Danos cromossômicos e alterações nucleares em células esfoliadas do epitélio gengival de indivíduos com periodontite crônica moderada / Chromosomal damage and nuclear alterations in exfoliated cells of the gingival epithelium of individuals with chronic moderate periodontitis

Introdução: a periodontite é uma doença multifatorial que resulta primariamente da ação de bactérias nos tecidos que compõem o periodonto. O processo inflamatório que se desenvolve em consequência está associado ao aumento da proliferação celular que propicia a ocorrência de danos no material genético. Objetivo: avaliar, com uso do teste de micronúcleo, a ocorrência de danos genéticos e alterações nucleares em indivíduos com periodontite crônica moderada. Metodologia: a amostra incluiu dez indivíduos portadores de periodontite crônica moderada em início do tratamento no Centro de Especialidades Odontológicas (CEO) de Senhor do Bonfim/BA. Os participantes responderam a um questionário e células esfoliadas da mucosa gengival de regiões sem alterações e com periodontite crônica moderada foram coletadas. Este material foi transferido para lâmina de vidro, fixado com metanol/ácido acético (3:1), corado com reativo de Shiff e fast green e analisado sob microscopia óptica. A análise estatística foi feita com o teste condicional para comparação de proporções em situações de eventos raros. Resultados: a ocorrência de micronúcleos, alterações nucleares degenerativas (cariorréxis, cariólise, cromatina condensada, picnose) e vacúolos nucleares foi significativamente maior em células da lesão. A ocorrência de broken eggs não diferiu estatísticamente. Conclusões: a periodontite crônica moderada é uma doença na qual há comprometimento da integridade genética das células do epitélio gengival. A maior ocorrência de alterações indicativas de apoptose na área da periodontite crônica aponta para a resposta biológica do organismo ao insulto genotóxico ao qual as células nesta lesão estão submetidas.

Introduction: periodontitis is a multifactorial disease that results primarily from the action of bacteria in the tissues that compose the periodontium. The inflammatory process that develops as a consequence is associated with increased cell proliferation which facilitates the occurrence of genetic damage. Objective: to evaluate, using the micronucleus test, the occurrence of genetic damage and nuclear alterations in individuals with chronic moderate periodontitis. Methodology: the sample included ten individuals with chronic moderate periodontitis beginning the treatment in the Centro de Especialidades Odontológicas (CEO) located in Senhor do Bonfim/BA. Participants answered a questionnaire and exfoliated cells of the gingival mucosal regions without alterations and with chronic moderate periodontitis were collected. This material was transferred to slide, fixed with metanol/acetic acid (3:1), stained with Schiff reactive and fast green and analyzed using optical microscope. The statistical analysis was done using the conditional test to compare proportions in a rare events situation. Results: the occurrence of micronuclei, degenerative nuclear alterations (karyorrhexis, karyolysis, condensed chromatin, pyknosis) and nuclear vacuoles was significantly higher in cells of the lesion. The occurrence of broken eggs did not differ statistically. Conclusions: the chronic moderate periodontitis is a disease that compromises the genetic integrity of the gingival epithelium cells. The higher occurrence of nuclear apoptotic cells in the area of chronic periodontitis is indicative of the biological response to genotoxic insult.

Trametes villosa Lignin Peroxidase (TvLiP): Genetic and Molecular Characterization.

White-rot basidiomycetes are the organisms that decompose lignin most efficiently, and Trametes villosa is a promising species for ligninolytic enzyme production. There are several publications on T. villosa applications for lignin degradation regarding the expression and secretion of laccase and manganese peroxidase (MnP) but no reports on the identification and characterization of lignin peroxidase (LiP), a relevant enzyme for the efficient breakdown of lignin. The object of this study was to identify and partially characterize, for the first time, gDNA, mRNA, and the corresponding lignin peroxidase (TvLiP) protein from T. villosa strain CCMB561 from the Brazilian semiarid region. The presence of ligninolytic enzymes produced by this strain grown in inducer media was qualitatively and quantitatively analyzed by spectrophotometry, qPCR, and dye fading using Remazol Brilliant Blue R. The spectrophotometric analysis showed that LiP activity was higher than that of MnP. The greatest LiP expression as measured by qPCR occurred on the 7th day, and the ABSA medium (agar, sugarcane bagasse, and ammonium sulfate) was the best that favored LiP expression. The amplification of the TvLiP gene median region covering approximately 50% of the T. versicolor LPGIV gene (87% identity); the presence of Trp199, Leu115, Asp193, Trp199, and Ala203 in the translated amplicon of the T. villosa mRNA; and the close phylogenetic relationship between TvLiP and T. versicolor LiP all indicate that the target enzyme is a lignin peroxidase. Therefore, T. villosa CCMB561 has great potential for use as a LiP, MnP, and Lac producer for industrial applications.

Mycelial development preceding basidioma formation in Moniliophthora perniciosa is associated to chitin, sugar and nutrient metabolism alterations involving autophagy.

We identified and characterized two chitinases, named MpCHIT1 and MpCHIT2, from the fungus Moniliophthora perniciosa - the etiologic agent of witches' broom disease in cacao tree (Theobroma cacao L.) - during its development, mainly in the mycelia phases preceding the basidioma formation. The expression of MpCHIT1 and MpCHIT2, together with MpCHS and MpATG8 (chitin synthase and autophagy genes, respectively), was analyzed during the M. perniciosa growth and development on bran-based solid medium as well as in liquid medium containing H2O2 or rapamycin (oxidative and nutritional related-autophagy stress agents, respectively). In order to link the expression of chitin metabolism-related genes to nutritional composition influencing fungus development, we also quantified total and reduced sugars, as well as macro- and micronutrients in the bran-based solid medium. The expression analysis showed that the MpCHS expression increased through mycelial development and then decreased in the primordium and basidioma phases, while the expression of MpCHIT1 and MpCHIT2 was higher in basidioma and primordium phases, respectively. Moreover, the expression pattern of MpCHIT1 and MpCHIT2 is distinct, the second correlated with the MpATG8 expression pattern and possibly with autophagy process, while the first may be related to the basidioma formation. The quantification of total and reduced sugars, as well as macro- and micronutrients supported the idea that the cell wall restructuration due to MpCHS, MpCHIT1 and MpCHIT2 is related to stress and fungal nutrient reallocation, allowing the formation and development of the basidioma. Experiments involving M. perniciosa growth on liquid medium containing H2O2 or rapamycin showed that MpCHIT1 and MpCHIT2 were over-expressed in response to oxidative but also to nutritional related-autophagy stresses. Interestingly, the expression level of MpCHS, MpCHIT1 and MpCHIT2 in presence of rapamycin is similar to the one observed in the primordium and basidioma from bran-based solid medium. The analysis of the overall data allowed designing a general scheme of chitin metabolism and autophagy during M. perniciosa development, focusing on the mycelium phases as crucial and environmentally influenced steps preceding the primordium and basidioma formation. These data support the idea that the nutritional environment of M. perniciosa influences its development and life cycle.

Transcriptomics and systems biology analysis in identification of specific pathways involved in cacao resistance and susceptibility to witches' broom disease.

This study reports on expression analysis associated with molecular systems biology of cacao-Moniliophthora perniciosa interaction. Gene expression data were obtained for two cacao genotypes (TSH1188, resistant; Catongo, susceptible) challenged or not with the fungus M. perniciosa and collected at three time points through disease. Using expression analysis, we identified 154 and 227 genes that are differentially expressed in TSH1188 and Catongo, respectively. The expression of some of these genes was confirmed by RT-qPCR. Physical protein-protein interaction (PPPI) networks of Arabidopsis thaliana orthologous proteins corresponding to resistant and susceptible interactions were obtained followed by cluster and gene ontology analyses. The integrated analysis of gene expression and systems biology allowed designing a general scheme of major mechanisms associated with witches' broom disease resistance/susceptibility. In this sense, the TSH1188 cultivar shows strong production of ROS and elicitors at the beginning of the interaction with M. perniciosa followed by resistance signal propagation and ROS detoxification. On the other hand, the Catongo genotype displays defense mechanisms that include the synthesis of some defense molecules but without success in regards to elimination of the fungus. This phase is followed by the activation of protein metabolism which is achieved with the production of proteasome associated with autophagy as a precursor mechanism of PCD. This work also identifies candidate genes for further functional studies and for genetic mapping and marker assisted selection.

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa.

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.

Efeitos genotóxicos de fatores considerados de risco para o câncer bucal / Genotoxic effects of factors considered of risk for the buccal cancer

A ocorrência de danos cromossômicos em células esfoliadas da mucosa bucal e sua associação com fatores de risco para desenvolvimento de câncer bucal foi avaliada em amostra de 40 indivíduos. Estes indivíduos foram distribuídos em dois grupos: Grupo 1 (N=20), formado por indivíduos expostos a fatores considerados de risco ao desenvolvimento do câncer bucal (fumo e álcool), atendidos no Centro de Referência de Lesões Bucais da Universidade Estadual de Feira de Santana (UEFS); Grupo 2 (N=20) constituído por indivíduos sem exposição a estes fatores, examinados na Clínica de Extensão em Dentística da UEFS em atendimento odontológico de rotina. A investigação da ocorrência, entre os grupos, dos fatores de risco foi avaliada a partir de informações obtidas em entrevista. Os danos genéticos foram avaliados utilizando o Teste de Micronúcleo com protocolo diferenciado para contagem de fenômenos nucleares degenerativos. Os resultados obtidos sugerem que o uso de antissépticos bucais está associado a uma maior ocorrência de micronúcleos. A freqüência significantemente maior de fenômenos nucleares degenerativos revela que mesmo a fraca exposição ao tabaco e álcool isoladamente pode produzir genotoxidade e que esta condição pode ser aumentada quando da combinação dos dois fatores.