TY  - CONF
AU  - Sibinčić, Nikolina
AU  - Krstić Ristivojević, Maja
AU  - Stojanović, Marijana
AU  - Mladenović Stokanić, Maja
AU  - Vasović, Tamara
AU  - Ćirković Veličković, Tanja
AU  - Stojadinović, Marija
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6318
AB  - The SARS-CoV-2 nucleocapsid (N) protein plays a significant role in the coronavirus life cycle and participates in a variety of critical events following viral invasion1. In infected patients, high titers of immunoglobulin G (IgG) targeting N protein were detected and correlated with the clinical course of the disease2. Therefore, N protein and anti-N protein IgGs were recognized as important diagnostic indicators of COVID-19 infection in serological and quick antigen tests3. In this study, we optimized the expression of the recombinant form of SARS-CoV-2 N protein in a mammalian cell line HEK293T by comparing the transfection efficiency between Polyethylenimine (PEI) and Calcium Phosphate (CaP) DNA-complexing agents. Transfection potency was tested at different cell confluence and passage number, in several cell culture media, pre-transfection and post-transfection media change and in conditions of reduced serum. Chloroquine and glycerol treatments were included to enhance transfection efficiency as they might inhibit DNA degradation in lysosomes or increase membrane permeability. Protein expression was monitored in cell supernatants up to 7 days post-transfection in dot-bot and Western blot using anti-N protein antibodies. Both transfection methods have shown moderate to relatively high transfection efficiency dependent on the applied conditions, making them affordable and easy to use techniques for recombinant N protein production on a small-scale in adherent mammalian systems. PEI acts as a good delivery system regardless of the presence of the fetal bovine serum (FBS), while CaP transfection is more dependent on the presence of FBS which in turn favors N protein degradation. However, we have optimized both methods to achieve optimal expression of unfragmented N-protein in serum-free conditions. Apart from setting up a cost-effective platform for expression of N protein in mammalian cells, we plan on investigating the mechanisms behind the PEI and CaP non-viral gene delivery systems as there are still some uncertainties in the scientific community.
PB  - Belgrade: Faculty of Chemistry
C3  - Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia
T1  - Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells
SP  - 91
EP  - 92
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6318
ER  - 

@conference{
author = "Sibinčić, Nikolina and Krstić Ristivojević, Maja and Stojanović, Marijana and Mladenović Stokanić, Maja and Vasović, Tamara and Ćirković Veličković, Tanja and Stojadinović, Marija",
year = "2023",
abstract = "The SARS-CoV-2 nucleocapsid (N) protein plays a significant role in the coronavirus life cycle and participates in a variety of critical events following viral invasion1. In infected patients, high titers of immunoglobulin G (IgG) targeting N protein were detected and correlated with the clinical course of the disease2. Therefore, N protein and anti-N protein IgGs were recognized as important diagnostic indicators of COVID-19 infection in serological and quick antigen tests3. In this study, we optimized the expression of the recombinant form of SARS-CoV-2 N protein in a mammalian cell line HEK293T by comparing the transfection efficiency between Polyethylenimine (PEI) and Calcium Phosphate (CaP) DNA-complexing agents. Transfection potency was tested at different cell confluence and passage number, in several cell culture media, pre-transfection and post-transfection media change and in conditions of reduced serum. Chloroquine and glycerol treatments were included to enhance transfection efficiency as they might inhibit DNA degradation in lysosomes or increase membrane permeability. Protein expression was monitored in cell supernatants up to 7 days post-transfection in dot-bot and Western blot using anti-N protein antibodies. Both transfection methods have shown moderate to relatively high transfection efficiency dependent on the applied conditions, making them affordable and easy to use techniques for recombinant N protein production on a small-scale in adherent mammalian systems. PEI acts as a good delivery system regardless of the presence of the fetal bovine serum (FBS), while CaP transfection is more dependent on the presence of FBS which in turn favors N protein degradation. However, we have optimized both methods to achieve optimal expression of unfragmented N-protein in serum-free conditions. Apart from setting up a cost-effective platform for expression of N protein in mammalian cells, we plan on investigating the mechanisms behind the PEI and CaP non-viral gene delivery systems as there are still some uncertainties in the scientific community.",
publisher = "Belgrade: Faculty of Chemistry",
journal = "Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia",
title = "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells",
pages = "91-92",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6318"
}

Sibinčić, N., Krstić Ristivojević, M., Stojanović, M., Mladenović Stokanić, M., Vasović, T., Ćirković Veličković, T.,& Stojadinović, M.. (2023). Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia
Belgrade: Faculty of Chemistry., 91-92.
https://hdl.handle.net/21.15107/rcub_ibiss_6318

Sibinčić N, Krstić Ristivojević M, Stojanović M, Mladenović Stokanić M, Vasović T, Ćirković Veličković T, Stojadinović M. Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia. 2023;:91-92.
https://hdl.handle.net/21.15107/rcub_ibiss_6318 .

Sibinčić, Nikolina, Krstić Ristivojević, Maja, Stojanović, Marijana, Mladenović Stokanić, Maja, Vasović, Tamara, Ćirković Veličković, Tanja, Stojadinović, Marija, "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells" in Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia (2023):91-92,
https://hdl.handle.net/21.15107/rcub_ibiss_6318 .

TY  - JOUR
AU  - Đokić, Jelena
AU  - Ninkov, Marina
AU  - Popov Aleksandrov, Aleksandra
AU  - Mirkov, Ivana
AU  - Subota, Vesna
AU  - Mihajlović, Luka
AU  - Stojadinović, Marija
AU  - Stanić-Vučinić, Dragana
AU  - Kataranovski, Dragan
AU  - Kataranovski, Milena
PY  - 2013
PY  - 2013
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/566
AB  - Warfarin (3-(α-acetonylbenzyl)-4-hydroxy coumarin) is a vitamin K (VK) antagonist that inhibits vitamin K-dependent (VKD) processes, such as blood coagulation. It also exerts an influence on some non-VKD-related activities. In this study, the effect of sub-acute (30-day) oral warfarin (2 and 1 mg L-1) intake on hematological parameters was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), that differ in their sensitivity to certain chemicals. Greater susceptibility to the anticoagulant effect of 2 mg L-1 of warfarin was observed in AO rats and was associated with an increase in the relevant hematological parameters in this strain. Although both strains responded to 2 mg L-1 of warfarin with quantitative changes in the peripheral blood leukocytes, differential bone marrow and lung responses were observed. Strain-related differences in the pro-inflammatory activity of peripheral blood granulocytes and in mononuclear cell IFN-γ production were observed. Recognition of differences in quantitative and qualitative effects of oral warfarin on processes other than hemostasis might be of relevance for those humans who are on warfarin therapy.
AB  - Varfarin (3-α-acetonilbenzil)-4–hidroksikumarin) je antagonist vitamina K (VK) koji inhibira procese zavisne od ovog vitamina, uključujući koagulaciju krvi. Osim toga, on ispoljava i aktivnosti koje ne zavise od vitamina K kao što su anti-tumorska i imunomodulatorna aktivnost. U ovom radu je ispitan efekat subakutnog (30 dana) oralnog unosa varfarina na hematološke parametre i aktivnost leukocita periferne krvi kod dva soja pacova Albino Oxford (AO) i Dark Agouti (DA) koji se raz- likuju u osetljivosti na iste hemijske agense. Kod jedinki AO soja zapažena je veća smrtnost nakon konzumiranja doze od 4 mg L–1 kao i veća osetljivost na antikoagulantno dejstvo varfarina pri nižim dozama (2 mg L–1) koje je praćeno povećanjem nekih hematoloških parametara. Iako kod jedinki oba soja dolazi do povećanja broja neutrofilnih leukocita periferne krvi pri dozi od 2 mg L–1, promene u osnovnim proinflamatornim aktivnostima ovih ćelija su zapažene samo kod jedinki DA soja. Promene u broju neutrofilnih leukocita u krvi DA jedinki su praćene povećanjem broja granulocitnih prekursora u koštanoj srži, dok prisustvo neutrofila u plućima AO jedinki ukazuje na razmenu ćelija između periferne krvi i plućnog intravaskularnog pula ćelija. Diferencijalne sojno–zavisne promene u aktivnosti mononuklearnih ćelija periferne krvi su takođe zapažene. Razlike u efektu oralno unetog varfarina mogu da imaju implikacije za osobe na oralnoj varfarinskoj terapiji.
T2  - Journal of the Serbian Chemical Society
T1  - Sojne razlike u toksičnosti antagoniste vitamina K varfarina kod pacova
T1  - Strain differences in the toxicity of the vitamin K antagonist warfarin in rats
IS  - 3
VL  - 78
SP  - 381
EP  - 394
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_566
ER  - 

@article{
author = "Đokić, Jelena and Ninkov, Marina and Popov Aleksandrov, Aleksandra and Mirkov, Ivana and Subota, Vesna and Mihajlović, Luka and Stojadinović, Marija and Stanić-Vučinić, Dragana and Kataranovski, Dragan and Kataranovski, Milena",
year = "2013, 2013",
abstract = "Warfarin (3-(α-acetonylbenzyl)-4-hydroxy coumarin) is a vitamin K (VK) antagonist that inhibits vitamin K-dependent (VKD) processes, such as blood coagulation. It also exerts an influence on some non-VKD-related activities. In this study, the effect of sub-acute (30-day) oral warfarin (2 and 1 mg L-1) intake on hematological parameters was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), that differ in their sensitivity to certain chemicals. Greater susceptibility to the anticoagulant effect of 2 mg L-1 of warfarin was observed in AO rats and was associated with an increase in the relevant hematological parameters in this strain. Although both strains responded to 2 mg L-1 of warfarin with quantitative changes in the peripheral blood leukocytes, differential bone marrow and lung responses were observed. Strain-related differences in the pro-inflammatory activity of peripheral blood granulocytes and in mononuclear cell IFN-γ production were observed. Recognition of differences in quantitative and qualitative effects of oral warfarin on processes other than hemostasis might be of relevance for those humans who are on warfarin therapy., Varfarin (3-α-acetonilbenzil)-4–hidroksikumarin) je antagonist vitamina K (VK) koji inhibira procese zavisne od ovog vitamina, uključujući koagulaciju krvi. Osim toga, on ispoljava i aktivnosti koje ne zavise od vitamina K kao što su anti-tumorska i imunomodulatorna aktivnost. U ovom radu je ispitan efekat subakutnog (30 dana) oralnog unosa varfarina na hematološke parametre i aktivnost leukocita periferne krvi kod dva soja pacova Albino Oxford (AO) i Dark Agouti (DA) koji se raz- likuju u osetljivosti na iste hemijske agense. Kod jedinki AO soja zapažena je veća smrtnost nakon konzumiranja doze od 4 mg L–1 kao i veća osetljivost na antikoagulantno dejstvo varfarina pri nižim dozama (2 mg L–1) koje je praćeno povećanjem nekih hematoloških parametara. Iako kod jedinki oba soja dolazi do povećanja broja neutrofilnih leukocita periferne krvi pri dozi od 2 mg L–1, promene u osnovnim proinflamatornim aktivnostima ovih ćelija su zapažene samo kod jedinki DA soja. Promene u broju neutrofilnih leukocita u krvi DA jedinki su praćene povećanjem broja granulocitnih prekursora u koštanoj srži, dok prisustvo neutrofila u plućima AO jedinki ukazuje na razmenu ćelija između periferne krvi i plućnog intravaskularnog pula ćelija. Diferencijalne sojno–zavisne promene u aktivnosti mononuklearnih ćelija periferne krvi su takođe zapažene. Razlike u efektu oralno unetog varfarina mogu da imaju implikacije za osobe na oralnoj varfarinskoj terapiji.",
journal = "Journal of the Serbian Chemical Society",
title = "Sojne razlike u toksičnosti antagoniste vitamina K varfarina kod pacova, Strain differences in the toxicity of the vitamin K antagonist warfarin in rats",
number = "3",
volume = "78",
pages = "381-394",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_566"
}

Đokić, J., Ninkov, M., Popov Aleksandrov, A., Mirkov, I., Subota, V., Mihajlović, L., Stojadinović, M., Stanić-Vučinić, D., Kataranovski, D.,& Kataranovski, M.. (2013). Sojne razlike u toksičnosti antagoniste vitamina K varfarina kod pacova. in Journal of the Serbian Chemical Society, 78(3), 381-394.
https://hdl.handle.net/21.15107/rcub_ibiss_566

Đokić J, Ninkov M, Popov Aleksandrov A, Mirkov I, Subota V, Mihajlović L, Stojadinović M, Stanić-Vučinić D, Kataranovski D, Kataranovski M. Sojne razlike u toksičnosti antagoniste vitamina K varfarina kod pacova. in Journal of the Serbian Chemical Society. 2013;78(3):381-394.
https://hdl.handle.net/21.15107/rcub_ibiss_566 .

Đokić, Jelena, Ninkov, Marina, Popov Aleksandrov, Aleksandra, Mirkov, Ivana, Subota, Vesna, Mihajlović, Luka, Stojadinović, Marija, Stanić-Vučinić, Dragana, Kataranovski, Dragan, Kataranovski, Milena, "Sojne razlike u toksičnosti antagoniste vitamina K varfarina kod pacova" in Journal of the Serbian Chemical Society, 78, no. 3 (2013):381-394,
https://hdl.handle.net/21.15107/rcub_ibiss_566 .