| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Devi, Priya, et al.
(författare)
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Activation of the Ca2+/NFAT pathway by assembly of hepatitis C virus core protein into nucleocapsid-like particles
- 2022
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 14:4
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The main virion component the core (C) protein has been implicated in several aspects of HCV pathology including oncogenesis, immune evasion, and stress responses. We and others have previously shown that the expression of C in different cell lines triggers Ca2+ signaling and alters Ca2+ homeostasis. In this study, we have identified two distinct C protein regions required for activation of the Ca2+/NFAT signaling. In the basic N-terminal domain, which has been implicated in the self-association of C, amino acids 1-68 were critical for NFAT activation. Sedimentation analysis of the four mutants in this domain revealed that association of the C protein into nucleocapsid-like particles correlated with NFAT-activated transcription. The internal, lipid droplet-targeting domain, was dispensable for NFAT-activated transcription. Finally, the C-terminal ER-targeting domain was required in extenso for the C protein function. Our results indicate that targeting of HCV C to the ER is a prerequisite, but not sufficient for inducing Ca2+/NFAT signaling. Taken together, our data are consistent with a model whereby proteolytic intermediates of C with intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane.
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| 3. |
- Devi, Priya, et al.
(författare)
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Development of a strand-specific RT qPCR assay for specific detection of genomic and antigenomic hepatitis C virus RNA
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA ((+ ss)RNA) virus replicating through a complementary negative stranded RNA intermediate. To understand the molecular mechanism of virus RNA replication and to monitor smoldering infections in minor reservoirs, it is necessary to accurately quantify both positive and negative RNA strands of HCV. In the present study, a strand-specific quantitative reverse transcriptase (RT) qPCR was developed to analyse HCV replication. Strand-specific detection of positive and negative sense HCV RNA was achieved using a unique non-viral tagged primer in the reverse transcription reaction. The tag sequence was then used to selectively amplify strand-specific cDNA in the PCR step. Amplification specificity was determined by quantifying one strand in the presence of up to 105 copies of competitor RNA of the opposite sense. The assay was also evaluated by analysis of plasma and PBMC samples from patients chronically infected with HCV.
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| 4. |
- Devi, Priya, et al.
(författare)
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Hepatitis C Virus Core Protein Down-Regulates Expression of Src-Homology 2 Domain Containing Protein Tyrosine Phosphatase by Modulating Promoter DNA Methylation
- 2021
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Ingår i: Viruses. - : MDPI. - 1999-4915. ; 13:12, s. 2514-2514
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been implicated in several aspects of HCV pathology including oncogenesis and immune subversion. Here we show that expression of the C protein induced specific tyrosine phosphorylation of the TCR-related signaling proteins ZAP-70, LAT and PLC-γ in the T cells. Stable expression of the C protein specifically reduced Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1) mRNA and protein accumulation. Quantitative CpG methylation analysis revealed a distinct CpG methylation pattern at the SHP-1 gene promoter in the C protein expressing cells that included specific hypermethylation of the binding site for Sp1 transcription factor. Collectively, our results suggest that HCV may suppress immune responses and facilitate its own persistence by deregulating phosphotyrosine signaling via repressive epigenetic CpG modification at the SHP-1 promoter in the T cells.
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| 5. |
- Devi, Priya
(författare)
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Molecular characterization of the hepatitis C virus core protein
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hepatitis C virus (HCV) is an RNA virus that causes chronic infection, which can lead to hepatocellular carcinomas in humans. Besides liver diseases, the chronic HCV infection causes a broad spectrum of extrahepatic complications such as lymphoproliferative, metabolic and autoimmune disorders. Notably, HCV encoded core (C) protein is the major virion component that is involved in the oncogenesis and immune subversion. Therefore, detailed molecular characterization of the C protein provides a rational starting point for identification of novel countermeasures against pathogenic HCV infections. In this thesis we have investigated the suppressive effect of the C protein on T cell functions in immortalized cell lines and clinical samples.In paper I, we found that the expression of the C protein enhanced overall tyrosine phosphorylation in immortalized T cells. Interestingly, stable expression of the C protein specifically reduced accumulation of the tyrosine phosphatase SHP-1 mRNA. Our detailed bisulfite sequencing (BS) studies revealed that the SHP-1 P2 promoter was particularly hypermethylated at CpG1 and proximal islands in these cells. In paper II, we presented a new high-throughput next generation bisulfite sequencing (NGS-BS) protocol for the analysis of locus specific CpG methylation in HCV-infected cells using SHP-1 P2 as a model promoter. In line with our data from the BS, the NGS-BS method showed similar methylation profile at CpG1 island in immortalized cells. Strikingly, peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and HCV-positive (HCV+) patients, showed much lower levels of methylation at the CpG1 island with no significant difference in DNA methylation pattern. In paper III, we investigated the mechanism of the C protein-mediated release of Ca2+ from intracellular stores. We identified two distinct regions in the N- and C-terminal parts of the protein that were essential for activation of the Ca2+/NFAT pathway. Of these, the N-terminal region was required for self-association of the C protein into nucleocapsid-like structures whereas the C-terminal part is essential for anchoring the protein to the ER-membrane. In paper IV, we presented a PCR based diagnostic method for the specific detection of positive and negative strand HCV RNA using primers with a non-viral tag. The method was evaluated by analysing the plasma and PBMC samples from chronic HCV+ patients.Taken together, our studies provide more detailed molecular characterization of the HCV C protein functions in immortalized as well as in HCV+ T cells. Importantly, specific DNA methylation pattern of the SHP-1 gene promoter may function as a potential prognostic marker for the disease progression in HCV-induced tumors. In addition, our updated PCR-based HCV diagnostic method may provide a more specific tool to monitor HCV infections in minor reservoirs.
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| 6. |
- Devi, Priya, et al.
(författare)
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Next-generation sequencing analysis of CpG methylation of a tumor suppressor gene SHP-1 promoter in HCV-positive patients
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Hepatitis C virus (HCV) is the major causative pathogen associated with hepatocellular carcinoma and liver cirrhosis. The main virion component, the Core (C) protein, is involved in multiple aspects of HCV pathology including oncogenesis and immune evasion. In this study, we established a next-generation bisulfite sequencing (NGS-BS) protocol to analyse the CpG methylation profile at the tumor suppressor protein SHP-1 P2 promoter as a model system. HCV C protein expression in the immortalized T cells correlated with a specific CpG methylation profile at the SHP-1 P2. The NGS-BS on HCV-positive (HCV+) patient-derived PBMCs revealed a considerably different CpG methylation profile compared to the HCV C protein immortalized T cells. Notably, CpG methylation profile was very similar in healthy and HCV+ PBMCs, suggesting that the SHP-1 P2 CpG methylation profile is not altered in the HCV+ individuals. Collectively, the NGS-BS is a sensitive method and can be used to quantitatively characterize the CpG methylation status at the level of individual CpG position.
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| 7. |
- Devi, Priya, et al.
(författare)
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Next-Generation Sequencing Analysis of CpG Methylation of a Tumor Suppressor Gene SHP-1 Promoter in Stable Cell Lines and HCV-Positive Patients
- 2022
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Ingår i: Viruses. - : MDPI. - 1999-4915. ; 14:11
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis C virus (HCV) is the major causative pathogen associated with hepatocellular carcinoma and liver cirrhosis. The main virion component, the Core (C) protein, is involved in multiple aspects of HCV pathology including oncogenesis and immune evasion. In this study, we established a next-generation bisulfite sequencing (NGS-BS) protocol to analyze the CpG methylation profile at the tumor suppressor gene SHP-1 P2 promoter as a model system. Our data show that HCV C protein expression in the immortalized T cells correlated with a specific CpG methylation profile at the SHP-1 P2. The NGS-BS on HCV-positive (HCV+) patient-derived PBMCs revealed a considerably different CpG methylation profile compared to the HCV C protein immortalized T cells. Notably, the CpG methylation profile was very similar in healthy and HCV+ PBMCs, suggesting that the SHP-1 P2 CpG methylation profile is not altered in the HCV+ individuals. Collectively, the NGS-BS is a highly sensitive method that can be used to quantitatively characterize the CpG methylation status at the level of individual CpG position and also allows the characterization of cis-acting effects on epigenetic regulation.
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| 8. |
- Gopinath, Madhumala, et al.
(författare)
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Role of Hippo Pathway Effector Tafazzin Protein in Maintaining Stemness of Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSC)
- 2018
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Ingår i: International Journal of Hematology-Oncology and Stem Cell Research. - : Tehran University of Medical Sciences. - 2008-3009 .- 2008-2207. ; 12:2, s. 153-165
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Forskningsöversikt (refereegranskat)abstract
- Tafazzin (TAZ) protein has been upregulated in various types of human cancers, although the basis for elevation is uncertain, it has been made definite that the effect of mutation in the hippo pathway, particularly when it is switched off, considerably activates tafazzin transcriptionally and thus this results in tissue or tumor overgrowth. Recent perceptions into the activity of tafazzin, have ascribed to it, a role as stem cell factor in mouse mesenchymal and as well as in neural stem cells. Being a downstream molecule in Hippo signalling, phosphorylation or dephosphorylation of tafazzin gene regulates its transcriptional activity and the stemness of mesenchymal stem cells. Commonly, extracellular matrix controls the stem cell fate commitment and perhaps tafazzin controls stemness through altering the extra cellular matrix. Extracellular matrix is generally made up of prime proteoglycans and the fate stabilization of the resulting lineages is surveilled by engineering these glycans. Tafazzin degradation and addition of proteoglycans affect physical attributes of the extracellular matrix that drives cell differentiation into various lineages. Thus, tafazzin along with major glycans present in the extracellular matrix is involved in imparting stemness. However, there are incoherent molecular events, wherein both tafazzin and the extracellular matrix components, together either activate or inhibit differentiation of stem cells. This review discusses about the role of tafazzin oncoprotein as a stemness factor.
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| 9. |
- Kases, Katharina, et al.
(författare)
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The RNA-binding protein ZC3H11A interacts with the nuclear poly(A)-binding protein PABPN1 and alters polyadenylation of viral transcripts
- 2023
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Ingår i: Journal of Biological Chemistry. - : Elsevier. - 0021-9258 .- 1083-351X. ; 299:8
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Tidskriftsartikel (refereegranskat)abstract
- Nuclear mRNA metabolism is regulated by multiple proteins, which either directly bind to RNA or form multiprotein complexes. The RNA-binding protein ZC3H11A is involved in nuclear mRNA export, NF-kappa B signaling, and is essential during mouse embryo development. Furthermore, previous studies have shown that ZC3H11A is important for nuclear-replicating viruses. However, detailed biochemical characterization of the ZC3H11A protein has been lacking. In this study, we established the ZC3H11A protein interactome in human and mouse cells. We demonstrate that the nuclear poly(A)-binding protein PABPN1 interacts specifically with the ZC3H11A protein and controls ZC3H11A localization into nuclear speckles. We report that ZC3H11A specifically interacts with the human adenovirus type 5 (HAdV-5) capsid mRNA in a PABPN1dependent manner. Notably, ZC3H11A uses the same zinc finger motifs to interact with PABPN1 and viral mRNA. Further, we demonstrate that the lack of ZC3H11A alters the polyadenylation of HAdV-5 capsid mRNA. Taken together, our results suggest that the ZC3H11A protein may act as a novel regulator of polyadenylation of nuclear mRNA.
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