scholarly journals An Aedes Aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1066
Author(s):  
Christina Scherer ◽  
Jack Knowles ◽  
Vattipally B. Sreenu ◽  
Anthony C. Fredericks ◽  
Janina Fuss ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Viral Replication ◽  
Cell Line ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Rna Sequences ◽  
Argonaute 2 ◽  
Si Rna ◽  
Sirna Pathway ◽  
The Impact

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

scholarly journals Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

2022 ◽  
Vol 18 (1) ◽  
pp. e1010202
Author(s):  
Rommel J. Gestuveo ◽  
Rhys Parry ◽  
Laura B. Dickson ◽  
Sebastian Lequime ◽  
Vattipally B. Sreenu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Antiviral Activity ◽  
Aedes Aegypti ◽  
Semliki Forest Virus ◽  
Mutational Analysis ◽  
Helicase Domain ◽  
Small Interfering Rnas ◽  
Rnase Iii ◽  
Argonaute 2 ◽  
Sirna Pathway

The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.

scholarly journals DEUS: an R package for accurate small RNA profiling based on differential expression of unique sequences

2019 ◽  
Vol 35 (22) ◽  
pp. 4834-4836
Author(s):  
Tim Jeske ◽  
Peter Huypens ◽  
Laura Stirm ◽  
Selina Höckele ◽  
Christine M Wurmser ◽  
...  
Keyword(s):  
Differential Expression ◽  
Small Rna ◽  
Sequence Similarity ◽  
Differential Expression Analysis ◽  
R Package ◽  
Supplementary Information ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Rna Sequences ◽  
Rna Profiling

Abstract Summary Despite their fundamental role in various biological processes, the analysis of small RNA sequencing data remains a challenging task. Major obstacles arise when short RNA sequences map to multiple locations in the genome, align to regions that are not annotated or underwent post-transcriptional changes which hamper accurate mapping. In order to tackle these issues, we present a novel profiling strategy that circumvents the need for read mapping to a reference genome by utilizing the actual read sequences to determine expression intensities. After differential expression analysis of individual sequence counts, significant sequences are annotated against user defined feature databases and clustered by sequence similarity. This strategy enables a more comprehensive and concise representation of small RNA populations without any data loss or data distortion. Availability and implementation Code and documentation of our R package at http://ibis.helmholtz-muenchen.de/deus/. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and ago-2 Expression

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Melanie McFarlane ◽  
Floriane Almire ◽  
Joy Kean ◽  
Claire L. Donald ◽  
Alma McDonald ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Antiviral Activity ◽  
Aedes Aegypti ◽  
Small Interfering Rna ◽  
Antiviral Response ◽  
Pathway Activity ◽  
Content Type ◽  
Argonaute 2 ◽  
Interfering Rna ◽  
Sirna Pathway

ABSTRACT Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus (Togaviridae) both in Aedes aegypti-derived cells and in vivo. Antiviral activity of p400 was also demonstrated against chikungunya virus (Togaviridae) and Bunyamwera virus (Peribunyaviridae) but not Zika virus (Flaviviridae). p400 was found to be expressed across mosquito tissues and regulated ago-2 but not dcr-2 transcript levels in A. aegypti mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein’s antiviral and RNA interference regulatory activities in Drosophila melanogaster. IMPORTANCE Female Aedes aegypti mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito’s antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that A. aegypti p400, whose ortholog Domino in Drosophila melanogaster is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls ago-2 expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in A. aegypti mosquitoes.

scholarly journals Identification of microRNAs in the West Nile virus vector Culex tarsalis

2021 ◽  
Author(s):  
Sultan Asad ◽  
Ahmed M Mehdi ◽  
Sujit Pujhari ◽  
Claudia Rueckert ◽  
Gregory D Ebel ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Cell Line ◽  
Rna Sequencing ◽  
Small Rna ◽  
The United States ◽  
Small Rna Sequencing ◽  
Culex Tarsalis ◽  
Sequencing Data ◽  
West Nile

Background: microRNAs (miRNAs) represent a group of small non-coding RNAs that are crucial gene regulators of important biological functions including development and pathogen defense in most living organisms. Presently, there is a lack of availability of information regarding the miRNAs in the mosquito Culex tarsalis, which is one of the most important vectors of West Nile virus (WNV) in the United States. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate the presence of a repertoire of miRNAs in Cx. tarsalis mosquitoes. Results: Using bioinformatic approaches we analyzed small RNA sequencing data from the Cx. tarsalis CT embryonic cell line to discover 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Cx. quinquefasciatus, mi-184 was found to be the most abundant miRNA in Cx tarsalis. We also identified an additional 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both cell line and adult Cx. tarsalis mosquitoes using RT-qPCR and sequencing. Conclusions: Cx. tarsalis is an important vector of many medically important pathogens including WNV and Western Equine encephalitis. Here we report a detailed insight into the miRNA population in Cx. tarsalis mosquitoes. These results will open new avenues of research deciphering the role of miRNAs in different Cx. tarsalis biological events such as development, metabolism, immunity and pathogen infection.

scholarly journals Characterizing the Cancer-Associated Microbiome with Small RNA Sequencing Data

2019 ◽  
Author(s):  
Wei-Hao Lee ◽  
Kai-Pu Chen ◽  
Kai Wang ◽  
Hsuan-Cheng Huang ◽  
Hsueh-Fen Juan
Keyword(s):  
Small Rna ◽  
The Cancer Genome Atlas ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Microbial Composition ◽  
Rna Sequences ◽  
Colon And Rectal Cancer ◽  
Microrna Sequencing ◽  
Cancer Genome Atlas ◽  
Inflammatory Immune Response

AbstractThe microbiome is recognized as a quasi-organ in the human body. In particular, the gut microbiome is correlated with immune function, metabolism, and tumorigenesis. When dysbiosis of the microbiome occurs, this variation may contribute to alterations in the microenvironment, potentially inducing an inflammatory immune response and providing a niche for neoplastic growth. However, there is limited evidence regarding the correlation and interaction between the microbiome and tumorigenesis. By utilizing microRNA sequencing data of patients with colon and rectal cancer from The Cancer Genome Atlas, we designed a novel analytical process to extract non-human small RNA sequences and align them with the microbial genome to obtain a comprehensive view of the cancer-associated microbiome. In the present study, we identified > 1000 genera among 630 colorectal samples and clustered these samples into three distinctive colorectal enterotypes. Each cluster has its own distinctive microbial composition and interactions. Furthermore, we found 12 genera from these clusters that are associated with cancer stages and revealed their putative functions. Our results indicate that the proposed analytical approach can effectively determine the cancer-associated microbiome. It may be readily applied to explore other types of cancer, in which specimens of the microbiome are difficult to collect.

scholarly journals Targeted Drug Repurposing Against the SARS-CoV-2 E channel Identifies Blockers With in vitro Antiviral Activity

2021 ◽  
Author(s):  
Prabhat Pratap Singh Tomar ◽  
Miriam Krugliak ◽  
Isaiah Tuvia Arkin
Keyword(s):  
Tissue Culture ◽  
Ion Channels ◽  
Antiviral Activity ◽  
Viral Replication ◽  
Drug Repurposing ◽  
Viral Spread ◽  
E Protein ◽  
Human Use ◽  
The Impact

It is difficult to overstate the impact that COVID-19 had on humankind. The pandemic’s etiological agent, SARS-CoV-2, is a member of the Coronaviridae, and as such, is an enveloped virus with ion channels in its membrane. Therefore, in an attempt to provide an option to curb the viral spread, we searched for blockers of its E protein viro-porin. Using three bacteria-based assays, we identified eight compounds that exhibited activity after screening a library of ca. 3000 approved-for-human-use drugs. Reassuringly, analysis of viral replication in tissue culture indicated that most of the compounds could reduce infectivity to varying extents. In conclusion, targeting a particular channel in the virus for drug repurposing may increase our arsenal of treatment options to combat COVID-19 virulence.Significance StatementThe goal of our study was to expand the treatment arsenal against COVID-19. To that end, we have decided to focus on drug therapy, and as a target - the E protein, an ion channel in the virus. Ion channels as a family are excellent drug targets, but viral channels have been underexploited for pharmaceutical point intervention. To hasten future regulatory requirements and focus the chemical search space, we screened a library of ca. 3000 approved-for-human-use drugs using three independent bacteria-based assays. Our results yielded eight compounds, which were subsequently tested for antiviral activity in tissue culture. Gratifyingly, most compounds were able to reduce viral replication, and as such, both validate our approach and potentially augment our anti-COVID tool kit.

scholarly journals Nm-Nano: Predicting 2′-O-methylation (Nm) Sites in Nanopore RNA Sequencing Data

2022 ◽  
Author(s):  
Doaa Hassan Salem ◽  
Aditya Ariyur ◽  
Swapna Vidhur Daulatabad ◽  
Quoseena Mir ◽  
Sarath Chandra Janga
Keyword(s):  
Hela Cell ◽  
Cell Line ◽  
Rna Sequencing ◽  
Hek293 Cell ◽  
Nanopore Sequencing ◽  
Hela Cell Line ◽  
Sequencing Data ◽  
Rna Sequences ◽  
Rna Sequence ◽  
Oxford Nanopore

Nm (2′-O-methylation) is one of the most abundant modifications of mRNAs and non-coding RNAs occurring when a methyl group (–CH3) is added to the 2′ hydroxyl (–OH) of the ribose moiety. This modification can appear on any nucleotide (base) regardless of the type of nitrogenous base, because each ribose sugar has a hydroxyl group and so 2′-O-methyl ribose can occur on any base. Nm modification has a great contribution in many biological processes such as the normal functioning of tRNA, the protection of mRNA against degradation by DXO, and the biogenesis and specificity of rRNA. Recently, the single-molecule sequencing techniques for long reads of RNA sequences data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications on the molecule that is being sequenced, but to our knowledge there was only one research attempt that applied this technology to predict the stoichiometry of Nm-modified sites in RNA sequence of yeast cells. To this end, in this paper, we extend this research direction by proposing a bio-computational framework, Nm-Nano for predicting Nm sites in Nanopore direct RNA sequencing reads of human cell lines. Nm-Nano framework integrates two supervised machine learning models for predicting Nm sites in Nanopore sequencing data, namely Xgboost and Random Forest (RF). Each model is trained with set of features that are extracted from the raw signal generated by the Oxford Nanopore MinION device, as well as the corresponding basecalled k-mer resulting from inferring the RNA sequence reads from the generated Nanopore signals. The results on two benchmark data sets generated from RNA Nanopore sequencing data of Hela and Hek293 cell lines show a great performance of Nm-Nano. In independent validation testing, Nm-Nano has been able to identify Nm sites with a high accuracy of 93% and 88% using Xgboost and RF models respectively by training each model with Hela benchmark dataset and testing it for identifying Nm sites on Hek293 benchmark dataset. Thus, Nm-Nano outperforms the Nm sites predictors existing in the literature (not relying on Nanopore technology) that were only limited to predict Nm sites on short reads of RNA sequences and unable to predict Nm sites on long RNA sequence reads. By deploying Nm-Nano to predict Nm sites in Hela cell line, it was revealed that a total of 196 genes was identified to have the most abundance of Nm modification among all other genes that have been modified by Nm in this cell line. Similarly, deploying Nm-Nano to predict Nm sites in Hek393 cell line revealed that a total of 196 genes line was identified to have the most abundance of Nm modification among all other genes that have been modified by Nm in this cell line. According to this, a significant enrichment of a wide range of functional processes like high confidences (adjusted p-val < 0.05) enriched ontologies that were more representative of Nm modification role in immune response and cellular homeostasis were revealed in Hela cell line, and "MHC class 1 protein complex", "mitotic spindle assembly", "response to glucocorticoid", and "nucleocytoplasmic transport" were revealed in Hek293 cell line. The source code of Nm-Nano can be freely accessed https://github.com/Janga-Lab/Nm-Nano.

scholarly journals Brilacidin Demonstrates Inhibition of SARS-CoV-2 in Cell Culture

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 271 ◽  
Author(s):  
Allison Bakovic ◽  
Kenneth Risner ◽  
Nishank Bhalla ◽  
Farhang Alem ◽  
Theresa L. Chang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antiviral Activity ◽  
Cell Line ◽  
Viral Entry ◽  
De Novo ◽  
Biological Properties ◽  
Defense Proteins ◽  
Culture Models ◽  
Different Strains ◽  
The Impact

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin—a de novo designed synthetic small molecule that captures the biological properties of HDPs—on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.

The Impact of Antioxidants from the Diet on Breast Cancer Cells Monitored by Raman Microspectroscopy

2018 ◽  
Vol 16 (2) ◽  
pp. 127-137
Author(s):  
Paula Sofia Coutinho Medeiros ◽  
Ana Lúcia Marques Batista de Carvalho ◽  
Cristina Ruano ◽  
Juan Carlos Otero ◽  
Maria Paula Matos Marques
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Breast Adenocarcinoma ◽  
Coumaric Acid ◽  
Human Breast ◽  
The Impact

Background: The impact of the ubiquitous dietary phenolic compound p-coumaric acid on human breast cancer cells was assessed, through a multidisciplinary approach: Combined biological assays for cytotoxicity evaluation and biochemical profiling by Raman microspectroscopic analysis in cells. </P><P> Methods: Para-coumaric acid was shown to exert in vitro chemoprotective and antitumor activities, depending on the concentration and cell line probed: a significant anti-invasive ability was detected for the triple-negative MDA-MB-231 cells, while a high pro-oxidant effect was found for the estrogen- dependent MCF-7 cells. A striking cell selectivity was obtained, with a more noticeable outcome on the triple-negative MDA-MB-231 cell line. Results: The main impact on the cellular biochemical profile was verified to be on proteins and lipids, thus justifying the compound´s anti-invasive effect and chemoprotective ability. Conclusion: p-Coumaric acid was thus shown to be a promising chemoprotective/chemotherapeutic agent, particularly against the low prognosis triple-negative human breast adenocarcinoma.

scholarly journals Upgrading the Repertoire of miRNAs in Gastric Adenocarcinoma to Provide a New Resource for Biomarker Discovery

2019 ◽  
Vol 20 (22) ◽  
pp. 5697 ◽  
Author(s):  
Michelle E. Pewarchuk ◽  
Mateus C. Barros-Filho ◽  
Brenda C. Minatel ◽  
David E. Cohn ◽  
Florian Guisier ◽  
...  
Keyword(s):  
Gastric Adenocarcinoma ◽  
Biomarker Discovery ◽  
The Cancer Genome Atlas ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Specific Expression ◽  
Novel Mirna ◽  
Cancer Genome Atlas ◽  
Context Specific ◽  
Independent Cohort

Recent studies have uncovered microRNAs (miRNAs) that have been overlooked in early genomic explorations, which show remarkable tissue- and context-specific expression. Here, we aim to identify and characterize previously unannotated miRNAs expressed in gastric adenocarcinoma (GA). Raw small RNA-sequencing data were analyzed using the miRMaster platform to predict and quantify previously unannotated miRNAs. A discovery cohort of 475 gastric samples (434 GA and 41 adjacent nonmalignant samples), collected by The Cancer Genome Atlas (TCGA), were evaluated. Candidate miRNAs were similarly assessed in an independent cohort of 25 gastric samples. We discovered 170 previously unannotated miRNA candidates expressed in gastric tissues. The expression of these novel miRNAs was highly specific to the gastric samples, 143 of which were significantly deregulated between tumor and nonmalignant contexts (p-adjusted < 0.05; fold change > 1.5). Multivariate survival analyses showed that the combined expression of one previously annotated miRNA and two novel miRNA candidates was significantly predictive of patient outcome. Further, the expression of these three miRNAs was able to stratify patients into three distinct prognostic groups (p = 0.00003). These novel miRNAs were also present in the independent cohort (43 sequences detected in both cohorts). Our findings uncover novel miRNA transcripts in gastric tissues that may have implications in the biology and management of gastric adenocarcinoma.

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