Interferons (IFN) are antiviral cytokines that mitigate the effects of invading viruses early on during the infection process. SARS-CoV and MERS induce weak IFN responses; hence, the clinical trials which included recombinant IFN accompanied with other antiviral drugs exhibited improved results in terms of shortening the duration of illness. The aim of the present study was to evaluate the type I IFN response in COVID-19 patients to determine whether it is sufficient to eliminate or reduce the severity of the infection, and whether it can be recommended as a potential therapy. Total RNA samples were converted to cDNA and used as templates to evaluate the gene expression levels of IFN regulatory factor (IRF)3 and IFN-β in COVID-19 patients or control. The results showed that IRF3 gene expression was upregulated ~250-fold compared with the negative samples. In contrast, IFN-β expression increased slightly in COVID-19 patients. Consistent with other coronaviruses, such as SARS-CoV and MERS, COVID-19 infection does not induce an efficient IFN response to reduce the severity of the virus. This may be attributed to an incomplete response of IRF3 in activating the IFN-β promoter in the infected patients. The results suggest IFN-β or α may be used as potential treatments.
Background The newly emerged technology, nanotechnology, represents a promising solution for many medical and industrial problems. Random targeting, resistance, and side effects are the main disadvantages of the available cancer chemotherapy which are critical aspects needed to be managed. So the aim of the study was to suggest the nanoparticles as an alternative therapy for the available therapies through detecting the cytotoxic effect of Ag nanoparticles against cancer and normal cell lines and how they affect the apoptotic function and the genes involved. Results Ag NPs exhibited a killing rate of 40% in MCF-7 cells (the cancer cell model) at a concentration of 100 μg/ml with almost no effect on Vero cells (the normal cell model). Concerning the phenotypic apoptotic changes that were analyzed by Acridine orange and eosin and hematoxylin, Ag NPs caused the apoptosis and Vacuole degeneration as well as cell formation and the emergence of Necrotic cells in MCF-7 cells, whereas in the normal cell line Vero, no change appears in its phenotype. Treating MCF-7 and Vero cells with Ag NPs upregulated the P53 and P21 gene expression in Vero cells, but their expression was downregulated in MCF-7 cells. PTEN was augmented in both MCF-7 and Vero cells compared to the control. Conclusions The AgNPs displayed selective effect in their cytotoxicity and both induced the apoptosis effect and might be suggested as a potential therapy since an increase in PTEN expression (up to 250-fold more compared to the control) due to the treatment with AgNPs augments the tumor suppressor effects of the PTEN.
The late phase of adenovirus gene expression is controlled by proteins made in the intermediate phase, including L4 proteins of 22,000- and 33,000-Da apparent molecular mass (L4-22K and -33K proteins) that are expressed initially from the L4 promoter (L4P). The L4P is activated by a combination of viral proteins and cellular p53 and is ultimately inhibited again by its own products. Here, we have examined the L4P of human adenovirus type 5 in detail and have defined its transcription start site, which our data suggest is positioned by a weak TATA box. Rather than contributing positively to promoter activity, a putative initiator element at the transcription start site acts as a target for negative regulation imposed on the L4P by cellular TFII-I. We show that this TFII-I inhibition is relieved by one of the previously defined viral activators of the L4P, the E4 Orf3 protein, which alters the pool of TFII-I in the cell. We also explore further the negative regulation of the L4P by its products and show that the L4-33K protein is more significant in this process than L4-22K. It is the combined actions of positive and negative factors that lead to the transient activation of the L4P at the onset of the late phase of adenovirus gene expression. IMPORTANCE The adenovirus replication cycle proceeds through multiple phases of gene expression in which a key step is the activation of late-phase gene expression to produce proteins from which progeny particles can be formed. Working with human adenovirus type 5, we showed previously that two proteins expressed from the L4 region of the viral genome perform essential roles in moving the infection on into the late phase; these two proteins are produced by the action of a dedicated promoter, the L4P, and without them the infection does not proceed successfully to progeny generation. In this new work, we delineate further aspects of L4P activity and regulation. Understanding how the L4P works, and how it contributes to activation of the late phase of infection, is important to our understanding of natural infections by the virus, in which late gene expression can fail to occur, allowing the virus to persist.
Promyelocytic leukemia (PML) proteins have been implicated in antiviral responses but PML and associated proteins are also suggested to support virus replication. One isoform, PML-II, is required for efficient transcription of interferon and interferon-responsive genes. We therefore investigated the PML-II contribution to human adenovirus 5 (Ad5) infection, using shRNAmediated knockdown. HelaDII cells showed a 2-3-fold elevation in Ad5 yield, reflecting an increase in late gene expression. This increase was found to be due in part to the reduced innate immune response consequent upon PML-II depletion. However, the effect was minor because the viral E4 Orf3 protein targets and inactivates this PML-II function. The major benefit to Ad5 in HelaDII cells was exerted via an increase in HSP70; depletion of HSP70 completely reversed this replicative advantage. Increased Ad5 late gene expression was not due either to the previously described inhibition of inflammatory responses by HSP70 or to effects of HSP70 on major late promoter or L4 promoter activity, but might be linked to an observed increase in E1B 55K, as this protein is known to be required for efficient late gene expression. The induction of HSP70 by PML-II removal was specific for the HSPA1B gene among the HSP70 gene family and thus was not the consequence of a general stress response. Taken together, these data show that PML-II, through its various actions, has an overall negative effect on the Ad5 lifecycle.
Background and aim of study: qPCR is a robust technique which quantifies the expressions of target genes in relation to reference genes. Stresses such as virus infection or heat shock change expressions of many cellular genes including the reference genes, so the aim was to introduce a constant calibrator to normalize the data to. Methodology: Constructed glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plasmid was transcribed to GAPDH RNA and used as spike RNA. Spiked RNA samples were subjected to qPCR at different conditions such as virus infection, IFN treatment, or mild heat shock.The results: Adenovirus hexon in interferon-deficient cells showed different expression levels when data were normalized to GAPDH or 18S. Consistently, hexon expression levels were different in untreated cells under the control or heat-shocked conditions when data were normalized to GAPDH or 18S. Promyelocytic leukemia protein II (PML-II) expression level was lower in HeLa-PML-II-deficient cells (PML-II-Kd) compared to the control when the data were normalized to GAPDH as a reference gene and also in GAPDH RNA spiked, which showed reasonable consistency. More consistent data were obtained when the GAPDH normalizer was added before the step of treating the extracted RNA with DNase compared to add it after the treatment or directly to the qPCR reaction. Conclusion:The internal controls that were chosen for this study completely changed the experimental results since they were affected with the experimental conditions. However, GAPDH spike RNA level was stable in its amplification at different kinds of stresses. So it can be an alternative for housekeeping gene due to its stability at these different conditions.
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