imagine (Institute of molecular genetics and genetic engineering)
Not a member yet
3088 research outputs found
Sort by
Importance of next generation sequencing for detection of novel pharmacogenomic markers in pediatric acute lymphoblastic leukemia
No full textAcute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. Therapy
causes side effects in 75% of patients and 1-3% of patients have a lethal outcome as a
consequence of treatment. Since treatment improvements of pediatric ALL have not been
achieved by introduction of novel drugs, but by avoiding the adverse effects of the drugs,
pharmacogenomics has become essential in pediatric ALL treatment. A leukemia patient has
two genomes: constitutional and leukemia genome. Variants in the constitutional genome
are responsible for the efficacy of the drugs and the side effects, while somatic mutations
in leukemia clone are responsible for the resistance to drugs. Next generation sequencing
(NGS) has made breakthroughs in pharmacogenomics by discovering novel genetic markers
which could be candidates for targeted therapy and predictors of efficacy and toxicity of
drugs.
We used NGS analysis to discover novel potential pharmacogenomic markers in pediatric
ALL. DNA samples from bone marrow of 17 pediatric ALL patients were analyzed for somatic
mutations using NGS TruSeq Amplicon–Cancer Panel (Ilumina). DNA samples from blood of
100 individuals were analyzed for germinative mutations using the NGS platform TruSightOne
(Ilumina). For prediction of effects of variants, we used software tools SIFT, PolyPhen-2 and
PROVEAN. For protein modeling we used STRUM method and i-TASSER server.
In the NGS study of somatic mutations in pediatric ALL, 9 novel variants have been identified.
Bioinformatic analysis has shown that STK11 c.1023G>T and ERBB2 c.2341C>T are candidates
for molecular targeted therapy. In the exome sequencing study, according to the in-house
virtual panel for GC response markers and the prediction algorithms, 3 new potential markers
in pharmacogenes related to GC response have been identified, ABCB1 c.947A>G, NCOA3
rs138733364 and TBX21 rs14059812. Pharmacogenenomic profiling of each pediatric ALL
patient is indispensable for efficient therapy design.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Genomic profiling as a tool for differential diagnosis of patients with pediatric lung diseases and discovery of novel disease-causing genes and genetic variants
No full textPrimary ciliary dyskinesia (PCD) is a rare disorder that affects lungs, reproductive organs
and the internal organs laterality. The disease is inherited in autosomal recessive or X-linked
manner. PCD is clinically and genetically heterogeneous disorder with overlapping symptoms
with other pediatric lung diseases. The aim of the study was genomic profiling of suspected
PCD patients in order to establish the genetic background of PCD in our patients, to confirm
clinical diagnosis, and to design a strategy for differential diagnosis of PCD patients among
patients with similar clinical presentation.
Using Clinical-Exome Sequencing Panel, we analysed 93 genes related to PCD and other
pediatric lung diseases in a cohort of 21 Serbian patients with clinically suspected PCD.
Analysis of obtained results revealed genetic variants in CCDC39, CCDC40, DNAI1, DNAL1,
DNAH5, DNAH11 and LRRC6 genes, and pointed and confirmed SPAG16 and SPAG17 as
novel PCD disease causing genes. Twenty variants in these genes were pathogenic, of which
fourteen (70 %) were novel. The PCD diagnosis was established in 54.55 % of patients.
Analysis of genes related to individual symptoms of PCD, revealed 6 pathogenic variants in
ABCA3, CFTR, MUC2, SCNN1A, and SLC26A9 genes, of which 5 (83.33%) were novel. This
enabled the diagnosis for additional 28.57% patients.
The analysis of extended list of genes enables mutation detection rate of 95.45% (21/22
patients), while the rate of established diagnosis reached 81.82% (18/22 patients).Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Antifungal potential of streptomycetes isolated from ethnomedicinal plants' rhizospheres: characterization and optimization of staurosporine biosynthesis by Streptomyces sp. BV410 strain
No full textZemljište predstavlja bogat izvor različitih mikroorganizama čiji produkti metabolizma mogu biti od izuzetnog značaja za čoveka. Dosadašnja ispitivanja mikrobnog diverziteta u zemljištu su otkrila bogati biosintetski potencijal za proizvodnju novih prirodnih proizvoda kod velikog broja mikroorganizama, naročito kada je u pitanju klasa Actinobacteria. Među zemljišnim izolatima, rod Streptomyces prednjači po broju identifikovanih bioaktivnih molekula u odnosu na sve ostale bakterije. Stoga je jedan od ciljeva u okviru ove doktorske disertacije izolacija streptomiceta iz rizosfera medicinski značajnih biljaka sakupljenih na teritoriji Republike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urtica dioica) i ispitivanje njihovog antifungalnog potencijala na različite vrste kandida. Morfološki različiti izolati (ukupno 103) su izolovani iz uzoraka rizosfera i okarakterisani kao streptomicete. Dve različite podloge i dve procedure za ekstrakciju su korišćene da bi se pospešila detekcija antifungalnih jedinjenja. Ispitan je uticaj ukupno 412 ekstrakata na rast Candida albicans disk difuzionim esejem pri čemu je utvrđeno da 42% (43/103) izolata imaju sposobnost proizvodnje antifungalnih jedinjenja pri ispitivanim uslovima. Pojedini ekstrakti su inhibirali rast važnih humanih patogena poput Candida krusei, Candida parapsilosis, i Candida glabrata. Na osnovu stepena i spektra antifungalne aktivnosti devet izolata je odabrano za dalja istraživanja. Ispitana je sposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnoj kulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na već formirane biofilmove kandide u koncentracijama od 8 do 250 pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovu metaboličku raznolikost dobijeni su korišćenjem tečne hromatografije visokih performansi. Tri ekstrakta sa specifičnom antifungalnom aktivnošću podvrgnuta su hemijskim analizama s ciljem da se detektuju i strukturno okarakterišu molekuli koji su nosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno- rezonantne spektroskopije otkriveno je da su aktivni molekuli genistein, daidzein i staurosporin. Genistein i daidzein koji su poznati fitoestrogeni poreklom iz sojinog brašna za koje je poznato da inhibiraju ključne enzime u biosintetskom putu steroida. Njihovo prisustvo je u ovom istraživanju detektovano usled korišćenja sojinog brašna u hranljivoj podlozi. Kako streptomicete u čijim ekstraktima su detektovani ovi molekuli pokazuju sposobnost oslobađanja ovih važnih jedinjenja iz kompleksne hranljive podloge, mogu se uzeti u razmatranje za biotehnološku proizvodnju fitoestrogena. Staurosporin je detektovan kao nosilac antifungalne aktivnosti kod ekstrakta soja Streptomyces sp. BV410. Staurosporin je inhibitor protein kinaza i njegovi derivati i analozi se koriste u kao antitumorski agensi. Biosinteza ovog molekula je optimizovana do prinosa od 36,94 mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadrži glukozu, skrob, manitol i sojino brašno (JS). Dalja optimizacija hranljive podloge za biosintezu staurosporina ukazala je na sledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba, 21,46 g/l manitola, 17,32 g/l sojinog brašna. Primenom definisanih optimalnih vrednosti i korišćenjem odgovarajućih matematičkih modela, predviđeno je da će se na ovaj način postići prinosi od 46,88 mg/l staurosporina i 12,05 mg/ml biomase. Validnost predviđenih rezultata potvrđena je izvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta). Ispitana je kinetika biosinteze staurosporina i produkcije biomase, kao i potrošnje izvora ugljenika i razvijeni su odgovarajući procesni modeli. Dodatna optimizacija je podrazumevala dodatak suplemenata koji prema literature stimulišu sekundarni metabolizam streptomiceta (joni cinka, gvožđa, fosfati, metil oleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pH vrednosti (6,5, 7,5 i 8,5) a uspešnost bioprocesa je procenjivana 7., 10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatka da dodatak soli gvožđa značajno pospešuje biosintezu staurosporina sa povećanjem prinosa od 25%. Dobijeni rezultati potvrđuju da su rizosfere medicinski značajnih biljaka značajan izvor streptomiceta koje proizvode komponente sa antifungalnom aktivnošću. Izolacija novog proizvođača staurosporina i optimizacija procesa njegove biosinteze omogućiće dalja istraživanja ovog jedinjenja koje može biti osnova za razvoj novih antifungalnih i jedinjenja koja inhibiraju angiogenezu. Rezultati dobijeni u okviru ovih istraživanja predstavljaju početni korak ka potencijalnoj industrijalizaciji proizvodnje staurosporina.Different soils are still a source of remarkable microbial diversity which also reflects in the unexplored chemical diversity. Recent advances in assessment of microbial diversity from soil have revealed the extraordinarily rich biosynthetic potential for the production of new natural products among different microbial strains, especially within the group of Actinobacteria. Among bacterial soil isolates, representatives of Streptomyces genus are the most prolific producers of bioactive compounds. One of the objectives of the present study was to isolate Streptomyces spp. from the rhizosphere soils of three ethno-medicinal plants collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and Urtica dioica) and to screen their antifungal activity against Candida spp. Morphologically different sporulating isolates (103 in total) were collected from rhizosphere soil samples and determined as Streptomyces spp. Two different media and two extraction procedures were used to induce the production and facilitate identification of antifungals. Overall, 412 crude cell extracts were tested against Candida albicans using disk diffusion assays, with 42% (43/103) of the strains showing the ability to produce antifungal agents. Also, extracts inhibited growth of other important human pathogens: Candida krusei, Candida parapsilosis, and Candida glabrata. Based on the established degree and range of antifungal activity, nine isolates were selected for further testing. Their ability to inhibit Candida growth in liquid culture, to inhibit biofilm formation, and to disperse pre-formed biofilms was assessed with active concentrations from 8 to 250 pg/ml. High-performance liquid chromatographic profiles of extracts derived from selected strains were recorded, revealing moderate metabolic diversity. The most potent extracts were subjected to comprehensive identification and structural characterization of antifungal compounds. Applying a bioactivity-guided isolation approach, active compounds of three extracts were separated, and based on NMR structure elucidation it was shown that active compounds were genistein, daidzein and staurosporine. Genistein and daidzein, soy phytoestrogens, are known to inhibit key enzymes in the steroid metabolism pathway and were coming from the fermentation medium containing soy flower. Since isolated Streptomyces spp. showed good ability to extract these molecules from complex medium, they can be further considered for biotechnological production of these phytoestrogens. One of the isolates, Streptomyces sp. BV410, was characterized as an efficient staurosporine producer. Staurosporine is a potent inhibitor of protein kinases and is considered in anticancer therapy. The biotechnological production of staurosporine by strain BV410 was optimized to yield 36.94 mg/l after 14 days of incubation in soy flowerglucose- starch-mannitol based fermentation medium (JS). Further optimization of medium for biosynthesis of staurosporine indicated the following optimal values of the examined factors: the content of glucose of 20 g/l, starch 0.36 g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying the defined optimal values and using the appropriate mathematical models, the following responses were predicted: concentration of staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. The validity of the results was confirmed by performing the biosynthesis of the staurosporine in the medium with optimal composition (JSSta). Kinetics of staurosporine and biomass production and carbon source consumption were examined and process models were developed. Additionally, optimization of staurosporine production was performed with different supplements which, according to literature data, had stimulative effect on secondary metabolism (Zn, Fe and P salts, methyl oleate, grape seed oil). In order to improve the production of staurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time (7, 10 and 14 days) were also examined. It was found that addition of FeS04 significantly improved the staurosporine yield in comparison to the starting conditions (increase of 25%). Our results proved that rhizosphere soils of ethno-medicinal plants are a prolific source of streptomycetes, producers of compounds with good antifungal activity. Isolation of the new staurosporine producing strain, allowed for its detailed bioactivity assessment. Staurosporine scaffold might serve as a lead structure for the development of new antifungal and antiangiogenic agents. Also, results obtained within this research represent the basis for the further scale-up and potential industrialization of the proposed production process
NEUROPROTECTIVE ROLE OF SELECTED ANTIOXIDANT AGENTS IN PREVENTING CISPLATIN-INDUCED DAMAGE OF HUMAN NEURONS IN VITRO
No full textChemotherapy-induced peripheral neuropathy (CIPN) is a side effect of
platinum-based chemotherapy and decreases the quality of life of cancer
patients. We compared neuroprotective properties of several agents using an in
vitro model of terminally differentiated human cells NT2-N derived from cell
line NT2/D1. Sodium azide and an active metabolite of amifostine (WR1065)
increase cell viability in simultaneous treatment with cisplatin. In addition,
WR1065 protects the non-dividing neurons by decreasing cisplatin caused
oxidative stress and apoptosis. Accumulation of Pt in cisplatin-treated cells was
heterogeneous, but the frequency and concentration of Pt in cells were lowered
in the presence of WR1065 as shown by X-ray fluorescence microscopy
(XFM). Transition metals accumulation accompanied Pt increase in cells; this
effect was equally diminished in the presence of WR1065. To analyze possible
chemical modulation of Pt-DNA bonds, we examined the platinum LIII near
edge spectrum by X-ray absorption spectroscopy. The spectrum found in
cisplatin-DNA samples is altered differently by the addition of either WR1065
or sodium azide. Importantly, a similar change in Pt edge spectra was noted in
cells treated with cisplatin and WR1065. Therefore, amifostine should be
reconsidered as a candidate for treatments that reduce or prevent CIPN.Abstract book: Immunology at the confluence of multidisciplinary approaches, Hotel Mona Plaza Belgrade December 6th-8th, 201
Next-generation genomics of rare diseases: on the road to new diagnostics and innovative therapies
No full textMajority of rare diseases (RD) are genetic diseases (80%). Therefore, the identification
of specific gene defect in each patient is important. Next generation sequencing (NGS)
methodology has enabled shortening the diagnostic odyssey of patients with RD, leading to
improved treatment and successful genetic counseling.
We have analyzed over 200 RD patients using Clinical-Exome Sequencing TruSightOne
Gene Panel (Illumina). VariantStudio and various in silico software tools were used for
bioinformatics analysis. Filtration and prioritization of variants were performed according to
“in-house” designed pipelines and virtual gene panels.
NGS studies enabled diagnosis of more than 50 different diseases (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. In our studies
mutation detection rate reached 80-100%. Our work enabled the establishment of RD
biobank collections containing DNA, RNA, mononuclear cells and tissue samples from over
2000 patients affected with RD. 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 Crisper/Cas9 edited cell lines.
Also, we used TruSeq-Amplicon Cancer Panel to analyze different childhood and adult rare
hematological malignancies. Our association studies revealed new diagnostic, prognostic
and pharmacogenomic markers, resulting in recommendations for personalized therapeutic
modalities in accordance with genomic profile of the patients.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Genetic layout and genetic structure of the greenhouse whitefly Trialeurodes vaporariorum from Serbia
No full textTrialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), a haplo-diploid sap
feeding insect species, is a vector of many plant viruses and a serious polyphagous pest of
greenhouse-grown vegetables and ornamental plants worldwide. To explore the populations
of this pest and recognize potential invasion routes, the genetic diversity and genetic structure
of T. vaporariorum from Serbia were analysed using six microsatellite loci. PCR multiplex
conditions were optimized and tested using 88 T. vaporariorum females collected from
natural populations in 10 sampling locations in Serbia: Apatin (AP), Beograd (BG), Kovin (KO),
Negotin (NE), Pirot (PI), Smederevska Palanka (SP), Subotica (SU), Svilajnac (SV), Trbušan (TR)
and Vranjska Banja (VB). Observed heterozygosity ranged from 0.105 to 0.796, deviations of
the frequency of genotypes from the expected Hardy–Weinberg equilibrium were detected in
all populations. The values of the Fst parameters ranged from 0.011 between populations of
AP and SU, up to 0.286 among the SV and BG populations. The obtained results for Fst values
were used to explore genetic structure using several analyzes: PAST, Principal Coordinate
Analysis – PCoA and SAMOVA. The analyzed T. vaporariorum populations were grouped in
three distinct genetic clusters based on these population-genetic analyses: (1) BG, (2) AP,
KO, SP, TR, VB and SU, and (3) SV, NE and PI. Also, BOTTLENECK identified reductions in
effective size of SP population in the recent past and the significant heterozygosity deficiency
was detected in the four populations VB, SU, BG and NE. These results demonstrate that
populations of T. vaporariorum in Serbia show significant genetic differentiation, indicating
the likelihood of multiple introductions of this important pest into Serbia, as well as the
influence of crop protection and pest management on their genetic variabilityAbstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Silicon alleviates copper (Cu) toxicity in cucumber by increased Cu-binding capacity
No full textAims Although silicon (Si) is known to increase plant resistance to metal toxicity stress, the mechanisms responsible for alleviation of copper (Cu) toxicity are still insufficiently clear. We investigated the role of Si on Cu-binding processes involved in buffering excessive Cu in cucumber (Cucumis sativus L.) tissues. Methods Cucumber plants were subjected to moderate Cu toxicity stress (10 mu M Cu) without (-Si) or with (+Si) supply of 1.5 mM Si. We analyzed total and cell wall concentrations of Cu and Cu-binding compounds (organic acids and Cu-proteins) along with parameters of oxidative stress (e.g. lipid peroxidation and lignification). Results Supply of Si decreased total Cu concentration in both root and leaf tissues, but increased the root cell wall Cu fraction. Also, Si increased superoxide dismutase (SOD) activity in 10 mu M Cu-treated plants. Concomitantly, protein levels of Cu/Zn SOD isoforms (CSD1 and CSD2) in root tissues also increased in +Si plants. The leaf Cu-binding compounds, such as aconitate and plastocyanin (including the expression of CsPC gene) were higher in the +Si plants. Consequently, Si supply effectively lowered lipid peroxidation in both roots and leaves of Cu-stressed plants. Conclusions Supply of Si enhanced both the accumulation of Cu-binding molecules (Zn/Cu SOD in roots; aconitate and plastocyanin in leaves), and the Cu-binding capacity of the root cell wall
Effects of two kits for amplification of EGFR single nucleotide polymorphisms:-191 C/A (rs712830) and 181946 G/A (rs2293347) which contained high GC rich region
No full textInherited thrombophilic risk factors in Serbian breast cancer patients
Get PDFBreast cancer is the leading cause of cancer-related death among women. An increased burden of thrombotic events among breast cancer patients, leading to higher mortality and morbidity rates, is well established. There are a number of genetic risk factors associated with thrombosis, but their contribution to thrombotic tendencies in patients with cancer is not completely elucidated. We aimed to investigate possible role of FV Leiden, FII G20210A, MTHFR C677T and PAI-1 4G/5G gene variants in etiopathology of breast cancer and accompanying thrombosis in cohort of Serbian patients. Our study included 316 subject divided in three groups: breast cancer patients with (97) or without (99) accompanying thrombosis and healthy control group (120). According to our results, the prevalence for all four prothrombotic gene variants were similar in cancer patients with and without thrombosis and no statistically significant difference was observed between these groups. We detected lower frequency of MTHFR 677TT genotype in breast cancer patients when compared to control group (P=0.014; OR=0.145 (95%CI 0.031-0.679)), indicated that MTHFR C677T homozygosity could play a protective role in breast cancer susceptibility. Our study noted the lack of association between common prothrombotic gene variants and increased prothrombotic risk in Serbian breast cancer patients. Also, our results point out possible role of MTHFR 677TT genotype in etiology of breast cancer, but further studies on larger cohort of patients are needed
Silicon and Iron Differently Alleviate Copper Toxicity in Cucumber Leaves
Get PDFCopper (Cu) toxicity in plants may lead to iron (Fe), zinc (Zn) and manganese (Mn) deficiencies. Here, we investigated the effect of Si and Fe supply on the concentrations of micronutrients and metal-chelating amino acids nicotianamine (NA) and histidine (His) in leaves of cucumber plants exposed to Cu in excess. Cucumber (Cucumis sativus L.) was treated with 10 mu M Cu, and additional 100 mu M Fe or/and 1.5 mM Si for five days. High Cu and decreased Zn, Fe and Mn concentrations were found in Cu treatment. Additional Fe supply had a more pronounced effect in decreasing Cu accumulation and improving the molar ratio between micronutrients as compared to the Si supply. However, the simultaneous supply of Fe and Si was the most effective treatment in alleviation of Cu-induced deficiency of Fe, Zn and Mn. Additional Fe supply increased the His but not NA concentration, while Si supply significantly increased both NA and His whereby the NA:Cu and His:Cu molar ratios exceeded the control values indicating that Si recruits Cu-chelation to achieve Cu tolerance. In conclusion, Si-mediated alleviation of Cu toxicity was directed toward Cu tolerance while Fe-alleviative effect was due to a dramatic decrease in Cu accumulation