imagine (Institute of molecular genetics and genetic engineering)
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Gene modifiers in β-thalassemia syndromes – a new therapy approach
Get PDFβ-talasemijski sindromi predstavljaju veoma heterogenu grupu naslednih oboljenja hemoglobina
koje odlikuje poremećena sinteza β-globinskog lanca. Kliničke manifestacije β-talasmijskih sindroma variraju
od asimptomatskog do teškog oblika ovih oboljenja koje odlikuje zavisnost pacijenata od čestih
transfuzija. Velika fenotipska varijabilnost karakteristična za β-talasemije koja se najčešće ne može objasniti
samo različitim alelskim varijantama unutar β-globinskog gena, usmerila je istraživanja ka genima modifikatorima
ovih sindroma. Naime, smatra se da bilo koji faktor koji dovodi do smanjenja disbalansa između
α - i β-globinskih lanaca predstavlja potencijalo važan modifikator β-talasemijskih sindroma. Tako, sinteza
fetalnog hemoglobina i u adultnom dobu, delimično ublažava kliničku sliku jer se nedostatak funkcionalnog
β-globinskog lanca kompenzuje viškom fetalnih, γ-globinskih lanaca. Brojne GWAS studije ukazale su
na postojanje varijanti i van β-globinskog genskog lokusa koje utiču na ekspresiju γ-globinskih ili stabilnost
α-globinskih lanaca, a samim tim i na fenotip β-talasemijskih sindroma (HBS1-MYB, BCL11A, AHSP,
FoP, KLF1).
Do skora, transplantacija kosne srži je bio jedini vid terapije β-talasemijskih sindroma, međutim usled
visoke stope morbititeta i mortaliteta ove vrste terapije, alternativne strategije kao što su genska terapija
i razvoj indukovanih pluripotentnih stem ćelija se ubrzano razvijaju.
Cilj genske terapije β-talasemijskih sindroma je indukcija produkcije β- ili γ-globinskih lanaca kao i
smanjenje α/β disbalansa u eritrocitima. Za transfer gena u genskoj terapiji koriste se različite vrste vektora,
kako ne-virusni, tako i virusni. Napredak na polju razvoja različitih vrsta vektora, transdukcije humanih
stem i progenitor ćelija, kao i različitih metoda editovanja samih gena, daje nadu da će u bliskoj budućnosti
genska terapija biti terapija izbora za β-talasemijske sindrome.The β-thalassemia syndromes are heterogeneous hereditary hemoglobin disorders, characterized by
defect in β-globin chain synthesis. Clinical manifestations of β-thalassemia vary from severe, transfusiondependent
thalassemia major to mild, asymptomatic thalassemia trait. This phenotypic variability characteristic
for β-thalassemia that usually cannot be explained only by variations affecting β-globin gene,
swayed researchers toward identifying genetic modifiers for these disorders. Namely, any factor that reduces
the imbalance between α- and β-globin polypeptide chains is considered to be an important modifier
of this disorder. Specifically, production of fetal hemoglobin through adulthood could ameliorate the
severity of β-thalassemia phenotype since γ-globin polypeptide chains compensate for the lack of the
functional β-globin ones. GWAS studies have shown that regions outside of the β-globin gene cluster are
also implicated in γ-globin gene expression regulation and α-globin chain stability, and hence, play an
important role in β-thalassemia phenotype (HBS1-MYB, BCL11A, AHSP, FoP, KLF1).
Until recently, a definitive cure for β-thalassemia could only be achieved through bone marrow transplantation.
However, since its significant risk of morbidity and mortality, an alternative strategies, such as
gene therapy and development of induced pluripotent stem cells have been explored. The goal of β-thalassemia
gene therapy is the induction of β- or γ-globin polypeptide chains, as well as the reduction of α/β-
chain disbalance in erythrocytes.Various types of vectors have been considered for gene transfer, including
non-viral and viral. Recent improvements in vector biology and development, human stem cell transduction,
as well as the availability of new gene-editing methodology, pave the way towards introduction of
gene therapy as a go-to therapy for β-thalassemia syndromes
Late embryogenesis abundant (LEA) proteins in Ramonda serbica Panc identification, classification and structural characterization
Get PDFAn ancient resurrection plant Ramonda serbica Panc. is able to survive a long desiccation period
and reestablish metabolic activity upon watering. A hallmark of desiccation tolerance in the resurrection
species is the accumulation of protective late embryogenesis abundant proteins (LEAPs).
These intrinsically disordered proteins (IDPs) may stabilize the correct structure of proteins and
membranes during cellular dehydration. The aim of our study was to assess LEA genes’ expression
levels in hydrated (HL) and desiccated leaves (DL) and to identify, characterise, and estimate
the potential role of R. serbica LEAPs in desiccation tolerance. In total, 318 LEAPs from HL and DL
were identified and classified into the seven LEA protein family groups ranging from LEA1-LEA5,
seed maturation proteins (SMPs), and dehydrins (DEH). Analysis of the physicochemical properties,
motif architecture, secondary structure, homology, and phylogenetic relationships demonstrated
that R. serbica LEAPs greatly differed among the LEA family groups. The most abundant LEA2
proteins (mostly downregulated upon desiccation) exhibited lower hydrophilicity and propensity
to fold into organised globular domains. Oppositely, hydrophilic LEA4 proteins tended to form
amphipathic, A-type, α-helices. Most of desiccation-upregulated LEA genes encoded highly disordered
DEH1, LEA1, LEA4.2, and LEA4.3 proteins. While dehydrins might chelate metals and bind
DNA under water deficit, other ID LEAPs (e.g. LEA1, LEA3, LEA4) might participate in forming intracellular
proteinaceous condensates or adopt amphipathic α-helical conformation, enabling them
to stabilise desiccation-sensitive proteins and membranes. Taken together, possible functions of
LEAPs are discussed with significant implications on drought tolerance improvement of crops
grown in arid areas.Book of Abstracts: 4th International Conference on Plant Biology [and] 23rd SPPS Meeting, 6-8 October 2022, Belgrad
The role of the sink tissue in variegated Pelargonium zonale under excess excitation energy pressure morphologic, photosynthetic and metabolic study
Get PDFIn the global warming era, we are facing extreme changes in environmental conditions such
as high and low temperatures, high light and ultraviolet radiation intensity, as well as drought
and increased CO2 levels. The effects of these factors on plants are often interrelated and usually
result in a disturbed balance between the amount of energy received and the ability to process
it. In order to balance energy input and prevent photooxidative damage, plants have evolved
multiple mechanisms for energy dissipation and photoprotection. To efficiently dissipate the excess
excitation energy (EEE), the additional electron sinks, such as photorespiration, biosynthesis
of phenolics and nitrate reduction are stimulated. Variegated leaves of Pelargonium zonale have
been proven to be a suitable model system for examining ‘source–sink’ interactions within the
same leaf, providing the same microenvironment conditions, unlike common root-shoot studies.
The aim of our study was to investigate carbon and nitrogen reallocation between photosynthetically
active (green leaf sectors) and photosynthetically inactive (white leaf sectors) under EEE
pressure induced by low temperature in combination with high light intensity. Besides the amino
acids and phenolics analyses, we monitored morphological and photosynthetic parameters of
P. zonale leaves. Our results revealed higher content of free proteogenic amino acids in the white
leaf sectors than in the green ones. Moreover, EEE triggered increased polyphenol synthesis, namely
anthocyanins, already on the fourth day of the experiment. Also, the leaf fresh/dry weight (FW/
DW) ratio was significantly lower in plants exposed to EEE, indicating possible cell wall stiffening.Book of Abstracts: 4th International Conference on Plant Biology and 23rd SPPS Meeting, 6-8 October 2022, Belgrad
Coumarin-palladium(II) complex acts as a potent and nontoxic anticancer agent against pancreatic carcinoma cells
Get PDFPancreatic carcinoma represents one of the most lethal malignant diseases in the world although some
progress has been made in treating the disease in the past decades. Current multi-agent treatment options
have improved the overall survival of patients, but more effective treatment strategies are still needed. In this
paper we have characterized anticancer potential of coumarin-palladium(II) complex against pancreatic
carcinoma cells. Cells viability, colony formation and migratory potential of pancreatic carcinoma cells were
assessed in vitro, followed by evaluation of apoptosis induction and in vivo testing on zebrafish. Presented
results showed remarkable reduction in pancreatic carcinoma cells growth both in vitro and in vivo, being
effective at micromolar concentrations (0.5 M). Treatments induced apoptosis, increased BAX/BCL-2 ratio
and suppressed the expression of SOX9 and SOX18, genes shown to be significantly up-regulated in
pancreatic ductal adenocarcinoma. Importantly, treatments of the zebrafish-pancreatic adenocarcinoma
xenografts resulted in significant reduction of tumor mass, while did not provoke any adverse toxic effects
including hepatotoxicity. Presented results indicate the great potential of tested compound and the
perspective of its further development towards pancreatic cancer therapy.BOOK OF ABSTRACTS: 10th JUBILEE INTERNATIONAL CONFERENCE ON RADIATION IN VARIOUS FIELDS OF RESEARCH Jun 13-17, 2022, Herceg Novi - Montenegr
Uticaj DOAK i DOAC-REMOVE® na testove koagulacije u toku testiranjatrombofilije kod bolesnika lečenih primenom DOAK
Get PDFDirect oral anticoagulants (DOACs) administration significantly interferes with coagulation as-says. The aim of the study was to evaluate the effect of DOACs and DOAC-Remove® on coagulation assays dur-ing thrombophilia testing. Methods. The study was car-ried out from January 2019 to the end of June 2020. It in-cluded 30 DOAC-treated patients, 14 females and 16 males aged 23 to 63 (median age 47.6 years), tested for thrombophilia due to venous thromboembolism (VTE). Thrombophilia testing was performed using DOAC-Remove® tablets (activated charcoal). The results before and after DOAC-Remove® were compared. Results. Posi-tive lupus anticoagulant (LA) results were observed in 20% apixaban, 100% dabigatran, and 70% rivaroxaban-treated patients, while in samples after DOAC-Remove®, the LA positivity was observed only in one from the apix-aban group. Before DOAC-Remove®, the activated pro-tein C (APC) resistance (APC-R) was measurable in 40% dabigatran and 80% rivaroxaban-treated patients, while, after using DOAC-Remove®, the APC-R was measurable in all cases. Comparing the results obtained from the sam-ples before and after DOAC-Remove®, a difference was noted in relation to all dilute Russell's viper venom time (dRVVT) coagulation tests, except for the dRVVT ratio in the apixaban group. Clot-based methods for detecting the APC resistance were significantly affected by dabigatran and less by rivaroxaban. Conclusion. DOACs were prac-tically inactivated after the addition of the DOAC-Remove®, which made it possible to perform analyses for the LA and APC-R testing freely and obtain relevant re-sults.Primena direktnih oralnih antikoagulansa (DOAK)značajno utiče na testove koagulacije. Cilj rada bio je da se pro-ceni uticaj DOAK i DOAC-Remove® tableta (aktivni ugalj) natestove koagulacije tokom ispitivanja trombofilije. Metode.Istraživanjem, sprovedenim od januara 2019. do juna 2020.godine, obuhvaćeno je 30 bolesnika lečenih DOAK-om itestiranih na trombofiliju zbog venskog tromboembolzma(VTE). Bilo je 14 žena i 16 muškaraca, starosti od 23 do 63godine (medijana 47,6 godina). Ispitivanje trombofilije izvršenoje upotrebom DOAC-Remove® tableta (aktivni ugalj).Upoređivani su rezultati pre i posle primene DOAC-Remove®.Rezultati. Pozitivni rezultati za lupus antikoagulantni (LA) testdobijeni su kod 20% bolesnika lečenih apiksabanom, kod100% bolesnika lečenih dabigatranom i kod 70% lečenih riva-roksabanom, a u uzorcima posle DOAC-Remove® pozitivnostna LA dobijena je samo kod jednog bolesnika iz grupe lečnihapiksabanom. Pre primene DOAC-Remove®, rezistencija naaktivisani protein C (activated protein C resistance – APC-R) bila jemerljiva kod 40% i 80% bolesnika lečenih dabigatranom, od-nosno rivaroksabanom, dok je posle primene DOAC-Remove®, APC-R bila merljiva u svim slučajevima.Upoređivanjem rezultata dobijenih iz uzoraka pre i posleprimene DOAC-Remove®, primećena je razlika u odnosu nasve testove vremena koagulacije izvršene razblaženim Russell-ovim zmijskim otrovom (dilute Russell’s viper venom time –dRVVT), osim dRVVT u grupi bolesnika lečenih apiksabanom.Na koagulacionu metodu za otkrivanje APC-R značajno je uti-cao dabigatran, a manje rivaroksaban. Zaključak. Nakonprimene DOAC-Remove® tableta, DOAK su praktičnoinaktivisani što je omogućilo izvođenje analiza za LA i APC-R idobijanje relevantnih rezultata testova
Bioaccessibility of Phenolic Compounds and Antioxidant Properties of Goat-Milk Powder Fortified with Grape-Pomace-Seed Extract after In Vitro Gastrointestinal Digestion
Get PDFThis study deals with the evaluation of the bioaccessibility and antioxidant properties of phenolic compounds from heat-treated skim goat-milk powder fortified with grape-pomace-seed extract, after in vitro gastrointestinal digestion. Ultra-high performance liquid chromatography coupled to diode array detection and mass spectrometry (UHPLC-DAD MS/MS) analysis confirmed the abundant presence of phenolic acids and flavan-3-ols in the grape-pomace-seed extract (SE) and heat-treated skim goat-milk/seed-extract powder (TME). After in vitro digestion of TME powder and recovery of total quantified phenolics, flavan-3-ols and phenolic acids were 18.11%, 24.54%, and 1.17%, respectively. Low recovery of grape-pomace-seed phenolics indicated strong milk protein–phenolic interactions. Electrophoretic analysis of a soluble fraction of digested heat-treated skim goat milk (TM) and TME samples showed the absence of bands originating from milk proteins, indicating their hydrolysis during in vitro gastrointestinal digestion. The digested TME sample had better antioxidant properties in comparison to the digested TM sample (except for the ferrous ion-chelating capacity, FCC), due to the presence of bioaccessible phenolics. Taking into account the contribution of the digestive cocktail, digested TME sample had lower values of total phenolic content (TPC), in vitro phosphomolybdenum reducing capacity (TAC) and ferric reducing power (FRP), compared to the undigested TME sample. These results could be attributed to low recovery of phenolic compounds. TME powder could be a good carrier of phenolics to the colon; thus, TME powder could be a promising ingredient in the formulation of functional food.Related to supp. material:[https://imagine.imgge.bg.ac.rs/handle/123456789/2071
Science on the move - from lab to streets & school (ScienceSCool)
Get PDF“Science on the move - from lab to streets & school” (ScienceSCool) is a two-year project to be
organized around the theme "Life in a science bulb (2022)/Life outside the science bulb (2023)"
by the Faculty of Physical Chemistry and Institute of Molecular Genetics and Genetic
Engineering in 12 cities and several rural areas in Serbia.
The objective of the project is to point out the importance of science for everyday life and to
break the prejudice related to researchers by enabling direct contact between participants and
researchers through attractive program that combines science and entertainment. At the same
time, researchers’ awareness of the public’s concerns related to their work will be raised.
The main topic of the project would be climate changes in accordance with the both EU and UN
missions and goals, with special attention directed towards young audience in accordance with
2022 being the Year of European Youth. The pre/post-event activities to be organized will
include Science Vans – mobile teams of researchers dispatched to rural areas to perform hands
on experiments with participants. The event activities will include Treasure Hunt (a set of hands
on experiments whose successful completion leads to a scientific award), and Open Day/Lab,
that became “trademarks” of the SCIMFONICOM project during the years. New activities such
as SCI-Circus and Fascination by Innovation will also be organised. Furthermore, a special
activity will be organized giving opportunities for successful researchers of Serbia that won
grants within highly competitive European calls to present their projects and their research, as
well as to bring researchers to schools for interaction with target audience.Principal Investigator: Biljana Šljukić PaunkovićCoordinator for IMGGE: Aleksandra NikolićDuration period: 2022-202
Indole-based hydrazone derivatives: Synthesis, cytotoxicity assessment, and molecular modeling studies
No full textIn the present study, we report the synthesis, cytotoxicity determination and molecular modeling studies of twelve novel indole-based hydrazone derivatives ( IH1 - IH12 ). To obtain the target molecules, initially, 1 H -indole-2-carboxylic acid and ethanol were heated in the presence of an acid catalyst to yield ethyl 1 H -indole-2-carboxylate. Following the hydrazinolyzation of the ester moiety, the resulting compound, 1 H -indole-2-carbohydrazide reacted with appropriate benzaldehyde derivatives to obtain IH1 - IH12 . The proposed chemical structures of all compounds were confirmed by their 1 H NMR, 13 C NMR, IR, and HRMS data. Additionally, the configuration of C = N bond in IH8 was determined as ( E ) by applying 2D NMR technique, NOESY. Subsequently, the compounds were tested against both colon cancer (HCT116) and lung cancer (A549), as well as healthy lung fibroblast (MRC-5) cell lines to determine their potential as anticancer agents and their selectivity indexes (SI). Based on the obtained data from the antiproliferative MTT assay, HCT116 cell line was more sensitive to our molecules compared to A549. Furthermore, lipophilic halogens were preferable substituents on the phenyl ring for the selective toxicity against cancer cell lines. Drug-likeness analysis carried out by calculating important physicochemical properties of IH1 - IH12 confirmed that they all obey Lipinski's rule of five. Finally, hypoxia inducible factor (HIF)-1 alpha was suggested as the potential biological target of the compounds through molecular docking studies
Identification of Genes Promoting Growth of Ustilago maydis on Biomolecules Released from Cells Killed by Oxidation
Get PDFMuch headway has been made in understanding the numerous strategies that enable microorganisms to counteract various types of environmental stress, but little is known about how microbial populations recover after a massive death caused by exposure to extreme conditions. Using the yeast-like fungus Ustilago maydis as a model, our recent post-stress regrowth under starvation (RUS) studies have demonstrated that this organism reconstitutes devastated populations with remarkable efficiency. Subsequently, we have identified four RUS-gene products. Two of these, Did4 and Tbp1, play parallel roles in protecting the genome. To identify additional molecular components, we took a molecular-genetic and a transcriptomic approach. By employing a simple and novel screening method, we identified five RUS-deficient mutants (snf8, slm1, vrg4, snf5, hsf1), three of which (snf8, slm1, and hsf1) displayed sensitivity to different genotoxic agents, indicating that the corresponding gene products have roles in genome protection. The global transcriptomic changes of cells grown in supernatants derived from peroxide-treated cell suspensions revealed sets of uniquely expressed genes. Importantly, among the genes induced by the substrates was Chk1, which encodes a protein kinase required for checkpoint-mediated cell cycle arrest in response to DNA damage. Mutants of U. maydis deleted of Chk1 are severely incapacitated in RUS
Crosstalk between Glycogen-Selective Autophagy, Autophagy and Apoptosis as a Road towards Modifier Gene Discovery and New Therapeutic Strategies for Glycogen Storage Diseases
Get PDFGlycogen storage diseases (GSDs) are rare metabolic monogenic disorders characterized by an excessive accumulation of glycogen in the cell. However, monogenic disorders are not simple regarding genotype-phenotype correlation. Genes outside the major disease-causing locus could have modulatory effect on GSDs, and thus explain the genotype-phenotype inconsistencies observed in these patients. Nowadays, when the sequencing of all clinically relevant genes, whole human exomes, and even whole human genomes is fast, easily available and affordable, we have a scientific obligation to holistically analyze data and draw smarter connections between genotype and phenotype. Recently, the importance of glycogen-selective autophagy for the pathophysiology of disorders of glycogen metabolism have been described. Therefore, in this manuscript, we review the potential role of genes involved in glycogen-selective autophagy as modifiers of GSDs. Given the small number of genes associated with glycogen-selective autophagy, we also include genes, transcription factors, and non-coding RNAs involved in autophagy. A cross-link with apoptosis is addressed. All these genes could be analyzed in GSD patients with unusual discrepancies between genotype and phenotype in order to discover genetic variants potentially modifying their phenotype. The discovery of modifier genes related to glycogen-selective autophagy and autophagy will start a new chapter in understanding of GSDs and enable the usage of autophagy-inducing drugs for the treatment of this group of rare-disease patients