The aim of our study was to analyze the presence of 5-methyl-cytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) in the genome of crustacean Daphnia pulex. First, the presence of 5-mC and 5-hmC in genomic DNA was demonstrated by using antibodies specific to either 5-mC or 5-hmC. Then, analysis of 5-mC and 5-hmC using pairs of restriction enzymes with different sensitivity to methylation and hydroxymethylation confirmed the presence of both modifications in selected regions of three genes (Cox4, Cand2 and Ephx1). To get a detailed picture of 5-hmC distribution over the D. pulex genome, we performed 5-hmC enrichment and sequenced the enriched fraction using next generation sequencing and non-enriched library (input) as a control. Comparison of input and enriched libraries showed that 5-hmC in exons is twice as frequent as in introns. Functional analysis indicated that 5-hmC abundance is associated with genes that are involved in the adenylate cyclase-activating G-protein-coupled receptor signaling pathway, molting cycles, morphogenesis and cell fate determination. Genes that lack 5-hmC tend to be involved in the regulation of the transforming growth factor beta receptor signaling pathway and in many mRNA-related processes. Our results suggest that epigenetic modifications are present in the genome of D. pulex and most likely are involved in the regulation of gene expression of this crustacean.

Altered expression of miRNAs in tumor tissue encourages the translation of this specific molecular pattern into clinical practice. However, the establishment of a selective biomarker signature for many tumor types remains an inextricable challenge. For this purpose, a preclinical experimental design, which could maintain a fast and sensitive discovery of potential biomarkers, is in demand. The present study suggests that the approach of 3D cell cultures as a preclinical cancer model that is characterized to mimic a natural tumor environment maintained in solid tumors could successfully be employed for the biomarker discovery and validation. Subsequently, in this study, we investigated an environment-dependent miRNA expression changes in colorectal adenocarcinoma DLD1 and HT29 cell lines using next-generation sequencing (NGS) technology. We detected a subset of 16 miRNAs differentially expressed in both cell lines cultivated in multicellular spheroids compared to expression levels in cells grown in 2D. Furthermore, results of in silico miRNA target analysis showed that miRNAs, which were differentially expressed in both cell lines grown in MCS, are involved in the regulation of molecular mechanisms implicated in cell adhesion, cell-ECM interaction, and gap junction pathways. In addition, integrins and platelet-derived growth factor receptors were determined to be the most significant target genes of deregulated miRNAs, which was concordant with the environment-dependent gene expression changes validated by RT-qPCR. Our results revealed that 3D microenvironment-dependent deregulation of miRNA expression in CRC cells potentially triggers essential molecular mechanisms predominantly including the regulation of cell adhesion, cell–cell, and cell–ECM interactions important in CRC initiation and development. Finally, we demonstrated increased levels of selected miR-142-5p in rectum tumor tissue samples after neoadjuvant long course treatment compared to miR-142-5p expression levels in tumor biopsy samples collected before the therapy. Remarkably, the elevation of miR-142-5p expression remained in tumor samples compared to adjacent normal rectum tissue as well. Therefore, the current study provides valuable insights into the molecular miRNA machinery of CRC and proposes a potential miRNA signature for the assessment of CRC in further clinical research.

In our previous work we showed that DNaseI-like protein from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix retained its activity at salt concentrations as high as 4 M NaCl and the key factor allowing this was the C-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs. The further investigations revealed that this domain originated from proteins related to bacterial competence ComEA/ComE proteins. It is likely that in the course of evolution the DNA-binding domain from these proteins was fused to a metallo-β-lactamase superfamily domain. Very likely such domain organization having proteins subsequently “donated” the DNA-binding domain to bacterial DNases. In this study we have mimicked this evolutionary step by fusing bovine DNaseI and DNA-binding domains. We have created two fusions: one harboring the DNA-binding domain of DNaseI-like protein from Thioalkalivibrio sp. K90mix and the second one harboring the DNA-binding domain of bacterial competence protein ComEA from Bacillus subtilis. Both domains enhanced salt tolerance of DNaseI, albeit to different extent. Molecular modeling revealed the essential differences between their interaction with DNA shedding some light on the differences in salt tolerance. In this study we have enhanced salt tolerance of bovine DNaseI; thus, we successfully mimicked the Nature’s evolutionary engineering that created the extremely halotolerant bacterial DNase. We have demonstrated that the newly engineered DNaseI variants can be successfully used in applications where activity of the wild type bovine DNaseI is impeded by buffers used.

Our study indicates that DNA binding domains are common in many halophilic or halotolerant bacterial DNases and they are potential activators of enzymatic activity at high ionic strength. Usually, proteins adapt to high ionic strength by increasing the number of negatively charged residues on the surface. However, in DNases such adaptation would hinder the binding to negatively charged DNA, a step critical for catalysis. In our study we demonstrate how evolution has solved this dilemma by engaging the DNA binding domain. We propose a mechanism, which enables the enzyme activity at salt concentrations as high as 4 M of sodium chloride, based on collected experimental data and domain structure analysis of a secreted bacterial DNase from the extremely halotolerant bacterium Thioalkalivibrio sp. K90mix. The enzyme harbors two domains: an N-terminal domain, that exhibits DNase activity, and a C-terminal domain, comprising a duplicate DNA binding helix-hairpin-helix motif. Here we present experimental data demonstrating that the C-terminal domain is responsible for the enzyme's resistance to high ionic strength.