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PCR and its variants (RT-PCR and qRT-PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID-19 pandemic, qPCR became the gold standard technique for detecting the SARS-CoV-2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In-house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT-qPCR test for detecting SARS-CoV-2 that could be used to diagnose COVID-19 accurately.
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ETHNOPHARMACOLOGICAL RELEVANCE: Cervical cancer is one of the most common malignancies in women that continues to be a public health problem worldwide. Human papillomavirus (HPV) infection is closely related as the causative agent of almost all cases of cervical cancer. Currently, there is no effective treatment for the persistence of HPV. Although vaccines have shown promising results in recent years, they are still a costly strategy for developing countries and have no therapeutic effect on existing infections, which is why the need arises to search for new strategies that can be used in treatment, suppressing oncogenic HPV and disease progression. Extracts of Schisandra Chinensis and Pueraria lobata have been used in traditional medicine, and it has been shown in recent years that some of their bioactive compounds have pharmacological, antioxidant, antitumor, apoptotic, and proliferation effects in HPV-positive cells. However, its mechanism of action has yet to be fully explored. AIM OF THE STUDY: The following study aimed to determine the chemical composition, antioxidant activity, and potential antiproliferative and viral oncogene effects of natural extracts of S. chinensis and P. lobata on HPV-18 positive cervical cancer cells. MATERIALS AND METHODS: The HPV-18-positive HeLa cells were treated for 24 and 48 h with the ethanolic extracts of S chinensis and P. lobata. Subsequently, cell viability was evaluated using the resazurin method, the effect on the cell cycle of the extracts (1.0, 10, and 100 µg/mL) was measured by flow cytometry, the gene of expression of the E6/E7, P53, BCL-2, and E2F-1 were determined by RT-PCR and the protein expression of p53, Ki-67, x|and Bcl-2 by immunohistochemistry. Additionally, the chemical characterization of the two extracts was carried out using LC-MS, and the total phenolics content (TPC), Total flavonoid content (TFC), and DPPH radical scavenging capacity were determined. Data were analyzed using the Mann-Whitney and Kruskal Wallis U test with GraphPad Prism 6 software. RESULTS: The natural extracts of Schisandra chinensis and Pueraria lobata induced down-regulation of E6 HPV oncogene (p<0.05) and a strong up-regulation of P53 (p<0.05), E2F-1 (p<0.05), and Bcl-2 (p<0.05) gene expression. Simultaneously, the natural extracts tend to increase the p53 protein levels and arrest the cell cycle of HeLa in the G1/S phase (p<0.05). Investigated extracts were characterized by the occurrence of bioactive lignans and isoflavones in S. chinensis and P. lobata, respectively. CONCLUSION: The extracts of S. chinensis and P. lobata within their chemical characterization mainly present lignan and isoflavone-type compounds, which are probably responsible for inhibiting the expression of the HPV E6 oncogene and inducing an increase in the expression of p53, Bcl -2 and E2F-1 producing cell cycle detection in S phase in HeLa cells. Therefore, these extracts are good candidates to continue studying their antiviral and antiproliferative potential in cells transformed by HPV.
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Infecciones por Papillomavirus , Pueraria , Schisandra , Neoplasias del Cuello Uterino , Humanos , Femenino , Células HeLa , Virus del Papiloma Humano , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Regulación hacia Abajo , Infecciones por Papillomavirus/tratamiento farmacológico , Oncogenes , Proteínas Proto-Oncogénicas c-bcl-2 , AntioxidantesRESUMEN
Paper-based microfluidic systems have emerged as one of the most promising technologies for developing point-of-care diagnostic platforms (POCT) for detecting and monitoring various diseases. Saliva is a non-invasive biofluid easily collected, transported, and stored. Due to its accessibility and connection to systemic diseases, saliva is one of the best candidates for medical advancement at the point of care, where people can easily monitor their health. However, saliva is a complex mixture of DNA, RNA, proteins, exosomes, and electrolytes. Thus, nucleic acid separation from the salivary components is essential for PCR applications. Paper membranes are a highly porous and foldable structure capable of transporting fluids without pumps and sophisticated systems. The current work presents an insight into simulations for nucleic acid extraction on three types of porous paper membranes for use in point-of-care devices. The flow fluid model is solved on a COMSOL Multiphysics 5.3 free version platform, and the results are compared with experimental assays. The results show that pore uniformity, wet strength, porosity, and functional groups of MF1™ and Fusion 5™ paper membranes are vital parameters affecting nucleic acid extraction and PCR amplification efficiency.
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Multicellular tumor spheroids are used as models in drug development due to their characteristics simulating in vivo tumors. Likewise, antiproliferative properties of extracts derived from fruits have been widely described. Peels and seeds can be used as a matrix to obtain different compounds. Recently, a study demonstrated the antiproliferative activity from a P. mollissima extract (PME) on human colon cancer cells; however, its effect on oral spheroids is unknown. Objective. To evaluate the antiproliferative potential of an extract obtained from P. mollissima seeds on the spheroid-type-3D culture model of CAL 27. Methods. CAL 27-spheroids were treated with three concentrations of PME (10, 50, and 100 µg/ml). After 72 hr incubation, morphology and cellular changes, cytotoxic and proapoptotic effect, gene expression, and metastasis were determined. Additionally, changes in the cell cycle phases responded to the PME concentrations. Comparisons between groups were made through a U Mann-Whitney test. Results. It was shown that 100 µg/ml PE affects CAL 27 cells proliferation grown in spheroids through cell cycle arrest and gene regulation of p53, HIF 1α, and CDH1. However, none of the treatments employed induced MMP9 gene expression. Conclusion. Our study shows that PME inhibits the growth and proliferation of oral tumor cells cultured in spheroids through the positive regulation of cell death and metastasis genes.
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La caries radicular es un proceso dinámico de destrucción localizada de las superficies radiculares, dependiente de biopelícula. Esta patología afecta principalmente a la población adulta mayor con superficies radiculares expuestas y altera su calidad de vida. Las vulnerabilidades y multimorbilidades, frecuentes en este grupo de edad, limitan el autocuidado oral y son criterios para considerar en la comprensión y manejo de la patología. El diagnóstico de las lesiones de caries radicular implica conocimiento de la estructura radicular sana, los factores indicadores o de riesgo, los mecanismos de desarrollo de las lesiones y los criterios para su detección y valoración. La elección correcta de opciones de manejo tanto del riesgo como de las lesiones de caries, enfocada en la preservación de la estructura, evidencia la comprensión de la caries radicular. La caries radicular plantea un desafío para la academia, los sistemas de salud, la práctica clínica dental y la población, debido a que sus indicadores están aumentando en relación con el envejecimiento poblacional y la mayor conservación de dientes naturales. El propósito de esta revisión de tema fue presentar el estado del entendimiento actual de caries radicular. Se incluyeron 81 artículos publicados entre 1971 y 2021, sobre embriogénesis de la raíz; prevalencia, etiopatogenia; características de las lesiones; diagnóstico y propuestas de manejo a nivel del riesgo individual y de las lesiones. Esta actualización es pertinente por el reto que conlleva la presencia cada vez mayor de esta patología, junto con sus consecuencias. El planteamiento de su estado del arte sugiere áreas de investigación.
Root caries is a dynamic process of localized destruction of the root surfaces, dependent on the biofilm. Its pathology mainly affects the elderly toothed population with exposed root surfaces and disrupts their quality of life. Vulnerabilities and multimorbidities, frequent in this age group, limit oral self-care and are criteria to be considered in understanding and managing the disease. The root caries lesions diagnosis implies knowledge about the healthy root structure, indicators or risk factors, caries lesions development mechanism, and the detection and assessment criteria. The correct selection of caries risk and management options preserving tooth structure shows the understanding of root caries. The root caries represents a challenge for academia, health systems, dental practice, and the population because its indicators are increasing related to aging population and greater conservation of natural teeth. This review aimed to present the state of the current understanding of root caries. Eighty-one papers published between 1971 and 2021 were included. This considered root embryogenesis; the current understanding of root caries, its prevalence and etiopathogenesis; characteristics of the lesions; diagnosis, and care proposals both at the individual risk level, and at the lesions level. This update is relevant due to the risk that the increasing presence of this pathology carries, together with its consequences. The statement of its state of the art suggests areas of research.