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The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium-mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.
Assuntos
Lactuca/genética , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/imunologia , Administração Oral , Animais , Feminino , Células HEK293 , Humanos , Imunidade Humoral , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas , Engenharia de Proteínas/métodos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/genéticaRESUMO
Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.
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Antígenos Virais/biossíntese , Cloroplastos/genética , Vírus da Dengue/imunologia , Técnicas Genéticas , Nicotiana/genética , Sequência de Aminoácidos , Antígenos Virais/química , Antígenos Virais/metabolismo , Etanol/farmacologia , Vetores Genéticos/metabolismo , Plantas Geneticamente Modificadas , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Regeneração , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
This year is a special year for plant biotechnology. It was 30 years ago, on January 18 1983, one of the most important dates in the history of plant biotechnology, that three independent groups described Agrobacterium tumefaciens-mediated genetic transformation at the Miami Winter Symposium, leading to the production of normal, fertile transgenic plants (Bevan et al. in Nature 304:184-187, 1983; Fraley et al. in Proc Natl Acad Sci USA 80:4803-4807, 1983; Herrera-Estrella et al. in EMBO J 2:987-995, 1983; Vasil in Plant Cell Rep 27:1432-1440, 2008). Since then, plant biotechnology has rapidly advanced into a useful and valuable tool and has made a significant impact on crop production, development of a biotech industry and the bio-based economy worldwide.
Assuntos
Biotecnologia/métodos , Abastecimento de Alimentos/legislação & jurisprudência , Alimentos Geneticamente Modificados/economia , Plantas Geneticamente Modificadas/fisiologia , Produtos Agrícolas/economia , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Produtos Agrícolas/fisiologia , Países em Desenvolvimento , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Aquaculture, the fastest growing food-producing sector, now accounts for nearly 50 % of the world's food fish (FAO in The state of world fisheries and aquaculture. FAO, Rome, 2010). The global aquaculture production of food fish reached 62.7 million tonnes in 2011 and is continuously increasing with an estimated production of food fish of 66.5 million tonnes in 2012 (a 9.4 % increase in 1 year, FAO, www.fao.org/fishery/topic/16140 ). Aquaculture is not only important for sustainable protein-based food fish production but also for the aquaculture industry and economy worldwide. Disease prevention is the key issue to maintain a sustainable development of aquaculture. Widespread use of antibiotics in aquaculture has led to the development of antibiotic-resistant bacteria and the accumulation of antibiotics in the environment, resulting in water and soil pollution. Thus, vaccination is the most effective and environmentally-friendly approach to combat diseases in aquaculture to manage fish health. Furthermore, when compared to >760 vaccines against human diseases, there are only about 30 fish vaccines commercially available, suggesting the urgent need for development and cost-effective production of fish vaccines for managing fish health, especially in the fast growing fish farming in Asia where profit is minimal and therefore given high priority. Plant genetic engineering has made significant contributions to production of biotech crops for food, feed, valuable recombinant proteins etc. in the past three decades. The use of plants for vaccine production offers several advantages such as low cost, safety and easy scaling up. To date a large number of plant-derived vaccines, antibodies and therapeutic proteins have been produced for human health, of which a few have been made commercially available. However, the development of animal vaccines in plants, especially fish vaccines by genetic engineering, has not yet been addressed. Therefore, there is a need to exploit plant biotechnology for cost effective fish vaccine development in plants, in particular, edible crops for oral fish vaccines. This review provides insight into (1) the current status of fish vaccine and vaccination in aquaculture, (2) plant biotechnology and edible crops for fish vaccines for oral administration, (3) regulatory constraints and (4) conclusions and future perspectives.
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Aquicultura/métodos , Doenças dos Peixes/prevenção & controle , Vacinas/economia , Administração Oral , Animais , Aquicultura/economia , Conservação dos Recursos Naturais/economia , Conservação dos Recursos Naturais/legislação & jurisprudência , Peixes/imunologia , Abastecimento de Alimentos/legislação & jurisprudência , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/imunologia , Transgenes , Vacinação , Vacinas/administração & dosagemRESUMO
Plants provide not only food and feed, but also herbal medicines and various raw materials for industry. Moreover, plants can be green factories producing high value bioproducts such as biopharmaceuticals and vaccines. Advantages of plant-based production platforms include easy scale-up, cost effectiveness, and high safety as plants are not hosts for human and animal pathogens. Plant cells perform many post-translational modifications that are present in humans and animals and can be essential for biological activity of produced recombinant proteins. Stimulated by progress in plant transformation technologies, substantial efforts have been made in both the public and the private sectors to develop plant-based vaccine production platforms. Recent promising examples include plant-made vaccines against COVID-19 and Ebola. The COVIFENZ® COVID-19 vaccine produced in Nicotiana benthamiana has been approved in Canada, and several plant-made influenza vaccines have undergone clinical trials. In this review, we discuss the status of vaccine production in plants and the state of the art in downstream processing according to good manufacturing practice (GMP). We discuss different production approaches, including stable transgenic plants and transient expression technologies, and review selected applications in the area of human and veterinary vaccines. We also highlight specific challenges associated with viral vaccine production for different target organisms, including lower vertebrates (e.g., farmed fish), and discuss future perspectives for the field.
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Through the last decades, environmentally and health-friendly production methods and conscientious use of resources have become crucial for reaching the goal of a more sustainable plant production. Protection of the environment requires careful consumption of limited resources and reduction of chemicals applied during production of ornamental plants. Numerous chemicals used in modern plant production have negative impacts on human health and are hazardous to the environment. In Europe, several compounds have lost their approval and further legal restrictions can be expected. This review presents the more recent progress of genetic engineering in ornamental breeding, delivers an overview of the biological background of the used technologies and critically evaluates the usefulness of the strategies to obtain improved ornamental plants. First, genetic engineering is addressed as alternative to growth retardants, comprising recombinant DNA approaches targeting relevant hormone pathways, e.g. the gibberellic acid (GA) pathway. A reduced content of active GAs causes compact growth and can be facilitated by either decreased anabolism, increased catabolism or altered perception. Moreover, compactness can be accomplished by using a natural transformation approach without recombinant DNA technology. Secondly, metabolic engineering approaches targeting elements of the ethylene signal transduction pathway are summarized as a possible alternative to avoid the use of chemical ethylene inhibitors. In conclusion, molecular breeding approaches are dealt with in a way allowing a critical biological assessment and enabling the scientific community and public to put genetic engineering of ornamental plants into a perspective regarding their usefulness in plant breeding.
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Cruzamento , Engenharia Genética/tendências , Plantas Geneticamente Modificadas/genética , DNA Recombinante , Etilenos/metabolismo , Engenharia Genética/métodos , Giberelinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais , Transformação GenéticaRESUMO
Despite the availability of improved antiviral therapies, infection with Hepatitis B virus (HBV) remains a3 significant health issue, as a curable treatment is yet to be discovered. Current HBV vaccines relaying on the efficient expression of the small (S) envelope protein in yeast and the implementation of mass vaccination programs have clearly contributed to containment of the disease. However, the lack of an efficient immune response in up to 10% of vaccinated adults, the controversies regarding the seroprotection persistence in vaccine responders and the emergence of vaccine escape virus mutations urge for the development of better HBV immunogens. Due to the critical role played by the preS1 domain of the large (L) envelope protein in HBV infection and its ability to trigger virus neutralizing antibodies, including this protein in novel vaccine formulations has been considered a promising strategy to overcome the limitations of S only-based vaccines. In this work we aimed to combine relevant L and S epitopes in chimeric antigens, by inserting preS1 sequences within the external antigenic loop of S, followed by production in mammalian cells and detailed analysis of their antigenic and immunogenic properties. Of the newly designed antigens, the S/preS116-42 protein assembled in subviral particles (SVP) showed the highest expression and secretion levels, therefore, it was selected for further studies in vivo. Analysis of the immune response induced in mice vaccinated with S/preS116-42- and S-SVPs, respectively, demonstrated enhanced immunogenicity of the former and its ability to activate both humoral and cellular immune responses. This combined activation resulted in production of neutralizing antibodies against both wild-type and vaccine-escape HBV variants. Our results validate the design of chimeric HBV antigens and promote the novel S/preS1 protein as a potential vaccine candidate for administration in poor-responders to current HBV vaccines.
Assuntos
Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Animais , Anticorpos Bloqueadores , Anticorpos Neutralizantes , Vacinas contra Hepatite B , Imunidade Humoral , Mamíferos , Camundongos , Camundongos Endogâmicos BALB C , Vacinas SintéticasRESUMO
Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a "green enzyme," laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda's gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7-8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants.
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The world population is expected to reach an estimated 9.2 billion by 2050. Therefore, food production globally has to increase by 70% in order to feed the world, while total arable land, which has reached its maximal utilization, may even decrease. Moreover, climate change adds yet another challenge to global food security. In order to feed the world in 2050, biotechnological advances in modern agriculture are essential. Plant genetic engineering, which has created a new wave of global crop production after the first green revolution, will continue to play an important role in modern agriculture to meet these challenges. Plastid genetic engineering, with several unique advantages including transgene containment, has made significant progress in the last two decades in various biotechnology applications including development of crops with high levels of resistance to insects, bacterial, fungal and viral diseases, different types of herbicides, drought, salt and cold tolerance, cytoplasmic male sterility, metabolic engineering, phytoremediation of toxic metals and production of many vaccine antigens, biopharmaceuticals and biofuels. However, useful traits should be engineered via chloroplast genomes of several major crops. This review provides insight into the current state of the art of plastid engineering in relation to agricultural production, especially for engineering agronomic traits. Understanding the bottleneck of this technology and challenges for improvement of major crops in a changing climate are discussed.
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Biotecnologia , Produtos Agrícolas , PlastídeosRESUMO
Aquaculture has undergone rapid development in the past decades. It provides a large part of high-quality protein food for humans, and thus, a sustainable aquaculture industry is of great importance for the worldwide food supply and economy. Along with the quick expansion of aquaculture, the high fish densities employed in fish farming increase the risks of outbreaks of a variety of aquatic diseases. Such diseases not only cause huge economic losses, but also lead to ecological hazards in terms of pathogen spread to marine ecosystems causing infection of wild fish and polluting the environment. Thus, fish health is essential for the aquaculture industry to be environmentally sustainable and a prerequisite for intensive aquaculture production globally. The wide use of antibiotics and drug residues has caused intensive pollution along with risks for food safety and increasing antimicrobial resistance. Vaccination is the most effective and environmentally friendly approach to battle infectious diseases in aquaculture with minimal ecological impact and is applicable to most species of farmed fish. However, there are only 34 fish vaccines commercially available globally to date, showing the urgent need for further development of fish vaccines to manage fish health and ensure food safety. Plant genetic engineering has been utilized to produce genetically modified crops with desirable characteristics and has also been used for vaccine production, with several advantages including cost-effectiveness, safety when compared with live virus vaccines, and plants being capable of carrying out posttranslational modifications that are similar to naturally occurring systems. So far, plant-derived vaccines, antibodies, and therapeutic proteins have been produced for human and animal health. However, the development of plant-made vaccines for animals, especially fish, is still lagging behind the development of human vaccines. The present review summarizes the development of fish vaccines currently utilized and the suitability of the plant-production platform for fish vaccine and then addresses considerations regarding fish vaccine production in plants. Developing fish vaccines by way of plant biotechnology are significant for the aquaculture industry, fish health management, food safety, and human health.
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Hepatitis B and C viruses chronically affect approximately 3.5% of the global population, causing more than 800,000 deaths yearly due to severe liver pathogenesis. Current HBV vaccines have significantly contributed to the reduction of chronic HBV infections, supporting the notion that virus eradication is a feasible public health objective in the near future. In contrast to HBV, a prophylactic vaccine against HCV infection is not available yet; however, intense research efforts within the last decade have significantly advanced the field and several vaccine candidates are shortlisted for clinical trials. A successful vaccine against an infectious disease of global importance must not only be efficient and safe, but also easy to produce, distribute, administer, and economically affordable to ensure appropriate coverage. Some of these requirements could be fulfilled by oral vaccines that could complement traditional immunization strategies. In this review, we discuss the potential of edible plant-based oral vaccines in assisting the worldwide fight against hepatitis B and C infections. We highlight the latest research efforts to reveal the potential of oral vaccines, discuss novel antigen designs and delivery strategies, as well as the limitations and controversies of oral administration that remain to be addressed to make this approach successful.
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The demand for animal protein has increased considerably worldwide, especially in China, where large numbers of livestock and poultry are produced. Antibiotics have been widely applied to promote growth and prevent diseases. However, the overuse of antibiotics in animal feed has caused serious environmental and health risks, especially the wide spread of antimicrobial resistance (AMR), which seriously affects animal and human health, food safety, ecosystems, and the sustainable future development of animal protein production. Unfortunately, AMR has already become a worldwide challenge, so international cooperation is becoming more important for combatting it. China's efforts and determination to restrict antibiotic usage through law enforcement and effective management are of significance. In this review, we address the pollution problems of antibiotics; in particular, the AMR in water, soil, and plants caused by livestock and poultry manure in China. The negative impact of widespread and intensive use of antibiotics in livestock production is discussed. To reduce and mitigate AMR problems, we emphasize in this review the development of antibiotic substitutes for the era of antibiotic prohibition.
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Propionate and propionyl-CoA accumulation have been associated with the development of mitochondrial dysfunction. In this study, we show that propionate induces intestinal damage in zebrafish when fed a high-fat diet (HFD). The intestinal damage was associated with oxidative stress owing to compromised superoxide dismutase 2 (Sod2) activity. Global lysine propionylation analysis of the intestinal samples showed that Sod2 was propionylated at lysine 132 (K132), and further biochemical assays demonstrated that K132 propionylation suppressed Sod2 activity. In addition, sirtuin 3 (Sirt3) played an important role in regulating Sod2 activity via modulating de-propionylation. Finally, we revealed that intestinal oxidative stress resulting from Sod2 propionylation contributed to compositional change of gut microbiota. Collectively, our results in this study show that there is a link between Sod2 propionylation and oxidative stress in zebrafish intestines and highlight the potential mechanism of intestinal problems associated with high propionate levels.
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Difenoconazole is a widely used triazole fungicide that has been frequently detected in the environment, but comprehensive study about its environmental fate and toxicity of potential transformation products (TPs) is still lacking. Here, laboratory experiments were conducted to investigate the degradation kinetics, pathways, and toxicity of transformation products of difenoconazole. 12, 4 and 4 TPs generated by photolysis, hydrolysis and soil degradation were identified via UHPLC-QTOF/MS and the UNIFI software. Four intermediates TP295, TP295A, TP354A and TP387A reported for the first time were confirmed by purchase or synthesis of their standards, and they were further quantified using UHPLC-MS/MS in all tested samples. The main transformation reactions observed for difenoconazole were oxidation, dechlorination and hydroxylation in the environment. ECOSAR prediction and laboratory tests showed that the acute toxicities of four novel TPs on Brachydanio rerio, Daphnia magna and Selenastrum capricornutum are substantially lower than that of difenoconazole, while all the TPs except for TP277C were predicted chronically very toxic to fish, which may pose a potential threat to aquatic ecosystems. The results are important for elucidating the environmental fate of difenoconazole and assessing the environmental risks, and further provide guidance for scientific and reasonable use.
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Solo , Poluentes Químicos da Água , Animais , Dioxolanos , Ecossistema , Cinética , Fotólise , Espectrometria de Massas em Tandem , Triazóis/toxicidade , Água , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
Diseases caused by viruses threaten the production industry and food safety of aquaculture which is a great animal protein source. Grass carp reovirus (GCRV) has caused tremendous loss, and the molecular function of viral proteins during infection needs further research, as for most aquatic viruses. In this study, interaction between GCRV major outer capsid protein VP4 and RIG-I, a critical viral RNA sensor, was screened out by GST pull-down, endogenous immunoprecipitation and subsequent LC-MS/MS, and then verified by co-IP and an advanced far-red fluorescence complementation system. VP4 was proved to bind to the CARD and RD domains of RIG-I and promoted K48-linked ubiquitination of RIG-I to degrade RIG-I. VP4 reduced mRNA and promoter activities of key genes of RLR pathway and sequential IFN production. As a consequence, antiviral effectors were suppressed and GCRV replication increased, resulting in intensified cytopathic effect. Furthermore, results of transcriptome sequencing of VP4 stably expressed CIK (C. idella kidney) cells indicated that VP4 activated the MyD88-dependent TLR pathway. Knockdown of VP4 obtained opposite effects. These results collectively revealed that VP4 interacts with RIG-I to restrain interferon response and assist GCRV invasion. This study lays the foundation for anti-dsRNA virus molecular function research in teleost and provides a novel insight into the strategy of immune evasion for aquatic virus.
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Proteínas do Capsídeo/metabolismo , Carpas/metabolismo , Carpas/virologia , Interferons/metabolismo , RNA Viral/metabolismo , Reoviridae/fisiologia , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Transporte Proteico , Reoviridae/genéticaRESUMO
Endogenous antimicrobial peptides (AMPs) are evolutionarily ancient factors of innate immunity, which are produced by all multicellular organisms and play a key role in their protection against infection. Red king crab (Paralithodes camtschaticus), also called Kamchatka crab, is widely distributed and the best known species of all king crabs belonging to the family Lithodidae. Despite their economic importance, the genetic resources of king crabs are scarcely known and no full-genome sequences are available to date. Therefore, analysis of the red king crab transcriptome and identification and characterization of its AMPs could potentially contribute to the development of novel antimicrobial drug candidates when antibiotic resistance has become a global health threat. In this study, we sequenced the P. camtschaticus transcriptomes from carapace, tail flap and leg tissues using an Illumina NGS platform. Libraries were systematically analyzed for gene expression profiles along with AMP prediction. By an in silico approach using public databases we defined 49 cDNAs encoding for AMP candidates belonging to diverse families and functional classes, including buforins, crustins, paralithocins, and ALFs (anti-lipopolysaccharide factors). We analyzed expression patterns of 27 AMP genes. The highest expression was found for Paralithocin 1 and Crustin 3, with more than 8,000 reads. Other paralithocins, ALFs, crustins and ubiquicidins were among medium expressed genes. This transcriptome data set and AMPs provide a solid baseline for further functional analysis in P. camtschaticus. Results from the current study contribute also to the future application of red king crab as a bio-resource in addition to its being a known seafood delicacy.
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Anomuros/genética , Perfilação da Expressão Gênica/veterinária , Proteínas Citotóxicas Formadoras de Poros/genética , Animais , Proteínas de Artrópodes/genética , Simulação por Computador , Bases de Dados Genéticas , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imunidade Inata , Análise de Sequência de RNA , Cauda/químicaRESUMO
Genetic instability could be provoked as an unintended consequence of genetic engineering in plants. Here, we report that the rice endogenous long terminal repeat (LTR) retrotransposon Tos17 was transpositionally activated only in transgenic calli and their regenerated plants produced by biolistic transformation in rice (Oryza sativa L.) ssp. japonica cv. Matsumae. Moreover, the transpositional activity of Tos17 was sustained after plant regeneration in the T0 generation, and produced new germinal insertions. In contrast, the element remained totally quiescent in calli and regenerated plants from tissue culture of this genotype. Nonetheless, transcriptional induction and cytosine demethylation of Tos17 were found to have occurred with no significant difference in both kinds of calli, tissue culture alone and transgenic. This suggests that callus culture is likely to have played an important role in destabilizing Tos17 in the direction towards transpositional activation, but that biolistic transformation is the direct causal factor.
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Biolística , Metilação de DNA , Oryza/genética , Retroelementos , DNA de Plantas/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Instabilidade Genômica , Plantas Geneticamente Modificadas/genética , Transformação GenéticaRESUMO
Cereal cyst nematode (CCN, Heterodera avenae) presents severe challenges to wheat (Triticum aestivum L.) production worldwide. An investigation of the interaction between wheat and CCN can greatly improve our understanding of how nematodes alter wheat root metabolic pathways for their development and could contribute to new control strategies against CCN. In this study, we conducted transcriptome analyses of wheat cv. Wen 19 (Wen19) by using RNA-Seq during the compatible interaction with CCN at 1, 3 and 8 days past inoculation (dpi). In total, 71,569 transcripts were identified, and 10,929 of them were examined as differentially expressed genes (DEGs) in response to CCN infection. Based on the functional annotation and orthologous findings, the protein phosphorylation, oxidation-reduction process, regulation of transcription, metabolic process, transport, and response process as well as many other pathways previously reported were enriched at the transcriptional level. Plant cell wall hydrolysis and modifying proteins, auxin biosynthesis, signalling and transporter genes were up-regulated by CCN infection to facilitate penetration, migration and syncytium establishment. Genes responding to wounding and jasmonic acid stimuli were enriched at 1 dpi. We found 16 NBS-LRR genes, 12 of which were down-regulated, indicating the repression of resistance. The expression of genes encoding antioxidant enzymes, glutathione S-transferases and UDP-glucosyltransferase was significantly up-regulated during CCN infection, indicating that they may play key roles in the compatible interaction of wheat with CCN. Taken together, the results obtained from the transcriptome analyses indicate that the genes involved in oxidation-reduction processes, induction and suppression of resistance, metabolism, transport and syncytium establishment may be involved in the compatible interaction of Wen 19 with CCN. This study provides new insights into the responses of wheat to CCN infection. These insights could facilitate the elucidation of the potential mechanisms of wheat responses to CCN.
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Grão Comestível/genética , Grão Comestível/parasitologia , Triticum/genética , Triticum/parasitologia , Tylenchoidea/patogenicidade , Animais , Grão Comestível/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Redes e Vias Metabólicas/genética , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Reguladores de Crescimento de Plantas , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/parasitologia , RNA-Seq , Triticum/metabolismoRESUMO
Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S) envelope protein are currently used in universal vaccination programs and achieve protective immune response in more than 90% of recipients. However, new vaccination strategies are necessary for successful immunization of the remaining non- or low-responders. We have previously characterized a novel HBV chimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L) envelope protein (genotype D). The S/preS121-47 chimera produced in mammalian cells and Nicotiana benthamiana plants, induced a significantly stronger immune response in parenterally vaccinated mice than the S protein. Here we describe the transient expression of the S/preS121-47 antigen in an edible plant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral administration of adjuvant-free Lactuca sativa expressing the S/preS121-47 antigen, three times, at 1⯵g/dose, was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies were able to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) more efficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results support the S/preS121-47 antigen as a promising candidate for future development as an edible HBV vaccine.
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Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/prevenção & controle , Precursores de Proteínas/imunologia , Administração Oral , Animais , Linhagem Celular Tumoral , Feminino , Células Hep G2 , Vacinas contra Hepatite B/administração & dosagem , Humanos , Lactuca/genética , Lactuca/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Proteínas do Envelope Viral/imunologiaRESUMO
Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most cost-effective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.