RESUMO
The emergence and spread of disease-causing viruses in shrimp aquaculture is not uncommon. Since 2016, unusual mortalities have been affecting the Brazilian shrimp industry and we have associated these unusual mortalities with a novel variant of infectious myonecrosis virus (IMNV). The transcriptome analysis of these diseased shrimp showed an additional divergent viral sequence that we have assigned to the family Solinviviridae. The novel virus has been tentatively termed Penaeus vannamei solinvivirus (PvSV) (GenBank accession: OP265432). The full-length genome of the PvSV is 10.44 kb (excluding the poly A tail) and codes for a polyprotein of 3326 aa. Five conserved domains coding for a helicase, RdRp, calicivirus coat protein, G-patch and tegument protein were identified. The genome organization of the PvSV is similar to other (Nylan deria fulva virus 1) solinvivirus. A unique feature of this virus that differs from other members of the Solinviviridae is the presence of putative nuclear localization signals. The tissue tropism of this virus is wide, infecting cells of the hepatopancreas, gastrointestinal tract, lymphoid organ and muscle tissue. Another unique feature is that it is the only RNA virus of penaeid shrimp that shows a nuclear localization by in situ hybridization. The PvSV has a wide distribution in Brazil and has been found in the states of Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State where it has been detected in coinfections with IMNV. The diagnostic methods developed here (real-time RT-PCR and in situ hybridization) are effective for the detection of the pathogen and should be employed to limit its spread. Furthermore, the identification of the PvSV shows the increasing host range of the relatively new family Solinviviridae.
Assuntos
Penaeidae , Vírus de RNA , Animais , Sinais de Localização Nuclear , Vírus de RNA/genética , RNA Polimerase Dependente de RNA , Poliproteínas , Poli ARESUMO
Poly(o-methoxyaniline) emeraldine-salt form (ES-POMA) was chemically synthesized using hydrochloric acid and subjected to a heat treatment (HT) process for 1 h at 100 °C (TT100) and 200 °C (TT200). The HT process promoted a progressive decrease in crystallinity. The Le Bail method revealed a decomposition from tetrameric to trimeric-folded chains after the HT process. The unheated POMA-ES presented a globular vesicular morphology with varied micrometric sizes. The heat treatment promoted a reduction in these globular structures, increasing the non-crystalline phase. The boundary length (S) and connectivity/Euler feature (χ) parameters were calculated from the SEM images, revealing that ES-POMA presented a wide distribution of heights. The TT100 and TT200 presented a narrow boundary distribution, suggesting smoother surfaces with smaller height variations. The UV-VIS analysis revealed that the transition at 343 nm (nonlocal π â π*) was more intense in the TT200 due to the electronic delocalization, which resulted from the reduced polymer chain caused by the HT process. In addition to the loss of conjugation, counter ion withdrawal reduced the ion-chain interaction, decreasing the local electron density. This result shows the influence of the chlorine counter ions on the peaks position related to the HOMO â LUMO transition, since the π â polaron transition occurs due to the creation of the energy states due to the presence of counter ions. Finally, the electrical conductivity decreased after the HT process from 1.4 × 10-4 S.cm-1 to 2.4 × 10-6 S.cm-1 as result of the polymer deprotonation/degradation. Thus, this paper proposed a systematic evaluation of the POMA molecular structure and crystallite size and shape after heat treatment.
Assuntos
Temperatura Alta , Poliaminas , Compostos de Anilina , Condutividade Elétrica , Poli A , Poliaminas/química , Polímeros/químicaRESUMO
Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.
Assuntos
Biossíntese de Proteínas/fisiologia , Sequência de Bases , Códon , Humanos , Poli A/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/genética , Cryptosporidium parvum/genética , Entamoeba histolytica/genética , Giardia lamblia/genética , Intestinos/parasitologia , RNA Mensageiro/genética , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Cryptosporidium parvum/metabolismo , Bases de Dados Genéticas , Entamoeba histolytica/metabolismo , Giardia lamblia/metabolismo , Humanos , Modelos Moleculares , Fases de Leitura Aberta , Poli A/química , Domínios Proteicos , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/química , RNA de Protozoário/química , RNA de Protozoário/genética , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Poly(A) tail shortening is a critical step in messenger RNA (mRNA) decay and control of gene expression. The carbon catabolite repressor 4 (CCR4)-associated factor 1 (CAF1) component of the CCR4-NOT deadenylase complex plays an essential role in mRNA deadenylation in most eukaryotes. However, while CAF1 has been extensively investigated in yeast and animals, its role in plants remains largely unknown. Here, we show that the Citrus sinensis CAF1 (CsCAF1) is a magnesium-dependent deadenylase implicated in resistance against the citrus canker bacteria Xanthomonas citri. CsCAF1 interacted with proteins of the CCR4-NOT complex, including CsVIP2, a NOT2 homologue, translin-associated factor X (CsTRAX) and the poly(A)-binding proteins CsPABPN and CsPABPC. CsCAF1 also interacted with PthA4, the main X. citri effector required for citrus canker elicitation. We also present evidence suggesting that PthA4 inhibits CsCAF1 deadenylase activity in vitro and stabilizes the mRNA encoded by the citrus canker susceptibility gene CsLOB1, which is transcriptionally activated by PthA4 during canker formation. Moreover, we show that an inhibitor of CsCAF1 deadenylase activity significantly enhanced canker development, despite causing a reduction in PthA4-dependent CsLOB1 transcription. These results thus link CsCAF1 with canker development and PthA4-dependent transcription in citrus plants.
Assuntos
Citrus sinensis/enzimologia , Citrus sinensis/microbiologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Ribonucleases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Citrus sinensis/genética , Citrus sinensis/imunologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Magnésio/farmacologia , Doenças das Plantas/genética , Proteínas de Plantas/genética , Poli A/metabolismo , Ligação Proteica/efeitos dos fármacos , Pirazóis/química , Pirazóis/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Xanthomonas/efeitos dos fármacos , Xanthomonas/fisiologiaRESUMO
Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.
Assuntos
Coffea/genética , MicroRNAs/genética , Nitrogênio/deficiência , RNA Mensageiro/genética , RNA de Plantas/genética , Pequeno RNA não Traduzido/genética , Aminoácidos/isolamento & purificação , Aminoácidos/metabolismo , Compostos de Amônio/metabolismo , Coffea/metabolismo , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/classificação , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Nitratos/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Poli A/genética , Poli A/metabolismo , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , RNA de Plantas/classificação , RNA de Plantas/metabolismo , Pequeno RNA não Traduzido/classificação , Pequeno RNA não Traduzido/metabolismo , Sementes/genética , Sementes/metabolismo , Estresse Fisiológico , TranscriptomaRESUMO
Rapid actions of triiodothyronine (T3) on thyrotropin (TSH) synthesis and secretion have been described in hypothyroid male rats. However, the molecular mechanisms remain unknown. TαT1 cells, a thyrotroph cell line, was used herein to characterize the possible non-genomic actions of T3 on the expression of alpha (Cga) and Tshb genes, and the posttranscriptional processing and translation of both transcripts. The involvement of αVß3 integrin was also assessed. T3 quickly reduced Tshb mRNA content, poly(A) tail length and its association with ribosomes. The effect of T3 on Tshb gene expression was detected even in the presence of a transcription inhibitor. The decrease in Tshb mRNA content and polyadenylation depend on T3 interaction with αVß3 integrin, while T3 reduced Cga mRNA content by transcriptional action. The translational rate of both transcripts was reduced by a mechanism, which does not depend on T3-αVß3 integrin interaction. Results indicate that, in parallel with the inhibitory transcriptional action in Cga and Tshb gene expression, T3 rapidly triggers additional posttranscriptional mechanisms, reducing the TSH synthesis. These non-genomic actions partially depend on T3-αVß3 integrin interaction at the plasma membrane of thyrotrophs and add new insights to the molecular mechanisms involved in T3 negative feedback loop.
Assuntos
Retroalimentação Fisiológica , Tireotropina Subunidade beta/genética , Transcrição Gênica/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Subunidade alfa de Hormônios Glicoproteicos/genética , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Integrina alfaVbeta3/metabolismo , Poli A/metabolismo , Poliadenilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Tireotrofos/efeitos dos fármacos , Tireotrofos/metabolismo , Tireotropina Subunidade beta/metabolismoRESUMO
The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.
Assuntos
Entamoeba histolytica/química , Leucina/química , Proteínas de Protozoários/química , RNA Bacteriano/química , RNA Mensageiro/química , Tirosina/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Leucina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Poli A/química , Poli A/genética , Poli A/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Termodinâmica , Tirosina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
OBJECTIVE: To examine attitudes and beliefs related to help-seeking for depression among an international sample of pregnant women, a majority of whom were Spanish-speakers residing in Latin America. METHODS: More than 6 000 (n = 6 672) pregnant women met eligibility criteria and consented to participate between 15 January 2009-12 August 2011. Of these, 1 760 with a Latino/Hispanic background completed a baseline survey as part of a larger study. Group comparisons analyzed attitudes and behaviors related to seeking help for depression, while a logistic regression was conducted to identify demographic characteristics related to help-seeking support. RESULTS: Of the participants, three-fourths reported experiencing depression during or after their current or past pregnancies. The majority of participants did not seek help, and generally reported ambivalence about their depressive symptoms and uncertainty as to the helpfulness of others. However, 44.8% did seek help, mostly by speaking to family or partners and reported feeling fear, shame, and embarrassment about their symptoms. A current major depressive episode and an income less than or equal to US$ 10 000 were significant predictors of help-seeking behaviors. CONCLUSIONS: Data from this study suggest that when feeling sad or depressed, perinatal Latinas tend to seek emotional support first from family and friends and may underutilize mental health services when needed. The Internet is an effective means for reaching perinatal women, especially those in areas of the world where there may be barriers to accessing psychological resources.
OBJETIVO: Analizar las actitudes y las creencias relacionadas con la búsqueda de ayuda para la depresión en una muestra internacional de mujeres embarazadas, la mayor parte de ellas hispanohablantes y residentes en América Latina. MÉTODOS: Más de 6 000 mujeres embarazadas (n = 6 672) cumplieron los criterios de selección y aceptaron participar entre el 15 de enero del 2009 y el 12 de agosto del 2011. De estas, 1 760 de origen latino o hispano completaron una encuesta básica que formaba parte de un estudio más amplio. Mediante comparaciones de grupo, se analizaron las actitudes y los comportamientos relacionados con la búsqueda de ayuda para la depresión, mientras que, mediante regresión logística, se determinaron las características demográficas relacionadas con la búsqueda de ayuda o apoyo. RESULTADOS: De todas las participantes, tres cuartas partes notificaron sentimientos de depresión durante o después de los embarazos actuales o pasados. La mayor parte de ellas no buscaron ayuda, y en general manifestaron ambivalencia acerca de sus síntomas depresivos e incertidumbre en cuanto a la capacidad de ayuda de otras personas. Sin embargo, 44,8% buscaron ayuda, principalmente hablando con familiares o compañeros, y notificaron sentimientos de temor, culpabilidad y vergüenza acerca de sus síntomas. Un episodio depresivo mayor actual y unos ingresos iguales o inferiores a US$ 10 000 fueron factores predictivos significativos de comportamientos de búsqueda de ayuda. CONCLUSIONES: Los datos de este estudio indican que, cuando se sienten tristes o deprimidas, las mujeres latinas en período perinatal tienden a buscar en primer lugar el apoyo emocional de la familia y los amigos, y podrían subutilizar los servicios de salud mental cuando son necesarios. La internet es un medio eficaz para llegar a las mujeres en período perinatal, especialmente a las que viven en zonas del mundo donde pueden existir barreras para el acceso a los recursos psicológicos.