Background: The COVID-19 disease caused by the SARS-CoV-2 virus has diverse symptoms, ranging from asymptomatic, mild symptoms such as flu-like illness and pneumonia to acute respiratory distress syndrome, which ends in death. Until now, the mechanism of the COVID-19 disease that causes widespread symptoms and the severity and factors that influence it are still unclear. During viral internalization, it needs to be cleaved by the serine protease encoded by the TMPRSS2 gene. It is hypothesized that higher expression of the TMPRSS2 gene causes higher virus internalization into cells, leading to more severe symptoms in patients. Methods: The Single Nucleotide Polymorphism Genotype Test was carried out to prove whether the TMPRSS2 gene affects the severity of COVID-19, as evidenced in other viral respiratory diseases. With a better understanding of gene expression related to this disease, it is hoped that we can better understand the mechanism of COVID-19 and establish better therapies and prevention against it. In this study, 68 COVID-19 patients participated and were categorized into two groups based on their clinical symptoms, namely mild symptoms without symptoms (n=12) and Moderate-Severe symptoms (n=56). PBMC cells were isolated from the patient. Then the DNA was extracted and used as a template in the SNP Genotyping of the TMPRSS2 rs2070788 gene variant. Results: The results showed that 35 samples had A/A homozygous genotypes, 29 A/G heterozygous samples, and 4 G/G homozygous samples. n=29) and heterozygous A/G (n=23), whereas only 4 were homozygous G/G. In addition, the homozygous G/G genotype was only detected in the moderate-severe group. Conclusions: A more significant number of samples from the asymptomatic mild symptom group is needed to statistically prove that homozygous G/G variants or G alleles are generally associated with the severity of COVID-19 patients.

COVID-19 is caused by the SARS-CoV-2 infection that attacked the human respiratory system. In severe conditions, it causes pneumonia, kidney failure, acute respiratory distress syndrome (ARDS), and even death. The SARS-CoV-2 infection triggers the immune cells to secrete an excess of proinflammatory cytokines lead to cytokine storm. It is believed to become one of the mechanisms that cause the ARDS condition. The level of pro-inflammatory cytokines will differ with each case severity. This study aimed to evaluate the profile of pro-inflammatory cytokines in COVID-19 patients with different severity. Therefore, it could be used as therapeutic approach for cytokine storm conditions. It was a cross sectional study using plasma samples of COVID-19 patients from Jakarta Islamic Hospital, Pondok Kopi and Dr. M. Goenawan Partowidigdo Hospital, Cisarua, Indonesia. The COVID-19 patients with severe (n=20) and mild to moderate (n=25) severity were involved in this study. As negative control plasma sample from healthy subjects (n=13) was used. Plasma IL-6 levels were measured using the ELISA technique and plasma lymphocyte levels were measured using a hematology analyzer. The results showed that no significant difference between severity and gender was observed (p=0.256). Meanwhile, there is a significant difference in IL-6 level between negative control, mild-moderate, and severe categories (p=0.015). The average IL-6 level in severe categories was higher than mild-moderate and negative control categories, with values 105.375, 59.75, and 64.577 pg/mL, respectively. This result becomes supporting evidence that there is a cytokine storm condition in severe COVID-19 patients. Furthermore, the lymphocyte level in the severe group is significantly lower than the mild to moderate group. This result may indicate lymphocytopenia in the severe group.

Type 2 diabetes mellitus, the most common diabetes type characterized by hyperglycemia, is caused by abnormal secretion and activity of pancreatic insulin enzymes. The extracellular matrix (ECM) plays a vital role in keeping β pancreatic cells intact and undissociated. The ECM in the pancreas can play a role in influencing insulin function and production. The most abundant ECM in the pancreas is collagen type VI. Collagen type VI has an essential role in the survival of pancreatic islet cells, including pancreatic β cells. Nowadays, Polymerase Chain Reaction (PCR) technology is widely utilized for molecular biology analysis. One of the most critical factors for successful PCR is designing the correct specific PCR primers. The objective of this study was to design a specific primer for collagen VI in the pancreas of Rattus norvegicus. The primer was designed and analyzed using MEGA.11, primer three-plus, and primer-BLAST. Five primer pairs were analyzed based on the characteristics of primer length, product amplicon length, Tm value, GC percentage, and secondary structure. Primer pair 3 (F:5’-TGTTTGGCTTTGTCGCGGGC-3’ and R:5’-TTGTTGCTGCCGACACTGGC-3’); Col6a2 (F:5’-TGTGGTCAACAGGCTGGGCG-3’ and R:5’-TCTGGCGCCGGCTCTCTTTG-3’) were considered as the best primer for the Collagen VI expression detection from the pancreas of Rattus norvegicus, which produce amplicon about 250pb and 245pb, respectively.

BACKGROUND Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like-3G (APOBEC3G) can abolish HIV infection by inducing lethal mutations in the HIV genome. The HIV protein virion infectivity factor (Vif) can interact with APOBEC3G protein and cause its degradation. Development of a method that can screen substances inhibiting the APOBEC3G-Vif interaction is necessary for identification of substances that potentially used in anti-HIV drug development. In order to increase expression of recombinant APOBEC3G protein that will be used in APOBEC3G-Vif interaction assay, we developed an optimized APOBEC3G gene for expression in Escherichia coli.  METHODS The gene coding APOBEC3G was codon-optimized in accordance with prokaryotic codon using DNA 2.0 software to avoid bias codons that could inhibit its expression. The APOBEC3G gene was synthesized and sub-cloned into pQE80L plasmid vector. pQE80L containing APOBEC3G was screened by polymerase chain reaction, enzyme restriction, and sequencing to verify its DNA sequence. The recombinant APOBEC3G was expressed in E. coli under isopropyl-β-D-thiogalactoside (IPTG) induction and purified by using nickel-nitrilotriacetic acid (Ni-NTA) resin.  RESULTS The synthetic gene coding APOBEC3G was successfully cloned into the pQE80L vector and could be expressed abundantly in E. coli BL21 in the presence of IPTG.  CONCLUSIONS Recombinant APOBEC3G is robustly expressed in E. coli BL21, and the APOBEC3G protein could be purified by using Ni-NTA. The molecular weight of the recombinant APOBEC3G produced is smaller than the expected value. However, the protein is predicted to be able to interact with Vif because this interaction is determined by a specific domain located on the N-terminal of APOBEC3G. 

Type 2 Diabetes Mellitus (T2DM) is one of the major health issues in the world. The cellular mechanism of T2DM is still not fully understood. It could be studied by using spheroid three-dimensional (3D) culture which is considered representative of the in vivo conditions. Several types of pancreatic β cell lines have been used, one of which is the insulin-GLase (iGL) cell line. This study aims to evaluate the effect of cell density and incubation time on spheroid morphology and cell viability in order to understand which one can be considered as the best option in studying T2DM using iGL cell. Spheroid was made by using the Hanging drop method. The variations of initial seeding cells were 50, 100, 200, and 400 cells/µL then incubated for 1, 2, 3, and 4 days. The evaluated parameters in this study are spheroid morphology and cell viability. Spheroid morphology was observed by using inverted phase contrast microscope integrated with camera (Nikon) and NIS-Elements Analysis D software. Cell viability was determined by using LUNA-II™ Automated Cell Counter (Logos Biosystem). The result of this study showed that spheroid in all of the group cell concentration have formed since the first day and its diameter was significantly increased on the following days (p<0,05). The spheroid size was positively correlated with the cell density in group 50-200 cells/µL. A single and stable spheroid morphology was observed in 50-100 cells/µL group. Cell viability in 3D culture system was lower and significantly decreased since day 3 compared to 2D culture (p <0.05; 0.01). In conclusion, spheroid derived from iGL cell line with a stable morphology and good viability could be obtained from a cell concentration of 50-100 cells / µL with two days of incubation.