RESUMO
Extracting genomic DNA of pathogenic agents from formalin-fixed specimens is inherently difficult. Storage of samples in formalin results in nucleic acid cross-linking and DNA fragmentation. In this study, DNA was extracted from 45 Giardia-positive stool samples stored in formalin and subjected to PCR amplification targeting the triose phosphate isomerase (tpi), beta gardin (bg) and glutamate dehydrogenase (gdh) genes. Samples were rehydrated by using a descending alcohol series before DNA extraction using a commercial kit. This was followed by EDTA-mediated inhibition of DNase activity and prolonged treatment with proteinase K to digest contaminating proteins. DNA was amplified at rates of 64.4% (29/45) at the tpi, 40% (18/45) at the bg and 20% (9/45) at the gdh loci as seen on nested PCR. DNA quality was subsequently tested in a genotyping experiment which produced high-quality sequences at the tpi (41.2%; 12/29) bg (50%; 9/18), and gdh (22.2%; 2/9) loci and enabled differentiation of Giardia strains at the subtype level. The modified extraction protocol was effective at removing inhibitors and reversing cross-linking of DNA. However, PCR amplification was limited to short fragments of DNA which resulted in highest success rate on amplification of the shortest (334 bp) gene fragment tested.
Assuntos
DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Fixadores/efeitos adversos , Formaldeído/efeitos adversos , Giardia/genética , Sequência de Bases , Proteínas do Citoesqueleto/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/normas , Etanol/administração & dosagem , Genótipo , Técnicas de Genotipagem , Giardia/química , Giardia/classificação , Giardia/enzimologia , Glutamato Desidrogenase/genética , Humanos , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Solventes/administração & dosagem , Fatores de Tempo , Triose-Fosfato Isomerase/genéticaRESUMO
The use of molecular biology in combination with morphological analysis is increasing because of the treatments by target therapies. However, to improve the methods for obtaining DNA for molecular analyses from formalin-fixed, paraffin-embedded (FFPE) tissue is a challenge. The aim of this study was to evaluate the DNA extracted from FFPE tissue blocks (non-tumoral liver, spleen, and brain), obtained from autopsy, 8-24 h post mortem, using three methods of DNA extraction. PCR of the ß-actin (136 pb) and human amelogenin (AMEL 212-218 bp/106-112 bp) genes, as well as short tandem repeat (STR) (100-400 bp fragments), reported in forensic scientific analysis, was performed to evaluate the effectiveness of the methods of DNA extraction. We used 28 archived (1 and 5 years) and 12 recent autopsy cases. The commercial kit showed reproducible and consistent results in the PCR amplification of the ß-actin and AMEL genes and in analysis by STR used in forensic analysis. This is the first report using non-tumoral samples from FFPE autopsy tissues, comparing the three most common methods of DNA extraction and using the STR previously described in forensics. Our study has clarified the challenges for pathologists in applying the molecular biology approach in combination with methods suited for morphology, which must be improved. The data provided here should be used in other molecular studies in FFPE samples.
Assuntos
Química Encefálica , DNA/isolamento & purificação , Fixadores , Formaldeído , Fígado/química , Inclusão em Parafina , Baço/química , Fixação de Tecidos/métodos , Actinas/genética , Amelogenina/genética , Autopsia , Feminino , Fixadores/efeitos adversos , Patologia Legal , Formaldeído/efeitos adversos , Técnicas Genéticas , Humanos , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Análise para Determinação do Sexo , Fatores de TempoRESUMO
OBJECTIVE: Keratocystic odontogenic tumors (KOTs) can be treated with Carnoy's solution, although this treatment modality is not free from complications. It is important to verify the incidence of complications after the use of Carnoy's solution and compare these with the literature. MATERIALS AND METHODS: This study verified the effects of a complementary treatment for KOTs and assessed the incidence of such complications as recurrence, infection, sequestrum formation, mandibular fracture, dehiscence, and neuropathy. RESULTS: Twenty-two KOTs treated with Carnoy's solution combined with peripheral ostectomy were included, and the follow-up period varied from 12 to 78months with a mean of 42.9months. Complications included recurrence (4.5%), dehiscence (22.7%), infection (4.5%), and paresthesia (18.2%). No difference was found among lesions associated (9.1%) or not (0%) with nevoid basal cell carcinoma syndrome (P>0.05). Dehiscence was influenced by marsupialization (P<0.05), and paresthesia was observed exclusively in cases of mandibular canal fenestration (P<0.01). CONCLUSIONS: Complementary treatment with Carnoy's solution and peripheral ostectomy appear to provide efficient treatment for KOTs. Complications originating from the use of the solution are less frequent and less serious than complications associated with cryotherapy. Neuropathy seems to be related to direct contact between the solution and the epineurium.
Assuntos
Ácido Acético/uso terapêutico , Clorofórmio/uso terapêutico , Etanol/uso terapêutico , Fixadores , Tumores Odontogênicos/tratamento farmacológico , Ácido Acético/efeitos adversos , Adolescente , Adulto , Idoso , Criança , Clorofórmio/efeitos adversos , Terapia Combinada , Etanol/efeitos adversos , Feminino , Fixadores/efeitos adversos , Seguimentos , Humanos , Masculino , Fraturas Mandibulares/etiologia , Neoplasias Mandibulares/tratamento farmacológico , Neoplasias Mandibulares/cirurgia , Nervo Mandibular/efeitos dos fármacos , Nervo Mandibular/fisiopatologia , Neoplasias Maxilares/tratamento farmacológico , Neoplasias Maxilares/cirurgia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Tumores Odontogênicos/cirurgia , Osteotomia/efeitos adversos , Parestesia/etiologia , Complicações Pós-Operatórias , Estudos Retrospectivos , Deiscência da Ferida Operatória/etiologia , Infecção da Ferida Cirúrgica/etiologia , Sensação Térmica/fisiologia , Fatores de Tempo , Tato/fisiologia , Traumatismos do Nervo Trigêmeo/etiologia , Adulto JovemRESUMO
Calcification of glutaraldehyde-treated bioprosthetic heart valves is a major cause of long-term failure. We studied porcine aortic valves treated by the L-Hydro process and implanted into 14 juvenile sheep (group 1). Another 10 sheep were implanted with glutaraldehyde-treated porcine bioprostheses (group 2). The animals were sacrificed after 150 days and the explanted valves were analyzed for calcification. Hemodynamic measurements by echocardiography and angiography were carried out prior to sacrifice. Macroscopic analysis showed calcification and loss of mobility of the leaflets in all group 2 implants and in one group 1 implant. Light microscopy showed foci of calcification in all group 2 implants and in 3 valves from group 1. A significant reduction in the level of calcification was found in porcine bioprostheses treated by the L-Hydro process and implanted into the juvenile sheep model.
Assuntos
Valva Aórtica/cirurgia , Bioprótese , Calcinose/induzido quimicamente , Fixadores/efeitos adversos , Glutaral/efeitos adversos , Doenças das Valvas Cardíacas/induzido quimicamente , Implante de Prótese de Valva Cardíaca/instrumentação , Próteses Valvulares Cardíacas , Fixação de Tecidos/métodos , Animais , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Calcinose/patologia , Calcinose/fisiopatologia , Análise de Falha de Equipamento , Feminino , Doenças das Valvas Cardíacas/patologia , Doenças das Valvas Cardíacas/fisiopatologia , Hemodinâmica , Masculino , Modelos Animais , Desenho de Prótese , Falha de Prótese , Ovinos , Resultado do TratamentoRESUMO
This investigation was conducted in an attempt to clarify the role of the organic matrix in bovine dentine demineralisation by 1% citric acid. Dentine slabs (n = 15) were treated for 2 min with 10% sodium hypochlorite (NaOCl, a strong protein solvent), 2% glutaraldehyde (GDA, a protein fixative) or deionised water (DIW, as negative control) prior to each of 5 demineralisation periods of 30 min. The mineral loss (DeltaZ), lesion depth (LD) and surface loss (SL) were determined after each period of demineralisation, by transverse microradiography. The NaOCl-treated group showed higher DeltaZ and LD than the GDA- and DIW-treated groups. No differences between GDA and DIW groups were found. The DeltaZ and LD profiles were explained by quadratic fits (r(2) >0.80; p = 0.001) in all groups. SL was detected only in the NaOCl-treated group. The data suggested that the maintenance of the organic matrix in the lesion might be important to reduce the erosion progression rate.
Assuntos
Quelantes/efeitos adversos , Ácido Cítrico/efeitos adversos , Dentina/patologia , Erosão Dentária/patologia , Raiz Dentária/patologia , Animais , Bovinos , Progressão da Doença , Fixadores/efeitos adversos , Glutaral/efeitos adversos , Microrradiografia , Minerais/análise , Distribuição Aleatória , Hipoclorito de Sódio/efeitos adversos , Solventes/efeitos adversos , Desmineralização do Dente/induzido quimicamente , Desmineralização do Dente/patologia , Erosão Dentária/induzido quimicamenteRESUMO
The periaqueductal gray matter (PAG) is an important center in the modulation of behavioral responses during nociception and stress. In the present experiment, extracellular excitatory amino acid overflow in the PAG was measured every 30 s during noxious stimulation. A combination of in vivo brain microdialysis in freely moving rats and capillary zone electrophoresis with laser induced-fluorescence detection allowed us to detect short lasting changes of excitatory amino acid in dialysates. A formalin injection in the hindpaw of the rat increased glutamate, arginine and aspartate concentration in PAG dialysates. This increase was calcium and nerve impulse-dependent, suggesting neuronal and glial origin of glutamate and arginine, respectively. Handling, pinching or saline injection in the hind paw did not increase glutamate showing that this neurochemical phenomenon is related to painful and persistent noxious stimulation. The results suggest that a rapid excitation of the PAG occurs during noxious stimulation. The role of glutamate and arginine in analgesia is discussed.