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- Appelberg, Sofia, et al.
(författare)
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Dysregulation in Akt/mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells
- 2020
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Ingår i: Emerging Microbes & Infections. - : Informa UK Limited. - 2222-1751. ; 9:1, s. 1748-1760
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Tidskriftsartikel (refereegranskat)abstract
- How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.
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| 2. |
- Soares, Ruben R. G., et al.
(författare)
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Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography
- 2020
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 166
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Tidskriftsartikel (refereegranskat)abstract
- Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies. Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of similar to 1 mu m-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes. The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.
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