Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages.
Abstract. Background Macrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understo…
Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing
AbstractAptamers have in recent years emerged as a viable alternative to antibodies. High-throughput sequencing (HTS) has revolutionized aptamer research by increasing the number of reads from a few (using Sanger sequencing) to millions (using an HTS approach). Despite the availability and advantages of HTS compared to Sanger sequencing, there are only 50 aptamer HTS sequencing samples available on public databases. HTS data in aptamer research are primarily used to compare sequence enrichment between subsequent selection cycles. This approach does not take full advantage of HTS because the enrichment of sequences during selection can be due to inefficient negative selection when using live…
Additional file 5: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages
Effect of SW480 and SW620-derived EVs on biomolecule secretion patterns of monocytes and M0, M1 and M2 macrophages. Luminex data analysis showing TNFα, IL-6, CXCL10, IL-23, IL-10, MMP9, IL-1β and CCL22 concentration in cell culture supernatants of monocytes (M) and M0, M1 and M2 macrophages following incubation with SW480 and SW620 EVs or without them (control). The graphs represent mean ± SD (n = 3). (PDF 39 kb)
Nano-engineered skin mesenchymal stem cells: potential vehicles for tumour-targeted quantum-dot delivery
Nanotechnology-based drug design offers new possibilities for the use of nanoparticles in imaging and targeted therapy of tumours. Due to their tumour-homing ability, nano-engineered mesenchymal stem cells (MSCs) could be utilized as vectors to deliver diagnostic and therapeutic nanoparticles into a tumour. In the present study, uptake and functional effects of carboxyl-coated quantum dots QD655 were studied in human skin MSCs. The effect of QD on MSCs was examined using a cell viability assay, Ki67 expression analysis, and tri-lineage differentiation assay. The optimal conditions for QD uptake in MSCs were determined using flow cytometry. The QD uptake route in MSCs was examined via fluore…
Additional file 3: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages
Effect of temperature on the SW480 EV uptake in THP-1 monocytes. Flow cytometry histograms showing Syto RNA Select fluorescence intensities of untreated (left) and Syto RNA Select-labeled SW480 EV-treated THP-1 monocytes following incubation at 4 °C (middle) and 37 °C (right). Histogram markers show the percentage of Syto RNA Select-positive cells. (PDF 53 kb)
Global effects of ade8 deletion on budding yeast metabolism
AbstractPurine auxotrophy is a typical marker for many laboratory yeast strains. Supplementation of additional purine source (like adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We tested purine starvation effects in budding yeast Saccharomyces cerevisiae ade8 knockout. We explored effects brought by purine starvation in cellular, central carbon metabolism and in the global transcriptome level.We observed that cells cultivated in purine depleted media became significantly more tolerant to severe thermal, oxidative and desiccation stresses when compared to the cells cultivated in media with all necessary supplements. When sta…
Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast
Purine auxotrophy is an abundant trait among eukaryotic parasites and a typical marker for many budding yeast strains. Supplementation with an additional purine source (such as adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We explored purine starvation effects in a model organism, a budding yeast Saccharomyces cerevisiae ade8 knockout, at the level of cellular morphology, central carbon metabolism, and global transcriptome. We observed that purine-starved cells stopped their cycle in G1/G0 state and accumulated trehalose, and the intracellular concentration of AXP decreased, but adenylate charge remained stable. Cells becam…
In vivophage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues
ABSTRACTIn vivophage display is widely used for identification of organ- or disease-specific homing peptides. However, the currentin vivophage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanningin vivo. The data fromin vivophage screen were analyzed using differential binding – relative representation of each peptide in the target orga…
Nanoparticle delivery to metastatic breast cancer cells by nanoengineered mesenchymal stem cells
We created a 3D cell co-culture model by combining nanoengineered mesenchymal stem cells (MSCs) with the metastatic breast cancer cell line MDA-MD-231 and primary breast cancer cell line MCF7 to explore the transfer of quantum dots (QDs) to cancer cells. First, the optimal conditions for high-content QD loading in MSCs were established. Then, QD uptake in breast cancer cells was assessed after 24 h in a 3D co-culture with nanoengineered MSCs. We found that incubation of MSCs with QDs in a serum-free medium provided the best accumulation results. It was found that 24 h post-labelling QDs were eliminated from MSCs. Our results demonstrate that breast cancer cells efficiently uptake QDs that a…
In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues.
Abstract In vivo phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current in vivo phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high-throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning in vivo. The data from in vivo phage screen were analyzed using differential binding—relative representation of each peptide in the target…
Additional file 2: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages
SW480 and SW620-derived EV effect on monocyte (M) and macrophage (M0, M1, M2) viability. a OD values at 450 nm which are in direct proportion of viable cell counts. b SW480 and SW620 EV cytotoxicity on THP-1 monocytes and M0, M1 and M2 macrophages. The graphs represent mean ± SEM (n = 3). Statistical analysis carried out with the t-test. *p ≤ 0.05, **p ≤ 0.01 vs. untreated cell control of the respective monocyte-macrophage cell subset. (PDF 50 kb)
Integrative Gene Expression and Metabolic Analysis Tool IgemRNA
ABSTRACTGenome scale metabolic modelling is widely used technique to research metabolism impacts on organism’s properties. Additional omics data integration enables a more precise genotype-phenotype analysis for biotechnology, medicine and life sciences. Transcriptome data amounts rapidly increase each year. Many transcriptome analysis tools with integrated genome scale metabolic modelling are proposed. But these tools have own restrictions, compatibility issues and the necessity of previous experience and advanced user skills. We have analysed and classified published tools, summarized possible transcriptome pre-processing, and analysis methods and implemented them in the new transcriptome…
Gaišo šūnu nieru karcinomas specifiski aptamēri
Nieru karcinomas skarto pacientu skaits pastāvīgi pieaug un joprojām nav efektīvas mērķētas terapijas, kas darbotos uz nieru karcinomas šūnām. Aptamēri ir oligonukleotīdu molekulas, kas saistās specifiski ar savu molekulāro mērķi to trīsdimensionālās struktūras dēļ un pēdējos gados pierādījuši sevi kā efektīvi mērķētās terapijas līdzekļi. Šūnu SELEX metode, kurā kā mērķis tiek izmantotas šūnas, uzskatāma par vienu daudzsološākajām aptamēru atlases metodēm, ar kuru identificēt terapeitiskos aptamērus. Darba rezultātā ir izstrādāts pētījuma protokols, ar kuru iespējams veikt specifisku aptamēru atlasi pret nieru karcinomas šūnām (RCC-MF), kā arī piesaistīti nepieciešamie finansiālie līdzekļi …
Additional file 4: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages
TNFα, IL-23, IL-6, IL-1 β, CXCL10, CCL22, IL-10 and MMP9 secretion profile at different monocyte-macrophage differentiation stages. The graphs represent average biomolecule concentrations SEM (n = 3). Statistical analysis carried out with one-way ANOVA test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and **** ≤ 0.0001 vs. untreated cell control of the respective monocyte-macrophage cell subset. (PDF 63 kb)
Additional file 1: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages
Experimental design of the THP-1 monocyte to macrophage differentiation showing the time points for the addition of EVs and stimulatory molecules. Below the experimental design, representative light microscopy images show morphology of THP-1 monocytes (M), M0 macrophages (M0), M1 macrophages (M1) and M2 macrophages (M2) (n = 4). Scale bar 100 μm. Representative flow cytometry dot plots show CD14, HLA-DR, CD206 and CD68 marker expression at M, M0, M1 and M2 stages. (JPG 1845 kb)