Prediction and validation of regulatory role of microRNAs in zebrafish (Danio rerio) responses to nanoparticle exposure with in silico and in vitro toxicological approaches

The release of engineered nanoparticles as by-product of human activities in the environment can interfere with normal biology and health of the exposed organisms. MicroRNAs have been suggested as potential toxicology biomarkers, however the information about expression and role of microRNA in regulation of signaling pathways in organisms exposed to nanoparticles (NP) is limited. Summary of reported biological and pathological outcomes of NP induced toxicity in zebrafish was followed with in silico analysis of the genes potentially responsible for observed toxicological effects. After identifying relevant genes, we constructed six miRNA-mRNA regulatory networks involved in nanoparticle induced toxicological responses in zebrafish. Based on our prediction and selection criteria, we identified six miRNAs that overlapped in networks with high prediction scores, and were validated by previous mammalian and zebrafish microRNA profiling studies: dre-miR-124, -144, -148, -155, -19a, -223. As the next step, we validated the expression of these six miRNAs in THP-1 human monocytic cell line after the exposure to Polystyrene (PS NPs) and ARS labeled Titanium dioxide nanoparticles (TiO2-ARS NPs). Also, identification of miRNAs expression post exposure to PLGA nanoparticles and E. coli BioParticles was used to exclude potential activation and engagement of miRNAs through phagocytosis or pro-inflammatory specific responses. In our study, miR-155-5p showed the most promise as biomarker for PS NPs and TiO2-ARS NPs induced adverse effects. To determine potential for PS NPs and TiO2-ARS NPs for genotoxicity, time and dose dependent DNA damage profile induced by PS NPs or TiO2-ARS NPs was established by comet assay. Results indicated the severe DNA damage was triggered by both PS NPs and TiO2-ARS NPs. However, we observed that the expression of DNA damage repairing genes was elevated post TiO2-ARS NPs but not post PS NPs exposure, questioning the utility of the comet assay as universal assessment tool for genotoxicity induced by nanoparticles in general. It was observed that after PS NPs exposure the successful transfection of miR-155-5p mimic induced the expression of ATM, TAOK1, TRIP13, and APAF-1 while the expression of ERCC1 was attenuated. The ATM, APAF-1 and RAD51 were strongly activated post TiO2-ARS NPs stimulation in mimic-transfected cells. These observations suggest there is significant involvement of miR-155-5p in PS NPs and TiO2-ARS NPs induced adverse effects.Für den menschlichen Gebrauch entwickelte Nanopartikel geraten auf Grund ihrer Verwendung in die Umwelt und gehen dort unter Umständen adverse Wechselwirkungen mit den biologischen Abläufen und der Gesundheit der angetroffenen Organismen ein. MicroRNAs sind bereits mögliche Biomarker für ähnliche toxikologische Fragestellungen. Es ist jedoch ungeklärt, wie sich microRNA Expressionsmuster und deren Regulation von Signalwegen nach einer Nanopartikel (NP) Exposition verhalten. Nachstehende Untersuchungen wurden daher angestellt: Die biologischen und pathologischen Folgen einer NP induzierten Intoxikation im Zebrafischmodell und daraufhin eine in silico Analyse von relevanten Genen, die potentiell mit toxikologischen Effekten im Zusammenhang stehen könnten. Nach einer Bestimmung dieser relevanten Gene, konnten wir sechs miRNA-mRNA Regelnetzwerke auffinden, die im Zusammenhang mit einer Nanopartikel induzierten Reaktion stehen können. Basierend auf dieser Vorhersage und der so getroffenen Auswahlkriterien war es möglich, diese miRNAs: dre-miR-124, -144, -148, -155, -19a, -223 zu identifizieren, die mit hohen Vorhersagewerten in den Regelnetzwerken auftreten und bereits in vorangegangen Profiling Studien bestätigt wurden. Im nächsten Schritt validierten wir die Expression dieser sechs miRNAs in THP-1 Monozyten nach einer Exposition mit Polystyrol (PS NP) und ARS markierten Titandioxid Nanopartikeln (TiO2-ARS NP). Ebenfalls wurde die miRNA Expression nach einer PLGA Nanopartikel und E. coli Bio-Partikel Exposition untersucht, um eine potentielle Verbindung der miRNA Aktivierung zu Phagozytose Vorgängen oder pro-inflammatorischen Reaktionen ausschließen zu können. Die microRNA miR-155-5p zeigte in dieser Studie das vielversprechendste Potential, um als Biomarker für PS NP und TiO2-ARS NP induzierte negative Effekte nutzbar zu sein. Zur Bestimmung des genotoxischen Potentials einer PS NP und TiO2-ARS NP Exposition wurde zusätzlich ein zeit- und dosisabhängiges DNA Schadensprofil durch einen Comet Assay erstellt. Die Ergebnisse zeigten, dass beide Nanopartikelarten in diesem erhebliche DNA Schäden provozieren. Ebenso konnten wir aber feststellen, dass auf eine TiO2-ARS NP Exposition hin, entsprechende DNA-Reparatur-Gene verstärkt exprimiert wurden, diese Reaktion blieb auf PS NP aber aus. Dieser Widerspruch stellt den Nutzen des Comet Assays als universelles Bewertungswerkzeug für genotoxische Geschehnisse durch Nanopartikel generell in Frage. Nach einer PS NP Exposition von THP 1 Zellen, bei den vorher eine erfolgreiche Transfektion mit einem miR-155-5p Mimic stattgefunden hat, wurde eine vermehrte induzierte Expression von ATM, TAOK1, TRIP13 und APAF-1 festgestellt, während die Expression von ERCC1 gedämpft wurde. Die Gene ATM, APAF-1 und RAD51 wurden auch stark in Zellen aktiviert, die das Mimic enthielten aber TiO2-ARS NP ausgesetzt wurden. Diese Beobachtungen lassen vermuten, dass die mircroRNA miR-155-5p eine signifikante Rolle bei den adversen Auswirkungen spielt, die durch PS und TiO2-ARS Nanopartikel in Organismen provoziert werden

Prediction and validation of regulatory role of microRNAs in zebrafish (Danio rerio) responses to nanoparticle exposure with in silico and in vitro toxicological approaches

The release of engineered nanoparticles as by-product of human activities in the environment can interfere with normal biology and health of the exposed organisms. MicroRNAs have been suggested as potential toxicology biomarkers, however the information about expression and role of microRNA in regulation of signaling pathways in organisms exposed to nanoparticles (NP) is limited. Summary of reported biological and pathological outcomes of NP induced toxicity in zebrafish was followed with in silico analysis of the genes potentially responsible for observed toxicological effects. After identifying relevant genes, we constructed six miRNA-mRNA regulatory networks involved in nanoparticle induced toxicological responses in zebrafish. Based on our prediction and selection criteria, we identified six miRNAs that overlapped in networks with high prediction scores, and were validated by previous mammalian and zebrafish microRNA profiling studies: dre-miR-124, -144, -148, -155, -19a, -223. As the next step, we validated the expression of these six miRNAs in THP-1 human monocytic cell line after the exposure to Polystyrene (PS NPs) and ARS labeled Titanium dioxide nanoparticles (TiO2-ARS NPs). Also, identification of miRNAs expression post exposure to PLGA nanoparticles and E. coli BioParticles was used to exclude potential activation and engagement of miRNAs through phagocytosis or pro-inflammatory specific responses. In our study, miR-155-5p showed the most promise as biomarker for PS NPs and TiO2-ARS NPs induced adverse effects. To determine potential for PS NPs and TiO2-ARS NPs for genotoxicity, time and dose dependent DNA damage profile induced by PS NPs or TiO2-ARS NPs was established by comet assay. Results indicated the severe DNA damage was triggered by both PS NPs and TiO2-ARS NPs. However, we observed that the expression of DNA damage repairing genes was elevated post TiO2-ARS NPs but not post PS NPs exposure, questioning the utility of the comet assay as universal assessment tool for genotoxicity induced by nanoparticles in general. It was observed that after PS NPs exposure the successful transfection of miR-155-5p mimic induced the expression of ATM, TAOK1, TRIP13, and APAF-1 while the expression of ERCC1 was attenuated. The ATM, APAF-1 and RAD51 were strongly activated post TiO2-ARS NPs stimulation in mimic-transfected cells. These observations suggest there is significant involvement of miR-155-5p in PS NPs and TiO2-ARS NPs induced adverse effects.Für den menschlichen Gebrauch entwickelte Nanopartikel geraten auf Grund ihrer Verwendung in die Umwelt und gehen dort unter Umständen adverse Wechselwirkungen mit den biologischen Abläufen und der Gesundheit der angetroffenen Organismen ein. MicroRNAs sind bereits mögliche Biomarker für ähnliche toxikologische Fragestellungen. Es ist jedoch ungeklärt, wie sich microRNA Expressionsmuster und deren Regulation von Signalwegen nach einer Nanopartikel (NP) Exposition verhalten. Nachstehende Untersuchungen wurden daher angestellt: Die biologischen und pathologischen Folgen einer NP induzierten Intoxikation im Zebrafischmodell und daraufhin eine in silico Analyse von relevanten Genen, die potentiell mit toxikologischen Effekten im Zusammenhang stehen könnten. Nach einer Bestimmung dieser relevanten Gene, konnten wir sechs miRNA-mRNA Regelnetzwerke auffinden, die im Zusammenhang mit einer Nanopartikel induzierten Reaktion stehen können. Basierend auf dieser Vorhersage und der so getroffenen Auswahlkriterien war es möglich, diese miRNAs: dre-miR-124, -144, -148, -155, -19a, -223 zu identifizieren, die mit hohen Vorhersagewerten in den Regelnetzwerken auftreten und bereits in vorangegangen Profiling Studien bestätigt wurden. Im nächsten Schritt validierten wir die Expression dieser sechs miRNAs in THP-1 Monozyten nach einer Exposition mit Polystyrol (PS NP) und ARS markierten Titandioxid Nanopartikeln (TiO2-ARS NP). Ebenfalls wurde die miRNA Expression nach einer PLGA Nanopartikel und E. coli Bio-Partikel Exposition untersucht, um eine potentielle Verbindung der miRNA Aktivierung zu Phagozytose Vorgängen oder pro-inflammatorischen Reaktionen ausschließen zu können. Die microRNA miR-155-5p zeigte in dieser Studie das vielversprechendste Potential, um als Biomarker für PS NP und TiO2-ARS NP induzierte negative Effekte nutzbar zu sein. Zur Bestimmung des genotoxischen Potentials einer PS NP und TiO2-ARS NP Exposition wurde zusätzlich ein zeit- und dosisabhängiges DNA Schadensprofil durch einen Comet Assay erstellt. Die Ergebnisse zeigten, dass beide Nanopartikelarten in diesem erhebliche DNA Schäden provozieren. Ebenso konnten wir aber feststellen, dass auf eine TiO2-ARS NP Exposition hin, entsprechende DNA-Reparatur-Gene verstärkt exprimiert wurden, diese Reaktion blieb auf PS NP aber aus. Dieser Widerspruch stellt den Nutzen des Comet Assays als universelles Bewertungswerkzeug für genotoxische Geschehnisse durch Nanopartikel generell in Frage. Nach einer PS NP Exposition von THP 1 Zellen, bei den vorher eine erfolgreiche Transfektion mit einem miR-155-5p Mimic stattgefunden hat, wurde eine vermehrte induzierte Expression von ATM, TAOK1, TRIP13 und APAF-1 festgestellt, während die Expression von ERCC1 gedämpft wurde. Die Gene ATM, APAF-1 und RAD51 wurden auch stark in Zellen aktiviert, die das Mimic enthielten aber TiO2-ARS NP ausgesetzt wurden. Diese Beobachtungen lassen vermuten, dass die mircroRNA miR-155-5p eine signifikante Rolle bei den adversen Auswirkungen spielt, die durch PS und TiO2-ARS Nanopartikel in Organismen provoziert werden

Response of microbial communities in the tobacco phyllosphere under the stress of validamycin

Validamycin, is classified as an environmentally friendly fungicide. It has high efficacy with little associated pollution risk, and it has been used in China on tobacco for many years especially during leaf spot season. To understand changes in microbial communities and functional aspects of the tobacco phyllosphere after exposure to validamycin, the chemical was sprayed on tobacco leaves during brown spot epidemic periods caused by Alternaria alternata, and asymptomatic and symptomatic leaves of tobacco were sampled at different times (0 day before, 5, 10, and 15 days after application). The fungal and bacterial population diversity and structure were revealed using Illumina NovaSeq PE250 high-throughput sequencing technology, and Biolog-ECO technology which analyzes the metabolic differences between samples by using different carbon sources as the sole energy source. The results showed that the microbial community structure of both asymptomatic and symptomatic tobacco leaves changed after the application of valproate, with the microbial community structure of the asymptomatic tobacco leaves being more strongly affected than that of the symptomatic leaves, and the diversity of bacteria being greater than that of fungi. Phyllosphere fungal diversity in asymptomatic leaves increased significantly after application, and bacterial abundance and diversity in both asymptomatic and symptomatic leaves first increased and then decreased. Validamycin treatment effectively reduced the relative abundance of Alternaria, Cladosporium, Kosakonia, and Sphingomonas in leaves showing symptoms of tobacco brown spot, while the relative abundance of Thanatephorus, Pseudomonas, and Massilia increased significantly after application. Furthermore, the ability to metabolize a variety of carbon sources was significantly reduced in both types of leaves after validamycin application, and both types had a weaker ability to metabolize α-Ketobutyric Acid after application. This study reveals phyllosphere micro-ecological changes in symptomatic and asymptomatic tobacco leaves during different periods after validamycin application and the effects on the metabolic capacity of phyllosphere microorganisms. It can provide some basis for exploring the effect of validamycin on the control of tobacco brown spot

Kupffer cell activation by different microbial lysates: Toll‐like receptor‐2 plays pivotal role on thromboxane A2 production in mice and humans

Thromboxane (TX) A2 has been identified as an important intrahepatic vasoconstrictor upon Kupffer cell (KC) activation during infections such as spontaneous bacterial peritonitis (SBP). The study aimed to investigate the role of TLRs in the TXA2 increase in liver nonparenchymal cells and their related mechanisms. Here, we identified TLR‐2 as a common pathway for different microbials: microbial lysates including Gram‐positive bacteria, Gram‐negative bacteria, and fungi all increased TXA2 secretion via activation of TLR‐2 in human KCs, accompanied by increased expression and phosphorylation of Myd88‐related pathway. Of all TLR agonists, only TLR‐1, ‐2, and ‐4 agonists increased TXA2 in human KCs. These results were further confirmed by mouse liver nonparenchymal cells. Comparing the effects of TLR‐1, ‐2, and ‐4 antagonists, only TLR‐2 antagonist showed inhibitory effects with all tested microbial lysates. Pretreatment with TLR‐2 antagonist in human KCs blocked the secretion of IL‐10, CXCL‐10, TNF‐α, and IL‐6 induced by Gram‐positive and Gram‐negative bacterial stimulation. IL‐23 and IL‐1β were only induced by Gram‐negative bacteria. Thus, TLR‐2 might be a potential marker and an attractive target for future treatment of patients with SBP. In addition, IL‐23 and IL‐1β might distinguish early between Gram‐positive and Gram‐negative SBP

In silico prediction of MicroRNA role in regulation of Zebrafish (Danio rerio) responses to nanoparticle exposure

The release of nanoparticles to the environment can affect health of the exposed organisms. MicroRNAs have been suggested as potential toxicology biomarkers, however the information about use of microRNA in aquatic organisms exposed to nanoparticles (NP) is limited. In silico analysis from publicly available gene expression data was performed. Data selection for the analysis was based on reported biological and pathological outcomes of NP induced toxicity in zebrafish. After identifying relevant genes, we constructed six miRNA-mRNA regulatory networks involved in nanoparticle induced toxicological responses in zebrafish. Based on our prediction and selection criteria we selected six miRNAs that overlapped in constructed networks with remarkable prediction score, and were validated by previous mammalian and zebrafish microRNA profiling studies: dre-miR-124, −144, −148, −155, −19a, −223. The results of this in silico analysis indicate that several highly conserved miRNAs likely have a regulatory role of organismal responses to nanoparticles, and can possibly be used as biomarkers of nanotoxicity in studies using zebrafish as model organism One health approaches.This is a manuscript of an article published as Hu, Moyan, Boris Jovanović, and Dušan Palić. "In silico prediction of MicroRNA role in regulation of Zebrafish (Danio rerio) responses to nanoparticle exposure." Toxicology in Vitro (2019). doi: 10.1016/j.tiv.2019.05.014. Posted with permission.</p

Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila.

The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens

Therapeutic potential of a triazole curcumin in inflammation: Decreased LPS-induced acute lung injury in mice by targeting MD2/TLR4

Curcumin has a wide range of biological activities. This study investigated the anti-inflammatory activity of a triazole curcumin derivative (TAC)—especially its effect on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and possible targets. In the xylene-induced ear edema experiment, the inhibition rate of TAC against mice ear edema was 23.8%–78.2% over a dose range of 2.5–40 mg/kg 4 h after administration. At a dose of 10 mg/kg, the anti-inflammatory activity of TAC reached a peak at 4 h with an inhibition rate of 73.1%. This was significantly better than the positive control drug sodium diclofenac (DCF). TAC can also effectively reduce the degree of pulmonary edema injury in mice. H&E and Masson staining showed that the inflammatory and pathological indicators of LPS-induced lung injury were significantly improved by TAC. MTT tests illustrated that TAC showed weak cytotoxicity against RAW264.7 cells, and inhibited TNF-α and IL-6 release induced by LPS. Western blotting indicated that TAC decreased the expression of NF-κB and AP-1 in LPS pre-treated RAW264.7 cells, but failed to influence the expression of NF-κB in IL-1β pre-treated HET293T-Myd88−/− cells. Docking studies show that TAC could bind to the hydrophobic pocket of the CD2-TLR4 complex and expressed a high binding affinity. In conclusion, TAC exerts its anti-inflammatory effects by targeting the MD2-TLR4 signaling pathway, thus suggesting that it may be a promising candidate for the treatment of acute lung injury

Blast X search analysis of all transcripts of <i>C</i>. <i>idella</i>.

<p>Blast X search analysis of all transcripts of <i>C</i>. <i>idella</i>.</p

Top 15 list of pathways related to immune system.

<p>Top 15 list of pathways related to immune system.</p