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22q11.2 Deletion syndrome as a tool for modelling and research of neurodevelopmental disorders
Get PDFIntroduction: Neurodevelopmental disorders (NDDs) are a group of complex and heterogeneous disorders that include autism spectrum disorders, intellectual disability, schizophrenia and bipolar disorder. However, underlying pathophysiological mechanisms are mostly unknown. In order to get better understanding of the underlying mechanisms and to discover potential therapeutics we have focused our research on 22q11.2 Deletion Syndrome (22q11.2DS), caused by microdeletion of the region q11.2 of chromosome 22 and associated with a high risk for NDDs. Methods: To study molecular mechanisms underlying intrafamilial phenotypic variability, we have identified families with the inherited form of 22q11.2DS with the aim of conducting the following analyses: whole genome sequencing in order to detect additional genetic variation(s) present in the affected child; generation of induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells; analysis of the effects of 22q11.2 microdeletion on neural differentiation including organoids as 3D model system; transcriptome analysis of iPSC-derived neurons and astrocytesto determine differentially expressed gene sets and dysregulated pathways; and testing the metabolic changes and drug responsiveness of neurons and astrocytes by high-throughput cell-based assays. Results: Peripheral blood mononuclear cells of the families with inherited form of 22q11.2DS were reprogrammed and established iPSCs were characterized. Generated iPSCs will be subjected to the further analyses. Conclusion: Currently, most of the treatments of NDDs are symptom-based due to limited understanding of underlying pathophysiological mechanisms. It is expected that patient-derived iPSCs will enable a deeper understanding of unique disease mechanisms and may also provide a significant contribution in preclinical drug development
Prognostic significance of cebpa mutations in patients with normal-karyotype - acute myeloid leukemia
Get PDFIntroduction: CEBPA gene encodes a transciptional factor with an essential role in granulocyte differentiation process. In acute myeloid leukemia (AML), mutationsin CEBPA gene have been associated with
favorable outcome and up to now only the presence of double mutated CEBPA gene (CEBPAdm) was included in WHO classification. Prognostic influence of CEBPA mutationsin C-terminal (bZIP) region recently
have been proposed as a marker for better overalsurvival (OS), higher probability of achieving complete
remission (CR) and a lower risk of relapse. Since AML with normal karyotype (AML-NK) is a group with intermediate prognosis with the need for new prognostic markers, we analyzed the influence of bZIP CEBPA
mutations as an additional molecular marker in Serbian AML-NK patients.
Methods: CEBPA mutational screening was performed using a multiplex polymerase chain reaction–
based fragment length analysis. A total of 61 bone marrow samples were collected from de novo AMLNK patients.
Results: In our analysis, frequency of CEBPA mutations in Serbian AML-NK patients was 15% (12/61 patients). Six out of 12 patients had mutation in bZIP region (CEBPAbZIP+). All six CEBPAbZIP+ patients (100%)
achieved CR after induction chemotherapy versus 62% of CEBPAbZIP- patients. CEBPAbZIP+ patients showed a significantly longer OS (CEBPAbZIP+ 31.5 months vs CEBPAbZIP- 10 months) and disease free survival
(DFS) (CEBPAbZIP+ 30 months vs CEBPAbZIP- 10.5 months).
Conclusion: Our analysis of Serbian AML-NK patients showed CEBPAdm was not associated with better
prognosis but our results indicate that CEBPAbZIP+ status is a good candidate for a prognostic molecular
marker
Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB
Get PDFIntroduction: Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by fasting hypoglycemia and the accumulation of glycogen in the liver, kidneys and intestinal mucosa. Recent studies revealed that chronic endoplasmic reticulum (ER) stress and increased apoptosis
play a role in the progression of disease manifestations. Although dietary control is commonly utilized
to manage hypoglycemia, there is still a lack of effective pharmacological therapy. Therefore, the establishment of proper model system is essential for testing novel treatment approaches.
Methods: To create GSD-Ib in vitro model system, CRISPR/Cas9-knockout (KO) method was used to introduce a deletion in SLC37A4 gene in the FlpInHEK293 cells. Characterization of CRISPR/Cas9-KO model
system was performed using Sanger sequencing, RT-qPCR and Western Blot. Additionally, the expression analysis of ER stress and apoptotic markers was performed.
Results: Sanger sequencing confirmed the presence of c.14_100del in SLC37A4 gene. The expression
level of SLC37A4 was decreased to 26.8% in the SLC37A4-/- cell line compared to the SLC37A4 wild-type
along with Western blot analysis, which confirmed reduced target protein level in SLC37A4-/- clones. Furthermore, ER stress (ATF4, DDIT3, HSPA5, XBP1s) and apoptotic (BCL2, BAX, CASP3, CASP7) markers expression levels were significantly altered in SLC37A4-/- clones compared to wild-type, which proved that
we created a suitable GSD-Ib in vitro model system.
Conclusion: Utilizing CRISPR/Cas9 technology, we established cellular GSD-Ib modelsystem that mirrors
increased ER stress and apoptosis. This model system could be used to facilitate a comprehensive understanding of disease mechanisms and enable testing of potential treatment effectiveness
Establishment of induced pluripotent stem cells from patients with 22q11.2 duplication syndrome as a model system for studying neurodevelopmental disorders
Get PDFIntroduction: Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD), schizophrenia, and intellectual disability, represent important public health challenge in modern societies
with a prevalence of about 10 to 15% of all births and the tendency of increasing worldwide. They are
caused by disruption of early brain development. Treatments of NDDs are focused on symptoms due to
a limited understanding of underlying pathophysiological mechanisms. Individuals with the 22q11.2
Duplication Syndrome (22q11.2dup), caused by heterozygous 22q11.2 microduplication, have an elevated risk of developing NDDs. Literature data revealed that ASD is detected in 14-25% of patients with
22q11.2dup while schizophrenia is less common in these patients than in the general population, suggesting that 22q11.2 duplication might be protective against schizophrenia.
Methods: Genomic and clinical findingsin patients with 22q11.2dup were analyzed and peripheral blood
mononuclear cells of patients with 22q11.2dup were reprogrammed.
Results: We formed a cohort of 8 patients with 22q11.2dup. The majority of patientsin our cohort have
microduplication of approximately 3Mb (80%). Also, the majority of them are familial cases and in 67%
of cases, the 22q11.2 microduplication is inherited from the mother. Congenital heart defects were detected in 25% of our patients, while all tested patients have facial dysmorphism. iPSCs were generated
from three patients with a familial form of 22q11.2dup and their mothers.
Conclusion: A cohort of patients with 22q11.2dup is formed and iPSCs were generated which can be
used as a model system for studying NDDs
The effect of UVB radiation onthe expression of SOX2 and SOX9 genes in human keratinocytes in vitro
Get PDFIntroduction: Prolonged exposure to sunlight, has a harmful effect on skin cells encompassing reduced
viability, morphological changes, and altered gene expression. The two most prevalent types ofskin cancer,squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC), arise from malignant transformation
of keratinocytes. UV radiation, among other factors, serves as the primary cause of these tumors. Previous data hasshown that changesin different SOX genes expression in these cancer types correlates with
disease progression, suggesting their role as oncogenes/tumor suppressors. The presented work is focused on examining the impact of UVB radiation on the expression of SOX2 and SOX9 genesin HaCaT cells
derived from human keratinocytes.
Methods: Using a custom-made UV solarsimulator for the irradiation of HaCaT cells with 150 mJ/cm2 or
300 mJ/cm2
, we analyzed SOX2 and SOX9 gene expression. In order to determine the protective effects
of quercetin, anti-inflammatory bioflavonoid, we treated irradiated HaCaT with quercetin, and analyzed
SOX gene expression.
Results: Our resultsindicate that UVB radiation induces a dose dependent decrease of SOX2 expression
while expression of SOX9 was increased at the dose of 150 mJ/cm2 in HaCaT. Treatment of cells with
quercetin increased the expression of both SOX2 and SOX9 genesin HaCaT cellsfollowing UVB radiation
at both doses compared to irradiated cells.
Conclusions: Further research is needed to understand the molecular mechanisms and significance of
SOX2 and SOX9 in UVB-induced cellular responses, in the context of nonmelanoma cancers with potential implications for targeted therapeutic strategies for nonmelanoma cancer
Novel Quinoline-Based Thiosemicarbazide Derivatives: Synthesis, DFT Calculations, and Investigation of Antitubercular, Antibacterial, and Antifungal Activities
Get PDFThe discovery of new antimicrobial agents as a means of treating drug-resistant microbial pathogens is of utmost significance to overcome their immense risk to human well-being. The current investigation involves the development, synthesis, and assessment of the antimicrobial efficacy of novel quinoline derivatives incorporating a thiosemicarbazide functionality. To design the target compounds (QST1–QST14), we applied the molecular hybridization approach to link various thiosemicarbazides to the quinoline core with a sulfonyl group. Upon the synthesis and completion of structural characterization via spectroscopic techniques (1H NMR, 13C NMR, 15N NMR, IR, and HRMS), the title molecules were extensively evaluated for their potential antitubercular, antibacterial, and antifungal activities. N-(3-Chlorophenyl)-2-(quinolin-8-ylsulfonyl)hydrazine-1-carbothioamide (QST4), the most effective compound against Mycobacterium tuberculosis H37Rv, was also tested on isoniazid-resistant clinical isolates with katG and inhA promoter mutations. Based on molecular docking studies, QST4 was also likely to demonstrate its antimycobacterial activity through inhibition of the InhA enzyme. Furthermore, three derivatives (QST3, QST4, and QST10) with preferable antimicrobial and drug-like profiles were also shown to be nontoxic against human embryonic kidney (HEK) cells. All compounds were optimized by the density functional theory method using B3LYP with the 6-31+G(d,p) basis set. Structural analysis, natural bond orbital calculations of donor–acceptor interactions, molecular electrostatic potential analysis, and frontier molecular orbital analysis were carried out. Quantum chemical descriptors and charges on the atoms were determined to compare the strengths of the intramolecular hydrogen bonds formed and their stabilities. We determined that the sulfur atom forms a stronger intramolecular hydrogen bond than the nitrogen, oxygen, and fluorine atoms in these sulfonyl thiosemicarbazide derivatives
NOVEL CELLULAR FACTORS INVOLVED IN REGULATION OF BRCA2-DRIVEN HOMOLOGOUS RECOMBINATION IN USTILAGO MAYDIS
Get PDFHomologous recombination (HR) is one of the most accurate mechanisms of preserving genome
integrity by precise repair of double strand breaks as the most deleterious type of DNA damage.
Mechanisms of HR are mostly studied in yeast which, unlike higher eukaryotes employs RAD52 as a
HR mediator, instead of BRCA2.
Ustilago maydis is a unicellular phytopathogen characterized by extreme radiation resistance
dependent on BRCA2-driven HR. The focus of our research is to uncover novel cellular factors that
regulate HR, by isolating suppressors of blm in U. maydis.
We have identified 3 new factors of unknown functions, as well as Rad55 and Mph. All mutations
suppress HU sensitivity of blm. Presence of truncated proteins caused by point mutations that
introduce the specific premature STOP codon and complete deletions of UMAG_01566 and
UMAG_01667, both lead to genotoxins sensitivity and altered growth rates on HU to a various extent.
Mutation in UMAG_03150 leads to slow growth which can be suppressed by truncated UMAG_01566.
Mitotic or meiotic recombination is also affected in some of the mutants.
We assume that these novel factors can provide insights into HR regulation, interactions among HR
participants and relations to other cellular processes.Fusion Conferences:Recombination Mechanisms 10-13 July 2023, Lisbon, Portuga
Exopolysaccharides from microorganisms which degrade BHET: Optimization of production
Get PDFNiska cena i jednostavna proizvodnja sintetičkih plastičnih materijala dovela je do
njihove raširene upotrebe za različita pakovanja, građevinarstvo, automobilsku i elektronsku
industriju i izradu proizvoda za domaćinstva. Zbog toga se proizvodnja plastike u poslednjih
pola veka povećala 20 puta [1]. Najviše se koristi polietilen tereftalat (PET), a procenjeno je
da je njegova proizvodnja 2022. godine dostigla 87,17 miliona kubnih metara. Visoka
otpornost PET-a, koja se ranije smatrala prednošću, danas dovodi do nekontrolisanog
nagomilavanja otpada u brojnim ekosistemima na planeti [2]. Bis-(2-hidroksietil)-tereftalat
(BHET) je komercijalni monomersa strukturom sličnom jezgru PET-a, pa se široko koristi
u ispitivanjima biodegradacije PET-a, jer je pokazano da pojedini mikroorganizmi imaju
sposobnost da ga degraduju [3]. Osim mogućnosti razgradnje različitih štetnih jedinjenja,
mikroorganizmi mogu da proizvode i egzopolisaharide (EPS), kao odgovor na različite
selektivne pritiske životne sredine. EPS su zbog svojih fizičko-hemijskih svojstava našli
primenu u farmaceutskoj, hemijskoj i prehrambenoj industriji [4].
Cilj ovog rada bio je selektovati mikroorganizme koji mogu da razgrade BHET kao
model jedinjenje za ispitivanje degradacije plastičnog otpada na bazi PET-a i sa visokim
prinosom produkuju EPS.
Sposobnost degradacije BHET-a ispitivana je na 100 bakterijskih izolata, pomoću
čvrstih podloga sa BHET-om kao glavnim izvorom ugljenika. Potom je selektovano 7 sojeva
sa najširim zonama prosvetljenja, koji su korišćeni za produkciju EPS-a u tečnim podlogama
sa saharozom. Nakon fermentacije i odvajanja biomase, EPS su taloženi dvostrukom
zapreminom etanola i njihova količina je određena gravimetrijskom metodom [5]. Masa
dobijenih EPS varirala je od 2,4 – 33,7 g/L, a odabran je soj sa visokom produkcijom za
dalje eksperimente. Pošto mikrobna produkcija polisaharida veoma zavisi od uslova
fermentacije, ispitan je uticaj pet faktora na prinos: temperature, aeracije, pH, količine
saharoze i različitih izvora azota. Rezultati su pokazali da najoptimalnija podloga za
dobijanje visokog prinosa EPS-a sadrži ekstrakt kvasca (izvor azota), 100 g/L saharoze
(izvor ugljenika), a pH vrednost joj je 7.
Budući ekperimenti će biti usmereni na ispitivanje interakcija između ključnih faktora
na proizvodnju EPS-a, radi naprednije optimizacije procesa i povećanja prinosa.KNJIGA IZVODA: 9. simpozijum Hemija i zaštita životne sredine Kladovo, 4-7. jun 2023. BOOK OF ABSTRACTS : 9th Symposium Chemistry and Environmental Protection Kladovo, 4-7th June 202
THE IMPACT OF NEXT-GENERATION SEQUENCING ON DIAGNOSIS AND TREATMENT OF RARE DISEASES
Get PDFIntroduction: Rare diseases are heterogeneous group of diseases, with one common characteristics, a
prevalence less than 1 in 2000 people. Vast majority of them are monogenic and finding pathogenic genetic
variants is needed to set the correct diagnosis, enable adequate treatment and provide genetic counselling to
members of affected family. This study is an overview of genomic studies of rare diseases in Serbia.
Methods: More than 1200 patients suspected to have a rare disease have been analyzed using sanger
sequencing, clinical-exome sequencing, whole-exome sequencing or whole-genome sequencing in order to
find disease-causing or disease-modifying variants. Novel variants were characterized using in silico modelling
or in in vitro eukaryotic assays (standard or CRISPR/Cas9 developed).
Results: Disease-causing variants were found in more than 150 different genes associated with a rare
disease. The most frequent were thalassemia syndromes (214 patients), followed by phenylketonuria (109
patients), congenital adrenal hyperplasia (>90 patients) and glycogen storage disease Ib (30 patients), while
majority of diseases is seen only in a single patient. More than 40 new genetic variants were comprehensively
characterized in silico or in vitro. For the first time, candidate modifiers (SHANK gene family) were identified
in a group of phenylketonuria patients with an unusual phenotype.
Conclusion: In the genomics era, next-generation sequencing significantly shortens time to diagnosis
and allows studying genetic modifiers of monogenic diseases and genotype-phenotype correlation. Furthermore,
characterization of novel genetic targets boosts development of precision medicine.ABSTRACT BOOK: “Genetic Diseases
from Diagnostics to Prevention and Therapy”
October 05-14th Balkan Congress of Human Genetics & 9th Rare Disease SEE Meeting 2023; Skopje, October 05-07, 202
Raw data for the article: Péter, G., Lukić, J., Brlás-Molnár, Z., Ardó, L., Horváth, Z., Rónyai, A., Bársony, P.,& Ljubobratović, U.. (2023). Effect of single-generation domestication of pikeperch on the performance of the offspring in conventional and pond recirculation aquaculture system. in aquaculture reports, 32, 101702. https://doi.org/10.1016/j.aqrep.2023.101702
No full textRaw data for: [https://doi.org/10.1016/j.aqrep.2023.101702]Related to published version:[https://imagine.imgge.bg.ac.rs/handle/123456789/2069