RESUMEN
Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain-of-function as an underlying disease mechanism in KIF5A-associated ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , ADN sin Sentido/genética , Drosophila melanogaster , Mutación con Ganancia de Función , Humanos , Cinesinas/genética , Neuronas Motoras/metabolismo , Mutación , Proteína 2 Similar al Factor de Transcripción 7/metabolismoRESUMEN
Functional mechanisms remain unknown for most genetic loci associated to complex human traits and diseases. In this study, we first mapped trans-eQTLs in a data set of primary monocytes stimulated with LPS, and discovered that a risk variant for autoimmune disease, rs17622517 in an intron of C5ORF56, affects the expression of the transcription factor IRF1 20 kb away. The cis-regulatory effect specific to IRF1 is active under early immune stimulus, with a large number of trans-eQTL effects across the genome under late LPS response. Using CRISPRi silencing, we showed that perturbation of the SNP locus downregulates IRF1 and causes widespread transcriptional effects. Genome editing by CRISPR had suggestive recapitulation of the LPS-specific trans-eQTL signal and lent support for the rs17622517 site being functional. Our results suggest that this common genetic variant affects inter-individual response to immune stimuli via regulation of IRF1. For this autoimmune GWAS locus, our work provides evidence of the functional variant, demonstrates a condition-specific enhancer effect, identifies IRF1 as the likely causal gene in cis, and indicates that overactivation of the downstream immune-related pathway may be the cellular mechanism increasing disease risk. This work not only provides rare experimental validation of a master-regulatory trans-eQTL, but also demonstrates the power of eQTL mapping to build mechanistic hypotheses amenable for experimental follow-up using the CRISPR toolkit.
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Enfermedades Autoinmunes/genética , Inmunidad/genética , Factor 1 Regulador del Interferón/genética , Adulto , Enfermedades Autoinmunes/metabolismo , Mapeo Cromosómico/métodos , ADN sin Sentido/genética , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Células HEK293 , Humanos , Factor 1 Regulador del Interferón/metabolismo , Masculino , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de RiesgoRESUMEN
The present study aimed to explore the biological functions and molecular mechanisms of the long noncoding RNA VIM antisense RNA 1 (VIMAS1) in gastric cancer (GC). The expression of VIMAS1 was analyzed in tissues from patients with GC and GC cell lines by reverse transcriptionquantitative (RTq)PCR. The relationship between VIMAS1 expression and overall survival time of patients with GC was also assessed. To determine the biological functions of VIMAS1, Cell Counting Kit8 assay, colony formation assay, flow cytometry, wound healing assay and Transwell assay were employed. The targeting relationship among VIMAS1, microRNA (miR)8052 and frizzled 1 (FZD1) was verified by the dual luciferase reporter gene assay. The underlying molecular mechanism of VIMAS1 on GC was determined by RTqPCR and western blotting. In addition, tumor formation was detected in nude mice. The results of the present study demonstrated that VIMAS1 was highly expressed in GC tissues and cells. In addition, VIMAS1 expression was demonstrated to be closely related to the prognosis of patients with GC. Notably, silencing VIMAS1 inhibited the proliferation, migration and invasion, and enhanced apoptosis of AGS and HGC27 cells. Silencing VIMAS1 significantly increased the protein expression levels of cleaved caspase3, Bax and Ecadherin, but decreased the protein expression levels of Bcl2, Ncadherin, vimentin, matrix metalloproteinase (MMP)2, MMP9, ßcatenin, cyclin D1, Cmyc and FZD1. Additionally, silencing VIMAS1 inhibited tumor growth in nude mice. Cumulatively, the present study demonstrated that VIMAS1 may promote cell proliferation, migration, invasion and epithelialmesenchymal transition by regulating FDZ1 and activating the Wnt/ßcatenin pathway in GC.
Asunto(s)
Neoplasias Gástricas/genética , Vimentina/genética , Adulto , Anciano , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , China , ADN sin Sentido/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , ARN Largo no Codificante/genética , Neoplasias Gástricas/metabolismo , Vimentina/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , ADN sin Sentido/genética , Fusión Génica/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Leucemia Mieloide Aguda/genética , Receptores de Ácido Kaínico/genética , Eliminación de Secuencia/genética , Translocación Genética/genética , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Factor Estimulante de Colonias de Granulocito/biosíntesis , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Receptor de Ácido Kaínico GluK2RESUMEN
Estrogen-responsive breast cancer cells exhibit both basal and estrogen-regulated transcriptional programs, which lead to the transcription of many different transcription units (i.e., genes), including those that produce coding and non-coding sense (e.g., mRNA, lncRNA) and antisense (i.e., asRNA) transcripts. We have previously characterized the global basal and estrogen-regulated transcriptomes in estrogen receptor alpha (ERα)-positive MCF-7 breast cancer cells. Herein, we have mined genomic data to define three classes of antisense transcription in MCF-7 cells based on where their antisense transcription termination sites reside relative to their cognate sense mRNA and lncRNA genes. These three classes differ in their response to estrogen treatment, the enrichment of a number of genomic features associated with active promoters (H3K4me3, RNA polymerase II, open chromatin architecture), and the biological functions of their cognate sense genes as analyzed by DAVID gene ontology. We further characterized two estrogen-regulated antisense transcripts arising from the MYC gene in MCF-7 cells, showing that these antisense transcripts are 5'-capped, 3'-polyadenylated, and localized to different compartments of the cell. Together, our analyses have revealed distinct classes of antisense transcription correlated to different biological processes and response to estrogen stimulation, uncovering another layer of hormone-regulated gene regulation.
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Adenocarcinoma/genética , Neoplasias de la Mama/genética , ADN sin Sentido/genética , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Adenocarcinoma/patología , Metabolismo Basal/efectos de los fármacos , Metabolismo Basal/genética , Neoplasias de la Mama/patología , ADN sin Sentido/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Humanos , Células MCF-7 , Análisis por Micromatrices , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , ARN sin Sentido/efectos de los fármacos , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , Transcripción Genética/efectos de los fármacos , Transcriptoma/efectos de los fármacosRESUMEN
Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.
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ADN sin Sentido/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Aminas/química , Animales , Cationes/química , ADN sin Sentido/química , ADN sin Sentido/genética , ADN sin Sentido/farmacocinética , Disulfuros/química , Silenciador del Gen , Células HeLa , Humanos , Masculino , Ratones Endogámicos ICR , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , Transfección/métodosRESUMEN
RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.
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ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/enzimología , Transcripción Genética , Adenosina Trifosfato/genética , Citidina Trifosfato/genética , ADN/genética , ADN/metabolismo , ADN sin Sentido/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Microscopía Fluorescente , Regiones Promotoras Genéticas , Imagen Individual de MoléculaRESUMEN
Shotgun expression of antisense cDNA, where each transformed cell expresses a different antisense cDNA, has been used for mutagenesis and gene identification in Dictyostelium discoideum. However, the method has two limitations. First, there were too few clones in the shotgun antisense cDNA library to have an antisense cDNA for every gene in the genome. Second, the unequal transcription level of genes resulted in many antisense cDNAs in the library for some genes but relatively few antisense cDNAs for other genes. Here we report an improved method for generating a larger antisense cDNA library with a reduced percentage of cDNA clones from highly prevalent mRNAs and demonstrate its utility by screening for signal transduction pathway components in D. discoideum.
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ADN sin Sentido/genética , ADN Complementario/genética , Mutagénesis/genética , ADN Complementario/química , ADN Protozoario/química , ADN Protozoario/genética , Dictyostelium/genética , Análisis de Secuencia de ADNRESUMEN
Gastric cancer (GC) is the second most prevalent carcinoma resulting in cancer-related deaths in the world, with differences among geographic areas. Although the incidence and mortality rates of GC in Asia are decreasing, the search for diverse and effective therapies of GC is still needed to be fully inquired. The present research explored the expression pattern, functional role and underlying mechanism of DLX6-AS1 in GC. Firstly, we measured DLX6-AS1 expression in GC and then found the elevated level of DLX6-AS1. To further inspect the function role of DLX6-AS1 involved in GC, we performed lost-of-function assays. The silencing of DLX6-AS1 suppressed cell proliferation, migration and EMT process of GC cells. Subsequently, we uncovered that MAP4K1 was also up-regulated in GC and could be positively regulated by DLX6-AS1. Moreover, MAP4K1 down-regulation similarly inhibited GC progression. In addition, DLX6-AS1 stabilized MAP4K1 via modulating FUS. In summary, DLX6-AS1 modulated GC progression through FUS-regulated MAP4K1. Our paper exposed the role and regulatory mechanism of DLX6-AS1 in GC, which suggested a novel and valid therapy for GC patients.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Largo no Codificante/genética , Proteína FUS de Unión a ARN/metabolismo , Neoplasias Gástricas/patología , Apoptosis , Biomarcadores de Tumor/genética , Movimiento Celular , Proliferación Celular , ADN sin Sentido/genética , Proteínas de Homeodominio/genética , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteína FUS de Unión a ARN/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: LpxB is an enzyme involved in the biosynthesis pathway of lipid A, a component of LPS. OBJECTIVES: To evaluate the lpxB gene in Acinetobacter baumannii as a potential therapeutic target and to propose antisense agents such as peptide nucleic acids (PNAs) as a tool to combat bacterial infection, either alone or in combination with known antimicrobial therapies. METHODS: RNA-seq analysis of the A. baumannii ATCC 17978 strain in a murine pneumonia model was performed to study the in vivo expression of lpxB. Protein expression was studied in the presence or absence of anti-lpxB (KFF)3K-PNA (pPNA). Time-kill curve analyses and protection assays of infected A549 cells were performed. The chequerboard technique was used to test for synergy between pPNA and colistin. A Galleria mellonella infection model was used to test the in vivo efficacy of pPNA. RESULTS: The lpxB gene was overexpressed during pneumonia. Treatment with a specific pPNA inhibited LpxB expression in vitro, decreased survival of the ATCC 17978 strain and increased the survival rate of infected A549 cells. Synergy was observed between pPNA and colistin in colistin-susceptible strains. In vivo assays confirmed that a combination treatment of anti-lpxB pPNA and colistin was more effective than colistin in monotherapy. CONCLUSIONS: The lpxB gene is essential for A. baumannii survival. Anti-lpxB pPNA inhibits LpxB expression, causing bacterial death. This pPNA showed synergy with colistin and increased the survival rate in G. mellonella. The data suggest that antisense pPNA molecules blocking the lpxB gene could be used as antibacterial agents.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Colistina/farmacología , ADN sin Sentido/genética , Ácidos Nucleicos de Péptidos/farmacología , Células A549 , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/genética , Animales , Proteínas Bacterianas/genética , Vías Biosintéticas , Sinergismo Farmacológico , Expresión Génica , Humanos , Lípido A/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/microbiología , RNA-SeqRESUMEN
Multidrug-resistant Acinetobacter baumannii isolates cause critical problems in health-care environments. AdeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically essential antibiotics in A. baumannii, such as ciprofloxacin. This study aimed to target adeB gene with antisense peptide nucleic acid (PNA) and investigate its effect on resistance to antibiotics. NCBI database was used to design appropriate PNA to target adeB gene, by connecting PNA to mRNA, the translation of mRNA can be prevented. Three clinical isolates and A. baumannii ATCC 17978 were treated with the designed PNA by electroporation and competence procedure. Minimum Inhibitory concentration (MIC) of ciprofloxacin, colistin, and tetracycline were determined by microbroth dilution method. In addition, the expression level of adeB gene was measured by quantitative real-time PCR (qRT-PCR). Isolates used in this study had mutations in gyrA and parC genes corresponding to resistance to ciprofloxacin. MIC of resistance to ciprofloxacin after treatment with PNA was reduced from 32⯵g/ml to16⯵g/ml in A. baumannii ATCC 17978 isolate. Susceptibility level of tetracycline, in the 2 clinical isolates was decreased from 64⯵g/ml to 32⯵g/ml and in the other isolate was reduced from 128⯵g/ml to 64⯵g/ml. The expression level of adeB gene was decreased in A. baumannii ATCC 17978 (Pâ¯>â¯0.01) but not in clinical isolate (Pâ¯=â¯0.107). Findings of the present study indicate overexpression of adeB efflux pump has extra effect on resistance to antibiotics in isolates with a defined mechanism of resistance. Antisense technology is a feasible technique to suppress the function of these genes, which may be further exploited to control multidrug-resistant isolates.
Asunto(s)
Acinetobacter baumannii/genética , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Técnicas de Silenciamiento del Gen , Proteínas de Transporte de Membrana/genética , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/aislamiento & purificación , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Ciprofloxacina/farmacología , Colistina/farmacología , ADN sin Sentido/genética , ADN sin Sentido/farmacología , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Ácidos Nucleicos de Péptidos/genética , Ácidos Nucleicos de Péptidos/farmacocinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tetraciclina/farmacologíaAsunto(s)
ADN sin Sentido/uso terapéutico , Industria Farmacéutica/organización & administración , Emprendimiento/organización & administración , Ingeniería Genética , Enfermedad de Huntington/terapia , Liderazgo , Ensayos Clínicos Fase III como Asunto , ADN sin Sentido/genética , Humanos , Enfermedad de Huntington/genética , Modelos OrganizacionalesRESUMEN
We aimed to detect Attention-deficit/hyperactivity (ADHD) risk-conferring genes in adults. In children, ADHD is characterized by age-inappropriate levels of inattention and/or hyperactivity-impulsivity and may persists into adulthood. Childhood and adulthood ADHD are heritable, and are thought to represent the clinical extreme of a continuous distribution of ADHD symptoms in the general population. We aimed to leverage the power of studies of quantitative ADHD symptoms in adults who were genotyped. Within the SAGA (Study of ADHD trait genetics in adults) consortium, we estimated the single nucleotide polymorphism (SNP)-based heritability of quantitative self-reported ADHD symptoms and carried out a genome-wide association meta-analysis in nine adult population-based and case-only cohorts of adults. A total of n = 14,689 individuals were included. In two of the SAGA cohorts we found a significant SNP-based heritability for self-rated ADHD symptom scores of respectively 15% (n = 3656) and 30% (n = 1841). The top hit of the genome-wide meta-analysis (SNP rs12661753; p-value = 3.02 × 10-7) was present in the long non-coding RNA gene STXBP5-AS1. This association was also observed in a meta-analysis of childhood ADHD symptom scores in eight population-based pediatric cohorts from the Early Genetics and Lifecourse Epidemiology (EAGLE) ADHD consortium (n = 14,776). Genome-wide meta-analysis of the SAGA and EAGLE data (n = 29,465) increased the strength of the association with the SNP rs12661753. In human HEK293 cells, expression of STXBP5-AS1 enhanced the expression of a reporter construct of STXBP5, a gene known to be involved in "SNAP" (Soluble NSF attachment protein) Receptor" (SNARE) complex formation. In mouse strains featuring different levels of impulsivity, transcript levels in the prefrontal cortex of the mouse ortholog Gm28905 strongly correlated negatively with motor impulsivity as measured in the five choice serial reaction time task (r2 = - 0.61; p = 0.004). Our results are consistent with an effect of the STXBP5-AS1 gene on ADHD symptom scores distribution and point to a possible biological mechanism, other than antisense RNA inhibition, involved in ADHD-related impulsivity levels.
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Trastorno por Déficit de Atención con Hiperactividad/genética , Proteínas del Tejido Nervioso/genética , Proteínas R-SNARE/genética , ARN Largo no Codificante/genética , Adulto , Animales , Trastorno por Déficit de Atención con Hiperactividad/metabolismo , Estudios de Cohortes , ADN sin Sentido/genética , ADN sin Sentido/metabolismo , Femenino , Predisposición Genética a la Enfermedad/genética , Genética de Población/métodos , Estudio de Asociación del Genoma Completo , Genotipo , Células HEK293 , Humanos , Masculino , Ratones , Fenotipo , Polimorfismo de Nucleótido Simple/genética , ARN Largo no Codificante/metabolismo , Factores de RiesgoRESUMEN
Transplanted bone marrow mononuclear cells (BMC) support the healing of large bone defects. Neutralization of microRNA (MiR) that negatively affects key processes of the reparative response in BMC might help to further improve the beneficial effect of transplanted BMC in bone healing. Hence, the aim of this study was to evaluate if the neutralization of MiR-92A (vascularization) and MiR-335-5p (osteogenic differentiation) in BMC using specific antiMiRs leads to a further improvement of the BMC-supported therapy of large bone defects. BMC transiently transfected with antiMiR- 92A, antiMiR-335, antiMiR-92A, and antiMiR-355 or control antiMiR were seeded on ß-TCP (beta-tricalcium phosphate) and placed in a femoral large bone defect (5 mm) in Sprague-Dawley rats. Ultimate load as well as osseous integration of the ß-TCP-scaffolds were significantly improved in the antiMiR-335 group compared to the control group after 8 weeks, whereas neutralization of antiMiR-92A lead to an improvement of early vascularization after 1 week, but not to enhanced bone healing after 8 weeks. We demonstrated that the targeted inhibition of MiRs in transplanted BMC is a new approach that enhances BMC-supported bone healing.
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Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , ADN sin Sentido/biosíntesis , Fracturas del Fémur/terapia , Curación de Fractura/efectos de los fármacos , MicroARNs/antagonistas & inhibidores , Transfección , Animales , Células de la Médula Ósea/patología , ADN sin Sentido/genética , Fracturas del Fémur/genética , Fracturas del Fémur/metabolismo , Fracturas del Fémur/patología , MicroARNs/genética , MicroARNs/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
A capsule-defective mutant strain PBA129 of Pasteurella multocida was constructed by electroporation of phagemid containing the coding region of the antisense RNA of the ompH gene into the wild type strain X-73 (serovar A:1) of P. multocida. The pathogenicity and protective potency of the mutant against homologous and heterologous challenge in mice and chickens were characterized. Greyish colonies of the mutant, indicating lower capsule thickness, on selective dextrose starch agar were observed under an obliquely transmitted light stereomicroscope and compared to iridescent colonies of the wild type strain X-73. Strain PBA129 had lower capsule thickness than the wild type strain as observed with an electron microscope. Strain PBA129 was apparently attenuated, as mice and chickens inoculated with the bacteria at 108 CFU survived. Protection was observed in both mice and chickens inoculated with strain PBA129 upon challenge exposure to avian P. multocida strains X-73 and P-1059 (serovar A:3), respectively. In conclusion, the mutant strain PBA129 of P. multocida strain X-73 was completely attenuated, and it was possible to induce sufficient protection against avian P. multocida strains.
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Proteínas de la Membrana Bacteriana Externa/genética , Infecciones por Pasteurella/veterinaria , Pasteurella multocida/patogenicidad , Animales , Pollos , ADN sin Sentido/genética , Femenino , Ratones , Mutación , Infecciones por Pasteurella/microbiología , Infecciones por Pasteurella/prevención & control , Pasteurella multocida/genética , Enfermedades de las Aves de Corral/microbiologíaRESUMEN
The majority of embryonic loss in cattle occurs within the first 3 to 4 wk of pregnancy, and there are currently no accurate predictors of pregnancy outcome. Existing embryo quality assessment methods include morphological evaluation and embryo biopsy. These methods are not accurate and carry some health risks to the developing embryo, respectively. Therefore, there is need to identify noninvasive biomarkers such as microRNA that can predict embryo quality and pregnancy outcome. Furthermore, researchers need a better understanding of the dynamic interaction between the mother and the embryo. The transcriptome of the uterus shows plasticity that depends on the embryo type so that the expression level of some genes for in vivo embryos would be different from that of in vitro-produced embryos. Similarly, the embryonic transcriptome and epigenome change in response to different environmental factors such as stress, diet, disease, and physiological status of the mother. This embryo-mother crosstalk could be better understood by investigating the molecular signaling that occurs at different stages of embryonic development. Although transcriptomics is a useful tool to assess the roles of genes and pathways in embryo quality and maternal receptivity, it does not provide the exact functions of these genes, and it shows correlation rather than causality. Therefore, an in-depth functional genomic analysis is needed for better understanding of the molecular mechanisms controlling embryo development. In this review, we discuss recent genomic technologies such as RNA interference, gapmer technology, and genome editing techniques used in humans and livestock to elucidate the molecular mechanisms of genes affecting embryo development.
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Bovinos/embriología , Bovinos/genética , Embrión de Mamíferos/fisiología , Desarrollo Embrionario/genética , Fertilidad/genética , Genómica , Animales , Enfermedades de los Bovinos/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN sin Sentido/genética , Pérdida del Embrión/genética , Pérdida del Embrión/veterinaria , Femenino , Edición Génica , Humanos , Embarazo , Interferencia de ARN , Transcriptoma/genética , ÚteroRESUMEN
BACKGROUND: Although the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum. METHODS: Sorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle. RESULTS: This study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum. CONCLUSION: In general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum.
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Bromovirus , Silenciador del Gen , Sorghum/genética , Bromovirus/genética , ADN sin Sentido/genética , Flores/virología , Hojas de la Planta/virología , Sorghum/metabolismo , Sorghum/virología , Temperatura , Ubiquitina/metabolismoRESUMEN
Stress conditions such as UV irradiation, exposure to genotoxic agents, stalled DNA replication, and even tumors trigger the release of cytosolic genomic DNA (cgDNA). Classically, cgDNA induces interferon response via its binding to proteins such as STING. In this study, we found previously reported cgDNA (cg721) exists in the cytosol of the mouse cell lines, cultured under no stress conditions. The overexpression of cg721 suppressed the complementary RNA expression using strand selection and knockdown of DNA/RNA hybrid R-loop removing enzyme RNase H and three prime repair exonuclease 1 TREX1 increased the expression levels of cg721 and thus, inhibited the target Naa40 transcript, as well as protein expression, with a phenotypic effect. In addition, cgDNA was incorporated into extracellular vesicles (EVs), and the EV-derived cg721 inhibited gene expression of the acceptor cells. Thus, our findings suggest that cg721 functions as a natural antisense DNA and play a role in cell-to-cell gene regulation once it secreted outside the cell as EVs.
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Citosol/metabolismo , Daño del ADN , ADN sin Sentido/metabolismo , Exodesoxirribonucleasas/metabolismo , Fosfoproteínas/metabolismo , Rayos Ultravioleta , Animales , Línea Celular Tumoral , ADN sin Sentido/genética , Exodesoxirribonucleasas/genética , Humanos , Ratones , Fosfoproteínas/genéticaRESUMEN
Dendritic cells (DC) are important in the onset and severity of inflammatory bowel disease (IBD). Tolerogenic DC induce T-cells to become therapeutic Foxp3+ regulatory T-cells (Tregs). We therefore asked if experimental IBD could be prevented by administration of bone marrow-derived DC generated under conventional GM-CSF/IL-4 conditions but in the presence of a mixture of antisense DNA oligonucleotides targeting the primary transcripts of CD40, CD80, and CD86. These cell products (which we call AS-ODN BM-DC) have demonstrated tolerogenic activity in preventing type 1 diabetes and preserving beta cell mass in new-onset type 1 diabetes in the NOD mouse strain, in earlier studies. In addition to measuring efficacy in prevention of experimental IBD, we also sought to identify possible mechanism(s) of action. Weight, behavior, stool frequency, and character were observed daily for 7-10 days in experimental colitis in mice exposed to dextran sodium sulfate (DSS) following injection of the AS-ODN BM-DC. After euthanasia, the colons were processed for histology while spleen and mesenteric lymph nodes (MLNs) were made into single cells to measure Foxp3+ Treg as well as IL-10+ regulatory B-cell (Breg) population frequency by flow cytometry. AS-ODN BM-DC prevented DSS-induced colitis development. Recipients of these cells exhibited significant increases in Foxp3+ Treg and IL-10+ Breg in MLN and spleen. Histological examination of colon sections of colitis-free mice remained largely architecturally physiologic and mostly free of leukocyte infiltration when compared with DSS-treated animals. Although DSS colitis is mainly an innate immunity-driven condition, our study adds to the growing body of evidence showing that Foxp3+ Treg and IL-10 Bregs can suppress a mainly innate-driven inflammation. The already-established safety of human DC generated from monocytic progenitors in the presence of the mixture of antisense DNA targeting the primary transcripts of CD40, CD80, and CD86 in humans offers the potential to adapt them for clinical IBD therapy.
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Traslado Adoptivo/métodos , Células de la Médula Ósea/inmunología , Colitis Ulcerosa/terapia , Células Dendríticas/trasplante , Adulto , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Linfocitos B Reguladores/inmunología , Linfocitos B Reguladores/metabolismo , Células Cultivadas , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Colon/inmunología , Colon/patología , ADN sin Sentido/genética , Células Dendríticas/inmunología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Voluntarios Sanos , Humanos , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos NOD , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Resultado del TratamientoRESUMEN
A logic circuit controlled multi-responsive sensing platform built on a three-way DNA junction (TWJ) is reported. It enabled the construction of novel fluorescent sensing platforms responsive to any target out of HIV gene, ATP and pH value, and furthermore were logically regulated by two other targets and then behaved as different logic circuits, which consist of two tandem AND gates or cascaded NAND and INH gates by varying the positions of the fluorescent tags.