Search

Abstract In recent years, the number of metagenomic studies increased significantly. Wide range of factors, including the tremendous community complexity and variability, is contributing to the challenge in reliable microbiome community profiling. Many approaches have been proposed to overcome these problems making hardly possible to compare results of different studies. The significant differences between procedures used in metagenomic research are reflected in a variation of the obtained results. This calls for the need for standardisation of the procedure, to reduce the confounding factors originating from DNA isolation, sequencing and bioinformatics analyses in order to ensure that the differences in microbiome composition are of a true biological origin. Although the best practices for metagenomics studies have been the topic of several publications and the main aim of the International Human Microbiome Standard (IHMS) project, standardisation of the procedure for generating and analysing metagenomic data is still far from being achieved. To highlight the difficulties in the standardisation of metagenomics methods, we thoroughly examined each step of the analysis of the human gut microbiome. We tested the DNA isolation procedure, preparation of NGS libraries for next-generation sequencing, and bioinformatics analysis, aimed at identifying microbial taxa. We showed that the homogenisation time is the leading factor impacting sample diversity, with the recommendation for a shorter homogenisation time (10 min). Ten minutes of homogenisation allows for better reflection of the bacteria gram-positive/gram-negative ratio, and the obtained results are the least heterogenous in terms of beta-diversity of samples microbial composition. Besides increasing the homogenisation time, we observed further potential impact of the library preparation kit on the gut microbiome profiling. Moreover, our analysis revealed that the choice of the library preparation kit influences the reproducibility of the results, which is an important factor that has to be taken into account in every experiment. In this study, a tagmentation-based kit allowed for obtaining the most reproducible results. We also considered the choice of the computational tool for determining the composition of intestinal microbiota, with Kraken2/Bracken pipeline outperforming MetaPhlAn2 in our in silico experiments. The design of an experiment and a detailed establishment of an experimental protocol may have a serious impact on determining the taxonomic profile of the intestinal microbiome community. Results of our experiment can be helpful for a wide range of studies that aim to better understand the role of the gut microbiome, as well as for clinical purposes.

Therapy for pancreatic ductal adenocarcinoma remains challenging, and the chances of a complete cure are very limited. As in other types of cancer, the expression and role of miRNAs in controlling the biological properties of this type of tumor have been extensively studied. A better insight into miRNA biology seems critical to refining diagnostics and improving their therapeutic potential. In this study, we focused on the expression of miR-21, -96, -196a, -210, and -217 in normal fibroblasts, cancer-associated fibroblasts prepared from a ductal adenocarcinoma of the pancreas, and pancreatic carcinoma cell lines. We compared these data with miRNAs in homogenates of paraffin-embedded sections from normal pancreatic tissues. In cancer-associated fibroblasts and cancer cell lines, miRNAs differed significantly from the normal tissue. In detail, miR-21 and -210 were significantly upregulated, while miR-217 was downregulated. Similar transcription profiles were earlier reported in cancer-associated fibroblasts exposed to hypoxia. However, the cells in our study were cultured under normoxic conditions. We also noted a relation to IL-6 production. In conclusion, cultured cancer-associated fibroblasts and carcinoma cells reflect miR-21 and -210 expression similarly to the cancer tissue samples harvested from the patients.

The aims of the study were to determine total mercury concentrations in “rainbow trout Oncorhynchus mykiss (Walbaum)” at their embryo-larval and juvenile stages and to assess mercury concentration dynamics in individual tissues. Samples of rainbow trout were collected at two-month intervals over a period of 18 months (one stock production cycle) at the Velká Losenice trout farm. Feedstuff samples were collected at the same time and analyzed for mercury concentrations. Tissue mercury concentrations were determined in muscle, liver, and kidneys. Analyses were performed using the AMA 254 atomic absorption spectrophotometer. The lowest mercury concentration was found in 14-day-old embryos (hard roe), and the highest concentrations in muscle tissue, liver, and kidneys at the end of monitoring, that is, in rainbow trout aged 18 months. The amount of mercury in feedstuffs showed an increasing trend and ranged between 0.0126 and 0.0859 mgkg−1. A significant effect (P