Cell insight最新文献

In vitro preparation of mRNA is a key step for mRNA therapeutics. The widely used T7 RNA polymerase (RNAP) was shown to have many by-products during in vitro transcription (IVT) process, among which double-stranded RNA (dsRNA) is the major by-product to activate the intracellular immune response. Here, we describe the use of a new VSW-3 RNAP that reduced dsRNA production during IVT and the resulting mRNA exhibited low inflammatory stimulation in cells. Compared to T7 RNAP transcripts, these mRNA exhibited superior protein expression levels, with an average of 14-fold increase in Hela cells and 5-fold increase in mice. In addition, we found that VSW-3 RNAP did not require modified nucleotides to improve protein production of IVT products. Our data suggest that VSW-3 RNAP could be a useful tool for mRNA therapeutics.

COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose an international public health threat and thus far, has resulted in greater than 6.4 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast-spreading COVID-19 variants that may elude vaccine efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through a luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5′CT, and dasabuvir. Dasabuvir (an FDA-approved drug) exhibited promising RdRp inhibitory activity among these inhibitors. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with EC₅₀ values 9.47 μM and 10.48 μM, for USA-WA1/2020 and B.1.617.2 variants, respectively. Our results suggest that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. Importantly, this system provides a robust, target-specific, and high-throughput screening compatible (z- and z’-factors of >0.5) platforms that will be a valuable tool for screening SARS-CoV-2 RdRp inhibitors.

As of 10 May 2022, at least 450 cases of pediatric patients with acute hepatitis of unknown cause have been reported worldwide. Human adenoviruses (HAdVs) have been detected in at least 74 cases, including the F type HAdV41 in 18 cases, which indicates that adenoviruses may be associated with this mysterious childhood hepatitis, although other infectious agents or environmental factors cannot be excluded. In this review, we provide a brief introduction of the basic features of HAdVs and describe diseases caused by different HAdVs in humans, aiming to help understand the biology and potential risk of HAdVs and cope with the outbreak of acute child hepatitis.

Interleukin-33 (IL-33) which belongs to the interleukin-1 (IL-1) family is an alarmin cytokine with critical roles in tissue homeostasis, pathogenic infection, inflammation, allergy and type 2 immunity. IL-33 transmits signals through its receptor IL-33R (also called ST2) which is expressed on the surface of T helper 2 (Th2) cells and group 2 innate lymphoid cells (ILC2s), thus inducing transcription of Th2-associated cytokine genes and host defense against pathogens. Moreover, the IL-33/IL-33R axis is also involved in development of multiple types of immune-related diseases. In this review, we focus on current progress on IL-33-trigggered signaling events, the important functions of IL-33/IL-33R axis in health and diseases as well as the promising therapeutic implications of these findings.

Epidermal growth factor receptor (EGFR) plays critical roles in cell proliferation and tumorigenesis. Autophagy has emerged as a potential mechanism involved in the acquired resistance to anti-EGFR treatments, however, the molecular mechanisms has not been fully addressed. In this study, we identified EGFR interacts with STYK1, a positive autophagy regulator, in EGFR kinase activity dependent manner. We found that EGFR phosphorylates STYK1 at Y356 site and STYK1 inhibits activated EGFR mediated Beclin1 tyrosine phosphorylation and interaction between Bcl2 and Beclin1, thus enhances PtdIns3K-C1 complex assembly and autophagy initiation. We also demonstrated that STYK1 depletion increased the sensitivity of NSCLC cells to EGFR-TKIs in vitro and in vivo. Moreover, EGFR-TKIs induced activation of AMPK phosphorylates STYK1 at S304 site. STYK1 S304 collaborated with Y356 phosphorylation to enhance the EGFR-STYK1 interaction and reverse the inhibitory effects of EGFR to autophagy flux. Collectively, these data revealed new roles and cross-talk between STYK1 and EGFR in autophagy regulation and EGFR-TKIs sensitivity in NSCLC.

Inflammatory bowel disease (IBD) is closely associated with dysregulation of genetic factors and microbial environment. Here, we report a susceptible role of ubiquitin-specific protease 2 (USP2) in experimental colitis and bacterial infections. USP2 is upregulated in the inflamed mucosa of IBD patients and in the colon of mice treated with dextran sulfate sodium salt (DSS). Knockout or pharmacologic inhibition of USP2 promotes the proliferation of myeloid cells to activate IL-22 and IFNγ production of T cells. In addition, knockout of USP2 in myeloid cells inhibits the production of pro-inflammatory cytokines to relieve the dysregulation of extracellular matrix (ECM) network and promote the gut epithelial integrity after DSS treatment. Consistently, Lyz2-Cre;Usp2^fl/fl mice exhibit hyper-resistance to DSS-induced colitis and Citrobacter rodentium infections compared to Usp2^fl/fl mice. These findings highlight an indispensable role of USP2 in myeloid cells to modulate T cell activation and epithelial ECM network and repair, indicating USP2 as a potential target for therapeutic intervention of IBD and bacterial infections in the gastrointestinal system.

Visualizing RNA dynamics is important for understanding RNA function. Catalytically dead (d) CRISPR-Cas13 systems have been established to image and track RNAs in living cells, but efficient dCas13 for RNA imaging is still limited. Here, we analyzed metagenomic and bacterial genomic databases to comprehensively screen Cas13 homologies for their RNA labeling capabilities in living mammalian cells. Among eight previously unreported dCas13 proteins that can be used for RNA labeling, dHgm4Cas13b and dMisCas13b displayed comparable, if not higher, efficiencies to the best-known ones when targeting endogenous MUC4 and NEAT1_2 by single guide (g) RNAs. Further examination of the labeling robustness of different dCas13 systems using the GCN4 repeats revealed that a minimum of 12 GCN4 repeats was required for dHgm4Cas13b and dMisCas13b imaging at the single RNA molecule level, while >24 GCN4 repeats were required for reported dLwaCas13a, dRfxCas13d and dPguCas13b. Importantly, by silencing pre-crRNA processing activity of dMisCas13b (ddMisCas13b) and further incorporating RNA aptamers including PP7, MS2, Pepper or BoxB to individual gRNAs, a CRISPRpalette system was developed to successfully achieve multi-color RNA visualization in living cells.

Alpha7 nicotinic acetylcholine receptor (α7 nAChR), a hub of the cholinergic anti-inflammatory pathway (CAP), is required for the treatment of inflammatory diseases. HIV-1 infection can upregulate the expression of α7 nAChR in T lymphocytes and affect the role of CAP. However, whether α7 nAChR regulates HIV-1 infection in CD4⁺ T cells is unclear. In this study, we first found that activation of α7 nAChR by GTS-21 (an α7 nAChR agonist) can promote the transcription of HIV-1 proviral DNA. Then, through transcriptome sequencing analysis, we found that p38 MAPK signaling was enriched in GTS-21 treated HIV-latent T cells. Mechanistically, activation of α7 nAChR could increase reactive oxygen species (ROS), reduce DUSP1 and DUSP6, and consequently enhance the phosphorylation of p38 MAPK. By co-immunoprecipitation and liquid chromatography tandem mass spectrometry, we found that p-p38 MAPK interacted with Lamin B1 (LMNB1). Activation of α7 nAChR increased the binding between p-p38 MAPK and LMNB1. We confirmed that knockdown of MAPK14 significantly downregulated NFATC4, a key activator of HIV-1 transcription. Taken together, activation of the α7 nAChR could trigger ROS/p-p38 MAPK/LMNB1/NFATC4 signaling pathway enhancing HIV-1 transcription. We have revealed an unrecognized mechanism of α7 nAChR-mediated neuroimmune regulation of HIV infection.

Annual and sporadic influenza outbreaks pose a great threat to human health and the economy worldwide. Moreover, the frequent mutation of influenza viruses caused by antigen drift complicates the application of antiviral therapeutics. As such, there is an urgent need for novel antiviral agents to tackle the problem of insufficient efficacy of licensed drugs. Inspired by the success of the newly emerged PROTACs (PROteolysis TArgeting Chimeras) strategy, we report herein the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks. Among these, several compounds showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity. The best compound, 8e, effectively induced influenza NA degradation in a dose-dependent manner and relied on the ubiquitin–proteasome pathway. Moreover, Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain (H1N1, H274Y). A molecular docking study demonstrated that Compound 8e had good hydrogen-bonding and hydrophobic interactions with both the active sites of NA and Von Hippel-Lindau (VHL) proteins, which could effectively drive the favorable interaction of these two proteins. Thus, as the first report of a successful anti-influenza PROTAC, this proof of concept will greatly widen the application range of the PROTAC technique to antiviral drug discovery.

Aging is characterized by progressive functional declines at the organismal, organic, and cellular levels and increased susceptibility to aging-related diseases. Epigenetic alteration is a hallmark of aging, senescent cells show epigenomic changes at multiple scales, such as 3D genome reorganization, alterations of histone modifications and chromatin accessibility, and DNA hypomethylation. Chromosome conformation capture (3C)-based technologies have enabled the generation of key information on genomic reorganizations during senescence. A comprehensive understanding of epigenomic alterations during aging will yield important insights into the underlying epigenetic mechanism for aging regulation, the identification of aging-related biomarkers, and the development of potential aging intervention targets.