A “Crossomics” Study Analysing Variability of Different Components in Peripheral Blood of Healthy Caucasoid Individuals

Background: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. Methodology/Principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex "crossomics'' analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4(+)T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset. Conclusions/Significance: Our study shows that the intra- individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective

Dynamics of Responses in Compatible Potato - Potato virus Y Interaction Are Modulated by Salicylic Acid

To investigate the dynamics of the potato – Potato virus Y (PVY) compatible interaction in relation to salicylic acid - controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVYNTN, the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid - analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level

GMO sampling strategies in food and feed chains

The sampling step is relevant whenever it is necessary to evaluate an analyte in a lot. However, sampling is by far the most crucial step when the analyte is heterogeneously distributed in the lot, especially at low concentration, because high sampling uncertainty could invalidate the overall analytical testing. The economic and legal implications of such failure could greatly exceed the very high cost of accurate sampling. When there is a legal requirement that the analyte be analytically traced, low sampling reliability at any step of the production and distribution chains could cause the entire traceability system to fail. These considerations also apply to the legally - imposed GMO requirements, particularly those for labelling and traceability. Improper sampling can mislead both food producers and consumers about the GM content in products. In addition, if GMO tests fail to detect an unauthorised GM crop, those unauthorised crops could pose potential risks to human and animal health. Failure of traceability could result in collapse of transgenic and conventional/organic co - existence in the fi eld, with subsequent environmental and economic consequences. A sampling plan should be reliable, cost - effective and feasible in the real life. ‘ Fit - for - purpose ’ sampling plans should be studied and applied, given the wide variety of field situations and ultimate goals. In addition, the uncertainty level of the ‘ overall analytical chain ’ is relevant in many respects, such as making decisions about the best procedures to put in place. Uncertainty in GMO sampling is high, therefore statistical studies should be performed to evaluate sampling uncertainty or at least generate insight into how to make responsible decisions on the most appropriate sampling plan to adopt at each step of the food and feed chain. Prior to the European research project Co - Extra, valuable and indispensible research had been conducted on topics such as the distribution of GMOs in a lot and the implementation of reliable software tools to reveal the most suitable sample size for GMO detection. Research conducted within the Co - Extra project fi lled in relevant theoretical gaps related to the assessment of control plans. All of this research represents a basis for selecting appropriate sampling plans that are best adapted to the operational conditions.JRC.I.3-Molecular Biology and Genomic

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals (‘measurement uncertainties’) were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling