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All Journal HAYATI Journal of Biosciences Jurnal Teknosains
Komang Alit Paramitasari
Department of Animal Production and Technology, Faculty of Animal Science, Bogor Agricultural University
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Civil Engineering, Building, Construction & Architecture Earth & Planetary Sciences Industrial & Manufacturing Engineering Mechanical Engineering

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Optimized condition for pei-based transient transfection of lifeact-gfp/nls-mcherry expressing plasmid used as cell barcode for syncytia live cell imaging Kumara, Dennaya; Harsan, Hayfa Salsabila; Novianti, Metta; Lestari, Dinda; Septisetyani, Endah Puji; Prasetyaningrum, Pekik Wiji; Paramitasari, Komang Alit; Meiyanto, Edy
Jurnal Teknosains Vol 13, No 1 (2023): December
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/teknosains.89479

Abstract

The transfection efficiency positively affects the successful plasmid DNA transfer into cells, with the highlight on the amount of plasmid DNA and its ratio to the transfection reagent. Polyethyleneimine (PEI) is a cost-effective transfection reagent that facilitates DNA transfer by forming positively charged DNA complexes. It allows DNA to interact with negatively charged cell surfaces and enter the cells by endocytosis. In this study, we optimized the condition for transient transfection of life act-GFP/NLS-mCherry-expressing plasmid in BHK-21 and 293T cells using PEI. This plasmid is helpful as a biosensor of the cytoskeleton and nucleus that enables live imaging observation using a fluorescence microscope, for instance, in the observation of syncytium. Here, we optimized two independent variables: the amount of DNA (0.5 and 1 µg) and the ratio of DNA-PEI (1:3 and 1:4). GFP and mCherry expressions were observed at 24, 48, and 72 h post-transfection. As a result, transfection efficiency achieved by using PEI in 293T cells is higher than in BHK-21 cells, which are ~90% and ~50%, respectively. Moreover, amongst four different transfection conditions, in both cell lines, 1 µg of plasmid DNA with a 1:3 DNA-PEI ratio yields the most efficiency with the least amount of toxicity. We used this condition for the syncytia observation in 293T cells as a model of the cell-to-cell transmission of SARS-CoV-2. Syncytia formation was successfully observed by detecting the giant cells expressing GFP/mCherry with multiple nuclei.
Naringin Effect on SARS-CoV-2 Pseudovirus Entry and Spike Mediated Syncytia Formation in hACE2-overexpressing Cells Septisetyani, Endah Puji; Prasetyaningrum, Pekik Wiji; Paramitasari, Komang Alit; Suyoko, Ahmad; Himawan, Alayna Lillahida Indri; Azzahra, Salsabila; Wisnuwardhani, Popi Hadi; Anam, Khairul; Ramadani, Ratna Dwi; Santoso, Adi; Ningrum, Ratih Asmana; Herawati, Neng; Rubiyana, Yana
HAYATI Journal of Biosciences Vol. 31 No. 2 (2024): March 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.2.336-347

Abstract

A molecular docking study demonstrates the interaction between naringin, a citrus flavonoid, with SARS-CoV-2 spike RBD. Nevertheless, in vitro investigation of the inhibitory effect of naringin on SARS-CoV-2 entry and syncytia models has yet to be carried out. We synthesized VSV∆G-GFP/Spike* pseudovirus (PSV) as a SARS-CoV-2 model by pseudotyping VSV∆G-GFP/S* in BHK-21 cells overexpressing the SARS-CoV-2 spike glycoprotein. In the SARS-CoV-2 PSV entry assay, we utilized CHO-K1 cells transfected with hACE2 plasmid, which were then treated with naringin and SARS-CoV-2 PSV/naringin. After 16-18 h incubation, PSV internalization represented by the GFP signal was observed under a fluorescence microscope. Immunofluorescence staining was also performed to probe the SARS-CoV-2 spike and confirm the PSV entry. We performed a syncytia assay using 293T cells co-transfected with SARS-CoV-2 spike/hACE2. Six hours after transfection, the cells were treated with naringin and incubated for another 16-18 hours. Then, we observed syncytia using a phase contrast microscope. Based on fluorescence foci quantification, the results indicated that naringin might inhibit SARS-CoV-2 PSV entry at a concentration of 100 µM (P<0.05). However, naringin did not prevent syncytia formation compared to solvent control. These PSV entry and syncytia assay results suggested that naringin potentially inhibited SARS-CoV-2 viral infection but not cell-to-cell viral transmission.
Co-Authors Anam, M. Khairul Azzahra, Salsabila Edy Meiyanto Endah Puji Septisetyani, Endah Puji Harsan, Hayfa Salsabila Himawan, Alayna Lillahida Indri Kumara, Dennaya Lestari, Dinda Neng Herawati, Neng Novianti, Metta Popi Hadi Wisnuwardhani, Popi Hadi Prasetyaningrum, Pekik Wiji Ramadani, Ratna Dwi RATIH ASMANA NINGRUM Santoso, Adi Suyoko, Ahmad Yana Rubiyana, Yana