TY - JOUR AU - Medeiros, Vasco AU - Pearl, Jennifer AU - Carboni, Mia AU - Zafeiri, Stamatia PY - 2024/11/5 TI - Exploring the Accuracy of Ab Initio Prediction Methods for Viral Pseudoknotted RNA Structures: Retrospective Cohort Study JO - JMIRx Bio SP - e58899 VL - 2 KW - pseudoknot KW - viral RNA KW - MFE KW - minimum free energy KW - MFE prediction KW - MEA KW - maximum expected accuracy KW - MEA prediction KW - virus KW - virology KW - computational biology N2 - Background: The prediction of tertiary RNA structures is significant to the field of medicine (eg, messenger RNA [mRNA] vaccines, genome editing) and the exploration of viral transcripts. Though many RNA folding software programs exist, few studies have condensed their locus of attention solely to viral pseudoknotted RNA. These regulatory pseudoknots play a role in genome replication, gene expression, and protein synthesis. Objective: The objective of this study was to explore 5 RNA folding engines that compute either the minimum free energy (MFE) or the maximum expected accuracy (MEA), when applied to a specified suite of viral pseudoknotted RNAs that have been previously confirmed using mutagenesis, sequence comparison, structure probing, or nuclear magnetic resonance (NMR). Methods: The folding engines used in this study were tested against 26 experimentally derived short pseudoknotted sequences (20-150 nt) using metrics that are commonplace while testing software prediction accuracy: percentage error, mean squared error (MSE), sensitivity, positive predictive value (PPV), Youden?s index (J), and F1-score. The data set used in this study was accrued from the Pseudobase++ database containing 398 RNAs, which was assessed using a set of inclusion and exclusion criteria following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Base pairings within a given RNA sequence were deemed correct or incorrect following Mathews? parameters. Results: This paper reported RNA prediction engines with greater accuracy, such as pKiss, when compared to previous iterations of the software and when compared to older folding engines. This paper also reported that when assessed using metrics such as the F1-score and the PPV, MEA folding software does not always outperform MFE folding software in prediction accuracy when applied to viral pseudoknotted RNA. Moreover, the results suggested that thermodynamic model parameters will not ensure accuracy if auxiliary parameters, such as Mg2+ binding, dangling end options, and hairpin-type penalties, are not applied. Conclusions: This is the first attempt at applying a suite of RNA folding engines to a data set solely comprising viral pseudoknotted RNA. The observations reported in this paper highlight the quality between different ab initio prediction methods, while enforcing the idea that a better understanding of intracellular thermodynamics is necessary for a more efficacious screening of RNAs. UR - https://bio.jmirx.org/2024/1/e58899 UR - http://dx.doi.org/10.2196/58899 UR - http://www.ncbi.nlm.nih.gov/pubmed/ ID - info:doi/10.2196/58899 ER - TY - JOUR AU - Suleiman, Khalid AU - Aljulidan, Mutaib AU - Hussein, Gamaleldin AU - Alkhalaf, Habib PY - 2024/7/18 TI - Establishment of a Novel Fetal Ovine Heart Cell Line by Spontaneous Cell Fusion: Experimental Study JO - JMIRx Bio SP - e53721 VL - 2 KW - fetal ovine heart KW - cell line KW - serial passage KW - spontaneous cell fusion KW - SNP genotyping KW - mitochondrial genome sequencing N2 - Background: The culture of immortal cell lines has become an indispensable tool in the field of modern biotechnology and has been used in the production of human and viral veterinary vaccines, therapeutic recombinant proteins, interferons, and monoclonal antibodies. Several approaches are used to immortalize cells in culture, such as transduction of cells with viral oncogenes, induced expression of telomerase reverse transcriptase, and spontaneous immortalization by serial passage of primary cell lines. Objective: This study aimed to establish an immortal cell line by serial passage of fetal ovine heart cells that could be used to produce veterinary viral vaccines. Methods: We serially passaged primary heart cells prepared from a fetal ovine heart till passage 140. We studied the events that led to the transformation and immortalization of the cell line under light and phase contrast microscopy. DNA samples of the cell line at passages 22 (before transformation) and 47 (after transformation) were genotyped according to single nucleotide polymorphisms (SNPs) using the OvineSNP50 BeadChip (Illumina). We sequenced the cytochrome b gene, control region, and tRNA-Phe and 12S rRNA genes of the mitochondrial genome of the cell line at passages 26 and 59 by Sanger sequencing. The susceptibility of the cell line to sheep pox, Peste des petits ruminants (PPR), lumpy skin disease (LSD), Rift Valley fever (RVF), and camel pox viruses was investigated. Results: We established a unique immortal cell line called fetal ovine heart?Saudi Arabia (FOH-SA) by serial passage of fetal ovine heart cells. We demonstrated that the transformation or immortalization of the cell line resulted from spontaneous cellular and nuclear fusion of 2 morphologically distinct cardiocytes at passage 29. Fused cells at passage 29 gave rise to progeny cells, which grew into multicellular filaments that persisted at passages 30, 31, and 32. Trypsinization of the filamentous multicellular growth at passage 32 gave an epithelial-type immortal heart cell line. SNP genotyping revealed 65% and 96% homozygosity in SNP genotypes of the cell line at passages 22 and 47, respectively. Partial sequencing of the mitochondrial genome of the cell line revealed mutational events in the control region and the tRNA-Phe and 12S rRNA genes of the mitochondrial genome of passage 59 cells. The cell line was found to be permissive to sheep pox, PPR, LSD, RVF, and camel pox viruses. Conclusions: We established an immortal cell line by serial passage of primary fetal ovine heart cells, which was permissive to many animal viruses. It could be used in animal virus isolation, vaccine production, and biotechnology. The study reported spontaneous cell fusion of cardiocytes as a method of cell immortalization. The findings of this study might help address the mystery of how the VERO cell line evolved. UR - https://bio.jmirx.org/2024/1/e53721 UR - http://dx.doi.org/10.2196/53721 UR - http://www.ncbi.nlm.nih.gov/pubmed/ ID - info:doi/10.2196/53721 ER -