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De Novo Transcriptome Sequencing of Ramonda serbica Identification of Late Embryogenesis Abundant Proteins
Get PDFAn extreme loss of cellular water or desiccation (5-10% of relative water content) leads to protein denaturation,
aggregation and degradation, and affects the fluidity of membrane lipids resulting in loss of membrane
integrity [1]. The essential constituents of vegetative desiccation tolerance in so-called resurrection plants
are late embryogenesis abundant proteins (LEAPs). This heterogeneous group of anhydrobiosis-related
intrinsically disordered proteins forms mostly random conformation when fully hydrated, turning into
compact α-helices during desiccation [2]. Based on in vitro studies, LEAPs can be involved in water binding,
ion sequestration, stabilization of both membrane and enzymes during freezing or drying, while by forming
intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells
during desiccation.
Here, we identify 164 members of LEA gene family in endemic and relict resurrection species Ramonda serbica
by integrating previously done de novo transcriptome and homologues protein motifs. Identified LEAPs
were classification into six groups according to Protein family (PFAM) database and the most populated
group was LEA4 containing 47% of total identified LEAPs. By using four secondary structure predictors, we
showed that this group exhibited a high propensity to form amphipathic α-helices (81% of total sequence
length is predicted to form α-helical structure). This implies that charged residues might be exposed to the
solvent, while hydrophobic amino acids might interact with lipid bilayers or with other target proteins in
the cell. In addition, as predicted by several bioinformatics tools, more than 70% of identified LEAPs were
found to be highly disordered. Structural characterization of LEAPs is a key to understand their function
and regulation of their intrinsic structural disorder-to-order transition during desiccation. These findings
will promote transformative advancements in various fields, such as the development of new strategies in
neurodegenerative disorders, cell preservation technology and the improvement of crop drought tolerance.Book of Abstracts: Belgrade BioInformatics Conference 202
De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins
Get PDFResurrection plant Ramonda serbica Panc. survives
desiccation for a long period and fully recovers
metabolic functions already within one day upon
watering. Desiccation (extreme dehydration) induces protein
unfolding and aggregation, destabilization or loss of
cellular membrane integrity. Besides, desiccation
provokes the accelerated generation of reactive
oxygen species.
HL
DL
Aim: To identify Late Embryogenesis Abundant Proteins
(LEAPs) that contribute to desiccation tolerance in
R. serbica by comparative transcriptomics of
hydrated (HL) and desiccated leaves (DL)
Analysis of the association of the CYP2C19 variants with the effects of clopidogrel therapy in patients underwent to carotid endarterectomy
Get PDFIntroduction. Despite proven clinical effect of clopidogrel, a
considerable number of patients do not have an adequate
response to this type of medication. Problems during therapy occur in the form of resistance, which is present in 11%
of patients or bleeding that occurs in about 9% of patients.
Pharmacogenomics studies demonstrated that variants of
the CYP2C19 gene significantly influence the interindividual heterogenity of the clopidogrel response. The American
Heart Association, US Food and Drug Administration and the
European Medicines Agency, cite the CYP2C19 gene as a significant factor which influences patients response to clopidogrel. Further, it has been shown that the contribution of
genetic and non-genetic factors affecting clopidogrel therapy
may vary between patients from different populations, which
justifies conducting population-specific studies.
The aim. The aim of our study was to examine the significance
of the CYP2C19*2 and the CYP2C19*17 variants in the individual response to clopidogrel, in Serbian patients.
Methods. The study involved 108 patients with carotid artery
stenosis who underwent endarterectomy and received clopidogrel for at least 30 days after the intervention. Also, 120
patients with myocardial infarction receiving clopidogrel after PCI (percutaneous coronary intervention) were included.
Commercial tests were used for standard laboratory testing.
Allelic discrimination was performed after Sanger sequencing.
Results were analysed using statistical tests.
Results. In patients undergoing endarterectomy CYP2C19*2
carriers had a higher risk for being clopidogrel low-responder
in comparison with non-carriers (1.250, 95% CI 1.695–1.658,
P<0.01). In the group of patients undergoing PCI, risk for reinfarction in patients who were carriers of CYP2C19*2 was higher
compared to patients with wild type genotype (OR 5.355, 95%
CI 0.955-31.08; P=0.038). Variant CYP2C19*17 showed no association with variations in response to clopidogrel therapy.
Conclusion. The CYP2C19*2 variant shows significant association with a poor response to clopidogrel and it should be
considered when planning therapy.XXIII Congress of the Cardiology society of Serbia, October 21-23, 2021, Belgrade, Serbia
Factors of the BRCA2-mediated Homologous Recombination: Uncovering New Players, Their Interplay, and Contribution to Genome Integrity and Stress Response (GENOVA)
Get PDFThis proposal addresses the maintenance of genome integrity which is one of the most
fundamental cellular processes that ensures genome stability by employing molecular factors
that are mostly conserved in all domains of life. Therefore, it is not surprising that inherited or
newly acquired defects within the mechanisms of genome protection are linked to life-
threatening conditions such as cancer, as well as to ageing and other disorders. Namely, DNA is
under constant challenge from both endogenous as well as exogenous genotoxins causing its
damage so that among the types of lesions, DNA double strand breaks are the most deleterious
ones. They are corrected with supreme accuracy via homologous recombination (HR)-mediated
repair. Our central contention is that a further breakthrough in understanding of the molecular
mechanisms of HR requires identification of new components of the molecular machinery
underlying the process.
Thus, focusing on discovery of novel HR-factors and on elucidating their collaboration with
known components of the HR machinery we propose here an inter-disciplinary approach using
an extremely radiation resistant (yeast-like) fungus Ustilago maydis as a model system. As a
microbial BRCA2 organism, it offers the advantage of simplicity in experimental design, plus a
perspective showing some surprising features in common with the human HR system.
Towards this goal we have already identified 2 novel cellular factors functionally related to HR,
supporting the validity of our approach and feasibility of the genetic screen we designed. We
will further isolate a collection of factors that regulate/modulate HR and characterize them in
considerable details using state-of-the-art molecular tools in experimentation. Given the
importance of DNA repair mechanisms for stress-protection in plants the investigations will be
extended to Arabidopsis thaliana. Collectively, this work promises new insight into the process
of HR-mediated DNA repair. The steps defined in the process could be points of intervention
not only for guiding new therapeutic strategies but also for crop breeding programs. Indeed,
detailed biological knowledge of the fundamental cellular processes, as it is nowadays painfully
evident, represents our best hope for efficient medical and other informed interventions.Principal Investigator: Dr Mira Milisavljevic, IMGGEDuration period: 2021-202
Precision medicine and COVID-19: importance of host genome profiling and bioinformatics
Get PDFClinical picture and course of the disease in patients with COVID-19 vary from asymptomatic to lethal.
Precision medicine could discover the cause of this phenomenon by analyzing the individual genomic
profiles of the patients.
We aimed to understand a host genetic component of COVID-19 focusing on variants in genes encoding
proteases and genes involved in innate immunity, important for susceptibility and resistance to SARS-CoV-2
infection. Also, we wanted to identify phamracogenes and pharmacogenomics markers associated with
drugs used for COVID-19 treatment in different clinical protocols in Serbia, and to compare the results
with various world populations.
Genotype information of 143 individuals of Serbian origin was extracted from database previously obtained
using TruSight One Gene Panel (Illumina). Variants in genes encoding proteases and genes involved in innate
immunity were identified and analysed in silico (PolyPhen-2, SIFT, MutPred2, Swiss-Pdb Viewer) to predict
the impact of the variants to the structure and/or function of proteins. Genotype data from Serbian population
was compared with European and 4 super-populations (total 2504 subjects). Data were extracted from
VCF files of Phase 3 variant calls of the 1000 Genomes Project (1kGP) sample collection via Ensembl Data
Slicer Tool. The level of population genetic variability at each selected loci was examined using the maximal
global differences in minor allele frequencies (delta MAF) calculated by subtracting the maximum and the
minimum MAF across analyzed population groups, and Fst statistics. Fisher exact test was used to measure
differences in genotypes distributions between Serbian and 1kGP populations, applying Bonferoni correction.
R software was utilized for genotype data manipulation and statistical calculations.
Based on high alternative allele frequencies in population and the functional effect of the variants, we identified
variants in genes encoding proteases and involved in the innate immunity that might be relevant for the
host response to SARS-CoV-2 infection. The potential pharmacogenomics markers in pharmacogenes relevant
for COVID-19 treatment were also identified. Bioinformatics tools integrated into precision medicine
could contribute to better understanding of inter-individual and population-specific genetic susceptibility and
resistance to the SARS-CoV-2 infection, therapy response inconsistencies, and could be applied to improve
the outcome of the COVID-19 patients.Book of Abstracts: Belgrade BioInformatics Conference 202
Bioinformatics pipeline for genotyping and genotype - phenotype association study in maize (Zea mays L.)
Get PDFMultidisciplinary research is today commonly used in plant breeding for improving important agronomic
traits. High throughput genotyping technologies and genotype – phenotype association studies as widely
used for improving breeding programs, depend on bioinformatics analysis for extracting information from
the gathered data. In this research, among plethora of widely used bioinformatics approaches, the custom
made one was chosen, based on the current recommendations in the field.
The material includes a set of 46 maize inbred lines commonly used in maize breeding programs. Phenotyping
was done for thirteen important quantitative agronomic traits in 8 environments during two years (2018
and 2019). For the purpose of genotyping, plants of all inbred lines were grown under optimal conditions
and sampled after completing the V4 growth stage. Total RNA was isolated from the third leaf of three plants
per inbred line and used for cDNA preparation by Illumina TruSeq Stranded RNA LT kit. Pair-end RNA-Seq
based on Next Generation Sequencing methodology was performed on MiSeq Illumina sequencer using
MiSeq Reagent kit, v2 (2 x 150bp). Raw sequencing data of maize leaves’ transcriptionally active genome
regions at the moment of sampling were used for identification of single nucleotide polymorphisms (SNPs)
in each of 46 inbred lines.
Bioinformatics pipeline for data manipulation and analysis was custom made and included FastQC (for
quality control (QC) of raw data), Trimmomatic tool v0.32 (for adapter and contaminants removal, as well
as for the removal of regions with QC below 30), TopHat (insert size 130, standard deviation 50, maximum
intron size 100.000 – for mapping filtered reads onto the B73 maize reference genome v3.0), Cufflinks
v2.2.1 (for reads assembly), Cuffmerge (for the final transcriptome assembly) and an intersection output of
two independent SNPs calling tools FreeBayes and BCFtools (to minimize false positive results). With the
aim to find SNP markers which show strongly statistically supported relationship with favorable values of
investigated quantitative traits, genotype - phenotype association analysis was conducted. It was performed
using two approaches – one relying on the TASSEL software, widely used in agronomics and the other based
on machine learning software like WEKA, rarely used in agronomics. The results of two approaches were
compared and discussed.Book of Abstracts: Belgrade BioInformatics Conference 202
alpha-Tocopherol Acetate Attenuates Mitochondrial Oxygen Consumption and Maintains Primitive Cells within Mesenchymal Stromal Cell Population
Get PDFWe present here the data showing, in standard cultures exposed to atmospheric O-2 concentration, that alpha-tocopherol acetate (alpha-TOA) has a positive impact on primitive cells inside mesenchymal stromal cell (MstroC) population, by maintaining their proliferative capacity. alpha-TOA decreases the O-2 consumption rate of MStroC probably by impacting respiratory chain complex II activity. This action, however, is not associated with a compensatory increase in glycolysis activity, in spite of the fact that the degradation of HIF-1 alpha was decreased in presence of alpha-TOA. This is in line with a moderate enhancement of mtROS upon alpha-TOA treatment. However, the absence of glycolysis stimulation implies the inactivity of HIF-1 alpha which might - if it were active - be related to the maintenance of stemness. It should be stressed that alpha-TOA might act directly on the gene expression as well as the mtROS themselves, which remains to be elucidated.This is the peer reviewed version of the paper: Loncarić, D., Rodriguez, L., Debeissat, C., Touya, N., Labat, V., Villacreces, A., Bouzier-Sore, A.-K., Pasquet, J.-M., de la Grange, P. B., Vlaski-Lafarge, M., Pavlović, S., & Ivanović, Z. (2021). Alpha-Tocopherol Acetate Attenuates Mitochondrial Oxygen Consumption and Maintains Primitive Cells within Mesenchymal Stromal Cell Population. Stem Cell Reviews and Reports, 17(4), 1390–1405.[ https://doi.org/10.1007/s12015-020-10111-9]Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1491
Design, synthesis, antibacterial activity evaluation and molecular modeling studies of new sulfonamides containing a sulfathiazole moiety
No full textSulfonamides represent the oldest synthetic antibacterial agents; however, their central position in controlling bacterial diseases has been seriously damaged by the development of widespread resistance. Herein, we revisited sulfathiazole, a commercial member of antibacterial sulfa drugs, intending to overcome sulfonamide resistance and identify new drug candidates through molecular modifications. We synthesized twelve sulfonamides (SA1-SA12) by replacing the amino group on the phenyl ring with various substituents and introducing a thiophene ring on the core scaffold of sulfathiazole. The obtained compounds and additionally two commercial sulfonamides, sulfathiazole and sulfadiazine, were extensively screened for their antimicrobial activities. The results indicated that new sulfonamides, unlike traditional ones, were selectively effective against various Staphylococcus aureus strains. Introducing a bulky lipophilic substituent at the para position of the phenyl ring significantly increased the antibacterial activities of the compounds against Staphylococcus aureus. The compounds demonstrating favourable selectivity indices were further evaluated for their membrane potential perturbation and DNA interaction properties. The obtained data showed that these are not supporting mechanisms for the antibacterial activities of the modified sulfathiazole derivatives. In order to rationalize the activity of the three most active compounds, SA7, SA11 and SA12, against S. aureus ATCC 25923, their binding hypotheses within the catalytic site of Staphylococcus aureus dihydropteroate synthase, the validated target enzyme of sulfonamides, were generated via molecular docking and further dissected using molecular dynamics simulations and dynamic 3D pharmacophores (dynophores)
Extracellular vesicles subtypes in sera of COVID-19 patients as indicators of immune dysregulation and disease severity
No full textCharacterization of antibiotic resistance in Escherichia coli isolates from Black-headed gulls (Larus ridibundus) present in the city of Novi Sad, Serbia
No full textDespite common resistance to antimicrobials in Escherichia coli isolates from farm animals in Serbia, no data are currently accessible on its occurrence in E. coli isolated from gulls. Therefore, 67 cloacal swabs and 70 fecal samples from black-headed gulls were investigated for the presence of antibiotic-resistant E. coli isolates. Ninety-nine isolates were obtained during the study. Resistotyping and resistance gene typing has shown that 44 isolates harbor resistance to one or more antibiotics. Multidrug resistance was detected in 24 E. coli isolates. Ten isolates were resistant to extended-spectrum cephalosporin antibiotics and were studied in detail including virulence gene typing, phylogenetic and multilocus sequence typing, and mating. These ten isolates belonged to phylogenetic groups B2 (five isolates), D (four isolates) and B1 (one isolate). Five different sequence types (ST38, ST2307, ST224, ST162 and ST34) were detected in E. coli isolates with AmpC phenotype and genotype. One isolate carried the Inc I2/FIB replicon type plasmid with the bla(CTX-M-1) gene. Nine isolates had bla(CMY-2) genes, which were detected on conjugative plasmids in seven isolates. The virulence genes hly, iroN, iss, ompT and cvaC were detected in one transconjugant. Ten isolates were found to be resistant to ciprofloxacin, whose MIC ranged from 4 to 32 mg/L. Genotyping revealed single or double mutations in the quinolone resistance determining region (QRDR) of the gyrA or gyrA, parC and parE genes, respectively. So, Black-headed gulls from Serbia may be colonized by multidrug-resistant E. coli, some of which are resistant to critically important antibiotics in medicine