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Retinoblastoma protein is central in signaling networks of fundamental cell decisions such as proliferation and differentiation in all metazoans and cancer development. Immunostaining and biochemical evidence demonstrated that during interphase retinoblastoma protein is in the nucleus and is hypophosphorylated, and during mitosis is in the cytoplasm and is hyperphosphorylated. The purpose of this study was to visualize in vivo in a non-diseased tissue, the dynamic spatial and temporal nuclear exit toward the cytoplasm of this protein during mitosis and its return to the nucleus to obtain insights into its potential cytosolic functions. Using high-resolution time-lapse images from confocal microscopy, we tracked in vivo the ortholog in plants the RETINOBLASTOMA RELATED (RBR) protein tagged with Green Fluorescent Protein (GFP) in Arabidopsis thaliana's root. RBR protein exits from dense aggregates in the nucleus before chromosomes are in prophase in less than 2 min, spreading outwards as smaller particles projected throughout the cytosol during mitosis like a diffusive yet controlled event until telophase, when the daughter's nuclei form; RBR returns to the nuclei in coordination with decondensing chromosomal DNA forming new aggregates again in punctuated larger structures in each corresponding nuclei. We propose RBR diffused particles in the cytoplasm may function as a cytosolic sensor of incoming signals, thus coordinating re-aggregation with DNA is a mechanism by which any new incoming signals encountered by RBR may lead to a reconfiguration of the nuclear transcriptomic context. The small RBR diffused particles in the cytoplasm may preserve topologic-like properties allowing them to aggregate and restore their nuclear location, they may also be part of transient cytoplasmic storage of the cellular pre-mitotic transcriptional context, that once inside the nuclei may execute both the pre mitosis transcriptional context as well as new transcriptional instructions.
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KEY MESSAGE: The miR822 together with of AGO9 protein, modulates monosporic development in Arabidopsis thaliana through the regulation of target genes encoding Cysteine/Histidine-Rich C1 domain proteins, revealing a new role of miRNAs in the control of megaspore formation in flowering plants. In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic cell differentiates into a megaspore mother cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana (Arabidopsis) undergoes a monosporic type of gametogenesis as three meiotically derived cells degenerate, and a single one-the functional megaspore (FM), divides mitotically to form the female gametophyte. The genetic basis and molecular mechanisms that control monosporic gametophyte development remain largely unknown. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the miR822, give rise to extranumerary surviving megaspores that acquire a FM identity and divides without giving rise to differentiated female gametophytes. The overexpression of three miR822 putative target genes encoding cysteine/histidine-rich C1 (DC1) domain proteins, At5g02350, At5g02330 and At2g13900 results in defects equivalent to those found in mutant mir822 plants. The three miR822 targets genes are overexpressed in ago9 mutant ovules, suggesting that miR822 acts through an AGO9-dependent pathway to negatively regulate DC1 domain proteins and restricts the survival of meiotically derived cells to a single megaspore. Our results identify a mechanism mediated by the AGO9-miR822 complex that modulates monosporic female gametogenesis in Arabidopsis thaliana.
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Late embryogenesis abundant (LEA) proteins are a large protein family that mainly function in protecting cells from abiotic stress, but these proteins are also involved in regulating plant growth and development. In this study, we performed a functional analysis of LEA13 and LEA30 from Arabidopsis thaliana. The results showed that the expression of both genes increased when plants were subjected to drought-stressed conditions. The insertional lines lea13 and lea30 were identified for each gene, and both had a T-DNA element in the regulatory region, which caused the genes to be downregulated. Moreover, lea13 and lea30 were more sensitive to drought stress due to their higher transpiration and stomatal spacing. Microarray analysis of the lea13 background showed that genes involved in hormone signaling, stomatal development, and abiotic stress responses were misregulated. Our results showed that LEA proteins are involved in drought tolerance and participate in stomatal density.
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Cutaneous drug-induced reactions are immune-mediated responses that can lead to life-threatening diseases such as drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome, and toxic epidermal necrolysis, collectively known as severe cutaneous adverse reactions (SCARs). Unfortunately, they cannot be predicted during drug development, and, at present, a prognostic biomarker is not available nor are validated in vitro assays for diagnosis. Thus, by using proteomic and microarray miRNA analysis, the cargo of extracellular vesicles obtained from SCARs patients was analyzed and correlated with the severity of the reaction. Confirmatory assays using Western blot and qRT-PCR were performed to validate findings, and bioinformatic tools were used to establish the correlation between protein and miRNAs expression between groups. The proteomic analysis showed an increase in the amount of pro-inflammatory proteins, von Willebrand factor, and C-reactive protein and a decrease in anti-inflammatory and protective proteins in the SCARs group compared with the control group. Additionally, histone protein H2A was enriched in DRESS patients. APO1 and SERPINA4 proteins, highly increased in the control group but absent in the SCARs group, are the target of several overexpressed miRNAs, suggesting that the regulation of these proteins might involve gene silencing and protein repressing mechanisms in the severe patients. According with previous reports showing its presence in plasma and T-cells, microRNA miR-18 was upregulated in extracellular vesicles obtained from the most severe patients. Determination of the unique cargo associated with different disease conditions will help to understand the pathophysiology of these complex reactions and might help to develop novel biomarkers for life-threatening iatrogenic cutaneous disease.
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Erupciones por Medicamentos/genética , Vesículas Extracelulares/genética , MicroARNs/genética , Erupciones por Medicamentos/diagnóstico , Vesículas Extracelulares/química , Vesículas Extracelulares/patología , Humanos , Proteoma/análisis , Proteoma/genética , Proteómica , TranscriptomaRESUMEN
Placentaderived exosomes play an important role in cellular communication both in the mother and the fetus. Their concentration and composition are altered in several pregnancy disorders, such as gestational diabetes mellitus (GDM). The isolation and characterization of placental exosomes from serum, plasma and tissues from patients with GDM have been previously described; however, to the best of our knowledge, to date, there is no study available on placental exosomes isolated from urine of patients with GDM. In the present study, placental exosomes were purified from urine the 1st, 2nd and 3rd trimester of gestation. Placental exosomes were characterized by transmission electron microscopy in cryogenic mode and by western blot analysis, confirming the presence of exosomal vesicles. The expression profile of five microRNAs (miR5165p, miR5173p, miR5185p, miR2223p and miR165p) was determined by RTqPCR. In healthy pregnant women, the expression of the miRNAs increased across gestation, apart from miR5165p, which was not expressed at the 2nd trimester. All the miRNAs examined were downregulated in patients with GDM at the 3rd trimester of gestation. The downregulated miRNAs affected several metabolic pathways closely associated with the pathophysiology of GDM. This provides further evidence of the regulatory role of miRNAs in the GDM. This also suggests that the of urinary exosomes may be an excellent source of biomarkers and therapeutic targets.
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Diabetes Gestacional/metabolismo , Exosomas/metabolismo , MicroARNs/metabolismo , Western Blotting , Diabetes Gestacional/genética , Femenino , Humanos , MicroARNs/genética , Microscopía Electrónica de Transmisión , Placenta/metabolismo , EmbarazoRESUMEN
miRNAs regulate post-transcriptional gene expression in metazoans, and thus are involved in many fundamental cellular biological processes. Extracellular miRNAs are also found in most human biofluids including plasma. These circulating miRNAs constitute a long distance inter cellular communication system and are potentially useful biomarkers. High throughput technologies like microarrays are able to scan a complete miRNome providing useful detection scores that are underexplored. We proposed to answer how many and which miRNAs are detectable in plasma or extracellular vesicles as these questions have not yet been answered. We set out to address this knowledge gap by analyzing the mirRNome in plasma and corresponding extracellular vesicles (EVs) from 12 children affected by retinoblastoma (Rb) a childhood intraocular malignant tumor, as well as from 12 healthy similarly aged controls. We calculated an average of 537 detectable miRNAs in plasma and 625 in EVs. The most miRNA enriched compartment were EVs from Rb cases with an average of 656 detectable elements. Using hierarchical clustering with the detection scores, we generated broad detection mirnome maps and identified a plasma signature of 19 miRNAs present in all Rb cases that is able to discriminate cases from controls. An additional 9 miRNAs were detected in all the samples; within this group, miRNA-5787 and miRNA-6732-5p were highly abundant and displayed very low variance across all the samples, suggesting both are good candidates to serve as plasma references or normalizers. Further exploration considering participant's sex, allowed discovering 5 miRNAs which corresponded only to females and 4 miRNAs corresponding only to males. Target and pathway analysis of these miRNAs revealed hormonal function including estrogen, thyroid signaling pathways and testosterone biosynthesis. This approach allows a comprehensive unbiased survey of a circulating miRNome landscape, creating the possibility to define normality in mirnomic profiles, and to locate where in these miRNome profiles promising and potentially useful circulating miRNA signatures can be found.
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Vesículas Extracelulares/metabolismo , MicroARNs/sangre , Neoplasias de la Retina/patología , Retinoblastoma/patología , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Preescolar , MicroARN Circulante/sangre , Análisis por Conglomerados , Análisis Discriminante , Femenino , Humanos , Lactante , Masculino , MicroARNs/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Retina/genética , Retinoblastoma/genéticaRESUMEN
Streptococcus pneumoniae is a causal agent of otitis media, pneumonia, meningitis and severe cases of septicemia. This human pathogen infects elderly people and children with a high mortality rate of approximately one million deaths per year worldwide. Antibiotic-resistance of S. pneumoniae strains is an increasingly serious health problem; therefore, new therapies capable of combating pneumococcal infections are indispensable. The application of gold nanoparticles has emerged as an option in the control of bacterial infections; however, the mechanism responsible for bacterial cell lysis remains unclear. Specifically, it has been observed that gold nanoparticles are capable of crossing different structures of the S. pneumoniae cells, reaching the cytosol where inclusion bodies of gold nanoparticles are noticed. In this work, a novel process for the separation of such inclusion bodies that allowed the analysis of the biomolecules such as carbohydrates, lipids and proteins associated with the gold nanoparticles was developed. Then, it was possible to separate and identify proteins associated with the gold nanoparticles, which were suggested as possible candidates that facilitate the interaction and entry of gold nanoparticles into S. pneumoniae cells.
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Antibacterianos/farmacología , Oro/farmacología , Nanopartículas del Metal , Infecciones Neumocócicas/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Farmacorresistencia Microbiana , Oro/química , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Viabilidad Microbiana/efectos de los fármacos , Streptococcus pneumoniae/fisiologíaRESUMEN
Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.
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Proteínas Bacterianas/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , NADH NADPH Oxidorreductasas/genética , Fosfitos/metabolismo , Fósforo/metabolismo , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Proteínas Bacterianas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Ingeniería Genética/métodos , Marcadores Genéticos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fosfitos/farmacología , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genética , Selección Genética , Transformación GenéticaRESUMEN
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.
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Adenina Fosforribosiltransferasa/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Chlamydomonas reinhardtii/genética , Genes Reporteros/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Electroporación/métodos , Edición Génica/métodos , Plásmidos/genética , ARN Guía de Kinetoplastida/genética , Ribonucleoproteínas/genéticaRESUMEN
microRNAs are noncoding RNAs of 20-24 nucleotides (nt) in length that act as repressors of genes and are important in key developmental processes in the entire life cycle of plants. To determine the function of a microRNA, the first step is to resolve its expression pattern; this can be achieved by in situ hybridization, RNA blot assays, or quantitative PCR. However, the study of the expression of a MIR gene is straightforward with the use of reporter proteins such as ß-D-glucuronidase (GUS), GFP, or mCherry. To do this, it is necessary to clone the promoter region of the MIR gene and place it upstream of the reporter gene; in this way the activity of the promoter will be a direct reflection of the expression of the MIR gene. Here, we indicate step by step how to make transcriptional fusion constructs from the cloning of a promoter region of a MIR gene fused to the classical reporter proteins GUS and mCherry, the latter with codon optimization for better expression in Arabidopsis thaliana. This method is particularly useful to dissect the promoter region of a MIR gene and to find its expression pattern in a tissue and developmental specific manner.
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Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , MicroARNs/genética , ARN de Planta/genética , Proteínas Recombinantes de Fusión/genética , Clonación Molecular , Genes de Plantas/genética , Genes Reporteros/genética , Glucuronidasa/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética/genéticaRESUMEN
KEY MESSAGE: Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3'-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.
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Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , ADN Intergénico/genética , Genes de Plantas/genética , Operón/genética , Plantas Modificadas Genéticamente/genética , Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas/genética , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , Ingeniería Metabólica/métodos , Plantas Modificadas Genéticamente/metabolismoRESUMEN
BACKGROUND: miRNAs exert their effect through a negative regulatory mechanism silencing expression upon hybridizing to their target mRNA, and have a prominent position in the control of many cellular processes including carcinogenesis. Previous miRNA studies on retinoblastoma (Rb) have been limited to specific miRNAs reported in other tumors or to medium density arrays. Here we report expression analysis of the whole miRNome on 12 retinoblastoma tumor samples using a high throughput microarray platform including 2578 mature miRNAs. METHODS: Twelve retinoblastoma tumor samples were analyzed using an Affymetrix platform including 2578 mature miRNAs. We applied RMA analysis to normalize raw data, obtained categorical data from detection call values, and also used signal intensity derived expression data. We used Diana-Tools-microT-CDS to find miRNA targets and ChromDraw to map miRNAs in chromosomes. RESULTS: We discovered a core-cluster of 30 miRNAs that were highly expressed in all the cases and a cluster of 993 miRNAs that were uniformly absent in all cases. Another 1022 miRNA were variably present in the samples reflecting heterogeneity between tumors. We explored mRNA targets, pathways and biological processes affected by some of these miRNAs. We propose that the core-cluster of 30 miRs represent miRNA machinery common to all Rb, and affecting most pathways considered hallmarks of cancer. In this core, we identified miR-3613 as a potential and critical down regulatory hub, because it is highly expressed in all the samples and its potential mRNA targets include at least 36 tumor suppressor genes, including RB1. In the variably expressed miRNA, 36 were differentially expressed between males and females. Some of the potential pathways targeted by these 36 miRNAs were associated with hormonal production. CONCLUSION: These findings indicate that Rb tumor samples share a common miRNA expression profile regardless of tumor heterogeneity, and shed light on potential novel therapeutic targets such as mir-3613 This is the first work to delineate the miRNA landscape in retinoblastoma tumor samples using an unbiased approach.
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MicroARNs/genética , Retinoblastoma/genética , Transcriptoma , Adolescente , Adulto , Niño , Análisis por Conglomerados , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Retinoblastoma/patología , Factores Sexuales , Adulto JovenRESUMEN
Light-up aptamers are practical tools to image RNA localization in vivo. A now classical light-up aptamer system is the combination of the 3,5-difluoro-4-hydroxybenzylidene (DFHBI) fluorogen and the RNA aptamer Spinach, which has been successfully used in bacterial and mammalian cells. However, light-up aptamers have not been used in algae. Here, we show that a simple vector, carrying Spinach, transcriptionally fused to the aphA-6 gene, can be effectively used to generate a functional light-up aptamer in the chloroplast of Chlamydomonas reinhardtii. After incubation with DFHBI, lines expressing the aphA-6/Spinach mRNA were observed with laser confocal microscopy to evaluate the functionality of the light-up aptamer in the chloroplast of C. reinhardtii. Clear and strong fluorescence was localized to the chloroplast, in the form of discrete spots. There was no background fluorescence in the strain lacking Spinach. Light-up aptamers could be further engineered to image RNA or to develop genetically encoded biosensors in algae.
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Aptámeros de Nucleótidos/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Compuestos de Bencilo , Fluorescencia , Colorantes Fluorescentes , Imidazolinas , Kanamicina Quinasa/genética , ARN Mensajero/genética , ARN de Planta/genéticaRESUMEN
Los exosomas son vesículas membranosas extracelulares esenciales en la comunicación intercelular a larga distancia, viajan en los fluidos corporales y entregan mensajes moleculares dirigidos a la mayoría de las células de todo el organismo. La liberación de mensajes vía exosomas ocurre en forma de ADN, ARN o proteínas; dicha liberación se ha asociado a diferentes condiciones fisiológicas normales y patológicas, como el cáncer. Por lo anterior, el aislamiento eficiente y caracterización celular de exosomas de plasma es clave para su uso como biomarcadores no invasivos de diversas enfermedades. En el presente estudio se purificaron exosomas a partir de muestras clínicas de plasmas de pacientes previamente diagnosticados con retinoblastoma y de individuos sanos como control. Los exosomas recuperados fueron caracterizados a nivel celular por microscopia electrónica de transmisión empleando una técnica de criogenia. Para demostrar la correcta purificación de exosomas se confirmó la presencia de las proteínas transmembranales CD63 y CD81 mediante immunoblot. Adicionalmente de los exosomas purificados, se identificaron ARNs pequeños no codificantes llamados microARNs. En general, se describe la purificación y caracterización celular de exosomas obtenidos de plasma humano para su potencial uso como biomarcadores.
Exosomes are small extracellular vesicles essential in intercellular communication; they act as vehicles of broad scope. They are travelling in body fluids and delivering molecular messages to cells in the organism. Messages released by exosomes like DNA, RNA and proteins are associated with different pathological conditions including cancer. Therefore, the efficient isolation and cellular characterization of exosomes from plasma is essential to use them as biomarkers in many diseases. Here, exosomes were purified from patients diagnosed with pediatric cancer and healthy individuals as control. The exosomes recovered were characterized using cryogenic transmission electron microscopy. Moreover, the presence of CD63 and CD81 transmembrane proteins was confirmed using Western blot. Besides, miRNAs presence was identified from exosomes. This work describes a complete technique to isolate and characterize exosomes from human plasma, recognizing their potential as biomarkers.
Os exossomos são vesículas membranosas extracelulares essenciais na comunicação intercelular de longa distância; eles viajam em fluidos corporais e entregam mensagens moleculares dirigidas à maioria das células de todo o organismo. A liberação de mensagens através dos exossomos ocorre em forma de DNA, RNA ou proteínas; essa liberação foi associada a diferentes condições fisiológicas normais e patológicas, tais como o câncer. Por tudo isso, o eficiente isolamento e caracterização celular de exossomos de plasma é chave para sua utilização como biomarcadores não invasivos de várias doenças. No presente estudo, exossomos foram purificados a partir de amostras clínicas de plasmas de pacientes que tinham sido diagnosticados previamente com retinoblastoma e de indivíduos saudáveis como controle. Os exossomos recuperados foram caracterizados a nível celular por microscopia eletrônica de transmissão usando uma técnica de criogenia. Para demonstrar a correta purificação dos exossomos, foi confirmada a presença de proteínas transmembranares CD63 e CD81 usando inmunoblot. Além dos exossomos purificados foram identificados ARNs não codificantes pequenos chamados microARNs. Em geral os métodos de purificação e caracterização celular de exossomos obtidos de plasma humano são descritos por seu potencial utilização como biomarcadores.
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Humanos , Masculino , Femenino , Lactante , Preescolar , Biomarcadores , Separación Celular/estadística & datos numéricos , Exosomas , Retinoblastoma/diagnóstico , DiagnósticoRESUMEN
Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures.