Topological capture of mRNA for silencing gene expression

We describe herein topological mRNA capture using branched oligodeoxynucleotides (ODNs) with multiple reactive functional groups. These fragmented ODNs efficiently formed topological complexes on template mRNA in vitro. In cell-based experiments targeting AcGFP mRNA, the bifurcated reactive ODNs showed a much larger gene silencing effect than the corresponding natural antisense ODN.journal articl

Potential and action mechanism of favipiravir as an antiviral against Junin virus

Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever

Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak.Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step-Direct RT-LAMP, which could detect more than 1.43 x 103 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries

Face validation and pharmacologic analysis of Sik3Sleepy mutant mouse as a possible model of idiopathic hypersomnia

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Anti-fibrotic efficacy of nintedanib in pulmonary fibrosis via the inhibition of fibrocyte activity

Background: Nintedanib, a tyrosine kinase inhibitor that is specific for platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR), and vascular endothelial growth factor receptors (VEGFR), has recently been approved for idiopathic pulmonary fibrosis. Fibrocytes are bone marrow-derived progenitor cells that produce growth factors and contribute to fibrogenesis in the lungs. However, the effects of nintedanib on the functions of fibrocytes remain unclear. Methods: Human monocytes were isolated from the peripheral blood of healthy volunteers. The expression of growth factors and their receptors in fibrocytes was analyzed using ELISA and Western blotting. The effects of nintedanib on the ability of fibrocytes to stimulate lung fibroblasts were examined in terms of their proliferation. The direct effects of nintedanib on the differentiation and migration of fibrocytes were also assessed. We investigated whether nintedanib affected the accumulation of fibrocytes in mouse lungs treated with bleomycin. Results: Human fibrocytes produced PDGF, FGF2, and VEGF-A. Nintedanib and specific inhibitors for each growth factor receptor significantly inhibited the proliferation of lung fibroblasts stimulated by the supernatant of fibrocytes. Nintedanib inhibited the migration and differentiation of fibrocytes induced by growth factors in vitro. The number of fibrocytes in the bleomycin-induced lung fibrosis model was reduced by the administration of nintedanib, and this was associated with anti-fibrotic effects. Conclusions: These results support the role of fibrocytes as producers of and responders to growth factors, and suggest that the anti-fibrotic effects of nintedanib are at least partly mediated by suppression of fibrocyte function

The potential role of Arhgef33 RhoGEF in foveal development in the zebra finch retina

The fovea is a pit formed in the center of the retina that enables high-acuity vision in certain vertebrate species. While formation of the fovea fascinates many researchers, the molecular mechanisms underlying foveal development are poorly understood. In the current study, we histologically investigated foveal development in zebra finch (Taeniopygia guttata) and found that foveal pit formation begins just before post-hatch day 14 (P14). We next performed RNA-seq analysis to compare gene expression profiles between the central (foveal and parafoveal) and peripheral retina in zebra finch at P14. We found that the Arhgef33 expression is enriched in the middle layer of the inner nuclear layer at the parafovea, suggesting that Arhgef33 is dominantly expressed in Müller glial cells in the developing parafovea. We then performed a pull-down assay using Rhotekin-RBD and observed GEF activity of Arhgef33 against RhoA. We found that overexpression of Arhgef33 in HEK293 cells induces cell contraction and that Arhgef33 expression inhibits neurite extension in Neuro 2A cells, which is partially recovered by a Rho-kinase (ROCK) inhibitor. Taken together, we used zebra finch as a model animal to investigate foveal development and identified Arhgef33 as a candidate protein possibly involved in foveal development through modulating RhoA activity

Genetic Diversity and Detection of Respiratory Viruses Excluding SARS-CoV-2 during the COVID-19 Pandemic in Gabon, 2020–2021

Acute respiratory infections are a major global burden in resource-limited countries, including countries in Africa. Although COVID-19 has been well studied since the pandemic emerged in Gabon, Central Africa, less attention has been paid to other respiratory viral diseases, and very little data are available. Herein, we provide the first data on the genetic diversity and detection of 18 major respiratory viruses in Gabon during the COVID-19 pandemic. Of 582 nasopharyngeal swab specimens collected from March 2020 to July 2021, which were SARS-CoV-2 negative, 156 were positive (26%) for the following viruses: enterovirus (20.3%), human rhinovirus (HRV) (4.6%), human coronavirus OC43 (1.2%), human adenovirus (0.9%), human metapneumovirus (hMPV) (0.5%), influenza A virus (IAV) (0.3%), and human parainfluenza viruses (0.5%). To determine the genetic diversity and transmission route of the viruses, phylogenetic analyses were performed using genome sequences of the detected viruses. The IAV strain detected in this study was genetically similar to strains isolated in the USA, whereas the hMPV strain belonging to the A2b subtype formed a cluster with Kenyan strains. This study provides the first complete genomic sequences of HRV, IAV, and hMPV detected in Gabon, and provides insight into the circulation of respiratory viruses in the country.Viruses, 16(5), art. no. 698; 2024journal articl

Re-emergence of dengue, chikungunya, and Zika viruses in 2021 after a 10-year gap in Gabon

Mosquito-borne viral infections are a major concern in endemic areas, such as Africa. Although outbreaks have been reported throughout Africa, only a few surveillance studies have been conducted in Gabon since the outbreaks of dengue virus (DENV) and chikungunya virus (CHIKV) in 2010. Therefore, the current situation is unknown. This study aimed to investigate the presence of arboviruses, especially DENV (serotypes 1–4), CHIKV, and Zika virus (ZIKV), in Gabon, Central Africa. Between 2020 and 2021, we collected 1060 serum samples from febrile patients and screened them against viruses using reverse transcription-quantitative PCR. We detected two DENV serotypes 1 (DENV-1), one CHIKV, and one ZIKV, and subsequently analyzed the genome sequences. To determine the genetic diversity and transmission route of the viruses, phylogenetic analysis was performed using complete or partial genome sequences. The DENV-1 and CHIKV strains detected in this study were closely related to the previous Gabonese strains, whereas the recent ZIKV strain was genetically different from a strain detected in 2007 in Gabon. This study provides new genomic information on DENV-1, CHIKV, and ZIKV that were detected in Gabon and insight into the circulation of the viruses in the country and their introduction from neighboring African countries