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1.
Ann Oncol ; 35(1): 98-106, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37871701

RESUMO

BACKGROUND: Treatment options are limited for patients with high-risk non-muscle-invasive bladder cancer (NMIBC) with disease recurrence after bacillus Calmette-Guérin (BCG) treatment and who are ineligible for/refuse radical cystectomy. FGFR alterations are commonly detected in NMIBC. We evaluated the activity of oral erdafitinib, a selective pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor, versus intravesical chemotherapy in patients with high-risk NMIBC and select FGFR3/2 alterations following recurrence after BCG treatment. PATIENTS AND METHODS: Patients aged ≥18 years with recurrent, BCG-treated, papillary-only high-risk NMIBC (high-grade Ta/T1) and select FGFR alterations refusing or ineligible for radical cystectomy were randomized to 6 mg daily oral erdafitinib or investigator's choice of intravesical chemotherapy (mitomycin C or gemcitabine). The primary endpoint was recurrence-free survival (RFS). The key secondary endpoint was safety. RESULTS: Study enrollment was discontinued due to slow accrual. Seventy-three patients were randomized 2 : 1 to erdafitinib (n = 49) and chemotherapy (n = 24). Median follow-up for RFS was 13.4 months for both groups. Median RFS was not reached for erdafitinib [95% confidence interval (CI) 16.9 months-not estimable] and was 11.6 months (95% CI 6.4-20.1 months) for chemotherapy, with an estimated hazard ratio of 0.28 (95% CI 0.1-0.6; nominal P value = 0.0008). In this population, safety results were generally consistent with known profiles for erdafitinib and chemotherapy. CONCLUSIONS: Erdafitinib prolonged RFS compared with intravesical chemotherapy in patients with papillary-only, high-risk NMIBC harboring FGFR alterations who had disease recurrence after BCG therapy and refused or were ineligible for radical cystectomy.


Assuntos
Neoplasias não Músculo Invasivas da Bexiga , Pirazóis , Quinoxalinas , Neoplasias da Bexiga Urinária , Humanos , Adolescente , Adulto , Vacina BCG/efeitos adversos , Adjuvantes Imunológicos/uso terapêutico , Recidiva Local de Neoplasia/tratamento farmacológico , Invasividade Neoplásica
2.
Hernia ; 26(4): 1053-1062, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-34591214

RESUMO

BACKGROUND: Enhanced-view total extra-peritoneal (eTEP) inguinal hernia repair is a technically demanding procedure with a steep learning curve. AIM: Examine the feasibility and effectiveness of an instructor approach to teaching residents how to perform laparoscopic eTEP independently following a dedicated course of individual teaching. METHODS: Prospective analysis of eTEP procedures performed by residents between March 2018 and September 2020. Six residents dispersed into three groups-Group A: two junior residents, Group B: two mid-level residents and Group C: two senior residents. All residents performed a unilateral IHR comprised of five core steps. Data reviewed for each procedure included the time of each step, total time and autonomy degree as assessment for every step: 1st degree-dependent (physical assistance), 2nd degree-partially dependent (vocal assistance) and 3rd degree-independent. Early and late procedures were divided at 50% of cases. RESULTS: Participants performed 44 procedures (220 steps). Late procedures presented with a significant improvement in all degrees of autonomy (1st degree p = 0.002, 2nd degree p = 0.007 and 3rd degree p < 0.0001) and in every step (Step 1 p = 0.015, Step 2 p = 0.006, Step 3 p < 0.0001, Step 4 p < 0.0001, Step 5 p = 0.002). There was no significant difference in surgery duration between early and late procedures (p = 0.32). At early procedures, junior residents needed significantly higher rates of physical intervention (1st degree) compared to the senior residents (p = 0.004). Conversely, there was no significant difference in 2nd degree of autonomy (p = 0.46), 3rd degree (p = 0.06) and surgery duration (p = 0.16). The last three procedures performed by all participants had no significant difference between the seniority groups in autonomy (1st degree p = 0.1, 2nd degree p = 0.18 and 3rd degree p = 0.1). CONCLUSION: Dedicated course with an individual instructor's approach is effective in achieving competence, autonomy and confidence in performing eTEP in a short time.


Assuntos
Cirurgia Geral , Hérnia Inguinal , Internato e Residência , Laparoscopia , Cirurgia Geral/educação , Hérnia Inguinal/cirurgia , Herniorrafia/métodos , Humanos , Laparoscopia/métodos , Curva de Aprendizado , Preceptoria
3.
Neuroimage ; 228: 117692, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33385546

RESUMO

Diffusion MRI (dMRI) represents one of the few methods for mapping brain fiber orientations non-invasively. Unfortunately, dMRI fiber mapping is an indirect method that relies on inference from measured diffusion patterns. Comparing dMRI results with other modalities is a way to improve the interpretation of dMRI data and help advance dMRI technologies. Here, we present methods for comparing dMRI fiber orientation estimates with optical imaging of fluorescently labeled neurofilaments and vasculature in 3D human and primate brain tissue cuboids cleared using CLARITY. The recent advancements in tissue clearing provide a new opportunity to histologically map fibers projecting in 3D, which represents a captivating complement to dMRI measurements. In this work, we demonstrate the capability to directly compare dMRI and CLARITY in the same human brain tissue and assess multiple approaches for extracting fiber orientation estimates from CLARITY data. We estimate the three-dimensional neuronal fiber and vasculature orientations from neurofilament and vasculature stained CLARITY images by calculating the tertiary eigenvector of structure tensors. We then extend CLARITY orientation estimates to an orientation distribution function (ODF) formalism by summing multiple sub-voxel structure tensor orientation estimates. In a sample containing part of the human thalamus, there is a mean angular difference of 19o±15o between the primary eigenvectors of the dMRI tensors and the tertiary eigenvectors from the CLARITY neurofilament stain. We also demonstrate evidence that vascular compartments do not affect the dMRI orientation estimates by showing an apparent lack of correspondence (mean angular difference = 49o±23o) between the orientation of the dMRI tensors and the structure tensors in the vasculature stained CLARITY images. In a macaque brain dataset, we examine how the CLARITY feature extraction depends on the chosen feature extraction parameters. By varying the volume of tissue over which the structure tensor estimates are derived, we show that orientation estimates are noisier with more spurious ODF peaks for sub-voxels below 30 µm3 and that, for our data, the optimal gray matter sub-voxel size is between 62.5 µm3 and 125 µm3. The example experiments presented here represent an important advancement towards robust multi-modal MRI-CLARITY comparisons.


Assuntos
Encéfalo/anatomia & histologia , Substância Cinzenta/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Imagem Multimodal/métodos , Neuroimagem/métodos , Substância Branca/anatomia & histologia , Animais , Imagem de Difusão por Ressonância Magnética/métodos , Humanos , Imageamento Tridimensional/métodos , Macaca , Imagem Óptica/métodos
4.
AJNR Am J Neuroradiol ; 40(2): 206-212, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30655254

RESUMO

Magnetic particle imaging is an emerging tomographic technique with the potential for simultaneous high-resolution, high-sensitivity, and real-time imaging. Magnetic particle imaging is based on the unique behavior of superparamagnetic iron oxide nanoparticles modeled by the Langevin theory, with the ability to track and quantify nanoparticle concentrations without tissue background noise. It is a promising new imaging technique for multiple applications, including vascular and perfusion imaging, oncology imaging, cell tracking, inflammation imaging, and trauma imaging. In particular, many neuroimaging applications may be enabled and enhanced with magnetic particle imaging. In this review, we will provide an overview of magnetic particle imaging principles and implementation, current applications, promising neuroimaging applications, and practical considerations.


Assuntos
Fenômenos Magnéticos , Neuroimagem/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Nanopartículas
5.
AJNR Am J Neuroradiol ; 38(11): 2119-2125, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28882863

RESUMO

BACKGROUND AND PURPOSE: Anterior communicating artery aneurysm rupture and treatment is associated with high rates of dependency, which are more severe after clipping compared with coiling. To determine whether ischemic injury might account for these differences, we characterized cerebral infarction burden, infarction patterns, and patient outcomes after surgical or endovascular treatment of ruptured anterior communicating artery aneurysms. MATERIALS AND METHODS: We performed a retrospective cohort study of consecutive patients with ruptured anterior communicating artery aneurysms. Patient data and neuroimaging studies were reviewed. A propensity score for outcome measures was calculated to account for the nonrandom assignment to treatment. Primary outcome was the frequency of frontal lobe and striatum ischemic injury. Secondary outcomes were patient mortality and clinical outcome at discharge and at 3 months. RESULTS: Coiled patients were older (median, 55 versus 50 years; P = .03), presented with a worse clinical status (60% with Hunt and Hess Score >2 versus 34% in clipped patients; P = .02), had a higher modified Fisher grade (P = .01), and were more likely to present with intraventricular hemorrhage (78% versus 56%; P = .03). Ischemic frontal lobe infarction (OR, 2.9; 95% CI, 1.1-8.4; P = .03) and recurrent artery of Heubner infarction (OR, 20.9; 95% CI, 3.5-403.7; P < .001) were more common in clipped patients. Clipped patients were more likely to be functionally dependent at discharge (OR, 3.2; P = .05) compared with coiled patients. Mortality and clinical outcome at 3 months were similar between coiled and clipped patients. CONCLUSIONS: Frontal lobe and recurrent artery of Heubner infarctions are more common after surgical clipping of ruptured anterior communicating artery aneurysms, and are associated with poorer clinical outcomes at discharge.


Assuntos
Aneurisma Roto/cirurgia , Infarto Cerebral/etiologia , Embolização Terapêutica/efeitos adversos , Embolização Terapêutica/instrumentação , Aneurisma Intracraniano/cirurgia , Adulto , Idoso , Aneurisma Roto/complicações , Infarto Cerebral/epidemiologia , Estudos de Coortes , Procedimentos Endovasculares/efeitos adversos , Procedimentos Endovasculares/instrumentação , Feminino , Humanos , Aneurisma Intracraniano/complicações , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Instrumentos Cirúrgicos , Resultado do Tratamento
6.
Fungal Genet Biol ; 105: 16-27, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28579390

RESUMO

Development of novel strategies to control fungal plant pathogens requires understanding of their cellular organisation and biology. Live cell imaging of fluorescent organelle markers has provided valuable insight into various aspects of their cell biology, including invasion strategies in plant pathogenic fungi. Here, we introduce a set of 17 vectors that encode fluorescent markers to visualize the plasma membrane, endoplasmic reticulum (ER), chromosomes, the actin cytoskeleton, peroxisomes and autophagosomes in the wheat pathogen Zymoseptoria tritici. We fused either enhanced green-fluorescent protein (eGFP) or a codon-optimised version of GFP (ZtGFP) to homologues of a plasma membrane-located Sso1-like syntaxin, an ER signalling and retention peptide, a histone H1 homologue, the LifeAct actin-binding peptide, a mitochondrial acetyl-CoA dehydrogenase, a peroxisomal import signal and a homologue of the ubiquitin-like autophagosomal protein Atg8. We expressed these markers in wildtype strain IPO323 and confirmed the specificity of these markers by counterstaining or physiological experiments. This new set of molecular tools will help understanding the cell biology of the wheat pathogen Z. tritici.


Assuntos
Ascomicetos/metabolismo , Biomarcadores/metabolismo , Corantes Fluorescentes/metabolismo , Organelas/metabolismo , Actinas/metabolismo , Ascomicetos/genética , Ascomicetos/ultraestrutura , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Peroxissomos/metabolismo , Triticum/microbiologia
7.
World J Urol ; 35(3): 367-378, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27342991

RESUMO

PURPOSE: To review the management of metastatic upper tract urothelial carcinoma (UTUC) including recent advances in targeted and immune therapies as an update to the 2014 joint international consultation on UTUC, co-sponsored by the Société Internationale d'Urologie and International Consultation on Urological Diseases. METHODS: A PubMed database search was performed between January 2013 and May 2016 related to the treatment of metastatic UTUC, and 54 studies were selected for inclusion. RESULTS: The management of patients with metastatic UTUC is primarily an extrapolation from evidence guiding the management of metastatic urothelial carcinoma of the bladder. The first-line therapy for metastatic UTUC is platinum-based combination chemotherapy. Standard second-line therapies are limited and ineffective. Patients with UTUC who progress following platinum-based chemotherapy are encouraged to participate in clinical trials. Recent advances in genomic profiling present exciting opportunities to guide the use of targeted therapy. Immunotherapy with checkpoint inhibitors has demonstrated extremely promising results. Retrospective studies provide support for post-chemotherapy surgery in appropriately selected patients. CONCLUSIONS: The management of metastatic UTUC requires a multi-disciplinary approach. New insights from genomic profiling using targeted therapies, novel immunotherapies, and surgery represent promising avenues for further therapeutic exploration.


Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células de Transição/terapia , Neoplasias Renais/patologia , Neoplasias Ureterais/patologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Bevacizumab/administração & dosagem , Carboplatina/administração & dosagem , Carcinoma de Células de Transição/secundário , Cisplatino/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Docetaxel , Humanos , Imunoterapia , Indóis/administração & dosagem , Pelve Renal , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Paclitaxel/administração & dosagem , Compostos de Fenilureia/administração & dosagem , Pirróis/administração & dosagem , Sorafenibe , Sunitinibe , Taxoides/administração & dosagem , Gencitabina
8.
Sci Rep ; 6: 35854, 2016 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-27775025

RESUMO

Strategies to identify tumors at highest risk for treatment failure are currently under investigation for patients with bladder cancer. We demonstrate that flow cytometric detection of poorly differentiated basal tumor cells (BTCs), as defined by the co-expression of CD90, CD44 and CD49f, directly from patients with early stage tumors (T1-T2 and N0) and patient-derived xenograft (PDX) engraftment in locally advanced tumors (T3-T4 or N+) predict poor prognosis in patients with bladder cancer. Comparative transcriptomic analysis of bladder tumor cells isolated from PDXs indicates unique patterns of gene expression during bladder tumor cell differentiation. We found cell division cycle 25C (CDC25C) overexpression in poorly differentiated BTCs and determined that CDC25C expression predicts adverse survival independent of standard clinical and pathologic features in bladder cancer patients. Taken together, our findings support the utility of BTCs and bladder cancer PDX models in the discovery of novel molecular targets and predictive biomarkers for personalizing oncology care for patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Idoso , Animais , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos SCID , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/cirurgia , Fosfatases cdc25/genética
9.
Fungal Genet Biol ; 79: 76-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092792

RESUMO

Many pathogenic fungi are genetically tractable. Analysis of their cellular organization and invasion mechanisms underpinning virulence determinants profits from exploiting such molecular tools as fluorescent fusion proteins or conditional mutant protein alleles. Generation of these tools requires efficient cloning methods, as vector construction is often a rate-limiting step. Here, we introduce an efficient yeast recombination-based cloning (YRBC) method to construct vectors for the fungus Zymoseptoria tritici. This method is of low cost and avoids dependency on the availability of restriction enzyme sites in the DNA sequence, as needed in more conventional restriction/ligation-based cloning procedures. Furthermore, YRBC avoids modification of the DNA of interest, indeed this potential risk limits the use of site-specific recombination systems, such as Gateway cloning. Instead, in YRBC, multiple DNA fragments, with 30bp overlap sequences, are transformed into Saccharomyces cerevisiae, whereupon homologous recombination generates the vector in a single step. Here, we provide a detailed experimental protocol and four vectors, each encoding a different dominant selectable marker cassette, that enable YRBC of constructs to be used in the wheat pathogen Z. tritici.


Assuntos
Ascomicetos/genética , Clonagem Molecular/métodos , Vetores Genéticos/isolamento & purificação , Genética Microbiana/métodos , Recombinação Homóloga , Biologia Molecular/métodos
10.
Fungal Genet Biol ; 79: 94-101, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092795

RESUMO

Pathogenic fungi are constantly emerging resistance to anti-fungal treatments. Therefore, identification of new fungicide targets is important. Good candidates are essential fungal proteins and their regulators. An efficient way to reveal the molecular environment of an essential protein is the search for interacting factors. Here, we establish three yeast two-hybrid libraries, covering yeast and hyphal stages of the wheat pathogen Zymoseptoria tritici. No detectable genomic DNA was present in any of the 3 libraries. Random amplification revealed that the libraries include cDNA fragments of up to 2000bp, suggesting that small-to-medium sized proteins are represented therein. Indeed, full-length cDNAs of five proteins were found in all libraries. The full-length cDNA of large chitin synthase gene mcs1 (5742bp with introns; 5568bp without introns) could not be amplified, but its 5' and 3' regions were represented, suggesting that even larger genes are covered in all libraries. Finally, we tested for the expected interaction of the autophagy proteins ZtAtg4 and ZtAtg8 in Z. tritici, and then used ZtAtg4 to screen one of the two-hybrid libraries. Indeed, we found ZtAtg8 as a positive interaction partner, confirming that interacting proteins can be identified. Thus, these molecular tools promise to be useful in identifying novel fungicide target proteins.


Assuntos
Ascomicetos/citologia , Ascomicetos/genética , Biblioteca Gênica , Genes Fúngicos , Testes Genéticos/métodos , Técnicas do Sistema de Duplo-Híbrido , Ascomicetos/crescimento & desenvolvimento , Hifas/genética , Hifas/crescimento & desenvolvimento , Mapeamento de Interação de Proteínas
11.
Fungal Genet Biol ; 79: 118-24, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092798

RESUMO

Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.


Assuntos
Ascomicetos/genética , Loci Gênicos , Genética Microbiana/métodos , Biologia Molecular/métodos , Mutagênese Insercional/métodos , Recombinação Genética , Expressão Gênica , Succinato Desidrogenase/genética
12.
Fungal Genet Biol ; 79: 125-31, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092799

RESUMO

Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.


Assuntos
Ascomicetos/fisiologia , Proteínas de Fluorescência Verde/análise , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Ascomicetos/genética , Ascomicetos/patogenicidade , Códon , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Virulência
13.
Fungal Genet Biol ; 79: 132-40, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092800

RESUMO

The use of fluorescent proteins (FPs) in plant pathogenic fungi provides valuable insight into their intracellular dynamics, cell organization and invasion mechanisms. Compared with green-fluorescent proteins, their red-fluorescent "cousins" show generally lower fluorescent signal intensity and increased photo-bleaching. However, the combined usage of red and green fluorescent proteins allows powerful insight in co-localization studies. Efficient signal detection requires a bright red-fluorescent protein (RFP), combined with a suitable corresponding filter set. We provide a set of four vectors, suitable for yeast recombination-based cloning that carries mRFP, TagRFP, mCherry and tdTomato. These vectors confer carboxin resistance after targeted single-copy integration into the sdi1 locus of Zymoseptoria tritici. Expression of the RFPs does not affect virulence of this wheat pathogen. We tested all four RFPs in combination with four epi-fluorescence filter sets and in confocal laser scanning microscopy, both in and ex planta. Our data reveal that mCherry is the RFP of choice for investigation in Z. tritici, showing highest signal intensity in epi-fluorescence, when used with a Cy3 filter set, and laser scanning confocal microscopy. However, mCherry bleached significantly faster than mRFP, which favors this red tag in long-term observation experiments. Finally, we used dual-color imaging of eGFP and mCherry expressing wild-type strains in planta and show that pycnidia are formed by single strains. This demonstrates the strength of this method in tracking the course of Z. tritici infection in wheat.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Genes Reporter , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Doenças das Plantas/microbiologia , Coloração e Rotulagem/métodos , Triticum/microbiologia , Ascomicetos/patogenicidade , Expressão Gênica , Vetores Genéticos , Microscopia de Fluorescência/métodos , Recombinação Genética , Transformação Genética , Virulência , Proteína Vermelha Fluorescente
14.
Fungal Genet Biol ; 79: 150-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092801

RESUMO

Hyphal growth in filamentous fungi is supported by the uptake (endocytosis) and recycling of membranes and associated proteins at the growing tip. An increasing body of published evidence in various fungi demonstrates that this process is of essential importance for fungal growth and pathogenicity. Here, we introduce fluorescent markers to visualize the endocytic pathway in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein (eGFP) to the actin-binding protein fimbrin (ZtFim1), which is located in actin patches that are formed at the plasma membrane and are participating in endocytic uptake at the cell surface. In addition, we tagged early endosomes by eGFP-labelling a Rab5-homologue (ZtRab5) and late endosomes and vacuoles by expressing eGFP-Rab7 homologue (ZtRab7). Using fluorescent dyes and live cell imaging we confirmed the dynamic behavior and localization of these markers. This set of molecular tools enables an in-depth phenotypic analysis of Z. tritici mutant strains thereby supporting new strategies towards the goal of controlling wheat against Z. tritici.


Assuntos
Ascomicetos/fisiologia , Endocitose , Genes Reporter , Proteínas de Fluorescência Verde/análise , Glicoproteínas de Membrana/análise , Proteínas dos Microfilamentos/análise , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Ascomicetos/genética , Proteínas de Fluorescência Verde/genética , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética
15.
Fungal Genet Biol ; 79: 166-73, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092803

RESUMO

Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen Zymoseptoria tritici. We fused the gene for enhanced green-fluorescent protein (egfp) to the promoter region of Z. tritici nitrate reductase (Pnar1; induced by nitrogen and repressed by ammonium), 1,4-ß-endoxylanase A (Pex1A; induced by xylose and repressed by maltodextrin), l-arabinofuranosidase B (PlaraB; induced by arabinose and repressed by glucose), galactose-1-phosphate uridylyltransferase 7 (Pgal7; induced by galactose and repressed by glucose) and isocitrate lyase (Picl1; induced by sodium acetate and repressed by glucose). This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that Pnar1, PlaraB and Pex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, Pgal7 and Picl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter Ptub2 revealed that PlaraB was the weakest promoter (∼20% of Ptub2), whereas Picl1 strongly expressed the reporter (∼250% of Ptub2). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici.


Assuntos
Ascomicetos/genética , Genes Essenciais , Genes Fúngicos , Regiões Promotoras Genéticas , Fusão Gênica Artificial , Ascomicetos/fisiologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Reporter , Genética Microbiana/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Biologia Molecular/métodos , Doenças das Plantas/microbiologia , Triticum/microbiologia
16.
Fungal Genet Biol ; 79: 158-65, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26092802

RESUMO

Fungal hyphae are highly polarized cells that invade their substrate by tip growth. In plant pathogenic fungi, hyphal growth is essential for host invasion. This makes polarity factors and secretion regulators potential new targets for novel fungicides. Polarization requires delivery of secretory vesicles to the apical Spitzenkörper, followed by polarized exocytosis at the expanding cell tip. Here, we introduce fluorescent markers to visualize the apical Spitzenkörper and the apical site of exocytosis in hyphae of the wheat pathogen Zymoseptoria tritici. We fused green fluorescent protein to the small GTPase ZtSec4, the myosin light chain ZtMlc1 and the small GTPase ZtRab11 and co-localize the fusion proteins with the dye FM4-64 in the hyphal apex, suggesting that the markers label the hyphal Spitzenkörper in Z. tritici. In addition, we localize GFP-fusions to the exocyst protein ZtExo70, the polarisome protein ZtSpa2. Consistent with results in the ascomycete Neurospora crassa, these markers did localize near the plasma membrane at the hyphal tip and only partially co-localize with FM4-64. Thus, these fluorescent markers are useful molecular tools that allow phenotypic analysis of mutants in Z. tritici. These tools will help develop new avenues of research in our quest to control STB infection in wheat.


Assuntos
Ascomicetos/fisiologia , Exocitose , Proteínas Fúngicas/análise , Genes Reporter , Proteínas de Fluorescência Verde/análise , Organelas/química , Coloração e Rotulagem/métodos , Ascomicetos/química , Ascomicetos/genética , Proteínas Fúngicas/genética , Proteínas de Fluorescência Verde/genética , Hifas/química , Hifas/genética , Hifas/fisiologia , Imagem Óptica/métodos , Doenças das Plantas/microbiologia , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Triticum/microbiologia
17.
Fungal Genet Biol ; 79: 141-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25857261

RESUMO

The microtubule cytoskeleton supports vital processes in fungal cells, including hyphal growth and mitosis. Consequently, it is a target for fungicides, such as benomyl. The use of fluorescent fusion proteins to illuminate microtubules and microtubule-associated proteins has led to a break-through in our understanding of their dynamics and function in fungal cells. Here, we introduce fluorescent markers to visualize microtubules and accessory proteins in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein to α-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located γ-tubulin ring complex, involved in the nucleation of microtubules. In vivo observation confirms the localization and dynamic behaviour of all three markers. These marker proteins are useful tools for understanding the organization and importance of the microtubule cytoskeleton in Z. tritici.


Assuntos
Ascomicetos/genética , Proteínas do Citoesqueleto/análise , Genes Reporter , Proteínas Luminescentes/análise , Microtúbulos/química , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Proteínas do Citoesqueleto/genética , Proteínas Luminescentes/genética , Microtúbulos/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética
18.
Clin Neurophysiol ; 126(3): 481-5, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25065300

RESUMO

OBJECTIVE: Electroencephalography is useful for evaluating transient neurological events in the setting of moyamoya disease. METHODS: EEG findings of adults with moyamoya seen at a large moyamoya referral center are summarized. Patients were identified by retrospective chart review. RESULTS: EEGs were ordered after cerebral revascularization for altered mental status, aphasia, limb shaking, or facial twitching. Among the study population of 103 patients having EEGs, 24% of adults with moyamoya had a history of clinical seizures. Ischemic or hemorrhagic strokes were associated with a twofold relative risk of seizures. Overall, 90% of EEGs were abnormal, most commonly focally (78%), or diffusely slow (68%). Epileptiform EEG discharges were seen in 24%. Whereas hemispheres with an ischemic stroke had a 19% risk of epileptiform discharges and an 8% risk of seizures on EEG, hemispheres with hemorrhagic stroke had a 35% risk of epileptiform discharges and 19% risk of seizures on EEG. Focal amplitude attenuation was seen in 19%, breach rhythm in 15%, rhythmic delta in 14%, and electrographic seizures in 12%. CONCLUSIONS: Seizures and epileptiform EEG changes are common in patients with moyamoya disease. SIGNIFICANCE: Transient events in patients with moyamoya can result from seizures as well as ischemia.


Assuntos
Afasia/fisiopatologia , Isquemia Encefálica/fisiopatologia , Encéfalo/fisiopatologia , Doença de Moyamoya/fisiopatologia , Convulsões/fisiopatologia , Acidente Vascular Cerebral/fisiopatologia , Adulto , Afasia/complicações , Isquemia Encefálica/complicações , Eletroencefalografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Moyamoya/complicações , Estudos Retrospectivos , Convulsões/complicações , Acidente Vascular Cerebral/complicações
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