The immunome and embryo quality in sea bream and sea bass

Gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) are teleosts belonging to Eupercaria and are the most important aquaculture fish species in the Mediterranean region. These two species are ranked second after the Atlantic salmon (Salmo salar) in production volume and value in the European Union (EU) aquaculture sector. Unpredictable fertilized egg/embryo quality and performance remain a bottleneck that threatens sustainability of sea bream and sea bass aquaculture, impeding the increased productivity of aquaculture that entirely depends on hatchery production. To address this issue, criteria and molecular markers linked to embryo quality that could be used to monitor and manage hatchery production were procured. Comparative molecular approaches using molecular biology, proteomics and transcriptomics were performed to analyze embryo performance and immunity in samples from several European commercial hatcheries. The core achievements were the: a) identification and characterization of lysozyme and complement 5 (C5) gene families and embryo and larval gene expression and enzyme activity from a diversity of hatcheries, b) characterization of the embryo proteome from three Mediterranean fish species [white sea bream (Diplodus sargus), meagre (Argyrosomus regius) and sea bream] 24h before hatch and at hatch and identification of common and species specific molecular patterns linked to biological function and putative quality-related proteins, c) comparative transcriptomics of good and poor quality sea bream embryos from several Mediterranean hatcheries. Qualitylinked transcripts and some elements of the regulatory epitranscriptome (non-coding RNA) were identified as well as the contribution of maternal proteins to embryos. Taken together, the results provide a comprehensive description of the molecular basis of sea bream and sea bass embryo development and reveal that immune-related molecules in fertilized eggs are low abundance. The development (quality)-related candidate markers identified will be of value for management of fish embryos in aquaculture hatcheries.O mar Mediterrâneo cobre apenas 0,7% da área oceânica mundial, mas é um dos principais reservatórios de biodiversidade marinha e costeira com cerca de 28% de espécies endémicas. A dourada (Sparus aurata) e o robalo (Dicentrarchus labrax), são duas espécies de peixe que pertencem à série Eupercaria e das mais comercializadas pela indústria de aquicultura nesta região. No sector da aquicultura da União Europeia (EU), estas duas espécies ocupam o segundo lugar no “ranking” da cadeia de valor, depois do salmão do Atlântico. Contudo, as suas características morfológicas e de crescimento são os parâmetros de qualidade relevantes considerados pela indústria. Esta abordagem deve-se à sua comercialização em formato de peixe inteiro, o que reduz o valor de mercado na cadeia de valor e também à ausência de critérios de qualidade para seleção de ovos e embriões, bem como, de marcadores moleculares de qualidade com maior grau de sensibilidade. Todos estes obstáculos, limitam substancialmente o desenvolvimento das indústrias associadas á comercialização destas duas espécies de peixe, impedindo a expansão da aquicultura e das “hatcheries” (maternidades incubadoras de ovos/embriões controlados artificialmente para fins comerciais). Para mitigar este problema, utilizou-se técnicas de biologia molecular e tecnologias ómicas e estabeleceu-se uma abordagem comparativa direcionada á descoberta de moléculas e vias metabólicas funcionais de importância crítica para o sistema imune dos peixes associada á “performance” de desenvolvimento de ovos e embriões. Esta tese está organizada em seis capítulos. Inicia-se com uma visão geral dos critérios morfológicos, físico-químicos e moleculares existentes para avaliar a qualidade de ovos e embriões para melhorar a gestão da aquicultura de peixes (Capítulo 1). Subsequentemente, caracterizou-se a família de genes do sistema imune: a) a das lisozimas em peixes teleósteos com enfase na sua caracterização molecular e funcional em dourada (Capítulo 2) e análises moleculares estruturais e evolutivas e b) a do complemento C5 (C5) em peixes especialmente em espécies da faamilia Cyprinidae (Capítulo 3). Os capítulos 4 e 5, integram abordagens de proteómica e transcriptómica em espécies de peixes mediterrânicos [pargo (Diplodus sargus), corvina (Argyrosomus regius) e dourada], focando os processos de desenvolvimento e de eclosão e na função da enzima “hatching enzyme” em dourada e robalo. Foi feita uma associação entre os capítulos e a análise integrada dos dados do transcritoma do embrião (Capítulo 5) revelou um padrão de expressão significativamente diferente (p-valor < 0,05) para o C5 (Capítulo 3) em diferentes lotes de embriões de dourada nas comparações entre graus de qualidade (Boa vrs Má) e entre estágios de desenvolvimento (Pré- eclosão vrs Eclosão). A variação do C5 em relação ao lote de embriões não foi afetada pela origem da “hatchery”, indicando que as prática de manejo ou os próprios reprodutores não influenciam a sua expressão. Os resultados sugerem que este gene e o seu produto proteico, são provavelmente importantes na proteção imunológica precoce e também em outras funções ainda não descritas na dourada ou em outras espécies de peixes. Também a integração dos resultados do proteoma (Capítulo 4) e do transcritoma (Capítulo 5) do embrião de dourada nos mesmos estágios de desenvolvimento, identificou um grupo de proteínas que se especula serem de origem materna. No último capítulo, sumarizou-se os resultados e são apresentadas perspetivas baseadas nos avanços e desafios atuais e propostas para o desenvolvimento de uma ferramenta integrada de monitorização da qualidade dos embriões e uma base biológica do desenvolvimento de ovos e embriões de peixes (Capítulo 6). Neste projeto foram: 1) identificadas duas importantes famílias de genes associadas à imunidade inata em peixes, a das lisozimas e a do C5. Caracterizou-se pela primeira vez a função das lisozimas através da sua expressão e atividade enzimática em embriões e em diferentes estágios larvares de uma diversidade de reprodutores de dourada. Estudou-se a função do C5 em peixes, através da construção de redes génicas, modelação por homologia e “docking” molecular entre o C5 e o seu receptor (C5R/CD88); 2) mapeou-se e caracterizou-se o proteoma do embrião de três espécies de peixes mediterrânicos (sargo, corvina e dourada) em duas fases do seu desenvolvimento (24h antes da eclosão e na eclosão) e identificou-se um grupo de proteínas potencialmente relacionadas com a imunidade e a qualidade dos embriões. Avaliou-se a função do gene para enzima “hatching enzyme”, com base na sua expressão em embriões de dourada e robalo nos estágios acima referidos; 3) mapeou-se e caracterizou-se o transcritoma de embriões de dourada com origem em diferentes “hatcheries” na região mediterrânica através de uma abordagem comparativa entre qualidade e estágios de desenvolvimento (qualidade- Boa vrs Má; estágios- Pré- eclosão vrs Eclosão) e identificou-se uma diversidade de transcritos, vias metabólicas e elementos do epitranscriptoma regulatório do RNA-não codificante. Foram identificados em comum 42 candidatos a marcadores de qualidade e enriquecidas duas vias metabólicas relacionadas com o sistema imunológico e associadas às “hatcheries”: a via de infeção por Salmonella (constituída por 7 genes relacionados com o sistema imune) e a via de sinalização MAPK (mitogen-activated protein kinase). Foram identificadas 543 proteínas que são expressas apenas no proteoma, sugerindo que podem ter origem materna e destas, 7 (diferencialmente expressas) estão potencialmente relacionadas com o sistema imune. Globalmente, os resultados forneceram um grupo de marcadores relacionados à imunidade e ao desenvolvimento (qualidade), com potencial de se traduzirem em critérios de qualidade de ovos e embriões para a indústria da aquicultura. Estes resultados, foram amplamente estudados para descrever a base molecular biológica entre os diferentes estágios de desenvolvimento de ovos e embriões de peixes e também, entre diferentes lotes de embriões de diferente qualidade. As ferramentas biológicas e critérios desenvolvidos neste trabalho, oferecem uma orientação para as “hatcheries” de peixes e a sua aplicação contribuirá para melhorar no futuro o sector da aquicultura.O apoio financeiro ao trabalho relatado na presente tese de doutoramento é reconhecido com gratidão, pois foi crucial para o progresso positivo e sucesso do trabalho científico. Uma diversidade de fontes de financiamento apoiou o trabalho desenvolvido nesta tese, quer diretamente através da compra de consumíveis/materiais (PerformFISH a European Union’s Horizon 2020 research and innovation grant, agreement Nº 727610), quer indiretamente através do financiamento do CCMAR de apoio a serviços e equipamentos utilizados durante a execução do trabalho (projetos da Fundação para a Ciência e Tecnologia (FCT) - UIDB/04326/2020, UIDP/04326/2020 e dos programas operacionais CRESC Algarve 2020 e COMPETE 2020 através do projeto EMBRC.PT ALG-01-0145-FEDER-022121)

Functional miR-142a-3p induces apoptosis and macrophage polarization by targeting tnfaip2 and glut3 in grass carp (Ctenopharyngodon idella)

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.31802285, CARROS-45-03info:eu-repo/semantics/publishedVersio

Hepatitis B virus inhibition in mice by lentiviral vector mediated short hairpin RNA

<p>Abstract</p> <p>Background</p> <p>Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication.</p> <p>Methods</p> <p>Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively.</p> <p>Results</p> <p>Co-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group.</p> <p>Conclusion</p> <p>Lentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.</p

The roles of inflammasomes in cancer

Inflammation is a key characteristic of all stages of tumor development, including tumor initiation, progression, malignant transformation, invasion, and metastasis. Inflammasomes are an important component of the inflammatory response and an indispensable part of the innate immune system. Inflammasomes regulate the nature of infiltrating immune cells by signaling the secretion of different cytokines and chemokines, thus regulating the anti-tumor immunity of the body. Inflammasome expression patterns vary across different tumor types and stages, playing different roles during tumor progression. The complex diversity of the inflammasomes is determined by both internal and external factors relating to tumor establishment and progression. Therefore, elucidating the specific effects of different inflammasomes in anti-tumor immunity is critical for promoting the discovery of inflammasome-targeting drugs. This review focuses on the structure, activation pathway, and identification methods of the NLRP3, NLRC4, NLRP1 and AIM2 inflammasomes. Herein, we also explore the role of inflammasomes in different cancers and their complex regulatory mechanisms, and discuss current and future directions for targeting inflammasomes in cancer therapy. A detailed knowledge of inflammasome function and regulation may lead to novel therapies that target the activation of inflammasomes as well as the discovery of new drug targets

Chromosome-level genome assembly of largemouth bass (Micropterus salmoides) using PacBio and Hi-C technologies

The largemouth bass (Micropterus salmoides) has become a cosmopolitan species due to its widespread introduction as game or domesticated fish. Here a high-quality chromosome-level reference genome of M. salmoides was produced by combining Illumina paired-end sequencing, PacBio single molecule sequencing technique (SMRT) and High-through chromosome conformation capture (Hi-C) technologies. Ultimately, the genome was assembled into 844.88 Mb with a contig N50 of 15.68 Mb and scaffold N50 length of 35.77 Mb. About 99.9% assembly genome sequences (844.00 Mb) could be anchored to 23 chromosomes, and 98.03% assembly genome sequences could be ordered and directed. The genome contained 38.19% repeat sequences and 2693 noncoding RNAs. A total of 26,370 protein-coding genes from 3415 gene families were predicted, of which 97.69% were functionally annotated. The high-quality genome assembly will be a fundamental resource to study and understand how M. salmoides adapt to novel and changing environments around the world, and also be expected to contribute to the genetic breeding and other research.info:eu-repo/semantics/publishedVersio

Genetic Polymorphisms in Enzymes Involved in One-Carbon Metabolism and Anti-epileptic Drug Monotherapy on Homocysteine Metabolism in Patients With Epilepsy

Aims: To investigate the effects of single nucleotide polymorphisms (SNPs) in genes of one-carbon metabolism (OCM) related enzymes and anti-epileptic drug (AED) monotherapy on homocysteine (Hcy) metabolism in patients with epilepsy, and to further explore specific SNPs that may increase patients' susceptibility to the effects of AEDs on the Hcy imbalance.Method: This case-control study analyzed 279 patients with epilepsy, including patients receiving monotherapy with valproate (VPA) (n = 53), oxcarbazepine (OXC) (n = 71), lamotrigine (LTG) (n = 55), or levetiracetam (LEV) (n = 35) and patients who had not taken any AEDs (controls, n = 65) for at least 6 months. Serum levels of vitamin B12 (vit B12), folate (FA) and Hcy were measured, and 23 SNPs in 13 genes of OCM-related enzymes were genotyped in all patients.Results: Methylenetetrahydrofolate reductase (MTHFR) rs1801133 was associated with elevated serum Hcy levels in patients with epilepsy (P &lt; 0.001), and patients presenting the TT genotype exhibited higher serum Hcy levels than patients with the CC (P &lt; 0.001) or CT (P &lt; 0.001) genotype. A subsequent multiple linear regression analysis showed that AED monotherapy with VPA (vs. control: P = 0.023) or OXC (vs. control: P = 0.041), and genotypes of MTHFR rs1801133 TT (vs. CC: P &lt; 0.001; vs. CT: P &lt; 0.001), transcobalamin 2 (TCN2) rs1801198 CC (vs. GC: P = 0.039) and folate receptor 1 (FOLR1) rs2071010 AA (vs. GA: P = 0.031) were independent risk factors for higher Hcy levels. In the subgroup analysis of patients taking OXC, we found that patients with genotypes of MTHFR rs1801133 TT (vs. CC: P = 0.001; vs. CT: P &lt; 0.001) and TCN2 rs1801198 CC (vs. GC: P = 0.021; vs. GG: P = 0.018) exhibited higher serum Hcy levels.Conclusions: VPA, OXC, and genotypes of MTHFR rs1801133 TT, TCN2 rs1801198 CC, and FOLR1 rs2071010 AA are all independent risk factors for elevated Hcy levels in patients with epilepsy. Moreover, genotypes of MTHFR rs1801133 TT and TCN2 rs1801198 CC may increase patients' susceptibility to the effect of OXC on disrupting Hcy homeostasis

Improved algorithm for time delay estimation in subway tunnel scenario

Based on the subway tunnel scenario,the time delay estimation problem generated by the multipath effect was transformed into the problem of solving the time difference among different paths in the three-dimensional space by the ray tracing method.An improved estimation method was proposed under the motion scenario considering the Doppler effect when the train running at high speed,which was different from the case of the relative static of the transmitter and receiver.The simulation results showed that the improved method was better matched with the actual scene than the method estimated in the relative static state of the receiver

Transcriptome Analysis Revealed GhWOX4 Intercedes Myriad Regulatory Pathways to Modulate Drought Tolerance and Vascular Growth in Cotton

Cotton is a paramount cash crop around the globe. Among all abiotic stresses, drought is a leading cause of cotton growth and yield loss. However, the molecular link between drought stress and vascular growth and development is relatively uncharted. Here, we validated a crucial role of GhWOX4, a transcription factor, modulating drought stress with that of vasculature growth in cotton. Knock-down of GhWOX4 decreased the stem width and severely compromised vascular growth and drought tolerance. Conversely, ectopic expression of GhWOX4 in Arabidopsis enhanced the tolerance to drought stress. Comparative RNAseq analysis revealed auxin responsive protein (AUX/IAA), abscisic acid (ABA), and ethylene were significantly induced. Additionally, MYC-bHLH, WRKY, MYB, homeodomain, and heat-shock transcription factors (HSF) were differentially expressed in control plants as compared to GhWOX4-silenced plants. The promotor zone of GhWOX4 was found congested with plant growth, light, and stress response related cis-elements. differentially expressed genes (DEGs) related to stress, water deprivation, and desiccation response were repressed in drought treated GhWOX4-virus-induced gene silencing (VIGS) plants as compared to control. Gene ontology (GO) functions related to cell proliferation, light response, fluid transport, and flavonoid biosynthesis were over-induced in TRV: 156-0 h/TRV: 156-1 h (control) in comparison to TRV: VIGS-0 h/TRV: VIGS-1 h (GhWOX4-silenced) plants. This study improves our context for elucidating the pivotal role of GhWOX4 transcription factors (TF), which mediates drought tolerance, plays a decisive role in plant growth and development, and is likely involved in different regulatory pathways in cotton