Determination of functional RNA binding sites for MBNL proteins using antisense oligonucleotides

Wydział BiologiiRodzinę białek Muscleblind-like tworzą trzy paralogi (MBNL1, 2 i 3) będące regulatorami alternatywnego splicingu kluczowymi podczas rozwoju wielu tkanek, głównie mięśni i komórek nerwowych. Białka MBNL mają również związek z patomechanizmem dystrofii miotonicznej (DM), choroby degeneracyjnej mięśni, w której dochodzi do sekwestracji białek MBNL na mRNA zmutowanego genu, co prowadzi do masowej deregulacji alternatywnego splicingu w komórkach pacjentów. W trzech publikacjach składających się na moją rozprawę doktorską przedstawiłem połączenie strategii AON i minigenów hybrydowych, jako wydajnej metody do weryfikacji potencjalnych miejsc wiązania dla MBNL wyłonionych na drodze głębokiego sekwencjonowania RNA. Podejście to umożliwiło też potwierdzenie hipotezy o rozpoznawaniu tych samych fragmentów RNA przez wszystkie paralogi MBNL. Konstrukty bazujące na minigenie hybrydowym zostały także wykorzystane w badaniach nad aranżacją strukturalną RNA miejsc wiązania dla białek MBNL jak i opracowaniu testu dla poszukiwania lub walidowania potencjalnych inhibitorów sekwestracji MBNL w DM. Ponadto, wykazano antagonistyczną rolę białek MBNL1 i SRSF1 w regulacji alternatywnego splicingu. Analiza dystrybucji trzech alternatywnych eksonów w transkryptach genów MBNL wykazała ich zróżnicowane włączanie do mRNA w poszczególnych tkankach na różnych etapach rozwoju oraz zaburzenie tego procesu w mięśniach pacjentów z DM. Z kolei w projekcie wykazującym wpływ organizacji miejsc wiązania w RNA na aktywność regulatorową MBNL dostarczyłem danych o poziomie ekspresji poszczególnych badanych białek MBNL.Muscleblind-like protein family gathering three paralogs (MBNL1, 2 and 3) is a group of alternative splicing regulators essential during the development of particular tissues, primarily, skeletal muscles and neuronal cells. MBNLs are also involved in the pathomechanism of myotonic dystrophy (DM), a disorder in which occurs the sequestration of MBNLs on the mRNA of a mutated gene leading to massive misregulation of alternative splicing in patients' cells. In three articles encompassed in my dissertation I presented combination of AONs and hybrid-type minigenes as an efficient method for validation of potential MBNL-binding sites extracted from high-throughput data. Using this approach I also confirmed the hypothesis that all MBNL paralogs recognize the same sequence in RNAs. Hybrid minigene-based constructs were also used in research on MBNL-binding sites spatial arrangement as well as in the development of assay for searching or validation of potential MBNL sequestration inhibitors in DM. Furthermore, I participated in experiments revealing antagonistic role of MBNL1 and SRSF1 proteins in alternative splicing regulation of exons via, either, structural rearrangements or competition for binding sites in RNA. In another project I analyzed the distribution of three alternative exons in the transcripts of MBNL genes which revealed differential inclusion among particular human tissues at different developmental stage and in DM patients' samples. In the project which focused on the impact of binding sites organization on MBNLs' activity I provided results quantifying expression level of particular MBNL proteins

Hyaluronic acid and multiwalled carbon nanotubes as bioink additives for cartilage tissue engineering

Abstract Articular cartilage and meniscus injuries are prevalent disorders with insufficient regeneration responses offered by available treatment methods. In this regard, 3D bioprinting has emerged as one of the most promising new technologies, offering novel treatment options. Additionally, the latest achievements from the fields of biomaterials and tissue engineering research identified constituents facilitating the creation of biocompatible scaffolds. In this study, we looked closer at hyaluronic acid and multi-walled carbon nanotubes as bioink additives. Firstly, we assessed the minimal concentrations that stimulate cell viability, and decrease reactive oxygen species and apoptosis levels in 2D cell cultures of normal human knee articular chondrocytes (NHAC) and human adipose-derived mesenchymal stem cells (hMSC-AT). In this regard, 0.25 mg/ml of hyaluronic acid and 0.0625 mg/ml of carbon nanotubes were selected as the most optimal concentrations. In addition, we investigated the protective influence of 2-phospho-L-ascorbic acid in samples with carbon nanotubes. Tests conducted on 3D bioprinted constructs revealed that only a combination of components positively impacted cell viability throughout the whole experiment. Gene expression analysis of COL1A1, COL6A1, HIF1A, COMP, RUNX2, and POU5F1 showed significant changes in the expression of all analyzed genes with a progressive overall loss of transcriptional activity in most of them

Novel Strategies in Artificial Organ Development: What Is the Future of Medicine?

The technology of tissue engineering is a rapidly evolving interdisciplinary field of science that elevates cell-based research from 2D cultures through organoids to whole bionic organs. 3D bioprinting and organ-on-a-chip approaches through generation of three-dimensional cultures at different scales, applied separately or combined, are widely used in basic studies, drug screening and regenerative medicine. They enable analyses of tissue-like conditions that yield much more reliable results than monolayer cell cultures. Annually, millions of animals worldwide are used for preclinical research. Therefore, the rapid assessment of drug efficacy and toxicity in the early stages of preclinical testing can significantly reduce the number of animals, bringing great ethical and financial benefits. In this review, we describe 3D bioprinting techniques and first examples of printed bionic organs. We also present the possibilities of microfluidic systems, based on the latest reports. We demonstrate the pros and cons of both technologies and indicate their use in the future of medicine

Bionic Organs: Shear Forces Reduce Pancreatic Islet and Mammalian Cell Viability during the Process of 3D Bioprinting

Background: 3D bioprinting is the future of constructing functional organs. Creating a bioactive scaffold with pancreatic islets presents many challenges. The aim of this paper is to assess how the 3D bioprinting process affects islet viability. Methods: The BioX 3D printer (Cellink), 600 μm inner diameter nozzles, and 3% (w/v) alginate cell carrier solution were used with rat, porcine, and human pancreatic islets. Islets were divided into a control group (culture medium) and 6 experimental groups (each subjected to specific pressure between 15 and 100 kPa). FDA/PI staining was performed to assess the viability of islets. Analogous studies were carried out on α-cells, β-cells, fibroblasts, and endothelial cells. Results: Viability of human pancreatic islets was as follows: 92% for alginate-based control and 94%, 90%, 74%, 48%, 61%, and 59% for 15, 25, 30, 50, 75, and 100 kPa, respectively. Statistically significant differences were observed between control and 50, 75, and 100 kPa, respectively. Similar observations were made for porcine and rat islets. Conclusions: Optimal pressure during 3D bioprinting with pancreatic islets by the extrusion method should be lower than 30 kPa while using 3% (w/v) alginate as a carrier