RESUMO
Plant-pathogen interaction is influenced by multiple environmental factors, including temperature and light. Recent works have shown that light modulates not only the defense response of plants but also the pathogens virulence. Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, an important plant disease worldwide. The Xcc genome presents four genes encoding putative photoreceptors: one bacteriophytochrome and three blue light photoreceptors, one LOV and two BLUFs (bluf1: XAC2120 and bluf2: XAC3278). The presence of two BLUFs proteins is an outstanding feature of Xcc. In this work we show that the bluf2 gene is functional. The mutant strain, XccΔbluf2, was constructed demonstrating that BLUF2 regulates swimming-type motility, adhesion to leaves, exopolysaccharide production and biofilm formation, features involved in the Xcc virulence processes. An important aspect during the plant-pathogen interaction is the oxidative response of the host and the consequent reaction of the pathogen. We observed that ROS detoxification is regulated by Xcc bluf2 gene. The phenotypes of disease in orange plants produced by WT and XccΔbluf2 strains were evaluated, observing different phenotypes. Altogether, these results show that BLUF2 negatively regulates virulence during citrus canker. This work constitutes the first report on BLUF-like receptors in plant pathogenic bacteria.
Assuntos
Citrus , Xanthomonas , Xanthomonas/genética , Xanthomonas/metabolismo , Citrus/metabolismo , Citrus/microbiologia , Virulência , Luz , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismoRESUMO
Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207+CD1a+ cells that can arise in almost any tissue and cause significant morbidity and mortality. Precursors of pathological Langerhans cells have yet to be defined. Our aim was to identify circulating CD207+CD1a+ cells and their inducers in LCH. Expression of CD207 and CD1a in the blood myeloid compartment as well as thymic stromal lymphopoietin (TSLP) and transforming growth factor ß (TGF-ß) plasma levels were measured in 22 pediatric patients with active disease (AD) or nonactive disease (NAD). In patients with AD vs those with NAD, the myeloid compartment showed an increased CD11b (CD11bhigh plus CD11b+) fraction (39.7 ± 3.6 vs 18.6 ± 1.9), a higher percentage of circulating CD11bhighCD11c+CD207+ cells (44.5 ± 11.3 vs 3.2 ± 0.5), and the presence of CD11chighCD207+CD1a+ cells (25.0 ± 9.1 vs 2.3 ± 0.5). Blood CD207+CD1a+ cells were not observed in adult controls or umbilical cord. Increased TSLP and TGF-ß levels were detected in patients with AD. Interestingly, plasma from patients with AD induces CD207 expression on CD14+ monocytes. We conclude that CD207+CD1a+ cells are circulating in patients with active LCH, and TSLP and TGF-ß are potential drivers of Langerhans-like cells in vivo.
Assuntos
Antígenos CD1/metabolismo , Antígenos CD/metabolismo , Histiocitose de Células de Langerhans/metabolismo , Histiocitose de Células de Langerhans/patologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Criança , Pré-Escolar , Citocinas/sangue , Feminino , Histiocitose de Células de Langerhans/sangue , Humanos , Lactente , Masculino , Fator de Crescimento Transformador beta/sangue , Linfopoietina do Estroma do TimoRESUMO
STUDY QUESTION: Does standardised treatments used in children and adolescents with haematologic malignancies, including acute lymphoblastic (ALL) or myeloid leukaemia (AML) and non-Hodgkin lymphoma (NHL), affect endocrine function of the developing testes? SUMMARY ANSWER: Therapy of haematologic malignancies do not provoke an overt damage of Sertoli and Leydig cell populations, as revealed by normal levels of anti-Müllerian hormone (AMH) and testosterone, but a mild primary testicular dysfunction may be observed, compensated by moderate gonadotropin elevation, during pubertal development. WHAT IS KNOWN ALREADY: Evidence exists on the deleterious effect that chemotherapy and radiotherapy have on germ cells, and some attention has been given to the effects on Leydig and Sertoli cells of the adult gonads, but information is virtually non-existent on the effects of oncologic treatment on testicular somatic cell components during childhood and adolescence. STUDY DESIGN, SIZE, DURATION: A retrospective, analytical, observational study included 97 boys with haematological malignancies followed at two tertiary paediatric public hospitals in Buenos Aires, Argentina, between 2002 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: Clinical records of males aged 1-18 years, referred with the diagnoses of ALL, AML or NHL for the assessment of gonadal function, were eligible. We assessed serum levels of AMH and FSH as biomarkers of Sertoli cell endocrine function and testosterone and LH as biomarkers of Leydig cell function. MAIN RESULTS AND THE ROLE OF CHANCE: All hormone levels were normal in the large majority of patients until early pubertal development. From Tanner stage G3 onwards, while serum AMH and testosterone kept within the normal ranges, gonadotropins reached mildly to moderately elevated values in up to 35.9% of the cases, indicating a compensated Sertoli and/or Leydig cell dysfunction, which generally did not require hormone replacement therapy. LIMITATIONS, REASONS FOR CAUTION: Serum inhibin B determination and semen analysis were not available for most patients; therefore, we could not conclude on potential fertility impairment or identify whether primary Sertoli cell dysfunction resulted in secondary depleted spermatogenesis or whether primary germ cell damage impacted Sertoli cell function. WIDER IMPLICATIONS OF THE FINDINGS: The regimens used in the treatment of boys and adolescents with ALL, AML or NHL in the past two decades seem relatively safe for endocrine testicular function; nonetheless, a mild primary testicular endocrine dysfunction may be observed, usually compensated by slightly elevated gonadotropin secretion by the pituitary in adolescents, and not requiring hormone replacement therapy. No clinically relevant risk factor, such as severity of the disease or treatment protocol, could be identified in association with the compensated endocrine dysfunction. STUDY FUNDING/COMPETING INTEREST(S): This work was partially funded by grants PIP 11220130100687 of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and PICT 2016-0993 of Fondo para la Investigación Científica y Tecnológica (FONCYT), Argentina. R.A.R., R.P.G. and P.B. have received honoraria from CONICET (Argentina) for technology services using the AMH ELISA. L.A.A. is part-time employee of CSL Behring Argentina. The other authors have no conflicts of interest to disclose.
Assuntos
Hormônio Antimülleriano/sangue , Antineoplásicos/efeitos adversos , Hormônio Foliculoestimulante/sangue , Leucemia/terapia , Linfoma não Hodgkin/terapia , Adolescente , Criança , Humanos , Masculino , Estudos RetrospectivosRESUMO
Hemoglobinopathies are the most common autosomal recessive disorders and are mostly inherited in a recessive manner. However, certain mutations can affect the globin chain stability, leading to dominant forms of thalassemia. The aim of this work was the molecular and structural characterization of two heterozygous in-frame deletions, leading to ß-globin variants in pediatric patients in Argentina. The HBB gene of the probands and their parents was sequenced, and other markers of globin chain imbalance were analyzed. Several structural analyses were performed, and the effect of the mutations on the globin chain stability was analyzed. In Hb JC-Paz, HBB:c.29_37delCTGCCGTTA (p.Ala10_Thr12del), detected in an Argentinean boy, one α-helix turn is expected to be lost. In Hb Tavapy, HBB:c.182_187delTGAAGG (p.Val60_Lys61del), the deleted residues are close to distal histidine (His63) in the heme pocket. Both mutations are predicted to have a destabilizing effect. The development of computational structural models and bioinformatics algorithms is expected to become a useful tool to understand the impact of the mutations leading to dominant thalassemia.
Assuntos
Substituição de Aminoácidos , Hemoglobinas Anormais/genética , Fases de Leitura , Deleção de Sequência , Globinas beta/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Índices de Eritrócitos , Feminino , Hemoglobinopatias/sangue , Hemoglobinopatias/diagnóstico , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Hemoglobinas Anormais/química , Humanos , Masculino , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Globinas beta/químicaRESUMO
Patients with Gaucher type 1 (GD1) throughout Argentina were enrolled in the Argentine bone project to evaluate bone disease and its determinants. We focused on presence and predictors of bone lesions (BL) and their relationship to therapeutic goals (TG) with timing and dose of enzyme replacement therapy (ERT). A total of 124 patients on ERT were enrolled in a multi-center study. All six TG were achieved by 82% of patients: 70.1% for bone pain and 91.1% for bone crisis. However, despite the fact that bone TGs were achieved, residual bone disease was present in 108 patients on ERT (87%) at time 0. 16% of patients showed new irreversible BL (bone infarcts and avascular osteonecrosis) despite ERT, suggesting that they appeared during ERT or were not detected at the moment of diagnosis. We observed 5 prognostic factors that predicted a higher probability of being free of bone disease: optimal ERT compliance; early diagnosis; timely initiation of therapy; ERT initiation dose ≥45 UI/kg/EOW; and the absence of history of splenectomy. Skeletal involvement was classified into 4 major phenotypic groups according to BL: group 1 (12.9%) without BL; group 2 (28.2%) with reversible BL; group 3 (41.9%) with reversible BL and irreversible chronic BL; and group 4 (16.9%) with acute irreversible BL. Our study identifies prognostic factors for achieving best therapeutic outcomes, introduces new risk stratification for patients and suggests the need for a redefinition of bone TG. Am. J. Hematol. 91:E448-E453, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Doenças Ósseas/diagnóstico , Doença de Gaucher/complicações , Adolescente , Adulto , Idoso , Argentina , Doenças Ósseas/etiologia , Doenças Ósseas/patologia , Criança , Diagnóstico Precoce , Terapia de Reposição de Enzimas , Doença de Gaucher/diagnóstico , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/epidemiologia , Humanos , Adesão à Medicação , Pessoa de Meia-Idade , Fenótipo , Prognóstico , Medição de Risco , Esplenectomia , Adulto Jovem , beta-Glucosidase/uso terapêuticoRESUMO
We describe here the molecular and hematological characteristics of novel frameshift mutations in exon 2 of the HBB gene (in heterozygous state) found in two Argentinean pediatric patients with dominant ß-thalassemia-like features. In Hb Wilde, HBB:c.270_273delTGAG(p.Glu90Cysfs*67), we detected the deletion of the third base of the codon 89 (T) and the codon 90 (GAG), whereas in Hb Patagonia, HBB:c.296_297dupGT(p.Asp99Trpfs*59), the frameshift mutation was due to a duplication of a 'GT' dinucleotide after the second base of codon 98 (GTG). The Hb Patagonia and Hb Wilde mutations would result in elongated ß-globin chains with modified C-terminal sequences and a total of 155 and 157 amino acids residues, respectively. Based on bioinformatics and structural analysis, as well as protein modeling, we predict that the elongated ß-globins would affect the formation of the αß dimers and their stability, which would further support the mechanism for the observed clinical features in both patients.
Assuntos
Variação Genética , Hemoglobinas Anormais/genética , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Adolescente , Adulto , Contagem de Células Sanguíneas , Pré-Escolar , Códon , Análise Mutacional de DNA , Índices de Eritrócitos , Éxons , Feminino , Mutação da Fase de Leitura , Hemoglobinas Anormais/química , Humanos , Masculino , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Multimerização Proteica , Globinas beta/químicaRESUMO
Ralstonia pseudosolanacearum GMI1000 (Rpso GMI1000) is a soil-borne vascular phytopathogen that infects host plants through the root system causing wilting disease in a wide range of agro-economic interest crops, producing economical losses. Several features contribute to the full bacterial virulence. In this work we study the participation of light, an important environmental factor, in the regulation of the physiological attributes and infectivity of Rpso GMI1000. In silico analysis of the Rpso genome revealed the presence of a Rsp0254 gene, which encodes a putative blue light LOV-type photoreceptor. We constructed a mutant strain of Rpso lacking the LOV protein and found that the loss of this protein and light, influenced characteristics involved in the pathogenicity process such as motility, adhesion and the biofilms development, which allows the successful host plant colonization, rendering bacterial wilt. This protein could be involved in the adaptive responses to environmental changes. We demonstrated that light sensing and the LOV protein, would be used as a location signal in the host plant, to regulate the expression of several virulence factors, in a time and tissue dependent way. Consequently, bacteria could use an external signal and Rpsolov gene to know their location within plant tissue during the colonization process.
Assuntos
Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno/fisiologia , Ralstonia/fisiologia , Solanum lycopersicum/microbiologia , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Luz , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Ralstonia/patogenicidadeRESUMO
Management, outcome, diagnosis, prognosis and treatment of immune thrombocytopenia are controversial. Several guidelines stating different experts' opinions have been published; however, no worldwide consensus regarding the management of the disease has still been reached. This guideline defines diagnostic criteria, states initial laboratory tests, establishes differential diagnosis, develops topics concerning outcome and prognosis, and enumerates available treatments for acute and chronic disease, as well as for management of life-threatening bleeding.
El manejo de la trombocitopenia inmune es motivo de discusión en lo concerniente a evolución, diagnóstico, pronóstico y tratamiento. Se han publicado varias guías que expresan distintas opiniones de expertos, pero no existe aún consenso mundial sobre cuál es el manejo más adecuado de la enfermedad. Esta guía establece los criterios para definir el diagnóstico; detalla el plan de estudios de laboratorio por realizar inicialmente; plantea los distintos diagnósticos diferenciales; desarrolla aspectos relativos a evolución y pronóstico, y enumera los tratamientos disponibles para las formas agudas y las crónicas, así como para el manejo de las emergencias y en algunas situaciones especiales.
Assuntos
Hemorragia/etiologia , Púrpura Trombocitopênica Idiopática/diagnóstico , Doença Aguda , Doença Crônica , Diagnóstico Diferencial , Hemorragia/terapia , Humanos , Prognóstico , Púrpura Trombocitopênica Idiopática/fisiopatologia , Púrpura Trombocitopênica Idiopática/terapiaRESUMO
The alternative oxidase (Aox) was studied at different levels (transcript, protein and capacity) in response to an osmotic shock applied to roots of cowpea (Vigna unguiculata). Two cultivars of V. unguiculata were used, Vita 3 and Vita 5, tolerant and sensitive to drought/saline stress respectively. The seedlings (17-day-old) were grown in hydroponic conditions and submitted to NaCl (100 and 200 mM) or 200.67 g L(-1) PEG 6000 (iso-osmotic condition to 100 mM NaCl). The VuAox1 and VuAox2a mRNA were not detected in either cultivar under all tested conditions while the VuAox2b gene was differently expressed. In the tolerant cultivar (Vita 3), the expression of VuAox2b gene was stimulated by an osmotic stress induced by PEG which was associated with a higher amount and capacity of the Aox protein. In the same cultivar, this gene was under-expressed in salt stress conditions with poor effect on the protein level. In the sensitive cultivar (Vita 5), the transcript level of the VuAox2b was unchanged in response to PEG treatment, even though the protein and the capacity tended to increase. Upon salt stress, the VuAox2b gene was over-expressed. At 100mM NaCl, this VuAox2b gene over-expression led to a higher amount and capacity of Aox. This effect was reduced at 200 mM NaCl. Overall, these results suggest complex mechanisms (transcriptional, translational and post-translational) for Aox regulation in response to osmotic stress.
Assuntos
Fabaceae/enzimologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Fabaceae/efeitos dos fármacos , Fabaceae/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Pressão Osmótica , Oxirredutases/genética , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/fisiologia , Polietilenoglicóis/farmacologia , RNA Mensageiro/metabolismo , Cloreto de Sódio/metabolismo , Cloreto de Sódio/farmacologiaRESUMO
Plants are continuously exposed to pathogen challenge. The most common defense response to pathogenic microorganisms is the nonhost response, which is usually accompanied by transcriptional changes. In order to identify genes involved in nonhost resistance, we evaluated the tobacco transcriptome profile after infection with Xanthomonas axonopodis pv. citri (Xac), a nonhost phytopathogenic bacterium. cDNA-amplified fragment length polymorphism was used to identify differentially expressed transcripts in tobacco leaves infected with Xac at 2, 8 and 24h post-inoculation. From a total of 2087 transcript-derived fragments (TDFs) screened (approximately 20% of the tobacco transcriptome), 316 TDFs showed differential expression. Based on sequence similarities, 82 differential TDFs were identified and assigned to different functional categories: 56 displayed homology to genes with known functions, 12 to proteins with unknown functions and 14 did not have a match. Real-time PCR was carried out with selected transcripts to confirm the expression pattern obtained. The results reveal novel genes associated with nonhost resistance in plant-pathogen interaction in tobacco. These novel genes could be included in future strategies of molecular breeding for nonhost disease resistance.
Assuntos
Regulação da Expressão Gênica de Plantas , Nicotiana/genética , Nicotiana/imunologia , Doenças das Plantas/imunologia , Transcriptoma , Xanthomonas axonopodis/imunologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , DNA Complementar/genética , Genes de Plantas/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Nicotiana/microbiologia , Transcriptoma/genética , Xanthomonas axonopodis/patogenicidadeRESUMO
Cowpea (Vigna unguiculata) alternative oxidase is encoded by a small multigene family (Aox1, 2a and 2b) that is orthologous to the soybean Aox family. Like most of the identified Aox genes in plants, VuAox1 and VuAox2 consist of 4 exons interrupted by 3 introns. Alignment of the orthologous Aox genes revealed high identity of exons and intron variability, which is more prevalent in Aox1. In order to determine Aox gene expression in V. unguiculata, a steady-state analysis of transcripts involved in seed development (flowers, pods and dry seeds) and germination (soaked seeds) was performed and systemic co-expression of VuAox1 and VuAox2b was observed during germination. The analysis of Aox transcripts in leaves from seedlings under different stress conditions (cold, PEG, salicylate and H2O2 revealed stress-induced co-expression of both VuAox genes. Transcripts of VuAox2a and 2b were detected in all control seedlings, which was not the case for VuAox1 mRNA. Estimation of the primary transcript lengths of V. unguiculata and soybean Aox genes showed an intron length reduction for VuAox1 and 2b, suggesting that the two genes have converged in transcribed sequence length. Indeed, a bioinformatics analysis of VuAox1 and 2b promoters revealed a conserved region related to a cis-element that is responsive to oxidative stress. Taken together, the data provide evidence for co-expression of Aox1 and Aox2b in response to stress and also during the early phase of seed germination. The dual nature of VuAox2b expression (constitutive and induced) suggests that the constitutive Aox2b gene of V. unguiculata has acquired inducible regulatory elements.
Assuntos
Fabaceae/enzimologia , Regulação da Expressão Gênica de Plantas , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Temperatura Baixa , Éxons , Fabaceae/genética , Germinação , Peróxido de Hidrogênio , Íntrons , Proteínas Mitocondriais , Dados de Sequência Molecular , Oxirredutases/genética , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Polietilenoglicóis , Regiões Promotoras Genéticas , Precursores de RNA , RNA Mensageiro/metabolismo , Sementes/enzimologia , Salicilato de SódioRESUMO
The higher plant mitochondrial electron transport chain contains an alternative pathway that ends with the AOX (alternative oxidase). The AOX proteins are encoded by a small gene family composed of two discrete gene subfamilies. Aox1 is present in both monocot and eudicot plants, whereas Aox2 is only present in eudicot plants. We isolated a genomic clone from Citrus sinensis containing the Aox1a gene. The orange Aox1a consists of four exons interrupted by three introns and its promoter harbours diverse putative stress-specific regulatory motifs including pathogen response elements. The role of the Aox1a gene was evaluated during the compatible interaction between C. sinensis and Xanthomonas axonopodis pv. citri and no induction of the Aox1a at the transcriptional level was observed. On the other hand, Aox1a was studied in orange plants during non-host interactions with Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria, which result in hypersensitive response. Both phytopathogens produced a strong induction of Aox1a, reaching a maximum at 8 h post-infiltration. Exogenous application of salicylic acid produced a slight increase in the steady-state level of Aox1a, whereas the application of fungi elicitors showed the highest induction. These results suggest that AOX1a plays a role during biotic stress in non-host plant pathogen interaction.