imagine (Institute of molecular genetics and genetic engineering)
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EFFECTS OF NEXT-GENERATION PROBIOTICS ON NEURAL GENE EXPRESSION IN Caenorhabditis elegans
No full textThere is growing body of evidences that the gut microbiota composition is associated with host
health making the human gut a unique environment to explore commensal microorganisms with
probiotic properties. The emerging subgroup of probiotics named Next-Generation Probiotics
represent a novel group of gut bacteria which are strict anaerobes, whose safety status is still not
fully determined, but could confer the specific healthy effect on the host. For several microbial
strains that are part of the gut ecosystem have been shown to improve behaviour-related functions
in the host. Therefore, the aim of this study was to evaluate the neuromodulating properties of gut
anaerobic bacteria isolated from healthy individuals in Caenorhabditis elegans model system
through assessment of the expression of the genes important for proper neural function of the
worms. Gut anaerobic bacteria including Dorea longicatena NGB204, Dorea formicigenerans
NGB229, Bacteroides xylanisolvens NGB220, Bacteroides cellulosilyticus NGB202, Roseburia
intestinalis NGB215 and Lancefieldella pravula NGB231 were isolated from human fecal material
in BACTEC media followed by serial dilutions spreading on Columbia Blood Agar supplemented
with cysteine and sodium thioglycolate in Whitley Anaerobic Workstation. All of tested gut
anaerobes showed capability to differently regulate the expression of the genes involved in
serotonin, dopamine and GABA synthesis (tph-1, cat-2, unc-25), neurotransmitters’ vesicle release
(unc-64, snb-1, snt-1), neuropeptide Y receptor homolog synthesis (npr-1), and different classes of
neuropeptide Y homologs (flp-18, flp-21) in C. elegans in comparison to worms fed with standard
laboratory food Escherichia coli OP50. These results imply that selected bacterial species could be
considered as potential novel Neurobiotics, the Next-Generation Probiotics with capability to target
neural function and modulate different neurodegenerative and psychiatric disorders.Book of Abstracts : The 3rd International UNIFood Conference, UNIFood2024 Conference, Belgrade, June 28-29, 202
Echinococcus spp. circulating in livestock in Serbia
No full textThe echinococcosis case burden in the human population of Serbia
is among the highest in Southeastern Europe, but comprehensive
data on the circulating tapeworm species is scarce. To
gain insight into the population genetics of Echinococcus spp. in
Serbia, the project WORM_PROFILER will collect samples from
livestock intended for human consumption at major abattoirs,
slaughterhouses and homesteads over the next two years. During
the first two months of sampling, 22 livers from pigs (n=15), lambs
(n=5) and calves (n=2) with clearly identifiable cysts and/or cystlike
formations as well as scar tissue were collected from major
abattoirs around the country. The cyst content and/or affected area
was sampled by direct isolation using pipette tips and/or excision
of the tissue and analyzed microscopically, followed by quick
DNA extraction by boiling in 0.02 M NaOH for 15 min and amplification
of the cox1 gene using primers which can differentiate
E. granulosus, E. multilocularis and E. canadensis via conventional
PCR. Several large cysts were present only in the liver of one boar.
Tapeworm larvae were microscopically confirmed in the cysts’
content and later identified by PCR as E. granulosus. PCR analysis
additionally revealed the presence of E. canadensis gDNA in a
tissue sample from another pig from a different farm. Although
these results indicate that most carcasses flagged for conditional
use based on liver examination by abattoir veterinarians are
not infected by Echinococcus spp., they nonetheless suggest that
pigs may play an important role in its transmission in Serbia.Sequencing of the cox1 and nad1 genes from the samples in which
Echinococcus spp. gDNA was detected and is currently underway.
This research was supported by the Science Fund of the Republic of
Serbia, #10841, Worm Profiler: Surveillance and population genetics
of Echinococcus in Serbia - WORM_PROFILER.Book of Abstracts:The XIV European
Multicolloquium
of Parasitology
Wrocław, Poland
August 26–30, 202
Genetic predisposition of suicidal behavior: variants in GRIN2B, GABRG2, and ODC1 genes in attempted and completed suicide in two Balkan populations
No full textCompleted suicide accounts for over 700,000 deaths worldwide annually, while attempted suicide is 20 times more frequent. Genetic background is an important factor contributing to suicidal behavior, including candidate genes in glutamate, γ-aminobutyric acid (GABA), and polyamine systems. Our aim was to differentiate genetic predispositions underlying different types of suicidal behavior, attempted and completed suicide, in two Balkan populations. Analysis of variants in the genes GRIN2B (rs2268115 and rs220557), GABRG2 (rs424740), and ODC1 (rs1049500 and rs2302614) was performed on a study sample including 173 suicide attempters with comorbid psychiatric disorders, 216 non-suicidal psychiatric patients and 172 healthy controls from Serbia, and 333 suicide completers and 356 non-suicidal autopsy controls from Slovenia. CA genotype of rs220557 in GRIN2B gene increased the risk for completed suicide (P = 0.021), and violent suicide (P = 0.037), compared to controls. In ODC1 gene, CA genotype of rs2302614 decreased the risk for completed suicide compared to suicide attempt (P = 0.012). Marginally, AC haplotype for variants rs1049500-rs2302614 in ODC1 gene decreased the risk for completed suicide compared to suicide attempt (P = 0.052). Specific genetic variants of glutamate and polyamine systems are differently distributed among diverse suicidal phenotypes, providing further information on the implication of these systems in suicidality
Polyhydroxyalkanoate bioplastics depolymerizing enzyme improvement for more robust biotechnological applications (PHOBioDepolyIMPROVE)
No full textThe growing global plastic production, reaching 7.82 billion metric tons by 2015, has prompted
a shift towards resource efficiency, recycling, and the adoption of biobased and biodegradable
polymers. Polyhydroxyalkanoates (PHAs), microbial polyesters, have garnered attention as
potential bioplastic alternatives. However, PHAs, including polyhydroxyoctanoate (PHO), have
demonstrated slow biodegradation rates in natural environments. This project aims to
enhance PHO depolymerase activity to address this issue. By combining experimental and
computational approaches, the project focuses on developing enzyme variants capable of
efficiently degrading PHAs. In silico modeling will be employed to predict promising enzyme
modifications, which will then be validated experimentally. The ultimate goal is to create a
scalable and efficient enzyme system for PHA degradation, ensuring a sustainable post-
consumer lifecycle for bioplastics.Principal Investigator: Dr Jelena Milovanović, IMGGEDuration period: 2024-202
Arabidopsis thaliana root cells interact with outer membrane vesicles (OMVs) produced by plant beneficial bacterial strain Paraburkholderia phytofirmans PsJN
No full textIntroduction
Plant growth-promoting bacteria (PGPB) play an essential role in the growth and development of
plants by facilitating the uptake of nutrients and increasing stress resistance. The interaction
between plants and their beneficial bacteria is multifaceted, but the role of extracellular vesicles
(EVs) in this interkingdom communication is poorly understood. To investigate the interaction of
outer membrane vesicles (OMVs) with plant cells, EVs produced by Gram-negative bacteria, we
isolated and characterized OMVs produced by Paraburkholderia phytofirmans PsJN, a PGPB strain
known for its plant-protective abilities.
Methods
The OMVs of P. phytofirmans PsJN were isolated using an affinity-based chromatography system
and characterized by Nanoparticle tracking analysis (NTA). To monitor the interaction of OMVs
with plant cells, the vesicles were labelled with the lipid-binding dye Vybrant™ DiD. To ensure the
specificity of the signals, different methods to remove the unbound dye were tested, namely
dialysis, ultracentrifugation, ultrafiltration and iodixanol density gradient purification. Arabidopsis
thaliana roots were incubated with DiD-labelled OMVs and observed by confocal laser scanning
microscopy (CLMS).
Results
NTA revealed 70nm to 180 nm size range of isolated OMVs. Regarding preparation of OMVs for
monitoring their interaction with plant cells, the most satisfactory removal of unbound dye was
obtained after the separation of DiD-OMVs in the density gradient. A. thaliana roots treated with
DiD-OMVs showed specific red signals in the root hairs and epidermal cells, while the signals were
absent in the control-treated roots.
Conclusions
The results suggest that PsJN OMVs directly interact with the root hairs and epidermal cells of A.
thaliana. As root hairs play a crucial role in plant nutrient uptake and interactions with microbes,
further investigation is needed to determine the potential contribution of PsJN OMVs to plant
recognition of beneficial bacteria, establishment of mutual interactions and the exertion of a
protective effect by PGPB.Book of abstract: 2nd MOV E Symposium, 8-11 October 2024, Belgrade, Serbi
Analysis of the subcellular localization of the transcripts CD81-205 and CD81-215
No full textIntroduction: Previous research suggested that transcripts CD81-205 and CD81-215 derived from the same gene promoter may be a long non-coding RNAs of stromal origin with potential tumor-promoting role in colon cancer. In silico analyses indicated non-coding potential of these transcripts and their elevated expression in tumor vs. non-tumor tissue of colon and rectum. The analysis of the transcripts' expression in tumor cells and tissue samples suggested low expression levels, especially in tumor cells, pointing to the stromal cells as a major source of these molecules. The aim of the study was to analyze the subcellular localization of transcripts CD81-205 and CD81-215. Material and Methods: The expression of transcripts CD81-205 and CD81-215 was analyzed in the subcellular compartments of the colon cancer cell line SW480 and the normal colon cell line with broblast morphology CCD-18Co. The RNA was extracted from nucleus and cytoplasm of SW480 and CCD-18Co cells, and also from exosomes collected from the cell culture media. The qPCR was performed using oligonucleotides targeting the sequence present in both transcripts of interest and not present in other transcripts from this gene. Results: The expression of transcripts CD81-205 and CD81-215 was undetectable in cytoplasms of cells SW480 and CCD-18Co, while their expression in nuclei of both cell lines and exosomes of SW480 was detectable, but low. The prominent expression was detected in exosomes of the CCD-18Co cells, and this expression was around 5 times elevated in comparison to the exosomes of the SW480 cells. Conclusion: The prominent expression of transcripts CD81-205 and CD81-215 in exosomes of the CCD-18Co cells con rms the suspected stromal origin of these molecules. Considering that non-coding RNAs may act as signaling molecules even at low concentrations, the study also points to the potential role of CD81-205 and CD81-215 in intercellular signaling between stromal and malignant cells in the tumor tissue.Book of abstract: 2nd MOV E Symposium, 8-11 October 2024, Belgrade, Serbi
Multiomic Profiling of Early Onset Colorectal Cancer
No full textEarly onset colorectal cancer (EOCRC), characterised by colorectal cancer (CRC) in
individuals under 50 years of age, has witnessed a concerning global rise over the past
three decades. While various risk factors such as smoking, alcohol consumption, and
dietary habits have been implicated, a definitive causal link remains to be fully understood.
Our research aims to explore the hypothesis that EOCRC represents a distinct clinical and
molecular entity compared to Late Onset CRC (LOCRC).
Through comprehensive omics profiling encompassing proteomics, genomics, and
transcriptomics, we aim to unravel the underlying mechanisms driving EOCRC. This
approach not only offers insights into disease pathogenesis, but also holds promise for
identifying novel biomarkers crucial for patient stratification and the development of
tailored treatment strategies.
In our study, we analysed healthy and matched tumour tissues from a cohort of 80 Irish
EOCRC and LOCRC patients diagnosed with microsatellite stable, resectable disease
and no familial syndromes, using mass spectrometry. Pathway enrichment analysis
unveiled alterations in molecular functions specific to EOCRC samples, including
dysregulated Granzyme A signalling and mitochondrial dysfunction indicative of potential
metabolic rewiring. Furthermore, downregulated NET signalling suggests unfavourable
immunomodulation, while observed deactivation of oxidative phosphorylation,
corroborated by transcriptomics analysis, may indicate a response to immune checkpoint
inhibitors.
By integrating these findings with mutational profiling and clinical data, we aim to develop
digital simulations capable of predicting patient outcomes and guiding personalised
treatment regimens. This multifaceted approach holds promise for advancing our
understanding of EOCRC and improving patient care outcomes.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
156P Phosphorylated neurofilament heavy chain in cerebrospinal fluid and plasma in clinically silent and childhood-onset SMA individuals from Serbia
No full textBiomarkers capable of reflecting disease onset and short- and long-term
therapeutic effects in individuals with spinal muscular atrophy (SMA) are still
an unmet need and phosphorylated neurofilament heavy chain (pNF-H) holds
significant promise. We conducted a longitudinal prospective study to evaluate
pNF-H levels in the cerebrospinal fluid (CSF) and plasma of 29 individuals with
childhood-onset SMA treated with Nuinersen (SMA type 1: n=6, type 2: n=17,
type 3: n=6). pNF-H levels before and during treatment were compared with
the levels of controls (n=22), patients with Duchenne muscular dystrophy (n=17),
myotonic dystrophy type 1 (n=11), untreated SMA individuals with chronic type 3
disease (n=8), and children with presymptomatic SMA (n=3). SMA type 1 showed
the highest mean CSF pNF-H levels before treatment initiation. All Nusinersentreated individuals (types 1, 2, and 3) showed significantly elevated mean baseline
CSF pNF-H compared to controls, which inversely correlated with age at disease
onset, age at first dose, disease duration and the initial CHOP INTEND result (for
SMA type 1 and 2). During 22 months of treatment, CSF pNF-H levels declined
during loading doses, stabilizing at reduced levels from the initial maintenance
dose in all individuals. Baseline plasma pNF-H levels in type 1 and 2 SMA were
significantly increased compared to other cohorts and decreased notably in type 1
after 2 months of treatment and type 2 after 14 months. Conversely, SMA type 3,
characterized by lower baseline pNF-H levels, did not show significant fluctuations
in plasma pNF-H levels after 14 months of treatment. Our findings suggest that
CSF pNF-H levels in untreated SMA individuals are significantly higher than in
controls and that monitoring of CSF pNF-H levels may serve as an indicator of rapid
short-term treatment response in childhood-onset SMA individuals, irrespective of
the subtype of the disease, while also suggesting its potential for assessing longterm suppression of neurodegeneration. Plasma pNF-H may serve as an appropriate
outcome measure for disease progression and/or response to treatment in types 1
and 2 but not in type 3. Clinically silent infants with SMA may show elevated pNFH levels, confirming early neuronal degeneration.Book of abdstracts:
29th International Congress
of the World Muscle Society
Prague, Czechia
8th-12th October 202
GENETIC SPECTRUM OF NOONAN SYNDROME IN PEDIATRIC POPULATION
No full textNoonan syndrome (NS) is an autosomal dominant hereditary disorder with a prevalence of
1:1000 to 1:2500 live births. First described in 1963 by pediatric cardiologist Jacqueline
Noonan, this syndrome is classified among RASopathies which arise from pathogenic variants
in genes disrupting RAS/MAPK signaling pathway. The most commonly affected genes in NS
are PTPN11, SOS1, RAF1, KRAS, and NRAS genes. The genetic diversity results in a
spectrum of clinical manifestations. The aim of this study is to explore genetic and clinical
heterogeneity of NS.
The patients included were monitored from 2010 to 2024 at the Mother and Child Health Care
Institute of Serbia “Dr Vukan Cupic”. Diagnosis was established on basis of suggestive clinical
findings while definitive diagnosis was confirmed through whole exome sequencing (WES) or
next-generation sequencing (NGS) panels for RASopathies.
We present the clinical features and genetic findings of 15 patients with NS with median age at
diagnosis of 8 years. Distribution of patients in regard to gender was balanced (f:m - 8:7). The
causative variants were found in the PTPN11, RAF1, SOS1, and LZTR1 genes, most frequently
in PTPN11. Typical facial dysmorphism was universally present, while developmental delay
was noted in 20%. The most common cardiac manifestations of NS in our group was
hypertrophic cardiomiopathy (53.3 %) and pulmonary artery stenosis (33.3%).
Identification of causative genetic variants in patients with Noonan syndrome through NGS
methodology provides insight into the pathomechanism thereby providing prognostic
information and potential therapeutic targets.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024
COMPARISON OF ADARB1 AND TPH2 VARIANTS BETWEEN SLOVENIAN AND SERBIAN CASES ON THE CONTINUUM OF SUICIDAL BEHAVIOR
No full textCompleted suicide and a 20 times more frequent attempted suicide are on the continuum of
suicidal behavior. Genetic background of suicidal behavior predominantly implicated
serotonergic system genes including TPH2, encoding tryptophan hydroxylase 2, a ratelimiting
serotonin synthetizing enzyme in the brain. TPH2 pre-mRNAs undergo adenosineto-
inosine RNA editing by ADAR and ADARB1 enzymes, which fine-tunes enzyme
function, and TPH2 rs4290270 variant has been shown to affect its own transcript alternative
splicing and editing. TPH2 rs4290270 in combination with childhood general traumas, and
main effects of ADARB1 rs9983925 and rs4819035 variants have been previously associated
with suicide attempt in psychiatric patients from Serbian population. Our current aim was to
compare our abovementioned findings with completed suicide in Slovenian population.
ADARB1 rs9983925 and rs4819035, and TPH2 rs4290270 variants were genotyped for 333
suicide completers (305 using violent and 28 using non-violent methods), and 357 nonsuicidal
autopsy controls from Slovenia. Genotypic data for 165 suicide attempters from
Serbia was taken from our previous study.
ADARB1 rs4819035 GT and GG carriers had an increased risk for non-violent completed
suicide compared to controls (P(χ2/adjusted) =0.012/0.012), and compared to violent
completed suicide (P(χ2/adjusted) =0.021/0.049). TPH2 rs4290270 AA genotype marginally
increased the risk for completed suicide compared to controls (P(χ2/adjusted) =0.038/0.086),
mostly due to violent completed suicide when compared to controls as well (P(χ2/adjusted)
=0.054/0.115), and for completed suicide compared to attempted suicide (P(χ2/adjusted)
=0.052/0.052).
ADARB1 and TPH2 variants differentiated genetic backgrounds between attempted and
completed suicide, as well as between violent and non-violent methods of completed suicide.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024