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Implementation of Dimer-based Screening System in Escherichia coli BL21(DE3) for Selection of Actinomycetes Compounds as Anti-HIV Candidate Kenia Permata Sukma; Putri Cahya Destiani; Azzania Fibriani
HAYATI Journal of Biosciences Vol. 29 No. 2 (2022): March 2022
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.29.2.192-203

Actinomycetes are reported to have inhibitory activity against several types of Human Immunodeficiency Virus proteases, enzyme with major role in the process of maturation of the virus thus it can infect new cells. Therefore, exploration of Indonesia’s actinomycetes species is expected to be a breakthrough for HIV treatment. In this study, selection of anti-HIV candidate compounds was conducted using a dimer-based screening system on recombinant Escherichia coli BL21(DE3). The construct includes the fusion of the AraC DNA binding domain + HIV-1 protease as the regulator and the green fluorescence protein as the reporter. Confirmation of the plasmid construct was carried out by PCR which showed size of ~1,076 bp. Sequencing analysis proved 100% similarity and identity between construct used in this study and one previously designed. SDS-PAGE showed the presence of band in the size of ~24 kDa equal to the size of the fusion protein. Compounds BLH 1-12 (2) EA, MAE 1-13 EA, BLH 1-1 EA, BLH 7-5 MetA, LC 98 (1) EA, exhibited consistent and significant protease-HIV inhibitory activity at certain concentrations. Thus, in this study, dimer-based screening system is considered to be able to detect actinomycetes as a new anti-HIV candidate for the protease inhibitor group.

Cloning and expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in Escherichia coli BL21(DE3) Fifi Fitriyah Masduki; Yeni Hotimah; Rosalia Rani; Arsyam Mawardi; Euniche R.P.F. Ramandey; Azzania Fibriani; Sony Suhandono
Acta Biochimica Indonesiana Vol. 2 No. 2 (2019): Acta Biochimica Indonesiana
Publisher : Indonesian Society for Biochemistry and Molecular Biology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.32889/actabioina.v2i2.39

Background: Immediate and accurate diagnosis of malaria is essential for effective control of this disease. Immunochromatographic based rapid diagnostic tests (RDTs) are economical, simple to perform, and provide results in a relative short time, can be useful to assist effective management of malaria. The commercially available malaria RDT in Indonesia is still imported. Therefore, an effort to produce malaria RDT independently is necessary. One of the biomarkers used in RDTs is Plasmodium lactate dehydrogenase pLDH. The production and accumulation of pLDH during asexual stage or blood-stage in all human infected malaria parasites can be used to indicate parasites viability, which is correlated with the number of parasites present in the plasma of infected patients. Objective: The aim of this research is to produce recombinant PfLDH in Escherichia coli BL21(DE3). Methods: PfLDH gene was cloned into pET30a expression vector to obtain a 6.2 kbp recombinant plasmid pET30a-PfLDH. E. coli BL21(DE3) was transformed with pET30a-PfLDH using the heat shock method. Then, E. coli BL21(DE3)- pET30a-PfLDH was cultured in LB broth containing 50 mg/mL kanamycin and was induced by 1mM IPTG at 37oC. Results: SDS-PAGE and Western Blot analysis showed that recombinant PfLDH was expressed with molecular mass ~30 kDa. Conclusion: Recombinant PfLDH is expressed in E. coli BL21(DE3) and can be used in further research for producing rPfLDH as a biomarker for malaria RDT development.

Selection of Indonesian Medicinal Plant Active Compounds as Inhibitor Candidates of Oncoproteins E6 and E7 Human Papillomavirus Type 16 by Molecular Docking Riyanti Weni Syafitri; Azzania Fibriani; Reza Aditama
3BIO: Journal of Biological Science, Technology and Management Vol. 3 No. 1 (2021)
Publisher : School of Life Sciences and Technology, Institut Teknologi Bandung

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5614/3bio.2021.3.1.2

Cervical cancer cases caused by infection with Human Papillomavirus (HPV), especially HPV 16 (60.5% of cases) continue to increase every year with a high mortality rate. The current anti-cancer drugs were not only specifically targeting cancer cells, but healthy cells and can cause serious side effects. Therefore, it is necessary to find safer alternative therapies, e.g., using active compounds from natural products. The purpose of this study was to find the active compounds of Indonesian medicinal plants potentially as an inhibitor of oncoprotein E6 and E7 HPV 16, the main protein causing cervical cancer by in silico method. In this study, 711 active compounds from 187 medicinal plant species were selected based on molecular weight, solubility, gastrointestinal absorption index, and drug-likeness. Compounds that meet the criteria were tested for their affinity and interaction profile with E6 and E7 proteins through the molecular docking method. The results of this study showed 164 compounds that met the criteria. The molecular docking analysis showed nine of the most potent compounds as E6 inhibitors on the E6AP binding site and six compounds on the p53 binding site. Besides that, there were eleven most potent compounds as E7 inhibitors. The results of this study indicate that there are natural compounds that can inhibit E6 and E7 proteins and have further potential to be used as anti-HPV drugs. However, further research is needed to test these compounds in vitro and in vivo.

Heterologous Production of Human Papillomavirus L1 Capsid Protein: Systematic Review and Meta-analysis Andre Hendrawan; Azzania Fibriani
3BIO: Journal of Biological Science, Technology and Management Vol. 4 No. 1 (2022)
Publisher : School of Life Sciences and Technology, Institut Teknologi Bandung

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5614/3bio.2022.4.1.5

The coverage of HPV vaccination in Indonesia remains low due to the high-cost vaccination. The vaccine prices were affected by the production rate of L1, the active substance of HPV vaccines. L1 has been produced using various organisms with varying L1 production rates and immunogenicity. A systematic review and meta-analysis were conducted to determine the organism producing L1 with the highest production, treatments affecting the L1 expression rate, and immunogenicity (represented by anti-L1 IgG titer in mice). The data of L1 titer, induction period, and IgG titer were extracted from 19 articles that have passed the articles screening. The L1 titer and induction period data were used to calculate the L1 production rate, while the IgG titer was used in the immunogenicity analysis. On a 95% confidence level, the meta-analysis revealed weak evidence that E. coli produced L1 at the highest rate. The highest IgG titer was induced using L1 expressed in Saccharomyces cerevisiae, albeit insufficient evidence on 95% confidence level. Pearson’s correlation analysis showed that the concentration of glucose, IPTG, NH4+, K+, Ca2+, Mn2+, Fe2+, Zn2+, B4O72−, H2PO4−, HPO42−, Mo7O246−, and citric acid had a positive correlation with L1 production rate in E. coli. The treatment injection doses positively correlated with IgG titer in S. cerevisiae. This study reveals the mineral salts as the potential treatments to increase L1 production rates.

Development of a dimer-based screening system for dimerization inhibitor of HIV-1 protease I Dewa Agung Panji Dwipayana; Yana Maolana Syah; Reza Aditama; Feraliana Feraliana; Azzania Fibriani
Journal of Microbial Systematics and Biotechnology Vol 2, No 2 (2020): December 2020
Publisher : Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.37604/jmsb.v2i2.42

An in vitro dimer-based screening system (DBSS) for selecting new HIV-1 protease dimerization inhibitor candidates from natural compounds had been established. This system utilizes a fusion between HIV-1 protease and dimer binding domain of AraC protein (proteaseHIV1-AraCDBD) where fluorescence signal will be emitted in the presence of HIV-1 protease inhibitor. However, this screening system had not been evaluated. Therefore, this study was aimed to evaluate it in recombinant Escherichia coli culture. The expression of proteaseHIV1-AraCDBD fusion gene was observed for 18 hours. Its crude lysate isolation was done once every 3 hours and analyzed using SDS PAGE. To test the DBSS, darunavir was used as positive control, and Nigella sativa extract (JH3) was used as the test compound. The results of SDS PAGE analysis on crude lysates presented a ~24.2 kDa band, which was the predicted size of the proteaseHIV1-AraCDBD fusion protein based on its amino acid sequence. The growth curve and protein expression profiles revealed that the 15 hours was the optimum culture age to be used in the screening system. Darunavir testing in DBSS showed an increase in fluorescence signal compared to the negative control. The same increase in fluorescence signal was also obtained from the JH3 compound test. In conclusion, DBSS could be used as an assay to screen for new HIV-1 protease inhibitors, and the JH3 compound demonstrated the ability to inhibit HIV-1 protease dimerization.

Analysis of the SARS-CoV-2 envelope (E), nucleocapsid (N), and non-structural protein12 (nsp12) genes from COVID-19 patients in West Java Azzania Fibriani; Irin Annisa Evitayani; Gusti Ayu Prani Pradani; Rebecca Stephanie; Ema Rahmawati; Ryan Bayusantika Ristandi; Cut Nur Cinthia Alamanda; Rifky Waluyajati Rachman; Rini Robiani; Isak Solihin
Journal of Microbial Systematics and Biotechnology Vol 3, No 1 (2021): August 2021
Publisher : Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.37604/jmsb.v3i1.66

According to World Health Organization, as of January 2021, Indonesia is the only Southeast Asian country in which COVID-19 is still occurring in community transmission. West Java is one of the provinces holding the highest positive cases number. With the envelope (E), nucleocapsid (N), and non-structural protein 12 (nsp12) being the target genes of SARS-CoV-2 diagnostic kits and several antiviral drugs, the study of genetic variations has become relevant and greatly important. Out of 267 oro-nasopharyngeal swab specimens that were previously confirmed positive for COVID-19 in qPCR diagnostic test in Laboratorium Kesehatan Provinsi Jawa Barat, ten samples with acceptable qualities were selected and three samples were sequenced using Sanger sequencing. Nonsynonymous mutations were observed in the envelope gene (L21F) and in the nucleocapsid genes (R203K, G204R, A211S, and S193I). Phylogenetic analysis showed that samples were clustered with other sequences carrying identical mutations, but clustered non-discriminatively with all sequences when carrying no mutation. No pattern in geographical areas and clades, except for R203K-G204R for being a marker for the GR clade. Protein structure analysis showed that mutations observed did not change the hydrophobicity and the secondary structure of the nucleocapsid, while stability change (ΔΔG) showed that all mutations, aside from the R203K-G204R, have neutral effect on the protein stability. Therefore, it can be concluded that mutations observed in this experiment did not impart preference to disperse in certain geographical areas or cause any significant structural change in the protein.

The Validity of Cross Priming Amplification to Detect SARS-CoV-2 Virus Luhung Budiailmiawan; Ryan Bayusantika Ristandi; Azzania Fibriani
INDONESIAN JOURNAL OF CLINICAL PATHOLOGY AND MEDICAL LABORATORY Vol 28, No 3 (2022)
Publisher : Indonesian Association of Clinical Pathologist and Medical laboratory

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24293/ijcpml.v28i3.1895

The standard molecular technique to detect the SARS-CoV-2 virus is The Real-Time Reverse-Transcription Polymerase Chain Reaction (rRT-PCR). It requires sophisticated equipment and a time-consuming sample process. The Cross Priming Amplification (CPA) is a nucleic acid amplification technique that amplifies DNA with high specificity and efficiency under constant thermal conditions. This technique is faster than rRT-PCR and doesn't require a biosafety level-2 (BSL-2) facility. The study aimed to determine the validity of CPA with rRT-PCR as a gold standard and to evaluate its performance as molecular rapid tests for detecting SARS-CoV-2 RNA from nasopharyngeal swab specimens. This study was a descriptive diagnostic test by using data retrospectively from swab nasopharyngeal patient samples who were treated at Palabuhan Ratu Hospital with COVID-19 from 01 January to 31 December 2021. The CPA was performed on a total of 52 nasopharyngeal samples at Pelabuhan Ratu Laboratory and rRT-PCR at Provincial Health Laboratory. The validity and correlation tests were performed. The majority of subjects were female between the ages of 34-50 years. The cut-off Tt-value is 3.25, 0.84 Area Under Curve (AUC), with a p-value <0.001. The CPA has good validity for COVID-19 diagnosis with 77% sensitivity, 94% specificity, 96% PPV, and 71% NPV. The sensitivity was increasing with Ct-value <30 (82%) and Ct-value <25 (87%). The CPA had a good validity for the COVID-19 diagnostic test. The CPA could be used as a rapid molecular test for detecting SARS-CoV-2 viral RNA from nasopharyngeal swab specimens.

The Validity of Cross Priming Amplification to Detect SARS-CoV-2 Virus Luhung Budiailmiawan; Ryan Bayusantika Ristandi; Azzania Fibriani
INDONESIAN JOURNAL OF CLINICAL PATHOLOGY AND MEDICAL LABORATORY Vol. 28 No. 3 (2022)
Publisher : Indonesian Association of Clinical Pathologist and Medical laboratory

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24293/ijcpml.v28i3.1895

The standard molecular technique to detect the SARS-CoV-2 virus is The Real-Time Reverse-Transcription Polymerase Chain Reaction (rRT-PCR). It requires sophisticated equipment and a time-consuming sample process. The Cross Priming Amplification (CPA) is a nucleic acid amplification technique that amplifies DNA with high specificity and efficiency under constant thermal conditions. This technique is faster than rRT-PCR and doesn't require a biosafety level-2 (BSL-2) facility. The study aimed to determine the validity of CPA with rRT-PCR as a gold standard and to evaluate its performance as molecular rapid tests for detecting SARS-CoV-2 RNA from nasopharyngeal swab specimens. This study was a descriptive diagnostic test by using data retrospectively from swab nasopharyngeal patient samples who were treated at Palabuhan Ratu Hospital with COVID-19 from 01 January to 31 December 2021. The CPA was performed on a total of 52 nasopharyngeal samples at Pelabuhan Ratu Laboratory and rRT-PCR at Provincial Health Laboratory. The validity and correlation tests were performed. The majority of subjects were female between the ages of 34-50 years. The cut-off Tt-value is 3.25, 0.84 Area Under Curve (AUC), with a p-value <0.001. The CPA has good validity for COVID-19 diagnosis with 77% sensitivity, 94% specificity, 96% PPV, and 71% NPV. The sensitivity was increasing with Ct-value <30 (82%) and Ct-value <25 (87%). The CPA had a good validity for the COVID-19 diagnostic test. The CPA could be used as a rapid molecular test for detecting SARS-CoV-2 viral RNA from nasopharyngeal swab specimens.

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