imagine (Institute of molecular genetics and genetic engineering)
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New trends in the diagnosis of the nature of thyroid nodules
No full textNodusi štitaste žlezde su lokalizovane morfološke promene koje se mogu klasifikovati kao benigne ili
maligne. Koloidni nodusi i folikulski adenomi predstavljaju uobičajene benigne varijante nodusa štitaste
žlezde, dok maligni nodusi uključuju papilarne, folikulske i medularne tumore. Istraživanja su pokazala da
se učestalost malignih nodusa, posebno papilarnog tiroidnog tumora, značajno povećala u poslednje
četiri decenije. Razlikovanje benignih od malignih nodusa štitaste žlezde predstavlja ključnu dijagnostičku
dilemu u medicinskoj praksi, usled nedostatka jedinstvenog dijagnostičkog testa ili biomarkera koji bi
pouzdano ukazao na prisustvo malignih nodusa. Genetičke modifikacije, kao što su mutacije u genima
BRAF, RET i TERT, igraju značajnu ulogu u patogenezi tumora štitaste žlezde, dok azot-monoksid (NO) i
mikroRNK (miRNK) predstavljaju obećavajuće biomarkere u dijagnostici malignih nodusa. U zavisnosti od
koncentracije u ciljnom tkivu, NO može imati i antitumorski i tumorogeni uticaj, dok su miRNK, kao što su
miR-146b, miR-221, miR-222 i miR-375, identifikovane kao indikatori maligniteta i prognostički biomarkeri
malignih nodusa. U ovom preglednom radu, sumirana su najnovija saznanja iz literature koja se odnose
na genetičke modifikacije, signalne puteve i uloge NO i miRNK u malignim oboljenjima štitaste žlezde, kao
i novi trendovi u dijagnostici prirode nodusa štitaste žlezde.Thyroid nodules are localized morphological changes that can be classified as either benign or malignant.
Colloid nodules and follicular adenomas represent common benign variants of thyroid nodules, while
malignant nodules include papillary, follicular, and medullary tumours. Research has shown that the
incidence of malignant nodules, particularly papillary thyroid carcinoma (PTC), has significantly increased
over the past four decades. Distinguishing between benign and malignant thyroid nodules presents a
critical diagnostic challenge in medical practice, due to the lack of a single diagnostic test or biomarker that
could reliably indicate the presence of malignancy. Genetic modifications, such as mutations in BRAF, RET,
and TERT genes, play a significant role in the pathogenesis of thyroid tumours, while nitric oxide (NO) and
microRNAs (miRNAs) are emerging as promising biomarkers for malignant thyroid nodules. NO can exhibit
anti-tumour and tumorigenic activities depending on its concentration in the target tissue. In contrast,
miRNAs such as miR-146b, miR-221, miR-222, and miR-375 have been identified as indicators of malignancy
and prognostic biomarkers for malignant thyroid nodules. In this review, we summarize recent literature
findings related to genetic modifications, signaling pathways, and the roles of NO and miRNAs in malignant
thyroid disorders, as well as new trends in the diagnosis of the nature of thyroid nodules
Influence of DIO1 i DIO2 gene variants on thyroid gland function
No full textTiroidna žlezda je endokrina žlezda čoveka, koja luči hormone tiroksin (T4) i trijodtironin (T3). Mada se T3
luči u značajno manjoj količini, T3 ima veću biološku aktivnost nego T4 i većinom se stvara dejodinacijom
T4 pomoću jodotironin dejodinaza tipa 1 ili 2 u perifernim tkivima. Metabolizam tiroidnih hormona, kojim
upravljaju enzimi dejonidaze, kodirani genima DIO1 i DIO2, igra ključnu ulogu u brojnim fiziološkim
procesima. Varijacije u ovim genima mogu uticati na nivoe tiroidnih hormona i doprineti patogenezi
velikog broja poremećaja.The thyroid gland is human endocrine gland, secreting the hormones thyroxine (T4) and triiodothyronine
(T3). Although T3 is secreted in significantly less quantity, T3 has greater biological activity than T4 and is
mostly created by deiodination of T4 by iodothyronine deiodinases type 1 or 2 in peripheral tissues. The
metabolism of thyroid hormones, regulated by deiodinase enzymes, encoded by the DIO1 and DIO2 genes,
plays a key role in numerous physiological processes. Variations in these genes can affect thyroid hormone
levels and contribute to the pathogenesis of a number of disorders
Alternative transcription initiation of the SMAD4 gene in colorectal cancer
No full textFenomen aberantne upotrebe alternativnih promotora i ekspresije tumor-specifičnih
transkripcionih izoformi uočen je u brojnim tipovima karcinoma. Cilj ovog istraživanja bio je
funkcionalna analiza alternativnih promotora tumor-supresora SMAD4, čiji proteinski produkt
ostvaruje važnu ulogu u kolorektalnom karcinomu (KRK), kao i analiza transkripata čija se
ekspresija inicira sa alternativnih promotora radi pronalaženja potencijalnih biomarkera ili
terapeutskih targeta. Transkriptomske, proteomske, in silico i in vitro funkcionalne analize
primenjene su kako bi se ispitao profil ekspresije i uloge SMAD4 transkrpata, kao i funkcije
alternativnih SMAD4 promotora u setu humanih ćelijskih linija i kliničkih uzoraka pacijenata.
Glavni transkript gena SMAD4, SMAD4-201, zastupljen je sa varijabilnim udelom. Transkript
SMAD4-209 predominantno je zastupljen u neizmenjenom tkivu, a predikciona analiza
pokazala je vezivanje onkogenih miRNK i RNK-vezujućih proteina koji regulišu splajsovanje.
SMAD4-213 zastupljen je prevashodno u tumoru, a predikciona analiza pokazala je ulogu u
kodiranju okrnjenog proteina koji narušava TGF-b signalizaciju i regulatornu ulogu
vezivanjem miRNK asociranih sa KRK. Alternativni promotori C i D pokazali su suprotan
profil aktivnosti u tumorskom u odnosu na okolno tkivo, a diferencijalno vezani regulatorni
proteini transkripcije predstavljaju jedan od mehanizama odgovornih za datu aktivnost
promotora. Predložena hipoteza alternativne regulacije ekspresije SMAD4 u KRK
podrazumeva postojanje parova promotora i transkripata koji pokazuju dieferencijalni obrazac
aktivnosti, odnosno ekspresije u malignim u odnosu na neizmenjene ćelije: promotor C i sa
njega prepisani SMAD4-209 dominantni su u neizmenjenim ćelijama, a promotor D i sa njega
prepisani SMAD4-213 dominanti su u malignim ćelijama. Ovo istraživanje potvrdilo je ulogu
alternativne inicijacije transkripcije gena SMAD4 u KRK. Buduća istraživanja trebalo bi da se
bave terapeutskim potencijalima administracije i utišavanja SMAD4-209 i SMAD4-213,
respektivno, u tumorskom tkivu.The phenomenon of aberrant use of alternative promoters and the expression of tumor-
specific transcriptional isoforms has been observed in numerous types of cancer. The aim of
this study was to conduct a functional analysis of alternative promoters of the tumor suppressor
SMAD4, whose role is well known in colorectal cancer, as well as the analysis of transcripts
transcribed from these promoters to identify potential biomarkers or therapeutic targets.
Transcriptomic, proteomic, in silico, and in vitro functional analyses were applied to examine
the expression profile and roles of SMAD4 transcripts, as well as the functions of alternative
SMAD4 promoters in a set of human cell lines and clinical samples. Although SMAD4-201 is
the main transcript of the SMAD4 gene, it is present at varying levels in tissues and cell lines.
The SMAD4-209 transcript is predominantly present in non-tumor tissue and prediction
analysis has shown binding of oncogenic miRNAs and RNA-binding proteins involved in
splicing regulation. SMAD4-213 is primarily present in tumors, and prediction analysis has
indicated its role in coding a truncated protein that disrupts TGF-β signaling and plays a
regulatory role by binding miRNAs associated with colorectal cancer. Alternative promoters C
and D showed opposite activity profiles in tumor compared to non-tumor environments, and
differentially bound transcriptional regulators are one of the mechanisms responsible for this
promoter activity. The proposed hypothesis of alternative regulation of SMAD4 expression in
colorectal cancer suggests the existence of promoter and transcript pairs that show differential
patterns of activity or expression in malignant versus non-malignant cells: promoter C and its
respective transcript SMAD4-209 are dominant in non-malignant cells, while promoter D and
its respective transcript SMAD4-213 are dominant in malignant cells. This study confirmed
the role of alternative initiation of SMAD4 gene transcription in colorectal cancer. Future
research should address the therapeutic potential of SMAD4-209 and SMAD4-213 by their
administration and silencing in tumor tissue, respectively.Datum odbrane
Beneficial Effects of Probiotic Lactobacillus paraplantarum BGCG11 on Pancreatic and Duodenum Function in Diabetic Rats
No full textDiabetes mellitus, as a chronic metabolic disorder, significantly impacts the pancreas and among other organs, affects duodenal function. Emerging evidence suggests that probiotics can exert beneficial effects on gut health and metabolism. In our previous research, we evaluated the probiotic Lactobacillus paraplantarum BGCG11 primarily for its protective properties against diabetic rats’ damaged liver and kidneys. In this work, we further examined the effects of probiotic strain BGCG11 on the function of the duodenum and pancreas in diabetic rats. We explored the potential mechanisms underlying the probiotic’s effects, focusing on general indicators of diabetes, the architecture and morphology of pancreatic islets, duodenal integrity (measuring the transfer of fluid and serum zonulin level), and the modulation of gut microbiota composition. Our findings reveal the protective and regulatory roles of L. paraplantarum BGCG11 in mitigating diabetes-induced pancreatic and duodenal dysfunction regardless of its application time (pre- or post-treatment), highlighting its therapeutic potential in managing diabetes-related gastrointestinal complications
Impact of Electronic Cigarette Components on Lung Cell Proteome: A High-Resolution Mass Spectrometry Analysis
No full textElectronic cigarettes (e-cigarettes) are a relatively new tobacco product, and still there
is much to be determined about the lasting consequences on health of these products.
Despite containing mainly non-harmful substances like polyethylene glycol and glycerol,
e-cigarettes can also include nicotine and various flavorings, which may contribute to
their potential harm.
Our aim was to determine the effects of different components of e-cigarettes on the
protein composition of lung cells.
We have performed the comprehensive proteome analysis of epithelial lung cells (BEAS
2B) exposed to e-cigarette vapor condensate. BEAS 2B cells were treated for 24 h with
sub-cytotoxic concentration of e-cigarette vapor condensate, made from different
e-cigarette liquids - with and without nicotine and with or without flavorings.
The proteome analysis was performed via high resolution mass spectrometry based
proteomics (Orbitrap Exploris 240, Thermo Scientific, USA). BEAS 2B proteins were
identified with the PEAKS X Pro platform (Bioinformatics Solution Inc., Ontario, Canada)
against a UniProtKB database of Homo sapiens and contamination database as common
Repository of Adventitious Protein entries. All qualitative gene products enrichment
analyses have been done with FunRich Software 1.3.1.
We have found reduction in the number of proteins in exposed cells. The most affected
cellular components were extracellular exosomes, mitochondria and ribosomes. Presence
of nicotine and flavoring together had most effect on the following biological processes:
protein translation and proteasome-mediated ubiquitin-dependent protein catabolic
processes, tricarboxylic acid cycle and cellular response to interelukin-7.
Our study showed that exposure to e-cigarettes affects critical cellular processes and
structures, which could have implications for cell function and overall lung health. Further
analysis on pathways identified will help us better understand health risks associated
with e-cigarette vaping.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Fine-tuning RNA-seq alignment parameters for Danio rerio genome
No full textIn RNA-seq analysis, mapping raw reads to the reference genome using splice-aware
aligners is a matter of personal preference. However, the distribution of reads among
different mapping metrics strongly depends on appropriate parameters selection.
In this study we conducted a thorough analysis of parameter impact on alignment metrics
using STAR aligner in 12 samples of PE150 RNA-seq data from zebrafish. When setting
parameters we considered factors such as insert size distribution of pre-processed library
data by fastp and the unique features of the reference genome (e.g., gene density and
intron size distribution).
Average number of input reads per sample were 25097912. Adjusting parameters led
to significant improvements in mapping metrics, notably an increase in the percentage
of uniquely mapped reads from 86.08% to 94.19%. A minor rise was observed in the
percentage of reads mapped to multiple loci, with figures of 4.72% and 3.62% respectively.
The quantification of reads by genomic origin has revealed an increase in uniquely mapped
reads allocated to exonic regions , on average by 2,034,563 reads per sample.
In addition, we’ve compared default and adjusted metrics of multi-sample 2-pass
mapping, which are important when analysing differential transcript usage. In the 2-pass
mode, we employed 1st pass junction files that were purged of probable false positives,
such as junctions within the mitochondrial genome or those crossed by multi-mapping
reads. Qualimap-rna analysis of 2-pass mapping revealed an increase in the usage of
novel splice sites, as expected. However, default parameters showed higher rates of
multimapping, noFeature, or ambiguous reads compared to adjusted parameters.
Running alignment with default options generally performs well initially. However, proper
parameter selection tailored to the characteristics of the raw library data and reference
genome significantly improves alignment metrics, especially for model organisms like
zebrafish.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Identification of Synonymous Genetic Variants Associated with Idiopathic Thrombosis using whole exome sequencing
No full textThe etiology of substantial number of thrombosis cases remains undetermined. Sequencing
and comprehensive analysis of the entire exome in patients with idiopathic thrombosis
enable the identification of novel gene variants potentially significant for disease onset and
the discovery of previously unknown molecular mechanisms underlying this multifactorial
condition.
This study aimed to investigate synonymous genetic variants potentially linked to
thrombosis. While previous studies predominantly focused on missense and stop gain
variants, the role of synonymous variants in thrombosis susceptibility remains relatively
overlooked.
The study included 50 subjects: 17 patients with recurrent idiopathic thrombosis and 33
controls from the general population. Whole exome sequencing (WES) was performed
according to protocol of the Beijing Genomics Institute and subsequent FASTQ files were
processed to obtain Variant Call Format (VCF) files with annotations. While the entire
exome was examined, a panel of 55 genes was selected for more focused analysis. The
data were filtered to extract only synonymous variants within the genes in this panel.
Statistical analyses were performed by creating contingency table counts in R and by
applying weights and utilizing chi-square or Fisher’s exact tests in Python. A p value of p
< 0.05 was defined as statistically significant.
A total of 15 synonymous variants exhibiting statistical significance in the idiopathic
thrombosis group were identified. Variants were detected in PLG, PROC, ABO, KNG1,
ADAMTS13, ACE, HIVEP1 and F2 genes.
In conclusion, although the study exhibits considerable limitations due to the small
sample size, the identification of significant synonymous variants underscored a potential
association between thrombosis and these genetic determinants. Further studies with
larger cohorts are imperative to validate and expand upon these initial findings.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Genome-wide association study identified genetic signal in cystatin genes associated with Long COVID-19
No full textacute phase of a SARS-CoV-2 infection. It can affect individuals who had both severe and mild
cases of COVID-19. The genetic predisposition to this condition remains largely unexplored.
This study aimed to identify genomic loci associated with Long COVID-19. The study included
92 patients with confirmed infection with SARS-CoV-2 during the delta variant wave and
treated in hospital settings. These patients were monitored for up to six months after the
acute infection. All patients were genotyped using the Illumina Infinium global screening
array, which covers over 700,000 genomic variants. Imputation was employed to expand
the number of variants to 12,001,939 using the 1kGP Phase 3 human reference panels.
We conducted a genome-wide association analysis using an in-house built pipeline on the
Cancer Genomic Cloud platform.
Our results identified association signals in the 20p11.21 genomic locus, with the lead variant
being rs1275745396 (p = 6.181x10-8), located within cystatin genes (CST3, CST4, CST1).
The cystatin family consists of cysteine protease inhibitors, involved in various physiological
processes. Previous literature has linked serum and saliva cystatin levels to COVID-19 severity
and outcomes. Moreover, cystatin’s role in taste perception has been recognized previously,
which is in line with the well-known perturbations in sensory perception caused by COVID-19.
This study is the first to find a genetic association between cystatin genes and Long COVID-19.
Further research is necessary to elucidate the role of the cystatin protein family and the
biological processes underlying Long COVID-19. A deeper understanding of Long COVID-19
can inform the development of preventive and therapeutic strategies.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Molecular genetic basis of childhood epilepsy in Serbia: utility of clinical and whole exome sequencing
No full textChildhood epilepsies are caused by heterogeneous underlying disorders where
approximately 40% of the origins of epilepsy can be attributed to genetic factors. The
application of next-generation sequencing (NGS) has revolutionized molecular diagnostics
and has enabled identification of disease-causing genes and variants in epilepsies.
In our study, 55 children with epilepsy of unknown etiology were analyzed combining
clinical-exome (CES) and whole-exome sequencing (WES). Novel variants were
characterized using various in silico algorithms for pathogenicity and structure prediction.
Molecular genetic cause of epilepsy was identified in 28 patients and the overall diagnostic
success rate was 50.9%. We identified variants in 22 different genes associated with
epilepsy that correlate well with the described phenotype. SCN1A gene variants were
found in 5 unrelated patients, while ALDH7A1 and KCNQ2 gene variants were found twice.
In the other 19 genes, variants were found only in a single patient. This includes genes:
ASH1L, CSNK2B RHOBTB2 and SLC13A5, which have only recently been associated with
epilepsy. Almost half of diagnosed patients (46.4%) carried novel variants. Interestingly,
identification of variants in ALDH7A1, KCNQ2, PNPO, SCN1A and SCN2A gene directed
therapy decision of 11 children from our study, including four children who all carry novel
SCN1A genetic variants.
Our study emphasizes the importance of NGS in diagnosing childhood epilepsy. With an
increasing number of genes associated with epilepsy, comprehensive analysis using CES
and WES is crucial for high diagnostic success. Given the expansion of molecular-based
approaches, each newly identified genetic variant could become a potential therapeutic target.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
BRIDGing the research and INnovation Gap for Rare Diseases in Europe by upgrading excellence of IMGGE (BRIDGING-RD)
No full textThere are over 330 million people living on the planet affected by one of over 6,000 identified
genetic rare diseases (RD). They urgently need timely diagnosis and development of specific
treatments. Although Europe leads the way in RD research, there is a clear gap in research and
innovation (R&I) between countries. To bridge this gap, networking and knowledge sharing
between IMGGE (Institute of Molecular Genetics and Genetic Engineering, Serbia) and 3 world-
class counterparts at EU level - KI (Karolinska Institute, Sweden), CNAG (National Center for
Genomic Analysis, Spain) and UAM (Autonomous University of Madrid, Spain) is envisioned.
BRIDGING-RD’s objectives are: (1) upgrade the IMGGE RD Biobank to reach full interoperability
of genetic and phenotypic data in order to increase participation in transnational research and
innovation projects related to human health; (2) upgrade bioinformatics pipelines specific to RD
to increase the rate of solved RD cases in IMGGE’s RD Biobank; (3) upgrade capacity for modelling
of metabolic diseases as well as capacity to test small molecule drugs, in order to increase the
number of translational studies; (4) upgrade research support offices in order to increase success
in obtaining funds from research and/or innovation agencies, industry, foundations etc.
Objectives will be achieved through staff exchanges, expert visits, trainings or workshops,
conference attendance, joint summer schools and dissemination/outreach activities. Crucially,
an exploratory R&I project, engaging all partners will focus on identifying an innovative drug,
pharmacochaperone, for a selected metabolic RD. BRIDGING-RD will have impact by significantly
improving the scientific excellence and innovation capacity of IMGGE, raising its research
reputation and research management skills. It will also impact networking and mobility of
qualified scientists in the RD field, thus boosting creativity and ultimately enhancing economic
growth at the European level.Principal Investigator: Dr. Maja Stojiljković, IMGGEDuration period: 2024-202