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1.
Pediatr Surg Int ; 40(1): 205, 2024 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-39033225

RESUMO

PURPOSE: This study systematically reviewed our team's research on the mechanism and assessment of liver fibrosis in BA, summarized our experience, and discussed the future development direction. METHODS: In this study, Pubmed and Wanfang databases were searched to collect the literature published by our team on the mechanisms of liver fibrosis in BA and the assessment of liver fibrosis in BA, and the above research results were systematically reviewed. RESULTS: A total of 58 articles were retrieved. Among the included articles, 25 articles related to the mechanism of liver fibrosis in BA, and five articles evaluated liver fibrosis in BA. This article introduces the key pathways and molecules of liver fibrosis in BA and proposes a new grading system for liver fibrosis in BA. CONCLUSIONS: The new BA liver fibrosis grading method is expected to assess children's conditions, guide treatment, and improve prognosis more accurately. In addition, we believe that the TGF-ß1 signaling pathway is the most important in the study of liver fibrosis in BA, and at the same time, the study of EMT occurrence in BA should also be deepened to resolve the controversy on this issue.


Assuntos
Atresia Biliar , Cirrose Hepática , Humanos , Atresia Biliar/complicações , Cirrose Hepática/diagnóstico , Fator de Crescimento Transformador beta1/metabolismo , Prognóstico
2.
Pediatr Surg Int ; 40(1): 24, 2023 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-38127131

RESUMO

PURPOSE: To determine the prevalent microbiological profile of biliary atresia (BA) patients at the time of its occurrence by studying their intestinal flora. METHODS: A total of 118 gut microbiota samples from three groups of 43 BA patients, 33 disease controls (DC) with other cholestatic diseases and 42 healthy controls (HC), were analyzed by deep mining of public data. Subsequently, a total of 23 fecal samples from three groups of clinically collected patients (11 BA, 6 DC and 6 HC) were sequenced for 16S rRNA gene amplification and analyzed for serum butyrate (BU) level by liquid chromatography. RESULTS: Taxonomic analysis revealed significant differences in the composition of the intestinal microbiota between BA patients and controls, with a reduction in diversity and a higher abundance of Proteobacteria, Streptococcus and Lactobacillus in the BA group. Database and clinical data analyses concluded that Streptococcus/Bacteroides (AUC = 0.9035, 95% CI 0.8347-0.9722, P < 0.0001) or Streptococcus/Eggerthella (AUC = 0.8333, 95% CI 0.6340-1.000, P = 0.027) was the best microbiota to differentiate between BA and DC. Serum butyrate levels were low in the BA and DC groups and differed from the HC group (P = 0.01, P = 0.04). Butyrate levels in BA were negatively correlated with jaundice clearance and cholangitis, but not statistically significant. CONCLUSIONS: Our study reveals changes in the composition of the gut microbiota in BA, especially the butyrate-producing microbiota, and suggests the potential for using gut microbiota as a noninvasive diagnostic benefit for BA. Low levels of serum butyrate in BA may indicate a poor prognosis.


Assuntos
Atresia Biliar , Microbioma Gastrointestinal , Criança , Humanos , Microbioma Gastrointestinal/genética , Butiratos , Prognóstico , RNA Ribossômico 16S
3.
BMC Pediatr ; 23(1): 589, 2023 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-37993876

RESUMO

OBJECTIVES: To validate an appropriate evaluation method of liver fibrosis assessment based on the unique pathological features of biliary atresia (BA) that could well predict its prognosis. METHODS: A total of 68 patients with BA who underwent Kasai procedure (KP) and an intraoperative liver biopsy, followed up from January 2019 to December 2021, were recruited in a retrospective analysis. Ishak, Metavir, and BA-specific staging systems in relation to outcomes were analyzed using logistic regression, COX proportional hazard regression, Kaplan-Meier analysis, etc. RESULTS: Kaplan-Meier analysis determined a significant difference in native liver survival according to the BA-specific stage (p = 0.002). The ROC curve analysis for predicting prognosis showed that the AUC of BA-specific staging combined with iBALF and severe bile duct proliferation (BDP) (0.811, 95% CI: 0.710-0.913, p < 0.0001) was higher than BA-specific staging alone (0.755, 95% CI: 0.639-0.872, p < 0.001). CONCLUSIONS: The BA-specific staging system reflects the condition of the liver fibrosis, and its combination with iBALF and severe BDP helps to better evaluate the prognosis of patients with BA.


Assuntos
Atresia Biliar , Humanos , Lactente , Atresia Biliar/cirurgia , Portoenterostomia Hepática , Prognóstico , Estudos Retrospectivos , Cirrose Hepática
4.
J Physiol Biochem ; 79(3): 613-623, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37145351

RESUMO

Glutamine (Gln) is the most widely acting and abundant amino acid in the body and has anti-inflammatory properties, regulates body metabolism, and improves immune function. However, the mechanism of Gln's effect on hyperoxic lung injury in neonatal rats is unclear. Therefore, this work focused on examining Gln's function in lung injury of newborn rats mediated by hyperoxia and the underlying mechanism. We examined body mass and ratio of wet-to-dry lung tissue weights of neonatal rats. Hematoxylin and eosin (HE) staining was performed to examine histopathological alterations of lung tissues. In addition, enzyme-linked immunoassay (ELISA) was conducted to measure pro-inflammatory cytokine levels within bronchoalveolar lavage fluid (BALF). Apoptosis of lung tissues was observed using TUNEL assay. Western blotting was performed for detecting endoplasmic reticulum stress (ERS)-associated protein levels. The results showed that Gln promoted body weight gain, significantly reduced pathological damage and oxidative stress in lung tissue, and improved lung function in neonatal rats. Gln reduced pro-inflammatory cytokine release as well as inflammatory cell production in BALF and inhibited apoptosis in lung tissue cells. Furthermore, we found that Gln could downregulate ERS-associated protein levels (GRP78, Caspase-12, CHOP) and inhibit c-Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) phosphorylation. These results in an animal model of bronchopulmonary dysplasia (BPD) suggest that Gln may have a therapeutic effect on BPD by reducing lung inflammation, oxidative stress, and apoptosis and improving lung function; its mechanism of action may be related to the inhibition of the IRE1α/JNK pathway.


Assuntos
Hiperóxia , Lesão Pulmonar , Ratos , Animais , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/etiologia , Lesão Pulmonar/prevenção & controle , Hiperóxia/complicações , Hiperóxia/metabolismo , Hiperóxia/patologia , Glutamina/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pulmão/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Apoptose , Citocinas/metabolismo , Estresse Oxidativo
5.
Int J Nanomedicine ; 17: 5851-5868, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36474527

RESUMO

Purpose: Maxillofacial infection is a common disease in stomatology and is difficult to treat owing to its high potential to spread to vital anatomical structures. Excessive levels of reactive oxygen species (ROS) in infected tissues lead to cellular damage and impede tissue regeneration. However, uncontrollable strategies to remove ROS have limited therapeutic efficacy. Nanoparticle systems for scavenging ROS and remodeling the inflammatory microenvironment offer much promise in the treatment of maxillofacial inflammation. Methods: Here, a novel microenvironment-stimuli-responsive drug delivery nanoplatform (HMPB@Cur@PDA) based on a polydopamine (PDA)-functionalized hollow mesoporous Prussian blue (HMPB) nanozyme was developed for the delivery of curcumin (Cur) in the treatment of maxillofacial infection. Low pH and excess ROS in the inflammatory microenvironment cause degradation of the outer PDA layer of the nanocomplex, exposing the HMPB nanozyme and loaded Cur, which synergistically act as a ROS scavenger and anti-inflammatory agent, respectively, and induce macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. Results: Experiments in vitro provided strong evidence for the application of novel nanocomplexes in scavenging multiple ROS and inhibiting lipopolysaccharide-induced inflammation. In addition, in vivo results obtained using a mouse maxillofacial infection model demonstrated that HMPB@Cur@PDA had excellent biocompatibility, significantly attenuated the inflammatory response in periodontal tissue, and improved the repair of damaged tissue. Conclusion: Our results indicate that HMPB@Cur@PDA nanocomposites have great potential for ROS regulation as well as having anti-inflammatory effects, providing new insights for the development of dual-response maxillofacial infection treatments.


Assuntos
Anti-Inflamatórios , Macrófagos , Preparações Farmacêuticas
6.
Exp Lung Res ; 48(4-6): 187-197, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35924334

RESUMO

Background: Insulin-like growth factor-1 (IGF-1), a member of the insulin family, has a high degree of homology with insulin and exhibits anti-inflammatory and anti-oxidative stress properties. However, the potential protective effect of IGF-1 on hyperoxia-induced lung injury remains unknown. In this study, we aimed to explore the effects and mechanism of action of IGF-1 in hyperoxia-induced lung injury in neonatal rats. Materials and Methods: Hematoxylin-eosin staining was used to observe pathological changes in lung tissue; transmission electron microscopy was used to examine the ultrastructure, and ELISA was used to detect the level of pro-inflammatory cytokines in bronchoalveolar lavage fluid. Further, malondialdehyde, glutathione, and superoxide dismutase activities in lung tissue were evaluated. TUNEL staining was used to detect cell apoptosis, and western blot analysis was used to detect the expression of Bax, Bcl-2, Caspase-3, p-PERK, p-eIF2α, ATF4, and CHOP in the lung tissue. Moreover, the wet/dry weight ratio of lung tissue was determined. Results: Intraperitoneal injection of IGF-1 effectively reduced lung tissue damage induced by hyperoxia; production of inflammatory cells and release of pro-inflammatory cytokines, oxidative stress, and cell apoptosis. Further, IGF-1 down-regulated the expression of ATF4, CHOP, and Bax/Bcl-2, and inhibited the phosphorylation of PERK and eIF2α. Conclusion: The results suggest that IGF-1 reduces hyperoxia-induced lung inflammation and oxidative stress in neonatal rats through the PERK/eIF2α/ATF4/CHOP signaling pathway and inhibits cell apoptosis.


Assuntos
Hiperóxia , Insulinas , Lesão Pulmonar , Pneumonia , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/farmacologia , Animais , Apoptose , Citocinas/metabolismo , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/farmacologia , Hiperóxia/complicações , Hiperóxia/tratamento farmacológico , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Insulinas/metabolismo , Insulinas/farmacologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/farmacologia , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologia , eIF-2 Quinase/metabolismo , eIF-2 Quinase/farmacologia
7.
PLoS One ; 17(7): e0271202, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35901060

RESUMO

BACKGROUND: Cell survival or death is one of the key scientific issues of inflammatory response. To regulate cell death during the occurrence and development of periodontitis, various forms of programmed cell death, such as pyroptosis, ferroptosis, necroptosis, and apoptosis, have been proposed. It has been found that ferroptosis characterized by iron-dependent lipid peroxidation is involved in cancer, degenerative brain diseases and inflammatory diseases. Furthermore, NCOA4 is considered one of ferroptosis-related genes (FRGs) contributing to butyrate-induced cell death in the periodontitis. This research aims to analyze the expression of FRGs in periodontitis tissues and to explore the relationship between ferroptosis and periodontitis. METHOD: Genes associated with periodontitis were retrieved from two Gene Expression Omnibus datasets. Then, we normalized microarray data and removed the batch effect using the R software. We used R to convert the mRNA expression data and collected the expression of FRGs. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), transcription factor (TF) and protein-protein interaction (PPI) network analyses were used. In addition, we constructed a receiver operating characteristic curve and obtained relative mRNA expression verified by quantitative reverse-transcription polymerase chain reaction (PCR). RESULTS: Eight and 10 FRGs related to periodontitis were upregulated and downregulated, respectively. GO analysis showed that FRGs were enriched in the regulation of glutathione biosynthetic, glutamate homeostasis, and endoplasmic reticulum-nucleus signaling pathway. The top TFs included CEBPB, JUND, ATF2. Based on the PPI network analysis, FRGs were mainly linked to the negative regulation of IRE1-mediated unfolded protein response, regulation of type IIa hypersensitivity, and regulation of apoptotic cell clearance. The expression levels of NCOA4, SLC1A5 and HSPB1 using PCR were significantly different between normal gingival samples and periodontitis samples. Furthermore, the diagnostic value of FRGs for periodontitis were "Good". CONCLUSIONS: We found significant associations between FRGs and periodontitis. The present study not only provides a new possible pathomechanism for the occurrence of periodontitis but also offers a new direction for the diagnosis and treatment of periodontitis.


Assuntos
Ferroptose , Periodontite , Sistema ASC de Transporte de Aminoácidos , Biologia Computacional , Ferroptose/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Antígenos de Histocompatibilidade Menor , Periodontite/genética , Periodontite/metabolismo , RNA Mensageiro/genética
8.
Front Plant Sci ; 13: 869178, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35586217

RESUMO

We adopted a systems-based approach to determine the role of two Candidatus Liberibacter asiaticus (CLas) proteins, LasP 235 and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these CLas proteins HLB pathogenesis, the exact structure-based mechanism of their action has not been elucidated. We conducted the following experiments to determine the structure-based mechanisms of action. First, we immunoprecipitated the interacting citrus protein partners of LasP 235 and Effector 3 from the healthy and CLas-infected Hamlin extracts and identified them by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Second, we performed a split green fluorescent protein (GFP) assay in tobacco to validate that the interactions observed in vitro are also retained in planta. The notable in planta citrus targets of LasP 235 and Effector 3 include citrus innate immune proteins. Third, in vitro and in planta studies were performed to show that LasP 235 and Effector 3 interact with and inhibit the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species, blocking of bactericidal lipid transfer protein (LTP), and induction of premature programed cell death (PCD), all of which are beneficial to CLas lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of LasP 235 and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked LasP 235 and rescued the bactericidal activity of LTP thereby proving that LasP 235 , indeed, participates in HLB pathogenesis.

9.
Exp Ther Med ; 23(3): 191, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35126694

RESUMO

Bronchopulmonary dysplasia (BPD) is a common respiratory disorder among preterm infants, particularly low-birth-weight infants (LBWIs) and very-low-birth-weight infants (VLBWIs). Although BPD was first reported 50 years ago, no specific drugs or efficient measures are yet available for prevention or treatment. Insulin-like growth factor-1 (IGF-1) belongs to the insulin family. It promotes mitosis and stimulates cell proliferation and DNA synthesis, the primary factors involved in pulmonary development during the fetal and postnatal periods. Several studies have reported that IGF-1 exerts certain effects on BPD genesis and progression by regulating BPD-related biological processes. In addition, exogenous addition of IGF-1 can alleviate lung inflammation, cell apoptosis and eliminate alveolar development disorders in children with BPD. These findings suggest that IGF-1 could be a new target for treating BPD. Here, we summarize and analyze the definition, pathogenesis, and research status of BPD, as well as the pathogenesis of IGF-1 in BPD and the latest findings in related biological processes.

10.
Front Immunol ; 13: 1054201, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36618395

RESUMO

Background: Predicting type 2 chronic rhinosinusitis with nasal polyps (CRSwNP) may help for selection of appropriate surgical procedures or pharmacotherapies in advance. However, an accurate non-invasive method for diagnosis of type 2 CRSwNP is presently unavailable. Methods: To optimize the technique for collecting nasal secretion (NasSec), 89 CRSwNP patients were tested using nasal packs made with four types of materials. Further, Th2low and Th2highCRSwNP defined by clustering analysis in another 142 CRSwNP patients using tissue biomarkers, in the meanwhile, inflammatory biomarkers were detected in NasSec of the same patients collected by the selected nasal pack. A diagnostic model was established by machine learning algorithms to predict Th2highCRSwNP using NasSecs biomarkers. Results: Considering the area under receiver operating characteristic curve (AUC) for IL-5 in NasSec, nasal pack in polyvinyl alcohol (PVA) was superior to other materials for NasSec collection. When Th2low and Th2highCRSwNP clusters were defined, logistic regression and decision tree model for prediction of Th2highCRSwNP demonstrated high AUCs values of 0.92 and 0.90 respectively using biomarkers of NasSecs. Consequently, the pre-pruned decision tree model; based on the levels of IL-5 in NasSec (≤ 15.04 pg/mL), blood eosinophil count (≤ 0.475*109/L) and absence of comorbid asthma; was chosen to define Th2lowCRSwNP from Th2highCRSwNP for routine clinical use. Conclusions: Taken together, a decision tree model based on a combination of NasSec biomarkers and clinical features can accurately define type 2 CRSwNP patients and therefore may be of benefit to patients in receiving appropriate therapies in daily clinical practice.


Assuntos
Pólipos Nasais , Rinite , Sinusite , Humanos , Pólipos Nasais/diagnóstico , Rinite/diagnóstico , Interleucina-5 , Sinusite/diagnóstico , Biomarcadores , Doença Crônica
11.
World J Pediatr Surg ; 4(1): e000209, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-36474636

RESUMO

Background: Although complete resolution and recovery occurs in most children with an initial attack of acute pancreatitis (AP), a subset of children may progress to recurrent AP (RAP). RAP has serious effects to the individual and the socioeconomic burden. The aim of this project was to identify the independent risk factors for pediatric RAP so as to provide evidence for its prevention, early diagnosis and treatment. Methods: A retrospective cohort study of children discharged from Tianjin Children's Hospital from June 2017 to January 2020 was performed. Demographic and clinical variables, treatment strategies, clinical course and outcomes were collected. Independent risk factors of RAP were identified using the logistic regression model. Results: Of the total 96 enrolled children, 30 (31.3%) developed RAP during the follow-up period. The majority (27/30, 90%) of the children with AP developed RAP within 6 months of their first AP attack. The presence of systemic inflammatory response syndrome (SIRS) [odds ratio (OR)=6.652, 95% confidence interval (CI) 1.989 to 22.247], fasting time (OR=1.267, 95% CI 1.104 to 1.583), whether meet all three AP diagnostic criteria (OR=7.438, 95% CI 1.346 to 41.103) and abnormal amylase/lipase value on the seventh day of hospitalization (OR=3.601, 95% CI 0.972 to 13.342) were independent risk factors of RAP in children. Conclusions: Most children who developed RAP had progressed within 6 months after their first episode of AP. RAP was more common in children who met all three AP diagnostic criteria at initial attack and in children with SIRS, long fasting time and abnormal amylase/lipase value on the seventh day of hospitalization.

12.
Pediatr Surg Int ; 36(7): 827-833, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32444895

RESUMO

BACKGROUND: Biliary atresia (BA) is an obstructive hepatobiliary disease which manifests during infancy. Kasai portoenterostomy (KPE) is the preferred operation for BA, supplemented with glucocorticoids, antibiotics, and choleretic agents. A great deal of research has been carried out regarding diagnosis, operation, and adjuvant therapies of BA, but no consensus had been reached. To understand the variation in diagnosis and treatment strategies of BA across mainland China and to help achieve a unified treatment strategy in the future, this investigation was carried out. METHODS: This investigation was conducted via electronic questionnaire. The centres were divided into three groups based on their annual caseload: low (0-20)-, mid (21-40)-, and high (≥ 41)-volume group. Differences in the clinical practice among three groups were analyzed by Chi-square test and considered statistically significant at P < 0.05. RESULTS: 41 Centres from 26 different administrative regions were involved. The average age at KPE was mainly 51-60 days (39%, 16/41) and 61-70 days (32%, 13/41). The annual caseload was 0-20 patients in 17 centres, 21-40 patients in 11 centres, and > 40 patients in 13 centres. Preoperative ultrasound and intraoperative cholangiography were performed in all centres. Low-volume centres had a high proportion of MRI (P = 0.005), while the high-volume group had a high proportion of LSM (P = 0.015). Open KPE without liver mobilisation is the most common surgical procedure (71%, 29/41). Open KPE without liver mobilisation was more commonly used in low-volume group (P = 0.044), and laparoscopic KPE was mainly used in high-volume group (P = 0.011). The spur anti-reflux intestinal valve was performed in more than half of the centres (51%, 21/41). The length of the Roux-en-Y loop was ≥ 30 cm in the majority of centres (78%, 32/41). Glucocorticoids and antibiotics were used in most centres (90%, 37/41; 100%, 41/41) with marked variations in type, administration, dose, and duration. Oral ursodeoxycholic acid (UDCA) was used in 38 centres, in varying doses of 10-20 mg/kg/day. The duration of oral UDCA was over a year in 19 centres. CONCLUSION: Mainland China has a large number of patients with biliary atresia. Diagnostic and surgical methods vary from centre to centre and are related to its caseload. In most centres, KPE is supplemented with glucocorticoids, antibiotics, and choleretic agents without a standard regimen.


Assuntos
Antibacterianos/uso terapêutico , Atresia Biliar/diagnóstico , Atresia Biliar/cirurgia , Colagogos e Coleréticos/uso terapêutico , Glucocorticoides/uso terapêutico , Pesquisas sobre Atenção à Saúde/métodos , Portoenterostomia Hepática/métodos , Atresia Biliar/tratamento farmacológico , China , Terapia Combinada , Feminino , Pesquisas sobre Atenção à Saúde/estatística & dados numéricos , Humanos , Lactente , Fígado/cirurgia , Masculino , Inquéritos e Questionários , Resultado do Tratamento
13.
Mol Plant Microbe Interact ; 31(12): 1312-1322, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29953333

RESUMO

The oxidative (H2O2) burst is a seminal feature of the basal plant defense response to attempted pathogen invasions. In 'Candidatus Liberibacter asiaticus' UF506, expression of the SC2 prophage-encoded secreted peroxidase (F489_gp15) increases bacterial fitness and delays symptom progression in citrus. Two chromosomal 1-Cys peroxiredoxin genes, CLIBASIA_RS00940 (Lasprx5) and CLIBASIA_RS00445 (Lasbcp), are conserved among all sequenced 'Ca. L. asiaticus' strains, including those lacking prophages. Both LasBCP and LasdPrx5 have only a single conserved peroxidatic Cys (CP/SH) and lack the resolving Cys (CR/SH). Lasprx5 appeared to be a housekeeping gene with similar moderate transcript abundance in both 'Ca. L. asiaticus'-infected psyllids and citrus. By contrast, Lasbcp was expressed only in planta, similar to the expression of the SC2 peroxidase. Since 'Ca. L. asiaticus' is uncultured, Lasbcp and Lasprx5 were functionally validated in a cultured surrogate species, Liberibacter crescens, and both genes significantly increased oxidative stress tolerance and cell viability in culture. LasBCP was nonclassically secreted and, in L. crescens, conferred 214-fold more resistance to tert-butyl hydroperoxide (tBOOH) than wild type. Transient overexpression of Lasbcp in tobacco suppressed H2O2-mediated transcriptional activation of RbohB, the key gatekeeper of the systemic plant defense signaling cascade. Lasbcp expression did not interfere with the perception of 'Ca. L. asiaticus' flagellin (flg22Las) but interrupted the downstream activation of RbohB and stereotypical deposition of callose in tobacco. Critically, LasBCP also protected against tBOOH-induced peroxidative degradation of lipid membranes in planta, preventing subsequent accumulation of antimicrobial oxylipins that can also trigger the localized hypersensitive cell death response.


Assuntos
Citrus/imunologia , Hemípteros/microbiologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Rhizobiaceae/patogenicidade , Animais , Citrus/microbiologia , Insetos Vetores , Doenças das Plantas/microbiologia , Rhizobiaceae/genética , Rhizobiaceae/imunologia
14.
PLoS One ; 12(10): e0186810, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29049366

RESUMO

Citrus Huanglongbing (HLB) associated with 'Candidatus Liberibacter asiaticus' (Las) and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs) in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri but not Las. Our results underscore the difficulty in controlling HLB compared to other bacterial diseases.


Assuntos
Bactérias/metabolismo , Peptídeos/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas syringae/patogenicidade , Xanthomonas/patogenicidade , Peptídeos Catiônicos Antimicrobianos , Citrus/genética , Citrus/microbiologia , Genes de Plantas , Vetores Genéticos , Plantas Geneticamente Modificadas , Nicotiana/genética , Nicotiana/microbiologia
15.
Hortic Res ; 4: 17041, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28904803

RESUMO

Identification of genes with differential transcript abundance (GDTA) in seedless mutants may enhance understanding of seedless citrus development. Transcriptome analysis was conducted at three time points during early fruit development (Phase 1) of three seedy citrus genotypes: Fallglo (Bower citrus hybrid (Citrus reticulata×C. reticulata×C. paradisi)×Temple (C. reticulata×C. sinensis)), grapefruit (C. paradisi), Pineapple sweet orange (C. sinensis), and their seedless mutants. Seed abortion in seedless mutants was observed at 26 days post anthesis (Time point 2). Affymetrix transcriptomic analysis revealed 359 to 1077 probe sets with differential transcript abundance in the comparison of seedless versus seedy fruits for each citrus genotypes and time points. The GDTA identified by 18 microarray probe sets were validated by qPCR. Hierarchical clustering analysis revealed a range of GDTA associated with development, hormone and protein metabolism, all of which may reflect genes associated with seedless fruit development. There were 14, 9 and 12 genes found exhibiting similar abundance ratios in all three seedless versus seedy genotype comparisons at time point 1, 2 and 3, respectively. Among those genes were genes coding for an aspartic protease and a cysteine protease, which may play important roles in seedless fruit development. New insights into seedless citrus fruit development may contribute to biotech approaches to create seedless cultivars.

16.
PLoS One ; 12(1): e0170931, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28141855

RESUMO

Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum-a synonym of X. campestris pv. malvacearum (Smith 1901-1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1-45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Parede Celular/metabolismo , Nicotiana/genética , Nicotiana/microbiologia , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Eletroforese em Gel de Poliacrilamida , Genoma de Planta , Proteínas Mutantes/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Deleção de Sequência , Solubilidade , Relação Estrutura-Atividade , Nicotiana/virologia , Vírus do Mosaico do Tabaco , Transcrição Gênica , Xanthomonas
17.
PLoS One ; 10(7): e0133796, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26218423

RESUMO

Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry.


Assuntos
Proteínas de Bactérias , Genoma Bacteriano , Doenças das Plantas/microbiologia , Sambucus/microbiologia , Fatores de Virulência , Vitis/microbiologia , Xylella , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Xylella/genética , Xylella/metabolismo , Xylella/patogenicidade
18.
Chin Med J (Engl) ; 121(11): 983-8, 2008 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-18706245

RESUMO

BACKGROUND: The relationship between cyclosporine-induced chronic nephrotoxicity (CAN) and renin-angiotensin II in humans is still contradictory. This study was conducted to detect the levels of renin and angiotensin II (ANGII) both in renal tissue and plasma from kidney transplantation patients suffering from CAN. METHODS: Twenty-six patients with allograft biopsy-proven CsA-related chronic nephrotoxicity (CAN group) and chronic rejection (control group) were enrolled in this study. Renal tissues were subjected to immunohistochemical staining with renin and ANGII antibodies. Renin and ANGII plasma levels were measured when the biopsy was performed. The relationship between expression of renin or ANGII and clinicopathological manifestations were also investigated. The cyclosporine plasma level was obtained 2 hours after morning dose (C2). In vitro, human umbilical vein endothelial cells (HUVEC) and rat mesangial cells (MC) were incubated with different concentrations of CsA (0, 250, 500, 1000 microg/L) for 24 hours. Secretion and expression of renin and ANGII was measured by radioimmunoassay or immunohistochemical staining. RESULTS: Renal pathological scores for renin and ANGII expression were significantly higher in specimens of CAN than in controls (P < 0.05). The plasma levels of renin, ANGII and C(2) in the CAN group were higher than the control group, but no significant difference was found ((0.37 +/- 0.12) ng x ml(-1)x h(-1) vs (0.20 +/- 0.10) ng x ml(-1) x h(-1), P = 0.076; (122.69 +/- 26.73) pg/ml vs (121.88 +/- 36.35) pg/ml, P = 0.977; (719.04 +/- 55.89) ng/ml vs (658.80 +/- 90.78) ng/ml, P = 0.196, respectively). In vitro, renin as well as ANGII expression increased significantly in both HUVEC and MC after the cells were incubated with CsA for 24 hours (P < 0.05). CsA also stimulated the secretion of ANGII in HUVEC and MC in a dose-dependent manner. CONCLUSIONS: Renal allograft biopsy is important to differentiate chronic CsA-related nephropathy from chronic rejection. The intrarenal renin angiotensin system plays an important role in CsA-related chronic nephropathy. The histological lesions of CsA nephrotoxicity fail to correspond spontaneously to either the change of C2 level or the change of renin and ANGII plasma level. CsA stimulates the secretion of ANGII and the expression of renin and ANGII in HUVEC and MC. Blockage of RAS may be helpful for therapeutic intervention in the progression of CsA-related chronic nephropathy.


Assuntos
Ciclosporina/efeitos adversos , Imunossupressores/efeitos adversos , Rim/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Adulto , Idoso , Angiotensina II/análise , Angiotensina II/sangue , Células Endoteliais/química , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Rim/patologia , Masculino , Pessoa de Meia-Idade , Renina/análise , Renina/sangue
19.
Phytopathology ; 96(10): 1052-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18943492

RESUMO

ABSTRACT HpaG(Xooc), produced by Xanthomonas oryzae pv. oryzicola, is a member of harpin group of proteins that stimulate plant growth, hypersensitive cell death (HCD), and pathogen defense. The protein contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Genetic modification generated the pro-tein mutants HpaG(Xooc)MG (MG) by deleting GRMs and HpaG(Xooc)C47T (C47T) by replacing cysteine with threonine. When applied to tobacco plants, C47T and MG were 1.2- and 1.7-fold stronger, respectively, than HpaG(Xooc) in inducing HCD, which occurred consistently with expression of the marker genes hin1 and hsr203. The proteins markedly alleviated infection of tobacco by Tobacco mosaic virus and Arabidopsis and tomato by Pseudomonas syringae. Treating tobacco plants with HpaG(Xooc), C47T, and MG decreased the viral infection by 58, 81, and 92%, respectively. In Arabidopsis and tomato plants treated with HpaG(Xooc), C47T, or MG, P. syringae multiplication was inhibited; bacterial population multiplied in 5 days in these plants were ca. 160-, 1,260-, or 15,860-fold smaller than that in control plants. So pathogen defense was induced in both plants. Defense-related genes Chia5, NPR1, and PR-1a were expressed consistently with resistance. In response to HpaG(Xooc), C47T, and MG, aerial parts and roots of tomato plants increased growth by 15 and 53%, 25 and 77%, and 46 and 106%, relative to controls. The expansin gene, EXP2, involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaG(Xooc) represses the effects of the protein in plants.

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