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Journal of Forensic Medicine ; (6): 478-486, 2023.
Article in English | WPRIM | ID: wpr-1009381


Skeleton and teeth are important biological samples. Due to their special structure and strong ability to resist degradation, they are ideal biological materials to retain DNA under natural condition. In many cases, such as historical figure identification, aged skeleton and teeth are usually the only biological samples. However, their DNA is in a state of trace, damage and degradation to different degrees, which requires special experimental treatment to achieve identification. This paper reviews the sample selection, DNA extraction, DNA enrichment and analysis approaches based on relevant research reports in recent years, aiming to promote the further development and improvement of the aged skeleton and teeth identification system.

Humans , Aged , Body Remains , DNA/analysis , Tooth , DNA Fingerprinting , Sequence Analysis, DNA
Journal of Forensic Medicine ; (6): 161-167, 2023.
Article in English | WPRIM | ID: wpr-981850


With the advance of molecular biology, DNA analysis technology has been widely applied in forensic science. Non-human DNA analysis can be used in some special cases and has unique forensic value to provide investigation clues and trial basis. Animal DNA typing plays a more prominent role in the detection of all kinds of non-human DNA related cases and is the main content of forensic non-human DNA analysis. This paper reviews the development history, present situation, advantages and disadvantages of animal DNA typing according to its technology, characteristic, challenges facing forensic science application scenarios, and also its future development.

Animals , DNA Fingerprinting , Forensic Medicine , DNA/analysis , Forensic Sciences , Molecular Biology , Forensic Genetics
Acta sci., Health sci ; 44: e56960, Jan. 14, 2022.
Article in English | LILACS | ID: biblio-1367539


Colorectal cancer is the 4thcause of cancer death; with considering the growth process of this cancer and the necessity of early diagnosis, the purpose of the research is to state the LncRNA 00970, LncRNA UCAI,and the Wntgene before and after the treatment by 5-Azacytidine epigenetic medicine, to reach the biomarker in the very first steps of colorectal cancer. In this experiment, the human colon cancer cell line (HT29) treated with different concentrations of 5-aza-2'-deoxycytidine (5-aza-dC) was utilized to induce DNA demethylation; Quantitative PCR (qPCR) was used to measure LncRNA UCA1and LncRNA LINC00970 and Wntexpression. There was a significant relationship between the expression of LncRNA 00970, LncRNA UCAI,and the Wntgene and its effects on colorectal (p < 0.05). The Wntgene was treated by 1 and 10 of 5-Azacytidine epigenetic medicine, which then experienced decreases. In LncRNA UCAI and LncRNA00970 in dose 1 micromolar of 5-Azacytidine had decrement and increment of expressionrespectively that explains their efficiency but in treatment by dose 10 mM of this medicine, no significant LncRNA expression difference was detected, 5-azacitidine has a direct impact on its target genes and LncRNAs.Therefore, it can be used in the early diagnosis of colorectal cancer.

In Vitro Techniques/methods , DNA/analysis , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/therapy , Colonic Neoplasms/diagnosis , Early Diagnosis , Azacitidine/analysis , Azacitidine/antagonists & inhibitors , Biomarkers , Colorectal Neoplasms/mortality , Cell Line/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/therapy , Epigenomics , RNA, Long Noncoding , RNA, Long Noncoding/drug effects , Genes
Braz. J. Pharm. Sci. (Online) ; 58: e20096, 2022. tab, graf
Article in English | LILACS | ID: biblio-1403677


Abstract Dexchlorpheniramine is a first-generation classical antihistamine, clinically used to treat allergies. The main objective of our study was to evaluate the effects of the dexchlorpheniramine reference standard (DCPA Ref. St) and a pharmaceutical formula on DNA in human peripheral blood mononuclear cells (PBMCs). We exposed PBMCs to five different concentrations (0.5, 2.5, 5, 10, and 50 ng/mL) of DCPA Ref. St DCPA Ref. St and pharmaceutical formula in order to evaluate their cytotoxic, genotoxic, and mutagenic potential. The results showed that both dexchlorpheniramine formulations did not affect PBMC viability and CD3+, CD4+, or CD8+ lymphocyte subpopulations. The DCPA Ref. St and pharmaceutical formula neither induced genotoxic or mutagenic effects nor numerical or structural chromosomal alterations in PBMCs after 24 hours of exposure.

Humans , Leukocytes, Mononuclear/metabolism , Cytotoxicity, Immunologic , Drug Compounding , Genotoxicity , Mutagenicity Tests , DNA/analysis , Histamine Antagonists/adverse effects , Hypersensitivity/complications
Motriz (Online) ; 28: e10220004521, 2022. tab, graf
Article in English | LILACS | ID: biblio-1360604


Abstract Aim: This study analyzed the influences of ACE and ACTN3 gene variants in sprinters, jumpers, and endurance young athletes of track and field. Methods: 36 school-level competitors of both sex (15 girls and 21 boys; aged 16.4 ± 1.2 years; training experience 4 ± 1.2 years) practitioners of different sport disciplines (i.e., sprint, jump, and endurance athletes) participated in the study. The deoxyribonucleic acid (DNA) was extracted from peripheral blood using a standard protocol. Anthropometric measurements, 30 m sprint, squat jump (SJ), and maximal oxygen uptake (VO2max) tests were measured. Results: Genotype distribution of the ACE and ACTN3 genes did not differ between groups. In ACE DD and ACTN3 RX genotypes, the SJ test was bigger in sprinters and jumpers than in the endurance runners. In contrast, when analyzing the ACE ID genotype, sprinters had higher SJ than endurance athletes. Moreover, in the ACE DD genotype, the sprinters and jumpers' athletes had lower time in 30 m tests compared to endurance runners. However, the ACE ID and ACTN3 RX genotypes was greater aerobic fitness in endurance runners than in jumpers' athletes. Conclusion: Although the genetic profile is not a unique factor for determining athletic performance, the ACE DD and ACTN3 RX genotypes seem to favor athletic performance in power and sprint versus endurance sports. Thus, this study evidenced that assessing genetic variants could be used as an auxiliary way to predict a favorable profile for the identification of young talents of track and field.

Humans , Aptitude , Track and Field , Athletes , Genetic Profile , DNA/analysis
São Paulo; s.n; s.n; 2022. 117 p. tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-1416836


As variabilidades genotípicas que determinam algumas alterações fenotípicas e metabólicas podem ter seu diagnostico falho se baseado apenas nos dados genômicos. Na hipercolesterolemia familial (HF) pode-se observar que os dados de variantes nos genes da LDLR, PCSK9, APOB e LDLRAP1 sugeridos pelos consensos atuais para confirmar o diagnóstico, tem mostrado serem insuficientes, mostrando baixa porcentagem de confirmação, mesmo nos dos casos em que características fenotípicas apresentam dados sugestivos importantes. A complementação no auxílio diagnóstico com dados epigenéticos tem sido sugerida em muitas doenças, principalmente nas crônicos degenerativos. A metilação do DNA pode estar envolvida no mecanismo que regulam vários processos metabólicos, entre os quais os envolvidos na expressão de proteínas e neste estudo os que estão envolvidos no metabolismo do colesterol, que poderia explicar fenótipos hipercolesterolemicos sem demonstração clara de variantes nos genes de consenso. O objetivo do presente estudo foi comparar o perfil de metilação dos genes LDLR, PCSK9 e LDLRAP1 entre pacientes com diagnóstico de Hipercolesterolemia Familial confirmado através de variantes genéticas descritas na literatura e pacientes sem diagnóstico confirmado. Além da comparação com indíviduos normolipidêmicos. A seleção dos indivíduos foi realizada na Seção de Dislipidemia do Instituto Dante Pazzanese de Cardiologia (IDPC), do Departamento de Análises Clínicas e Toxicológicas da Universidade Federal do Rio Grande do Norte (UFRN), da Universidade Estadual de Campinas (UNICAMP) e da Faculdade de Medicina de São José do Rio Preto (FAMERP). Através dos critérios MEDPED foram selecionados 133 pacientes para a realização do sequenciamento de um painel de genes relacionados ao fenótipo de HF e a homeostasia do colesterol a fim de confirmar o diagnóstico. Todos os pacientes tiveram o DNA purificado, que foi submetido ao tratamento com bissulfito, amplificado, purificado, desnaturado e sequenciado no sistema PyroMark Q24. Avaliou-se o perfil de metilação, em sítio CpG dos genes da LDLR, PCSK9 e LDLR AP1. A análise estatística foi realizada utilizando o software SPSS v.19, GraphPad Prism, versão 1.03 e o e o software R. 4.1.0. Os pacientes foram classificados em Grupo I: Pacientes com diagnóstico molecular confirmado pelo estudo fenotípico e genotipico (n=40); Grupo II: Pacientes fenotipicamente determinados como hipercolesterolemico, mas sem diagnóstico molecular confirmado pelo estudo genomico (n=93); Grupo III: indivíduos fenotipicamente determinados normolipidêmicos de acordo com a V Diretriz Brasileira de Dislipidemia (n=23). A análise comparativa entre os grupos I x II e II x III, demonstrou diferença estatísta significativa em 13 sítios CpG do total de 28 sítios CpG analisados nos três genes. Além disso, foi possível concluir que alterações nos sítios CpG presentes no gene LDLR influenciaram na presença de xantomas e arco córneo. Houve correlação positiva entre a idade e perfil de metilação do gene PCSK9, assim como, alterações nos sítios CpG deste gene influenciaram na presença de arco córneo e IAM. Além disso, alterações no sítio CPG presente no gene LDLRAP1 influenciou no desenvolvimento de DAC, IAM e RM, além da presença de xantelasma

The genotypic variabilities that determine some phenotypic and metabolic alterations can be misdiagnosed if based only on genomic data. In familial hypercholesterolemia (FH) it can be observed that the data of variants in the genes of LDLR, PCSK9, APOB and LDLR AP1 suggested by the current consensus to confirm the diagnosis, has shown to be insufficient, showing a low percentage of confirmation, even in the cases in which phenotypic characteristics present important suggestive data. Complementing the diagnostic aid with epigenetic data has been suggested in many diseases, especially in chronic degenerative diseases. DNA methylation may be involved in the mechanisms that regulate several metabolic processes, including those involved in the expression of proteins that ,in this study, are involved in cholesterol metabolism, which could explain hypercholesterolemic phenotypes without a clear demonstration of variants in consensus genes. The aim of the present study was to compare the methylation profile of LDLR, PCSK9 and LDLRAP1 genes between patients with a diagnosis of Familial Hypercholesterolemia confirmed through genetic variants described in the literature and patients without a confirmed diagnosis. In addition to the comparison with normolipidemic individuals. The selection of individuals was carried out at the Dyslipidemia Section of the Instituto Dante Pazzanese de Cardiologia (IDPC), in the Department of Clinical and Toxicological Analyzes of the Federal University of Rio Grande do Norte (UFRN), in the State University of Campinas (UNICAMP) and in the Faculdade of Medicine of São José do Rio Preto (FAMERP). Through the MEDPED criteria, 133 patients were selected to perform the sequencing of a panel of genes related to the FH phenotype and cholesterol homeostasis in order to confirm the diagnosis. All patients had their DNA purified, which were subjected to bisulfite treatment, amplified, purified, denatured and sequenced on the PyroMark Q24 system. The methylation profile in the CpG site of the LDLR, PCSK9 and LDLRAP1 genes were evaluated. Statistical analysis were performed using SPSS v.19 software, GraphPad Prism, version 1.03 and R. 4.1.0 software. Patients were classified into Group I: Patients with a molecular diagnosis confirmed by phenotypic and genotypic studies (n=40); Group II: Patients phenotypically determined to be hypercholesterolemic, but without a molecular diagnosis confirmed by the genomic study (n=93); Group III: phenotypically determined normolipidemic individuals according to the V Brazilian Dyslipidemia Directive (n=23). The comparative analysis between groups I x II and II x III showed a statistically significant difference in 13 CpG sites of the total of 28 CpG sites analyzed in the three genes of the project. Furthermore, it was possible to conclude that alterations in the CpG sites present in the LDLR gene influenced the presence of xanthomas and arc corneum. There was a positive correlation between age and PCSK9 gene methylation profile, as well as changes in the CpG sites of this gene influenced the presence of arc corneum and AMI. In addition, alterations in the site present in the LDLRAP1 gene are influencing the development of CAD, AMI and MR, in addition to the presence of xanthelasma

Humans , Male , Female , DNA/analysis , Proprotein Convertase 9/analysis , Hyperlipoproteinemia Type II/diagnosis , Coronary Artery Disease/classification , Clinical Laboratory Techniques/methods , Molecular Diagnostic Techniques/methods , Diagnosis
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 | VETINDEX, LILACS | 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
Journal of Forensic Medicine ; (6): 825-831, 2021.
Article in English | WPRIM | ID: wpr-984081


OBJECTIVES@#To evaluate the effects of different pretreatment methods and preservation time on RNA quality of peripheral blood samples, and to optimize the preservation method of peripheral blood samples.@*METHODS@#Eight pretreatment methods were used to preprocess the peripheral blood from 3 healthy unrelated individuals and the treated samples were stored at -80 ℃. Total RNA of samples was extracted using Quick-RNATM Miniprep Plus kit. DNA/RNA ShieldTM was added to peripheral blood and total RNA was extracted after preservation at -80 ℃ for 0, 5, 10, 15, 30 and 60 days, respectively. The concentration, purity and integrity of RNA were determined. Statistical analyses were performed by SPSS 22.0 software to compare the differences in RNA yield, purity and integrity among the eight pretreatment methods.@*RESULTS@#In terms of purity, leukocyte pretreated with RNAlaterTM and directly cryopreservation peripheral blood showed the worst purity. The other six methods showed better purity. In terms of yield, blood cells with DNA/RNA ShieldTM came out with the highest yield, followed by peripheral blood with DNA/RNA ShieldTM. In terms of integrity, peripheral blood preserved in PAXgene Blood RNA tube method had the best integrity. Except for peripheral blood pretreated with DNA/RNA ShieldTM and blood cells pretreated with DNA/RNA shieldTM, the other five methods had statistical differences when compared to the method by keeping peripheral blood in PAXgene Blood RNA tube. The purity of RNA stored at six-time gradients ranged from 1.815 to 1.952. With the increase of storage time, RNA yield decreased from 4.516 ng to 1.039 ng, and RNA integrity decreased from 8.533 to 7.150.@*CONCLUSIONS@#According to the results of total RNA's yield, purity and integrity, peripheral blood pretreated with DNA/RNA ShieldTM was the best pretreatment method. After the pretreatment, samples can be preserved for up to 60 days in low temperature.

Humans , Blood Specimen Collection/methods , Cryopreservation , DNA/analysis , RNA
Bol. micol. (Valparaiso En linea) ; 35(2): 15-24, dic. 2020. ilus
Article in Spanish | LILACS | ID: biblio-1437201


El término de onicomicosis se emplea para describir las infecciones de las uñas causadas por diferentes grupos taxonómicos fúngicos ya sea filamentosos como levaduriformes. A pesar de que estas patologías son causadas en los vertebrados principalmente por integrantes de la Familia Artrodermatáceae (Onygenales), la micología médica aplicó para ellos la terminología más específica de dermatofitosis, por ser un grupo ecológico de mayor importancia y presencia clínica. Las dermatomicosis de piel y fanéreos, representan un conjunto de infecciones producidas por especies fúngicas distribuidas en ambientes diversos, capaces de crecer a temperaturas de 37° y que actúan usualmente como patógenos oportunistas cuando existe generalmente un factor predisponente en el huésped. Se destaca la colonización en una uña de los pies en un hombre de 49 años por Neoscytalidium dimidiatum(Penz.) Crous & Slippers, un reconocido fitopatógeno de rápido crecimiento, común en zonas tropicales y subtropicales, que presentó la capacidad de invadir tejidos queratinizados con un aspecto clínico indistinguible de los causadas por dermatofitos. Por la rara presencia de este hongo en nuestra zona geográfica (provincia de Valparaíso, Chile), se aportan los principales datos morfofisiológicos,taxonómicos y moleculares utilizados en su diagnóstico. (AU)

The term onychomycosis is used to describe nail infections caused by different fungal taxonomic groups, either filamentous or yeast. Despite the fact that these pathologies are caused in vertebrates mainly by members of the Artrodermatáceae Family (Onygenales), medical mycology applied the more specific terminology of dermatophytosis for them, as it is an ecological group of greater importance and clinical presence. Skin and pharynx dermatomycosis represent a set of infections produced by fungal species distributed in diverse environments, capable of growing at temperatures of 37° and that usually act as opportunistic pathogens when there is a predisposing factor in the host. The colonization on a toenail in a 49-year-old man by Nesoscytalidium dimidiatumis highlighted (Penz.) Crous & Slippers, a recognized fast-growing phytopathogen, common in tropical and subtropical areas, which presented the ability to invade keratinized tissues with a clinical appearance indistinguishable from those caused by dermatophytes. Due to the rare presence of this fungus in our geographical area (Valparaíso province, Chile), the main morphophysiological, taxonomic and molecular data used in its identificationare provided. (AU)

Humans , Male , Middle Aged , Ascomycota/pathogenicity , Onychomycosis/etiology , Ascomycota/cytology , Ascomycota/classification , Ascomycota/physiology , DNA/analysis , Chile , Genome Components , Dermatomycoses/diagnosis
Rev. biol. trop ; 68(2)jun. 2020.
Article in Spanish | LILACS, SaludCR | ID: biblio-1507694


Introducción: Recientemente ha tomado relevancia el uso de especímenes de museo como fuente de información genética para desarrollar estudios que resuelven preguntas taxonómicas, ecológicas, demográficas y evolutivas a diversas escalas temporales y geográficas. Sin embargo, material genético obtenido a partir de ejemplares depositados en colecciones biológicas es poco usado, debido al deterioro natural del ADN preservado en dichos ejemplares, de manera que la obtención de material genético de calidad es demandante en términos de tiempo y dinero. Objetivo: Usando material de museo, identificar una secuencia mini-barcode que pueda ser empleada en la determinación taxonómica, y que a su vez suministre información que permita la estimación de relaciones filogenéticas de especies del género Bombus. Métodos: Se estandarizó el protocolo de extracción de ADN a partir de la extremidad mesotoracica derecha y/o una muestra de músculo torácico de 96 especímenes depositados en la colección LABUN entre 7 y 38 años atrás. Las diferentes combinaciones de oligonucleótidos evaluadas permitieron amplificar fragmentos de 152 a 407 pares de bases (pb) del gen mitocondrial Cytochrome Oxidase I (COI). Usando como plantilla un grupo de 31 secuencias amplificadas a partir de especímenes recolectados recientemente, los fragmentos obtenidos de los especímenes del museo fueron ensamblados y analizados en un marco filogenético. Además, se realizó un análisis de red de haplotipos para evaluar en detalle las relaciones entre los haplotipos mitocondriales resultantes. Resultados: Se determinó un mayor éxito de extracción de ADN a partir de muestras de extremidad depositadas a partir del año 1982.Entretanto, la amplificación exitosa de fragmentos de más de 300 pares de bases (pb) se logró principalmente en muestras depositadas en fechas posteriores a 1999, lo que indica una mayor integridad del material genético recuperado de individuos de 19 años de recolección en adelante. Aunque todos los fragmentos evaluados pueden ser empleados como mini-barcode, solo con uno se obtiene una topología similar a la observada con el fragmento completo. Se detectó una gran variacion genética, particularmente al interior de las especies Bombus atratus y B. funebris, en las que se reveló una clara estructura filogeográfica. Conclusiones: Se obtuvieron nuevas secuencias de códigos de barras mediante extracción de ADN y protocolo de amplificación de muestras de museos. Además, se generó nueva información sobre la variabilidad genética intraespecífica, detectando la presencia de haplotipos mitocondriales únicos que podrían constituir Unidades Significativas Evolutivas sujetas a conservación. Dicha información es de vital importancia para formular estrategias de conservación para estos polinizadores en Colombia.

Introduction: The use of museum specimens as a source of genetic information to develop studies that resolve taxonomic, ecological, demographic, and evolutionary questions at various temporal and geographic scales, has recently become relevant. However, genetic material obtained from specimens deposited in biological collections is not used frequently due to the natural deterioration of the DNA preserved in these specimens. Getting quality genetic material is demanding in terms of time and money. Objective: By using museum material,to identify a mini-barcode sequence that can be used in the taxonomic determination and provides information that allows the estimation of phylogenetic relationships of species of the genus Bombus. Methods: The DNA extraction protocol for museum samples was standardized using the mesothoracic right leg and / or a sample of thoracic muscle of 96 specimens deposited in the LABUN collection between 7 and 38 years ago. Different combinations of oligonucleotides allowed to amplify fragments from 152 to 407 base pairs (bp) of the mitochondrial gene Cytochrome Oxidase I (COI). Using as a template a group of 31 sequences amplified from recently collected specimens, the fragments obtained from the museum specimens were assembled and analyzed in a phylogenetic framework. Additionally, a haplotype network analysis was performed in order to evaluate in detail the relationships between the resulting mitochondrial haplotypes. Results: The greatest success of DNA extraction was achieved from limb samples deposited since the year 1982 on. Meanwhile, successful amplification of fragments longer than 300 base pairs (bp) was achieved mostly in samples deposited on dates after 1999, which indicates greater integrity of the genetic material recovered from individuals of 19 years of collection and onwards. Although all the fragments evaluated can be used as mini-barcode, only with one primer pair, it was possible to obtain a topology similar to that observed with the complete fragment. A large genetic variation was detected, particularly within the Bombus atratus and B. funebris species, in which a clear phylogeographic structure was revealed. Conclusions: New barcode sequences were obtained through DNA extraction and amplification protocol from museum samples. Furthermore, new information on intraspecific genetic variability was generated, detecting the presence of unique mitochondrial haplotypes that could constitute management units subject of conservation. Such information is of vital importance to formulate conservation strategies for these pollinators in Colombia.

Animals , Bees/genetics , DNA/analysis , Hymenoptera/genetics , Colombia , Collection
Mem. Inst. Oswaldo Cruz ; 115: e200157, 2020. tab, graf
Article in English | 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. ciênc. vet ; 26(4): 152-157, out./dez. 2019. il.
Article in English | LILACS, VETINDEX | ID: biblio-1380143


The seasons influence the production of buffalos' milk. Because of this, the producers may produce a mixture of buffalo and bovine milk during cheese production in periods of low production. Therefore, the present work aimed to investigate fraud in buffalo cheese and the relationship between seasonality and different physicochemical properties of buffalo cheeses produced and marketed in eastern Amazonia. We obtained commercial samples of buffalo cheese during two Amazonian climatic periods from commercial points of Marajó-Pará, Brazil. After collection, there were lipid, protein, ash, and humidity analyses. Determination of carbohydrates and energy values was also performed for the nutritional characterization of samples, as well as for mPCR analysis to detect buffalo and/or bovine DNA. DNA extraction protocol of the samples was standardized and two pairs were used for the mPCR reaction, amplifying fragments of approximately 220 bp for Bubalus bubalis DNA and 346 bp fragments for Bos taurus DNA. Among the samples acquired in the rainy season, we observed that 33% were inadequately labeled, indicating fraud from cow's milk incorporation and fraud from substitution of raw material. From the nine samples obtained in the dry season, all the samples showed cow's milk incorporation fraud. The highest fraud rate coincided with the period of low milk production from buffalo and there was a difference in composition between fraudulent and non-fraudulent cheeses. Therefore, seasonality influences increase in cattle milk for the production of buffalo cheese, and this adulteration may decrease the nutritional content of the product.

As estações climáticas influenciam a produção de leite de búfala. Isso pode levar os produtores a misturarem os leites de búfala e bovino durante a produção de queijo em períodos de baixa produção. Portanto, o presente trabalho teve como objetivo verificar fraudes em queijo de búfala, a relação com a sazonalidade e as diferenças físico-químicas de queijos de origem bubalina, produzidos e comercializados no leste da Amazônia. Foram coletadas amostras comerciais de queijo de búfala em dois períodos climáticos da Amazônia em pontos comerciais do Marajó-Pará, Brasil. Após a coleta foram realizadas análises de lipídios, proteínas, cinzas e umidade. A determinação dos carboidratos e do valor energético também foi feita para a caracterização nutricional das amostras, bem como a análise de mPCR para a detecção de DNA de búfalo e/ou bovino. Para isso, padronizou-se um protocolo de extração de DNA das amostras e utilizou-se dois pares na reação mPCR, amplificar fragmentos de aproximadamente 220 pb para o DNA de Bubalus bubalis e fragmentos de 346 pb para o Bos taurus. Entre as amostras adquiridas na estação chuvosa, observou-se que 33% foram rotuladas inadequadamente, caracterizando fraude por incorporação de leite de vaca e fraude por substituição de matéria-prima. Das 9 amostras coletadas no período seco, todas as amostras apresentaram fraude na incorporação do leite de vaca. Este estudo revelou que a maior taxa de fraude coincide com o período de baixa produção de leite e que há uma diferença na composição entre queijos fraudulentos e não fraudulentos. Portanto, a sazonalidade influencia no acréscimo de leite de bovinos na produção de queijo de búfala e que esta adulteração pode diminuir o conteúdo nutricional do produto.

DNA/analysis , Buffaloes , Food Production , Cheese/analysis , Polymerase Chain Reaction , Dairying/ethics , Milk , Fraud/prevention & control
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
Journal of Forensic Medicine ; (6): 526-531, 2018.
Article in English | WPRIM | ID: wpr-984968


OBJECTIVES@#To investigate the genetic polymorphisms of 18 autosomal short tandem repeats (STR) loci in Changsha Han population, and explore the population genetic relationships and evaluate its application value in forensic medicine.@*METHODS@#The DNA of 2 004 unrelated individuals in Changsha Han population were amplified using Goldeneye®DNA ID System BASIC, and the PCR products were analyzed by electrophoresis using 3130xl genetic analyzer. The fragment sizes of alleles were analyzed subsequently by GeneMapper® ID v3.2. The frequency data and forensic genetic parameters [observed heterozygosity (Ho), expected heterozygosity (He), power of discrimination (DP) and polymorphic information content (PIC)] of 18 STR loci were statistically analyzed. Total probability of discrimination (TDP), probability of exclusion in trio cases (PEtrio) and probability of exclusion in duo cases (PEduo) were calculated by Cervus 3.0. Hardy-Weinberg equilibrium and linkage disequilibrium of the loci were detected by Arlequin v3.5. The results were compared with the available data of other populations from different races and regions.@*RESULTS@#The power of discrimination (DP), and the polymorphic information content (PIC) of each locus of Changsha Han population ranged from 0.783 6 to 0.987 9 and 0.549 4 to 0.914 5, respectively. The TDP, cumulative probability of exclusion in trio cases (CPEtrio) and cumulative probability of exclusion in duo cases (CPEduo) were 0.999 999 999 999 999 999 999 865 2, 0.999 999 979 and 0.999 988 325, respectively. According to the Nei's DA genetic distance, the genetic distance between Changsha Han and Hunan Han populations was the smallest (0.014 1), while it was the largest (0.041 8) between Changsha Han and Xinjiang Kazakh populations.@*CONCLUSIONS@#The 18 STR loci shows abundant genetic polymorphisms in Changsha Han population. The study of genetic diversity among different populations has an important meaning for the research of their origins, migrations and their relationships.

Humans , Male , Alleles , Asian People/genetics , China , DNA/analysis , Gene Frequency , Genetics, Population , Microsatellite Repeats , Polymorphism, Genetic
Journal of Forensic Medicine ; (6): 401-404, 2018.
Article in English | WPRIM | ID: wpr-984952


OBJECTIVES@#To collect single piece of dandruff with microscopes to improve the regular EZ-tape method for DNA extraction and genotyping, increase the utilization of samples, reduce the miss rate as well as the proportion of genotyping results of mixed stains.@*METHODS@#The insides of the hats worn by two volunteers were stuck by EZ-tape and scotch tape respectively. DNA on EZ-tape was directly extracted using traditional method. Single piece of dandruff was collected from the scotch tapes under microscope. The two kinds of methods were both performed under continuous oscillation and standing digestion, respectively. DNA was extracted through Chelex-100 method, and STR genotypes were obtained after amplification and electrophoresis. The results of STR genotypes obtained by EZ-tape method and single piece of dandruff analytical method were compared.@*RESULTS@#Miss detections happened in 11 samples (45.8%) by EZ-tape method and only single-source typing results were obtained. Ten samples (41.7%) showed the genotype results of mixed stain and six of which showed allele insertions and deletions. The genotype results were obtained successfully using the single piece of dandruff analytical method, and two samples showed mixed stain genotype. The number of exact typing processed by oscillation was higher than that by standing digestion ( P<0.05).@*CONCLUSIONS@#The oscillation during the DNA extraction process is in favour of the DNA releasing. Single piece of dandruff analytical method can be used to obtain single-source STR genotype with high successful ratio and low miss rate. This method can be a collection method of special samples such as dandruff in forensic practice.

Humans , Alleles , DNA/analysis , DNA Fingerprinting/methods , Dandruff/genetics , Genotype , Microsatellite Repeats , Resins, Synthetic