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Prensa méd. argent ; 107(3): 135-142, 20210000. tab
Article in English | LILACS, BINACIS | ID: biblio-1359564


Antecedentes: el síndrome de ovario poliquístico (SOP) es un trastorno endocrino reproductivo común, se puede identificar por hiperandrogenismo, oligomenorrea o anovulación y ovarios poliquísticos en la ecografía. Los polimorfismos de la metilentetrahidrofolato reductasa (MTHFR) C677T asociados con la hiperhomocisteinemia se encuentran entre los factores de riesgo del síndrome de ovario poliquístico. Objetivo: El presente estudio de casos y controles tiene como objetivo explorar la relación entre los polimorfismos C677T de la metilenotetrahidrofolato reductasa (MTHFR) como factor de riesgo y el síndrome de ovario poliquístico entre los pacientes jordanos que padecen esta enfermedad. Métodos: Se inscribieron en el estudio 306 sujetos (146 pacientes con SOP y 160 sujetos sanos como grupo de control). Se extrajo ADN de una muestra de sangre venosa extraída de cada participante para analizar los polimorfismos de MTHFR C677T utilizando la reacción en cadena de la polimerasa (PCR) en combinación con digestión con enzima de restricción (PCRRFLP). Posteriormente, los productos de PCR-RFLP se digirieron con la enzima HinfI, luego se sometieron a electroforesis en un gel de agarosa al 2%, se tiñeron y se examinaron bajo luz ultravioleta. Los niveles de homocisteína en plasma se analizaron utilizando el método ELISA. Resultados: Se observó una diferencia significativa en los niveles plasmáticos de homocisteína entre los pacientes con SOP frente a los sujetos de control y entre los diferentes polimorfismos de los pacientes con SOP. No se detectaron diferencias significativas en la distribución y frecuencia alélica de los polimorfismos MTHFR C677T en pacientes con SOP en comparación con los controles. El genotipo 677 / TT y el alelo T se asociaron con un aumento de 1,54 y 1,46 veces en la susceptibilidad al síndrome de ovario poliquístico. Conclusión: El estudio ha demostrado que el polimorfismo MTHFR T677T y el alelo T son posibles factores de riesgo de SOP entre las mujeres jordanas y pueden desempeñar un papel en la patogenia de la enfermedad

Background: Polycystic ovary syndrome (PCOS) is a common endocrine reproductive disorder, it can be identified by hyperandrogenism, oligomenorrhea or anovulation and polycystic ovaries on ultrasound. Methylenetetrahydrofolate Reductase (MTHFR) C677T polymorphisms associated with hyperhomocysteinemia are among the risk factors for PCOS. Objective: The present case control study aims to explore the relationship between Methylenetetrahydrofolate Reductase (MTHFR) C677T polymorphisms as a risk factor and PCOS among Jordanian patients suffering from this disease. Methods: 306 subjects (146 PCOS patients and 160 healthy subjects as a control group) were enrolled in the study. DNA was extracted from venous blood sample withdrawn from each participant for analyzing MTHFR C677T polymorphisms using Polymerase Chain Reaction (PCR) in combination with restriction enzyme fragment length polymorphism (PCR-RFLP). Later, PCR-RFLP products were digested with hinfI enzyme, then, electrophoresed on a 2% agarose gel, stained and examined under UV light. Plasma homocysteine levels were assayed using ELISA method. Results: A significant difference was observed in plasma homocysteine levels among PCOS patients versus the control subjects and in between the different polymorphisms of PCOS patients. No significant difference was detected in the distribution and allelic frequency of MTHFR C677T polymorphisms in PCOS patients compared to the controls. 677/TT genotype and T allele were associated with 1.54 and 1.46 folds increase in the susceptibility for PCOS. Conclusion: The study has shown that MTHFR T677T polymorphism and T allele are possible risk factors for PCOS among Jordanian women and may play a role in the pathogenesis of the disease.

Humans , Female , Polycystic Ovary Syndrome/diagnosis , Polycystic Ovary Syndrome/pathology , DNA/analysis , Polymerase Chain Reaction , Risk Factors , Genotype , Homocysteine/blood
Electron J Biotechnol ; 49: 29-33, Jan. 2021. tab, ilus
Article in English | LILACS | ID: biblio-1291632


BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Animals , Polymerase Chain Reaction/methods , Agkistrodon/genetics , Cytochromes b/genetics , Mitochondria/genetics , Snakes , Species Specificity , DNA/analysis , Cloning, Molecular , Medicine, Chinese Traditional
J. venom. anim. toxins incl. trop. dis ; 27: e20200183, 2021. graf
Article in English | LILACS, VETINDEX | ID: biblio-1287097


The COVID-19 pandemic brought attention to studies about viral infections and their impact on the cell machinery. SARS-CoV-2, for example, invades the host cells by ACE2 interaction and possibly hijacks the mitochondria. To better understand the disease and to propose novel treatments, crucial aspects of SARS-CoV-2 enrolment with host mitochondria must be studied. The replicative process of the virus leads to consequences in mitochondrial function, and cell metabolism. The hijacking of mitochondria, on the other hand, can drive the extrusion of mitochondrial DNA (mtDNA) to the cytosol. Extracellular mtDNA evoke robust proinflammatory responses once detected, that may act in different pathways, eliciting important immune responses. However, few receptors are validated and are able to detect and respond to mtDNA. In this review, we propose that the mtDNA and its detection might be important in the immune process generated by SARS-CoV-2 and that this mechanism might be important in the lung pathogenesis seen in clinical symptoms. Therefore, investigating the mtDNA receptors and their signaling pathways might provide important clues for therapeutic interventions.(AU)

DNA/analysis , Genes, Mitochondrial , COVID-19 , Cytokines
Mem. Inst. Oswaldo Cruz ; 115: e200157, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135268


BACKGROUND In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved. OBJECTIVES Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.

Animals , Female , Psychodidae/parasitology , DNA/analysis , Insect Vectors/genetics , Leishmania/genetics , Psychodidae/classification , Brazil , Polymerase Chain Reaction , DNA, Protozoan/analysis , Leishmaniasis, Cutaneous/transmission , Insect Vectors/classification , Insect Vectors/parasitology , Leishmania/isolation & purification
Prensa méd. argent ; 105(11): 816-826, dic2019. fig, tab, graf
Article in English | LILACS, BINACIS | ID: biblio-1049970


Background: To investigate the potential of the phage display-identified tumor cellbinding peptide as a biomarker of epithelial ovarian cancer using phage display technology. Method: The Ph.D.-7 Phage Display Peptide Library was used to identify the specific conjugated phages with SKOV3 epithelial ovarian cancer cells, while Chinese hamster ovary cells formed the basis. After employing the rapid differential screening method invitro, the enzyme-linked immunosorbent assay (ELISA), DNA sequencing, immunohistochemistry, immunofluorescence, and the competitive inhibition test of synthetic peptides were used to determine the affinity and specificity of the phages with SKOV3 cells. Results: Using bio panning, we screened the phages, showing a 3590-fold increase after the third round. A total of 61 titers of the phage were randomly selected for ELISA and 10 kinds of the phages with an optical density >0.5 were used for DNA sequencing. Clones of the phage TRRNIPN were derived from DNA sequencing based on ELISA, exhibiting both the brown granular phenomenon and green fluorescence. The specific targeted peptide TRRNIPN was incorporated in tumor cells through the competitive inhibition test. Conclusion: The results of our study indicate that the phage display identified polypeptide TRRNIPN may be an effective biomarker for the early diagnosis and targeted therapy of ovarian cancer

Humans , Female , Bacteriophages , DNA/analysis , Enzyme-Linked Immunosorbent Assay , Biomarkers, Tumor , Mass Screening/methods , Peptide Library , Early Diagnosis , Research Report , /therapy
Rev. bras. parasitol. vet ; 27(1): 60-65, Jan.-Mar. 2018. tab
Article in English | LILACS | ID: biblio-899315


Abstract This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

Resumo Este trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. em 498 amostras fecais de canários (Serinus canaria) criados em cativeiro, utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR seguida de sequenciamento dos fragmentos amplificados e PCR duplex em tempo real específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade total para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%; 15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidium spp. A PCR duplex em tempo real apresentou maior sensibilidade que a nested PCR/sequenciamento para detectar as espécies/genótipos gástricos de Cryptosporidium.

Animals , Canaries/parasitology , Cryptosporidiosis/diagnosis , Cryptosporidiosis/parasitology , Cryptosporidium/isolation & purification , Brazil , DNA/analysis , Cryptosporidium/genetics , Molecular Diagnostic Techniques , Animals, Domestic
Neotrop. ichthyol ; 16(1): e170068, 2018. graf
Article in English | LILACS, VETINDEX | ID: biblio-895117


The Caribbean Red Snapper (Pargo) Lutjanus purpureus is the most economically important snapper in Brazil, which is sold, among other forms, as frozen fillets. During the process of transformation into fillets there is the removal of the distinctive morphological traits, being able to favor the substitution by less valued species. In addition, there is no national legislation requiring the insertion of the specific name on the product label. However, according to a Normative Instruction (IN N ° 29/2015 MAPA) that correlates the common and specific names of the products destined to the national trade, in Brazil only L. purpureus and L. campechanus can be denominated "Pargo". Thus, the DNA barcode tool was used to identify the fillets sold in north of Brazil, labeled "Pargo", with the aid of sequences from the public and control databases. The results showed that among 142 fillets examined, 78% was identified as L. purpureus and 22% as Rhomboplites aurorubens, a snapper with low commercial value in the country, revealing commercial fraud. The molecular identification method successfully used in this study to authenticate fillets snappers may also be used by surveillance authorities in the quality control of processed fish products, towards ensuring consumer rights.(AU)

O Pargo Lutjanus purpureus, lutjanídeo mais importante economicamente no Brasil, é vendido, entre outras formas, como filés congelados. Durante a transformação em filés, há a remoção das características morfológicas distintivas, podendo favorecer a substituição por espécies menos valorizadas. Além disso, não há legislação nacional que exija a inserção do nome específico no rótulo. Porém, de acordo com uma Instrução Normativa (IN N° 29 /2015 MAPA) que correlaciona os nomes comuns e específicos dos produtos destinados ao comércio nacional, no Brasil somente L. purpureus e L. campechanus podem ser denominados "Pargo". Assim, a ferramenta DNA barcode foi usada para identificar os filés vendidos no norte do Brasil, rotulados como "Pargo", com o auxílio de sequências dos bancos de dados públicos e banco controle. Os resultados mostraram que entre os 142 filés examinados, 78% foi identificado como L. purpureus e 22% como Rhomboplites aurorubens, um lutjanídeo com baixo valor comercial no país, revelando fraude comercial. O método de identificação molecular, utilizado com êxito neste estudo para autenticar filés de lutjanídeos, pode também ser utilizado pelas autoridades de vigilância no controle de qualidade de produtos processados derivados de peixes em geral, para garantir os direitos dos consumidores.(AU)

Animals , DNA/analysis , Economics/trends , Perciformes/genetics
Braz. J. Pharm. Sci. (Online) ; 54(1): e00265, 2018. tab, graf
Article in English | LILACS | ID: biblio-951915


ABSTRACT In recent years, non-viral delivery systems for plasmid DNA have become particularly important. They can overcome the disadvantages of viral systems such as insertional mutagenesis and unpredicted immunogenicity. Some additional advantages of non-viral gene delivery systems are; good stability, low cost, targetability, delivery of a high amount of genetic materials. The aim of the study was to develop novel non-viral nanosystems suitable for gene delivery. Two formulations were developed for this purpose: water-in-oil microemulsion (ME) and solid lipid nanoparticles (SLN). The microemulsion was composed of Peceol, Tween 80, Plurol oleique, ethanol and water. The SLN was consisting of Precirol, Esterquat-1 (EQ1), Tween 80, Lecithin, ethanol and water. Characterization studies were carried out by measuring particle size, zeta potential, viscosity and pH. TEM imaging was performed on SLN formulations. Protection against DNase I degradation was examined. Cytotoxicity and transfection efficacy of selected formulations were tested on L929 mouse fibroblast cells. Particle sizes of complexes were below 100 nm and with high positive zeta potential. TEM images revealed that SLNs are spherical. The SLN:DNA complexes have low toxicity and good transfection ability. All results showed that the developed SLN formulations can be considered as suitable non-viral gene delivery systems.

DNA/analysis , Genes/genetics , Transfection/statistics & numerical data , Genetic Therapy/classification
Int. j. odontostomatol. (Print) ; 11(2): 173-177, June 2017. ilus
Article in English | LILACS | ID: biblio-893247


The aim of this study was to evaluate the extraction of dental DNA exposed to different chemical solutions. The sample was composed by 15 subjects, from which 5 samples of oral mucosal cells (reference population) and 15 teeth (experimental population) were collected. The experimental population was divided in three equal parts, which were exposed to different chemical solutions, namely hydrochloric acid (HCl) at 37 %, formaldehyde (CH2O) at 10 % and sodium hypochlorite (NaOCl) at 2.5 %. The DNA from the oral mucosa was extracted using organic method, while the dental DNA was extracted using the AFDIL method, including amplification by PCR and sequencing through capillary electrophoresis. The DNA exposed to hydrochloric acid dissolved, lacking extraction. The exposure of teeth to formaldehyde and sodium hypochlorite did not interfere in the extraction of DNA, once the amplification was visible in both experimental populations. The present outcomes demonstrated that DNA extraction may be limited under exposure to chemical solutions.

El objetivo de este estudio fue evaluar la extracción de ADN dental expuesto a diferentes soluciones químicas. La muestra estuvo compuesta por 15 sujetos, de los cuales se recogieron 5 muestras de células de la mucosa oral (población de referencia) y 15 dientes (población experimental). La población experimental se dividió en tres partes iguales, que fueron expuestas a diferentes soluciones químicas, a saber, ácido clorhídrico (HCl) al 37 %, formaldehído (CH2O) al 10 % e hipoclorito de sodio (NaOCl) al 2,5 %. El ADN de la mucosa oral se extrajo utilizando el método orgánico, mientras que el ADN dental se extrajo utilizando el método AFDIL, incluyendo la amplificación por PCR y la secuenciación a través de electroforesis capilar. El ADN expuesto al ácido clorhídrico se disolvió, careciendo de extracción. La exposición de los dientes al formaldehído e hipoclorito de sodio no interfirió en la extracción de ADN, una vez que la amplificación era visible en ambas poblaciones experimentales. Los resultados actuales demostraron que la extracción de ADN puede ser limitada bajo la exposición a soluciones químicas.

Humans , DNA Fingerprinting/methods , Forensic Dentistry/methods , Solutions , Tooth , DNA/analysis , Forensic Genetics
Rev. paul. pediatr ; 35(1): 25-32, jan.-mar. 2017. tab, graf
Article in Portuguese | LILACS | ID: biblio-845724


RESUMO Objetivo: Validar a quantificação de T-cell receptor excision circles (TRECs) e kappa-deleting recombination circles (KRECs) por reação em cadeia de polimerase (polymerase chain reaction, PCR) em tempo real (qRT-PCR), para triagem neonatal de imunodeficiências primárias que cursam com defeitos nas células T e/ou B no Brasil. Métodos: Amostras de sangue de recém-nascidos (RN) e controles foram coletadas em papel-filtro. O DNA foi extraído e os TRECs e KRECs foram quantificados por reação duplex de qRT-PCR. O valor de corte foi determinado pela análise de Receiver Operating Characteristics Curve, utilizando-se o programa Statistical Package for the Social Sciences (SSPS) (IBM®, Armonk, NY, EUA). Resultados: 6.881 amostras de RN foram analisadas quanto à concentração de TRECs e KRECs. Os valores de TRECs variaram entre 1 e 1.006 TRECs/µL, com média e mediana de 160 e 139 TRECs/µL, respectivamente. Três amostras de pacientes diagnosticados com imunodeficiência grave combinada (severe combined immunodeficiency, SCID) apresentaram valores de TRECs abaixo de 4/µL e um paciente com Síndrome de DiGeorge apresentou TRECs indetectáveis. Os valores de KRECs encontraram-se entre 10 e 1.097 KRECs/µL, com média e mediana de 130 e 108 KRECs/µL, e quatro pacientes com diagnóstico de agamaglobulinemia tiveram resultados abaixo de 4 KRECs/µL. Os valores de corte encontrados foram 15 TRECs/µL e 14 KRECs/µL, e foram estabelecidos de acordo com a análise da Receiver Operating Characteristics Curve, com sensibilidade de 100% para detecção de SCID e agamaglobulinemia, respectivamente. Conclusões: A quantificação de TRECs e KRECs foi capaz de diagnosticar crianças com linfopenias T e/ou B em nosso estudo, validando a técnica e dando o primeiro passo para a implementação da triagem neonatal em grande escala no Brasil.

ABSTRACT Objective: To validate the quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) by real-time polymerase chain reaction (qRT-PCR) for newborn screening of primary immunodeficiencies with defects in T and/or B cells in Brazil. Methods: Blood samples from newborns and controls were collected on filter paper. DNA was extracted and TRECs, and KRECs were quantified by a duplex real-time PCR. The cutoff values were determined by receiver operating characteristic curve analysis using SPSS software (IBM®, Armonk, NY, USA). Results: Around 6,881 samples from newborns were collected and TRECs and KRECs were quantified. The TRECs values ranged between 1 and 1,006 TRECs/µL, with mean and median of 160 and 139 TRECs/µL, respectively. Three samples from patients with severe combined immunodeficiency (SCID) showed TRECs below 4/µL and a patient with DiGeorge syndrome showed undetectable TRECs. KRECs values ranged from 10 to 1,097 KRECs/µL, with mean and median of 130 and 108 KRECs/µL. Four patients with agammaglobulinemia had results below 4 KRECs/µL. The cutoff values were 15 TRECs/µL and 14 KRECs/µL and were established according to the receiver operating characteristic curve analysis, with 100% sensitivity for SCID and agammaglobulinemia detection, respectively. Conclusions: Quantification of TRECs and KRECs was able to diagnose children with T- and/or B-cell lymphopenia in our study, which validated the technique in Brazil and enabled us to implement the newborn screening program for SCID and agammaglobulinemia.

Humans , Infant, Newborn , Infant , Neonatal Screening/methods , Severe Combined Immunodeficiency/diagnosis , Severe Combined Immunodeficiency/blood , Brazil , DNA/analysis , Receptors, Antigen, B-Cell/genetics , Pilot Projects , Cross-Sectional Studies , Severe Combined Immunodeficiency/genetics , Real-Time Polymerase Chain Reaction
Mem. Inst. Oswaldo Cruz ; 111(7): 423-432, tab, graf
Article in English | LILACS | ID: lil-787560


In this study, we identified the phlebotomine sandfly vectors involved in the transmission of American Cutaneous Leishmaniasis (ACL) in Assis Brasil, Acre, Brazil, which is located on the Brazil-Peru-Bolivia frontier. The genotyping of Leishmania in phlebotomines was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. A total of 6,850 sandflies comprising 67 species were captured by using CDC light traps in rural areas of the municipality. Three sandfly species were found in the state of Acre for the first time: Lutzomyia georgii, Lu. complexa and Lu. evangelistai. The predominant species was Lu. auraensis/Lu. ruifreitasi and Lu. davisi (total 59.27%). 32 of 368 pools were positive for the presence of Leishmania DNA (16 pools corresponding to Lu. davisi, and 16 corresponding to Lu. auraensis/Lu. ruifreitasi), with a minimal infection prevalence of 1.85% in Lu. davisi and 2.05% in Lu. auraensis/Lu. ruifreitasi. The Leishmania species found showed maximum identity with L. (Viannia) guyanensis and L. (V.) braziliensis in both phlebotomine species. Based on these results and similar scenarios previously described along the Brazil/Peru/Bolivia tri-border, the studied area must take into consideration the possibility of Lu. davisi and Lu. auraensis/Lu. ruifreitasi as probable vectors of ACL in this municipality.

Humans , Animals , Female , DNA/analysis , Insect Vectors/genetics , Leishmania/genetics , Psychodidae/genetics , Biodiversity , Bolivia , Brazil , DNA, Kinetoplast , Genotype , Insect Vectors/classification , Insect Vectors/parasitology , Leishmaniasis, Cutaneous/transmission , Peru , Polymerase Chain Reaction , Population Density , Psychodidae/classification , Psychodidae/parasitology
Rev. bras. oftalmol ; 75(3): 223-227, tab, graf
Article in Portuguese | LILACS | ID: lil-787695


RESUMO Objetivo: Avaliar a ocorrência de mutação em locus gênico candidato e sua relação com ceratocone em pacientes atendidos no Brasil comparados a voluntários saudáveis, através da análise de polimorfismo de nucleotídeo único no gene DOCK9. Métodos: Neste estudo clínico foram avaliados 108 indivíduos, sendo 46 pacientes com ceratocone e 62 voluntários saudáveis (controles). Amostras de DNA foram obtidas do sangue coletado de pacientes com ceratocone e controles para a realização de análise de genotipagem. O genótipo do polimorfismo de nucleotídeo único rs7995432 no gene DOCK9 foi determinado através de reação em cadeia da polimerase em tempo real (qPCR). Resultados: A frequência do alelo mutante (C) foi de 4,8% para os pacientes e 7,6% para os controles. Para o alelo selvagem (T), as frequências foram de 95,2% para os pacientes e 92,4% para os controles. O genótipo heterozigótico esteve presente em 9,5% dos pacientes e 11% dos controles, enquanto o genótipo homozigótico para o alelo selvagem (TT) foi encontrado em 90,5% e 87% para os pacientes e controles, respectivamente. Conclusão: Não foram observadas diferenças significativas na frequência e discriminação dos alelos mutante e selvagem entre os pacientes com ceratocone e os controles. Portanto, não foi possível fazer uma associação destas mutações no gene DOCK9 com a ocorrência do ceratocone para esta população.

ABSTRACT Objective: To evaluate the occurrence of a mutation in candidate genetic loci and its relation with keratoconus in patients treated in Brazil compared to healthy volunteers, through analysis of single nucleotide polymorphism in the DOCK9 gene. Methods: In this clinical study, 108 participants were evaluated, including 46 keratoconus patients and 62 healthy volunteers (controls). DNA samples were extracted from collected blood from keratoconus patients and controls. The genotyping of the single nucleotide polymorphism rs7995432 in the DOCK9 gene was determined through a real-time polymerase chain reaction (qPCR). Results: The frequency of the mutant allele (C) was 4.8% in patients and 7.6% in controls. For the wild allele (T), the frequencies were 95.2% in patients and 92.4% in controls. The heterozygous genotype was present in 9.5% of patients and 11% of controls, while the homozygous genotype for the wild allele (TT) was found in 90.5% and 87% for patients and controls, respectively. Conclusion: There were no significant differences un the frequency and discrimination of the mutant and wild alleles between patients and controls. Therefore, these results confirm no association of these mutations in the DOCK9 gene and the occurrence of keratoconus for this population.

Humans , Male , Female , Middle Aged , Guanine Nucleotide Exchange Factors/genetics , Polymorphism, Single Nucleotide/genetics , Keratoconus/genetics , Mutation/genetics , DNA/analysis , DNA/blood , Polymerase Chain Reaction , Alleles , Genotyping Techniques , Genotype
Article in English | WPRIM | ID: wpr-56706


BACKGROUND: Currently, the hypertension (HTN) patients undergo appropriate medical treatment, and traditional risk factors are highly controlled. Therefore, potential risk factors of atherosclerotic vascular diseases (AVD) and venous thromboembolisms (VTE) in HTN should be reconsidered. We investigated thrombophilic genetic mutations and existing biomarkers for AVD or VTE in HTN patients receiving treatment. METHODS: A total of 183 patients were enrolled: AVD with HTN (group A, n=45), VTE with HTN (group B, n=62), and HTN patients without any vascular diseases (group C, n=76). The lipid profile, homocysteine (Hcy) levels, D-dimers, fibrinogen, antithrombin, lupus anticoagulant, and anti-cardiolipin antibody (aCL) were evaluated. Prothrombin G20210A, Factor V G1691A, and methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C were analyzed. RESULTS: All patients revealed wild type prothrombin G20210A and Factor V G1691A polymorphisms. The frequency of MTHFR polymorphisms was 677CT (n=84, 45.9%); 677TT (n=46, 25.1%); 1298AC (n=46, 25.1%); and 1298CC (n=2, 1.1%). The MTHFR 677TT genotype tended to increase the odds ratio (OR) to AVD events in HTN patients (OR 2.648, confidence interval 0.982-7.143, P=0.05). The group A demonstrated significantly higher Hcy levels (P=0.009), fibrinogen (P=0.004), and platelet counts (P=0.04) than group C. Group B had significantly higher levels of D-dimers (P=0.0001), platelet count (P=0.0002), and aCL (P=0.02) frequency than group C. CONCLUSIONS: The MTHFR 677TT genotype and Hcy level could be potential risk factors associated with development of AVD in HTN patients receiving treatment. D-dimer and aCL might be useful to estimate the occurrence of VTE in them.

Adult , Aged , Antihypertensive Agents/therapeutic use , DNA/analysis , Factor V/genetics , Female , Fibrin Fibrinogen Degradation Products/analysis , Genotype , Homocysteine/blood , Humans , Hypertension/complications , Lipids/blood , Male , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Middle Aged , Odds Ratio , Platelet Count , Polymorphism, Single Nucleotide , Prothrombin/genetics , Real-Time Polymerase Chain Reaction , Republic of Korea , Risk Factors , Vascular Diseases/etiology , Venous Thrombosis/etiology
Gut and Liver ; : 204-211, 2016.
Article in English | WPRIM | ID: wpr-25629


Colorectal cancer screening dates to the discovery of pre-cancerous adenomatous tissue. Screening modalities and guidelines directed at prevention and early detection have evolved and resulted in a significant decrease in the prevalence and mortality of colorectal cancer via direct visualization or using specific markers. Despite continued efforts and an overall reduction in deaths attributed to colorectal cancer over the last 25 years, colorectal cancer remains one of the most common causes of malignancy-associated deaths. In attempt to further reduce the prevalence of colorectal cancer and associated deaths, continued improvement in screening quality and adherence remains key. Noninvasive screening modalities are actively being explored. Identification of specific genetic alterations in the adenoma-cancer sequence allow for the study and development of noninvasive screening modalities beyond guaiac-based fecal occult blood testing which target specific alterations or a panel of alterations. The stool DNA test is the first noninvasive screening tool that targets both human hemoglobin and specific genetic alterations. In this review we discuss stool DNA and other commercially available noninvasive colorectal cancer screening modalities in addition to other targets which previously have been or are currently under study.

Biomarkers, Tumor/genetics , Colorectal Neoplasms/diagnosis , DNA/analysis , Early Detection of Cancer/methods , Feces , Humans , Mass Screening/methods
Femina ; 43(4): 181-184, jul.-ago. 2015.
Article in Portuguese | LILACS | ID: lil-771210


A infecção pelo papilomavírus humano (HPV) é extremamente comum e está associada a várias condições clínicas, que variam de infecções assintomáticas a doenças benignas e malignas da mucosa genital, como as verrugas genitais, a neoplasia intraepitelial cervical e o câncer do colo do útero. O objetivo deste estudo foi apresentar as técnicas de biologia molecular por captura híbrida (CH) e reação em cadeia da polimerase (PCR), utilizadas no diagnóstico do HPV e suas aplicações. Métodos mais precisos, quando aplicados em situações especiais, principalmente no caso de divergência entre outros métodos diagnósticos, podem ser aplicados às políticas de saúde pública, visando diminuir a mortalidade causada pelo câncer do colo do útero em consequência do HPV.(AU)

HPV infection is extremely common and is associated with various clinical conditions, ranging from asymptomatic infections to benign and malignant diseases of the genital mucosa, such as genital warts, cervical intraepithelial neoplasia and cervical cancer. The aim of this study was to present the techniques of molecular biology by hybrid capture and PCR, used in the diagnosis of HPV and its applications. More accurate methods when applied in special situations, especially in the case of divergence between other diagnostic methods can be applied to public health policies in order to reduce mortality caused by cervical cancer because of HPV.(AU)

Female , DNA/analysis , Uterine Cervical Neoplasms/diagnosis , Polymerase Chain Reaction/methods , Papillomavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Hybridization/methods , Papillomaviridae/pathogenicity , Databases, Bibliographic
São Paulo; s.n; s.n; 2015. 107 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-847457


O DNA está constantemente exposto a danos causados tanto por agentes endógenos quanto exógenos. Estes podem causar diferentes tipos de lesões incluindo modificações de bases e do açúcar, além de quebras de fitas simples ou duplas. As quebras de duplas fitas, quando comparadas às demais, constituem as mais citotóxicas e podem resultar em deleções no DNA e instabilidade genética. Deleções no DNA mitocondrial (mtDNA) causam diversas doenças e estão envolvidas no processo de envelhecimento. No núcleo, as quebras de duplas fitas no DNA podem ser reparadas por recombinação homóloga (HR), ligação de pontas não homólogas (NHEJ) e anelamento de fita simples (SSA). No entanto, em mitocôndrias de células de mamíferos, o reparo de quebras de duplas fitas ainda não foi completamente caracterizado. Experimentos in vitro usando extratos mitocondriais de células de roedores mostraram que estes são capazes de reparar essas quebras, no entanto pouco é sabido sobre quais proteínas são responsáveis por cada etapa de reparo, bem como sua implicação na manutenção da integridade do genoma mitocondrial. Sendo assim, nesse trabalho investigamos a localização e função mitocondrial das proteínas ATM, Rad51, Rad52, Ku70/86 e DNA-PKCs, que são sabidamente envolvidas em reparo de quebras de duplas fitas no núcleo. Para identificar essas proteínas em mitocôndrias de células de mamíferos, mitocôndrias foram isoladas a partir de células da linhagem HEK293T, usando centrifugação diferencial seguida por gradiente de Percoll. Para as proteínas de recombinação homóloga, ATM e Rad51, imunodetectamos isoformas semelhantes em todos os compartimentos celulares. Já para a proteína Rad52 o mesmo anticorpo imunodetectou duas bandas distintas na mitocôndria ao passo que no núcleo foram quatro. Além disso, verificamos que baixos níveis de proteína Rad52, induzidos pela expressão de shRNA (short hairping RNA) específico, resultam em diminuição do número de cópias de mtDNA bem como acúmulo de deleções no genoma mitocondrial. Para as proteínas de NHEJ, DNA-PKCs e a subunidade Ku70, identificamos isoformas semelhantes em todos os compartimentos celulares. Já para a subunidade 86 do heterodímero Ku70/86 o anticorpo detectou, somente em mitocôndrias, uma banda menor de 50 kDa, a qual difere na região N-terminal da subunidade detectada no núcleo (86 KDa). Experimentos de co-imunprecitação de proteínas mostraram que essa isoforma menor compõe o heterodímero mitocondrial juntamente com a subunidade 70 (mtKu70/50) e que esse interage com DNA ligase III mitocondrial. Nossos resultados também mostraram que a estabilidade proteica de mtKu70/50 é regulada por ATM. Tratamento das células com peróxido de hidrogênio, que induz quebras de duplas fitas, aumentou a associação do heterodímero mtKu70/50 com o mtDNA, de forma independente de aumento da concentração proteica intra-mitocondrial. Já a diminuição dos níveis proteicos de Ku, induzida através de shRNA, resultou em diminuição do número de cópias de mtDNA e acumulo de danos nesse genoma. Extratos mitocondriais de células knockdown para Ku apresentaram menor atividade de reparo NHEJ em um ensaio in vitro, sugerindo que o acúmulo de danos nestas células é provavelmente devido a deficiências na via de NHEJ. Em conjunto, nossos dados sugerem que tanto HR quanto NHEJ operam em mitocôndrias. Além disso, a via de NHEJ mitocondrial utiliza o heterodímero mitocondrial Ku70/50 o qual está envolvido na manutenção do mtDNA. Ademais, nossos resultados mostram uma grande conservação molecular e funcional entre as vias de reparo de NHEJ e HR no núcleo e na mitocôndria, o que reforça sua importância para a manutenção da estabilidade genômica mitocondrial e, provavelmente a função mitocondrial

DNA is constantly exposed to damaging agents from both endogenous and exogenous sources. These can cause different types of DNA lesions that include base and sugar modifications and single and double strand breaks. DNA doublestrand breaks (DSBs) are among the most cytotoxic DNA lesions, which can result in deletions and genetic instability. Deletions in the mitochondrial DNA (mtDNA) cause numerous human diseases and drive normal aging. DSBs in the nuclear DNA are repaired by non-homologous DNA end joining (NHEJ), homologous recombination (HR) or Single Strand Annealing (SSA). Yet, repair of DSBs in mammalian mitochondria has not been fully characterized. Mitochondrial extracts from rodent cells are proficient in ligating DNA ends in vitro, but little is known about which proteins are responsible for each enzymatic step and its implication in mitochondrial genome maintenance. Thus, we investigated mitochondrial localization and function of DSBR (double strand break repair) proteins ATM, Rad51, Rad52, the Ku70/86 heterodimer and DNA-PKCs.To identify DSBR proteins in mammalian mitochondria, highly purified mitochondria from HEK293T cells were isolated using differential centrifugation followed by Percoll gradient. For HR proteins, we detected similar isoforms for ATM and Rad51 proteins in all cellular compartments. Two mitochondriaspecific isoforms of Rad52 were detected, while the same antibody detected four isoforms in the nucleus. In addition, lower Rad52 protein levels, induced by specific shRNA expression, result in decreased mtDNA copy number and accumulation of deleted mitochondrial genomes. For NHEJ proteins, similar isoforms of DNA-PKcs and the Ku70 subunit were detected in all cellular compartments. On the other hand, antibodies against the Ku86 subunit detected a smaller band in mitochondrial extracts (50 KDa), lacking the N-terminal region of the canonical isoform detected in the nucleus (86 KDa). The mitochondrial Ku70/50 heterodimer interacts with mitochondrial DNA ligase III, suggesting a role in DSBR. Moreover, stability of the mtKu heterodimer is regulated by ATM. Hydrogen peroxide treatment, which induces DSBs, increases mtKu70/50 association with the mtDNA and cells with reduced Ku levels, also induced by shRNA transfection, have lower mtDNA copy number and accumulate mtDNA damage. Moreover, mitochondrial extracts from Ku knockdown cells show lower NHEJ repair activity in an in vitro assay, suggesting that damage accumulation in these cells is likely due to deficiencies in NHEJ. Together, our data suggest that both HR and NHEJ operate in mitochondria. Also, mtNHEJ requires the Ku heterodimer and is involved in mtDNA maintenance. Moreover, our results indicate that there is a significant molecular and functional conservation between NHEJ and HR repair pathways in the nucleus and in mitochondria, which reinforces their importance for maintenance of mitochondrial genomic stability and, likely mitochondrial function

DNA End-Joining Repair/genetics , DNA, Mitochondrial/genetics , DNA/analysis , Ataxia Telangiectasia Mutated Proteins , Ku Autoantigen , Rad51 Recombinase , Rad52 DNA Repair and Recombination Protein
Article in English | WPRIM | ID: wpr-95306


Cowden syndrome is an uncommon, autosomal dominant disease which is characterized by multiple hamartomas of the skin, mucous membrane, brain, breast, thyroid, and gastrointestinal tract. The diagnosis of Cowden syndrome implicates an increased risk of developing breast cancer. We report a case of a 22-year-old woman with Cowden syndrome that presented as breast cancer with concomitant bilateral exuberant benign masses in both breasts.

Arteriovenous Malformations/diagnostic imaging , Breast Neoplasms/complications , DNA/analysis , DNA Mutational Analysis , Diagnosis, Differential , Female , Hamartoma Syndrome, Multiple/complications , Humans , PTEN Phosphohydrolase/genetics , Thyroid Neoplasms/diagnostic imaging , Tomography, X-Ray Computed , Young Adult
Article in English | WPRIM | ID: wpr-70754


The combined array comparative genomic hybridization plus single-nucleotide polymorphism microarray (CGH+SNP microarray) platform can simultaneously detect copy number alterations (CNA) and copy-neutral loss of heterozygosity (LOH). Eighteen children with acute myeloid leukemia (AML) (n=15) or myelodysplastic syndrome (MDS) (n=3) were studied using CGH+SNP microarray to evaluate the clinical significance of submicroscopic chromosomal aberrations. CGH+SNP microarray revealed CNAs at 14 regions in 9 patients, while metaphase cytogenetic (MC) analysis detected CNAs in 11 regions in 8 patients. Using CGH+SNP microarray, LOHs>10 Mb involving terminal regions or the whole chromosome were detected in 3 of 18 patients (17%). CGH+SNP microarray revealed cryptic LOHs with or without CNAs in 3 of 5 patients with normal karyotypes. CGH+SNP microarray detected additional cryptic CNAs (n=2) and LOHs (n=5) in 6 of 13 patients with abnormal MC. In total, 9 patients demonstrated additional aberrations, including CNAs (n=3) and/or LOHs (n=8). Three of 15 patients with AML and terminal LOH>10 Mb demonstrated a significantly inferior relapse-free survival rate (P=0.041). This study demonstrates that CGH+SNP microarray can simultaneously detect previously cryptic CNAs and LOH, which may demonstrate prognostic implications.

Adolescent , Child , Child, Preschool , Chromosome Aberrations , Comparative Genomic Hybridization , DNA/analysis , DNA Copy Number Variations , Female , Hematopoietic Stem Cell Transplantation , Humans , Infant , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/diagnosis , Loss of Heterozygosity , Male , Myelodysplastic Syndromes/diagnosis , Oligonucleotide Array Sequence Analysis , Polymorphism, Single Nucleotide , Real-Time Polymerase Chain Reaction , Transplantation, Homologous
Electron. j. biotechnol ; 16(6): 14-14, Nov. 2013. ilus, tab
Article in English | LILACS | ID: lil-696555


Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/µl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/µl group, 5 in 38 alive pups in the 1 ng/µl group and 1 in 21 alive pups in the 4 ng/µl group ( p < 0.1). The overall transgenic efficiency was higher for the 1 ng/µl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.

Animals , Mice , Gene Transfer Techniques , Chemokine CXCL2 , Luciferases/genetics , In Vitro Techniques , DNA/analysis , Promoter Regions, Genetic , Cloning, Molecular , Cell Culture Techniques , Embryo Transfer , Genotype , Microinjections