35 research outputs found

    MAPK ERK Signaling Regulates the TGF-β1-Dependent Mosquito Response to Plasmodium falciparum

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    Malaria is caused by infection with intraerythrocytic protozoa of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Although a variety of anti-parasite effector genes have been identified in anopheline mosquitoes, little is known about the signaling pathways that regulate these responses during parasite development. Here we demonstrate that the MEK-ERK signaling pathway in Anopheles is controlled by ingested human TGF-β1 and finely tunes mosquito innate immunity to parasite infection. Specifically, MEK-ERK signaling was dose-dependently induced in response to TGF-β1 in immortalized cells in vitro and in the A. stephensi midgut epithelium in vivo. At the highest treatment dose of TGF-β1, inhibition of ERK phosphorylation increased TGF-β1-induced expression of the anti-parasite effector gene nitric oxide synthase (NOS), suggesting that increasing levels of ERK activation negatively feed back on induced NOS expression. At infection levels similar to those found in nature, inhibition of ERK activation reduced P. falciparum oocyst loads and infection prevalence in A. stephensi and enhanced TGF-β1-mediated control of P. falciparum development. Taken together, our data demonstrate that malaria parasite development in the mosquito is regulated by a conserved MAPK signaling pathway that mediates the effects of an ingested cytokine

    Anopheles stephensi p38 MAPK signaling regulates innate immunity and bioenergetics during Plasmodium falciparum infection.

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    BackgroundFruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum.MethodsA. stephensi p38 MAPK (AsP38 MAPK) was identified and patterns of signaling in vitro and in vivo (midgut) were analyzed using phospho-specific antibodies and small molecule inhibitors. Functional effects of AsP38 MAPK inhibition were assessed using P. falciparum infection, quantitative real-time PCR, assays for reactive oxygen species and survivorship under oxidative stress, proteomics, and biochemical analyses.ResultsThe genome of A. stephensi encodes a single p38 MAPK that is activated in the midgut in response to parasite infection. Inhibition of AsP38 MAPK signaling significantly reduced P. falciparum sporogonic development. This phenotype was associated with AsP38 MAPK regulation of mitochondrial physiology and stress responses in the midgut epithelium, a tissue critical for parasite development. Specifically, inhibition of AsP38 MAPK resulted in reduction in mosquito protein synthesis machinery, a shift in glucose metabolism, reduced mitochondrial metabolism, enhanced production of mitochondrial reactive oxygen species, induction of an array of anti-parasite effector genes, and decreased resistance to oxidative stress-mediated damage. Hence, P. falciparum-induced activation of AsP38 MAPK in the midgut facilitates parasite infection through a combination of reduced anti-parasite immune defenses and enhanced host protein synthesis and bioenergetics to minimize the impact of infection on the host and to maximize parasite survival, and ultimately, transmission.ConclusionsThese observations suggest that, as in mammals, innate immunity and mitochondrial responses are integrated in mosquitoes and that AsP38 MAPK-dependent signaling facilitates mosquito survival during parasite infection, a fact that may attest to the relatively longer evolutionary relationship of these parasites with their invertebrate compared to their vertebrate hosts. On a practical level, improved understanding of the balances and trade-offs between resistance and metabolism could be leveraged to generate fit, resistant mosquitoes for malaria control

    MAPK ERK Signaling Regulates the TGF-β1-Dependent Mosquito Response to Plasmodium falciparum

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    Malaria is caused by infection with intraerythrocytic protozoa of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Although a variety of anti-parasite effector genes have been identified in anopheline mosquitoes, little is known about the signaling pathways that regulate these responses during parasite development. Here we demonstrate that the MEK-ERK signaling pathway in Anopheles is controlled by ingested human TGF-β1 and finely tunes mosquito innate immunity to parasite infection. Specifically, MEK-ERK signaling was dose-dependently induced in response to TGF-β1 in immortalized cells in vitro and in the A. stephensi midgut epithelium in vivo. At the highest treatment dose of TGF-β1, inhibition of ERK phosphorylation increased TGF-β1-induced expression of the anti-parasite effector gene nitric oxide synthase (NOS), suggesting that increasing levels of ERK activation negatively feed back on induced NOS expression. At infection levels similar to those found in nature, inhibition of ERK activation reduced P. falciparum oocyst loads and infection prevalence in A. stephensi and enhanced TGF-β1-mediated control of P. falciparum development. Taken together, our data demonstrate that malaria parasite development in the mosquito is regulated by a conserved MAPK signaling pathway that mediates the effects of an ingested cytokine

    Expression of miR-29, miR-125, and miR-181 in the anterior kidneys of Streptococcus-infected Nile tilapia (Oreochromis niloticus)

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    MicroRNAs (miRNAs) are a group of small non-coding RNAs which posttranscriptionally regulate gene expression in eukaryotic cells. In this study, three miRNAs participating in the immune function in mammalian cells: miR-29, miR-125, and miR-181 have been characterized in Nile tilapia (Oreochromis niloticus). A comparison of all three miRNAs in Nile tilapia and other organisms, including zebrafish, mouse, rat, chicken, pig, and human revealed identical sequence of mature miRNA sequences. The genomic sequences of miR-29, miR-125, and miR-181 of Nile tilapia have been amplified using polymerase chain reaction (PCR) and cloned into pGT-19T plasmid. The plasmids containing the sequences of miR-29, miR-125, and miR-181 were used for standardization and validation of quantitative PCR (qPCR) methods. Moreover, the qPCR methods have been applied as a tool to determine the expression of all three miRNAs in the anterior kidneys of Streptococcus agalactiae-infected tilapia. Specifically, expression of miR-29, miR-125, and miR-181 reduced significantly at 3 days post bacterial challenge. In silico analysis of three miRNA targets revealed that multiple genes could be regulated by these miRNAs including Ifi30, Cd276, Lfng, Cdc42se1, Ddx3x, Il1a, and Atg5. In conclusion, these results demonstrated the expression level and molecular targets of miRNAs in Nile tilapia which could be applied for the understanding of these miRNAs during pathogen-host interaction

    The Modulation of Immune Responses in <i>Tilapinevirus tilapiae</i>-Infected Fish Cells through MAPK/ERK Signalling

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    Tilapia lake virus (TiLV) is a novel RNA virus that has been causing substantial economic losses across the global tilapia industry. Despite extensive research on potential vaccines and disease control methods, the understanding of this viral infection and the associated host cell responses remains incomplete. In this study, the involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in the early stages of TiLV infection was investigated. The results showed a distinct pattern of ERK phosphorylation (p-ERK) upon TiLV infection in two fish cell lines, E-11 and TiB. Specifically, the p-ERK levels in the TiB cells decreased substantially, while the p-ERK levels in the E-11 cells remained constant. Interestingly, a large number of cytopathic effects were observed in the infected E-11 cells but none in the infected TiB cells. Furthermore, when p-ERK was suppressed using the inhibitor PD0325901, a significant reduction in the TiLV load and decrease in the mx and rsad2 gene expression levels were observed in the TiB cells in days 1–7 following infection. These findings highlight the role of the MAPK/ERK signalling pathway and provide new insights into the cellular mechanisms during TiLV infection that could be useful in developing new strategies to control this virus

    Expressions of miR-155 and miR-181 and predictions of their structures and targets in pigs (Sus scrofa)

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    Background and Aim: MicroRNAs (miRNAs) are responsible for gene expression control at the post-transcription level in many species. Several miRNAs are required in the regulation of immune responses, such as B-cell differentiation, T-cell receptor signaling pathway, CD4+ T cell selection, and so on. Studies on miRNAs have been extensively conducted in humans and mice; however, reports relevant to miRNAs, especially miR-155 and miR-181, in pigs are limited. Consequently, the present study aimed to investigate the structures, target genes, and expressions of miR-155 and miR-181 in various porcine cells and tissues. Materials and Methods: Five healthy male pigs from a porcine reproductive and respiratory syndrome virus-negative farm were studied. Before slaughter, blood samples were collected for peripheral blood mononuclear cell isolation. After slaughter, samples of spleen, lymph nodes, and forelimb muscles were collected. Both miR-155 and miR-181 were investigated for their structures with RNAfold web server, for their target genes from three online web servers, and for their expressions using polymerase chain reaction (PCR). Results: The structures of miR-155 and miR-181 contained hairpins with free energies of –35.27 and –35.29 kcal/mole, respectively. Target gene prediction revealed that miR-155 had perfect complementarity with Socs1 and Mapk3k14, while miR-181 had perfect complementarity with Ddx3x, Nfat5, Foxp1, and Mpp5. PCR showed that both miRNAs were detectable from all investigated cells and tissues. Moreover, the highest expression of both miRNAs was found from the lymph node of the pigs. Conclusion: Both miR-155 and miR-181 might be involved with the regulation of porcine immune functions as both miRNAs were detected in several cells and tissues of the pigs. In addition, they had very high complementarities with the seed regions of several immune-related genes

    Effects of sample preservation and storage times on the detection of tilapia lake virus (TiLV) RNA in tilapia tissues

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    Tilapia lake virus (TiLV) is a novel orthomyxo-like virus, and has genomic RNA material that is easily degradable. Hence, proper sample storage and preservation should be applied for accurate diagnostic results. In this study, we investigated various conditions for preserving tilapia tissues for the detection of TiLV. Our results revealed that RNAlater® and deep-freezing at −20 °C are the best practices to maintain TiLV genomic RNA for subsequent diagnosis. Samples stored in these conditions could maintain TiLV genomic RNA for 365 days with minimal reduction. In contrast, TiLV genomic RNA is substantially degraded in ethanol and on Whatman® FTA® classic cards with a reduction of TiLV genomic material of 2 logs and up to 3 logs within 30 days of storage, respectively. Besides, all preservation methods showed a difference in the amount of TiLV genomic RNA between the initial day, during, and after 30 days of storage. The benefits of this study can be applied to preserve samples efficiently and to transport samples properly from remote areas to a laboratory that is suitably-equipped for disease examination.The Faculty of Veterinary Medicine and the Center for Advanced Studies for Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand, under the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Ministry of Education, Thailand.http://www.elsevier.com/ locate/aqua-online2021-12-05hj2021Paraclinical Science

    ICTV Virus Taxonomy Profile: Amnoonviridae 2023

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    Amnoonviridae is a family of negative-sense RNA viruses with genomes totalling about 10.3 kb. These viruses have been found in fish. The amnoonvirid genome consists of 10 segments, each with at least 1 open reading frame (ORF). The RNA1–3 ORFs encode the three subunits of the viral polymerase. The RNA4 ORF encodes a nucleoprotein. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Amnoonviridae, which is available at ictv.global/report/amnoonviridae

    Tilapia lake virus causes mitochondrial damage: a proposed mechanism that leads to extensive death in fish cells

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    Background Tilapia lake virus (TiLV), also known as Tilapinevirus tilapiae, poses a significant threat to tilapia aquaculture, causing extensive mortality and economic losses. Understanding the mechanisms and pathogenesis of TiLV is crucial to mitigate its impact on this valuable fish species. Methodology In this study, we utilized transmission electron microscopy to investigate the ultrastructural changes in E-11 cells following TiLV infection. We also examined the presence of TiLV particles within the cells. Cellular viability and mitochondrial functions were assessed using MTT and ATP measurement assays and mitochondrial probes including JC-1 staining and MitoTracker™ Red. Results Our findings provide novel evidence demonstrating that TiLV causes cytotoxicity through the destruction of mitochondria. Transmission electron micrographs showed that TiLV particles were present in the cytoplasm of E-11 cells as early as 1 h after infection. Progressive swelling of mitochondria and ultrastructural damage to the cells were observed at 1, 3 and 6 days post-infection. Furthermore, losses of mitochondrial mass and membrane potential (MMP) were detected at 1 day after TiLV inoculation, as determined by mitochondrial probes. The results of the MTT assay also supported the hypothesis that the cell deaths in E-11 cells during TiLV infection may be caused by the disruption of mitochondrial structure and function. Conclusions Our study reveals the significant role of mitochondrial disruption in contributing to cellular death during the early stages of TiLV infection. These findings advance the understanding of TiLV pathogenesis and further enhance our knowledge of viral diseases in fish
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