imagine (Institute of molecular genetics and genetic engineering)
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Pharmacogenetics of Clopidogrel Therapy and Neurointerventional Procedures: We Need Precision Data for Precision Medicine
No full textGenetics has given a real boost to personalized and precision medicine, providing data used either for precise diagnostics, prediction of the course of illness, or for selecting therapy and tailoring it. Pharmacogenetics, as a discipline researching connection between genetic background of an individual and the effect of a certain drug, has created new possibilities in medicine. One of the most researched drugs in pharmacogenetics is certainly clopidogrel
Hypoxia affects the expression of SOX genes and induction of neural differentiation of human embryonal carcinoma NT2/D1 cells
Get PDFThe family of SOX genes encodes proteins that display properties of both classical transcription factors and architectural components of chromatin. During development of nervous system, as well as adult neurogenesis, SOX transcription factors govern diverse cellular processes such as maintaining the multipotency of neural stem cells, cell proliferation, cell fate decision, migration as well as terminal differentiation of neurons. Despite their well-known function in development and brain homeostasis, the expression and role of these genes in pathology- induced neural stem cell plasticity is poorly understood. Reduction in oxygen supply or ischemia are involved in various pathological conditions, such as stroke, traumatic brain injury and cardiac arrest, which promotes neurogenesis, angiogenesis, cell proliferation and other cell mechanisms for survival under the stress. The aim of the present study was to analyze the expression of SOX genes during in vitro neurogenesis following chemical hypoxia. Neuronal differentiation of human pluripotent embryonal carcinoma stem cell line NT2/D1 was used as an in vitro model system for studying the process of human neurogenesis. Depending on different concentration, RA directed the differentiation of NT2/D1 cells into neurons with a different phenotype. The effect of stress caused by hypoxia on the properties of pluripotent cells as well as the induction of neural differentiation was monitored in vitro by culturing NT2/D1 cells in the presence of cobalt chloride, a chemical inducer of hypoxia. The results of the analysis showed that the effect of hypoxia on the expression of SOX2 and OCT4 proteins involved in maintaining the pluripotency of cells depends on the duration of action of cobalt chloride. After short-term exposure of the cells, an increase in the levels of expression of SOX2 and OCT4 proteins was detected, while long-term treatment of the cells led to a decrease in the expression of these proteins. Furthermore, results showed that depending of duration of cobalt chloride treatments, the level of expression of miR-21 in undifferentiated NT2/D1 cells significantly changed. In addition, long-term pretreatment of pluripotent cells with cobalt chloride resulted in increased expression levels of SOX2, SOX3 and GAD67 proteins in neural progenitors induced for 7 days in the presence of, either low or high concentration of retinoic acid, indicating that hypoxia causes increased efficiency of NT2/D1 cell neural differentiation. Damage of brain tissue caused by reduction of oxygen and/or blood flow to the tissue is the leading cause of death worldwide and the leading cause of disability in humans. Our results contributes to the research focused on discovering the roles of SOX TFs and their gene targets in ischemia related pathologies, making them promising biomarkers and potential targets for future diagnostic and therapeutic strategies.BOOK OF ABSTRACTS: 10th JUBILEE INTERNATIONAL CONFERENCE ON RADIATION IN VARIOUS FIELDS OF RESEARCH Jun 13-17, 2022, Herceg Novi - Montenegr
Biodegradation of poly(epsilon-caprolactone) (PCL) and medium chain length polyhydroxyalkanoate (mcl-PHA) using whole cells and cell free protein preparations of Pseudomonas and Streptomyces strains grown on waste cooking oil
Get PDFPetrochemical plastics are generally recalcitrant to microbial degradation and accumulate in the environment. Biodegradable polymers obtained synthetically like poly(epsilon-caprolactone) (PCL) or poly-hydroxyalkanoates (PHA), obtained biotechnologically, have shown great potential as a replacement for petroleum-based plastics. Nevertheless, their biodegradation and environmental faith have been less examined. In this study, thin films of PCL (200 mu m) and medium chain length PHA (mcl-PHA, 70 M fraction of 3-hydroxyoctanoate and 30 M fraction of 3-hydroxydecanoate, 600 mu m) were exposed to total protein preparations (extracellular proteins combined with a crude cell extract) of soil isolates Pseudomonas chiororaphis B-561 and Streptomyces sp. BV315 that had been grown on waste cooking oil as a sole carbon source. Biodegradation potential of two polyesters was evaluated in buffer with total protein preparations and in a laboratory compost model system augmented with selected bacteria. Overall, PCL showed better biodegradation properties in comparison to mcl-PHA. Both materials showed surface erosion after 4-weeks of exposure to total protein preparations of both strains, with a moderate weight loss of 1.3% when P. chlororaphis13-561 was utilized. In laboratory compost model system PCL and mcl-PHA showed significant weight loss ranging from 13 to 17% when Streptomyces sp. BV315 culture was used. Similar weight loss of PCL and mcl-PHA was achieved for 4 and 8 weeks, respectively indicating slower degradation of mcl-PHA. Growth on waste cooking oil as a sole carbon source increased the potential of both tested strains to degrade PCL and mcl-PHA, making them good candidates for augmentation of compost cultures in waste management of both waste cooking oils and biodegradable polymers.Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1246]This is the peer reviewed version of the paper: Mandić, M., Spasić, J., Ponjavić, M., Nikolić, M. S., Cosović, V. R., O’Connor, K., Nikodinović-Runić, J., Đokić, L., & Jeremić, S. (2019). Biodegradation of poly(epsilon-caprolactone) (PCL) and medium chain length polyhydroxyalkanoate (mcl-PHA) using whole cells and cell free protein preparations of Pseudomonas and Streptomyces strains grown on waste cooking oil. Polymer Degradation and Stability, 162, 160–168.[ https://doi.org/10.1016/j.polymdegradstab.2019.02.012
Hyperphenylalaninemia: from complex genetics to complex phenotypes
No full textHyperphenylalaninemia (HPA) is the most frequent inborn error of amino acid metabolism
and it is characterized by an increased level of blood phenylalanine. Excessive phenylalanine
has toxic effect on brain development, and leads to severe and irreversible mental retardation
of a patient. In 98% of all cases, HPA is caused by mutations in phenylalanine hydroxylase
(PAH) gene, known as phenylketonuria (PKU). In other 2%, HPA results from deficiency of
PAH enzyme cofactor, tetrahydrobiopterin (BH4). BH4 deficiency results from mutations in
genes responsible either for BH4 biosynthesis (PTS and GCH1), or BH4 metabolism (DHPR
and PCBD). To ensure an accurate differential diagnosis of HPA in a patient and to introduce
adequate treatment, an appropriate genetic diagnosis is necessary. By combining Sanger
sequencing, MLPA and next generation sequencing (NGS), using our virtual gene panel, we
reached mutation detection rate of 99,34% for HPA in Serbia. We detected 29 known and 1
novel mutation, p.Gln226Lys PAH, which we functionally characterized as a disease causing
mutation.
Although variants in a single gene are the main determinant of PKU phenotype, it is not
always possible to predict the phenotype based only on PAH genotype. Genetic factors which
attribute to the development of final PKU phenotype are still poorly understood. In the
research which aimed to identify new modifier genes of cognitive PKU phenotype, complete
genome sequence of 10 untreated PKU patients (from 6 families) with normal cognitive
development was analyzed using NGS. After bioinformatics analysis, variats in SHANK genes
were selected as possible modifiers which contributed to the atypical phenotype in our
patients. Computational analysis showed that SHANK proteins encoded by identified variant
genes could gain new characteristics related to synaptic transmission, and could have a
protective modifier effect on development of cognitive dysfunction in PKU patients.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Identification of genes involved in steroid alkaloid biosynthesis in Fritillaria imperialis via de novo transcriptomics
Get PDFCrown imperial (CI) has been used in traditional medicine. Today it is known that such beneficial effects are due to its richness in steroidal alkaloids (SA). Using de novo transcriptomics, orthologues/paralogues finder, phylogenetic analysis and tissue- and developmental stage-specific expression analysis, we identified ten genes and several TFs involved in the biosynthesis of SA in CI. The comparative analysis of ten genes expression profiles revealed the possibility of their co-regulation, which may imply the possibility of their organization in metabolic gene clusters. Having in mind convergent evolution of steroidal biosynthetic pathways in flowering plants and records of convergent evolution of specific proteins, observed expression patterns open a reasonable interest to investigate the possibility of the existence of genes cluster organization in SA pathway in the family Liliaceae or at least in some species of genus Fritillaria. Obtained results support transcriptomics as useful approach in elucidating genes underlying complex biochemical pathways
Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm formation of Candida albicans
No full textRhamnolipids are biodegradable low toxic biosurfactants which exert antimicrobial and anti-biofilm properties. They have attracted much attention recently due to potential applications in areas of bioremediation, therapeutics, cosmetics and agriculture, however, the full potential of these versatile molecules is yet to be explored. Based on the facts that many naturally occurring lipopeptides are potent antimicrobials, our study aimed to explore the potential of replacing rhamnose in rhamnolipids with amino acids thus creating lipopeptides that would mimic or enhance properties of the parent molecule. This would allow not only for more economical and greener production but also, due to the availability of structurally different amino acids, facile manipulation of physico-chemical and biological properties. Our synthetic efforts produced a library of 43 lipopeptides revealing biologically more potent molecules. The structural changes significantly increased, in particular, anti-biofilm properties against Candida albicans, although surface activity of the parent molecule was almost completely abolished. Our findings show that the most active compounds are leucine derivatives of 3-hydroxy acids containing benzylic ester functionality. The SAR study demonstrated a further increase in activity with aliphatic chain elongation. The most promising lipopeptides 15, 23 and 36 at 12.5 mu g/mL concentration allowed only 14.3%, 5.1% and 11.2% of biofilm formation, respectively after 24 h. These compounds inhibit biofilm formation by preventing adhesion of C. albicans to abiotic and biotic surfaces
Pyrimethanil: Between efficient fungicide against Aspergillus rot on cherry tomato and cytotoxic agent on human cell lines
No full textPyrimethanil, a synthetic fungicide widely used for the treatment of pre- and postharvest fungal diseases on different agricultural crops, was explored for its antifungal activity on different fungal strains. The effect of pyrimethanil on fungal ergosterol was tested by using Aspergillus niger as a model organism. Furthermore, it was investigated, if pyrimethanil can effectively reduce the appearance of Aspergillus rot in wounded cherry tomato fruits. The fungicide cytotoxic effect on different human cell lines was evaluated. In addition, its influence on cell proliferation was studied. A. niger was the most resistant fungal strain (MFC 1.88 mu g mu L-1) to the effect of pyrimethanil. Addition of ergosterol doubled the MFC on A. niger, indicating that the compound might interfere with ergosterol, main sterol of fungal cell membrane. Disease incidence of A. niger in wounded cherry tomato fruits was not detected with pyrimethanil treatment of 0.75 mg/wound. Some cytotoxic effects of pyrimethanil on human cell lines were recorded already at 50 ng mu L-1, while the expression of Ki67 marker of proliferation was decreased with 150 ng mu L-1. These results altogether indicate that pyrimethanil is effective in reducing various fungal pathogens, but further use of this fungicide should be reevaluated because of its cytotoxicity
Aspergillus fumigatus branching complexity in vitro: 2D images and dynamic modeling
No full textBackground Aspergillus fumigatus causes serious infections in humans, and its virulence correlates with hyphal growth, branching and formation of the filamentous mycelium. The filamentous mycelium is a complex structure inconvenient for quantity analysis. In this study, we monitored the branching of A. fumigatus filamentous mycelium in vitro at different points in time in order to assess the complexity degree and develop a dynamic model for the branching complexity. Method: We used fractal analysis of microscopic images (FAMI) to measure the fractal dimensions (D) of the branching complexity within 24 h of incubation. Results: By photographing the filamentous mycelium dynamically and processing the images, the D variation curve of A. fumigatus complexity degree was obtained. We acquired the D variation curve which contained initial exponential period and stationary period of A. fumigants branching. Further, the obtained data of D was modeled via the logistic model (LM) to develop a dynamic model of A. fumigatus branching for the prediction of the specific growth rate of branching value (0.23 h(-1)). Conclusions: Developed FAMI and LM models present a simple and non-destructive method of predicting the evolution of branching complexity of A. fumigatus. These models are useful as laboratory measurements for the prediction of hyphal and mycelium development, especially relevant to the pathogenesis study of aspergillosis, as well as pathogenesis of other diseases caused by moulds
Nutraceutical properties and phytochemical characterization of wild Serbian fruits
No full textWild fruits grown in Serbia, i.e., elderberry (Sambucus nigra), hawthorn (Crataegus monogyna), cornelian cherry (Cornus mas), and blackthorn (Prunus spinosa), are rich in secondary metabolites. In this study, the polyphenolic composition of wild fruit extracts and their antioxidant capacity were investigated by in vitro assays. Elderberry was characterized by the presence of arbutin (a skin protector), and cornelian cherry by syringic and gallic acids. In blackthorn, at least 11 different phenolic compounds were reported for the first time, including vanillic acid and naringin, the metabolite present in the highest amount. Blackthorn extracts were the richest in polyphenols (11.24-18.70g GAE/kgFW) and had the highest activity in the DPPH radical test (180.93-267.11mMTE/mL), while cornelian cherry extracts showed the most effective ferric ion chelating (81.37-90.66%) and antityrosinase inhibition capacities (21.75-74.23%). No sample was able to scavenge NO. Using the principal component analysis, wild fruit samples were classified into four separate clusters due to distinctive phenolic profiles and antioxidant capacity. Our investigation showed how every fruit could be considered unique in terms of its phytonutrient content. Thus, Serbian wild fruits may be a great source of bioactive natural compounds and could be therefore considered particularly useful in food supplement production. Particularly, as a source of natural antioxidants, these species could be used to extend the shelf life of food products and replace synthetic antioxidants, avoiding potential health risks and toxicity
Synthesis and initial biological evaluation of myxocoumarin B
No full textThe myxocoumarins A and B from Stigmatella aurantiaca MYX-030 are natural products featuring unusual nitro-and long-chain alkyl substitution. While myxocoumarin A was shown to exhibit strong antifungal properties, the antifungal potential of myxocoumarin B was not yet assessed due to low production titers during initial isolation. We therefore developed a total synthesis of myxocoumarin B that involves a late-stage Pd-catalyzed nitration of the coumarin core. The availability of synthetic material facilitated the initial evaluation of the bioactivity of myxocoumarin B, which revealed a lack of activity against medically relevant Candida sp. and low cytotoxicity in vitro against human fibroblasts (MRC-5) and in vivo (zebrafish)