We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.
In the human brain, microRNAs (miRNAs) from the microRNA-376 (miR-376) cluster undergo programmed "seed" sequence modifications by adenosine-to-inosine (A-to-I) editing. Emerging evidence suggests a link between impaired A-to-I editing and cancer, particularly in high-grade gliomas. We hypothesized that disruption of A-to-I editing alters expression of genes regulating glioma tumor phenotypes. By sequencing the miR-376 cluster, we show that the overall miRNA editing frequencies were reduced in human gliomas. Specifically in high-grade gliomas, miR-376a* accumulated entirely in an unedited form. Clinically, a significant correlation was found between accumulation of unedited miR-376a* and the extent of invasive tumor spread as measured by magnetic resonance imaging of patient brains. Using both in vitro and orthotopic xenograft mouse models, we demonstrated that the unedited miR-376a* promoted glioma cell migration and invasion, while the edited miR-376a* suppressed these features. The effects of the unedited miR-376a* were mediated by its sequence-dependent ability to target RAP2A and concomitant inability to target AMFR. Thus, the tumordependent introduction of a single base difference in the miR-376a* sequence dramatically alters the selection of its target genes and redirects its function from inhibiting to promoting glioma cell invasion. These findings uncover a new mechanism of miRNA deregulation and identify unedited miR-376a* as a potential therapeutic target in glioblastoma cells. IntroductionMicroRNAs (miRNAs) are short, noncoding RNAs that mediate post-transcriptional silencing of a set of target genes. The target gene specificity of each miRNA is dictated by sequence-dependent interaction between approximately 22-nt-long mature miRNAs, especially their 6-or 7-nucleotide "seed" sequences at the 5′ end, and the 3′ untranslated regions of mRNAs (1). It has been shown that the epigenetic process of adenosineto-inosine (A-to-I) editing of certain miRNAs can lead to a single base substitution in their seed sequence and generate variant "edited" mature miRNA species with target gene specificity drastically different from that of the unedited, genomically encoded miRNA (2). This was demonstrated for miR-376 cluster transcripts collected from normal human and mouse tissues. The miR-376 cluster encodes 4 primary miRNAs (primiRs), including pri-miR-376a1, -376a2, -376b, and -376c, that are processed to 5 distinct mature miRNAs, miR-376a, -376a*, -376a2-5p, -376b, and -376c. In the human brain, 9 adenosines within this miRNA cluster are subject to specific and high-level A-to-I RNA editing (2).In gliomas, the most frequent primary brain tumors, Alu repeats and several protein-coding substrates of adenosine deaminases acting on RNA (ADARs; a family of enzymes that mediate A-to-I editing of RNAs) have been found to be edited to lower than normal frequencies (3-5). Particularly in high-grade gliomas, glioblastoma multiforme (GBMs), emerging lines of evidence suggest a
Transcriptional targeting using a tissue-specific cellular promoter is proving to be a powerful means for restricting transgene expression in targeted tissues. In the context of cancer suicide gene therapy, this approach may lead to cytotoxic effects in both cancer and nontarget normal cells. Considering microRNA (miRNA) function in post-transcriptional regulation of gene expression, we have developed a viral vector platform combining cellular promoter-based transcriptional targeting with miRNA regulation for a glioma suicide gene therapy in the mouse brain. The therapy employed, in a single baculoviral vector, a glial fibrillary acidic protein (GFAP) gene promoter and the repeated target sequences of three miRNAs that are enriched in astrocytes but downregulated in glioblastoma cells to control the expression of the herpes simplex virus thymidine kinase (HSVtk) gene. This resulted in significantly improved in vivo selectivity over the use of a control vector without miRNA regulation, enabling effective elimination of human glioma xenografts while producing negligible toxic effects on normal astrocytes. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of gene delivery vectors with high targeting specificity for cancer therapy.
Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.