Ústav biomedicínského inženýrství
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- ItemAcidogenesis, solventogenesis, metabolic stress response and life cycle changes in Clostridium beijerinckii NRRL B-598 at the transcriptomic level(Springer Nature, 2019-02-04) Patáková, Petra; Branská, Barbora; Sedlář, Karel; Vasylkivska, Maryna; Šabatová, Kateřina; Kolek, Jan; Cicková, Pavlína; Provazník, ValentýnaClostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABE) fermentative pathway. The pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. The description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. The study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. Surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium.
- ItemA transcriptional response of Clostridium beijerinckii NRRL B-598 to a butanol shock(BioMed Central, 2019-10-13) Sedlář, Karel; Kolek, Jan; Gruber, Markus; Šabatová, Kateřina; Branská, Barbora; Csaba, Gergely; Vasylkivska, Maryna; Zimmer, Ralf; Patáková, Petra; Provazník, ValentýnaBackground One of the main obstacles preventing solventogenic clostridia from achieving higher yields in biofuel production is the toxicity of produced solvents. Unfortunately, regulatory mechanisms responsible for the shock response are poorly described on the transcriptomic level. Although the strain Clostridium beijerinckii NRRL B-598, a promising butanol producer, has been studied under different conditions in the past, its transcriptional response to a shock caused by butanol in the cultivation medium remains unknown. Results In this paper, we present a transcriptional response of the strain during a butanol challenge, caused by the addition of butanol to the cultivation medium at the very end of the acidogenic phase, using RNA-Seq. We resequenced and reassembled the genome sequence of the strain and prepared novel genome and gene ontology annotation to provide the most accurate results. When compared to samples under standard cultivation conditions, samples gathered during butanol shock represented a well-distinguished group. Using reference samples gathered directly before the addition of butanol, we identified genes that were differentially expressed in butanol challenge samples. We determined clusters of 293 down-regulated and 301 up-regulated genes whose expression was affected by the cultivation conditions. Enriched term “RNA binding” among down-regulated genes corresponded to the downturn of translation and the cluster contained a group of small acid-soluble spore proteins. This explained phenotype of the culture that had not sporulated. On the other hand, up-regulated genes were characterized by the term “protein binding” which corresponded to activation of heat-shock proteins that were identified within this cluster. Conclusions We provided an overall transcriptional response of the strain C. beijerinckii NRRL B-598 to butanol shock, supplemented by auxiliary technologies, including high-pressure liquid chromatography and flow cytometry, to capture the corresponding phenotypic response. We identified genes whose regulation was affected by the addition of butanol to the cultivation medium and inferred related molecular functions that were significantly influenced. Additionally, using high-quality genome assembly and custom-made gene ontology annotation, we demonstrated that this settled terminology, widely used for the analysis of model organisms, could also be applied to non-model organisms and for research in the field of biofuels.
- ItemThe Effect of Rhodamine-Derived Superparamagnetic Maghemite Nanoparticles on the Motility of Human Mesenchymal Stem Cells and Mouse Embryonic Fibroblast Cells(MDPI, 2019-03-27) Chmelíková, Larisa; Skopalík, Josef; Chmelík, Jiří; Zumberg, Inna; Čmiel, Vratislav; Poláková, Kateřina; Provazník, ValentýnaNanoparticles have become popular in life sciences in the last few years. They have been produced in many variants and have recently been used in both biological experiments and in clinical applications. Due to concerns over nanomaterial risks, there has been a dramatic increase in investigations focused on safety research. The aim of this paper is to present the advanced testing of rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R), which are used for their nontoxicity, biocompatibility, biodegradability, and magnetic properties. Recent results were expanded upon from the basic cytotoxic tests to evaluate cell proliferation and migration potential. Two cell types were used for the cell proliferation and tracking study: mouse embryonic fibroblast cells (3T3) and human mesenchymal stem cells (hMSCs). Advanced microscopic methods allowed for the precise quantification of the function of both cell types. This study has demonstrated that a dose of nanoparticles lower than 20 microg-cm -2 per area of the dish does not negatively affect the cells’ morphology, migration, cytoskeletal function, proliferation, potential for wound healing, and single-cell migration in comparison to standard CellTracker Green CMFDA (5-chloromethylfluorescein diacetate). A higher dose of nanoparticles could be a potential risk for cytoskeletal folding and detachment of the cells from the solid extracellular matrix.
- ItemStool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform(Springer Nature, 2019-09-25) Vídeňská, Petra; Šmerková, Kristýna; Zwinsová, Barbora; Popovici, Vlad; Micenková, Lenka; Sedlář, Karel; Budinská, EvaMany studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.
- ItemQuantitative Phase Dynamics of Cancer Cell Populations Affected by Blue Light(MDPI, 2020-04-09) Feith, Marek; Vičar, Tomáš; Gumulec, Jaromír; Raudenská, Martina; Gjörloff Wingren, Anette; Masařík, Michal; Balvan, JanIncreased exposition to blue light may induce many changes in cell behavior and significantly affect the critical characteristics of cells. Here we show that multimodal holographic microscopy (MHM) within advanced image analysis is capable of correctly distinguishing between changes in cell motility, cell dry mass, cell density, and cell death induced by blue light. We focused on the effect of blue light with a wavelength of 485 nm on morphological and dynamical parameters of four cell lines, malignant PC-3, A2780, G361 cell lines, and the benign PNT1A cell line. We used MHM with blue light doses 24 mJ/cm2, 208 mJ/cm2 and two kinds of expositions (500 and 1000 ms) to acquire real-time quantitative phase information about cellular parameters. It has been shown that specific doses of the blue light significantly influence cell motility, cell dry mass and cell density. These changes were often specific for the malignant status of tested cells. Blue light dose 208 mJ/cm2 × 1000 ms affected malignant cell motility but did not change the motility of benign cell line PNT1A. This light dose also significantly decreased proliferation activity in all tested cell lines but was not so deleterious for benign cell line PNT1A as for malignant cells. Light dose 208 mJ/cm2 × 1000 ms oppositely affected cell mass in A2780 and PC-3 cells and induced different types of cell death in A2780 and G361 cell lines. Cells obtained the least damage on lower doses of light with shorter time of exposition.