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1.
J Tissue Eng ; 11: 2041731420921482, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32742631

RESUMO

Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.

2.
J Appl Oral Sci ; 28: e20200131, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32725049

RESUMO

Objective There is increasingly common the consumption more times a day of foods and acidic drinks in the diet of the population. The present study aimed to evaluate and compare the effects of a calcium mesoporous silica nanoparticle single application of other calcium and/or fluoride products in reducing the progression of dental erosion. Methodology Half of the eroded area was covered of 60 blocks of enamel, after which the block was submitted to the following treatments: (Ca2+-MSN), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP); CPP-ACP/F-(900 ppm F-); titanium tetrafluoride (TiF4 1%) (positive control); sodium fluoride (NaF 1.36%) (positive control); and Milli-Q® water (negative control) before being submitted to a second erosive challenge. A surface analysis was performed via a three-dimensional (3D) noncontact optical profilometry to assess the volumetric roughness (Sa) and tooth structure loss (TSL) and and through scanning electron microscopy (MEV). An analysis of variance (ANOVA) and Tukey's test were performed. Results Regarding Sa, all experimental groups exhibited less roughness than the control (p<0.05). The TSL analysis revealed that the Ca2+-MSN and NaF groups were similar (p>0.05) and more effective in minimizing tooth loss compared with the other groups (p<0.05). Conclusions The Ca2+-MSN and NaF treatments were superior compared with the others and the negative control.


Assuntos
Nanopartículas , Erosão Dentária , Remineralização Dentária , Cálcio , Caseínas , Fluoretos , Humanos , Dióxido de Silício , Fluoreto de Sódio
3.
Arch Oral Biol ; 110: 104619, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31805483

RESUMO

OBJECTIVE: The aim of this study was to evaluate the morphological and chemical effect of in-office and at-home desensitising agents containing sodium fluoride (NaF) on eroded root dentine in vitro. METHODS: Fifty bovine dentine samples were pre-eroded and randomised into five groups (n = 10): G1 (Control) - milli-Q water; G2 - fluoride varnish containing NaF 22,500 ppm; G3 - desensitising cream containing NaF 9,000 ppm associated with 20% nanohydroxyapatite; G4 - toothpaste with NaF 5,000 ppm associated to tricalcium phosphate; G5 - toothpaste containing NaF 900 ppm and casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF). The specimens were submitted to erosive challenge for three days. The analyses were performed using non-contact profilometry for volumetric (Sa) and linear roughness (Ra) followed by scanning electron microscopy (SEM) and Energy Dispersive X-ray Spectrometry (EDS). The data were analysed by Kruskal-Wallis and Wilcoxon tests (α = 0.05). RESULTS: There was a significant reduction of Ra and Sa for the eroded samples from the G2 and G5 (p < 0.05) after an erosive challenge. The dentine surface topography pattern showed partially or totally occluded dentinal tubules after treatments, except in the control group. The control, G4 and G3 groups showed a reduction in the dentine inorganic content percentage of Ca (Calcium) and P (Phosphorus) minerals. CONCLUSION: The fluoride varnish and CPP-ACPF toothpaste were able to prevent morphological changes and were the only materials that showed the Ca and P content increased after treatment. These materials may be promising alternatives in the clinical control of dentin erosion.


Assuntos
Dentina , Fluoreto de Sódio , Erosão Dentária , Raiz Dentária , Animais , Bovinos , Dentina/efeitos dos fármacos , Fluoretos , Fluoreto de Sódio/farmacologia , Erosão Dentária/tratamento farmacológico , Raiz Dentária/efeitos dos fármacos , Cremes Dentais
4.
Indian J Dent Res ; 31(6): 924-929, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33753666

RESUMO

Context: The side effects of bleaching products are still incompletely known. Aims: This work aims to evaluate the effects of bleaching regimens on colour variation, microstructure, roughness, composition and nanohardness of human dental enamel until 8 weeks. Settings and Design: : Twenty teeth were cross-sectioned to obtain eighty enamel fragments (50 × 50 mm) divided into four groups: CN (control Negative-artificial saliva), CP10 (10% carbamide peroxide), HP10 (10% hydrogen peroxide), and WS (whitening strips-10% hydrogen peroxide). Methods and Material: Roughness (atomic force microscopy-AFM and 3-D surface scanning), morphology (confocal laser scanning microscopy-CLSM and AFM), hardness and elastic modulus (nanoindentation), and composition (Raman microspectroscopy) were analysed before the therapy and after 4 and 8 weeks. Colour measures were performed weekly. Statistical Analysis Used: : Two-way ANOVA with repeated measures (P < 0.05). Results: Bleaching stabilizes after 3 weeks for HP10 and after 4 weeks for CP10 and WS. Roughness evaluation showed statistical difference for HP10 after 8 weeks for Sa and Sq, for HP10 and WS after 4 weeks and for CP10 after 8 weeks. The same occurred for hardness and elastic modulus. The morphological evaluation demonstrated the most significant effects after 8 weeks of treatment for HP10 and WS. Composition analysis revealed modifications in peaks related to the organic content spectra (protein) with an increase in detection after 4 weeks, followed by a decrease after 8 weeks. Conclusions: H2O2-based products caused morphological and compositional alterations on enamel.


Assuntos
Clareamento Dental , Peróxido de Carbamida , Esmalte Dentário , Dureza , Humanos , Peróxido de Hidrogênio , Peróxidos , Propriedades de Superfície , Clareamento Dental/efeitos adversos , Ureia
5.
J. appl. oral sci ; 28: e20200131, 2020. tab, graf
Artigo em Inglês | LILACS, BBO - Odontologia | ID: biblio-1134780

RESUMO

Abstract Objective There is increasingly common the consumption more times a day of foods and acidic drinks in the diet of the population. The present study aimed to evaluate and compare the effects of a calcium mesoporous silica nanoparticle single application of other calcium and/or fluoride products in reducing the progression of dental erosion. Methodology Half of the eroded area was covered of 60 blocks of enamel, after which the block was submitted to the following treatments: (Ca2+-MSN), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP); CPP-ACP/F-(900 ppm F−); titanium tetrafluoride (TiF4 1%) (positive control); sodium fluoride (NaF 1.36%) (positive control); and Milli-Q® water (negative control) before being submitted to a second erosive challenge. A surface analysis was performed via a three-dimensional (3D) noncontact optical profilometry to assess the volumetric roughness (Sa) and tooth structure loss (TSL) and and through scanning electron microscopy (MEV). An analysis of variance (ANOVA) and Tukey's test were performed. Results Regarding Sa, all experimental groups exhibited less roughness than the control (p<0.05). The TSL analysis revealed that the Ca2+-MSN and NaF groups were similar (p>0.05) and more effective in minimizing tooth loss compared with the other groups (p<0.05). Conclusions The Ca2+-MSN and NaF treatments were superior compared with the others and the negative control.


Assuntos
Humanos , Erosão Dentária , Remineralização Dentária , Nanopartículas , Fluoreto de Sódio , Caseínas , Cálcio , Dióxido de Silício , Fluoretos
6.
Indian J Dent Res ; 29(5): 651-656, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30409948

RESUMO

Context: Despite the relevance of the sandwich technique, there are still doubts about the best adhesive strategy and surface treatment for glass ionomer cements (GICs). Aim: The aim of this study is to evaluate the best surface treatment for GIC to ensure an effective and durable adhesion to resin, through micro-shear test, using an alternative method to build up test specimens. Subjects and Methods: Eighty GIC samples were divided into eight groups (n = 10) according to five surface treatments (none, etching, air drying, grinding, and grinding plus etching) and according to the adhesive system (conventional or self-etch). Five starch tubes were positioned on each sample, and a flowable composite was inserted generating 50 resin test bodies per group and a total of 400 tested areas. All specimens were submitted to the micro-shear test: half immediately and half after thermal cycling (10,000 cycles of 20 s each/5° and 55°C). All samples were analyzed to evaluate fracture. Representative samples were also analyzed by scanning electron microscopy and energy dispersive spectroscopy. Data were analyzed with two-way ANOVA and Tukey's honest significant difference post hoc test (P <.05). Results: The bond strengths in the thermal cycled specimens were lower and showed a statistically significant difference (P = 0). The "grinding" groups showed the highest bond strength. Conclusions: The alternative method to build up test specimens was effective and easy to execute. Grinding of the GIC surface, which is not normally performed before the use of the adhesive system, represented the best option of surface treatment.


Assuntos
Resinas Compostas , Colagem Dentária/métodos , Análise do Estresse Dentário/métodos , Cimentos de Ionômeros de Vidro , Teste de Materiais/métodos , Resistência ao Cisalhamento , Humanos , Cimentos de Resina , Propriedades de Superfície
7.
Microsc Res Tech ; 81(9): 1071-1076, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30295354

RESUMO

Human dental enamel is organized by prisms that are structured between 3 and 6 µm in diameter. Determining the relationships between different treatments on the surface of enamel using ultrastructural analysis is the purpose of many in vitro experiments. Different sample pretreatments have been reported in the literature. Grinding and polishing are common procedures for enamel preparation. They provide a flat and standardized surface, which is imperative for the use of some techniques such as ATR-FTIR. However, for morphological analysis, SEM and AFM represent easier methods to measure and reduce the biological sample variation. Therefore, the objective of this study was to establish how different forms of enamel preparation can influence the advent of artifacts during ultrastructural observation, especially by AFM analysis. Four groups (n = 10) were tested: (a) without preparation; (b) polishing with a diamond paste; (c) grinding with decreasing granulations of silicon carbide papers; (d) grinding with polishing. Images were obtained using the Peak-Force Tapping mode. After the first images were obtained, all fragments were acid etched with 37% phosphoric acid for 30 seconds, rinsed for 60 seconds, and dried intensively. Upon grinding and polishing, the exposure of the inner enamel surfaces provided a less mineralized layer that was marked by scratches and a higher susceptibility to treatments. Moreover, using native enamel provided more valuable information on the surface and the roughness changes for clinical applications. In addition, phosphoric acid is an option for observing the prismatic arrangement after grinding and/or polishing changes the morphology. RESEARCH HIGHLIGHTS: The use of native enamel samples to investigate the effects of different treatments on surface should be preferred in research, when the technique allows it.

8.
Biomed J ; 41(3): 184-193, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-30080658

RESUMO

BACKGROUND: Fusobacterium nucleatum is a Gram-negative anaerobic bacterium associated with periodontal disease. Some oral bacteria, like Porphyromonas gingivalis, evade the host immune response by inhibiting inflammation. On the other hand, F. nucleatum triggers inflammasome activation and release of danger-associated molecular patterns (DAMPs) in infected gingival epithelial cells. METHODS: In this study, we characterized the pro-inflammatory response to F. nucleatum oral infection in BALB/c mice. Western blots and ELISA were used to measure cytokine and DAMP (HMGB1) levels in the oral cavity after infection. Histology and flow cytometry were used to observe recruitment of immune cells to infected tissue and pathology. RESULTS: Our results show increased expression and production of pro-inflammatory cytokines during infection. Furthermore, we observe that F. nucleatum infection leads to recruitment of macrophages in different tissues of the oral cavity. Infection also contributes to osteoclast recruitment, which could be involved in the observed bone resorption. CONCLUSIONS: Overall, our findings suggest that F. nucleatum infection rapidly induces inflammation, release of DAMPs, and macrophage infiltration in gingival tissues and suggest that osteoclasts may drive bone resorption at early stages of the inflammatory process.


Assuntos
Reabsorção Óssea/etiologia , Polpa Dentária/imunologia , Infecções por Fusobacterium/imunologia , Fusobacterium nucleatum , Macrófagos/fisiologia , Doenças da Boca/imunologia , Animais , Movimento Celular , Citocinas/biossíntese , Citocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos/fisiologia
9.
J Phycol ; 53(6): 1294-1304, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28990189

RESUMO

Approximately half of the Padina (Dictyotales, Phaeophyceae) species mineralize aragonite needles over the adaxial thallus surface, where mineral bands are interspersed with nonmineralized regions along the thallus from the apical to basal end. However, this calcification pattern and the related algal properties are not well understood. Therefore, this work was performed to elucidate a potential role of cell walls in the inhibition/induction of mineralization in the brown alga Padina gymnospora. In a comparison of specific thallus regions, differences were identified in the cellulose distribution, microfibrils arrangement and thickness, distribution and abundance of phenolic substances, and physical differences among the surfaces of the thallus (deformation, adhesion, topography, and nano-rugosity). In vitro mineralization assays indicated that phenolic substances are strong modulators of calcium carbonate crystals growth. In addition, de novo mineralization assays over cell wall surfaces that were used as templates, even without cellular activity, indicated that the cell wall remains a key factor in the induction/inhibition of mineralization. Overall, the current findings indicate a strong correlation between the physico-chemical and structural properties of the cell wall and the alternation pattern of the mineralization bands over the thallus of P. gymnospora.


Assuntos
Calcificação Fisiológica , Carbonato de Cálcio/metabolismo , Feófitas/fisiologia , Brasil , Parede Celular/fisiologia , Parede Celular/ultraestrutura , Feófitas/ultraestrutura
10.
Sci Rep ; 7: 46768, 2017 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-28440301

RESUMO

Invasive fungal infections, including cryptococcosis, are a growing threat to immunocompromised patients. Although Cryptococcus neoformans and Cryptococcus gattii are the main agents of human cryptococcosis, opportunistic infections by environmental species, such as C. liquefaciens, have been observed recently. The main Cryptococcus virulence factor is the production and secretion of polysaccharides (PS). Previously, we showed that both species produce PS of similar composition. Here, we examined the ultrastructure and biological activity of capsular and secreted PS from C. liquefaciens, and yeast pathogenicity to an invertebrate host, in comparison with C. neoformans. Ultrastructural analysis by high-resolution microscopy showed that both species produce large and complex capsules. PS from both species had indistinguishable effects on phagocytosis levels, NO production and the secretion of a variety of immune mediators. Challenge with C. liquefaciens or C. neoformans led to complete lethality of G. mellonella larvae. Treatment with C. liquefaciens PS could not protect mice against infection with C. neoformans. We conclude that polysaccharides of the environmental yeast C. liquefaciens have strikingly similar ultrastructural and biological properties to those of C. neoformans, highlighting the importance of monitoring the emergence of new fungal pathogens for which thermotolerance may be an important transitional step towards pathogenesis in humans.


Assuntos
Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Polissacarídeos Fúngicos/efeitos adversos , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Mariposas/crescimento & desenvolvimento , Fagocitose , Animais , Criptococose/metabolismo , Cryptococcus neoformans/classificação , Cryptococcus neoformans/ultraestrutura , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Óxido Nítrico/metabolismo , Células THP-1
11.
J Prosthet Dent ; 118(5): 666-671, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28385437

RESUMO

STATEMENT OF PROBLEM: Universal adhesives combine silane and various monomers in a single bottle to make them more versatile. Their adhesive performance is unclear. PURPOSE: The purpose of this in vitro study was to assess the effects of an additional silane application before using a universal adhesive on the adhesion between a disilicate glass ceramic and a composite resin by using a microshear bond strength test (µSBS) and fracture analysis immediately and after thermocycling. MATERIAL AND METHODS: One hundred lithium disilicate glass ceramic disks were divided into 10 groups for bond strength testing according to the following 3 surface treatments: silane application (built-in universal adhesive or with additional application), adhesive (Adper Single Bond Plus [SB, 3M ESPE], Scotchbond Universal Adhesive [U, 3M ESPE], and mixed U with Dual Cure Activator [DCA, 3M ESPE]); or thermocycling (half of the specimens were thermocycled 10000 times). After surface treatment, 5 resin cylinders were bonded to each disk and submitted to a µSBS test. The failure mode was analyzed under a stereomicroscope and evaluated by scanning electron microscope and energy-dispersive x-ray spectroscopy. Data from the µSBS test were analyzed by 3-way ANOVA followed by the Tukey HSD post hoc test (α=.05). RESULTS: An additional silane application resulted in a higher µSBS result for all adhesive groups (P<.05). CONCLUSIONS: Ceramic surface treatment influenced the performance of adhesives, which may be improved with an additional silane application.


Assuntos
Cerâmica/uso terapêutico , Colagem Dentária/métodos , Cimentos Dentários/uso terapêutico , Porcelana Dentária/uso terapêutico , Silanos/uso terapêutico , Falha de Restauração Dentária , Análise do Estresse Dentário , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Espectrometria por Raios X
12.
J Struct Biol ; 193(1): 75-82, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26655746

RESUMO

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. Its main virulence factor is an extracellular polysaccharide capsule whose structure, assembly and dynamics remain poorly understood. In this study, we apply improved protocols for sample preparation and recently-developed scanning microscopy techniques to visualize the ultrastructure of the C. neoformans capsule at high-resolution (up to 1 nm) and improved structural preservation. Although most capsule structures in nature consist of linear polymers, we show here that the C. neoformans capsule is a 'microgel-like' structure composed of branched polysaccharides. Moreover, we imaged the capsule-to-cell wall link, which is formed by thin fibers that branch out of thicker capsule filaments, and have one end firmly embedded in the cell wall structure. Together, our findings provide compelling ultrastructural evidence for a branched and complex capsule conformation, which may have important implications for the biological activity of the capsule as a virulence factor.


Assuntos
Parede Celular/ultraestrutura , Cryptococcus neoformans/ultraestrutura , Polissacarídeos/metabolismo , Parede Celular/metabolismo , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Microscopia , Fatores de Virulência
13.
Mem Inst Oswaldo Cruz ; 109(2): 220-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24714966

RESUMO

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus.


Assuntos
Antifúngicos/metabolismo , Candida/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Fusarium/metabolismo , Nanopartículas Metálicas , Prata/metabolismo , Antifúngicos/uso terapêutico , Candida/classificação , Candida/ultraestrutura , Extratos Celulares , Cryptococcus/classificação , Cryptococcus/ultraestrutura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Inibidores do Crescimento , Nanopartículas Metálicas/uso terapêutico , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Prata/análise , Prata/uso terapêutico
14.
Mem. Inst. Oswaldo Cruz ; 109(2): 220-228, abr. 2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-705813

RESUMO

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .


Assuntos
Antifúngicos/metabolismo , Candida/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Fusarium/metabolismo , Nanopartículas Metálicas , Prata/metabolismo , Antifúngicos/uso terapêutico , Extratos Celulares , Candida/classificação , Candida/ultraestrutura , Cryptococcus/classificação , Cryptococcus/ultraestrutura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Inibidores do Crescimento , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Nanopartículas Metálicas/uso terapêutico , Prata/análise , Prata/uso terapêutico
15.
Micron ; 41(8): 939-44, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20719525

RESUMO

In many cells, motility is mediated by flagellar beating. Protist parasites are capable of highly coordinated motility which contributes to their pathogenicity in mammalian hosts. Understanding the structural aspects of the flagellum may be important to the identification of novel targets for therapeutic intervention. Our group used atomic force microscopy (AFM) to examine the ultrastructure of Trypanosoma cruzi, obtaining valuable information on the organisation of the flagellar sub-structure. AFM images revealed novel flagellar components such as the presence of periodically-spaced protrusions organised along a flagellar furrow and oriented through the major flagellar axis between the axoneme and the paraflagellar rod. The nature and functional role of this structure are still unknown, although the hypothesis that the furrow might physically separate the two distinct domains of the flagellar membrane that comprise the surface of the axoneme and the paraflagellar rod (PFR) has been raised. To test whether the furrow was present or not only in PFR-bearing flagella, different protists containing or lacking the PFR, were analysed by AFM. Analysis of T. cruzi, Trypanosoma brucei and Herpetomonas megaseliae, which present distinct PFRs, showed similar and equivalent furrows along the main axis of their flagella, whereas Crithidia deanei, Giardia lamblia and Tritrichomonas foetus (in which the PFR is reduced or absent) lacked a furrow. Our results strongly suggest that the flagellar furrow is a characteristic feature of PFR-containing flagella and opens new perspectives for its functional role in the definition of sub-domains on the flagellar membrane.


Assuntos
Flagelos/ultraestrutura , Trypanosoma cruzi/ultraestrutura , Membrana Celular/ultraestrutura , Microscopia de Força Atômica
16.
Parasitol Int ; 59(4): 629-33, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20670692

RESUMO

The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.


Assuntos
Endocitose/fisiologia , Flagelos/metabolismo , Organelas/ultraestrutura , Transferrina/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/ultraestrutura , Animais , Flagelos/química , Flagelos/ultraestrutura , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Ouro/metabolismo , Cinética , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética
17.
PLoS One ; 5(6): e11407, 2010 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-20613980

RESUMO

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea. CONCLUSIONS/SIGNIFICANCE: Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating.


Assuntos
Flagelos/ultraestrutura , Trypanosoma cruzi/fisiologia , Animais , Técnica de Fratura por Congelamento , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão
18.
J Biol Chem ; 283(17): 11714-20, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18276595

RESUMO

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.


Assuntos
Membrana Basal/metabolismo , Laminina/química , Polímeros/química , Animais , Bioquímica/métodos , Concentração de Íons de Hidrogênio , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica , Modelos Biológicos , Conformação Molecular , Eletricidade Estática , Fatores de Tempo
19.
Parasitol Res ; 102(5): 1059-67, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18224488

RESUMO

In a previous work, we have investigated the effects of piperine and several of its chemical derivatives on the proliferation of the protozoan parasite Trypanosoma cruzi. It was observed that natural piperine is more active against intracellular amastigotes than axenically grown epimastigotes with IC50 values of 4.91 and 7.36 microM, respectively. Despite its superior trypanocidal activity against the intracellular amastigotes, here, we show that piperine did not enhance microbiocidal characteristics of murine peritoneal macrophages (Mø) based on nitric oxide production. As shown by light and electron microscopy analysis, epimastigotes treated with sublethal concentrations of piperine presented a reversible cell cycle arrestment and become round shaped, with swelling of the mitochondrion matrix and intense intracellular vacuolization with structures displaying complex membrane invaginations. Similar to the effects of exposing epimastigotes to the antitumor and microtubule stabilizer taxol, multiplication of cell organelles such as the flagellum, kinetoplast, and nucleus occurred, but division into daughter cells was impaired. Unlike the effects caused by the anti-microtubular vinca alkaloids vincristine and vinblastine, which also induce cytokinesis arrestment in T. cruzi epimastigotes, piperine did not induce the formation of giant multinucleated cells. The data reinforce the selectivity of the mechanisms of action of piperine against T. cruzi.


Assuntos
Alcaloides/farmacologia , Benzodioxóis/farmacologia , Citocinese/efeitos dos fármacos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/ultraestrutura , Animais , Células Cultivadas , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Óxido Nítrico/biossíntese , Testes de Sensibilidade Parasitária , Trypanosoma cruzi/crescimento & desenvolvimento
20.
Microsc Res Tech ; 71(2): 133-9, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17992694

RESUMO

Most advances in atomic force microscopy (AFM) have been accomplished in recent years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites.


Assuntos
Microscopia de Força Atômica , Trypanosoma cruzi/ultraestrutura , Animais , Axonema/ultraestrutura , Flagelos/ultraestrutura , Microscopia Eletrônica de Varredura
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