SummaryReprogramming to induced pluripotent stem cells (iPSCs) and differentiation of pluripotent stem cells (PSCs) are regulated by epigenetic machinery. Tripartite motif protein 28 (TRIM28), a universal mediator of Krüppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate both processes; however, the exact mechanism and identity of participating KRAB-ZNF genes remain unknown. Here, using a reporter system, we show that TRIM28/KRAB-ZNFs alter DNA methylation patterns in addition to H3K9me3 to cause stable gene repression during reprogramming. Using several expression datasets, we identified KRAB-ZNFs (ZNF114, ZNF483, ZNF589) in the human genome that maintain pluripotency. Moreover, we identified target genes repressed by these KRAB-ZNFs. Mechanistically, we demonstrated that these KRAB-ZNFs directly alter gene expression of important developmental genes by modulating H3K9me3 and DNA methylation of their promoters. In summary, TRIM28 employs KRAB-ZNFs to evoke epigenetic silencing of its target differentiation genes via H3K9me3 and DNA methylation.

BackgroundRNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.ResultsUsing the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.ConclusionsUsing three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.

Wound fluids (WF) are believed to play a role in the local recurrences by inducing an inflammatory process in scar tissue area. Given that most local relapse in primary breast cancer patients occur within the scar tissue area, researchers have investigated whether localized radiotherapy, such as intraoperative radiotherapy (IORT), could be more effective than postoperative RT in inhibiting local tumor recurrence. The epithelial-mesenchymal transition (EMT) program plays a critical role in promoting metastasis in epithelium-derived carcinoma. Given this background the main aim of the present study was to determine the mechanisms by which IORT decreases the tumorigenic potential of WF. We assumed that postoperative fluids from patients would activate the radiation-induced bystander effect (RIBE) in treated cells, thus altering the tumor microenvironment. To confirm this hypothesis, WF collected from patients after breast conserving surgery (BCS) alone, after BCS followed by IORT treatment or WF from BCS patients together with RIBE medium were incubated with MCF7 and MDA-MB-468 cells. Changes in the CSC phenotype, in EMT program and potential to migrate were performed to determine the possible role of WF on the migration of breast cancer cells. Our findings show that wound fluids stimulate the CSC phenotype and EMT program in breast cancer cell lines. This effect was partially abrogated when the cells were incubated in wound fluids collected from patients after breast-conserving surgery followed by IORT. Additionally, we confirmed the role of radiation-induced bystander effect in altering the properties of the WF to induce the CSC phenotype and EMT program.

Die folgenden Verbindungen werden untersucht: CaTeO3 (2 Modifikatione CaTe2O5 (monoklin), BaTeO3 (rhombisch), BaTe2O5 (niedrige Symmetrie), BaTe4O9 sowie α‐ und β‐CdTeO3.

Invasive oncological procedures affect the remaining tumor cells by increasing their survival, proliferation, and migration through the induction of wound healing response. The phenomena of local relapse after breast-conserving surgery (BCS) has resulted in a series of research and clinical trials with the aim of assessing whether localized intraoperative radiotherapy (IORT), may be beneficial in inhibiting local recurrences. Therefore, it is essential to assess the impact of intraoperative radiotherapy in modulating the immunological response and wound healing process. Thus, we decided to perform a quantitative analysis of the composition of surgical wound fluids (SWF) in two groups of breast cancer (BC) patients: those treated with BCS followed by IORT, and those who underwent BCS alone. We found that several cytokines, which are believed to have anti-tumor properties, were highly expressed in the luminal A breast cancer subtype in the IORT treatment group. Interestingly, we also found significant differences between IORT patients with tumors of different molecular subtypes. Based on these findings, we hypothesized that IORT treatment might be beneficial in changing the tumor bed microenvironment, making it less favorable for tumor recurrence due to decreased concentration of tumor-facilitating cytokines, especially in the luminal A subtype of BC.

Organization of a mammahan egg at the beginning of development has been the subject of several experimental as well as descriptive cytological and cytochemical investigations. The regulative capacities of blastomeres isolated at the 2-cell stage have been studied in the rat (Nicholas & Hall, 1942), rabbit (Seidel, 1952,1960) and mouse (Tarkowski, 1959a, b; Mulnard, 1965a, b). Investigations by Seidel and Tarkowski have shown that although the majority of 1/2 blastomeres can regulate and develop into smaller but otherwise normal blastocysts, som e of them give rise to purely trophoblastic vesicles devoid of the inner cell mass. In the mouse, Tarkowski (1959 a, b) observed in addition in a few cases multicellular morulae which had not undergone cavitation. The findings of Tarkowski have been recently confirmed by Mulnard (1965 a, b) who studied cytochemically mouse 1/2 blastomeres developing in vitro.

Background: Uveal melanoma (UM) is the most common intraocular tumour in adults with a poor prognosis and extremely high mortality rate due to the development of metastatic disease. However, despite relatively good knowledge about the histological and genetic risk factors for metastasis development, there is no specific biomarker that would allow early detection of UM progression. Recently, exosomes and their molecular cargo have been widely studied in the search for potential biomarkers in several cancers. The purpose of this study was to analyze the inflammation-related protein cargo of exosomes derived from the serum of primary and metastatic UM patients and healthy donors. Methods: The exosomes were isolated from the serum of primary and metastatic UM patients and healthy donors. Using multiplex immunoassay technology, we analyzed the concentration of 37 inflammation-related proteins in obtained exosomes. Results: The analysis of protein cargo showed several molecules related to inflammation, such as interferon-gamma, interleukin 2, 22 and 12(p40), Pentraxin-3, TNFSF13B and TNFSF8 which were significantly enriched in metastatic UM exosomes. We showed a significant correlation between the disease stage and the concentration of these inflammation-related proteins from exosomal cargo. Conclusions: Based on the obtained results, we propose the panel of exosomal proteins for early detection of uveal melanoma progression into metastatic disease.

MicroRNAs (miRNA) play an essential role in the regulation of gene expression and influence signaling networks responsible for several cellular processes like differentiation of pluripotent stem cells. Despite several studies on the neurogenesis process, no global analysis of microRNA expression during differentiation of induced pluripotent stem cells (iPSC) to neuronal stem cells (NSC) has been done. Therefore, we compared the profile of microRNA expression in iPSC lines and in NSC lines derived from them, using microarray-based analysis. Two different protocols for NSC formation were used: Direct and two-step via neural rosette formation. We confirmed the new associations of previously described miRNAs in regulation of NSC differentiation from iPSC. We discovered upregulation of miR-10 family, miR-30 family and miR-9 family and downregulation of miR-302 and miR-515 family expression. Moreover, we showed that miR-10 family play a crucial role in the negative regulation of genes expression belonging to signaling pathways involved in neural differentiation: WNT signaling pathway, focal adhesion, and signaling pathways regulating pluripotency of stem cells.