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1.
J. Health Biol. Sci. (Online) ; 10(1): 1-5, 01/jan./2022. ilus
Article in English | LILACS | ID: biblio-1411474

ABSTRACT

Objective: The study aimed to evaluate molecular and immunological methods and to propose a workflow using them for tuberculosis (TB) diagnosis routine. Methods: A cross-sectional retrospective study was performed, including 121 liquid cultures from a TB laboratory located in the extreme south of Brazil. All cultures were positive for Mycobacterium tuberculosis complex (MTBC) by in-house Polymerase Chain Reaction (PCR) using DNA extracted by the CTAB method (PCR-CTAB) for IS6110 detection. These cultures were subjected to faster tests than this one, the immunological MPT64 assay and the PCR using DNA extracted by thermal lysis method (PCR-TL), and these were evaluated for MTBC identification using PCR-CTAB as a reference method. Results: The sensitivity of MPT64 assay and PCR-TL to identify MTBC in positive cultures by PCR-CTAB were 73.6% (89/121) and 98.3% (119/121), respectively. We proposed a workflow based on the use of MPT64 assay in liquid cultures suggestive of MTBC, and in case of a negative result, we suggest the performance of PCR-TL. The PCR-CTAB is suggested only if faster tests are negative. Conclusions: Methods capable of confirming MTBC in cultures should continue to be standardized, tested, and optimized to meet the ideal requirements of simplicity, quickness, and effectiveness. The molecular and immunological methods evaluated have differences in the execution and detection of MTBC in cultures, but they are rapid tools for laboratory TB diagnosi


Objetivos: O estudo objetivou avaliar métodos molecular e imunológico e propor um fluxo de trabalho utilizando-os para a rotina de diagnóstico da tuberculose (TB). Métodos: Foi realizado um estudo transversal retrospectivo, incluindo 121 culturas líquidas de um laboratório de TB localizado no extremo sul do Brasil. Todas as culturas foram positivas para o complexo Mycobacterium tuberculosis (CMTB) por Reação em Cadeia da Polimerase (PCR) in-house para detecção do IS6110, usando DNA extraído pelo método CTAB (PCR-CTAB). Essas culturas foram submetidas a testes mais rápidos que este, o ensaio imunológico MPT64 e a PCR com DNA extraído pelo método de lise térmica (PCR-LT), e estas foram avaliadas para identificação de CMTB usando PCR-CTAB como método de referência. Resultados: A sensibilidade do ensaio MPT64 e da PCR-LT para identificar o CMTB em culturas positivas pela PCRCTAB foi de 73,6% (89/121) e 98,3% (119/121), respectivamente. Propusemos um fluxo de trabalho baseado no uso do ensaio MPT64 em culturas líquidas sugestivas de CMTB e, em caso de resultado negativo, sugerimos a realização de PCR-LT. Sugere-se a PCR-CTAB apenas se os testes mais rápidos forem negativos. Conclusões: Os métodos capazes de confirmar o CMTB em culturas devem continuar sendo padronizados, testados e otimizados para atender aos requisitos ideais de simplicidade, rapidez e eficácia. Os métodos molecular e imunológico avaliados apresentam diferenças na execução e detecção do CMTB em culturas, mas são ferramentas rápidas para o diagnóstico laboratorial da TB.


Subject(s)
Mycobacterium tuberculosis , Tuberculosis , DNA , Polymerase Chain Reaction , Diagnostic Tests, Routine , Cetrimonium , Mycobacterium
2.
Rev. peru. med. exp. salud publica ; 39(3): 312-320, jul.-sep. 2022. tab, graf
Article in Spanish | LILACS | ID: biblio-1410010

ABSTRACT

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.


ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.


Subject(s)
Humans , Bordetella pertussis , DNA , Molecular Diagnostic Techniques , Cellulose , Whooping Cough , Evaluation Study , Real-Time Polymerase Chain Reaction , Point-of-Care Testing
3.
Poblac. salud mesoam ; 19(2)jun. 2022.
Article in Spanish | LILACS-Express | LILACS, SaludCR | ID: biblio-1386950

ABSTRACT

Resumen Introducción: las radiaciones ionizantes (RI) son capaces de perjudicar el ADN; para evaluar este fenómeno es posible utilizar la formación de micronúcleos como biomarcador de efecto temprano del daño radioinducido. El ensayo de micronúcleos con bloqueo de la citocinesis (MNBC) es una técnica citogenética que permite demostrar el impacto de agentes genotóxicos. Propósito: en el presente trabajo se describieron mecanismos moleculares involucrados en la radioinducción de micronúcleos, la técnica del MNBC, los criterios de análisis, sus aplicaciones dentro de la investigación biológica y su extensión a la clínica, con énfasis en su empleo como biomarcador del daño genético en grupos sobreexpuestos a RI. Argumentos para la discusión: el MNBC se considera un método confiable, simple y rápido y existe evidencia de su aplicabilidad para el estudio de los efectos biológicos en casos de riesgo ocupacional y en accidentes radiológicos aislados o a gran escala. Conclusiones: el MNBC es una herramienta valiosa que posibilita estimar las consecuencias por dosis bajas de RI en poblaciones involucradas y, a la vez, orientar la toma de decisiones en cuanto a su prevención o atenuación . De igual forma, puede ser utilizado en análisis del campo de la radiobiología, a fin de detallar las incidencias de las radiaciones ionizantes sobre el ADN.


Abstract Introduction. Ionizing radiation (IR) is capable of causing DNA damage. For the evaluation of this phenomenon it is possible to use chromosomal aberrations as biomarkers. The Cytokinesis-Block Micronucleus assay (CBMN) is a cytogenetic technique that allows to demonstrate the effect of genotoxic agents.Proposition:in the present review, we will describe the molecular mechanisms involved in micronucleus radioinduction, the micronucleus technique and criteria for analysis, its applications within biological research and its extension in clinical research, with emphasis on its application as a biomarker of radioinduced genetic damage. Arguments for discussion: the CBMN is considered a reliable, simple and fast technique and there is evidence of its applicability in the evaluation of biological effects in occupationally exposed personnel and in isolated or large-scale radiological accidents. Conclusions: the CBMN a valuable tool in estimating radiological risk in populations exposed to low doses of IR, allowing to guide decision-making regarding prevention or mitigation of exposure to IR in populations involved. Similarly, the cbmn can be used in research in the field of radiobiology, as a means to describe the effects of ionizing radiation on DNA.


Subject(s)
Humans , Radiation, Ionizing , DNA , Cytogenetic Analysis
4.
An. Fac. Cienc. Méd. (Asunción) ; 55(1): 27-38, 20220401.
Article in Spanish | LILACS, BDNPAR | ID: biblio-1366663

ABSTRACT

Introducción: Procesos como la mutagénesis, la carcinogénesis y la teratogénesis son producto de la interacción de agentes de origen endógeno como exógeno que interactúan con la molécula de ADN en forma crónica produciendo rupturas en la doble hélice, y en cromosomas completos resultando en la inestabilidad genómica. El estrés oxidativo al que se encuentran sometidas las células al formarse las especies reactivas de oxígeno (ROS) y también las especies reactivas de nitrógeno (RNS), que pueden provenir de radicales producidos a consecuencia de la diabetes o en estados iniciales de la enfermedad renal crónica o como respuesta a procesos inflamatorios en estados avanzados de estas patologías, actúan como agentes genotóxicos endógenos.Objetivos: Esta investigación tuvo como objetivo determinar el daño basal en la molécula de ADN de pacientes diabéticos hemodializados, a través del ensayo del Cometa, como un bioindicador de inestabilidad genómica., durante seis meses de tratamiento. Materiales y métodos: Se planteó un estudio longitudinal prospectivo de cohorte para comparar los diferentes niveles de daño antes y durante los primeros seis del tratamiento de hemodiálisis. Se evaluó con el test del cometa o electroforesis de células individuales, el daño basal en muestras de sangre venosa de pacientes diagnosticados con Diabetes de tipo II como control negativo y en pacientes diabéticos con enfermedad renal crónica antes de iniciar el tratamiento de diálisis y luego durante el tratamiento. Se utilizó el test de t- Student para muestras independientes y emparejadas. Resultados: Se observó un aumento significativo de daño basal y oxidativo en el material genético de pacientes diabéticos con enfermedad renal crónica, comparados con los controles negativos (p< 0.005) y se observó, además, que el daño celular aumenta con el tratamiento de hemodiálisis (p<0.005). Conclusión: Los resultados obtenidos en esta investigación permiten concluir que el estrés oxidativo tiene un efecto genotóxico y que el nivel de daño genético es un buen bioindicador del avance de la enfermedad renal crónica y que la hemodiálisis induce a un aumento de daño a nivel del material genético, aumentando el riesgo de carcinogénesis.


Introduction: Processes such as mutagenesis, carcinogenesis and teratogenesis are the product of the interaction of agents of endogenous and exogenous origin that interact with the DNA molecule in a chronic way producing ruptures in the double helix, and in complete chromosomes resulting in genomic instability. The oxidative stress to which the cells are subjected when reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed, which may come from radicals produced as a result of diabetes or in initial stages of chronic kidney disease or in response to inflammatory processes in advanced stages of these pathologies, act as endogenous genotoxic agents. Objectives: This research aimed to determine the basal damage in the DNA molecule of hemodialyzed diabetic patients, through the Comet assay, as a bioindicator of genomic instability, during six months of treatment. Materials and methods: For this research, a prospective longitudinal cohort study was proposed to compare the different levels of genetic damage before and during the first six of hemodialysis treatment. Baseline damage was evaluated with the comet test or single cell electrophoresis, in venous blood samples from patients diagnosed with Type II Diabetes as a negative control and in diabetic patients with chronic kidney disease before starting dialysis treatment and then during treatment. Results: A significant increase in basal and oxidative damage was observed in the genetic material of diabetic patients with chronic kidney disease, compared to negative controls (p< 0.005) and it was also observed that cell damage increases with hemodialysis treatment (p<0.005). The t-Student test was used for independent and paired samples. Conclusion: The results obtained in this research allow us to conclude that oxidative stress has a genotoxic effect and that the level of genetic damage is a good bioindicator of the progression of chronic kidney disease and that hemodialysis induces an increase in damage at the level of the genetic material, increasing the risk of carcinogenesis.


Subject(s)
Renal Dialysis , Comet Assay , Dialysis , Research , DNA , Oxidative Stress
5.
Acta sci., Health sci ; 44: e56960, Jan. 14, 2022.
Article in English | LILACS | ID: biblio-1367539

ABSTRACT

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.


Subject(s)
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
6.
São Paulo; s.n; s.n; 2022. 116 p. tab, graf.
Thesis in English | LILACS | ID: biblio-1378343

ABSTRACT

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies


Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC


Subject(s)
Stem Cells , Biomarkers/analysis , SELEX Aptamer Technique/instrumentation , Mesenchymal Stem Cells/classification , ADAM17 Protein/pharmacology , Patient Isolation , Mass Spectrometry/methods , Staining and Labeling/methods , Transplantation/adverse effects , Umbilical Cord , DNA/agonists , Transforming Growth Factors/agonists , Cell Separation/instrumentation , Cytokines/adverse effects , Adipocytes/metabolism , Chondrocytes/classification , Scientists for Health and Research for Development , Adult Stem Cells/classification , Fibroblasts/chemistry , Flow Cytometry/instrumentation , Germ Layers , Antigens/adverse effects
7.
São Paulo; s.n; s.n; 2022. 172 p. tab, graf.
Thesis in English | LILACS | ID: biblio-1378625

ABSTRACT

The solar ultraviolet (UV) radiation that reaches the Earth is composed of 95% of UVA (320 to 400 nm) and 5% of UVB (280 to 320 nm) radiation. UVB is carcinogenic, generating potentially mutagenic DNA lesions. The solar UVA radiation also causes DNA damage, but this fact does not fully account for its biological impact. UVA is absorbed by non-DNA cellular chromophores, generating reactive oxygen species such as singlet oxygen. Knowing the proteome mediates stress responses in cells, here we investigated the cellular effects of a non-cytotoxic dose of UVA radiation, equivalent to about 20 minutes of midday sun exposure, on the proteome of human keratinocytes. Using a combination of mass spectrometry-based proteomics, bioinformatics, and conventional biochemical assays, we analyzed two aspects of UVA-induced stress: spatial remodeling of the proteome in subcellular compartments 30 minutes after stress and long-term changes in protein levels and secretion (24 hours and 7 days postirradiation). In the first part of this thesis, we quantified and assigned subcellular localization for over 3000 proteins, of which about 600 potentially redistribute upon UVA exposure. Protein redistributions were accompanied by redox modulations, mitochondrial fragmentation and DNA damage. In the second part of the work, our results showed that primary human keratinocytes enter senescence upon exposure to a single dose of UVA, mounting antioxidant and inflammatory responses. Cells under UVA-induced senescence further elicit paracrine responses in neighboring premalignant HaCaT epithelial cells via inflammatory mediators. Altogether, these results reiterate the role of UVA radiation as a potent metabolic stressor in the skin


A radiação ultravioleta (UV) solar que atinge a superfície terrestre é composta por 95% de radiação UVA (320 a 400 nm) e 5% de radiação UVB (280 a 320 nm). A radiação UVB é carcinogênica e gera lesões potencialmente mutagênicas no DNA. A radiação UVA solar também gera danos no DNA, mas a genotoxicidade dessa radiação não explica inteiramente o seu impacto biológico. Atualmente, sabe-se que a radiação UVA é absorvida por cromóforos celulares, gerando espécies reativas de oxigênio, como o oxigênio singlete. Sabendo que o proteoma é um mediador de respostas ao estresse celular, nós investigamos os efeitos celulares de uma dose não-citotóxica de radiação UVA, equivalente a cerca de 20 minutos de exposição ao sol, no proteoma de queratinócitos humanos. Utilizando espectrometria de massas, bioinformática e ensaios bioquímicos convencionais, nós analisamos dois aspectos do estresse induzido por radiação UVA: o remodelamento espacial do proteoma 30 minutos depois do estresse e alterações nos níveis e na secreção de proteínas no longo prazo (24 horas e 7 dias depois da irradiação). Na primeira parte desta tese, nós quantificamos e atribuímos classificações de localização subcelular a mais de 3000 proteínas. Dentre essas proteínas, 600 tem potencialmente a sua distribuição subcelular alterada em resposta à radiação. As redistribuições subcelulares são acompanhadas de modulações redox, fragmentação mitocondrial e danos no DNA. Na segunda parte da tese, os nossos resultados mostraram que queratinócitos humanos primários entram em senescência sob exposição a uma única dose de radiação UVA, montando respostas antioxidantes e pró-inflamatórias. Células sob senescência induzida por UVA, por sua vez, desencadeiam respostas parácrinas em queratinócitos pré-tumorais (células HaCaT) por meio de mediadores inflamatórios. Em conjunto, esses resultados reiteram o papel da radiação UVA como um potente estressor metabólico em células da pele


Subject(s)
Skin , Ultraviolet Rays/adverse effects , Keratinocytes/chemistry , Proteomics/classification , Radiation Dosage , Mass Spectrometry/methods , DNA , Epithelial Cells/classification , Genotoxicity/adverse effects , HaCaT Cells/classification , Antioxidants/adverse effects
8.
Med. lab ; 26(3): 215-218, 2022. ilus
Article in Spanish | LILACS | ID: biblio-1412313

ABSTRACT

Para el Editor: Cerca de 4.500 casos de viruela del mono o viruela símica (MPX, del inglés, Monkeypox), una enfermedad zoonótica rara, han sido reportados en el mundo, tres de ellos en Colombia, lo cual ha puesto en alerta a los médicos y científicos que buscan respuestas. El virus de la viruela del mono (MPXV) fue aislado por primera vez en 1958 a partir de unos monos enfermos, y en 1970 se confirmó el primer caso en humanos en un niño en la República Democrática del Congo [1]. El MPXV es un virus que pertenece a la familia de los Poxviridae, género Orthopoxvirus, con genoma de cadena doble de ADN, y apariencia oval o en forma de ladrillo bajo el microscopio electrónico (figura 1), que causa una enfermedad en humanos similar a la viruela, pero con menor mortalidad [2]. Es endémico en África central y occidental, con brotes esporádicos en el hemisferio occidental asociados con el comercio de animales exóticos y viajes a esas regiones. El reservorio aún se desconoce, pero se sospecha que es un roedor o un mamífero pequeño [3]. La inmunidad parcial contra este virus se consiguió previamente con la vacuna contra la viruela, pero la suspensión de su uso rutinario como resultado de la erradicación de la viruela, abrió el escenario para la aparición de brotes de viruela del mono con importancia clínica


Subject(s)
Humans , Animals , Monkeypox , DNA , Zoonoses , Infections
9.
Motriz (Online) ; 28: e10220004521, 2022. tab, graf
Article in English | LILACS | ID: biblio-1360604

ABSTRACT

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.


Subject(s)
Humans , Aptitude , Track and Field , Athletes , Genetic Profile , DNA/analysis
10.
Protein & Cell ; (12): 316-335, 2022.
Article in English | WPRIM | ID: wpr-929165

ABSTRACT

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.


Subject(s)
Animals , CRISPR-Cas Systems/genetics , DNA/genetics , Embryo, Mammalian/metabolism , Endonucleases/metabolism , Gene Editing , Mammals/metabolism
11.
Article in English | WPRIM | ID: wpr-929068

ABSTRACT

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.


Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , DNA , Gene Editing/methods , Humans , Streptococcus pyogenes/metabolism
12.
Article in English | WPRIM | ID: wpr-939831

ABSTRACT

Neutrophils are predominant leukocytes in the circulation, which are essential for killing invading pathogens via the activation of effector responses and the production of reactive oxygen species (ROS), also named as "oxidative burst." When infected, activated neutrophils fight bacteria, fungi, and viruses through oxidative burst, phagocytosis, degranulation, and the production of neutrophil extracellular traps (NETs) in a neutrophil death process named as "NETosis" (Mutua and Gershwin, 2021). NETs, consisting of DNA fibers decorated with modified histones and numerous antimicrobial proteins from cytoplasmic granules and the nucleus, can either be beneficial or detrimental (Mutua and Gershwin, 2021). Several pathways can lead to this death process. In response to various stimuli, NETosis traps and clears pathogens, facilitating phagocytosis by other neutrophils and phagocytes. However, excessive NETosis often results in disease due to increasing the pro-inflammatory response and perpetuating the inflammatory condition (Hellebrekers et al., 2018; Hidalgo et al., 2019; Klopf et al., 2021). Accordingly, inhibiting aberrant NETosis may alleviate the severity of various autoimmune and inflammatory diseases.


Subject(s)
DNA , Extracellular Traps/metabolism , Neutrophils/metabolism , Reactive Oxygen Species/metabolism , Respiratory Burst
13.
Article in Chinese | WPRIM | ID: wpr-936145

ABSTRACT

OBJECTIVE@#To investigate the consistency of cytomegalovirus deoxyribo nucleic acid (CMV-DNA) and immunoglobulin M (IgM) antibody detections in patients with different clinical characteristics and their guiding value for clinical practice.@*METHODS@#From December 2014 to November 2019, a total of 507 patients who were detected with both CMV-IgM and CMV-DNA were collected in Peking University International Hospital. Their general information, such as gender, age and clinical data, including the patient's diagnosis, medication, and outcome were also collected. The groups were stratified according to whether CMV-DNA was negative or positive, CMV-IgM was negative or positive, age, gender, and whether they received immunosuppressive therapy or not. The Pearson Chi-square test or Fisher's exact test was used for comparison of the rates between the groups. P < 0.05 means the difference is statisti-cally significant.@*RESULTS@#Of the 507 patients submitted for examination, 55 (10.85%) were positive for CMV-DNA, 74 (14.60%) were positive for CMV-IgM, and 20 (3.94%) were positive for both CMV-DNA and CMV-IgM. Of the 55 patients with CMV-DNA positive, 37 were male, accounting for 67.27%. In addition, 25 patients were older than 60 years, accounting for 45.45% and 33 patients received immunosuppressive therapy, accounting for 60%. The rates were higher than that of CMV-DNA negative group, 47.35% (P=0.005), 68.14% (P=0.043), 46.02% (P=0.050), respectively. Of the patients with both CMV-DNA and IgM positive, 45% received immunosuppressive threapy, which was lower than that of CMV-DNA positive but IgM negative patients (68.57%, P=0.086), and also lower than CMV-DNA negative but IgM positive patients (68.52%, P=0.064). In the patients with both CMV-DNA and IgM positive, 91.67% showed remission after receiving ganciclovir, whereas in the patients with CMV-DNA positive but IgM negative, the rate was only 60% (P=0.067).@*CONCLUSION@#CMV-IgM antibody detection is affected by age, gender, and immune status. It is not recommended to use CMV-IgM alone to determine CMV infection in patients with immunosuppressive status and those older than 60 years. CMV-DNA and CMV-IgM combined detection may help to predict patients' immune status and outcomes of antiviral therapy.


Subject(s)
Antibodies, Viral , Cytomegalovirus/genetics , Cytomegalovirus Infections/drug therapy , DNA , Female , Humans , Immunoglobulin M , Immunosuppressive Agents/therapeutic use , Male , Nucleic Acids
14.
Chinese Journal of Epidemiology ; (12): 692-695, 2022.
Article in Chinese | WPRIM | ID: wpr-935445

ABSTRACT

Objective: To analyze the dynamic changes and influencing factors of HIV-1 DNA load in HIV-1 infected individuals under antiretroviral therapy (ART) in Dehong Dai and Jingpo autonomous prefecture, Yunnan province, and provide information support for the clinical use of HIV-1 DNA quantitative detection. Methods: The HIV infection cases in recent infection cohort from Dehong Center for Disease Control and Prevention during 2009-2018 were selected as study subjects. The dynamic curve of HIV-1 DNA load varrying with time was generated and logistic regression analysis was conducted to identify the risk factors for HIV-1 load in the recent follow up after ART and statistical analysis was performed by using SPSS 17.0. Results: Among the 113 HIV infection cases detected from the recent infection cohort, the recent HIV infection rate were 49.6%(56/113) males, sexual transmission cases and drug injection transmission cases accounted for 53.1% (60/113), 80.5% (91/113) and 19.5% (22/113), respectively. The dynamic changes curve showed that HIV-1 DNA load was relatively high (>800 copies /106 PBMCs) before ART, and droped rapidly (<400 copies /106 PBMCs) after ART for 1 year. However, HIV-1 DNA load decreased insignificantly from the second year of ART, and remained to be 269 copies/106 PBMCs after ART for 6 years. Univariable logistic regression analysis indicated that OR (95%CI) of CD8, CD4/CD8 and HIV-1 DNA load were 1.00 (1.00-1.00), 0.30 (0.09-1.05) and 1.01 (1.00-1.01), respectively. Multivariable logistic regression analysis showed that OR value of HIV-1 DNA load base was 1.00 (1.00-1.01). Conclusions: HIV-1 DNA load decreased significantly in the first year of ART, then remained stable for years. HIV-1 DNA load base was the key factor associated with the decrease of HIV-1 DNA load, the lower the HIV-1 DNA load base, the lower HIV-1 DNA load. Therefore, earlier ART can contribute to the decrease of HIV-1 DNA load.


Subject(s)
China/epidemiology , DNA/therapeutic use , HIV Infections/drug therapy , HIV Seropositivity , HIV-1/genetics , Humans , Male , Viral Load
15.
Article in English | WPRIM | ID: wpr-928922

ABSTRACT

DNA hypermethylation is an epigenetic modification that plays a critical role in the oncogenesis of myelodysplastic syndromes (MDS). Aberrant DNA methylation represses the transcription of promotors of tumor suppressor genes, inducing gene silencing. Realgar (α-As4S4) is a traditional medicine used for the treatment of various diseases in the ancient time. Realgar was reported to have efficacy for acute promyelocytic leukemia (APL). It has been demonstrated that realgar could efficiently reduce DNA hypermethylation of MDS. This review discusses the mechanisms of realgar on inhibiting DNA hypermethylation of MDS, as well as the species and metabolisms of arsenic in vivo.


Subject(s)
Arsenicals/therapeutic use , DNA , DNA Methylation/genetics , Humans , Myelodysplastic Syndromes/genetics , Sulfides
16.
Article in Chinese | WPRIM | ID: wpr-928399

ABSTRACT

OBJECTIVE@#To assess the application value of noninvasive prenatal testing (NIPT) based on cell-free fetal DNA.@*METHODS@#The results of 2777 cases of basic and extended NIPT were retrospectively analyzed. The clinical data and outcome of pregnancy were analyzed, in addition with the diagnosis rate and testing efficiency.@*RESULTS@#Among the 2777 pregnant women, 1192 (42.9%) had accepted basic NIPT and 1585 (57.1%) accepted extended NIPT. With a failure rate of 0.1%, 8 and 6 cases were reported respectively as high-risk pregnancies for trisomy 21 and sex chromosomal abnormalities. Other genetic abnormalities were detected in 32 cases. The positive predictive value for trisomy 21 was 85.7%, and one case of 47,XXX was diagnosed among 3 women with high risks for sex chromosomal abnormalities. For those with a high risk for other genetic abnormalities, pregnant diagnosis rates of basic and extended NIPT were 71.4% (5/7) and 68.2% (15/22), respectively. Seven copy number variations (CNVs) were confirmed, including one pathogenic CNV, one likely pathogenic CNV and 5 variants of unknown significance. Among 6 cases with high-risk of maternal CNVs, 5 fetuses and the mothers were confirmed to be carriers. No CNV was detected in the remainder fetus by chromosomal microarray analysis, while its mother was a carrier of the corresponding CNV.@*CONCLUSION@#NIPT has shown a relatively high positive predictive value for the screening of trisomy 21 and maternal CNVs but with a limited efficiency for the discovery of fetal CNVs. For other genetic abnormalities signaled by NIPT, informed choice by the pregnant women during pre-testing consultation is recommended. Invasive prenatal diagnosis should be considered in the combination of NIPT reports and fetal ultrasound, while the residual risks should be fully informed.


Subject(s)
Aneuploidy , Cell-Free Nucleic Acids/genetics , DNA/genetics , DNA Copy Number Variations , Female , Fetus , Humans , Noninvasive Prenatal Testing , Pregnancy , Retrospective Studies
17.
Article in Chinese | WPRIM | ID: wpr-928375

ABSTRACT

OBJECTIVE@#To assess the value of re-sampling for patients who had failed non-invasive prenatal testing (NIPT) due to low cell-free fetal DNA (cffDNA) fraction.@*METHODS@#Clinical data of 20 387 patients undergoing NIPT test was reviewed. The patients were re-sampled when initial blood test did not yield a result due to cffDNA fraction. The results were analyzed, and the outcome of pregnancy was followed up.@*RESULTS@#Among all samples, 17 (0.08%) had failed to yield a result due to low cffDNA fraction, all of which accepted re-sampling. A result was attained in 16 cases, with a success rate of 94.12%. Only one sample had failed the re-test.@*CONCLUSION@#For patients who had failed the initial NIPT due to low cffDNA fraction, re-sampling should be considered with gestational week and ultrasound results taken into consideration.


Subject(s)
Aneuploidy , Cell-Free Nucleic Acids/genetics , DNA/genetics , Female , Fetus , Humans , Pregnancy , Prenatal Diagnosis
18.
Chinese Journal of Biotechnology ; (12): 1837-1846, 2022.
Article in Chinese | WPRIM | ID: wpr-927821

ABSTRACT

We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.


Subject(s)
African Swine Fever Virus/genetics , Animals , DNA , Immunity, Innate , Nucleotidyltransferases/metabolism , Signal Transduction , Swine
19.
Chinese Journal of Biotechnology ; (12): 915-924, 2022.
Article in Chinese | WPRIM | ID: wpr-927754

ABSTRACT

Group Ⅱ introns are self-splicing ribozymes, which insert directly into target sites in DNA with high frequency through "retrohoming". They specifically and efficiently recognize and splice DNA target sites, endowing themselves with great potential in genetic engineering. This paper reviewed the gene targeting principle of group Ⅱ introns and the application in microbial genetic modification, and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the "retrohoming" characteristics and the dependence on high Mg2+ concentration. Finally, we dissected the potential of group Ⅱ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns, hoping to provide a reference for the application of group Ⅱ introns in biotechnology.


Subject(s)
DNA , Eukaryota , Gene Targeting , Introns/genetics , RNA, Catalytic/genetics
20.
Chinese Journal of Biotechnology ; (12): 411-426, 2022.
Article in Chinese | WPRIM | ID: wpr-927719

ABSTRACT

Adulteration in meat products is a widespread issue that could lead to serious threats to public health and religious violations. Technology that offers rapid, sensitive, accurate and reliable detection of meat species is the key to an effectual monitoring and control against meat adulteration. In recent years, high-throughput sequencing-based DNA metabarcoding technology has developed rapidly. With the characteristics of being high-throughput, highly precise and high-speed, this technology can simultaneously identify multiple species in complex samples, thus offering pronounced advantages in the surveillance of adulteration in meat and meat products. Starting with an introduction of the major developments in the high-throughput sequencing technology in the past two decades, this review provides an overview of the technical characteristics and research methods of DNA metabarcoding, summarizes the application of DNA metabarcoding technology in meat adulteration detection over the last few years, discusses the challenges of using DNA metabarcoding technology in the detection of meat adulteration, and provides future prospects on the development of this technology.


Subject(s)
DNA , Food Contamination/analysis , High-Throughput Nucleotide Sequencing/methods , Meat/analysis , Meat Products , Technology
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