The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and ...Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.
Gene expression analysis is increasingly important in biological research, with reverse transcription-quantitative PCR (RT-qPCR) becoming the method of choice for high-throughput and accurate ...expression profiling of selected genes. Considering the increased sensitivity, reproducibility and large dynamic range of this method, the requirements for proper internal reference gene(s) for relative expression normalization have become much more stringent. Given the increasing interest in the functional genomics of Eucalyptus, we sought to identify and experimentally verify suitable reference genes for the normalization of gene expression associated with the flower, leaf and xylem of six species of the genus. We selected 50 genes that exhibited the least variation in microarrays of E. grandis leaves and xylem, and E. globulus xylem. We further performed the experimental analysis using RT-qPCR for six Eucalyptus species and three different organs/tissues. Employing algorithms geNorm and NormFinder, we assessed the gene expression stability of eight candidate new reference genes. Classic housekeeping genes were also included in the analysis. The stability profiles of candidate genes were in very good agreement. PCR results proved that the expression of novel Eucons04, Eucons08 and Eucons21 genes was the most stable in all Eucalyptus organs/tissues and species studied. We showed that the combination of these genes as references when measuring the expression of a test gene results in more reliable patterns of expression than traditional housekeeping genes. Hence, novel Eucons04, Eucons08 and Eucons21 genes are the best suitable references for the normalization of expression studies in the Eucalyptus genus.
Huanglongbing (HLB) is a destructive disease of citrus. The disease is caused by the phloem-limited fastidious proteobacterium
Candidatus
Liberibacter asiaticus, which is transmitted by the Asian ...citrus psyllid (
Diaphorina citri
). The symptoms of HLB have been related by callose accumulation in the phloem sieve plates. The key class of enzymes for callose synthesis is the Callose Synthases. The callose synthase genes (
calS
) expression is modulated for biotic and abiotic stresses. In this study, nine
C. sinensis calS
genes (
CscalS
) were identified and the expression patterns were analyzed in
Ca
Las inoculated and healthy plants. At 120 days after inoculation (dpi),
CscalS2
and
CscalS7
were significantly up-regulated in the HLB positive plants. At 360 dpi
CscalS7
and
CscalS12
were significantly up-regulated in the HLB positive plants. Our results confirmed that
Ca
Las infection is accompanied by the increased deposition of callose in the phloem sieve tubes and accumulation of starch in the leaves. It is suggested that the increased deposition of callose in the phloem sieve tubes is a hypersensitivity reaction, inhibiting phloem transport to consequently reducing the bacterial colonization via phloem, what possibly contribute to the starch accumulation in the leaves and the development of HLB symptoms. It is also proposed that
CscalS2
,
CscalS7
and
CscalS12
are involved in citrus defense against
Ca
Las, forming a complex in the phloem.
Citrus leprosis virus C (CiLV-C) causes a severe disease affecting citrus orchards in the Western hemisphere. This study reveals the molecular variability of the virus by analyzing four genomic ...regions (p29, p15, MP and RNA2-intergenic region) distributed over its two RNAs. Nucleotide diversity (π) values were relatively low but statistically different over the analyzed genes and subpopulations, indicating their distinct evolutionary history. Values of πp29 and πMP were higher than those of πp15 and πRNA2-IR, whereas πMP was increased due to novel discovered isolates phylogenetically clustered in a divergent clade that we called SJP. Isolate BR_SP_SJP_01 RNA1 and RNA2 sequences, clade SJP, showed an identity of 85.6% and 88.4%, respectively, with those corresponding to CiLV-C, the type member of the genus Cilevirus, and its RNA2 5'-proximal region was revealed as a minor donor in a putative inter-clade recombination event. In addition to citrus, BR_SP_SJP_01 naturally infects the weed Commelina benghalensis and is efficiently transmitted by Brevipalpus yothersi mites. Our data demonstrated that negative selection was the major force operating in the evaluated viral coding regions and defined amino acids putatively relevant for the biological function of cilevirus proteins. This work provides molecular tools and sets up a framework for further epidemiological studies.
Drug induced liver injury (DILI) is an adverse drug reaction that causes liver damage in a predictable (dose-dependent) or an unpredictable (idiosyncratic) fashion. We performed an assessment of DILI ...in Portugal, by analyzing the reports, sent to the Portuguese Pharmacovigilance System (PPS).
A search was performed on the PPS database, in a 10-year time frame, from 1 January 2010 to 31 December 2019.
There was not a prevalence of either sex in any age group. Most reports (
= 1120, 55.0%) belonged to patients in the age group 19-64 years old. Hepatitis (
= 626, 26.7%) was the most common adverse drug reaction in our study. Hepatotoxicity (
= 362, 15.5%) and hepatitis (
= 333, 14.2%) were more frequent in age group 19-64 years old. Cholestasis was more prevalent in adults independently of age. Hepatic fibrosis and encephalopathy were more common in the elderly. Most patients consumed between one and four suspected drugs (
= 1867, 92%). Most patients in our study evolved to "cure" (
= 796; 39%). Hepatotoxicity (
= 23; 13.8%) and hepatitis (
= 610; 25.9%) had a female predominancy while choluria (
= 8; 4.8%) and splenomegaly (
= 8; 4.8%) were of male predominance.
DILI is rare but can be fatal. As such, an active search of DILI is necessary.
•In the liver and porta hepatis of a subset of patients with BA, there is an activation of the HIF-1alpha pathway in cholangiocytes.•HIF-1alpha signals in cholangiocytes involve the progenitor cell ...niches and the endothelium of the PVP.•HIF-1alpha activation in biliary structures suggests a role for ischemic cholangiopathy.
Biliary atresia is a neonatal disease characterized by choledochal obstruction and progressive cholangiopathy requiring liver transplantation in most patients. Hypoxia-ischemia affecting the biliary epithelium may lead to biliary obstruction. We hypothesized that ischemic cholangiopathy involving disruption of the peribiliary vascular plexus could act as a triggering event in biliary atresia pathogenesis.
Liver and porta hepatis paraffin-embedded samples of patients with biliary atresia or intrahepatic neonatal cholestasis (controls) were immunohistochemically evaluated for HIF-1alpha-nuclear signals. Frozen histological samples were analyzed for gene expression in molecular profiles associated with hypoxia-ischemia. Prospective clinical-laboratory and histopathological data of biliary atresia patients and controls were reviewed.
Immunohistochemical HIF-1alpha signals localized to cholangiocytes were detected exclusively in liver specimens from biliary atresia patients. In 37.5% of liver specimens, HIF-1alpha signals were observed in biliary structures involving progenitor cell niches and peribiliary vascular plexus. HIF-1alpha signals were also detected in biliary remnants of 81.8% of porta hepatis specimens. Increased gene expression of molecules linked to REDOX status, biliary proliferation, and angiogenesis was identified in biliary atresia liver specimens. In addition, there was a trend towards decreased GSR expression levels in the HIF-1alpha-positive group compared to the HIF-1alpha-negative group.
Activation of the HIF-1alpha pathway may be associated with the pathogenesis of biliary atresia, and additional studies are necessary to confirm the significance of this finding. Ischemic cholangiopathy and REDOX status disturbance are putative explanations for HIF-1alpha activation. These findings may give rise to novel lines of clinical and therapeutic investigation in the BA field.
A indústria de base florestal é estratégica para o Brasil devido ao seu perfil fortemente exportador. O setor responde pela segunda posição na balança comercial do agronegócio brasileiro, ficando ...atrás somente da soja em grão. Atualmente, a área ocupada com florestas de eucaliptos no Brasil atinge 1,9 milhões de ha. Diante da importância sócio-econômica que a silvicultura desempenha no mercado brasileiro e do aumento progressivo das áreas plantadas com florestas de Eucalyptus, o grande desafio para o melhoramento do eucalipto está na integração da biotecnologia mais avançada ao seu cultivo, o que compreende a identificação de genes controladores das características de importância econômica e ambiental e a transferência destes genes entre árvores por meio de cruzamentos controlados ou modificação direcionada. Portanto, os objetivos deste estudo foram apresentados em 3 capítulos distintos: I - a identificação de genes expressos em flores de E. grandis em processo de antese; II - o estudo mais refinado de mineração e identificação de genes potencialmente codificadores de fatores de transcrição presentes no genoma de E. grandis; e III - seleção de 50 genes cujas expressões mostraram-se constitutivas entre folhas e xilemas de E. grandis e xilema de E. globulus, pela técnica de hibridização de microarranjos de DNA. No capítulo I é apresentada uma breve fundamentação teórica sobre as flores de E. grandis e a importância do estudo da expressão gênica e da identificação de genes envolvidos em determinados processos metabólicos e fisiológicos das plantas. Nos resultados, apresentados juntamente com a discussão, estão apresentados o conjunto de transcritos identificados e a anotação dos mesmos conforme as bibliotecas geradas. As sequências de genes expressos estão fundamentalmente envolvidas na manutenção do órgão, na senescência e em respostas a estímulos ambientais. Também são mostrados resultados obtidos por RT-qPCR para genes selecionados a partir das anotações cujo perfil de transcrição foi avaliado para as partes da flor, folha e xilema. Ao final do capítulo, é feita uma breve descrição da metodologia e das conclusões referente a este estudo. A partir dos dados anotados dos genes expressos nas bibliotecas de flores e botões florais descritos no primeiro capítulo, foram encontradas algumas famílias de fatores de transcrição, dentre elas, a família Dof, encontrada nas bibliotecas de carpelos/receptáculos florais. Assim, um segundo capítulo foi redigido na forma de manuscrito de artigo científico a ser submetido ao periódico BMC Plant Biology, em língua inglesa. Após a fundamentação teórica sobre os fatores Dof em plantas, foram apresentados os resultados obtidos de um estudo mais refinado de mineração e identificação de genes potencialmente codificadores destes fatores de transcrição presentes no genoma de E. grandis. Posteriormente, uma quantificação dos níveis de mRNA para alguns dos genes Dof de E. grandis a partir da técnica de RT-qPCR foi realizada. A análise foi realizada para órgãos diferentes da planta e em plântulas submetidas a estresses abióticos e sinalização por reguladores de crescimento vegetais. Os resultados e a discussão deste capítulo também são mostrados em uma única sessão, assim como a descrição da metodologia e as conclusões sobre esta etapa. O terceiro e último capítulo, também apresentado na forma de manuscrito de artigo científico a ser submetido ao periódico BMC Plant Biology, em língua inglesa, é composto de um estudo paralelo aos temas citados acima, realizado a partir da seleção de 50 genes cujas expressões mostraram-se constitutivas entre folhas e xilemas de E. grandis e xilema de E. globulus, pela técnica de hibridização de microarranjos de DNA. Dos 50 genes selecionados e anotados, oito foram selecionados para a validação por RT-qPCR em seis espécies de Eucalyptus e em três órgãos diferentes (flor, folha e xilema). As expressões destes genes candidatos foram comparadas àquelas de sete genes normalizadores tradicionais, usados frequentemente em estudos de avaliação da expressão gênica em plantas. Como nos demais capítulos, as conclusões referentes a este estudo, bem como a metodologia empregada, são apresentadas ao final do capítulo. Na parte final desta tese, constam as conclusões gerais sobre a totalidade do trabalho conduzido.
The forest industry is strategic to Brazil due to its strong export profile. The sector accounts for the second position in the trade brazilian agribusiness balance. Currently, the area planted with Eucalyptus forests in Brazil reached 1.9 million/ha. Given the socioeconomic importance that forestry plays in the brazilian market and the gradual increase in areas planted with Eucalyptus forest, the great challenge for the Eucalyptus breeding is the integration of advanced biotechnology to cultivation, which includes the identification of economic importance genes controlling environmental effects and transfer of genes between trees through intersections controlled or directed modification. The objectives of this study were presented in three distinct chapters: I - the identification of expressed genes in E. grandis flowers in the anthesis process; II – more refined study, mining and identification of genes potentially encoding transcription factors present in the E. grandis genome and III - selection of 50 genes whose expressions were shown to be constitutive in E. grandis xylem and leaves and E. globulus xylem, by DNA microarray hybridization tecnique. In Chapter I, we present a brief theoretical background on the E. grandis flowers and the importance of studying gene expression and identifying genes involved in metabolic and physiological processes of plants. The results, presented transcripts sets identified and annotation the libraries generated. The sequences of expressed genes are mainly involved in organ maintaining, senescence and in responses to environmental stimuli. Also shown are results obtained by RT-qPCR for selected genes from the notes whose transcription profile was assessed for part of the flower, leaf and xylem. At the end of the chapter is a brief description of the methodology and conclusions about this study. From the data recorded in the libraries of genes expressed in flowers and flower buds described in the first chapter, we found some families of transcription factors, among them, the Dof family, found in the libraries of carpel/floral receptacles. Thus, a second chapter was drafted as a manuscript of a scientific paper to be submitted to the journal BMC Plant Biology. After the theoretical DOF on the factors in plants, we presented the results of a more refined mining and identification of genes potentially encoding these transcription factors present in the E. grandis genome. Subsequently, a quantification of mRNA levels for some of E. grandis Dof genes from the RT-qPCR was performed. The analysis was performed for different plant organs and in seedlings subjected to abiotic stress signaling and plant growth regulators. The results and discussion of this chapter are also shown in a single session, as well as a description of the methodology and conclusions about this step. The third and final chapter, also presented as a manuscript of a scientific paper to be submitted to the journal BMC Plant Biology, is composed of a parallel study to the subjects mentioned above, performed by the selection of 50 genes whose expression showed is constitutive of E. grandis leaves and xylem and E.globulus xylem, by the DNA microarray hybridization tecnique. Of the 50 selected genes and annoted, eight were selected for validation by RT-qPCR in six Eucalyptus species and three different organs (flower, leaf and xylem). The expression of these candidate genes were compared to those of traditional standard-seven genes, often used in evaluation studies of gene expression in plants. As in other chapters, the findings of this study and the methodology employed, are included at the end of the chapter. In the final part of this thesis, contains the general conclusions about the totality of the work conducted.
La co-présence massive du texte et de l’image dans les arts et les lettres du Moyen Âge n’est plus à démontrer. Des manuscrits aux vitraux, des édifices aux objets du quotidien, des reliquaires aux ...documents administratifs et juridiques, les interactions et la complémentarité de l’iconographie et du texte ont fait l’objet, au xxe siècle, d’une interrogation constante, d’abord hésitante puis de plus en plus systématisée, en même temps que des champs épistémologiques neufs se sont affirmés. Ces trente dernières années, le devant de la scène a été largement occupé en France par les études des historiens, dans une orientation résolument anthropologique. Analyse sérielle, prise en compte des contextes textuels, spatiaux, sociaux, politiques, théologiques ont renouvelé l’approche de la dynamique texte-image, la sortant du pré carré de l’histoire de l’art traditionnelle dont elle n’est plus aujourd’hui la prérogative. Pour autant, cette approche renouvelée épuise-t-elle et circonscrit-elle tous les discours possibles sur les relations entre le texte et l’image ? Les autres champs disciplinaires actuels – comme la littérature, la linguistique, la philosophie, l’histoire de l’art, la musicologie, mais aussi la psychanalyse, la sociologie ou encore le droit – ont sans doute leur mot à dire sur le sujet, mais lequel ? Par ailleurs, l’approche contextualisée qui a renouvelé les discours critiques n’a-t-elle pas minoré l’interprétation du texte ou celle de l’image au profit de leur interaction ? Le contexte pris en compte est-il aussi aisé à définir qu’il n’y paraît ? Les outils numériques et la mise en série des images ne donnent-ils par l’illusion fallacieuse d’un accès illimité aux sources au détriment de la compréhension de leur contexte large de création et d’usage ? L’extrême rareté de manuels universitaires sur ce sujet révèle la difficulté toujours réelle qu’il y a à définir des théories et des méthodes d’analyse qui permettent la formation d’un consensus épistémologique et pédagogique dans un domaine qui ne possède encore pas d’autonomie académique et dont le caractère trans-disciplinaire rend délicate l’émergence. La diversité du corpus à envisager n’étant pas le moindre des obstacles. Ce nouveau numéro de Perspectives médiévales a ouvert ses pages à des auteurs en leur proposant de mettre en perspective les pratiques, les nouvelles réflexions théoriques, les approches actuelles des relations texte-image dans les horizons critiques et les champs disciplinaires contemporains. Sébastien Douchet & Maud Pérez-Simon
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