imagine (Institute of molecular genetics and genetic engineering)
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Polyphenols: the role of food bioactive agents in combating microbial virulence
No full textPolyphenols are well-known group of bioactive molecules distributed in various foods such as
citruses and honey. They have been recognized mainly due to their antioxidant properties but recent
insight into their antimicrobial spectrum implies potential novel application for this group.
Polyphenols are able not only to block growth of pathogenic microorganisms, but they can also
interfere with microbial ability to induce disease, microbial virulence. With the abundant studies
conducted worldwide we now know about range of virulence factors that are necessary for disease
induction and though we have new antimicrobial targets. One of the most studied virulence factors
is microbial ability to form biofilms. We have recently studied rutin, flavonoid glycoside found in
citruses. This molecule has the ability to block biofilm formation of Pseudomonas aeruginosa IBRS
P001 and Staphylococcus aureus IBRS MRSA 011, strains that are resistant to antimicrobial
therapeutics. In addition to reduction of biofilm biomass, rutin antibiofilm mechanisms also
included reduction in cell viability, exopolysaccharide, and extracellular DNA levels. Moreover,
moderate inhibition of bacterial adhesion to keratinocytes upon rutin treatment was observed. Rutin
antivirulence mechanisms involved inhibition of P. aeruginosa protease, pyocyanin, rhamnolipid,
and elastase production and the downregulation of the lasI, lasR, rhlI, rhlR, pqsA and mvfR genes.
This research has proven wide antivirulence potential of rutin. Moreover, we have tested range of
flavonoids as inhibitors of fungal virulence and resistance. Rutin, but also apigenin and apigetrin,
have shown promising antibiofilm and anti-hyphal properties in several clinical Candida albicans
strains examined, which was also confirmed on molecular level.
Polyphenols have wide antivirulence capacity employing a range of mechanisms and might be used for
the development of novel antimicrobial strategies. Intake of food polyphenols might have some health
benefits in the terms of limiting microbial pathogenicity, which is the possibility that should be explored
in more detail.Book of Abstracts : The 3rd International UNIFood Conference, UNIFood2024
Conference, Belgrade, June 28-29, 2024
Generation of Induced Pluripotent Stem Cells Carrying 22q11.2 CNVs as a Model System for Studying Neurodevelopmental Disorders
No full textCopy number variations (CNVs) at 22q11.2 are associated with elevated risk for neurodevelopmental
psychiatric disorders and they represent a powerful genetics-first approach to delineate molecular mechanisms underlying
these disorders. Many clinical presentations are shared between 22q11.2 deletion and duplication carriers, including elevated
risk for developmental delays, intellectual disability, and autism spectrum disorders. However, 22q11.2 microdeletion is a
high-risk factor for schizophrenia, while 22q11.2 microduplication is less common in patients with this disorder than in the
general population. Differences in brain structure between 22q11.2 deletion and duplication carriers are also reported.
Although many animal models mimicking human diseases have been available for the research, only limited success has
been achieved in revealing molecular mechanisms underlying human brain diseases. Thus, iPSCs derived from patients with
specific disorders, such as neurodevelopmental disorders (NDDs), represent a powerful in vitro model system for studying
molecular mechanisms underlying their pathophysiology. Methods: Control subjects and patients with 22q11.2 CNVs were
recruited from the University Children's Hospital, and their peripheral blood mononuclear cells were reprogrammed using
CytoTune™-iPS 2.0 Sendai Reprogramming Kit. The pluripotency of generated iPSCs was analyzed by
immunofluorescence, qPCR, and trilineage differentiation using STEMdiff Trilineage Differentiation Kit. The iPSC lines
were genotyped to identify any additional pathogenic CNVs. Results: We have generated iPSC cell lines from five patients
with 22q11.2 microdeletion, six patients carrying 22q11.2 microduplication, and three healthy individuals. Pluripotency
was confirmed by the expression analysis of pluripotency markers, while the capacity of generated iPSCs to differentiate
into all three germ layers was revealed by STEMdiff Trilineage Differentiation Kit. Conclusions: Generated patient-specific
iPSCs carrying 22q11.2 CNVs represent a model system that will enable further studies on molecular mechanisms
underlying NDDs.Book of abstract: Praia D’El Rey Golf and Beach Resort Óbidos, Portugal | July 16 - July 18, 202
A comparative transcriptomic analysis of mouse DM1 models’ skeletal muscles
No full textMyotonic dystrophy type 1 (DM1) is a rare, incurable multisystemic disease, with the
main symptoms being skeletal muscle weakness, atrophy, and myotonia. It is caused by
CTG expansion in the 3’ UTR of the DMPK gene whose RNA acquires toxic functions and
sequesters MBNL proteins, resulting in globally altered RNA metabolism. Despite having
many mouse models with different phenotypes, none of them has been able to fully
recapitulate the phenotype and molecular pathogenesis of DM1. To map transcriptomic
differences among various mouse DM1 models, we systematically analyzed gene
expression in their skeletal muscles.
We retrieved all publicly available RNA-seq datasets from mouse models expressing
expanded CTG repeats and Mbnl knockout models. Our workflow with unified parameters
consisted of preprocessing, and differential gene expression analysis (DESeq2).
Additionally, gene co-expression networks (WGCNA), were focused on the CTG repeatexpressing
model that was most commonly used and had the largest number of biological
replicates (HSALR), where network nodes were represented by a union of dysregulated
genes from all analyzed datasets.
In models expressing CTG repeats, the average number of up- (787) and down-regulated
(642) genes was greater compared to Mbnl knockouts (676 and 380; log2FC>1, padj>0.05).
WGCNA recovered three modules of strongly co-expressed genes (adjacency>0.5). The
turquoise module, sharply correlated with muscle type, was associated with extracellular
space and muscle development. The midnightblue module consisted of Mup gene family
members, while the brown module was associated with the immune response.
Our results revealed pathway changes in DM1 skeletal muscles, where immune pathways
in muscle homeostasis and development are intriguing as molecular targets for further
investigation. Furthermore, gene expression patterns separated Mbnl knockouts from
models expressing CTG repeats, indicating the significance of mouse model choice for
basic and preclinical research.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Dysregulation of transcripts SMAD4-209 and SMAD4-213 and their respective promoters in colon cancer cell lines
No full textBackground: The pervasive role of alternative promoters in context-specific isoform expression and
the importance of promoter choice over its level of transcriptional activity have been recently implied
based on pan-cancer in silico studies. We aimed to explore this phenomenon at the cellular level on the
example of a major tumor suppressor SMAD4 in search of molecular mechanisms in colorectal cancer
that could be exploited for novel biomarkers or therapeutic approaches.
Methods: Multi-omics technologies, in silico tools and in vitro functional assays were applied to analyze
the transcripts expression and the alternative promoters’ function of the SMAD4 gene in colon cell lines
HCEC-1CT, HCT116, DLD-1, SW480 and SW620.
Results: High expression of the transcript SMAD4-213 emerged as a hallmark of colon cancer cells,
while in silico tools point to its possible additional role and potential for sponging miRNAs. Based on the
observed dysregulation of SMAD4-209 and SMAD4-213 in malignant vs. non-malignant colon cells, we
propose that their expression ratio might be a solid biomarker candidate for colorectal cancer detection.
Conclusions: A differential pattern of the respective promoters’ activity was observed that corresponds
to the expression of transcripts, confirming the role of alternative promoters in context-specific isoform
expression. The investigated SMAD4 promoters and transcripts harbor translational potential that should
be further investigated
PRENATAL ORIGIN OF PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA: TRACING BACK LEUKEMIA TO BIRTH USING LEUKEMIC CLONE-SPECIFIC IMMUNOGLOBULIN HEAVY CHAIN REARRANGEMENTS
No full textBackground: Several lines of evidence strongly support the view that a significant proportion of
childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) originates in utero, with the
preleukemic “first hit” occurring during fetal hematopoiesis. In this study we investigated prenatal
origin of BCP-ALL using immunoglobulin heavy chain (IGH) rearrangements as a leukemic clone-specific
marker for the detection of preleukemic clones at birth.
Methods: This retrospective study was conducted on a cytogenetically and clinically heterogeneous
cohort of 24 pediatric BCP-ALL patients. Identification of leukemic clone-specific IGH rearrangements
was performed in diagnostic bone marrow samples using polymerase chain reaction (PCR) and Sanger
sequencing. Based on the sequences of clonotypic IGH rearrangements, 2 patient-specific primers
were designed for each patient and used in semi-nested PCR for the detection of preleukemic clones
in patients’ neonatal blood spots (Guthrie cards).
Results: The study cohort enrolled 12 male and 12 female subjects, aged 1 to 9.6 years (median=3.1
years) at diagnosis. The majority of patients (20/24; 83.3%) had common-ALL, three patients (12.5%)
had pre-B, and one patient (4.2%) pro-B immunophenotypic subtype. Chromosomal translocations
were detected in 9 patients (37.5%); t(12;21) (ETV6/RUNX1) in 7 patients and t(1;19) (TCF3/PBX1) in 2
patients. The analysis of leukemic clone-specific IGH rearrangements’ composition revealed
preferential usage of IGHV3, IGHD2 and IGHD3 family genes, IGHJ6 and IGHJ4 genes, and the
predominance of unproductive IGH rearrangements (18/27; 66.7%). Leukemic clone-specific IGH
rearrangements were detected in neonatal blood spots of 54.2% of patients (13/24). In two cases that
had double IGH rearrangements at diagnosis, only one rearrangement was present at birth, while in
the third case both clonotypic rearrangements were detected in neonatal blood. Guthrie card-positive
findings were significantly more frequent in children ≤5 years of age at diagnosis than in older children
(p=0.011). Regarding patients’ characteristics at birth and at diagnosis, Guthrie card-positivity was not
associated with sex, birth weight and mother’s age, as well as with white blood cell count, percentage
of bone marrow blasts, karyotype, immunophenotype and the presence of ETV6/RUNX1 and
TCF3/PBX1 fusion genes.
Conclusion: Our study confirms that a large number of childhood BCP-ALL cases have prenatal origin,
regardless of the molecular subtype defined by chromosomal aberrations. Younger age at diagnosis of
BCP-ALL in patients with positive findings on Guthrie cards indicates a relatively short latency period
during which the "second hit" necessary for the conversion of preleukemic to leukemic clone and the
development of clinically overt disease occurs.32nd Meeting of the European Society of Paediatric Clinical Research (ESPCR) in Opatija, Croatia, May 24-25, 2024
Antibacterial and immunostimulatory effect of ivACME: a new approach to combat respiratory infections (DefensivACME)
No full textThe development of antimicrobial resistance to available medicines (with antibiotics as the greatest concern) is declared as one of the biggest challenges in healthcare worldwide and the development of alternative treatments as top priority. Respiratory infections, as the main contributor to the misuse/overuse of antibiotics, are among the most suitable for alternative therapies considering the mild/self-limiting infections nature. DefensivACME goal is to create a novel therapeutic avenue as an alternative to antibiotics to combat mild respiratory infections, by using plant extract ivACME, in more efficient, safe and sustainable manner than available alternatives. Biotechnology-based technique for plant propagation that increased bioactive compounds yield, will ensure the chemical consistency of ivACME and absence of environmental hazards as common shortcomings of plant products. Scientific validation of ivACME's combined efficacy in antibacterial activity against the most common respiratory pathogens and in stimulation of immune functions important for fighting infections will be ensured by analysis of ivACME effect on respiratory bacterial pathogens and on macrophages' function (immunostimulatory and bactericidal) in vitro. Preclinical assessment on efficiency (to enhance host defence against respiratory infections) and safety of orally applied ivACME will be provided by evaluation of ivACME effect on immune/oxidative activity by macrophages and lung cells in rats, together with toxicological examination. Proving ivACME as unique, safe, and efficient alternative/complement for antibiotics against respiratory infections will result in application for IP protection and will lead us to the next step of product definition, development, process scale-up and clinical assessment. Having environmentally sustainable and safe plant product with combined efficiency (antibacterial and immunoenhancing) will allow taping into local and global markets, but open door for new applicationsPrincipal Investigator: Dr Aleksandra Popov Aleksandrov, Institute for Biological Research "Sinisa Stankovic" - National Institute of the Republic of Serbia, University of BelgradeParticipant from IMGGE: Dr Milica ŽivkovićDuration period: 2024-202
Hypoxia decreases the efficiency of neuronal differentiation of human pluripotent stem cells
No full textBrain trauma leads to the induction of neural stem cell proliferation and the
migration of young neurons to injured areas. The capacity of adult neurogenesis has
limited potential to fully restore a neuronal network and function. SOX transcription
factors govern diverse cellular processes during embryonic and adult neurogenesis,
such as maintaining the multipotency of neural stem cells, cell proliferation, cell fate
decision as well as terminal differentiation of neurons and glia. This study aimed to
investigate the effect of hypoxia on the differentiation potential and terminal phases
of differentiation of NT2/D1 cell line. We analyzed the expression of SOX genes
and microRNAs as they control a variety of cellular processes during neuronal
differentiation, including cell proliferation and cell fate determination. Neuronal
differentiation of human pluripotent embryonal carcinoma stem cell line NT2 / D1
was used as an in vitro model system for studying the process of human
neurogenesis. Chemical hypoxia was induced with cobalt chloride (CoCl2).
The results of the analysis showed that, following hypoxia, the efficiency as well as
terminal differentiation were significantly decreased. In contrast, the expression
level of miR-21 was significantly increased.
Our findings advance the study of SOX TFs, miR-21, and their possible interplay in
ischemia-related pathologies, establishing them as prospective biomarkers and
possible targets for future diagnostic and therapeutic approaches.Book of abstracts: Belgrade Neuroscience Next Hub 2024 with international participation 24-25 May 2024. Belgrade, Serbi
Transcriptome Profiling of Phenylalanine-Treated Human Neuronal Model: Spotlight on Neurite Impairment and Synaptic Connectivity
No full textPhenylketonuria (PKU) is the most common inherited disorder of amino acid metabolism, characterized by high levels of phenylalanine (Phe) in the blood and brain, leading to cognitive impairment without treatment. Nevertheless, Phe-mediated brain dysfunction is not fully understood. The objective of this study was to address gene expression alterations due to excessive Phe exposure in the human neuronal model and provide molecular advances in PKU pathophysiology. Hence, we performed NT2/D1 differentiation in culture, and, for the first time, we used Phe-treated NT2-derived neurons (NT2/N) as a novel model for Phe-mediated neuronal impairment. NT2/N were treated with 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 30 mM Phe and subjected to whole-mRNA short-read sequencing. Differentially expressed genes (DEGs) were analyzed and enrichment analysis was performed. Under three different Phe concentrations (2.5 mM, 5 mM, and 10 mM), DEGs pointed to the PREX1, LRP4, CDC42BPG, GPR50, PRMT8, RASGRF2, and CDH6 genes, placing them in the context of PKU for the first time. Enriched processes included dendrite and axon impairment, synaptic transmission, and membrane assembly. In contrast to these groups, the 30 mM Phe treatment group clearly represented the neurotoxicity of Phe, exhibiting enrichment in apoptotic pathways. In conclusion, we established NT2/N as a novel model for Phe-mediated neuronal dysfunction and outlined the Phe-induced gene expression changes resulting in neurite impairment and altered synaptic connectivity
Molecular characterization of CYP21A2 mutations and clinical findings in Macedonian patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency – twenty years experience
No full textCongenital adrenal hyperplasia (CAH) is a heterogeneous autosomal recessive disease which has a different
prevalence in different populations and ethnicities. The disease is caused by a variety of CYP21A2 gene
mutations. The most common mutations affect the enzyme 21-hydroxylase. CAH has variable clinical
presentation depending on the amount of the active enzyme. Where no enzyme is produced, the lifethreatening
disease occurs in newborn child, whereas milder mutations can produce some percentage of
21-hyroxylase causing milder clinical presentation with precocious puberty in childhood, or
oligomenorrhea and sterility later in life. In female, additional signs are virilization which can be present
as complete sexual ambiguity and problems with sexual identity which can occur despite the surgical
correction.
In this paper we have presented the 20 years of work with clinical and molecular issues in 96 Macedonian
CAH patients with all forms of severity. The diagnosis and clinical care were provided following the
contemporary international guidelines. Nine most common CYP21A2 point mutations were explored and
the findings are classified through the severity of mutations and their role in the clinical presentation.
The mutations distribution in our population corresponds to most of the European countries with
exception of the p.P30L mutation which has higher prevalence. Genotype-phenotype concordance was
good in our cohort except for a stronger role of p.P30L mutation which delivered stronger virilization in
NC form or even complete SV form of the disease. We have presented a novel 9 bp deletion
(p.G424_R426del) in exon 10 of the CYP21A2 gene. We have provided our finding that some mutations
such as p.P30L and intron 2 are among the main causes of genotype-phenotype discrepancy. We had
presented also several rare genotypes and the genotypes in Roma ethnicity for the first time.
We believe that genetic testing in patients with CAH is important for the exact diagnosis, understanding
the role of mutations and for genetic counseling
Actinomycins from Streptomyces anulatus BV365 efficiently functionalize silk for biomedical applications
Get PDFA newly isolated strain Streptomyces sp. BV365 producing high amounts of orange
extracellular pigments on mannitol-soy flour agar was identified as Streptomyces anulatus
BV365. The producing strain Streptomyces anulatus BV365 was isolated from the
ectomycorrhizosphere soil of the black truffle Tuber melanosporum. Crude cell extract of this
strain was fractionated and orange pigment fraction showed very strong antimicrobial and
cytotoxic activities. On further analysis, the strain was found to produce metabolites
actinomycin D, C2 and C3 and nonactin. The application of purified actinomycins in the
dyeing of multifiber fabric was assessed. Actinomycins exhibited a high affinity towards
protein fibers (silk and wool), but washing durability was maintained only with silk. The
morphologies and chemical components of the treated silk fabrics were analyzed using
scanning electron microscopy and Fourier transform infrared spectroscopy. In addition, a
skin irritation test in 3D-reconstructed human epidermis model was conducted to evaluate
the biocompatibility of the tested fabrics. The results showed that the dyed silk had a safe
biological properties.Book of abstracts: 6th Symposium on Biotransformations for Pharmaceutical and Cosmetic Industry June 17-21, 2024, Kraków, Polan