imagine (Institute of molecular genetics and genetic engineering)
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SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis
Get PDFThe SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)
No full textIn 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field
Host-commensal interaction promotes health and lifespan in Caenorhabditis elegans through the activation of HLH-30/TFEB-mediated autophagy
Get PDFGut homeostasis is maintained by the close interaction between commensal intestinal microbiota and the host, affecting the most complex physiological processes, such as aging. Some commensal bacteria with the potential to promote healthy aging arise as attractive candidates for the development of pro-longevity probiotics. Here, we showed that heat-inactivated human commensal Lactobacillus fermentum BGHV110 (BGHV110) extends the lifespan of Caenorhabditis elegans and improves age-related physiological features, including locomotor function and lipid metabolism. Mechanistically, we found that BGHV110 promotes HLH30/TFEB-dependent autophagy to delay aging, as longevity assurance was completely abolished in the mutant lacking HLH-30, a major autophagy regulator in C. elegans. Moreover, we observed that BGHV110 partially decreased the content of lipid droplets in an HLH-30-dependent manner and, at the same time, slightly increased mitochondrial activity. In summary, this study demonstrates that specific factors from commensal bacteria can be used to exploit HLH-30/TFEB-mediated autophagy in order to promote longevity and fitness of the host
Prostaglandin E2 differentially regulate the suppressive mechanisms in myeloid derived suppressor cells subsets in vitro
No full textAssociation of Vitamin D, Zinc and Selenium Related Genetic Variants With COVID-19 Disease Severity
Get PDFBackground: COVID-19 pandemic has proved to be an unrelenting health threat for more than a year now. The emerging amount of data indicates that vitamin D, zinc and selenium could be important for clinical presentation of COVID-19. Here, we investigated association of genetic variants related to the altered level and bioavailability of vitamin D, zinc and selenium with clinical severity of COVID-19. Methods: We analyzed variants in genes significant for the status of vitamin D (DHCR7/NADSYN1 rs12785878, GC rs2282679, CYP2R1 rs10741657, and VDR rs2228570), zinc (PPCDC rs2120019) and selenium (DMGDH rs17823744) in 120 Serbian adult and pediatric COVID-19 patients using allelic discrimination. Furthermore, we carried out comparative population genetic analysis among European and other worldwide populations to investigate variation in allelic frequencies of selected variants. Results: Study showed that DHCR7/NADSYN rs12785878 and CYP2R1 rs10741657 variants were associated with severe COVID-19 in adults (p = 0.03, p = 0.017, respectively); carriers of DHCR7/NADSYN TG+GG and CYP2R1 GG genotypes had 0.21 and 5.9 the odds for developing severe disease, OR 0.21 (0.05-0.9) and OR 5.9 (1.4-25.2), respectively. There were no associations between selected genetic variants and disease severity in pediatric patients. Comparative population genetic analysis revealed that Serbian population had the lowest frequency of CYP2R1 rs10741657 G allele compared to other non-Finish Europeans (0.58 compared to 0.69 and 0.66 in Spanish and Italian population, respectively), suggesting that other populations should also investigate the relationship of CYP2R1 variant and the COVID-19 disease course. Conclusion: The results of the study indicated that vitamin D related genetic variants were implicated in severe COVID-19 in adults. This could direct prevention strategies based on population specific nutrigenetic profiles
Bacterial Nanocellulose as a Scaffold for In Vitro Cell Migration Assay
Get PDFBacterial nanocellulose (BNC) stands out among polymers as a promising biomaterial due to its mechanical strength, hydrophilicity, biocompatibility, biodegradability, low toxicity and renewability. The use of scaffolds based on BNC for 3D cell culture has been previously demonstrated. The study exploited excellent properties of the BNC to develop an efficient and low-cost in vitro cell migration assay. The BNC scaffold was introduced into a cell culture 24 h after the SW480 cells were seeded, and cells were allowed to enter the scaffold within the next 24-48 h. The cells were stained with different fluorophores either before or after the introduction of the scaffold in the culture. Untreated cells were observed to enter the BNC scaffold in significant numbers, form clusters and retain a high viability after 48 h. To validate the assay's usability for drug development, the treatments of SW480 cells were performed using aspirin, an agent known to reduce the migratory potential of this cell line in culture. This study demonstrates the application of BNC as a scaffold for cell migration testing as a low-cost alternative to commercial assays based on the Boyden chamber principle. The assay could be further developed for routine use in cancer research and anticancer drug development
alpha-Tocopherol Acetate Attenuates Mitochondrial Oxygen Consumption and Maintains Primitive Cells within Mesenchymal Stromal Cell Population
No full textWe present here the data showing, in standard cultures exposed to atmospheric O-2 concentration, that alpha-tocopherol acetate (alpha-TOA) has a positive impact on primitive cells inside mesenchymal stromal cell (MstroC) population, by maintaining their proliferative capacity. alpha-TOA decreases the O-2 consumption rate of MStroC probably by impacting respiratory chain complex II activity. This action, however, is not associated with a compensatory increase in glycolysis activity, in spite of the fact that the degradation of HIF-1 alpha was decreased in presence of alpha-TOA. This is in line with a moderate enhancement of mtROS upon alpha-TOA treatment. However, the absence of glycolysis stimulation implies the inactivity of HIF-1 alpha which might - if it were active - be related to the maintenance of stemness. It should be stressed that alpha-TOA might act directly on the gene expression as well as the mtROS themselves, which remains to be elucidated.Peer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1754
Tailoring copper(II) complexes with pyridine-4,5-dicarboxylate esters for anti-Candida activity
Get PDFFive novel copper(ii) complexes with pyridine-4,5-dicarboxylate esters as ligands, [Cu(NO3)(py-2tz)(H2O)(3)]NO3 (1), [Cu(NO3)(2)(py-2metz)(H2O)] (2), [Cu(NO3)(2)(py-2py)(H2O)]center dot H2O (3), [CuCl2(py-2tz)](2) (4) and [CuCl2(py-2metz)](n) (5) (py-2tz is dimethyl 2-(thiazol-2-yl)pyridine-4,5-dicarboxylate, py-2metz is dimethyl 2-(4-methylthiazol-2-yl)pyridine-4,5-dicarboxylate and py-2py is dimethyl 2,2 '-bipyridine-4,5-dicarboxylate), were synthesized and structurally characterized by different spectroscopic and electrochemical methods. The structure of these complexes was determined by single-crystal X-ray diffraction analysis, confirming the bidentate coordination mode of the corresponding pyridine-4,5-dicarboxylate ester to the Cu(ii) ion through the nitrogen atoms. The antimicrobial potential of copper(ii) complexes 1-5 was assessed against two bacterial and two Candida species. These complexes showed better growth inhibiting activity against Candida spp. with respect to the tested bacterial species, also being moderately toxic towards normal human lung fibroblast cells (MRC-5). Complexes 1 and 4 showed the greatest ability to inhibit the filamentation of C. albicans, which is an important process during fungal infection, and these two complexes efficiently inhibited the biofilm formation of C. albicans at subinhibitory concentrations. Complex 4 also successfully prevented the adhesion of C. albicans in an in vitro epithelial cell model. The mechanism of the antifungal activity of copper(ii) complexes 1-5 was studied through their interaction with ct-DNA, as one of the possible target biomolecules, by fluorescence spectroscopy and gel electrophoresis. Finally, the ability of these complexes to bind to bovine serum albumin (BSA) was studied by fluorescence emission spectroscopy
Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients
Get PDFLiquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups-patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/mu L, 5.17 ng/mu L, and 0.29 ng/mu L for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be interpreted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted personalized CRC patient management
Optimization of 3d cancer cell culture conditions by application of chemical engineering principles
No full textCancer cell immobilization in polymer hydrogels serving as extracellular matrices and cultivation in perfusion bioreactors that provide appropriate chemical signals, efficient mass transfer and hydrodynamic shear stresses is a promising strategy for development of physiologically relevant tumor models. In this work, perfusion cultures of 2 cancer cell types (C6 rat glioma and embryonal carcinoma NT2/D1 cells) immobilized in alginate microgels were established, while static cultures served as controls. Continuous perfusion had different effects on the cultured cells inducing enhanced proliferation of the glioma cells immobilized in microfibers (8x10^6 cell/ml), while reducing the viability of the NT2/D1 cells immobilized in microbeads (1x10^6 cell/ml). In order to elucidate the observed effects, chemical engineering principles were applied to assess mass transfer and hydrodynamic conditions. The second Fick’s law was solved analytically while the diffusionadvection-reaction equation was solved numerically to model mass transport in the static and bioreactor cultures, respectively. Moreover, Reynolds numbers, pressure drops and shear stresses in bioreactor cultures were calculated for assessment of flow regime and hydrodynamic conditions. The modeling results have indicated that oxygen transport is diffusion-controlled through the alginate hydrogel, while medium perfusion improves mass transfer of larger compounds having smaller diffusion coefficients (∼10^(-13) m^2/s), which possibly stimulated glioma cell proliferation. On the other hand, the obtained shear stress (~50 mPa) in the perfused packed bed of microbeads was above physiological levels, which provided the explanation of the poor NT2/D1 cell survival. This study stresses the importance of multidisciplinary approach in addressing such multifactorial diseases as cancer.Book of abstracts: 6th World Congress of the Tissue-Engineering-and-Regenerative-Medicine-International-Society (TERMIS