RESUMEN
Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.
Asunto(s)
Chlamydomonas reinhardtii , Cloroplastos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas , Transcripción Genética , Genes Reporteros , Fotosíntesis/genética , ARN sin Sentido/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismoRESUMEN
SUMMARY: Overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in various tumor tissues and cell lines was found to promote tumor cell proliferation, migration, and invasion. However, the role of MALAT1 in gastric cancer (GC) is still unclear. We aimed to investigate the correlation between long-chain non-coding RNAs (lncRNAs), MALAT1, MicroRNAs (miRNA) and vascular endothelial growth factor A (VEGFA) in gastric cancer and to disclose underlying mechanism. The correlation between MALAT1 levels and clinical features was analyzed by bioinformatics data and human samples. The expression of MALAT1 was down regulated in AGS cells to detect the cell proliferation, migration, and invasion characteristics, as well as the effects on signal pathways. Furthermore, we validated the role of MALAT1/miR-330-3p axis in GC by dual luciferase reporter gene assays. Expression of MALAT1 was higher in cancer tissues than in para-cancerous tissues. The high MALAT1 level predicted malignancy and worse prognosis. Down-regulation of MALAT1 expression in AGS cells inhibited cell proliferation, migration, and invasion by targeting VEGFA. By dual luciferase reporter gene assay and miR-330-3p inhibitor treatment, we demonstrate that MALAT1 sponged miR-330-3p in GC, leading to VEGFA upregulation and activation of the mTOR signaling pathway. The MALAT1/miR-330-3p axis regulates VEGFA through the mTOR signaling pathway and promotes the growth and metastasis of gastric cancer.
Se descubrió que la sobreexpresión del transcrito 1 de adenocarcinoma de pulmón asociado a metástasis (MALAT1) en varios tejidos tumorales y líneas celulares promueve la proliferación, migración e invasión de células tumorales. Sin embargo, el papel de MALAT1 en el cáncer gástrico (CG) aún no está claro. Nuestro objetivo fue investigar la correlación entre los ARN no codificantes de cadena larga (lncRNA), MALAT1, los microARN (miARN) y el factor de crecimiento endotelial vascular A (VEGFA) en el cáncer gástrico y revelar el mecanismo subyacente. La correlación entre los niveles de MALAT1 y las características clínicas se analizó mediante datos bioinformáticos y muestras humanas. La expresión de MALAT1 se reguló negativamente en las células AGS para detectar las características de proliferación, migración e invasión celular, así como los efectos sobre las vías de señales. Además, validamos el papel del eje MALAT1/miR- 330-3p en GC mediante ensayos de genes indicadores de luciferasa dual. La expresión de MALAT1 fue mayor en tejidos cancerosos que en tejidos paracancerosos. El alto nivel de MALAT1 predijo malignidad y peor pronóstico. La regulación negativa de la expresión de MALAT1 en células AGS inhibió la proliferación, migración e invasión celular al apuntar a VEGFA. Mediante un ensayo de gen indicador de luciferasa dual y un tratamiento con inhibidor de miR-330-3p, demostramos que MALAT1 esponjaba miR-330-3p en GC, lo que lleva a la regulación positiva de VEGFA y la activación de la vía de señalización mTOR. El eje MALAT1/miR-330-3p regula VEGFA a través de la vía de señalización mTOR y promueve el crecimiento y la metástasis del cáncer gástrico.
Asunto(s)
Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Factor A de Crecimiento Endotelial Vascular , Serina-Treonina Quinasas TOR , ARN Largo no Codificante , ARN/genética , Transducción de Señal , Regulación Neoplásica de la Expresión Génica , Movimiento Celular , Western Blotting , Apoptosis , Genes Reporteros , Proliferación Celular , Reacción en Cadena en Tiempo Real de la Polimerasa , Invasividad NeoplásicaRESUMEN
BACKGROUND: Hsa_circ_0001535 is involved in biological processes in various tumors. However, the biological effects and related mechanism of hsa_circ_0001535 in ovarian cancer (OC) is unclear. This work is aimed to probe the biological function and underlying mechanism of hsa_circ_0001535 in OC, especially sponged with mi-RNA, require further elucidation. METHODS: Hsa_circ_0001535 expression in OC tissues and cell lines were examined by qRT-PCR. Hsa_circ_0001535 overexpression model was constructed by lentivirus-mediated transfection in two OC cell lines, and the biological functions of hsa_circ_0001535 were evaluated by CCK-8, transwell assay and Western blot. Dual luciferase reporter gene assay was respectively used to explore the relationship between hsa_circ_0001535 and miR-593-3p, as well as miR-593-3p and PTEN. The expression of miR-593-3p and PTEN were detected by qRT-PCR in two OC cell lines and OC tissues. RESULTS: Hsa_circ_0001535 was down-regulated in OC tissues and cell lines. Hsa_circ_0001535 overexpression inhibited proliferation, migration and EMT marker expression in OC cells. Of interest, hsa_circ_0001535 targeted miR-593-3p and reduced its RNA level in OC cells. PTEN was a target gene of miR-593-3p, which was up-regulated by inhibiting miR-593-3p in OC cells. Furthermore, miR-593-3p mimic treatment reversed the up-regulation of PTEN by hsa_circ_0001535 overexpression in OC cells. CONCLUSIONS: The above results showed that hsa_circ_0001535 acted as a molecular sponge for miR-593-3p to repress miR-593-3p expression, and promoted the expression of PTEN, thus inhibited proliferation and migration of OC cells. Our research provides a potential therapeutic target for ovarian cancer patients.
Asunto(s)
MicroARNs , Neoplasias Ováricas , Femenino , Humanos , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , MicroARNs/genética , Neoplasias Ováricas/genética , Fosfohidrolasa PTEN/genética , ARN Circular/genéticaRESUMEN
MicroRNAs (miRs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. Single-nucleotide variants (SNVs) in primary miRs (pri-miRs), precursor miRs (pre-miRs), promoters of pri-miRs, and seed regions can affect miR stability or processing, may influence mature miR expression, and can affect target gene identification, respectively. The present protocol tests the binding and activity of miRs on 3'-UTR target sequences based on the expression of luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miR of interest to test in vitro cell culture assay.
Asunto(s)
MicroARNs , MicroARNs/genética , Genes Reporteros , Bioensayo , Técnicas de Cultivo de Célula , Regiones no Traducidas 3'/genética , NucleótidosRESUMEN
Soil quality is usually determined by its physical-chemical characteristics without taking into account the bacterial communities that play a fundamental role in the chemical decomposition of plant nutrients. In this context, the objective of the study was to evaluate bacterial diversity in high Andean grassland soils disturbed with Lepidium meyenii cultivation under different gradients of use (first, second and third use) and crop development (pre-sowing, hypocotyl development and post-harvest). The sampling was carried out in the Bombón plateau in the central Andes of Peru, during the rainy and low water seasons, by the systematic method based on a specific pattern assigned in a geometric rectangular shape at a depth of 0 - 20 cm. The characterization of the bacterial communities was carried out through the metagenomic sequencing of the 16S rRNA. 376 families of bacteria were reported, of which it was determined that there was a significant change in bacterial composition and distribution in relation to use pressure. There were no major changes due to the development of Lepidium meyenii. The families most sensitive to use pressure and soil poverty indicators were Verrucomicrobiaceae, Acidobacteraceae and Aakkermansiaceae.(AU)
A qualidade do solo é normalmente determinada pelas suas características físico-químicas sem ter em conta as comunidades bacterianas que desempenham um papel fundamental na decomposição química dos nutrientes das plantas. Neste contexto, o objetivo do estudo foi avaliar a diversidade bacteriana em solos de prados andinos elevados perturbados pelo cultivo de Lepidium meyenii sob diferentes gradientes de utilização (primeira, segunda e terceira utilizações) e desenvolvimento das culturas (pré-semeadura, desenvolvimento do hipocótilo e pós colheita). A amostragem foi realizada no planalto de Bombón, nos Andes centrais do Peru, durante as estações das chuvas e das águas baixas, pelo método sistemático baseado num padrão específico atribuído em forma geométrica retangular a uma profundidade de 0 - 20 cm. A caracterização das comunidades bacterianas foi realizada através da sequenciação metagenômica do rRNA 16S. Foram relatadas 376 famílias de bactérias, das quais se verificou uma alteração significativa na composição e distribuição bacteriana em relação à pressão de utilização. Não se registaram grandes alterações devido ao desenvolvimento do Lepidium meyenii. As famílias mais sensíveis à utilização de indicadores de pressão e pobreza do solo foram as Verrucomicrobiaceae, Acidobacteraceae e Aakkermansiaceae.(AU)
Asunto(s)
Animales , Regiones Promotoras Genéticas , Genes Reporteros , Microbiología del Suelo , LepidiumRESUMEN
Soil quality is usually determined by its physical-chemical characteristics without taking into account the bacterial communities that play a fundamental role in the chemical decomposition of plant nutrients. In this context, the objective of the study was to evaluate bacterial diversity in high Andean grassland soils disturbed with Lepidium meyenii cultivation under different gradients of use (first, second and third use) and crop development (pre-sowing, hypocotyl development and post-harvest). The sampling was carried out in the Bombón plateau in the central Andes of Peru, during the rainy and low water seasons, by the systematic method based on a specific pattern assigned in a geometric rectangular shape at a depth of 0 - 20 cm. The characterization of the bacterial communities was carried out through the metagenomic sequencing of the 16S rRNA. 376 families of bacteria were reported, of which it was determined that there was a significant change in bacterial composition and distribution in relation to use pressure. There were no major changes due to the development of Lepidium meyenii. The families most sensitive to use pressure and soil poverty indicators were Verrucomicrobiaceae, Acidobacteraceae and Aakkermansiaceae.
A qualidade do solo é normalmente determinada pelas suas características físico-químicas sem ter em conta as comunidades bacterianas que desempenham um papel fundamental na decomposição química dos nutrientes das plantas. Neste contexto, o objetivo do estudo foi avaliar a diversidade bacteriana em solos de prados andinos elevados perturbados pelo cultivo de Lepidium meyenii sob diferentes gradientes de utilização (primeira, segunda e terceira utilizações) e desenvolvimento das culturas (pré-semeadura, desenvolvimento do hipocótilo e pós colheita). A amostragem foi realizada no planalto de Bombón, nos Andes centrais do Peru, durante as estações das chuvas e das águas baixas, pelo método sistemático baseado num padrão específico atribuído em forma geométrica retangular a uma profundidade de 0 - 20 cm. A caracterização das comunidades bacterianas foi realizada através da sequenciação metagenômica do rRNA 16S. Foram relatadas 376 famílias de bactérias, das quais se verificou uma alteração significativa na composição e distribuição bacteriana em relação à pressão de utilização. Não se registaram grandes alterações devido ao desenvolvimento do Lepidium meyenii. As famílias mais sensíveis à utilização de indicadores de pressão e pobreza do solo foram as Verrucomicrobiaceae, Acidobacteraceae e Aakkermansiaceae.
Asunto(s)
Animales , Genes Reporteros , Lepidium , Microbiología del Suelo , Regiones Promotoras GenéticasRESUMEN
Antihistamines and glucocorticoids (GCs) are often used together in the clinic to treat several inflammation-related situations. Although there is no rationale for this association, clinical practice has assumed that, due to their concomitant anti-inflammatory effects, there should be an intrinsic benefit to their co-administration. In this work, we evaluated the effects of the co-treatment of several antihistamines on dexamethasone-induced glucocorticoid receptor transcriptional activity on the expression of various inflammation-related genes in A549 and U937 cell lines. Our results show that all antihistamines potentiate GCs' anti-inflammatory effects, presenting ligand-, cell- and gene-dependent effects. Given that treatment with GCs has strong adverse effects, particularly on bone metabolism, we also examined the impact of antihistamine co-treatment on the expression of bone metabolism markers. Using MC3T3-E1 pre-osteoblastic cells, we observed that, though the antihistamine azelastine reduces the expression of dexamethasone-induced bone loss molecular markers, it potentiates osteoblast apoptosis. Our results suggest that the synergistic effect could contribute to reducing GC clinical doses, ineffective by itself but effective in combination with an antihistamine. This could result in a therapeutic advantage, as the addition of an antihistamine may reinforce the wanted effects of GCs, while related adverse effects could be diminished or at least mitigated. By modulating the patterns of gene activation/repression mediated by GR, antihistamines could enhance only the desired effects of GCs, allowing their effective dose to be reduced. Further research is needed to correctly determine the clinical scope, benefits, and potential risks of this therapeutic strategy.
Asunto(s)
Antiinflamatorios/farmacología , Dexametasona/farmacología , Regulación de la Expresión Génica , Antagonistas de los Receptores Histamínicos/farmacología , Inflamación/genética , Receptores de Glucocorticoides/metabolismo , Animales , Biomarcadores/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Ratones , FN-kappa B/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Ftalazinas/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
PURPOSE: The GEO database and KEGG database-based analyses identified the differential expression of cyclin-dependent kinase 1 (CDK1) in cervical cancer and its involvement in the cell cycle pathway. In the present study, we aim to clarify the role of CDK1 in cervical cancer and the function of upstream microRNA (miR)-143-3p/miR-495-3p. METHODS: The expression of miR-143-3p, miR-495-3p, and CDK1 in cervical cancer tissues and cells was determined using RT-qPCR. Cell bioactivities were examined by CCK-8 and flow cytometry. The binding affinity between CDK1 and miR-143-3p/miR-495-3p was investigated using dual luciferase gene reporter assay. A xenograft mouse model of cervical cancer was then established to explore their effect on the tumorigenicity of cervical cancer cells in vivo. RESULTS: CDK1 was found to be the common target gene of miR-143-3p and miR-495-3p. CDK1 overexpression occurred in cervical cancer tissues and cells, while expression of miR-495-3p and miR-143-3p was down-regulated. The viability was inhibited while the apoptosis was promoted in cervical cancer cells in response to miR-143-3p or miR-495-3p overexpression, or CDK1 silencing. Further, miR-143-3p or miR-495-3p overexpression was also substantiated to inhibit the tumorigenicity of cervical cancer cells in vivo, while CDK1 overexpression counteracted their effect. CONCLUSION: Taken together, miR-143-3p and miR-495-3p co-target CDK1, thereby inhibiting the occurrence and development of cervical cancer.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , MicroARNs/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Animales , Apoptosis , Proteína Quinasa CDC2/genética , Ciclo Celular , Movimiento Celular , Proliferación Celular , Cuello del Útero/metabolismo , Bases de Datos Genéticas , Regulación hacia Abajo , Femenino , Silenciador del Gen , Genes Reporteros , Células HeLa , Xenoinjertos , Humanos , Luciferasas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Distribución Aleatoria , Regulación hacia Arriba , Neoplasias del Cuello Uterino/patologíaRESUMEN
OBJECTIVE: Emerging studies highlight the crucial effects of microRNAs on cancer initiation and malignant progression of various tumors. This study focused on the biological effect of miR-377-3p on CUL1 and epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin pathways in osteosarcoma (OS). METHODS: We performed quantitative real-time polymerase chain reaction (qRT-PCR) to analyze miR-377-3p and CUL1 expression levels in OS tissues and MG-63 cells. Then, cell counting kit (CCK)-8 and Transwell assay were used to examine the functions of miR-377-3p in OS cell growth and metastasis abilities. Meanwhile, luciferase reporter assay was used to validate CUL1 as direct target of miR-377-3p. qRT-PCR and Western blot were then carried out to detect the impact of miR-377-3p on EMT and Wnt/ß-catenin pathways. Tumor xenograft models were established to further examine the effects of miR-377-3p on OS tumorigenesis in vivo. RESULTS: miR-377-3p downregulation was frequently identified in OS tissues and cells, which was associated with worse prognosis of OS patients. Functional experiments showed miR-377-3p restoration could dramatically repress OS cell growth and migration by regulation of EMT and Wnt/ß-catenin pathways. Moreover, luciferase reporter assay revealed that CUL1 acted as a functional target of miR-377-3p. Additionally, the elevated CUL1 expressions in OS tissues also indicated poor prognosis of OS patients. Furthermore, the OS tumor growth was also obviously inhibited by miR-377-3p overexpression in vivo. CONCLUSIONS: Collectively, all the above findings revealed that miR-377-3p exerted anti-OS functions via CUL1 and EMT and Wnt/ß-catenin pathways. These results may contribute to the development of clinical OS treatment.
Asunto(s)
Neoplasias Óseas/metabolismo , Proteínas Cullin/metabolismo , MicroARNs/metabolismo , Osteosarcoma/metabolismo , Vía de Señalización Wnt , Animales , Neoplasias Óseas/mortalidad , Neoplasias Óseas/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Genes Reporteros , Xenoinjertos , Humanos , Luciferasas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Osteosarcoma/mortalidad , Osteosarcoma/patología , Osteosarcoma/secundario , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hijacking the autophagic machinery is a key mechanism through which invasive pathogens such as Staphylococcus aureus replicate in their host cells. We have previously demonstrated that the bacteria replicate in phagosomes labeled with the autophagic protein LC3, before escaping to the cytoplasm. Here, we show that the Ca2+-dependent PKCα binds to S. aureus-containing phagosomes and that α-hemolysin, secreted by S. aureus, promotes this recruitment of PKCα to phagosomal membranes. Interestingly, the presence of PKCα prevents the association of the autophagic protein LC3. Live cell imaging experiments using the PKC activity reporter CKAR reveal that treatment of cells with S. aureus culture supernatants containing staphylococcal secreted factors transiently activates PKC. Functional studies reveal that overexpression of PKCα causes a marked inhibition of bacterial replication. Taken together, our data identify enhancing PKCα activity as a potential approach to inhibit S. aureus replication in mammalian cells.
Asunto(s)
Autofagia , Interacciones Huésped-Patógeno , Fagosomas/metabolismo , Proteína Quinasa C-alfa/metabolismo , Infecciones Estafilocócicas/etiología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/fisiología , Animales , Autofagia/inmunología , Células CHO , Línea Celular , Células Cultivadas , Cricetulus , Susceptibilidad a Enfermedades , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Interacciones Huésped-Patógeno/inmunología , Modelos Biológicos , Fagosomas/inmunología , Proteína Quinasa C-alfa/genéticaRESUMEN
Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.
Asunto(s)
Técnicas Biosensibles , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Potenciales de la Membrana , Microscopía Fluorescente , Imagen Óptica , Animales , Retículo Endoplásmico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Células MCF-7 , Optogenética , Células PC12 , RatasRESUMEN
Intracellular cAMP (i-cAMP) levels play an important role in acute myeloid leukemia (AML) cell proliferation and differentiation. Its levels are the result of cAMP production, degradation, and exclusion. We have previously described histamine H2 receptors and MRP4/ABCC4 as two potential targets for AML therapy. Acting through histamine H2 receptors, histamine increases cAMP production/synthesis, while MRP4/ABCC4 is responsible for the exclusion of this cyclic nucleotide. In this study, we show that histamine treatment induces MRP4/ABCC4 expression, augmenting cAMP efflux, and that histamine, in combination with MRP inhibitors, is able to reduce AML cell proliferation. Histamine, through histamine H2 receptor, increases i-cAMP levels and induces MRP4 transcript and protein levels in U937, KG1a, and HL-60 cells. Moreover, histamine induces MRP4 promoter activity in HEK293T cells transfected with histamine H2 receptor (HEK293T-H2 R). Our results support that the cAMP/Epac-PKA pathway, and not MEK/ERK nor PI3K/AKT signaling cascades, is involved in histamine-mediated upregulation of MRP4 levels. Finally, the addition of histamine potentiates the inhibition of U937, KG1a, and HL-60 cell proliferation induced by MRP4 inhibitors. Our data highlight that the use of a poly-pharmacological approach aimed at different molecular targets would be beneficial in AML treatment.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Histamina/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Receptores Histamínicos H2/genética , Benzotiazoles/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Leucémica de la Expresión Génica , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HL-60 , Histamina/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Terapia Molecular Dirigida/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Probenecid/farmacología , Regiones Promotoras Genéticas , Propionatos/farmacología , Quinolinas/farmacología , Receptores Histamínicos H2/metabolismo , Transducción de Señal , Triazoles/farmacología , Células U937RESUMEN
The Target of Rapamycin (TOR) protein kinase plays a pivotal role in metabolism and gene expression, which enables cell proliferation, growth and development. Lipopolysaccharides (LPS) are a class of complex glycolipids present in the cell surface of Gram-negative bacteria and mediate plant-bacteria interactions. In this study, we examined whether LPS from Azospirillum brasilense Sp245 affect Arabidopsis thaliana growth via a mechanism involving TOR. A. thaliana plants were treated with LPS and plant growth and development were analyzed in mature plants. Morphological and molecular changes as well as TOR expression and activity were analyzed in root tissues. LPS increased total fresh weight, root length and TOR::GUS expression in the root meristem. Phosphorylation of S6k protein, a downstream target of TOR, increased following LPS treatment, which correlated with increased or decreased expression of CycB1;1::GUS protein upon treatment with LPS or TOR inhibitor AZD-8055, respectively. Long term LPS treatment further increased the rosette size as well as the number of stems and siliques per plant, indicating an overall phytostimulant effect for these signaling molecules. Taken together, the results suggest that A. brasilense LPS play probiotic roles in plants influencing TOR-mediated processes.
Asunto(s)
Arabidopsis/efectos de los fármacos , Azospirillum brasilense/química , Lipopolisacáridos/farmacología , Probióticos/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomasa , Genes Reporteros , Fosforilación , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrolloRESUMEN
The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tandem dimer fluorescent protein reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of-function approaches.
Asunto(s)
Genes Reporteros , Ingeniería Genética , Trypanosoma cruzi , Enfermedad de Chagas , Electroporación , Histonas , Humanos , Integrasas/genética , Plásmidos , Trypanosoma cruzi/genéticaRESUMEN
Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas de la Membrana/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación Alostérica , Animales , Animales Modificados Genéticamente , Membrana Celular/ultraestructura , Polaridad Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Embrión no Mamífero , Células Epiteliales/enzimología , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Larva/citología , Larva/enzimología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C/química , Proteína Quinasa C/genética , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.
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Antivirales/farmacología , Descubrimiento de Drogas/métodos , Genes Reporteros/fisiología , Virus ARN/efectos de los fármacos , Replicón/fisiología , Animales , Antivirales/química , Línea Celular , Chlorocebus aethiops , Cricetinae , Humanos , Virus ARN/genética , Transfección , Células VeroRESUMEN
Dengue is the single most important human viral infection transmitted by insects. The function of the viral proteins andtheir interactions with the host cell is under exhaustive investigation with the aim of identifying antiviral strategies. Here,using recombinant full-length dengue virus genomes, carrying a fluorescent mCherry fused to capsid, we studied biophysicalproperties of the viral protein during one infectious cycle in living cells. Dengue virus capsid protein associates to differentcellular compartments but its function in these locations is largely unknown. We evaluated the diffusion of capsid inside the celland determined a higher effective diffusion coefficient in the cytoplasm than in the nucleus. Using advanced fluorescencecorrelation methods, including the recently developed two-dimensional pair correlation analysis, we constructed for the first timehigh resolution maps of capsid mobility in an infected cell. We observed that the motion of capsid in the nucleoplasm-nucleolusinterface was highly organized, indicating an obstacle in this interface. Although nucleoli are membraneless structures, theydisplayed liquid-liquid phase separation. Once inside nucleoli, the protein showed isotropic mobility, indicating free diffusion orimmobilized capsid inside these structures. This is the first study presenting spatial and temporal dynamics of the dengue viruscapsid protein during infection.
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Proteínas de la Cápside/metabolismo , Virus del Dengue/fisiología , Dengue/virología , Animales , Proteínas de la Cápside/genética , Compartimento Celular , Línea Celular , Sistemas de Computación , Cricetinae , Difusión , Fibroblastos , Genes Reporteros , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Mesocricetus , Microscopía Confocal , Movimiento (Física) , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Fluorescencia , Fracciones Subcelulares/química , Imagen de Lapso de Tiempo , Proteína Fluorescente RojaRESUMEN
Pluripotency maintenance requires transcription factors (TFs) that induce genes necessary to preserve the undifferentiated state and repress others involved in differentiation. Recent observations support that the heterogeneous distribution of TFs in the nucleus impacts on gene expression. Thus, it is essential to explore how TFs dynamically organize to fully understand their role in transcription regulation. Here, we examine the distribution of pluripotency TFs Oct4 and Sox2 in the nucleus of embryonic stem (ES) cells and inquire whether their organization changes during early differentiation stages preceding their downregulation. Using ES cells expressing Oct4-YPet or Sox2-YPet, we show that Oct4 and Sox2 partition between nucleoplasm and a few chromatin-dense foci which restructure after inducing differentiation by 2i/LIF withdrawal. Fluorescence correlation spectroscopy showed distinct changes in Oct4 and Sox2 dynamics after differentiation induction. Specifically, we detected an impairment of Oct4-chromatin interactions whereas Sox2 only showed slight variations in its short-lived, and probably more unspecific, interactions with chromatin. Our results reveal that differentiation cues trigger early changes of Oct4 and Sox2 nuclear distributions that also include modifications in TF-chromatin interactions. This dynamical reorganization precedes Oct4 and Sox2 downregulation and may contribute to modulate their function at early differentiation stages.
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Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Células Madre Embrionarias/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transcripción Genética , Animales , Ciclo Celular , Diferenciación Celular , Núcleo Celular/ultraestructura , Células Cultivadas , Doxiciclina/farmacología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Ratones , Microscopía Fluorescente , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción SOXB1/genética , TransfecciónRESUMEN
MAIN CONCLUSION: The structure of the cotton uceA1.7 promoter and its modules was analyzed; the potential of their key sequences has been confirmed in different tissues, proving to be a good candidate for the development of new biotechnological tools. Transcriptional promoters are among the primary genetic engineering elements used to control genes of interest (GOIs) associated with agronomic traits. Cotton uceA1.7 was previously characterized as a constitutive promoter with activity higher than that of the constitutive promoter from the Cauliflower mosaic virus (CaMV) 35S gene in various plant tissues. In this study, we generated Arabidopsis thaliana homozygous events stably overexpressing the gfp reporter gene driven by different modules of the uceA1.7 promoter. The expression level of the reporter gene in different plant tissues and the transcriptional stability of these modules was determined compared to its full-length promoter and the 35S promoter. The full-length uceA1.7 promoter exhibited higher activity in different plant tissues compared to the 35S promoter. Two modules of the promoter produced a low and unstable transcription level compared to the other promoters. The other two modules rich in cis-regulatory elements showed similar activity levels to full-length uceA1.7 and 35S promoters but were less stable. This result suggests the location of a minimal portion of the promoter that is required to initiate transcription properly (the core promoter). Additionally, the full-length uceA1.7 promoter containing the 5'-untranslated region (UTR) is essential for higher transcriptional stability in various plant tissues. These findings confirm the potential use of the full-length uceA1.7 promoter for the development of new biotechnological tools (NBTs) to achieve higher expression levels of GOIs in, for example, the root or flower bud for the efficient control of phytonematodes and pest-insects, respectively, in important crops.
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Gossypium/genética , Regiones no Traducidas 5' , Arabidopsis/genética , Caulimovirus/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genes Reporteros , Ingeniería Genética , Gossypium/anatomía & histología , Gossypium/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Regiones Promotoras GenéticasRESUMEN
The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.