for organisation of life science project-associated research data and metadata. pISA-tree was initiated by end-user requirements thus its strong points are practicality and low maintenance cost. It enables on-the-fly creation of enriched directory tree structure (project/Investigation/Study/Assay) based on the ISA model, in a standardised manner via consecutive batch files. Templates-based metadata is generated in parallel at each level enabling guided submission of experiment metadata. pISA-tree is complemented by two R packages, pisar and seekr. pisar facilitates integration of pISA-tree datasets into bioinformatic pipelines and generation of ISA-Tab exports. seekr enables synchronisation with the FAIRDOMHub repository. Applicability of pISA-tree was demonstrated in several national and international multi-partner projects. The system thus supports findable, accessible, interoperable and reusable (FAIR) research and is in accordance with the Open Science initiative. Source code and documentation of pISA-tree are available at https://github.com/NIB-SI/pISA-tree. [ABSTRACT FROM AUTHOR]

Bois noir (BN) is the most widespread European grapevine yellows disease caused by ' Candidatus Phytoplasma solani'. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology - a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ' Ca. P. solani' infection of cv. 'Chardonnay' grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ' Ca. P. solani'. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ' Ca. P. solani' biology and its interactions with grapevine host. [ABSTRACT FROM AUTHOR]

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework. [ABSTRACT FROM AUTHOR]

Objectives: To identify transcription markers that uniquely determine specific Chinese hamster ovary (CHO) cell lines and can be used for the identification of cell lines in the process of biopharmaceutical cell-line development. Results: Five CHO cell lines with different origins were extensively characterised at the transcriptomic level and the results were compared to their karyotype characterisation. The analysed cell lines differ in their karyotype but, due to the genome instability observed during parental and recombinant cell-line establishment, karyotyping is not the preferred method for accurate identification of the various CHO cell lines. Marker genes unique to a specific cell line were identified by microarrays, and their expression was validated by reverse-transcription quantitative real-time PCR. The analysed cell lines can be differentiated by the presence/absence of detectable marker gene expression. Additionally, the similarity of the transcriptional profiles is dependent on cell-line history but independent of the manipulation steps involved in the recombinant cell-line development process. Conclusions: Certain transcripts can be used as markers for the identification of a CHO cell line undergoing recombinant development and thus represent a powerful tool for ensuring the maintenance of high quality standards. [ABSTRACT FROM AUTHOR]

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize ( Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 ( Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In order to comply with the European Union regulatory threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, it is important to trace GMOs from the field. Appropriate sampling methods are needed to accurately predict the presence of GMOs at the field level. A 2-year field experiment with two maize varieties differing in kernel colour was conducted in Slovenia. Based on the results of data mining analyses and modelling, it was concluded that spatial relations between the donor and receptor field were the most important factors influencing the distribution of outcrossing rate (OCR) in the field. The approach for estimation fitting function parameters in the receptor (non-GM) field at two distances from the donor (GM) field (10 and 25 m) for estimation of the OCR (GMO content) in the whole receptor field was developed. Different sampling schemes were tested; a systematic random scheme in rows was proposed to be applied for sampling at the two distances for the estimation of fitting function parameters for determination of OCR. The sampling approach had already been validated with some other OCR data and was practically applied in the 2009 harvest in Poland. The developed approach can be used for determination of the GMO presence at the field level and for making appropriate labelling decisions. The importance of this approach lies in its possibility to also address other threshold levels beside the currently prescribed labelling threshold of 0.9% for food and feed. [ABSTRACT FROM AUTHOR]

Abstract: Postembryonic development of nine species of Niphargus (Crustacea: Amphipoda) was studied, with spine development and shifts in allometric growth being combined in developmental sequences that were compared across species. The developmental sequences show high diversity with respect to the position of individual events in the sequence, as well as a high frequency of events being inapplicable in some species. Within Niphargus, the highest degree of independence between events occurs mainly in early mid-aged instars, where the shifts in position are both the largest in magnitude and also the most frequent in occurrence. Constructive troglomorphic features of subterranean species were inferred to develop more because of accelerated growth rather than a delayed offset of growth. Shifts in both relative timing and growth rate appeared to have played a role in the evolution of sexually dimorphic elongated appendages. Growth patterns differed greatly between species for individual sexually dimorphic and troglomorphic traits, hinting at their possible independent origin. The independence between developmental events that was generally apparent might indicate the existence of an important genetic basis for the extreme intrageneric morphological variation arising from numerous highly variable body parts being combined in a ‘mosaic’ manner. Moreover, we suggest that the overall diversity of the genus might actually be underestimated in light of possible convergent features accompanied by ‘cryptic’ speciation. We conclude that both sequential and growth heterochrony appear to have played a key role in the evolution of Niphargus, the most diverse genus of freshwater amphipods. A preliminary list of heterochronic characters is provided. [Copyright &y& Elsevier]

A series of sampling sites in 6 caves was established in order to assess the importance of the epikarst as a habitat of high diversity of meiofauna. Special attention was given to the stygobiotic species of copepods (Crustacea), which was the most abundant group of animals in water percolating from the epikarst. In total, 37 species of Copepoda were identified from nearly 4000 individuals. To understand epikarst as a habitat, 14 structural and physico-chemical factors were analysed and compared for two types of habitats, i.e., trickles and pools filled with percolation water. Results of canonical correspondence analysis indicated that the significant factors influencing faunal composition in trickles were the thickness of the cave ceiling and the concentrations of sodium, nitrate, and potassium ions, while in pools correlations between individual species and pH, temperature, volume, conductivity and thickness of the cave ceiling were statistically significant. Aquatic copepod assemblages in epikarst encompass species of different taxonomic affinities, living in severe environmental conditions. The pattern of the system reflects both high taxonomic diversity and environmental severity. [ABSTRACT FROM AUTHOR]

An statistical method for analysis of event data analysis, common in electrophysiological investigations of activities of olfactory receptor neurons, is proposed. The method is based on the analysis of the slopes of cumulative frequency distribution function. During the spontaneous activity the distribution function is linear which indicate that probability density distribution is uniform. During the chemical stimulation, the density of action potentials is either increased (excitation - E) or decreased (suppression -S), causing the observable change of the slope. The slope at each event is evaluated by piecewise linear regression. The type (E or S) and time of change of the slope can be determined, providing information for further statistical analyses. Graphically supported computer program (in S-PLUS) is available for interactive or routine use. [ABSTRACT FROM AUTHOR]