imagine (Institute of molecular genetics and genetic engineering)
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Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis
Get PDFBackground: Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. Results: We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average similar to 62 million reads (53.4% of similar to 116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR lt 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR lt 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter (R) we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Conclusions: Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF
Complementary approaches for the evaluation of biocompatibility of Y-90-labeled superparamagnetic citric acid (Fe,Er)(3)O-4 coated nanoparticles
No full textMagnetic nanoparticles (MNPs) are of immense interest for diagnostic and therapeutic applications in medicine. Design and development of new iron oxide-based MNPs for such applications is of rather limited breadth without reliable and sensitive methods to determine their levels in body tissues. Commonly used methods, such as ICP, are quite problematic, due to the inability to decipher the origin of the detected iron, i.e. whether it originates from the MNPs or endogenous from tissues and bodily fluids. One of the approaches to overcome this problem and to increase reliability of tracing MNPs is to partially substitute iron ions in the MNPs with Er. Here, we report on the development of citric add coated (Fe,Er)(3)O-4 nanopartides and characterization of their physico-chemical and biological properties by utilization of various complementary approaches. The synthesized MNPs had a narrow (6-7 nm) size distribution, as consistently seen in atomic pair distribution function, transmission electron microscopy, and DC magnetization measurements. The particles were found to be superparamagnetic, with a pronounced maximum in measured zero-field cooled magnetization at around 90 K. Reduction in saturation magnetization due to incorporation of 1.7% Er3+ into the Fe3O4 matrix was clearly observed. From the biological standpoint, citric acid coated (Fe,Er)(3)O-4 NPs were found to induce low toxicity both in human cell fibroblasts and in zebrafish (Danio rerio) embryos. Biodistribution pattern of the MNPs after intravenous administration in healthy Wistar rats was followed by the radiotracer method, revealing that Y-90-labeled MNPs were predominantly found in liver (7533% ID), followed by lungs (16.70% ID) and spleen (2.83% ID). Quantitative agreement with these observations was obtained by ICP-MS elemental analysis using Er as the detected tracer. Based on the favorable physical, chemical and biological characteristics, citric add coated (Fe,Er)(3)O-4 MNPs could be further considered for the potential application as a diagnostic and/or therapeutic agent. This work also demonstrates that combined application of these techniques is a promising tool for studies of pharmacokinetics of the new MNPs in complex biological systems.Peer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1763]Supplementary data: [https://imagine.imgge.bg.ac.rs/handle/123456789/1764
In vitro antimicrobial activity and cytotoxicity of nickel(II) complexes with different diamine ligands
Get PDFThree diamines, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-propanediamine (2,2-diMe-1,3-pd) and (+/-)-1,3-pentanediamine (1,3-pnd), were used for the synthesis of nickel(II) complexes 1-3, respectively, of the general formula [Ni(L)(2)(H2O)(2)]Cl-2. The stoichiometries of the complexes were confirmed by elemental microanalysis, and their structures were elucidated by spectroscopic (UV-Vis and IR) and molar conductivity measurements. The complexes 1-3, along with NiCl2 center dot 6H(2)O and the diamine ligands, were evaluated against a panel of microbial strains that are associated with skin, wound, urinary tract and nosocomial infections. The obtained results revealed no significant activity of 1-3 against the investigated bacterial strains. On the other hand, they showed good antifungal activity against pathogenic Candida strains, with minimum inhibitory concentration (MIC) values in the range from 15.6 to 62.5 mu g mL(-1). The best anti-Candida activity was observed for complex 2 against C. parapsilosis, while the least susceptible to the effect of the complexes was C. krusei. The antiproliferative effect on normal human lung fibro-blast cell line MRC-5 was also evaluated in order to determine the therapeutic potential of nickel(II) complexes 1-3. These complexes showed lower negative effects on the viability of the MRC-5 cell line than the clinically used nystatin and comparable selectivity indexes to that of this antifungal drug
Antibacterial potential of electrochemically exfoliated graphene sheets
Get PDFElectrochemically exfoliated graphene is functionalized graphene with potential application in biomedicine. Two most relevant biological features of this material are its electrical conductivity and excellent water dispersibility. In this study we have tried to establish the correlation between graphene structure and its antibacterial properties. The exfoliation process was performed in a two electrode-highly oriented pyrolytic graphite electrochemical cell. Solution of ammonium persulfate was used as an electrolyte. Exfoliated graphene sheets were dispersed in aqueous media and characterized by atomic force microscopy, scanning electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, X photoelectron spectroscopy, X-ray diffraction, electron paramagnetic resonance, zeta potential, contact angle measurements and surface energy. Antibacterial assays have shown lack of the significant antibacterial activity. Major effect on bacteria was slight change of bacteria morphology. Membrane remained intact despite significant change of chemical content of membrane components.This is the peer reviewed version of the paper: Marković, Z. M., Matijašević, D. M., Pavlović, V. B., Jovanović, S. P., Holclajtner-Antunović, I. D., Špitalský, Z., Mičušik, M., Dramićanin, M. D., Milivojević, D. D., Nikšić, M. P., & Todorović Marković, B. M. (2017). Antibacterial potential of electrochemically exfoliated graphene sheets. Journal of Colloid and Interface Science, 500, 30–43. [https://doi.org/10.1016/j.jcis.2017.03.110][https://www.sciencedirect.com/science/article/abs/pii/S0021979717303776?via%3Dihub
Long-Chain 4-Aminoquinolines as Quorum Sensing Inhibitors in Serratia marcescens and Pseudomonas aeruginosa
No full textAntibiotic resistance has become a serious global threat to public health; therefore, improved strategies and structurally novel antimicrobials are urgently needed to combat infectious diseases. Here we report a new type of highly potent 4-aminoquinoline derivatives as quorum sensing inhibitors in Serratia marcescens and Pseudomonas aeruginosa, exhibiting weak bactericidal activities (minimum inhibitory concentration (MIC) gt 400 mu M). Through detailed structure-activity study, we have identified 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines (5 and 10) as biofilm formation inhibitors with 50% biofilm inhibition at 69 mu M and 63 mu M in S. marcescens and P. aeruginosa, respectively. These two compounds, 5 and 10, are the first quinoline derivatives with anti-biofilm formation activity reported in S. marcescens. Quantitative structure-activity relationship (QSAR) analysis identified structural descriptors such as Wiener indices, hyper-distance-path index (HDPI), mean topological charge (MTC), topological charge index (TCI), and log D(o/w)exp as the most influential in biofilm inhibition in this bacterial species. Derivative 10 is one of the most potent quinoline type inhibitors of pyocyanin production described so far (IC50 = 2.5 mu M). While we have demonstrated that 5 and 10 act as Pseudomonas quinolone system (PQS) antagonists, the mechanism of inhibition of S. marcescens biofilm formation with these compounds remains open since signaling similar to P. aeruginosa PQS system has not yet been described in Serratia and activity of these compounds on acylhomoserine lactone (AHL) signaling has not been detected. Our data show that 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines present the promising scaffolds for developing antivirulence and anti-biofilm formation agents against multidrug-resistant bacterial species.Supplementary material: [https://imagine.imgge.bg.ac.rs/handle/123456789/2227]Related to accepted verison: [https://imagine.imgge.bg.ac.rs/handle/123456789/2226
Supplementary data for article: Warzajtis, B.; Glišić, B. D.; Savić, N. D.; Pavic, A.; Vojnovic, S.; Veselinović, A.; Nikodinovic-Runic, J.; Rychlewska, U.; Djuran, M. I. Mononuclear Gold(Iii) Complexes with l-Histidine-Containing Dipeptides: Tuning the Structural and Biological Properties by Variation of the N-Terminal Amino Acid and Counter Anion. Dalton Transactions 2017, 46 (8), 2594–2608. https://doi.org/10.1039/c6dt04862e
Get PDFSupplementary material for: [https://doi.org/10.1039/c6dt04862e]Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1050]Related to accepted version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1807
Stimulation of Various Phenolics in Plants Under Ambient UV‐B Radiation
No full textUnder natural conditions, plants are constantly exposed to dynamic changes of solar
radiation, which mainly consists of infrared (IR, >700 nm), photosynthetically active
radiation (PAR, 400–700 nm) and minor portion of ultraviolet (UV) radiation (UV-B,
290–315 nm and UV-A, 315–400 nm). Besides being the primary source of energy
in photosynthesis, sunlight is an important signal which regulates plant growth and
development. In addition to light quantity, plants are able to monitor the quality,
periodicity and direction of light (reviewed in Caldwell et al., 2007; Jiao et al., 2007).
Plants perceive light signals through several protein photoreceptors: five phy-
tochromes (PHY A‐E), which are sensitive to red and far red light (600–750 nm), and
two cryptochromes (CRY1 and CRY2), two phototropins (PHOT1 and PHOT2) and
zeitlupe proteins (ZTLs) for blue and UV‐A radiation (315–500 nm), while UV‐B
radiation is sensed by UV Resistant Locus 8 (UVR8) (reviewed in Jiao et al., 2007;
Heijde and Ulm, 2012).
During the period from the 1970s to 1990s, investigations on UV‐B effects on organ-
isms were in the centre of attention, due to alarming depletion of stratospheric ozone
layer and increased UV‐B radiation reaching the Earth’s surface. However, the results
of numerous studies that explored the impact of high UV‐B radiation on plants were
often contradictory. In following years, this was explained by different unrealistic
UV‐B : UV‐A : PAR ratios, high UV‐B doses applied, different spectral distribution in
the UV‐B region, as well as simultaneous effects of other environmental stressors
(drought, high temperature, nutrient deprivation), and previous plant exposure to
UV‐B radiation (plant history). Inconsistent reports on UV‐B effects on photosynthe-
sis and stomata conductance were a result of different UV‐B doses applied, species‐
specific, and even genotype‐specific responses, but also plant history and overall plant
metabolism. In the light of these findings, during the last decade, research on UV‐B radiation
effects on biological systems has advanced towards more controlled conditions aiming
to imitate ambient solar radiation. Using sun simulators with realistic balance of UV-B,
UV-A and PAR, is a very good solution to achieve realistic and reproducible experi-
mental conditions (Döhring et al., 1996; Aphalo et al., 2012). Contrary to previous
widely accepted beliefs, in the last several years it has been demonstrated that UV‐B
radiation, at low and ecologically relevant doses, presents an important regulator of
plant growth and development (Jenkins, 2009; Hideg et al., 2013). Plants grown in the
open field, exposed to natural UV‐B doses, have higher nutritional and pharmacologi-
cal value than plants grown in polytunnels and glasshouses, which are non‐transpar-
ent to UV radiation (Jansen et al., 2008; Behn et al., 2010). Moreover, it has been shown
that UV‐B radiation improves plant adaptive capacity to drought, high temperatures,
pathogen and insect attack, and nutrient deficiency conditions (Schmidt et al., 2000;
Caputo et al., 2006). These findings have a strong impact on the agricultural, pharma-
ceutical and food industries.
A hallmark of UV‐B response in plants is accumulation of secondary metabolites, such as
phenolic compounds (particularly flavonoids and phenylpropanoids), alkaloids and terpe-
noids. Phenolics are the most abundant secondary metabolites in plants, and 20% of carbon
fixed in photosynthesis is directed to their biosynthesis (Hernández and Van Breusegem,
2010). Phenolic compounds in plants are involved in many processes, from growth and
development, to flowering, reproduction and seed dispersion, defence against pathogens,
plant–insect interactions and protection against numerous abiotic stresses (Gould and
Lister, 2005; Sedlarević et al., 2016). The most well‐studied mechanism of UV‐B induction
of phenolic metabolism is certainly the UVR8 pathway, which will be discussed in detail in
this chapter. However, regarding UV‐B and sunlight exposure in general, antioxidative vs.
UV‐B‐absorbing (screening) functions of phenolics remain debatable (Agati et al., 2013).
Genes encoding UVR8‐like proteins are highly conserved, and have been identified in a
large number of plants, algae and mosses, suggesting the importance of this pathway for the
adaptation of autotrophic organisms to sunlight (Tilbrook et al., 2013).
In this chapter, we have provided overview of publications reporting phenolics
induction by supplementary UV‐B radiation in the last decade. Plant response depends
on UV‐B fluence rate and spectrum. Therefore, it is important to standardize UV‐B
exposure experimental designs to adequately compare the responses of phenolic
metabolism obtained in different studies. In order to interpret morphological and
physiological changes in plants, phenolics function and distribution on the cellular,
tissue and plant level should be understood. Moreover, recent findings on relationship
between photosynthesis and storage molecules, such as starch, and stimulated flavo-
noid biosynthesis under UV‐B radiation are considered
Supplementary data for article: Antic, B.; Boskovic, M.; Nikodinovic-Runic, J.; Ming, Y.; Zhang, H.; Bozin, E. S.; Janković, D.; Spasojevic, V.; Vranjes-Djuric, S. Complementary Approaches for the Evaluation of Biocompatibility of 90Y-Labeled Superparamagnetic Citric Acid (Fe,Er)(3)O4 Coated Nanoparticles. Materials Science and Engineering C 2017, 75, 157–164. https://doi.org/10.1016/j.msec.2017.02.023
Get PDFSupplementary material for: [https://doi.org/10.1016/j.msec.2017.02.023]Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1082]Related to accepted version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1763
Mineralized agar-based nanocomposite films: Potential food packaging materials with antimicrobial properties
Get PDFNew mineralized, agar-based nanocomposite films (Zn-carbonate and Zn-phosphate/agar) were produced by a combination of in situ precipitation and a casting method. The presence of minerals significantly influenced the morphology, properties and functionality of the obtained nanocomposites. Reinforcement with the Zn-mineral phase improved the mechanical properties of the carbonate-mineralized films, but had a negligible effect on the phosphate-mineralized samples. Both nanocomposites showed improved optical and thermal properties, better Zn(II) release potential in a slightly acidic environment and exhibited antimicrobial activity against S. aureus. These results suggest that Zn-mineralized agar nanocomposite films could be potentially used as affordable, eco-friendly and active food packaging materials.This is the peer reviewed version of the paper: Malagurski, I., Levic, S., Nesic, A., Mitric, M., Pavlovic, V., & Dimitrijevic-Brankovic, S. (2017). Mineralized agar-based nanocomposite films: Potential food packaging materials with antimicrobial properties. Carbohydrate Polymers, 175, 55–62. [https://doi.org/10.1016/j.carbpol.2017.07.064][https://www.sciencedirect.com/science/article/abs/pii/S0144861717308408?via%3Dihub
Bactericidal activity of Cu-, Zn-, and Ag-containing zeolites toward Escherichia coli isolates
No full textTwo types of zeolites-natural clinoptilolite (NZ) and synthetic zeolite A (A)-were enriched with approx. 0.25 mmol of Cu(II), Zn(II), or Ag(I) ions, and the obtained materials (M-Z) were tested against three different isolates of Escherichia coli. Two isolates were environmental isolates from waters in Serbia whereas the third one was DSM 498. Antibacterial activity was studied in different water media-nutrient-rich media (peptone water), water from Sava Lake, and commercially available spring water. The Ag-containing zeolites showed bactericidal activity in the nutrient-rich peptone water after 1 h of contact. Cu- and Zn-containing zeolites showed bactericidal activity in real water samples. Antibacterial activity of the M-Z decreases in all three examined water media in the following order: Ag-NZ ae Ag-A gt Cu-NZ ae Cu-A gt Zn-NZ gt gt gt Zn-A, suggesting that mainly the metal type and not the zeolite type have a role in the antibacterial activity. Leaching experiments showed small amounts of the leached Cu(II) and Zn(II) ions, indicating that the antibacterial activity is not due to the metal ions but should be attributed to the M-Z itself. However, leached amounts of Ag(I) from Ag-NZ and Ag-A in peptone water indicate that the released Ag(I) could be mainly responsible for the bactericidal effect of the Ag(I)-containing zeolites. Since no loss of cellular material was found, the antibacterial activity is not attributed to cytoplasmic membrane damage