imagine (Institute of molecular genetics and genetic engineering)
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Differentiation of stem cells from apical papilla into neural lineage using graphene dispersion and single walled carbon nanotubes
Get PDFStem cell-based therapies are considered a promising treatment modality for many medical conditions. Several types of stem cells with variable differentiation potentials have been isolated from dental tissues, among them stem cells from apical papilla (SCAP). In parallel, new classes of biocompatible nanomaterials have also been developed, including graphene and carbon nanotube-based materials. The aim of the study was to assess whether graphene dispersion (GD) and water-soluble single walled carbon nanotubes (ws-SWCNT), may enhance SCAPs capacity to undergo neural differentiation. SCAPs cultivated in neuroinductive medium supplemented with GD and ws-SWCNT, separately and in combination, were subjected to neural marker analysis by real-time polymerase chain reaction (neurofilament medium [NF-M], neurogenin-2 [ngn-2], III-tubulin, microtubule-associated protein 2) and immunocytochemistry (NeuN and III-tubulin). GD, ws-SWCNT, and their combination, had neuro-stimulatory effects on SCAPs, as judged by the production of neural markers. Compared to cells grown in nanomaterial free medium, cells with GD showed higher production of B3T, cells with ws-SWCNT had higher production of ngn-2 and NF-M, while the combination of nanomaterials gave similar levels of both B3T and NF-M as the neuroinductive medium aloal ne, but with the finest neuron-like morphology. In conclusion, GD and ws-SWCNT seem to enhance neurdifferentiation of SCAP.This is the peer reviewed version of the paper: Simonovic, J., Toljic, B., Nikolic, N., Peric, M., Vujin, J., Panajotovic, R., Gajic, R., Bekyarova, E., Cataldi, A., Parpura, V., & Milasin, J. (2018). Differentiation of stem cells from apical papilla into neural lineage using graphene dispersion and single walled carbon nanotubes. Journal of Biomedical Materials Research Part A, 106(10), 2653–2661. [https://doi.org/10.1002/jbm.a.36461
Ascorbate as a key player in plant abiotic stress response and tolerance
No full textDuring their lifespan, plants are frequently exposed to adverse environmental conditions such as high solar irradiance, drought, heat, chilling, salinity, metal excess, and nutrient deficiency. The effects of these factors on plants are often interrelated and usually result in a decreased capacity of carbon fixation in photosynthesis, disturbed redox homeostasis, and growth arrest. Under severe conditions, increased excitation pressure in the chloroplasts exceeds the antioxidative capacity of plant cells leading to oxidative damage of cellular constituents. Although the plant ascorbate (Asc) level varies depending on external factors, developmental stage, diurnal rhythm, and light, its redox status is related to redox homeostasis in the cell. In chloroplasts, peroxisomes, and cytosol, Asc has a key role in hydrogen peroxide (H2O2) scavenging via Asc peroxidase and is efficiently recycled via the ascorbate-glutathione (Asc-GSH) cycle and directly by monodehydroascorbate reductase activity. In apoplast and vacuoles, Asc is the main reductant of phenolic radicals generated under oxidative stress. Besides its antioxidative role, Asc has an important role in a complex and well-orchestrated plant response network to environmental stress, performing multiple tasks in redox signalling, regulation of enzymatic activities, modulation of gene expression, biosynthesis of phytohormones, and growth regulation. The content of Asc and its redox state is tightly related to cellular compartments. Therefore, it is important to emphasize Asc cellular distribution, which has a great impact on reactive oxygen species regulation and signalling. Numerous studies on transgenic plants with altered endogenous Asc levels and redox status were done with the aim to influence plant growth and improve tolerance to various abiotic stressors. In this chapter, we discuss the current understanding of the involvement of Asc metabolism in abiotic stress response. Moreover, the improved resilience to stressors in transgenic plants with altered enzymes involved in Asc biosynthesis and recycling will be discussed
The Role of Macrophage Migration Inhibitory Factor in the Function of Intestinal Barrier
Get PDFMacrophage migration inhibitory factor (MIF) is a multifunctional protein that is involved in the development of gut-related inflammation. To investigate the role of MIF in the function of the intestinal barrier, we have explored intestinal permeability and gut-associated immune response in MIF-deficient (MIF-KO) mice. The absence of MIF provoked impairment of tight and adherens epithelial junctions in the colon through the disturbance of E-cadherin, zonula occludens-1, occludin and claudin-2 expression, which lead to the increase of intestinal barrier permeability. In these circumstances the diversity and content of gut microbiota in MIF-KO mice was considerably different compared to wild type mice. This change in microbiota was accompanied by an increased intestinal IgA concentration and a higher production of pro-inflammatory cytokines TNF and IFN-gamma in mesenteric lymph nodes of MIF-KO mice. The forced changes of microbiota executed by antibiotics prevented the "leakage" of the barrier in MIF-KO mice, probably through up-regulation of occludin expression and normalization of cellular pore diameters. In addition, cytokine secretion was normalized after the treatment with antibiotics. These results suggest that MIF participates in the maintenance of physiological microbiota diversity and immunosurveillance, which in turn enables the proper intestinal barrier function
The Characterization of the Selected Trees Damaged During Severe Weather Episode on the Mountain Avala (Serbia) Using IR Thermography, ICP-OES, and Microbiological Analysis
No full textSelected plants of white fir and lime, damaged during severe weather episode on the mountain Avala (Serbia) in summer 2014, were analyzed and characterized (including their spatial soil samples) by inductively coupled plasma optical emission spectroscopy (ICP-OES), infrared (IR) thermography, and microbiological method such as enumeration of cultivable microorganisms. The results obtained from chemical and microbiological analyses provided valuable information on possible biotic and abiotic stressors such as soil fungi and heavy metals, which could affect the health status of trees, while IR thermography visualized this status in a very specific and effective way. The results of ICP-OES analysis clearly showed that the investigated heavy metals (Cu, Zn, Pb, As, Cd, and Ni) were less likely crucial factors responsible for ruined health status of damaged trees. The role of soil fungi was not clear, since the results of microbiological analysis only provided evidence that their amounts in all investigated soil samples were within normal ranges as well as that their amounts in the corresponding samples of the uprooted trees were much greater than in the case of snapped trees. Therefore, further molecular characterization of microorganisms should be performed to identify if pathogenic species are present and clarify their role. Nevertheless, all used methods, especially IR thermal imaging as a totally non-invasive, fast and very comfortable technique, can be recommended as very useful in preventive screening of the trees' health status and for early detection of tissue decay that usually hamper trees survival or resistance to extreme weather events
Bisphenol A decreases progesterone synthesis by disrupting cholesterol homeostasis in rat granulosa cells
No full textBisphenol A (BPA) is an endocrine disruptor used in a variety of consumer products. Exposure to BPA leads to alterations in steroidogenesis of ovarian granulosa cells. Here, we analyzed the mechanism by which BPA alters progesterone biosynthesis in immature rat granulosa cells. BPA increased expression of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase in granulosa cells; however, BPA prevented the basal and the FSH-induced progesterone production. BPA caused sequestration of cholesterol to the perinuclear area, as evident by the Filipin staining. BPA decreased mRNA expression of ATP binding cassette transporter-A1 (Abca1) and increased level of sterol regulatory element binding protein 1. Addition of exogenous cell-permeable cholesterol restored the effect of BPA on Abca1 and Star mRNA expression and partially reversed BPA's effect on progesterone production. These results indicate that exposure to BPA disrupts cholesterol homeostasis leading to decreased progesterone production in immature rat granulosa cells
Utilization of supercritical carbon dioxide in fabrication of cellulose acetate films with anti-biofilm effects against Pseudomonas aeruginosa and Staphylococcus aureus
Get PDFThis study discusses utilization of supercritical carbon dioxide for impregnation of cellulose acetate films with thymol in order to produce material with anti-biofilm activity against Pseudomonas aeruginosa and Staphylococcus aureus. Analysis of anti-biofilm activity of cellulose acetate beads impregnated with thymol suggested that optimal thymol loading was in the range from 26% to 30% for efficient reduction of biofilm formation and eradication of pre-formed biofilms. Polymer films were fabricated by the solvent casting method from polymer solutions of different contents, and loaded with thymol using supercritical carbon dioxide at 15.5 MPa and 35 degrees C. The film containing 30% of thymol (F1 30%) exhibited substantial anti-adhesion properties inhibiting biofilm formation on its surface and considerably reduced formation of biofilms on the surrounding surfaces (up to 80%) by all tested strains including antibiotic resistant P. aeruginosa DM50 and methicillin-resistant S. aureus.This is the peer reviewed version of the paper: Zizovic, I., Senerovic, L., Moric, I., Adamovic, T., Jovanovic, M., Krusic, M. K., Misic, D., Stojanovic, D., & Milovanovic, S. (2018). Utilization of supercritical carbon dioxide in fabrication of cellulose acetate films with anti-biofilm effects against Pseudomonas aeruginosa and Staphylococcus aureus. The Journal of Supercritical Fluids, 140, 11–20.[ https://doi.org/10.1016/j.supflu.2018.05.025]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1143
Influence of bioactive molecules of lactobacilli on autophagy and inflamation processes in in vitro and in vivo systems
Get PDFIzolacija i karakterizacija bakterijskih biomolekula koji interaguju sa receptorima ćelija domaćina predstavlja kljuĉ za razumevanje mehanizama probiotiĉkog dejstva laktobacila. Savremena istraţivanja probiotiĉkih bakterija usmerena su ka identifikaciji biomolekula koji mogu modulisati razliĉite signalne puteve u humanim ćelijama. Svi molekuli koji su poreklom iz probiotika i odgovorni su za njihov pozitivan efekat na zdravlje domaćina nazvani su postbiotici. Zbog slabijeg imunostimulišućeg potencijala, njihova primena predstavlja bezbednu alternativu primeni ţivih bakterija. Ovo istraţivanje je imalo za cilj da testira mogućnost primene postbiotika u ublaţavanju simptoma bola i neţeljenih efekata koji nastaju kao posledica primene analgetika. Taĉnije, definisana su dva specifiĉna cilja istraţivanja: (i) ispitati uticaj bioaktivnih molekula odabranih sojeva laktobacila na proces autofagije u hepatocitima in vitro, kao i protektivan efekat ovih molekula kod toksiĉnosti izazvane paracetamolom i (ii) ispitati potencijalni imunomodulatorni efekat egzopolisaharida (EPS-CG11) izolovanog iz soja Lactobacillus paraplantarum BGCG11, u in vivo eksperimentalnim modelima inflamacije. Eksperimenti u kulturi hepatocita ukljuĉili su praćenje stepena oštećenja HepG2 ćelija izloţenih toksiĉnoj koncentraciji paracetamola i procesa autofagije, sa ciljem identifikacije potencijalnog mehanizma delovanja postbiotika. Metodološki, vijabilnost HepG2 ćelija analizirana je MTT i LDH esejima. Autofagija je praćena Western blot analizom odreĊivanjem ekspresije p62/SQSTM1 proteina i akumulacijom liposolubilne forme LC3 proteina. Dodatno, autofagija je analizirana praćenjem ekspresije BECN1, Atg5, p62/SQSTM1 i PINK1 gena i autofagnog fluksa. Za analizu imunomodulatornog efekta EPS-CG11 korišćena su dva in vivo modela inflamacije izazvane karageninom: model inflamatornog bola i model peritonitisa kod pacova Wistar soja. U eksperimentu inflamatornog bola praćeni su: vremenski tok razvoja hiperalgezije i edema šapica nakon profilaktiĉke sistemske primene EPS-CG11, ekspresija medijatora inflamacije (IL-1β, TNF-α, IL-6 i iNOS), infiltracija neutrofila (ekspresija MPO enzima) i aktivacija/infiltracija monocita (ekspresija CD14 markera)...Isolation and characterization of bacterial biomolecules involved in the interaction with the receptors of the host cells represent the key factor for understanding the mechanisms of probiotic action of lactobacilli. Novel studies regarding probiotic bacteria have been focused on the identification of biomolecules which can modulate different signaling pathways in human cells. All molecules that originate from probiotics which are responsible for its positive effects on the host’s health are called postbiotics. Their application represents the safe alternative to the use of live bacteria and its immunostimulating potential. This research aimed to test the possibility of using postbiotics in alleviation of pain symptoms and analgesics side effects. More precisely, two main objectives of this research were: (i) to examine the influence of bioactive molecules of selected strains of lactobacilli on the autophagy process in the hepatocytes, in vitro, as well as protective effect of these molecules in paracetamol-induced toxicity and (ii) to examine the potential immunomodulatory effect of exopolysaccharide (EPS-CG11) isolated from Lactobacillus paraplantarum BGCG11 strain, in in vivo experimental models of inflammation. Experiments in the hepatocytes culture included monitoring the degree of damage of HepG2 cells exposed to the toxic paracetamol concentration and the autophagy process, with the aim of identification of potential mechanism of postbiotic action. Methodologically, the cell viability was monitored by MTT and LDH assays. Autophagy was monitored by Western blot analysis, in order to determine the expression of the p62/SQSTM1 protein and the accumulation of the liposoluble form of the LC3 protein. Further, the autophagy was analyzed by monitoring the expression of BECN1, Atg5, p62/SQSTM1 and PINK1 genes and the autophagy flux. For the analysis of the immunomodulatory effect of EPS-CG11, two in vivo models of carrageenan-induced inflammation were used: an inflammatory pain model and a peritonitis model in the Wistar rats..
Light and sex interplay: differential herbivore damage in sun and shade in dioecious Mercurialis perennis
Get PDFInteractions between plants and herbivorous insects can be shaped by light environment, resulting in differential herbivory in sun and shade. In dioecious species, the combination of plant sex and light-induced changes in defense traits and nutritive value can alter the patterns of foliar damage. We explored the combined effects of light environment and plant sex on natural herbivore damage and plant traits in the dioecious understory forb Mercurialis perennis on Mt. Kopaonik (Serbia). The role of plant traits in predicting the extent of damage was examined as well. Male and female plants from contrasting light environments (shade vs. sun) were analyzed with respect to leaf damage, as well as plant morphological and biochemical traits (size, specific leaf area, carbon-based defensive compounds and nutritional quality). We found prominent differences in herbivore damage between sun and shade conditions, but not between the sexes. Plants from the sun-exposed site had a significantly larger leaf area removed. The specific leaf area co-varied with herbivore damage in an inverse fashion, while leaf nutritional value had a moderate effect. Contrasting light conditions influenced the patterns of intersexual differences in the contents of condensed tannins and soluble proteins, with females exhibiting higher trait values. We found that factors defining risk of consumption were related to plant morphological traits and nutritive value rather than to chemical defenses
Pharmacogenomic markers of glucocorticoid response in the initial phase of remission induction therapy in childhood acute lymphoblastic leukemia
Get PDFBackground. Response to glucocorticoid (GC) monotherapy in the initial phase of remission induction treatment in childhood acute lymphoblastic leukemia (ALL) represents important biomarker of prognosis and outcome. We aimed to study variants in several pharmacogenes (NR3C1, GSTs and ABCB1) that could contribute to improvement of GC response through personalization of GC therapy. Methods. Retrospective study enrolling 122 ALL patients was carried out to analyze variants of NR3C1 (rs33389, rs33388 and rs6198), GSTT1 (null genotype), GSTM1 (null genotype), GSTPI (rs1695 and rs1138272) and ABCB1 (rs1128503, rs2032582 and rs1045642) genes using PCR-based methodology. The marker of GC response was blast count per microliter of peripheral blood on treatment day 8. We carried out analysis in which cut-off value for GC response was 1000 (according to Berlin-Frankfurt-Munster [BFM] protocol), as well as 100 or 0 blasts per microliter. Results. Carriers of rare NR3C1 rs6198 GG genotype were more likely to have blast count over 1000, than the noncarriers (p = 0.030). NR3C1 CAA (rs33389-rs33388-rs6198) haplotype was associated with blast number below 1000 (p = 0.030). GSTP1 GC haplotype carriers were more likely to have blast number below 1000 (p = 0.036), below 100 (p = 0.028) and to be blast negative (p = 0.054), while GSTP1 GT haplotype and rsl 138272 T allele carriers were more likely to be blasts positive (p = 0.034 and p = 0.024, respectively). ABCB1 CGT (rs1128503-rs2032582-rs1045642) haplotype carriers were more likely to be blast positive (p = 0.018). Conclusions. Our results have shown that NR3C1 rs6198 variant and GSTP1 rs1695-rs1138272 haplotype are the most promising pharmacogenomic markers of GC response in ALL patients
Pathophysiology and Genetics of Bronchiectasis Unrelated to Cystic Fibrosis
No full textBronchiectasis is characterized by deregulated inflammatory response and recurrent bacterial infection resulting in progressive lung damage and an irreversible dilatation of bronchi and bronchioles. Generally accepted model of the development of bronchiectasis is the "vicious cycle hypothesis" that proposes compromising of the mucociliary clearance by an initial event, which leads to the infection of the respiratory tract followed by further impairment of mucociliary function, bacterial proliferation, and more inflammation. Bronchiectasis is a very common symptom in patients with cystic fibrosis (CF), while bronchiectasis unrelated to CF is heterogeneous pathology of unknown cause with a large number of potential contributory factors and poorly understood pathogenesis. It is presumed that bronchiectasis unrelated to CF is a multifactorial condition predisposed by genetic factors. Different molecules have been implicated in the onset and development of idiopathic bronchiectasis, as well as modulation of the disease severity and response to therapy. Most of these molecules are involved in the processes that contribute to the homeostasis of the lung tissue, especially mucociliary clearance, protease-antiprotease balance, and immunomodulation. Evaluation of the studies performed towards investigation of the role these molecules play in bronchiectasis identifies genetic variants that may be of potential importance for clinical management of the disease, and also of interest for future research efforts. This review focuses on the molecules with major roles in lung homeostasis and their involvement in bronchiectasis unrelated to CF