The Journal of Experimental Life Science

Optimization of Real Time-Polymerase Chain Reaction (RT-PCR) Annealing Temperatures for the Detection of Superoxide Dismutase (SOD) in Wistar Rat (Rattus novergicus) Liver

Erryana Martati — 2024-06-30

Free radicals exposure causes oxidative stress in the human body and leads to various diseases, including cardiovascular, neurodegenerative disorders, cancer, diabetes, and aging. The endogenous enzymatic antioxidant superoxide dismutase (SOD) acts directly to reduce free radicals, and thus, its levels in tissue organs represent an important biomarker for oxidative stress in humans. One of the most reliable methods for detecting SOD is real-time polymerase chain reaction (RT-PCR). Still, the annealing temperatures and their results can vary widely depending on the samples. This study aims to optimize the annealing temperature of RT-PCR to detect SOD levels in liver tissue from Wistar rats (Rattus norvegicus). Total RNA was extracted from the liver tissue of one healthy Wistar rat using a cell lysis reagent. Purified RNA was reverse-transcribed into cDNA. The RT-PCR annealing temperature was optimized for detecting the expression of SOD with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference housekeeping gene. The optimum RT-PCR annealing temperature for detecting SOD was 50°C, and GAPDH was 60°C. The optimization of annealing temperatures in RT-PCR is essential to obtain single peak readouts (higher specificity) and lowest Ct values possible (higher sensitivity).

Keywords: Annealing temperature, Free radicals, RT-PCR, Superoxide dismutase, Wistar rat liver.

Identification of Microplastics in The Outer Ambon Bay, Mollucas

Vanela Chatrin Lekatompessy — 2024-06-30

Marine plastic debris that enters the sea can be fragmented by physical and chemical factors, then float in the water column or accumulate in sediments, which have the potential to be ingested by marine biota, causing digestive system disorders, fecundity, eating capacity, reproduction, and death. This study aimed to identify the accumulation of microplastics in water and sediments in Outer Ambon Bay using a purposive sampling method and descriptive analysis for data related to microplastics' type, color, and size, as well as follow-up tests of the least significant difference. The results show that the waters of Outer Ambon Bay have been polluted by microplastics. The number of microplastic particles found in water samples at three different depths (0, 50, and 100 cm) was 201 particles L^-1, and in sediment samples, it was 325 particles g^-1. It is supported by the fact that the type of fiber has a thin shape and size and can float on the surface of the water and by the existence of beaches where there are fishing activities, boat ports, rivers, and densely populated areas, as well as sandy and muddy sediments that can trap more microplastics during the tidal period. Therefore, it is necessary to prevent the waste problem in the waters of Ambon Bay.

Keywords: Identification, Marine Pollution, Microplastics, Outer Ambon Bay.

Purple Roselle Fortified Yogurt Supplementation Prevents Dioxin-Induced Oxidative Testicular Impairment in Rats

Ani Setianingrum — 2024-06-30

Dioxins are highly toxic and hurt multiple organs and systems, including the reproductive organs. This study determined the supplementation of purple roselle (Hibiscus sabdariffa var. altissima) extract into yogurt to prevent testicular deterioration induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The fortification of purple roselle extract into yogurt (RY) was used as a supplement because bioactive components are known to be efficacious in preventing cell damage. Five groups of male Wistar rats were group as follows: 1) negative control (C), 2) TCDD (T), 3) TCDD and 0.5% RY (P₁), 4) TCDD and 1% RY (P₂), 5) TCDD and 1.5% RY (P₃). All treatments were carried out orally for 12 days. Testicular Malondialdehyde (MDA) level and histopathological changes were described quantitatively by one-way ANOVA. The study's results showed that treatment groups P₁ (0.5% RY) and P₂ (1% RY) had significantly (P<0,05) lower MDA levels than the T group, while group P₃ had no significant decrease in MDA level. It proved that supplementation of purple roselle yogurt reduced the formation of MDA in testicular tissue. Histopathology changes show that all groups exposed to TCDD have depleted spermatogenic, Sertoli, and Leydig cells compared to the C group. Suggests that dioxin exposure can have adverse effects on male reproductive organs. This research concludes that supplementing yogurt fortified with purple roselle extract can prevent increased testicular MDA levels. The decrease in testicular cells due to TCDD toxicity was inevitable. However, antioxidant activity from roselle yogurt supplementation can prevent further damage in rat testicular cells.

Keywords: Histopathology, MDA, Purple Roselle, Testis, TCDD, Yogurt.

Effect of Ethyl Methane Sulfonate on Bulbil Explant Growth and In Vitro Shoot Formation in Porang (Amorphophallus muelleri Blume)

Vera Febriyanti — 2024-06-30

Porang is one of the plant commodities with high economic value due to its high glucomannan content in the tubers. Currently, the global demand for porang tubers continues to rise, but domestic production has not yet met this demand due to the limited supply of superior porang seeds. Mutation breeding with Ethyl Methane Sulfonate (EMS) can be one of the alternative methods for developing superior porang to enhance tuber production. This study aims to determine the response of porang bulbil explants to EMS mutagen treatment in vitro. The research used a Randomized Complete Block Design consisting of six EMS concentrations (0, 0.02, 0.04, 0.06, 0.08, and 0.1%). The research stages included pre-culture of bulbil explants on MS + BAP 3 mg.L^-1 + NAA 0.1 mg.L^-1 for two weeks in dark conditions and EMS mutagen treatment on bulbil cultures for four weeks. The results showed that adding EMS mutagen to the medium for four weeks caused the explants browning, inhibited shoots forming, and decreased the fresh weight and growth index. The higher the EMS concentration in the medium, the increased percentage of browning explants and reduced explant growth and shoot formation. The EMS concentration (≥0.06%) inhibited explant growth and shoot formation. While EMS concentration (0.02-0.04%) increased explant growth, shoot formation was not significantly different from control, even though there was an increase. Up to four weeks of culture, there was no explant death, so LD₅₀ is not yet known. The LD₅₀ EMS can also be calculated by explant browning and shoot proliferation. Based on explant browning and shoot proliferation, the LD50 EMS in porang bulbil explants was 0.07% and 0.09%. The EMS concentration and length of culture age need to be increased to obtain an LD₅₀ survival rate.

Keywords: Amorphophallus muelleri Blume, bulbil, EMS, In Vitro mutagenesis.

Resistance Test of Potential Mercury-Reducing Bacteria Using Agar Plate Method

Fitri Yola Amandita — 2024-07-30

The restoration of mercury-contaminated sites is a global concern, and countless studies have been conducted to formulate the best remediation practice. Mercury pollution in Indonesia has been a national issue. Thus, the government has enforced the reduction and elimination of this toxic and persistent chemical element and its compounds since 2019. As claimed to be the most environmentally friendly and cost-efficient technique, bioremediation using indigenous bacteria offers excellent potential to be implemented in developing countries such as Indonesia. This study is a preliminary part of four-year research at formulating a bioremediation technique to restore mercury-contaminated soils, especially in agricultural areas. In this investigation, fifteen indigenous bacterial isolates were tested for their resistance to mercury using two different techniques: the agar plate method, namely the disk-diffusion technique, and a modified version of the cup-plate technique. Inhibitory zone diameter (IZD) was measured and compared using Bland-Altman analysis between two techniques with supplementation of HgCl₂ 2-100 ppm range to the Mueller-Hinton agar media. The disk-diffusion technique appeared to give inconsistent measurements of IZD along the increase of HgCl₂ concentration, in which the highest IZD mean was obtained from samples with 8 and 10 ppm HgCl₂. Meanwhile, the cup-plate technique shows a lower average of IZD compared to the disk-diffusion technique. This report provides insights on the resistance of potential bacteria for mercury bioremediation. Further study should consider the accuracy of the agar plate method to test the bacterial resistance to any particular compound.

Keywords: bioremediation, cup-plate, disk-diffusion, inhibitory zone diameter, method accuracy.

An Assessment of Polar Solvent Extraction on Free Scavenging Activity of Sonneratia caseolaris

Feni Iranawati — 2024-08-26

Degenerative diseases such as stroke and cancer arise due to oxidative stress by a large number of reactive oxygen species (ROS) in the body. Free radical scavenging (antioxidant) compounds are needed to neutralize the ROS. The natural antioxidant potential of mangrove species Sonneratia caseolaris has been widely explored. However, its extraction is usually carried out with polar solvents such as methanol. Besides being more toxic than ethanol, methanol can cause blindness and death when inhaled in large quantities. Additionally, Indonesia, as the country with the largest Muslim population, requires the use of halal products for food and cosmetics. This study aims to analyze the scavenging activities of S. caseolaris with methanol and distilled water as the polar solvents for extraction. Antioxidant activity was tested with the free radical assay method of DPPH (2,2-diphenyl-1-picrylhydrazyl radicals). The test results were calculated as the IC₅₀ value, a concentration capable of reducing free radicals by 50%. The antioxidant activity test showed that the IC₅₀ values of methanol and distilled water were 18.9 ppm and 36 ppm, respectively. Therefore, both are considered very strong antioxidants. It indicates that distilled water is an acceptable substitute for methanol in the extraction process.

Keywords: antioxidant, extraction, mangrove, solvent, Sonneratia.

Prevalence of Anemia, Iron Level, Total Iron Binding Capacity Level, and Body Mass Index of Medical Students Living in Hostels in South India

Kalimuthu Senthilkumar — 2024-08-29

Studies have evidenced that hostel-staying medical students are at increased risk for anemia due to improper diet and long periods of studies that ultimately affect their performance. The nature of medical education, student efforts, and stress also raise the risk of anemia. The objective of this study is to find out the prevalence of anemia in medical students and iron levels, total iron binding capacity (TIBC) levels, and body mass index (BMI) in residing medical students in the hostel. This study is a descriptive cross-sectional study conducted at the Swamy Vivekanandha Medical College Hospital and Research Institute (SVMCHRI). The Institutional Ethical Committee (IEC) was obtained from IEC-SVMCHRI. The total number of students who participated in this study was 115 first-year medicine students (female = 76, male = 39) aged 18-24 years and were boarded in the hostel. Samples were collected after obtaining informed consent from students. Demographic parameters such as name, age, sex, and anthropometric parameters including weight, height, and BMI were recorded. Haemoglobin (Hb) was analyzed by a cell counter analyzer, iron levels, and TIBC levels were measured by a semi-autoanalyzer, and a blood smear study was performed. Anemia prevalence was higher in females than males; therefore, statistical analysis for male and female associations was observed through the Chi-squared test. The Hb cut-off followed the anemia classification per the World Health Organization (WHO). Overall, students had severe anemia (0.9%), moderate (8.7%), and mild (26.1%) anemia in hostels studying medicine. Females had a higher prevalence of anemia than males. The comparison of Hb, iron levels, and TIBC levels between men and women was highly significant: Hb 14.93 and 11.85 g.dL^-1 in men vs. women (P<0.001); iron levels 122.07 and 77.5289 µg.dL^-1 in men vs. women (P<0.001); and TIBC levels 323.02 and 349.92 µg.dL^-1 in men vs. women (P<0.008), consecutively. BMI was not significant between male and female associations. Overall, 18 participants had microcytic and hypochromic anemia in the blood smear study. This research finding concludes that the anemia prevalence is more common in female medical students with a tendency to microcytic and hypochromic anemia. Therefore, they may suffer from anemia due to nutritional deficiencies and benefit from iron supplementation.

Keywords: Anemia, Iron, Hemoglobin, Microcytic and Hypochromic, Total Ion Binding Capacity.