imagine (Institute of molecular genetics and genetic engineering)
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Characterizing Somatic Mutation Clusters in Cancers Enriched with APOBEC Mutagenesis
No full textThe APOBEC family of cytidine deaminases plays an active role in the human immune
system, combating viruses and transposable elements. These enzymes convert cytosine
to uracil, which often leads to C->T or C->G mutations within the specific nucleotide
contexts (TCN). Clusters of such mutations have been identified in the genomes of various
cancer types, including bladder, breast, cervical, head and neck, and lung cancers. Previous
studies have shown that these APOBEC-induced mutation clusters are not uniformly
distributed across the genome. To analyze their distribution, we developed a method for
detecting APOBEC-induced clusters in cancer genomes.
The method presented was developed using a subset of cancer samples from the
PCAWG project, which exhibited a strong enrichment of APOBEC-signature mutations.
Mutations were iteratively merged into clusters using a distance threshold determined
through chromosome-specific simulations that matched the actual number of mutations.
We constructed distributions of distances between neighboring mutations and derived
distance thresholds for several significance levels (5%, 1%, 0.1%). We also separately
estimated the heterogeneity of APOBEC mutagenesis along the genome, adjusting the
distance threshold accordingly. This method was applied to the entire PCAWG dataset,
identifying traces of APOBEC mutagenesis in additional cancer types.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Impact of 3D chromatin structure on cancer mutation patterns and tissue-of-origin prediction
No full textTissue-of-origin (TOO) detection poses a challenge for effective therapy selection due to the
heterogeneity and distinct genomic and molecular characteristics of carcinoma of unknown
primary site (CUP). Machine-learning algorithms utilizing mutational landscape data from
whole-genome sequencing and normal tissue epigenetic features have shown promise in
predicting TOO. These models leverage the association between mutation densities, regional
histone modifications, and the non-uniform distribution of mutations across 1 MB genomic
regions and tumor types. However, some cancer types remain difficult to classify accurately.
To address this, we developed TOO models utilizing tissue-specific topologically associated
domains (TADs). Since TADs represent fundamental units of 3D genome architecture, we
investigated whether the TOO prediction can be improved by using TADs (TAD model) or
genes clustered based on their location in TADs (TAD gene model).
We analyzed publicly available liver cancer cohorts from the International Cancer Genome
Consortium, obtaining ChIP-seq data for six histone modifications and input controls from
the Roadmap Epigenomics project. Tissue-specific TADs were downloaded from TADBK
and the 3D Genome Browser. We used a multiple linear regression model with 10-fold
cross-validation to compute the amount of variance of aggregated mutations across
various TADs or TAD-based gene clusters explained by the epigenome of each normal
tissue. The model with the highest variance represents the TOO for a specific cancer type.
The results demonstrated consistent correct TOO prediction across all tissue-specific
TADs identified using various tools. Although the overall accuracy of TAD models did
not significantly differ from the original model developed using 1 MB region-based
predictions, TAD gene models showed a significant increase in correct TOO prediction
compared to other gene models we developed. Genes located in TADs where the number
of predicted mutations was lower than the observed number were associated with cancer
development and progression, indicating that this type of analysis can facilitate the
identification of structural units that influence carcinogenesis.
Overall, the results show that we can use the cell’s epigenome and cancer’s mutation
profile based on TADs to predict the tissue-of-origin and use the developed models to
analyze the mechanisms of cancer initiation and progression.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Supplementary information for the article: Lukić, J., Vass, N., Ardó, L., Stanivuk, J., Lengyel-Kónya, É., Golić, N., Jakabné Sándor, Z., & Ljubobratović, U. (2024). Evaluation of weaning diets for sustainable indoor largemouth bass (Micropterus salmoides) larviculture. Czech Journal of Animal Science, 69(12), 471–483. https://doi.org/10.17221/129/2024-CJAS
No full textSupplementary material for:[http://dx.doi.org/10.17221/129/2024-CJAS]Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/2576]Related to preanalyzed data: [https://imagine.imgge.bg.ac.rs/handle/123456789/2759
MORPHOLOGICAL AND FUNCTIONAL ANALYSIS OF PANCREATIC CARCINOMA CELLS IN 2D MIXED CULTURES
No full textPancreatic carcinoma is the third leading cause of cancer-related deaths, following lung and colon
carcinomas. A key characteristic is its well-developed desmoplastic stroma, which contributes to
chemoresistance and creating a tumor-promoting microenvironment. This complex stroma consists
of activated fibroblasts, immune, and endothelial cells embedded in a collagen-rich, dense
extracellular matrix. Attempts to remove the stroma with drugs have failed in clinical trials, where
patients showed equal or reduced survival compared to standard therapy. This suggests that the
stroma has a complex dual role. Our research was focused on the role of fibroblasts in the
proliferation, migration, and morphology of pancreatic carcinoma cells PANC-1 and MIA PaCa2. We used non-contact and contact cultivation methods. We found that fibroblasts, whether in coculture or through conditioning medium, had a moderate inhibitory or no effect on the viability of
PANC-1 and MIA PaCa-2 cells, and did not change the expression of the proliferation marker
Ki67. However, fibroblast conditioning medium significantly decreased colony formation and
influenced cellular morphology. Results suggest that fibroblasts are not inducers of pancreatic
carcinoma cell proliferation in 2D cultures. Pancreatic cancer cells showed greater migratory
potential in the presence of fibroblasts in wound healing and transwell assays. To better understand
the role of fibroblasts, our future research will focus on creating 3D mixed culture models to better
depict in vivo conditionsVII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024
DEVELOPMENT OF A MICROBIAL-CELL-FACTORY CONVERTING PLASTIC WASTE TO BIODEGRADABLE POLYMERS
No full textAs plastic waste accumulation continues to surge uncontrollably, a planetary crisis is unfolding
beneath our feet. Heightened concerns about the environmental impact of plastics underscore the
need for urgent eco-friendly strategies, overcoming the conventional management practices.
Despite plastics' durability and resistance to degradation [1,2], recent emphasis has shifted towards
enzymatic breakdown, the ability of enzymes to target and shatter polymer bonds, leading to
breakdown into oligo- and monomers [2–4]
. The combination of chemical and biological valorization
of these molecules generated through plastic degradation offers the potential for diverse product
production, including lubricants, fuels, and innovative polymers[5]
. Recent attention has increasingly
focused on the biological valorization of the oligo- and monomers, serving as a carbon source for
microorganisms. Engineered microorganisms, expressing plastic-degrading enzymes, have
demonstrated the ability to metabolize plastic monomers like ethylene glycol (EG), terephthalic acid
(TPA), or 6-hydroxyhexanoic acid, deriving from the degradation of polyethylene terephthalate
(PET) and polycaprolactone (PCL), respectively. The valorization process holds the potential to yield
value-added products such as polymers or chemicals [6–8]
. In the present study, we focus on the
creation of a consolidated bioprocess through the development of a microbial-cell-factory with
plastic waste as feedstock. Bacterial strains of Bacillus genus, B. subtilis BPM12 and B. subtilis RIK,
were utilized as platform. Strains were evolved for monomer (TPA and 6-hydroxyhexanoic acid)
metabolism, through adaptive laboratory evolution (ALE) technique and transformed with
recombinant plasmid, carrying expression gene of a recently discovered thermophilic polyesterase,
DmPETase [9] and phaCAB operon, for polyhydroxyalkanoates (PHAs) production. The engineered
strains were tested for recombinant expression in Terrific Broth (TB) medium. Following up, the
ability of recombinant bacteria to break down virgin polyesters (PCL and PET) as well as a mixture
of different polyesters considered as post-consumer plastic waste, was monitored, through High-
Performance Liquid Chromatography and mass loss, in liquid cultures of mineral salts medium
(MSM), with polymers as the only carbon source. Eventually, the evaluation of PHAs synthesis was
conducted utilizing Nile red staining, under fluorescent microscope or by Gas Chromatography-Mass
Spectrometry (GS-MS) analysis.Book of abstract: 14o Πανελλήνιο Επιστημονικό Συνέδριο Χημικής Μηχανικής Θεσσαλονίκη, 29-31 Μαΐου 202
Silicon Affects The Expression Of Conserved And Novel Cucumber miRNAs In Response To Copper Stres
No full textEnvironmental pollution with heavy metals such as copper (Cu) severely hinders optimal
plant growth and development. Silicon (Si) is a plant nutrient that can improve plant health
through diverse mechanisms. microRNAs (miRNAs) are recognized as effective regulators
of various processes in plants, including stress responses, however, their involvement in
Si-protective effect remains unrevealed. To clarify the molecular pathways involved in the
protective effect of Si, small RNA-seq analyses (Illumina Sequencing SE50) was performed
on cucumber plants treated with silicon (Si) alone or with a high concentration of Cu (Cu+Si).
The small RNA tags were mapped to the reference sequence by Bowtie tool, following
no-mismatch or one mismatch criterion, to analyze their expression and distribution.
Identification of known miRNAs among the mapped small RNA tags was accomplished by
miRBase22.0 database. miREvo and mirdeep2 were exploited to predict novel miRNAs by
analyzing the secondary structure, Dicer cleavage site, and minimum free energy of the
small RNA tags unannotated in the previous steps. The prediction of the target gene of
miRNA was performed by psRobot. Differential expression analysis of two treatments was
done using edgeR, with the default threshold qvalue 1.
A total of 71 miRNAs were identified in cucumber plants. Twenty miRNAs that were
significantly differentially expressed, including seven novel miRNAs were clustered to find
similar expression patterns. miR398 and miR408, which are known to target Cu-proteins
important for Cu metabolism and transport, were significantly downregulated in Cu+Si
treatment. In contrast, miR156, miR164, mir167, miR394, and miR477 were upregulated
in Cu+Si. Additionally, two novel miRNAs (Novel_15 and Novel_19) were highly expressed
and specific to Cu+Si, while, the Novel_12 miRNA was specific to Si treatment. The putative
target genes of the differentially expressed miRNAs were subjected to Gene Ontology (GO)
enrichment analysis, which revealed localization and transport as the most significant GO
terms in this study.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Aggregation of LEA proteins from Ramonda serbica: in silico vs. in vitro
No full textProteins known as Late Embryogenesis Abundant Proteins (LEAPs) are intrinsically
disordered and play a crucial role in desiccation tolerance. Resurrection plants can
withstand prolonged desiccation and fully recover their metabolic function the next day
after watering. We identified, structurally characterized in silico, and categorized LEAPs in
hydrated and desiccated leaves of the ancient resurrection plant Ramonda serbica to get
more insights into their physiological functions.
A representative LEA4 protein highly accumulated under water scarcity was
recombinantly produced and purified. It was predicted to be a highly disordered and polar
protein. Structural models of dimers obtained by AlfaFold2 served as input for molecular
simulation dynamics (MDS). Further, coarse-grain MDS using the GROMACS simulation
package showed a high propensity of LEA4 protein to form dimers mostly by hydrophobic
interactions. After the production phase, the obtained trajectory was analyzed for
chain-chain contact for both cases λ=1.06 and 1.10. The dissociation constants Kd were
calculated and were Kd ≈ 0.000075 M (75 μM) for the case λ=1.06 and Kd ≈ 0.0000758 M
(75.8 μM) for the case λ=1.10., for the protein concentration Cp=8.91*10-5 M.
The results obtained by size exclusion chromatography, dynamic light scattering, and
atomic forced microscopy confirmed that the selected LEA4 protein is aggregation-prone.
Our results are important for further elucidating protective LEAP’s mechanism during
desiccation. Structural flexibility and aggregation propensity of LEAP might be crucial in
direct, or indirect (e.g. via LLPS) buffering free cellular water and maintaining the native
conformations of biomolecules. We suggest that native or bioengineered LEAPs can be
used to improve the drought resistance of crops.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Transcriptome-wide detection of RNA cleavage sites revealed tRNA cleavage by target-activated CRISPR-Cas13a effector
No full textfeature of Type VI systems is collateral RNA damage. Specifically, the binding of a target
transcript by Cas13a, charged with cognate CRISPR RNA (crRNA), activates the Cas13a
enzyme, turning it into an active ribonuclease that mediates the cleavage of noncomplementary
RNA molecules. Previously, Cas13a-mediated collateral RNA cleavage
was observed in in vitro experiments and was described to be nonspecific. In Escherichia
coli, targeting of nonessential transcripts by heterologously expressed Leptotrichia shahii
Cas13a enzyme (LshCas13a) leads to cell growth retardation, which was proposed to be a
consequence of collateral degradation of essential cellular transcripts. However, the direct
link between collateral RNA cleavage and cell growth retardation was not established.
Specifically, the products of collateral RNA cleavage mediated by target-activated Cas13a
enzyme were not identified in living cells.
To detect RNA cleavage sites associated with collateral Cas13a activity, a specific
approach based on high-throughput RNA sequencing was developed. This approach
was successfully applied to detect RNA cleavage sites introduced by target-activated
LshCas13a enzyme in both in vivo and in vitro experiments. In E. coli cells, the targetactivated
LshCas13a enzyme cleaves tRNA molecules within anticodon loops, leading
to protein synthesis inhibition and slowing down cell growth. Additionally, LshCas13amediated
collateral tRNA cleavage indirectly activates cellular ribonucleases encoded by
Type II toxin-antitoxin systems.
Together, the results suggest that the L. shahii Type VI CRISPR-Cas system mediates the
immune response by inhibiting translation through collateral tRNA cleavage.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Transcriptome profiling of pharmacological manipulation of zebrafish tailfin regeneration
No full textZebrafish (Danio rerio) have a remarkable regenerative capacity of various tissues and
organs, making them a widely used model organism for studying tissue regeneration
and therapeutic development through pharmacological screens. The zebrafish larval fin
consists of a two-layered, infolded epithelium and its regeneration after amputation is
completed within just 3 days. We utilized this model to study the effect of human protein
alpha-1 antitrypsin (AAT) on wound healing. This study aimed to identify the effect of AAT
on transcriptional profiles during zebrafish larvae fin regeneration at 24h post amputation.
Caudal fin was amputated at 48h post fertilization and larvae were treated with 2mg/ml
AAT for 24h.
Pools of 24 larvae were collected at 24h post amputation and total RNAs were extracted
using TRIzol Reagent. RNAseq was performed using Illumina Novaseq6000. Adapter
trimming and low-quality reads were removed from raw data with fastp and reads were
aligned to Danio rerio genome assembly GRCz11 by STAR aligner. Counts of differentially
expressed transcripts were calculated at gene level with FeatureCount. Differential
expression was analysed by DESeq2 package after correction for batch effect by ComBatseq
from sva bioconductor package. The threshold for significantly differential expression
was set as the adjusted p-value < 0.01.
At 24hpa there were 185 differentially expressed genes (89 upregulated and 96
downregulated) in treated larvae compared to the untreated. Mgst2, Apobb.2, and
Prss59.2 were in the top 10 downregulated genes while Col9a1b, Col8a1a, and Matn3a
were in the top 10 upregulated genes. Enrichment analysis by Enrichr showed that key
gene onthologies in AAT treated zebrafish larvae were intermediate filament, collagen
containing extracellular matrix, cellular response to glucocorticoid and endoplasmic
reticulum lumen among others. Our data support further evaluation of AAT as a potential
promoter of wound healing.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
LEA4 protein group member from resurrection plant Ramonda serbica Panč. – production and in silico characterization
No full textRamonda serbica Panč., an ancient resurrection plant, can withstand extended periods
of desiccation and reestablish metabolic activity shortly upon watering. One of the key
players of desiccation resistance in resurrection species is the accumulation of protective
late embryogenesis abundant proteins (LEAPs). During cellular dehydration, these
intrinsically disordered proteins (IDPs) may stabilize the native structures of proteins and
membranes. Differential transcriptome and proteome analyses revealed that members of
the LEA4 protein family represent the majority of desiccation-inducible LEAPs.
The aim of this work was to in silico characterize and recombinantly produce a member of
the LEA4 protein family group – LEA_301. This protein is predicted to be highly disordered
(above 85%). Its sequence is composed of predominantly charged and polar amino acids
(39% of the sequence is charged, 12% of the sequence is lysine), with acidic pI (5.92).
According to secondary structure predictors (JPred, PsiPred, Phyer2, Sopma, and FELLS),
LEAP_301 exhibits high propensity to form amphipathic α-helix (above 95 %). This can be
important for their function during desiccation, when this protein may undergo a disorderto-
order transition, and stabilize biomolecules. To experimentally validate the secondary
structure of this protein and assume its physiological role, we produced high yield and
purity of LEA_301 by recombinant DNA technology.
Bacterial cells (Escherichia coli BL21(DE3)) were transformed with the vector with the
LEA_301-6xHis gene, and optimization of protein expression was done. Final purification
was done by immobilized metal ion affinity chromatography. The sequence was
validated by Western blot and mass spectrometry. Obtained LEA_301 will be structurally
characterized using methods for secondary structure determination such as Fouriertransform
infrared spectroscopy and circular dichroism spectroscopy and compared with
the in silico results.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024