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      A new circular RNA–encoded protein BIRC6-236aa inhibits transmissible gastroenteritis virus (TGEV)–induced mitochondrial dysfunction

      research-article
      , , , , , , ,
      The Journal of Biological Chemistry
      American Society for Biochemistry and Molecular Biology
      transmissible gastroenteritis virus, mitochondrial permeability transition pore, circBIRC6-2, BIRC6-236aa, voltage-dependent anion-selective channel protein 1 (VDAC1), AM, acetoxymethyl ester, BCF, back-circular frame, CCK8, cell counting kit-8, cDNA, complementary DNA, CypD, Cyclophilin D, DAPI, 4, 6-diamidino-2-phenylindole, FCF, front-circular frame, gDNA, genomic DNA, HA, hemagglutinin, IEM, immunoelectron microscopy, IMM, inner mitochondrial membrane, IP, immunoprecipitation, IPEC-J2, intestinal epithelial cell line jejunum 2, IRES, internal ribosomal entrance site, LR, London Resin, lv, lentiviruse, MMP, mitochondrial membrane potential, MOI, multiplicity of infection, mPTP, mitochondrial permeability transition pore, MS, mass spectrometry, PLA, proximity ligation assay, qRT-PCR, quantitative real-time PCR, Rhod-2, rhodamine-2, TGEV, transmissible gastroenteritis virus

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          Abstract

          Transmissible gastroenteritis virus (TGEV), a member of the coronavirus family, is the pathogen responsible for transmissible gastroenteritis, which results in mitochondrial dysfunction in host cells. Previously, we identified 123 differentially expressed circular RNAs (cRNA)from the TGEV-infected porcine intestinal epithelial cell line jejunum 2 (IPEC-J2). Previous bioinformatics analysis suggested that, of these, circBIRC6 had the potential to regulate mitochondrial function. Furthermore, mitochondrial permeability transition, a key step in the process of mitochondrial dysfunction, is known to be caused by abnormal opening of mitochondrial permeability transition pores (mPTPs) regulated by the voltage-dependent anion-selective channel protein 1 (VDAC)–Cyclophilin D (CypD) complex. Therefore, in the present study, we investigated the effects of circBIRC6-2 on mitochondrial dysfunction and opening of mPTPs. We found that TGEV infection reduced circBIRC6-2 levels, which in turn reduced mitochondrial calcium (Ca 2+) levels, the decrease of mitochondrial membrane potential, and opening of mPTPs. In addition, we also identified ORFs and internal ribosomal entrance sites within the circBIRC6-2 RNA. We demonstrate circBIRC6-2 encodes a novel protein, BIRC6-236aa, which we show inhibits TGEV-induced opening of mPTPs during TGEV infection. Mechanistically, we identified an interaction between BIRC6-236aa and VDAC1, suggesting that BIRC6-236aa destabilizes the VDAC1–CypD complex. Taken together, the results suggest that the novel protein BIRC6-236aa encoded by cRNA circBIRC6-2 inhibits mPTP opening and subsequent mitochondrial dysfunction by interacting with VDAC1.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            The PRIDE database and related tools and resources in 2019: improving support for quantification data

            Abstract The PRoteomics IDEntifications (PRIDE) database (https://www.ebi.ac.uk/pride/) is the world’s largest data repository of mass spectrometry-based proteomics data, and is one of the founding members of the global ProteomeXchange (PX) consortium. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2016. In the last 3 years, public data sharing through PRIDE (as part of PX) has definitely become the norm in the field. In parallel, data re-use of public proteomics data has increased enormously, with multiple applications. We first describe the new architecture of PRIDE Archive, the archival component of PRIDE. PRIDE Archive and the related data submission framework have been further developed to support the increase in submitted data volumes and additional data types. A new scalable and fault tolerant storage backend, Application Programming Interface and web interface have been implemented, as a part of an ongoing process. Additionally, we emphasize the improved support for quantitative proteomics data through the mzTab format. At last, we outline key statistics on the current data contents and volume of downloads, and how PRIDE data are starting to be disseminated to added-value resources including Ensembl, UniProt and Expression Atlas.
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              Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ *

              Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity determination and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein separation prior to LC-MS analysis. Protein abundance profiles are assembled using the maximum possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technology that is readily applicable to many biological questions; it is compatible with standard statistical analysis workflows, and it has been validated in many and diverse biological projects. Our algorithms can handle very large experiments of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button.
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                Author and article information

                Contributors
                Journal
                J Biol Chem
                J Biol Chem
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology
                0021-9258
                1083-351X
                19 July 2022
                September 2022
                19 July 2022
                : 298
                : 9
                : 102280
                Affiliations
                [1]College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
                Author notes
                []For correspondence: Dewen Tong dwtong@ 123456nwafu.edu.cn
                [‡]

                These authors contributed equally to this work.

                Article
                S0021-9258(22)00722-0 102280
                10.1016/j.jbc.2022.102280
                9400091
                35863430
                12378941-a6aa-4e17-81f5-44b90afa6f24
                © 2022 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 13 October 2021
                : 4 July 2022
                Categories
                Research Article

                Biochemistry
                transmissible gastroenteritis virus,mitochondrial permeability transition pore,circbirc6-2,birc6-236aa,voltage-dependent anion-selective channel protein 1 (vdac1),am, acetoxymethyl ester,bcf, back-circular frame,cck8, cell counting kit-8,cdna, complementary dna,cypd, cyclophilin d,dapi, 4, 6-diamidino-2-phenylindole,fcf, front-circular frame,gdna, genomic dna,ha, hemagglutinin,iem, immunoelectron microscopy,imm, inner mitochondrial membrane,ip, immunoprecipitation,ipec-j2, intestinal epithelial cell line jejunum 2,ires, internal ribosomal entrance site,lr, london resin,lv, lentiviruse,mmp, mitochondrial membrane potential,moi, multiplicity of infection,mptp, mitochondrial permeability transition pore,ms, mass spectrometry,pla, proximity ligation assay,qrt-pcr, quantitative real-time pcr,rhod-2, rhodamine-2,tgev, transmissible gastroenteritis virus

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