imagine (Institute of molecular genetics and genetic engineering)
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Biomarker potential of the transcript PHF19-207 in colon cancer
Get PDFIntroduction: Recent data from a comprehensive pan-cancer
transcriptome analysis demonstrated differential activity of
two alternative PHF19 gene promoters in malignant vs.
non-malignant gut mucosa. The promoter found to be upregulated
in colon and rectal cancer gives rise to the
transcript PHF19-207. This finding has pointed to the
biomarker potential and possible tumor-promoting role of
this transcript. Our study aimed to evaluate the expression
of PHF19-207 in colon cancer, as well as to investigate its
potential function using in silico tools.EACR 2023: Innovative Cancer Science, 12-15 June 2023, Torino, Ital
CADDIE - An online knowledge base for network-based mechanism exploration and drug repurposing in oncolog
Get PDFDrug repurposing is the use of previously developed and tested pharmaceutical agents in new
application cases and lately often used as a solution to the increasing drug development costs.
Cancers are extremely heterogeneous disorders demonstrating a wide variability of drug responses
due to diverse subtypes, quickly evolving and acquiring drug resistance. Therefore, the identification
of compounds that can effectively combat a specific tumor type is crucial. Drug candidates that are
potentially effective against a specific tumor can be chosen based on the set of driver mutations
acquired by this tumor. For optimal treatment, it is important to consider targeted anti-cancer
therapies and drugs initially developed to treat non-cancerous diseases.
To overcome this hurdle, we present CADDIE (Cancer Driver Drug Interaction Explorer), a web platform
to identify oncological drug repurposing candidates. CADDIE’s biomedical knowledge base integrates
a multitude of gene-gene and drug-gene interaction datasets, detailed anticancer drug information
and cancer biology data such as cancer driver genes, mutation frequencies and gene expressions. For
the purpose of locating drug targets and candidates for drug repurposing, CADDIE makes network
medicine algorithms available to the researchers. It guides the users from the choice of seed genes
through the discovery of therapeutic targets or drug candidates. Network medicine also provides
indirect strategies that take into account other functionally relevant targets in the gene interaction
network since potential cancer driver genes may be inaccessible for direct targeting. We demonstrate
the application of CADDIE in different cancer subtypes such as sarcoma and ovarian cancer with a
detailed analysis of the found drug targets and chemical compounds. CADDIE is available online at
https://exbio.wzw.tum.de/caddie/ and as a python package at https://pypi.org/project/caddiepy/.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Cell-type-specific mechanistic drivers of progressive multiple sclerosis lesions
Get PDFUnderstanding the drivers of compartmentalized and sustained inflammation in the
brain of progressive multiple sclerosis (PMS) remains elusive. To investigate the interplay
between inter- and intra-cellular molecular mechanisms in white matter (WM) lesions, we
integrated single-cell transcriptome and chromatin accessibility data from PMS lesions
with spatial transcriptomics of chronic active lesion borders. We identified a PMS-specific
oligodendrocyte genetic program governed by the Krüppel-like factor and specificity
protein (KLF/SP) gene family, implicated in myelination and stress-induced iron uptake.
Additionally, we found high expression of transferrin gene (TF) and its receptor megalin
(LRP2) across lesion types, suggesting autocrine communication of iron uptake potential
related to iron rim lesion in smoldering MS. Additionally, inflammatory phenotype of
oligodendrocytes expressing osteopontin gene and complement were observed at
chronic active lesion edges. Inside the chronic active lesion, the axonal damage biomarker,
neurofilament light (NFL) gene expression was upregulated, and an astrocytic-neuronal
axis through fibroblast growth factor (FGF) signaling (FGFR3-FGF13) was present.
Additionally, a metabolic astrocyte phenotype at the lesion border potentially segregates
inflammation areas. We also identified two distinct B cell co-expression networks with
different locations and gene expressions, preferring different lesion types. Overall, singlecell
multi-omics enabled the identification of specific cell types with unique molecular
profiles, cell-cell communications, and spatial context, contributing to lesion fate.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Alternative splicing impacts microRNA regulation within coding regions
Get PDFMicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional
gene expression by binding to specific target sites. Approximately 95% of human multi-exon
genes can be spliced alternatively, which enables the production of functionally diverse
transcripts and proteins from a single gene. In complex diseases, such as cancer, gene but also
miRNA dysregulation plays a significant role. According to most studies miRNAs preferably
bind to 3’-untranslated regions of mRNA. However, through alternative splicing, transcripts
might lose exons harboring miRNA target sites and, hence, become unresponsive to miRNA
regulation.
To check this hypothesis, we studied the role of miRNA target sites in both coding and noncoding
regions using six cancer data sets from The Cancer Genome Atlas (TCGA). First, we predicted
miRNA target sites on mRNAs from their sequence using TarPmiR. For our analysis, we focused
on miRNAs whose expression was negatively correlated with gene expression (as evidence
for active regulation) as well as genes that were at least moderately expressed and showed
evidence of alternative splicing. We chose different subsets of transcripts to differentiate the
effects of target sites in different gene regions. To check whether alternative splicing interferes
with miRNA regulation, we trained linear regression models to predict miRNA expression from
transcript expression. Using nested models, we compared the predictive power of transcripts
with miRNA target sites to that of transcripts without target sites in the investigated gene
region. For all six cancer data sets and all subsets, models containing transcripts with target
sites predicted miRNA abundance significantly better.
We conclude that alternative splicing does interfere with miRNA regulation by skipping exons
with miRNA target sites within the coding region.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Seven miRNAs potentially included in the chilling response of maize plants in early stages of development
Get PDFMicro RNAs (miRNAs) are known regulators of various processes in plants, including growth,
development and stress responses. They achieve this through mRNA cleavage or translational
inhibition, in a process called RNA interference. Herein, their role in chilling stress response in
young maize seedlings (Zea mays L.) is examined, using high-throughput sequencing methods.
Bringing light to all aspects of chilling stress response in maize is necessary since earlier
sowing, during colder periods, is one of the most promising strategies of avoiding maize yield
loss due to effects of climate change in these areas.
Sterilized seeds of two maize genotypes (tolerant - T and sensitive - S to low temperatures)
were germinated in the dark for five days (optimal conditions), after which the 5-d old seedlings
were exposed to chilling conditions for 6h (10° C). Samples for RNA isolation and cDNA library
preparation were taken after the treatment ended, and single-end 50 bp sequencing was
performed (Illumina® Novaseq 6000). The miRNAs were then filtered, mapped, identified
and quantified using adequate bioinformatics tools; and the differential expression analysis
was carried out using the DEGseq R package. The analysis was performed on 859 miRNAs,
after previously executed TPM normalization using the MA-plot-based method with random
sampling model (MARS). The threshold for significantly differential expression was set as the
Bayesian adjusted p-value, or q-value 1.
A total of 612 were expressed differentially, but only 55 miRNAs were common for both
genotypes and at the same time differentially expressed between control and treatment
conditions – 40 novel and 15 known. Half of the common miRNAs showed the same
expression patterns in both genotypes, while the other half did not. Among them, seven known
miRNAs showed opposing expression patterns between the genotypes (zma-miR167b-3p
zma-miR167e-3p, zma-miR159c-5p, zma-miR164g-3p, zma-miR166a-5p, zma-miR398a-
3p, and zma-miR528a-3p). These miRNAs were shown to have a role in various abiotic stress
responses, including drought, waterlogging, high salts – but not chilling. While the results point
to their potential role in establishing chilling tolerance in maize seedlings, further research is
necessary to confirm it and connect the miRNAs to their potential targets.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Elongation factor P (-like) protein and polyproline motifs
Get PDFTwo or more consecutive prolines induce ribosome stalling during translation. In bacteria
the elongation factor P (EF-P) efficiently rescues the ribosome stalling and allows the
protein biosynthesis to continue. A seven amino acids long loop between beta-strands
β3/β4 is crucial for EF-P function. The residue at the tip of the loop is subjected to the
post-translational modifications: lysine is lysylated or arginine is rhamnosylated. We have
demonstrated that only those enzymes that are needed for specific post-translational
modification of the tip are coded in the bacterial genome (EpmA, EpmB and EpmC
proteins for EF-P with lysine and EarP- for those with arginine). Phylogenetic analysis
has also unveiled an invariant proline in the -2 position of the tip of the loop in EF-Ps that
utilize lysine modifications such as Escherichia coli. Bacteria with the arginine modification
like Pseudomonas putida on the contrary have selected against it. Combining these
observations with experimental evidence, we conclude that β3/β4 loop composition is
important for functionalization of EF-P by chemically distinct modifications.
Some bacterial genomes also code the elongation factor P-like (EfpL) protein that shares
the same domain architecture with EF-P and has an extended loop of eight amino acid
residues long. The evolution, sequence and the structure of EfpL protein have been
extensively characterized. Using the assay based on luminescence emission and ribosomal
profiles we have shown that EfpL can also relieve the arrest of the ribosome induced by
polyproline motifs.
We have also observed the negative correlation between the occurrence of the motif in
the proteome of Escherichia coli and its stalling strength measured in luminescence assay.
We hypothesize that motifs that cause strong ribosome stalling are disfavored in the
protein sequences during evolution due to their impact on the dynamics of translation.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Combined experimental and theoretical study of Type-II toxin-antitoxin system response to antibiotics
Get PDFBacterial Type-II toxin-antitoxin (TA) systems, including kacAT in Klebsiella pneumoniae,
respond to antibiotics. We investigated kacAT’s regulation relevant to antibiotic
persistence, which refers to the survival of antibiotic exposure by dormant bacterial cells.
Elevated toxin levels may induce dormancy. KacAT complex binds and represses the
kacAT promoter cooperatively, leading to highly non-linear negative feedback. Antibiotics
increase transcription of the kacA and kacT genes by inducing KacA degradation and
consequently reducing the KacA:KacT ratio. Our model reproduced experimental findings,
explaining increased kacAT transcription and reduced [KacA]:[KacT] ratio. Interestingly,
KacAT overexpression induces antibiotic stress tolerance, while deleting kacAT has no
effect, which our model can also explain. KacAT, therefore, cannot induce spontaneous (in
the absence of antibiotics) persister formation. Earlier theoretical models, which predicted
spontaneous persistence in Type-II TA systems, assumed the cooperative action of
multiple TA systems. Our bioinformatics analysis, however, reveals a limited occurrence
of multiple TA instances within clades and that cross-talk between clades is disfavored.
These challenges the assumption of cooperativity in TA action, possibly explaining the
absence of spontaneous persister generation in kacAT.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Data mining for long-non coding RNAs deregulated in colon cancer through analysis of Gene Expression Omnibus database
Get PDFColorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. Lack
of specific CRC symptoms is a challenge for clinicians, as the symptoms overlap with other
non-cancerous diseases, leading to 20-25% of newly diagnosed CRC patients already
having liver metastasis. Thus, discovering reliable early-disease biomarkers is of high
importance. Non-coding RNAs (ncRNAs) have been demonstrated to be involved in CRC
development and progression. Long non-coding RNAs (lncRNAs) can interact with RNA,
DNA and proteins, forming complexes that are involved in regulation of gene expression
via multiple mechanisms, affecting every stage of colon carcinogenesis and making them
top candidates for novel biomarker discovery.
The aim of our study was to conduct data mining of Gene Expression Omnibus (GEO)
database by using “colon cancer” and “ncRNA” keywords, and identify differentially
expressed lnRNAs present in different GEO datasets.
GEO database which collects submitted high-throughput gene expression data was queried
for all datasets that studied colon cancer and ncRNA. Over 60 datasets were manually
inspected in order to identify those where analysis of colon and normal tissue originating
from the same patient was done. Each dataset was analyzed by GEO2R software to
discover differentially expressed lncRNAs. LncRNAs were considered significant if they
appeared in more than one GEO dataset. Parts of lncRNAs sequences available in GEO2R
analysis results were run through BLAST in order to identify full length lncRNAs.
Five GEO datasets matched our criteria. We discovered 12 sequences that appeared in
more than one dataset and we identified them through BLAST analysis. Six sequences
originated from lncRNAs (RYR3 divergent transcript, long intergenic non-protein coding
RNA 595, TOX divergent transcript, FLVCR2 antisense RNA 1, LHRI_LNC744.1 lncRNA
gene, and ELFN1 antisense RNA 1), while six sequences represented partial sequences
of various mRNAs. Four lncRNAs were down-regulated in colon cancer; one was upregulated,
while one showed different expression patterns in different GEO datasets.
In this study, we have identified six lncRNAs that have potential significance for colorectal
cancer etiology and will be a subject of further in silico and in vitro study.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Transcriptome analysis of Pseudomonas aeruginosa after MhqO dioxygenase treatment
Get PDFPseudomonas aeruginosa is an opportunistic pathogen that can cause severe chronic
infections due to its exceptional ability to form a biofilm. Regulation of biofilm formation
is very sophisticated and involves multiple bacterial systems and regulatory pathways.
We found an enzyme MhqO dioxygenase from Bacillus paralicheniformis ZP1, which
was effective in the inhibition of biofilm formation and disruption of mature biofilm
of P. aeruginosa. Our results suggest that MhqO exerts its effect at the adhesion level,
preventing cells from attaching to the surface. We have also shown that the enzyme
stimulates the rhamnolipids synthesis.
To elucidate the mechanism of enzyme action, we analyzed the transcriptome of the P.
aeruginosa PAO1 strain treated with MhqO. Since cell adhesion occurs at the beginning
of the stationary phase growth, the PAO1 strain was treated with MhqO for four hours,
followed by total RNA isolation and cDNA synthesis. Transcriptome sequencing was
performed by Illumina NovaSeq 6000 and data were analyzed by Novogene Bioinformatics
Technology Co., Ltd. (Beijing, China).
Obtained data showed that 122 genes were up-regulated, 41 genes were down-regulated,
and the expression of 5947 genes was not changed. Five genes whose expression was
altered are directly related to biofilm formation. MhqO increased the expression of the
RsmA post-transcriptional regulator in P. aeruginosa. Transcriptome data revealed that pili
IV biosynthesis genes were up-regulated, which is in accordance with literature data that
RsmA positively regulates these genes. The inhibition of cells’ attachment to the surface
could be explained by these results. In addition, RsmA positively regulates rhamnolipid
production but negatively regulates biofilm matrix synthesis, which was supported by
expression levels in the sequenced transcriptome.
Data obtained from transcriptome analysis suggest that P. aeruginosa treated with MhqO
dioxygenase should be more sensitive to oxidative and osmotic stress, as well as to
beta-lactam antibiotics. Our further investigations should confirm these effects at the
phenotypic level as well.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Biological and physiochemical studies of electrospun polylactid/polyhydroxyoctanoate PLA/ P(3HO) scaffolds for tissue engineering applications
Get PDFPolyhydroxyoctanoate, as a biocompatible and biodegradable biopolymer, represents an ideal candidate for biomedical applications. However, physical properties make it unsuitable for electrospinning, currently the most widely used technique for fabrication of fibrous scaffolds. To overcome this, it was blended with polylactic acid and polymer blend fibrous biomaterials were produced by electrospinning. The obtained PLA/PHO fibers were cylindrical, smaller in size, more hydrophilic and had a higher degree of biopolymer crystallinity and more favorable mechanical properties in comparison to the pure PLA sample. Cytotoxicity evaluation with human lung fibroblasts (MRC5 cells) combined with confocal microscopy were used to visualize mouse embryonic fibroblasts (MEF 3T3 cell line) migration and distribution showed that PLA/PHO samples support exceptional cell adhesion and viability, indicating excellent biocompatibility. The obtained results suggest that PLA/PHO fibrous biomaterials can be potentially used as biocompatible, biomimetic scaffolds for tissue engineering applications.Related to supplementary material: [https://imagine.imgge.bg.ac.rs/handle/123456789/2060