Optimization of multiplex PCR composition to screen for SARS-CoV-2 variants of concern

Background: The ongoing COVID-19 pandemic has led to the emergence of several variants of concern. To rapidly identify those variants, screening samples for whole-genome sequencing (WGS) prioritization could be performed. Objective: We optimized the polymerase chain reaction (PCR) screening method to identify the mutation in spike and ORF1a regions. Methods: We adopted primers targeting mutation in spike and ORF1a region from another study. We optimized the PCR screening method using kits readily available in Indonesia. Firstly, we compared N1 and N2 primers as internal positive control. We also compared GoTaq® 1-Step RT-qPCR System and Indonesia TFRIC-19 BioCOV-19 for the multiplex reaction. We used the optimized composition to screen SARS-CoV-2 positive samples from April – June 2021. Samples with spike and/or ORF1a target failure were subjected to whole genome sequencing (WGS). Results: The results demonstrated the N2 BioCOV-19 reaction as the optimized multiplex PCR composition for spike and ORF1a mutations screening. Whole-genome sequencing has shown that a sample with spike and ORF1a targets failure to be Alpha variant, while other samples with single target failure as non-variants of concern. Therefore, a multiplex RT-PCR composition has been optimized to detect mutation in spike and ORF1a regions. Conclusion: We have optimized a multiplex RT-PCR composition to detect mutation in spike and ORF1a regions.

The role of senescence-associated secretory phenotype (SASP) in cellular senescene

Cellular senescence is one of the defense mechanisms of cells against oncogenic signals by permanently stopping the proliferation of the cell. Senescence cells show a similar characteristic, one of them is senescence-associated secretory phenotype (SASP). SASPs secrete various components, divided according to the type of molecule secreted and based on their mechanism of action against target cells. The main components of SASP are pro-inflammatory mediators. SASP performs dual and contradictory roles, which concurrently provides beneficial effects such as tumor suppression due to the termination of proliferation, recruitment of immune cells, and tissue repair. On the other hand, SASP produces detrimental effects on cells undergoing the senescence process as well as cells in the surrounding environment by increasing tumorigenesis. This review article explains the various components of the SASP, the role of SASP in the inflammatory process, tumor suppression, and tumorigenesis.

Genetic polymorphism of ALDH2 in Indonesia’s Minang ethnic

Background: In some people, acetaldehyde, a toxic product from ethanol oxidation, cannot be oxidized to acetate. The excess of acetaldehyde could cause facial flushing, dizziness, and hypertension when they consume ethanol. This ethanol sensitivity is caused by a deficiency of ALDH2. Objective: This study aims to analyze and count the polymorphism frequency of the ALDH2 gene in Indonesia’s Minang ethnic. Methods: DNA samples were taken randomly from hair bulbous of 60 subjects (male and female, 3rd generation). A nested polymerase chain reaction was conducted to amplify the ALDH2 in the samples. Afterward, restriction fragment length polymorphism (RFLP) was conducted to the amplicons using the EcoRI restriction enzyme. The measured parameters were the distribution of the wildtype, atypical homozygote, and heterozygote. Results: Results showed that out of 60 subjects, 53.33% have an atypical homozygote gene (subjects prone to hypersensitive to alcohol), 28.33% have a heterozygote gene, and 18.33% have a wildtype gene. The frequency of the atypical alleles in Minang ethnic is 0.675. Conclusion: The atypical ALDH2 allele was much higher than the normal ALDH2 allele, in which most participants have atypical homozygote ALDH2, suggesting the samples are sensitive to alcohol.

Increased level of malondialdehyde in preterm labor

Background: The pathophysiological mechanism associated with spontaneous preterm delivery is oxidative stress through the increased formation of reactive oxygen species (ROS) due to lipid peroxidation. Malondialdehyde (MDA) is one of the biomarkers of oxidative stress produced through the lipid peroxidation process. Objective: The aim of this study is to observe the difference in MDA levels among preterm labor compared to full-term labor. Methods: Observational research was conducted with a comparative cross-sectional design. Maternal venous blood samples were taken from private hospitals and midwives in Padang city and Aro Suka Hospital Solok Regency. Samples were selected by consecutive sampling and divided into two groups with a total of 40 samples. MDA level was measured using the spectrophotometry method. Results: MDA levels in preterm delivery were 3,6±0.42 nmol/mL and in full-term delivery were 2.9±0.33 nmol/mL. Conclusion: There was a significant difference in MDA levels between preterm labor and full-term delivery. MDA levels in preterm childbirth were higher than MDA levels in full-term delivery.

Effect of glucose on reduced glutathione level in Malay uncomplicated type 2 diabetes patients

Background: Increasing blood sugar level may increase free radical compounds in type 2 diabetes. Free radical compounds can cause oxidative stress, thereby decreasing endogenous antioxidants such as reduced glutathione (GSH). Objective: This study aimed to determine whether random blood glucose levels affect GSH in type 2 diabetes patients within the Malay race. Methods: This study was observational with case-control, involving 25 patients with uncomplicated type 2 diabetes (receiving metformin and/or glimipiride) and 25 healthy controls. Random blood glucose levels were determined using ACCU-CHECK® Kit. Blood GSH levels were determined by Sigma GSH Assay Kit. Results: Results show that type 2 diabetes patients have a significantly lower random blood glucose level compared with those of age-matched normal subjects (p<0.0001). Type 2 diabetic patients had significantly lower levels of GSH (p=0.00) than those of age-matched normal subjects. We found a moderate negative correlation (r=-0.437 and p=0.02) between the level of random blood glucose and the level of GSH. Conclusion: The depletion of GSH during hyperglycemia may neutralize the free radicals indirectly generated by the abundant of glucose.

The effect of fermented tempeh aerobic anaerobic (FETAA) on pancreatic duodenal homeobox 1 (Pdx1) gene expression and HOMA-beta index in diabetic mice

Background: Diabetes is a result of oxidative stress which causes the impaired function of pancreatic beta-cells. Fermented tempeh aerobic anaerobic (FETAA) containing gamma-aminobutyric acid and isoflavones can reduce oxidative stress in diabetes. Objective: The aim of this study is to evaluate FETAA in improving pancreatic β-cell function in diabetic mice. Methods: Twenty streptozotocin-induced diabetic mice, divided into four groups (n = 5 each group): DM, DM + FETAA 10 mg/100 g BW, DM + FETAA 20 mg/100 g BW, DM + FETAA 40 mg/100 g BW as well as normal group (n = 5). DM mice were treated with FETAA for 21 days. Fasting glucose was determined using the GOD-PAP method, while insulin level was determined by ELISA. The homeostasis model assessment of β-cell function (HOMA-β) was calculated using the HOMA2 calculator, and the Pdx1 mRNA level was determined by Real Time-PCR. Results: The DM mice group treated with FETAA had lower glucose levels than the DM mice group. FETAA dosage of 40 mg/100 g BW was able to reduce the highest blood glucose levels (p<0.05). DM mice group treated with FETAA had higher levels of insulin and HOMA-β than the DM mice group (p <0.05). Treatment of FETAA 10 mg/100 g BW produced the highest insulin content of 57.44 ± 8.132 pmol/L, while treatment of FETAA 40 mg/100 g BW had a HOMA-β value of 72.86 ± 21.85%. Pdx1 mRNA expression in group FETAA-treated DM mice was higher than the DM mice group, although it was not statistically significant (p> 0.05). Conclusion: FETAA could improve HOMA-β, blood glucose levels, but did not affect Pdx1 mRNA expression.

In silico recombinant plasmid design of pHA171 with phdABCD insertion for ethidium bromide degradation

Background: Ethidium bromide is a common reagent that is used in nucleic acid staining. However, ethidium bromide has toxic and carcinogenic properties that are harmful to the environment. Phenanthrene dioxygenase (encoded by phdA, phdB, phdC, and phdD genes) in Nocardioides sp. KP7 can oxidize the phenanthridine structure aim to eliminate carcinogenic properties. Objective: This study aims to visualize and predict the structure, active site, and characteristics of the phenanthrene dioxygenase using bioinformatics tools. Methods: Plasmid design were prepared by inserting genes of interest phdA, phdB, phdC, and phdD from the NCBI database. Furthermore, several protein analysis tools were used for structure visualization, active site enzyme improvement, and protein characteristic of phenanthrene dioxygenase. Results: The prediction results found that phenanthrene dioxygenase reacts with the ethidium bromide substrate through the interaction of Fe3+ ions with water. The solubility level of phenanthrene dioxygenase protein is 0.404, suggesting that the protein has low solubility. The protein isoelectric point (pI) is between 5.17 to 5.36, and the protein molecular weight is 121.143 kDa. Conclusion: In silico analysis has supported that recombinant plasmid met characteristics for the construct which consists of gene interest and protein library.

CYTOTOXIC ACTIVITY AND SELECTIVITY INDEX OF BINAHONG (Anredera cordifolia) EXTRACTS ON MCF-7 BREAST CANCER CELLS AND VERO NORMAL CELLS LINE

Background: Breast cancers occur because of an impaired balance between proliferation, differentiation, and apoptosis of breast glands. Natural products have potency as cytotoxic agents with less side effects than chemotherapy. One of the potential plants is Anredera cordifolia (Ten.) Stennis (binahong), which contains flavonoid 8-glucopyranosyl- 4’,5,7-trihydroxy flavone compounds.Objective: This study aims to determine the potency of binahong leaves extract as an anticancer for breast cancer in vitro and in silico.Methods: Preliminary tests using molecular docking of 8-glucopyranosyl-4’,5,7-trihydroxyflavone compounds on Bcl-2 and HER-2 proteins. The extraction and fractionation were to obtain binahong extract. Thin layer chromatography to identify flavonoid compounds in the extract. DPPH assay was performed to evaluate the antioxidant activity. MTT assay was performed to evaluate cytotoxic activity on MCF-7 breast cancer cells and Vero cells.Results: In silico test showed a stable bond between 8-glucopyranosyl- 4’,5,7-trihydroxylflavone, and Bcl-2 and HER-2 with a docking score of -7.5 kcal/mol and -8.0 kcal/mol, respectively. The binahong extract contain flavonoid compounds that had the retention factor value 0.78; 0.49; 0.35. Antioxidant test resulted IC50 value of 4940 μg/mL. Cytotoxic test resulted in IC50 value of 1073 μg/mL and 486 μg/mL for Vero cells and MCF-7 breast cancer cells, respectively. The comparison between IC50 produced a selectivity index value of 2.149, which shows that binahong extract was selective against MCF-7 breast cancers.Conclusion: This study concluded that binahong extract has weak potency as anticancer agent on MCF-7 cells.

SUBACUTE TOXICITY TEST OF Rhizophora apiculata BARK EXTRACT ON LIVER AND PANCREAS HISTOPATHOLOGY OF RATS

Background: Rhizophora apiculata, one of the mangrove plant widely spread in Indonesia, can be developed as a medicinal plant. The extract of the bark has been found to have antioxidant and anti-inflammatory. However, the toxicity of Rhizophora apiculata has not been established yet.Objective: This research aims to evaluate the toxicity of ethanolic extract of Rhizophora apiculata bark on histopathological changes in rat’s liver and pancreas.Methods: Subacute toxicity study of the ethanol extract of Rhizophora apiculata bark was performed in healthy male rats by administering the extract at doses of 57, 114, 228, 456, and 918 mg/kg of body weight daily for 28 days. The subacute toxicity in rats was determined by histological analyses.Results: No significant adverse effect of the extract at dose 57 mg/kg was found. However at and over 114 mg/kg dose of the extract exhibited toxicities to the rats’ liver. In addition, the toxic effect appeared in rats’ pancreas at and over 228 mg/kg dose.Conclusions: Rhizophora apiculata bark extract showed no toxicity at or below 57 mg/kg. The ethanol extract from bark of Rhizophora apiculata showed toxicity at 114 mg/kg by subchronic toxicity.

ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY OF MANGROVE Brugueira gymnorrhiza STEM EXTRACTS AGAINST PATHOGENIC BACTERIA Vibrio cholerae

Background : The treatment of some diseases caused by free radicals and pathogenic bacteria usually by using antioxidants and antibiotics. Due to excessive use of antibiotics and other environmental cues, some bacteria are now resistant to certain antibiotics or even to multiple antibiotics. Some Vibrio cholerae bacterial strains are multiresistant to many antibiotics.Objective : The antioxidant and antibacterial activities of Brugueira gymnorrhiza stem extracts against pathogenic bacteria V. cholerae.Method : The B. gymnorrhiza stem was extracted by gradient maceration method. The DPPH method was used to determine the antioxidant activity and the disc diffusion method was used to determine the antibacterial activities. The column chromatography method was used to fractionate the selective extract with the best activity. The LC-MS/MS method was used to identify the compound obtained from the fraction with the best antioxidant and antibacterial activity.Result : Ethyl acetate extract of B. gymnorrhiza stem had the best antibacterial activity with MIC and MBC values of 62.50 mg/L. Ethyl acetate extract also showed the best value of antioxidant activity as indicated by an IC50 value of 255.03 mg/L. The results of fractions test showed that fraction 3 had the best antibacterial and the best antioxidant activities with both the MIC and MBC values of 7.90 mg/L and IC50 value of 348.91 mg/L, respectively.Conclusion : Ethyl acetate extract of B. gymnorrhiza stem has good potential as antioxidant and antibacterial. The compound which is thought as antioxidant and antibacterial from Ethyl acetate extract is 2-Ethyl-4-methyl-1H-imidazole.