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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.

It has been suggested that both niche-based and neutral mechanisms are important for biological communities to evolve and persist. For communities in extreme and isolated environments such as caves, theoretical and empirical considerations (low species turnover, high stress, strong convergence owing to strong directional selection) predict neutral mechanisms and functional equivalence of species. We tested this prediction using subterranean amphipod communities from caves and interstitial groundwater. Contrary to expectations, functional morphological diversity within communities in both habitats turned out to be significantly higher than the null model of randomly assembled communities. This suggests that even the most extreme, energy-poor environments still maintain the potential for diversification via differentiation of niches.

The primary patterns of racial differentiation in the water louse, Asellus aquaticus (L.) sensu RACOVITZA, were assessed by the analysis of geographic variation in 59 morphometric characters. Males from 20 localities in Slovenia and 18 in other countries were analyzed. In Multivariate Discriminant Function Analysis, Minimum Spanning Tree and in Cluster Analysis, samples comprising specimens without eye and body pigmentation are separated from the samples with pigmented specimens, even though these two troglomorphic traits are not being considered. Only 7 traits are needed for the separation. In the cline of the samples with pigmented specimens, 3 groups of samples with the concentric distribution ranges may be distinguished. The results confirm the existence of extensive racial diversification in Slovenia and indicate some inconsistencies in the presently applied classification. The results of separate analysis of male gonopod structure are consistent with the results of multivariate statistical analysis.

Abstract: Proteolytic enzymes have been proposed as new biological prognostic indicators to facilitate decisions about treatment of breast cancer patients following surgery. We reported earlier that the activities of cysteine proteinases (CP), cathepsin (Cat) B and cathepsin (Cat) L and the expression of stefin A might be associated with breast tumor progression and prognosis. Here, the protein concentrations of Cats D, B and L and stefin A have been measured in a series of 60 matched pairs of breast tumours and control adjacent tissues, using ELIS As developed in our laboratory. Median tumor concentrations of Cat D (47 pm/mg), Cat B (222 ng/mg) and Cat L (88 ng/mg) were significantly (p<0.0005) increased by 7 fold, 27 fold and 6 fold, respectively. Much greater increases in the activities of Cat B (63 fold) and of Cat L (274 fold) were found, indicating enhanced activation of cysteine proteinases in tumors, due either to proteolytic activation of proCat B and proCat L and/or to a decrease in specific endogenous cystatins. However, the 1.6-fold decreased (p<0.0001) levels of inhibition by cystatins could not be entirely responsible for more than 100-fold increased ratio of CP:cystatins activity. Moreover, stefin A was either increased or decreased in tumor samples, resulting in a 1.4-fold median increase in tumors. Comparing the biological parameters with the established histo-pathological prognosticators, we found that the increased protein concentration of Cat B was associated with lymph node involvement (p<0.009) and higher stage (p<0.003), and both Cat B and Cat L activities were more increased in high grade tumours (p<0.05). Survival analysis revealed that stefin A was the most significant prognostic factor for disease-free (p<0.008) and overall survival (p<0.02), followed by increased Cat B activity and protein concentration. Cat L was of borderline significance while Cat D was not significant for prognosis. We conclude that enhanced activation of CP, due partially to an imbalance between cysteine proteinases and inhibitors is linked to the progression of breast cancer. Larger sample size is needed to confirm the prognostic significance of stefin A, Cat B and Cat L.