imagine (Institute of molecular genetics and genetic engineering)
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Vortioxetine reduces the development of pain-related behaviour in a knee osteoarthritis model in rats: Involvement of nerve growth factor (NGF) down-regulation
No full textBackground and Purpose Vortioxetine, a multimodal-acting antidepressant, has recently shown analgesic properties. We aimed to investigate its prophylactic effect in the osteoarthritis (OA) model and gain insights into the underlying molecular mechanisms. Duloxetine was studied as a reference. Experimental Approach In the monoiodoacetate (MIA)-induced rat model of knee OA, pain-related behaviour was assessed in weight-bearing and Von Frey tests. Antidepressants were administered orally once daily for 28 days. Gene expression of pain-related mediators (Ngf, Il-1β, Tnf-α, Bdnf, and Tac1 encoding substance P) and oxidative stress parameters were determined after completion of the treatment/behavioural testing protocol. Key Results Vortioxetine and duloxetine dose dependently reduced weight-bearing asymmetry and mechanical hyperalgesia of the paw ipsilateral to the MIA-injected knee. Vortioxetine reduced the increased Ngf mRNA expression in the MIA-injected knees to the level in sham-injected counterparts. It reduced oxidative stress parameters in the affected knees, more effectively in females than males. Duloxetine showed no effect on Ngf mRNA expression and oxidative stress. Both antidepressants decreased mRNA expression of pain-related mediators in the lumbar L3–L5 ipsilateral DRGs and spinal cords, which were up-regulated in MIA-injected rats. This effect was male-specific. Conclusion and Implications Vortioxetine may be effective against the development of chronic pain in OA. Its antihyperalgesic effect may be mediated, at least in part, by normalization of NGF expression in the affected joint. Decrease of localized oxidative stress and of expression of pain-related mediators that contribute to central sensitization are also involved in vortioxetine's antihyperalgesic effect, in a sex-specific pattern
SUPPRESSORS OF BLM-DEFICIENCY IDENTIFY FOUR NOVEL PROTEINS AFFECTING DNA REPAIR AND HOMOLOGOUS RECOMBINATION IN USTILAGO MAYDIS
No full textThe maintenance of genome integrity is a fundamental cellular process and it’s highly
conserved among all domains of life. Homologous recombination (HR) is one of the DNA
repair mechanisms and it is essential for the error-free repair of DNA double strand breaks,
which are the most deleterious lesions. Mechanisms of HR are mostly studied in yeast which,
unlike higher eukaryotes employs RAD52 as a HR mediator, instead of BRCA2.
The focus of our research is to uncover novel cellular factors that regulate HR, by isolating
suppressors of blm in Ustilago maydis, a unicellular phytopathogen, which is extremely
resistant to radiation and has DNA repair system similar to that in human, with highly conserved
BRCA2 (named Brh2).
We have identified 4 novel factors of unknown functions (named Rec3, Zdr1, Bls9 and Bls2),
and 4 known factors – Rad51, Dna2, Mph and paraplegin. Mutations in each of these genes
suppress hydroxyurea sensitivity of blm.
Three of these four novel proteins are involved in DNA Repair, and play critical roles in mitotic
and/or meiotic recombination. Rec3 is a member of the family of Rad51 ATPases, Zdr1 is Cys2-
His2 zinc finger protein, whereas Bls9 and Bls2 are completely uncharacterized proteins.
Deletion of Rec3, Zdr1 and Bls2 can also suppress HU-sensitivity of Δgen1, and Δmus81
mutants, but loss of Bls9 does not rescue HU-sensitivity of Δgen1.
These novel factors can provide insights into HR regulation, interactions among HR participants
and relations to other cellular processes.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024
Biological control of soil pathogens of tomato with streptomycetes from different soil samples
No full textNedostatak efikasnih i ekološki održivih tretmana za kontrolu zemljišnih patogena paradajza nametnuo je potrebu za istraživanjem alternativnih rešenja. Stoga smo iz poljoprivrednog zemljišta na otvorenom polju i u stakleniku, kao i iz šumskog zemljišta, prikupljenog iz zone rizosfere na području Smederevske Palanke, izolovali vrste roda Streptomyces i proučavali njihovu biološku aktivnost prema gljivi Fusarium oxysporum f. sp. lycopersici (FOL) i bakteriji Ralstonia solanacearum (RS). Na osnovu odgajivačkih odlika na bogatoj podlozi (Mannitol Soy Flour) izolovano je 30 različitih izolata: 7 iz uzorka sa otvorenog polja, 2 iz staklenika i 21 iz šumskog zemljišta. Razlike u broju izolata ukazuju na veliki uticaj kvaliteta zemljišta, poljoprivrednih aktivnosti i uslova spoljne sredine na brojnost populacije streptomiceta. Biološka aktivnost ovih izolata testirana je in vitro korišćenjem metode dvostruke kultivacije. Od 30 proučavanih izolata, 16 je ispoljilo biološku aktivnost, 6 prema FOL i 6 prema RS, dok su 4 izolata prouzrokovala inhibiciju razvoja oba patogena. Rezultati su statistički obrađeni analizom varijanse i testirani LSD i Tukey testom. Svih 16 biološki aktivnih izolata pokazalo je statistički značajnu aktivnost u poređenju sa kontrolom. Najviši procenat inhibicije porasta FOL izazvao je izolat BJO11 - 26,67%, dok je najjači antagonizam prema RS (29 mm) ispoljio izolat BJŠ19b. Analiza sekvence 16S RNK potvrdila je da pripadaju rodu Streptomyces. Rezultati su pokazali da se u našem poljoprivrednom i nepoljoprivrednom zemljištu nalazi populacija streptomiceta iz koje se pojedini izolati mogu iskoristiti kao biološki agensi za zaštitu paradajza od zemljišnih patogena.The deficiency of effective and sustainable treatments for the control of soil-borne pathogens of tomato has created a need to research alternative solutions. Therefore, from agricultural soil in an open field and in a greenhouse, as well as from forest soil, collected from the rhizosphere zone in the area of Smederevska Palanka, we isolated species of the genus Streptomyces and studied their biological activity against the fungus Fusarium oxysporum f. sp. lycopersici (FOL) and bacteria Ralstonia solanacearum (RS). Based on their growth characteristics, 30 different isolates were isolated on rich medium (Mannitol Soy Flour): 7 from the sample from the open field, 2 from the greenhouse and 21 from the forest soil. Differences in the number of isolates indicated a great influence of soil quality, agricultural activities and environmental conditions on the abundance of the streptomycete population. The biological activity of these isolates was tested in vitro using the double cultivation method. Out of the 30 studied isolates, 16 showed biological activity, 6 against FOL and 6 against RS, while 4 isolates inhibited the development of both pathogens. The results were statistically processed by analysis of variance and tested by LSD and Tukey test. All 16 biologically active isolates showed statistically significant activity compared to the control. The highest percentage of FOL growth inhibition was caused by isolate BJO11 - 26.67%, while the isolate BJŠ19b was the most antagonistic towards RS (29 mm). Sequence analysis 16S RNA confirmed that they belong to the genus Streptomyces. The results showed that in our agricultural and non- agricultural soil there is a population of streptomycetes that can be isolated and used as biological agents to protect tomato from soil pathogens.Zbornik izvoda : Nacionalni naučni skup sa međunarodnim učešem Biotehnologija i savremeni pristup gajenju i oplemenjivanju bilja, Smederevska Palanka 2024, 6. novemba
New model compounds for the efficient colorimetric screening of medium chain length polyhydroxyalkanoate (mcl-PHA) depolymerases reveal mechanism of activity
No full textPlastic pollution presents a significant environmental problem contributing to increased CO2 emissions and persistently accumulation in ecosystems. Biobased polymers, like polyhydroxyalkanoates (PHAs), offer a part of a solution with their biodegradability and reduced carbon footprint. However, effective end-of-life strategies, such as controlled enzymatic depolymerization, are crucial for sustainability, relying on efficient PHA depolymerases (PHAases). Here we describe the synthesis of two new chromogenic compounds derived from polyhydroxyoctanoate (PHO) and their application in a continuous, quantitative spectrophotometric assay for PHO depolymerase and other medium chain lengths PHAase activity within 10 min. These substrates allow activity measurement at temperatures above 45 °C, simplifying the comparison of PHAases and aiding enzymatic degradation progress. The study also explores enzyme specificity and identifies key amino acids involved in PHO recognition by PfPHOase. The 3-hydroxyoctanoyl moieties of both compounds were found to bind specifically to a groove formed by the amino acids Phe96, Phe125, Ile171, and Val230, which are highly conserved in known mcl-PHA depolymerases
Butyric acid-producing Faecalimonas sp. NGB245 strain attenuates the symptoms of experimental autoimmune encephalomyelitis
No full textPurpose: The bacterial strains residing in the anaerobic environment of the colon have been shown to produce shortchain
fatty acid (SCFA) essential for preserving homeostasis in the gut, and in the host more broadly. The reduction in
the abundance of butyric acid (BA)-producing bacteria associated with multiple sclerosis (MS). Hence, our study aimed
to isolate the bacterial strains with the high capacity to produce BA from the feces of healthy donors and to test the effects
of supplementation in a mice model of MS.
Methods: By using different media in anaerobic conditions and measuring BA by HPLC we selected Faecalimonas sp.
NGB245 strain. To overcome the sensitivity of this strict anaerobe to oxygen, the NGB245 overnight culture (NGB245-
postbiotic) was used as the supplement. Myelin oligodendrocyte glycoprotein peptide/complete Freund's
adjuvant/pertussis toxin-induced experimental autoimmune encephalomyelitis (EAE) in C57BL6 mice was used as a
model of MS. Mice consumed NGB245-postbiotic in a 16h/day (instead of the water, ad libitum) regime during the 15
days. The control group of EAE mice consumed PYG medium enriched with cellobiose and starch, used for the NGB245
cultivation, in the same regime. The effects of NGB245 strain on EAE symptoms, immunological responses, and
microbiota properties were analyzed by flow cytometry, ELISA, microscopy, and new-generation sequencing,
respectively.
Results: NGB245 strain was selected for the study based on the high production of BA (15mM). The supplementation
with NGB245-postbiotic alleviated daily clinical scores, maximal clinical scores, and the duration of the EAE in
comparison to the control group. This was associated with the lower abundance of Th1 and Th17 cells, myeloid dendritic
cells, inflammatory monocytes, macrophages, and activated microglia, but a higher level of myeloid-derived suppressor
cells in the CNS of treated EAE mice. These immunomodulatory effects were associated with the higher diversity of
microbiota in the colon.
Conclusion: These results support the idea of targeting the microbiota in autoimmune diseases to preserve the
homeostatic functions of the immune system and microbiota and thus restrain the autoimmune process development.Special Issue: 7th European Conference of Immunology, 1–4 September, 2024, Dublin, Irelan
Bioprospecting of microbial enzymes for industial application
No full textBiofilms have a major impact on human and animal health, pose a problem
for food safety and contaminate drinking water supplies. They are one of the main
causes of tolerance to antibiotics or disinfectants and thus represent a persistent bacterial
reservoir for the spread of infections or contamination of food. A promising strategy to
combat biofilms is to combine antimicrobial agents with enzymes that inhibit the
formation of biofilms or are able to disintegrate components of the biofilm matrix, thus
facilitating the penetration of antimicrobial agents and increasing their efficacy. New
anti-biofilm enzymes in a unique proprietary collection of non-pathogenic environmental
bacteria is searched. The enzymes candidates are heterologously expressed, purified and
thoroughly analysed for their biological activity and stability. This approach has proven
successful in the bioprospecting of enzymes that combat biofilms of pathogenic bacteria
and can be used in the food and feed industry or as therapeutics
CHARACTERISATION OF MICROBIAL COMMUNITIES IN PETROLEUM-CONTAMINATED SOIL
No full textThe widespread use of petroleum and its derivatives has led to substantial environmental
contamination through extraction, processing, transport, and utilization of this fuel. Despite
exposure to toxic stress, some microorganisms can tolerate these conditions, and those capable of
rapid pollutant decomposition are more likely to survive. This is a report on the members of the
microbial community in soil polluted with petroleum and its derivatives, outlining the
physicochemical and biological characteristics of these unique habitats. Most of the examined
bacteria showed sensitivity only to higher concentrations of heavy metals, indicating their resistance
and adaptation to high concentrations of these metals. The clustering analysis aligned with the
phylogenetic diversity of strains, with strains exhibiting the highest bioremediation capabilities,
such as Planomicrobium sp. RNP01 and Planococcus sp. RNP07 and Rhodococcus sp. RNP05
clustered tightly.17th International Conference on Fundamental
and Applied Aspects of Physical Chemistry, 23–27 September 2024
Palace Hotel — Belgrade — Serbi
GENETIC LANDSCAPE OF PHENYLKETONURIA IN SERBIA
No full textPhenylketonuria (PKU) is the most frequent inborn disorder of amino acid metabolism caused by variants
in human phenylalanine hydroxylase gene (PAH). In this study, a total of 109 PKU patients from Serbia
were included, who were classified into three phenotypic categories in accordance with pre-treatment
plasma phenylalanine level: classic PKU, mild PKU and mild hyperphenylalaninemia. For genetic
analyses, we combined Sanger sequencing, MLPA and next generation sequencing to identify diseasecausing
variants in PAH gene, which were further classified using ACMG classification. Additionally,
we used in silico and/or eukaryotic expression studies to assess the effect of novel genetic variants
identified in our patients. Disease-causing variants were identified in 217 of 218 alleles, reaching
detection rate of 99.5%. We detected a total of 32 different variants, of which 29 previously described
and three novel ones: p.Gln226Lys, p.Pro244His and p.Pro416Leu. In silico and/or eukaryotic expression
studies confirmed pathogenic effect of all novel genetic variants. The most frequent variant was
p.Leu48Ser (31.2%), followed by p.Arg408Trp (13.8%), p.Ile306Val (9.2%). p.Glu390Gly (5%),
p.Pro281Leu (4.6%), and p.Arg261Gln (3.2%). All detected disease-causing variants were classified as
pathogenic using ACMG classification. Our study brings the updated spectrum of molecular genetic data,
variant classification and detailed phenotypic characteristics for PKU patients from Serbia. Therefore,
our study contributes to better understanding of molecular landscape of PKU in Europe and to general
knowledge on genotype–phenotype correlation in PKU.Book of abstracts: 2nd B&H Symposium of Laboratory Geneticists and Molecular Biologists (with International Participation) May, 202
Higher-Order Connectivity and Synchronization Patterns in Human Connectome Networks
No full textRecent advances in the physics of complex systems aim at understanding the emergence
of new features at a larger scale---the central idea of physical notion of complexity, linking
the features of collective dynamical behaviors with higher-order interactions. Mapping the
brain imaging data onto brain networks and revealing their detailed structures enables
the application of these approaches in the study of complex brain functioning. Increasing
evidence shows that connections between many brain regions play a role in specific brain
processes and help describe pathways of neurodegeneration. In this context, studies
highlight the importance of synchronization processes and the role of central brain
regions (hubs).
In this lecture, we describe some representative examples that motivated our research.
We then present more details of our results obtained by mapping human connectome data
onto brain networks and their higher-order structures described by simplicial complexes.
Furthermore, we present a phenomenological model of the phase synchronization
processes simulated on the core network consisting of simplexes of all orders around
eight brain hubs. Our results reveal partial synchronization patterns explain how the brain
avoids pathological states of full synchronization. Co-evolving phases at groups of nodes
(brain regions) result in multifractal fluctuations of the order parameter, which measures
the degree of synchrony.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Determining the efficiency of the miTAR neural network in searching for microRNA target genes
No full textIntroduction: Atherosclerosis is a cardiovascular disease characterized by a chronic
inflammatory process of the intima of elastic arteries, which is known to be one of the
leading causes of death in developed countries. Recent studies have revealed a correlation
between mtDNA mutations and pathological disorders leading to atherosclerosis.
Moreover, increasing number of research focuses on the microRNA-mediated gene
expression suppression. miRNAs play an important role in the development and
regulation of diseases of the cardiovascular system. Consequently, the study of possible
linkage between the mitophagy disruption and the miRNA-mediated gene suppression is
of interest.
Objective: To assess the effectiveness of neural network based search for target genes for
miRNA regulation of atherosclerosis development.
Materials and methods: The list of mitophagy associated genes was selected by analyzing
scientific databases. Then, each of the genes was passed through a specialized miRDB
database in order to find corresponding miRNA. The resulting miRNA list was analyzed
through the web interface, which transmitted data to the miTAR neural network, in order
to estimate affinity values. The final stage of the experiment included the identification
of the obtained through miTAR sequences through the NCBI Blast web service. Next, the
list of obtained genes was compared with the list of genes found manually using miRDB.
Results and discussion: Based on the results of the comparison we came to the conclusion
that the neural network based miTAR surpassingly copes with the task of predicting
microRNA target genes, albeit the results for miRNA with lower affinity index significantly
varied from the ones found manually. However, this reservation could be attributed to
upsides of the model as it reveals new potential target genes for further evaluation.
Conclusion: Our results indicate that neural network models can serve as an effective
tool in the search for miRNA target genes. In addition, the found genes could be furtherly
studied to identify their linkage to atherosclerosis. This will also assist in the study of
possible drug substances in the treatment of atherosclerosis.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024