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Biocatalytic versatility of engineered and wild-type tyrosinase from R-solanacearum for the synthesis of 4-halocatechols
No full textWe evaluated the kinetic characteristics of wild type (WT) and three engineered variants (RVC10, RV145, and C10_N322S) of tyrosinase from Ralstonia solanacearum and their potential as biocatalysts to produce halogenated catechols. RV145 exhibited a 3.6- to 14.5-fold improvement in catalytic efficiency (k (cat)/K (m)) with both reductions in K (m) and increases in k (cat) compared to WT, making it the best R. solanacearum tyrosinase variant towards halogenated phenols. RVC10 also exhibited increases in catalytic efficiency with all the tested phenols. A single-mutation variant (C10_N322S) exhibited the greatest improvement in k (cat) but lowest improvement in catalytic efficiency due to an increase in K (m) compared to WT. Consistent with kinetic characteristics, biotransformation experiments showed that RV145 was a superior biocatalyst in comparison to WT. To prevent through conversion of the catechol to quinone, ascorbic acid (AA) was added to the biotransformation medium in 1:2 (substrate:AA) ratio resulting in a catechol yield of gt 90%. Flask experiments with 10 mM 4-iodophenol and 10 mu g/mL of the RV145 enzyme yielded 9.5 mM 4-iodocatechol in the presence of 20 mM AA in 30 min. Similarly, 10 mM 4-fluorophenol was completely consumed by 20 mu g/mL of RV145 enzyme and yielded 9.2 mM 4-fluorocatechol in the presence of 20 mM AA in 80 min. The biotransformation of 20 mM 4-fluorphenol was incomplete (93%) and the yield of 4-flurocatechol was 87.5%. The 4-halophenol conversion rates and product yields obtained in this study are the highest reported using tyrosinase or any other enzyme
Expression of TLR7, TLR9, JAK2, and STAT3 genes in peripheral blood mononuclear cells from patients with systemic sclerosis
No full textSystemic sclerosis (SSc) is a rare, chronic, multisystem autoimmune disease clinically characterized by progressive fibrosis of the skin and internal organs. The basic mechanism appears to involve endothelial cell injury, overproduction of extracellular matrix proteins, and aberrant immune activation. So far, there have been a few attempts to find genetic biomarkers for monitoring disease activity or for correlation with certain symptoms. In order to reveal reliable biomarkers, we analyzed the expression of four genes representing three important signaling pathways, TLR7, TLR9, and JAK2-STAT3. Using RT-qPCR technique, we analyzed the expression of TLR7, TLR9, JAK2, and STAT3 genes in peripheral blood mononuclear cells of 50 SSc patients and 13 healthy individuals. We detected significant upregulation of TLR7 gene expression in a group of SSc patients compared to non-SSc group. Receiver operating characteristic (ROC) analysis showed that TLR7 expression efficiently discriminates SSc cases from healthy individuals. High TLR7 expression positively correlated with the late form of disease, active SSc, and the presence of digital ulcers. Decreased levels of TLR9 and JAK2 mRNA were found in the patient's cohort in comparison to non-SSc individuals, but showed no correlation with specific clinical outcomes. The expression level of the STAT3 gene did not differ between the analyzed groups. This is the first study on the expression of TLR7, TLR9, and STAT3 genes in SSc patients. Our results show that TLR7, TLR9, and JAK2 genes are potential biomarkers for SSc. The results obtained in this study could contribute to better classification, monitoring, and outcome prediction of patients with SSc based on genetics
Analysis of the expression and role of the human gene SOX14 in neural differentiation in vitro and in the regulation of malignant phenotype
Get PDFVisok stepen očuvanosti tokom evolucije i činjenica da do danas nije opisan nijedan klinički sindrom povezan sa mutacijama u genu SOX14, ukazuju na značajnu ulogu ovog gena tokom embrionalnog razvića. Iako je prošlo dvadeset godina od otkrića gena SOX14, funkcija ovog gena još uvek nije dovoljno istražena. U ovoj doktorskoj disertaciji analizirana je ekspresija i uloga gena SOX14 u procesu neuralne diferencijacije in vitro i regulaciji malignog fenotipa ćelija poreklom od karcinoma grlića materice. Tokom neuralnog razvića kod miša i pileta, gen Sox14 je eksprimiran u određenom setu neuralnih progenitorskih ćelija i u određenim tipovima zrelih neurona u mozgu. Za članove SOXB1 podgrupe, kojoj pripadaju geni SOX1, SOX2 i SOX3, poznato je da doprinose održavanju neuralnih progenitorskih ćelija u stanju proliferacije, sprečavajući proces neuralne diferencijacije, dok se za članove SOXB2 podgrupe, koja obuhvata gene SOX14 i SOX21, pretpostavlja da imaju ulogu u određivanju sudbine neuralnih progenitora, indukujući proces neurogeneze. Smatra se da nivo ekspresije SOX14 i SOX21 proteina tokom neuralnog razvića određuje da li će ćelija ostati neuralni progenitor ili će započeti proces neurogeneze. Iako je okarakterisan kao marker neurona, u ovoj tezi je pokazano da se gen SOX14 eksprimira u pluripotentnim ćelijama, neuronima, kao i u drugim tipovima ćelija koje se dobijaju nakon neuralne diferencijacije pluripotentnih NT2/D1 i P19 ćelija in vitro. Takođe, u ovoj tezi je pokazano da je profil ekspresije SOX14 proteina tokom neuralne diferencijacije sličan profilu ekspresije markera postmitotičkih neurona β-III-Tubulina kod NT2/D1 i P19 ćelija. Rezultati prikazani u ovoj doktorskoj disertaciji su pokazali da izlaskom ćelija iz stanja pluripotentnosti i ulaskom u proces neuralne diferencijacije dolazi do povećanja nivoa ekspresije SOX14 proteina. Pokazano je da SOX14 protein ima preklapajući profil ekspresije sa SOXB1 proteinima tokom procesa neuralne diferencijacije in vitro, a dobijeni rezultati su ukazali na specifičnu regulaciju ekspresije gena SOX14 na post-transkripcionom nivou u pluripotentnim ćelijama. Novija istraživanja ukazuju da je ekspresija gena SOX14 izmenjena u maligno transformisanim ćelijama. Naime, istraživanja su pokazala da je gen SOX14 metilovan u tkivima poreklom od tumora, i to naročito u tkivima poreklom od karcinoma grlića materice. Dok studije metilacionog statusa na uzorcima tkiva pacijentkinja obolelih od ovog karcinoma ukazuju na odsustvo ekspresije gena SOX14 i njegovu potencijalnu ulogu tumor supresora, in vitro studije na ćelijama poreklom od karcinoma grlića materice ukazuju na onkogeni potencijal ovog gena. Upravo je kontradiktornost ovih podataka otvorila nove pravce istraživanja usmerene na detaljnu analizu ekspresije i uloge gena SOX14 u regulaciji malignog fenotipa ćelija poreklom od karcinoma grlića materice. Rezultati prikazani u ovoj doktorskoj disertaciji su pokazali da SOX14 ostvaruje funkciju tumor supresora u HeLa ćelijama aktivacijom p53 signalnog puta koji se smatra jednim od najvažnijih signalnih puteva u ćeliji...SOX14 might have an essential role during development due to the high evolutionary conservation and lack of any known mutated phenotype associated with this gene. Although the first SOX14 gene in vertebrates was cloned and characterized two decades ago, the function of this gene is largely unknown. This doctoral dissertation provides an insight into SOX14 expression and its roles during neural differentiation in vitro and in regulation of malignant phenotype of cervical carcinoma cells. During the neural development, the expression of Sox14 gene is restricted to a defined set of neural progenitors and the precise subset of neurons in the brain. Because of its specific expression in the brain, SOX14 is recognised as a marker of neurons. While SOXB1 subgroup of genes, including SOX1, SOX2 and SOX3 genes, maintains neural progenitors' identity, it is proposed that SOXB2 subgroup, comprising SOX14 and SOX21 genes, are important for induction of neurogenesis. It is suggested that the fine balance between expressions of these proteins during early stages of neural development determines neural progenitor identity. Results presented in this thesis show that SOX14 is expressed in pluripotent cells, as well as in neurons and non-neuronal differentiated cells. Also, it is shown that the expression profile of SOX14 correlates to the expression profile of neuronal marker β-III-Tubulin and that the exit of cells from the pluripotent state toward neural differentiation is accompanied with the increased expression of SOX14. It is also shown that the expression of SOX14 overlaps with the expression of SOXB1 proteins during neural differentiation in vitro. The presented results also imply the specific post-transcriptional mechanism of regulation of SOX14 expression in pluripotent cells. Recently it became evident that SOX14 has a role during malignant transformation of the cells. Previous studies showed that SOX14 is methylated in tissues originating from several tumors, including cervical carcinoma. Results obtained by the analysis of the methylation status of SOX14 gene in tissues from cervical carcinoma suggested its potential role as a tumor suppressor, while, on the other hand, in vitro studies suggested that SOX14 exerts oncogenic potential in cervical carcinoma cells. These contradictory data opened up the possibility for further study of the role of SOX14 gene in cervical carcinoma. Results presented in this thesis show that SOX14 exerts tumor suppressor activity in HeLa cells through activation of p53 signaling pathway, one of the most important signaling pathways in cell. In particular, increased expression of SOX14 leads to stabilization of p53 protein by increasing the level of phosphorylated form of p53 protein in HeLa cells. Phosphorylated p53 acts as a transcriptional regulator of target genes that have an important role in tumor growth prevention. The increased expression level of SOX14 and stabilization of p53 are followed by induction of the expression of p53 target genes, mainly involved in apoptosis and cell cycle regulation, such as pro-apoptotic BAX gene and cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1. Increased expression of SOX14, through activation of a p53 signaling pathway, leads to decreased proliferation, cell cycle arrest and induction of apoptosis..
Evaluation of selected virulence factors in Pseudomonas aeruginosa isolates and their susceptibility to silver complexes
Get PDFPseudomonas aeruginosa je ubikvitaran, oportunistički patogen, uzročnik infekcija kod životinja i ljudi, koje su najčešće povezane sa kompromitovanim imunološkim sistemom domaćina. Patogeneza pseudomonasne infekcije je multifaktorijalan proces koji uključuje produkciju faktora virulencije vezanih za ćeliju, odnosno faktora virulencije koji se sekretuju, složenu međubakterijsku komunikaciju putem četiri sistema, kao i sposobnost prelaska iz planktonske životne forme u formu biofilma. Značaj ove vrste za medicinu i veterinu potvrdila je Svetska zdravstvena organizacija u izveštaju objavljenom 2017. godine, po kom P. aeruginosa pripada kritičnoj grupi bakterijskih vrsta za koje je potrebno hitno razviti nove terapeutike jer, zbog sve učestalije pojave multirezistentnih sojeva, ova vrsta predstavlja ozbiljnu pretnju javnom zdravlju. U okviru ove disertacije uporedno su analizirani faktori virulencije izolata P. aeruginosa izolovanih iz uzoraka obolelih životinja i ljudi, kao što su sposobnost formiranja biofilma (ukupan biofilm i biofilm na granici vazduh-tečnost), proizvodnja piocijanina, hemolitička aktivnost, sposobnost manifestovanja tri tipa pokretljivosti (plivanje, rojenje i grčenje), kao i citotoksični efekat na ćelijama iz ćelijske linije A549. Filogenetska srodnost i grupisanje odabranih izolata P. aeruginosa proučavana je na osnovu parcijalne sekvence gena za 16S rRNK. Ispitivanje postojanja korelacija između odabranih faktora virulencije urađeno je primenom robusne rang korelacije. Grupisanje izolata je izvedeno metodom hijerarhijske klasterizacije na osnovu euklidske udaljenosti normalizovane vrednostima sedam ispitivanih faktora virulencije i citotoksičnosti. Metodom mašinskog učenja konstruisan je prediktivni model na osnovu koga je određena značajnost ispitivanih faktora virulencije u predviđanju citotoksičnog potencijala izolata P. aeruginosa. Ispitivanje razlika između faktora virulencije izolata različitog porekla (izolati poreklom od ljudi u odnosu na izolate poreklom od životinja, odnosno izolati poreklom iz uzorka tečnosti u odnosu na izolate poreklom iz briseva) je izvedeno Wilcoxon Rank-Sum testom i Relative effect testom..
Silicon mediates sodium transport and partitioning in maize under moderate salt stress
No full textSilicon (Si) is known to alleviate salt stress in various crops; however, the influence of Si on sodium (Na) transport and partitioning at the tissue, cell and organelle levels is poorly understood. Maize (Zea mays L.) hybrid sensitive to salt stress was exposed to moderate salt stress (40 mM NaCl; simulating conditions in salinized agricultural soils) without or with supply of 1.5 mM Si(OH)4. We investigated the expression of SOS genes encoding Na+ efflux transporter in various root tissues of maize, paralleled by measurements of tissue Na concentration. In addition, subcellular localization of Na (using Na fluorescent dye) within the leaf mesophyll cells was also performed. Silicon supplied plants accumulate less Na in both root apex and cortex, but allocate more Na+ to the leaves via the xylem. This was accompanied by increased expression of ZmSOS1 and ZmSOS2 in the root apex and cortex facilitating Na+ exclusion, and in the root stele for enhanced Na+ loading into the xylem. Also, Si down-regulated the expression of ZmHKT1 in the root stele, which further decreased Na+ unloading from the xylem. Consequently, Si increased accumulation of Na in leaves, but also enhances sequestration of Na+ into the vacuoles thereby decreasing Na+ accumulation in the chloroplasts. In response to moderate salt stress in maize, Si shifts the typical glycophyte behavior of this species towards that of halophytes.Peer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1154][https://www.sciencedirect.com/science/article/abs/pii/S0098847218309493?via%3Dihub
Can untreated PKU patients escape from intellectual disability? A systematic review
Get PDFBackground: Phenylketonuria (PKU) is often considered as the classical example of a genetic disorder in which severe symptoms can nowadays successfully be prevented by early diagnosis and treatment. In contrast, untreated or late-treated PKU is known to result in severe intellectual disability, seizures, and behavioral disturbances. Rarely, however, untreated or late-diagnosed PKU patients with high plasma phenylalanine concentrations have been reported to escape from intellectual disability. The present study aimed to review published cases of such PKU patients. Methods: To this purpose, we conducted a literature search in PubMed and EMBASE up to 8th of September 2017 to identify cases with 1) PKU diagnosis and start of treatment after 7 years of age; 2) untreated plasma phenylalanine concentrations gt = 1200 mu mol/l; and 3) IQ gt = 80. Literature search, checking reference lists, selection of articles, and extraction of data were performed by two independent researchers. Results: In total, we identified 59 published cases of patients with late-diagnosed PKU and unexpected favorable outcome who met the inclusion criteria. Although all investigated patients had intellectual functioning within the normal range, at least 19 showed other neurological, psychological, and/or behavioral symptoms. Conclusions: Based on the present findings, the classical symptomatology of untreated or late-treated PKU may need to be rewritten, not only in the sense that intellectual dysfunction is not obligatory, but also in the sense that intellectual functioning does not (re) present the full picture of brain damage due to high plasma phenylalanine concentrations. Further identification of such patients and additional analyses are necessary to better understand these differences between PKU patients
Application of Next-Generation Sequencing Technology and Establishment of Biobanks
Get PDFInstitute of Molecular Genetics and Genetic Engineering has
become widely recognized as an expert centre for rare diseases
(RD). It is the first institution is Serbia that applied NGS methodology in research and diagnostics of RD. We are also the institution
with RD biobank collections containing DNA, RNA, mononuclear
cells and tissue samples from over 2000 of patients affected with
50 different diseases.
An accurate diagnosis was provided to over than 100 RD patients who were undiagnosed for years. We used Clinical-Exome
Sequencing TruSightOne Gene Panel (4813 clinicaly-relevant
genes), Illumina MiSeq instrument and Illumina VariantStudio.
For monogenic diseases, filtration and prioritization of variants
were performed according to “in-house” pipeline, using virtual
gene panels. Variants were analyzed by various in silico softwares
and classified according to ACMG guidelines. Variants selected by
these criteria were confirmed by conventional Sanger sequencing
and parents’ samples were analyzed whenever available.
Furthermore, novel variants in DNAI1, MUT, PAH, PCCB,
SLC37A4, SPAG16 and SPAG17 genes were functionally characterized in adequate in vitro systems such as immortalized patients’
fibroblasts or CRISPR /Cas9 edited commercial cell lines.
Clinical-exome sequencing enabled diagnosis of more than 50
different diagnosis (hematological, metabolic, endocrinological,
pulmonary, immunological, orthopedic, dermatological, ophthalmological, cardiological, epileptic encephalopathies etc.). It
was particularly important for genetically heterogeneous diseases,
such as glycogen storage diseases, branched-chain organic acidurias, primary ciliary dyskinesia, MODY or mitochondriopathies.
Moreover, different diseases with overlapping clinical manifestations were accurately diagnosed.
Also, we used TruSeq-Amplicon Cancer Panel to analyse different childhood and adult rare hematological malignancies. Besides studying diagnostic and prognostic malignancy markers, we
designed “in-house” virtual pharamocogenomic panel, and performed association studies of pharmacogenomic markers and the
course and outcome of rare hematological malignancies, resulting in recommendations for therapeutic modalities in accordance
with genomic profile of the patien
Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel
Get PDFP2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATP gamma S or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating
Patterns of herbivore damage, developmental stability, morphological and biochemical traits in female and male Mercurialis perennis in contrasting light habitats
No full textLight environments can influence variation in plant morphology, development and susceptibility to herbivores. Our research interest was to investigate the patterns of herbivore damage and developmental stability in dioecious understory forb Mercurialis perennis in contrasting light habitats, located at 1700m a.s.l. on Mt. Kopaonik. Male and female plants from two light habitats, open (a sun-exposed field) and shaded (a spruce forest) were examined with respect to: herbivore damage (percentage of leaf area loss), fluctuating asymetry (FA) as a measurement of developmental stability, plant morphological and, specifically, leaf size traits, as well as biochemical traits relating to nutritional quality and defence, taking into account the possible presence of intersexual differences. Our results show that herbivore damage was significantly higher in open habitat, as well as one out of four univariate FA indices and the multivariate index. Morphological and biochemical traits, apart from defensive compounds, had higher values in the shade, pointing to sun-exposed habitat being more stressful for this species. Intersexual differences were observed for foliar damage, defensive compounds (phenolics and tannins), all leaf size traits, total leaf area, and protein content. Contrasting light habitats affected most of the analysed traits. Both foliar damage and FA were higher in a more stressful habitat; within habitats, no positive correlations were found. Herbivore damage was significantly male biased in open habitat. The analysis of intersexual differences in developmental stability measured by leaf asymmetry levels provided no evidence that female plants were more sensitive to environmental stress
Synthesis, cytotoxic activity and DNA-binding properties of copper(II) complexes with terpyridine
No full textMononuclear copper(II) complexes with 2,2':6',2 ''-terpyridine (terpy), [Cu(terpy)(ClO4)(2)(H2O)] (1) and [Cu(terpy())2](CF3SO3)(2)center dot 2H(2)O (2), were synthesized and structurally characterized by UV-Vis and IR spectroscopy, ESI mass spectrometry and single-crystal X-ray diffraction analysis. In vitro study of cytotoxicity of the complexes demonstrated good antiproliferative properties in the case of human non-small cell lung cancer (A549), as well as in lung fibroblast (MRC5) cell line. Copper(II) complexes with terpy showed significant ability to interact with the high molecular weight double stranded DNA, without induction of DNA damage. On the other side, they caused nicking of plasmid DNA without presence of co-oxidant, indicating moderate nucleolytic activity. Circular dichroism spectra confirmed intercalation of the complexes to double-stranded DNA. Molecular docking studies also indicated strong binding affinity of the complexes with DNA revealing that two forms of 1 (1a and 1b with and without coordinated perchlorate ion, respectively) and 2 bind to the major groove of DNA