ABSTRACT
PURPOSE: Quantitative PCR (qPCR) is a reliable and robust technique for gene expression analysis, but its efficacy is dependent on the normalization of qPCR data with the stably expressed reference gene. Selection of a suitable reference gene is mandatory for accurate gene expression analysis, till data the most appropriate reference gene during chikungunya virus infection has not been elucidated. METHOD: In this study the expression of reference genes(GAPDH, GUSB, HPRT, Beta-actin, 18S rRNA) was analysed during chikungunya virus infection by quantitative PCR. The stability of the house-keeping genes was evaluated with three bioinformatics softwares: BestKeeper, NormFinder and GeNorm. RESULT: The significant variation in the expression of house-keeping genes (GusB, Beta-actin, HPRT) was observed during chikungunya virus infection; whereas GAPDH and 18S rRNA was most stable. The stability of reference genes analysed by the bioinformatics software further corroborate the results of qPCR. CONCLUSION: This is first study that identifies and validates the most suitable reference gene for normalization of qPCR data during chikungunya based gene expression analysis. This could serve as a reference study for the researchers working on different aspects of chikungunya virus infections.
Subject(s)
Chikungunya Fever , Humans , Genes, Essential , Actins , RNA, Ribosomal, 18S/genetics , Hypoxanthine Phosphoribosyltransferase , Real-Time Polymerase Chain Reaction/methods , Gene Expression Profiling/methods , Reference StandardsABSTRACT
Abstract: Viruses invade the host cells and maneuver the cellular translation machinery to translate the viral proteins in substantial amounts, which may disturb Endoplasmic Reticulum homeostasis leading to induction of Unfolded Protein Response (UPR), a host response pathway involved in viral pathogenesis. Here, we investigated the effect of UPR pathways on the pathogenesis of chikungunya virus infection. We observed that chikungunya virus mediated the modulation of UPR. A positive modulation was observed in the activation of IRE1 and ATF6 branch while the PERK branch of UPR observed suppressed upon virus infection. We further investigated the effect of the inhibition of UPR pathways on chikungunya virus replication using inhibitors for each branch. Cells treated with 3-ethoxy-5,6-dibromosalicylaldehyde (IRE1 inhibitor) and AEBSF (ATF6 inhibitor) significantly inhibits the viral replication process. This study has provided a novel perspective in designing antivirals against chikungunya virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01046-5.
ABSTRACT
Unfolded protein response (UPR) is an evolutionarily conserved pathway triggered during perturbation of endoplasmic reticulum (ER) homeostasis in response to the accumulation of unfolded/misfolded proteins under various stress conditions like viral infection, diseased states etc. It is an adaptive signalling cascade with the main purpose of relieving the stress from the ER, which may otherwise lead to the initiation of cell death via apoptosis. ER stress if prolonged, contribute to the aetiology of various diseases like cancer, type II diabetes, neurodegenerative diseases, viral infections etc. Understanding the role of UPR in disease progression will help design pharmacological drugs targeting the sensors of signalling cascade acting as potential therapeutic agents against various diseases. The current review aims at highlighting the relevance of different pathways of UPR in disease progression and control, including the available pharmaceutical interventions responsible for ameliorating diseased state via modulating UPR pathways.
Subject(s)
Diabetes Mellitus, Type 2 , Diabetes Mellitus, Type 2/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Humans , Signal Transduction , Unfolded Protein ResponseABSTRACT
AIMS: The aim of this study is to compare in vitro the fracture resistance of the endodontically treated tooth restored with a novel design Sharonlay, with the two component restorative method, i.e., post with separate onlay, and onlay without post. SUBJECTS AND METHODS: 45 single-rooted mandibular second premolars were taken, and root canal treatment was performed. Teeth were then randomly divided into three groups (n = 15) based on the type of restoration given: Group I - metal onlay with cast post extension (Sharonlay), Group II - metal onlay with prefabricated metal post (Mani post system), Group III - metal onlay over endodontically treated tooth obturated with Gutta-percha (control group). Fracture resistance was checked using the Instron universal testing machine and the fracture patterns were analyzed. RESULTS: According to the values recorded, Group I showed maximum mean fracture resistance followed by Groups II and III. Statistically significant difference was found between Groups I and II and Groups I and III and statistically significant difference was found between Groups II and III. CONCLUSION: A single unit component Sharonlay, gives higher fracture resistance to a premolar as compared to (a) metal onlay with prefabricated metal post and (b) metal onlay over endodontically treated tooth.
