RESUMO
Mexico has the in-house technical and regulatory capacity to undertake human genome editing (HGE) governance. However, its regulatory framework must be reformed to be more targeted and govern the application of any emerging HGE technologies, leaving no room for unethical or unsafe practices for reproductive purposes.
Assuntos
Edição de Genes , Genoma Humano , Humanos , México , Edição de Genes/legislação & jurisprudência , Edição de Genes/ética , Edição de Genes/métodos , Genoma Humano/genéticaRESUMO
OBJECTIVES: This study aimed to evaluate whether the expression of circulating dystromiRs and a group of oxidative stress-related (OS-R) miRNAs is associated with muscle injury and circulating metabolic parameters in Duchenne muscular dystrophy (DMD) patients. METHODS: Twenty-four DMD patients were included in this cross-sectional study. Clinical scales to evaluate muscle injury (Vignos, GMFCS, Brooke, and Medical Research Council), enzymatic muscle injury parameters (CPK, ALT, and AST), anthropometry, metabolic indicators, physical activity, serum dystromiRs (miR-1-3p, miR-133a-3p, and miR-206), and OS-R miRNAs (miR-21-5p, miR-31-5p, miR-128-3p, and miR-144-3p) levels were measured in ambulatory and non-ambulatory DMD patients. RESULTS: DystromiRs (except miR-1-3p) and miRNAs OS-R levels were lower (p-value <.05) in the non-ambulatory group than the ambulatory group. The expression of those miRNAs correlated with Vignos scale score (For instance, rho = -0.567, p-value <0.05 for miR-21-5p) and with other scales scores of muscle function and strength. CPK, AST, and ALT concentration correlated with expression of all miRNAs (For instance, rho = 0.741, p-value <.05 between miR-206 level and AST concentration). MiR-21-5p level correlated with glucose concentration (rho = -0.369, p-value = .038), and the miR-1-3p level correlated with insulin concentration (rho = 0.343, p-value = .05). CONCLUSIONS: Non-ambulatory DMD patients have lower circulating dystromiRs and OS-R miRNAs levels than ambulatory DMD patients. The progressive muscle injury is associated with a decrease in the expression of those miRNAs, evidencing DMD progress. These findings add new information about the natural history of DMD.
Assuntos
MicroRNA Circulante , Insulinas , MicroRNAs , Distrofia Muscular de Duchenne , Biomarcadores , Estudos Transversais , Glucose , Humanos , Músculos/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismoRESUMO
Currently, there are over 230 different COVID-19 vaccines under development around the world. At least three decades of scientific development in RNA biology, immunology, structural biology, genetic engineering, chemical modification, and nanoparticle technologies allowed the accelerated development of fully synthetic messenger RNA (mRNA)-based vaccines within less than a year since the first report of a SARS-CoV-2 infection. mRNA-based vaccines have been shown to elicit broadly protective immune responses, with the added advantage of being amenable to rapid and flexible manufacturing processes. This review recapitulates current advances in engineering the first two SARS-CoV-2-spike-encoding nucleoside-modified mRNA vaccines, highlighting the strategies followed to potentiate their effectiveness and safety, thus facilitating an agile response to the current COVID-19 pandemic.
Assuntos
Engenharia Biomédica , Vacinas contra COVID-19 , COVID-19 , Desenvolvimento de Medicamentos/métodos , Descoberta de Drogas/métodos , SARS-CoV-2 , Vacina de mRNA-1273 contra 2019-nCoV , Engenharia Biomédica/métodos , Engenharia Biomédica/tendências , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/classificação , Vacinas contra COVID-19/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Humanos , Imunogenicidade da Vacina , Lipossomos/farmacologia , Nanopartículas , Nucleosídeos/farmacologia , Nucleosídeos/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/farmacologiaRESUMO
MicroRNAs (miRNAs) have a prominent role in virtually every aspect of cell biology. Due to the small size of mature miRNAs, the high degree of similarity between miRNA family members, and the low abundance of miRNAs in body fluids, miRNA expression profiling is technically challenging. Biosensors based on electrochemical detection for nucleic acids are a novel category of inexpensive and very sensitive diagnostic tools. On the other hand, after recognizing the target sequence, specific CRISPR-associated proteins, including orthologues of Cas12, Cas13, and Cas14, exhibit collateral nonspecific catalytic activities that can be employed for specific and ultrasensitive nucleic acid detection from clinically relevant samples. Recently, several platforms have been developed, connecting the benefits of enzyme-assisted signal amplification and enzyme-free amplification biosensing technologies with CRISPR-based approaches for miRNA detection. Together, they provide high sensitivity, precision, and fewer limitations in diagnosis through efficient sensors at a low cost and a simple miniaturized readout. This review provides an overview of several CRISPR-based biosensing platforms that have been developed and successfully applied for ultrasensitive and specific miRNA detection.
Assuntos
Sistemas CRISPR-Cas/genética , MicroRNAs/análise , Animais , Técnicas Biossensoriais , Colorimetria , Eletroquímica , Engenharia Genética , Humanos , MicroRNAs/genéticaRESUMO
The genome of the SARS-CoV-2 virus, the causal agent of the COVID-19 pandemic, has diverged due to multiple mutations since its emergence as a human pathogen in December 2019. Some mutations have defined several SARS-CoV-2 clades that seem to behave differently in terms of regional distribution and other biological features. Next-generation sequencing (NGS) approaches are used to classify the sequence variants in viruses from individual human patients. However, the cost and relative scarcity of NGS equipment and expertise in developing countries prevent studies aimed to associate specific clades and variants to clinical features and outcomes in such territories. As of March 2021, the GR clade and its derivatives, including the B.1.1.7 and B.1.1.28 variants, predominate worldwide. We implemented the post-PCR small-amplicon high-resolution melting analysis to genotype SARS-CoV-2 viruses isolated from the saliva of individual patients. This procedure was able to clearly distinguish two groups of samples of SARS-CoV-2-positive samples predicted, according to their melting profiles, to contain GR and non-GR viruses. This grouping of the samples was validated by means of amplification-refractory mutation system (ARMS) assay as well as Sanger sequencing.
Assuntos
COVID-19/virologia , Técnicas de Genotipagem/métodos , SARS-CoV-2/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Desnaturação de Ácido Nucleico , RNA Viral/isolamento & purificaçãoRESUMO
OBJECTIVE: The aim of this study was to examine the association of three TNFSF4 single nucleotide variants (SNVs) with systemic lupus erythematosus (SLE) susceptibility in Mexican patients. METHODS: Genotypes of the TNFSF4 rs1234315T/C, rs2205960G/T, and rs704840T/G SNVs were determined using a TaqMan assay. In our study, we included 395 patients with SLE and 500 controls. RESULTS: Our information shows a significant difference in the allelic and genotypic frequency of the three TNFSF4 SNVs between cases and controls. Thus, our data showed an association between TNFSF4 rs1234315T/C (T vs. C, OR 1.40, p = 0.00087), rs2205960G/T (G vs. T, OR 1.32, p = 0.0037), and rs704840T/G (T vs. G, OR 1.41, p = 0.0003) and SLE susceptibility in Mexican subjects. Besides, we conducted a meta-analysis to determine the role of TNFSF4 rs2205960G/T and SLE susceptibility; our results showed that this variant is a risk factor for SLE in Latin Americans and Asians. CONCLUSION: Our results show that TNFSF4 rs1234315T/C, rs2205960G/T, and rs704840T/G are risk factors to SLE in Mexicans. This is the first study to document an association between TNFSF4 rs704840T/G and SLE in a Latin American population. In addition, our meta-analysis showed that TNFSF4 rs2205960G/T is a risk factor for Asians and Latin Americans. Key Point ⢠The TNFSF4 rs1234315T/C, rs2205960G/T, and rs704849T/G SNVs are risk factors to SLE in patients from Mexico.
Assuntos
Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico , Genótipo , Humanos , América Latina/epidemiologia , Lúpus Eritematoso Sistêmico/genética , México , Ligante OX40/genética , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
Resumen La revisión por pares tradicional atraviesa por crecientes cuestionamientos, dado el aumento en el fraude científico detectado y la crisis de replicación que recientemente se ha presentado en la investigación biomédica. Investigadores, instituciones académicas y agencias de financiamiento activamente promueven el análisis del registro científico y se han desarrollado múltiples herramientas para lograrlo. Diferentes revistas biomédicas se fundaron con la revisión por pares pospublicación como característica; existen varias plataformas digitales que hacen posible este proceso. Asimismo, cada vez hay más revistas biomédicas que permiten comentar artículos publicados en sus sitios web, lo cual también es posible en repositorios de preimpresiones. Sumado a esto, las casas editoriales y los investigadores están usando ampliamente las redes sociales para la difusión y discusión de artículos, lo cual a veces culmina en refutaciones y retracciones.
Abstract Traditional peer review is undergoing increasing questioning, given the increase in scientific fraud detected and the replication crisis biomedical research is currently going through. Researchers, academic institutions, and research funding agencies actively promote scientific record analysis, and multiple tools have been developed to achieve this. Different biomedical journals were founded with post-publication peer review as a feature, and there are several digital platforms that make this process possible. In addition, an increasing number biomedical journals allow commenting on articles published on their websites, which is also possible in preprint repositories. Moreover, publishing houses and researchers are largely using social networks for the dissemination and discussion of articles, which sometimes culminates in refutations and retractions.
Assuntos
Humanos , Editoração/normas , Revisão da Pesquisa por Pares/métodos , Controle de Qualidade , Fatores de Tempo , Má Conduta Científica/estatística & dados numéricosRESUMO
BACKGROUND: Focal dermal hypoplasia (FDH) is rare X-linked dominant disease characterized by atrophy and linear pigmentation of the skin, split hand/foot deformities and ocular anomalies. FDH is caused by mutations of the Porcupine (PORCN) gene, which encodes an enzyme that catalyzes the palmitoylation of Wnt ligands required for their secretion. High resolution melting analysis (HRM) is a technique that allows rapid, labor-efficient, low-cost detection of genomic variants. In the present study, we report the successful implementation of HRM in the molecular diagnosis of FDH. METHODS: Polymerase chain reaction and HRM assays were designed and optimized for each of the coding exons of the PORCN gene, processing genomic DNA samples form a non-affected control and a patient complying with the FDH diagnostic criteria. The causal mutation was characterized by Sanger sequencing from an amplicon showing a HRM trace suggesting heterozygous variation and was validated using an amplification-refractory mutation system (ARMS) assay. RESULTS: The melting profiles suggested the presence of a variant in the patient within exon 1. Sanger sequencing revealed a previously unknown C to T transition replacing a glutamine codon for a premature stop codon at position 28, which was validated using ARMS. CONCLUSIONS: Next-generation sequencing facilitates the molecular diagnosis of monogenic disorders; however, its cost-benefit ratio is not optimal when a single, small or medium size causal gene is already identified and the clinical diagnostic presumption is strong. Under those conditions, as it is the case for FDH, HRM represents a cost- and labor-effective approach.
Assuntos
Aciltransferases/genética , Éxons/genética , Hipoplasia Dérmica Focal/diagnóstico , Hipoplasia Dérmica Focal/genética , Proteínas de Membrana/genética , Desnaturação de Ácido Nucleico/genética , Sequência de Aminoácidos , Códon sem Sentido , Feminino , Hipoplasia Dérmica Focal/fisiopatologia , Heterozigoto , Humanos , Lactente , Mutação , Filogenia , Reação em Cadeia da Polimerase/métodos , Alinhamento de SequênciaRESUMO
Traditional peer review is undergoing increasing questioning, given the increase in scientific fraud detected and the replication crisis biomedical research is currently going through. Researchers, academic institutions, and research funding agencies actively promote scientific record analysis, and multiple tools have been developed to achieve this. Different biomedical journals were founded with post-publication peer review as a feature, and there are several digital platforms that make this process possible. In addition, an increasing number biomedical journals allow commenting on articles published on their websites, which is also possible in preprint repositories. Moreover, publishing houses and researchers are largely using social networks for the dissemination and discussion of articles, which sometimes culminates in refutations and retractions.La revisión por pares tradicional atraviesa por crecientes cuestionamientos, dado el aumento en el fraude científico detectado y la crisis de replicación que recientemente se ha presentado en la investigación biomédica. Investigadores, instituciones académicas y agencias de financiamiento activamente promueven el análisis del registro científico y se han desarrollado múltiples herramientas para lograrlo. Diferentes revistas biomédicas se fundaron con la revisión por pares pospublicación como característica; existen varias plataformas digitales que hacen posible este proceso. Asimismo, cada vez hay más revistas biomédicas que permiten comentar artículos publicados en sus sitios web, lo cual también es posible en repositorios de preimpresiones. Sumado a esto, las casas editoriales y los investigadores están usando ampliamente las redes sociales para la difusión y discusión de artículos, lo cual a veces culmina en refutaciones y retracciones.
Assuntos
Revisão da Pesquisa por Pares/métodos , Editoração/normas , Humanos , Controle de Qualidade , Má Conduta Científica/estatística & dados numéricos , Fatores de TempoRESUMO
BACKGROUND: MicroRNAs (miRNAs) modulate gene expression through destabilization or translational inhibition of cytoplasmic transcripts or by transcriptional regulation through binding to genomic DNA. Although miRNAs are globally down-regulated in cancer, some are overexpressed in neoplastic tissues, playing key roles in tumorigenesis (oncomiRs), sometimes behaving as effective cancer markers. METHODS: Using total RNA from human uterus adenocarcinoma and non-neoplastic uterus, we conducted a small RNA-sequencing experiment followed by prediction of novel miRNAs using MirDeep* software. Synteny analysis and whole genome alignments were performed using BLAST. We also evaluated expression by a reverse transcriptase-polymerase chain reaction (RT-PCR) in normal tissues of the FSD2 gene, which spans the human miR-1839-5p gene in the opposite direction. RESULTS: MirDeep* analysis predicted a miRNA not previously annotated in databases, identical to and likely the orthologue of mouse miR-1839-5p. Whole-genome local alignments of this miRNA revealed a single perfect hit that is indeed syntenic to mouse miR-1839-5p. Alignments with other mammalian orthologues showed considerable conservation. We validated the prediction via a stem-loop RT-PCR assay, also employed to screen RNA samples from several additional normal and cancer tissues, showing increased expression in neoplastic tissues compared to their respective non neoplastic counterparts. Human heart tissue expresses both miR-1839-5p and FSD2. CONCLUSIONS: Human tissues express an orthologue of mouse miR-1839-5p and, given its expression pattern, we suggest that this miRNA could be explored as a potential oncomiR or cancer marker. Also, according to the genomic organization of miR-1839-5p and FSD2, perfect complementarity exists between the two elements, making possible miRNA-directed cleavage in human cardiac tissue.
Assuntos
Biomarcadores Tumorais , MicroRNAs , Neoplasias/genética , RNA Interferente Pequeno , Sequência de Aminoácidos , Animais , Biologia Computacional/métodos , Sequência Conservada , Perfilação da Expressão Gênica , Genoma Humano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
BACKGROUND: Hundreds of microRNAs (miRNAs), comprising small non-coding RNAs of 20-24 nucleotides, have been discovered, although the entirety of their biological functions is poorly understood. Overexpression or suppression approaches are commonly performed to investigate the function of specific miRNAs. In the present study, we focused on generating a lentiviral vector-based strategy that enables hsa-miR-223-3p (miR-223) overexpression and suppression in the target cells for functional analysis of this miRNA easily and rapidly. METHODS: The sequence that gives rise to miR-223 and the sequence generating the sponge RNA with four binding sites for miR-223 were cloned in pLVX-shRNA2 vector. The functionality of the vector to overexpress miR-223 was evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays, whereas the post-transcriptional regulation exerted by miR-223 was evaluated by luciferase reporter assays in AD-293 cells. The anti-miR-223 sponge activity with one binding site for miR-223 (pmCherry-anti-miR-223) was confirmed by qRT-PCR and the restoration of its target (IKKα) was evaluated by western blot assays in Jurkat cells. RESULTS: The pLVX-miR-223 vector is functional for over-expressing miR-223 and regulates the mRNA of MDR1/ABCB1 at the post-transcriptional level in AD-293 cells. The anti-miR-223 sponge with one miR-223 binding site efficiently modulates the miR-223 availability and not the one with four sites. The over-expression of anti-miR-223 correlated with a decrease in the levels of miR-223 and, consequently, with an increase in the expression level of the IKKα protein in Jurkat cells. CONCLUSIONS: This single miRNA and miRNA sponge expression system specifically alters the availability of miR-223 in mammalian cells.
Assuntos
Expressão Gênica , Vetores Genéticos/metabolismo , MicroRNAs/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Células Jurkat , Lentivirus/genética , Luciferases/genética , Células MCF-7 , MicroRNAs/genéticaRESUMO
Medulloblastomas are among the most frequently diagnosed pediatric solid tumors, and drug resistance remains as the principal cause of treatment failure. Hypoxia and the subsequent activation of hypoxiainducible factor 1α (HIF1α) are considered key factors in modulating drug antitumor effectiveness, but the underlying mechanisms in medulloblastomas have not yet been clearly understood. The aim of the present study was to determine whether hypoxia induces resistance to cyclophosphamide (CPA) and ifosfamide (IFA) in DAOY medulloblastoma cells, whether the mechanism is dependent on HIF1α, and whether involves the modulation of the expression of cytochromes P450 (CYP)2B6, 3A4 and 3A5 and the control of cell proliferation. Monolayer cultures of DAOY medulloblastoma cells were exposed for 24 h to moderate (1% O2) or severe (0.1% O2) hypoxia, and protein expression was evaluated by immunoblotting. Cytotoxicity was studied with the MTT assay and by Annexin V/PI staining and flow cytometry. Cell proliferation was determined by the trypanblue exclusion assay and cell cycle by propidium iodide staining and flow cytometry. Hypoxia decreased CPA and IFA cytotoxicity in medulloblastoma cells, which correlated with a reduction in the protein levels of CYP2B6, CYP3A4 and CYP3A5 and inhibition of cell proliferation. These responses were dependent on hypoxiainduced HIF1α activation, as evidenced by chemical inhibition of its transcriptional activity with 2methoxyestradiol (2ME), which enhanced the cytotoxic activity of CPA and IFA and increased apoptosis. Our results indicate that by stimulating HIF1α activity, hypoxia downregulates the expression of CYP2B6, CYP3A4 and CYP3A5, that in turn leads to decreased conversion of CPA and IFA into their active forms and thus to diminished cytotoxicity. These results support that the combination of HIF1α inhibitors and canonical antineoplastic agents provides a potential therapeutic alternative against medulloblastoma.
Assuntos
Neoplasias Cerebelares/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Resistencia a Medicamentos Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Meduloblastoma/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Ciclofosfamida/farmacologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ifosfamida/farmacologiaRESUMO
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) system has been rapidly harnessed to perform various genomic engineering tasks. Recently, it has been demonstrated that a novel RNA-targeting CRISPR effector protein, called Cas13, binds and cleaves RNA rather than DNA substrates analogously to the eukaryotic RNA interference system. The known Cas13a-Cas13d effectors are able to efficiently cleave complementary target single-stranded RNAs, which represent a potentially safer alternative to deoxyribonuclease Cas9, because it induces loss-of-function phenotypes without genomic loss of the targeted gene. Furthermore, through the improvement in Cas13 effector functionalities, a system called REPAIR has been developed to edit full-length transcripts containing pathogenic mutations, thus providing a promising opportunity for precise base editing. Moreover, advanced engineering of this CRISPR effector also permits nucleic acid detection, allowing the identification of mutations in cell-free tumor DNA through a platform termed Specific High Sensitivity Enzymatic Reporter Unlocking. All of these properties give us a glimpse about the potential of the CRISPR toolkit for precise transcriptome engineering, possibly leading to an expansion of CRISPR technologies for cancer therapeutics and diagnostics. Here, we examine previously unaddressed aspects of the CRISPR-based RNA-targeting approach as a feasible strategy for globally interrogating gene function in cancer in a programmable manner. Cancer Res; 78(15); 4107-13. ©2018 AACR.
Assuntos
Sistemas CRISPR-Cas/genética , Neoplasias/genética , Transcriptoma/genética , DNA/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Genoma/genética , Humanos , RNA/genéticaRESUMO
The peer-review system has allowed the quality control of the manuscripts submitted for publication to scientific journals for over three centuries. However, due to its relative slowness and other drawbacks, some researchers, mainly in the areas of Physics and Mathematics, started some decades ago to propagate, by electronic means, manuscripts not yet submitted to a journal for formal publication. The dissemination of this practice led to the establishment of permanent repositories like ArXiv, to which preprints can be sent to be published whitou charge, allowing also the search and download of the works they contain with no payment required from the reader. In biomedical sciences, the adoption of the system has been slower than in the exact sciences and previous attempts like e-biomed, Netprints, and Nature Precedings did not prosper. A new generation of repositories like bioRXiv, inspired by ArXiv, seems to enjoy an increasing acceptance among biomedical researchers. Here, we discuss the potential role of this emerging system to establish discovery priority in biomedicine and to improve manuscripts before they are submitted to scientific journals besides other applications which could be implemented in the extent that the model becomes more popular.
La revisión por pares es un sistema que ha permitido el control de calidad de los manuscritos enviados para publicación en revistas científicas durante más de tres siglos. Sin embargo, debido a su relativa lentitud y otras desventajas, algunos investigadores (principalmente en las áreas de la física y las matemáticas) iniciaron hace algunas décadas la difusión electrónica de manuscritos aún no sometidos a una revista de publicación formal. La popularización de esta práctica condujo al establecimiento de repositorios permanentes como ArXiv, a los que es posible enviar preimpresiones de forma gratuita y que a la vez permiten la búsqueda y descarga de los trabajos que contienen sin cargo para el lector. En las ciencias biomédicas la adopción de este sistema ha sido más lenta que en las ciencias exactas e intentos previos como e-biomed, Netprints y Nature Precedings no prosperaron. Una nueva generación de repositorios como bioRXiv, inspirado en ArXiv, parece gozar de una creciente aceptación entre investigadores biomédicos. Aquí discutimos el potencial papel de este sistema emergente para establecer la primicia de descubrimientos en biomedicina y el mejoramiento de manuscritos antes de su sometimiento a revistas científicas, así como para otras aplicaciones que podrían implementarse en la medida en que el modelo se popularice.