RESUMO
The accumulation of misfolded and aggregated proteins is a hallmark of neurodegenerative proteinopathies. Although multiple genetic loci have been associated with specific neurodegenerative diseases (NDs), molecular mechanisms that may have a broader relevance for most or all proteinopathies remain poorly resolved. In this study, we developed a multi-layered network expansion (MLnet) model to predict protein modifiers that are common to a group of diseases and, therefore, may have broader pathophysiological relevance for that group. When applied to the four NDs Alzheimer's disease (AD), Huntington's disease, and spinocerebellar ataxia types 1 and 3, we predicted multiple members of the insulin pathway, including PDK1, Akt1, InR, and sgg (GSK-3ß), as common modifiers. We validated these modifiers with the help of four Drosophila ND models. Further evaluation of Akt1 in human cell-based ND models revealed that activation of Akt1 signaling by the small molecule SC79 increased cell viability in all models. Moreover, treatment of AD model mice with SC79 enhanced their long-term memory and ameliorated dysregulated anxiety levels, which are commonly affected in AD patients. These findings validate MLnet as a valuable tool to uncover molecular pathways and proteins involved in the pathophysiology of entire disease groups and identify potential therapeutic targets that have relevance across disease boundaries. MLnet can be used for any group of diseases and is available as a web tool at http://ssbio.cau.ac.kr/software/mlnet.
Assuntos
Doença de Alzheimer , Doença de Huntington , Deficiências na Proteostase , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Glicogênio Sintase Quinase 3 beta , Doença de Huntington/genética , Transdução de SinaisRESUMO
The Lingulidae are often considered living fossils, because they have shown little morphological change since the Paleozoic. Limited morphological variation has also made the taxonomic study of living lingulids challenging. We investigated species diversity and phylogenetic relationships of extant lingulids and show that they are substantially more diverse than realized, demonstrating that morphological stasis was commonly accompanied by speciation. Species delimitation based on cytochrome c oxidase subunit I (COI) gene sequences from 194 specimens sampled from East Asia, Australia, Oceania, and the Americas suggested 14-22 species in the lingulids (9-17 species in Lingula and 4-5 species in Glottidia), in contrast to the 11-12 species currently recognized globally in the family. Four-gene phylogenetic analyses supported the sister relationship between Lingula and Glottidia. Within Lingula, L. adamsi, which possesses large, brownish shells, was recovered as sister to all remaining Lingula species, which have more or less greenish shells. Within the greenish Lingula clade, the 'L. anatina' complex was sister to the clade that includes the 'L. reevei' complex. The 'L. anatina' complex was further separated into two major clades with partly separate ranges centered on (i) temperate East Asia, and (ii) the tropical west-central Pacific. Within Glottidia, Pacific species were nested within Atlantic species. Time-calibrated phylogenetic analyses suggested that Lingula likely originated in the early Cretaceous contrary to a previously proposed hypothesis advocating a Cenozoic origin. The separation of Lingula and Glottidia appears to date from the Mesozoic, not from the Carboniferous, contrary to a previous hypothesis. Overall, our results uncovered substantial cryptic diversity in lingulids, which will form the basis for conservation and further taxonomic revision.
Assuntos
Fósseis , Hidrozoários , Animais , Ásia Oriental , Invertebrados/genética , FilogeniaRESUMO
The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, we performed cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, in Drosophila S2 cells. We found that regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Moreover, we provide evidence that miR-252-5p targets Abelson interacting protein ( Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, our data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila.-Lim, D.-H., Lee, S., Han, J. Y., Choi, M.-S., Hong, J.-S., Seong, Y., Kwon, Y.-S., Lee, Y. S. Ecdysone-responsive microR-252-5p controls the cell cycle by targeting Abi in Drosophila.
Assuntos
Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Ecdisona/metabolismo , MicroRNAs/metabolismo , Animais , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Larva/metabolismo , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Multiple etiologies of liver injury are associated with fibrosis in which the key event is the activation of hepatic stellate cells (HSCs). Although microRNAs (miRNAs) are reportedly involved in fibrogenesis, the complete array of miRNA signatures associated with the disease has yet to be elucidated. Here, deep sequencing analysis revealed that compared to controls, 80 miRNAs were upregulated and 21 miRNAs were downregulated significantly in the thioacetamide (TAA)-induced mouse fibrotic liver. Interestingly, 58 of the upregulated miRNAs were localized to an oncogenic miRNA megacluster upregulated in liver cancer. Differential expression of some of the TAA-responsive miRNAs was confirmed, and their human orthologs were similarly deregulated in TGF-ß1-activated HSCs. Moreover, a functional analysis of the experimentally validated high-confidence miRNA targets revealed significant enrichment for the GO terms and KEGG pathways involved in HSC activation and liver fibrogenesis. This is the first comprehensive report of miRNAs profiles during TAA-induced mouse liver fibrosis.
Assuntos
Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/genética , Fígado/metabolismo , MicroRNAs/genética , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular Transformada , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Transdução de Sinais , Tioacetamida , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The rhizocephalan Sacculina shiinoi sp. nov. parasitizes three species of Upogebia in Japan. It is described morphologically and compared with another Upogebia parasite, Sacculina upogebiae Shiino, 1943 from Japan and Korea. These two species are the only sacculinids that parasitize mud shrimps. DNA analyses clearly show the two species to be separate and not closely related. The cuticle differs in being provided with close-set, branched, and spiny excrescences in S. shiinoi, while it lacks excrescences, but forms small scales in S. upogebiae. In S. upogebiae, the bulbous sperm-producing part and the narrow receptacle duct are separated by a compartmentalized mid portion, which is missing in S. shiinoi. A ridge, having a thickened, fluffy cuticle with a U-shaped course, passes across the visceral mass between the two receptacle openings in S. shiinoi. Such a structure has never been described in other rhizocephalans, and its function is uncertain.
Assuntos
Crustáceos/anatomia & histologia , Crustáceos/classificação , Animais , Crustáceos/parasitologia , Crustáceos/fisiologia , Interações Hospedeiro-Parasita , Especificidade da EspécieRESUMO
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which leads to serious economic losses in the pig industry worldwide. While the molecular basis of PCV2 replication and pathogenicity remains elusive, it is increasingly apparent that the microRNA (miRNA) pathway plays a key role in controlling virus-host interactions, in addition to a wide range of cellular processes. Here, we employed Solexa deep sequencing technology to determine which cellular miRNAs were differentially regulated after expression of each of three PCV2-encoded open reading frames (ORFs) in porcine kidney epithelial (PK15) cells. We identified 51 ORF1-regulated miRNAs, 74 ORF2-regulated miRNAs, and 32 ORF3-regulated miRNAs that differed in abundance compared to the control. Gene ontology analysis of the putative targets of these miRNAs identified transcriptional regulation as the most significantly enriched biological process, while KEGG pathway analysis revealed significant enrichment for several pathways including MAPK signaling, which is activated during PCV2 infection. Among the potential target genes of ORF-regulated miRNAs, two genes encoding proteins that are known to interact with PCV2-encoded proteins, zinc finger protein 265 (ZNF265) and regulator of G protein signaling 16 (RGS16), were selected for further analysis. We provide evidence that ZNF265 and RGS16 are direct targets of miR-139-5p and let-7e, respectively, which are both down-regulated by ORF2. Our data will initiate further studies to elucidate the roles of ORF-regulated cellular miRNAs in PCV2-host interactions.
Assuntos
Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Regulação da Expressão Gênica , MicroRNAs/genética , Síndrome Definhante Multissistêmico de Suínos Desmamados/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Infecções por Circoviridae/genética , Infecções por Circoviridae/virologia , Circovirus/genética , Ontologia Genética , MicroRNAs/metabolismo , Fases de Leitura Aberta , Síndrome Definhante Multissistêmico de Suínos Desmamados/virologia , Suínos , Proteínas Virais/metabolismoRESUMO
MicroRNAs (miRNAs) are short (about 18-24 nucleotides) non-coding RNAs and have emerged as potential biomarkers for various diseases, including cancers. Due to their short lengths, the specificity often becomes an issue in conventional amplification-based methods. Next-generation sequencing techniques could be an alternative, but the long analysis time and expensive costs make them less suitable for routine clinical diagnosis. Therefore, it is essential to develop a rapid, selective, and accurate miRNA detection assay using a simple, affordable system. In this work, we report a CRISPR/Cas13a-based miRNA biosensing using point-of-care dark-field (DF) imaging. We utilized magnetic-gold nanoparticle (MGNPs) complexes as signal probes, which consist of 200 nm-sized magnetic beads and 60 nm-sized gold nanoparticles (AuNPs) linked by DNA hybridization. Once the CRISPR/Cas13a system recognized the target miRNAs (miR-21-5p), the activated Cas13a cleaved the bridge linker containing RNA sequences, releasing 60 nm-AuNPs detected and quantified by a portable DF imaging system. The combination of CRISPR/Cas13a, MGNPs, and DF imaging demonstrated amplification-free detection of miR-21-5p within 30 min at a detection limit of 500 attomoles (25 pM) and with single-base specificity. The CRISPR/Cas13a-assisted MGNP-DF assay achieved rapid, selective, and accurate detection of miRNAs with simple equipment, thus providing a potential application for cancer diagnosis.
RESUMO
Label-free detection of multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. First, we characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles are required.
Assuntos
Nanopartículas , Humanos , Nanopartículas/química , Bicamadas Lipídicas/química , Holografia/métodos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Microfluídica/métodos , Microfluídica/instrumentação , Feminino , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Linhagem Celular Tumoral , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Biomarcadores/análiseRESUMO
MicroRNAs are a class of small non-coding RNA molecules that repress gene expression primarily at the post-transcriptional level. Genetic variations in microRNA genes may contribute to phenotypic differences by altering the expression of microRNAs and their targets. Here, we identified 12 single nucleotide polymorphisms (SNPs) in the genomic region of the porcine MIR206 / MIR133B cluster, 10 and 2 of which were associated with MIR206 and MIR133B respectively. All 12 SNPs were located within primary microRNAs. Allele frequency determination in different pig breeds (Berkshire, n = 153; Landrace, n = 125; Yorkshire, n = 173) and association studies of muscle fiber characteristics, lean meat production and meat quality traits were performed on the MIR206 and MIR133B SNPs. The MIR206 SNPs were associated with the percentage of type IIa and IIb fibers for muscle fiber area composition, meat quality traits including drip loss and lightness, and backfat thickness, a parameter of lean meat production. In addition, we found significant association of the MIR133B SNPs with total muscle fiber number, loin eye area, and muscle pH. Furthermore, these SNPs significantly affected the levels of mature MIR206 and MIR133B , respectively, primarily by regulating the processing of primary microRNAs into precursor microRNAs. Interestingly, altered MIR206 levels correlated with phenotypic variability among genotypes of the MIR206 SNP. Our data suggest that polymorphisms in the porcine MIR206 / MIR133B cluster are a genetic factor affecting muscle and meat quality traits.
Assuntos
Carne/normas , MicroRNAs/genética , Fibras Musculares Esqueléticas/metabolismo , Sus scrofa/genética , Sus scrofa/metabolismo , Animais , Feminino , Frequência do Gene , Genótipo , Masculino , MicroRNAs/metabolismo , Fenótipo , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles in cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for cancer diagnosis and longitudinal monitoring. Traditional PCR-based methods, however, require multi-step procedures and remain as bulk analysis. Here, the authors introduce an amplification-free and extraction-free EV miRNA detection method using a CRISPR/Cas13a sensing system. CRISPR/Cas13a sensing components are encapsulated in liposomes and delivered them into EVs through liposome-EV fusion. This allows for accurately quantify specific miRNA-positive EV counts using 1 × 108 EVs. The authors show that miR-21-5p-positive EV counts are in the range of 2%-10% in ovarian cancer EVs, which is significantly higher than the positive EV counts from the benign cells (<0.65%). The result show an excellent correlation between bulk analysis with the gold-standard method, RT-qPCR. The authors also demonstrate multiplexed protein-miRNA analysis in tumor-derived EVs by capturing EpCAM-positive EVs and quantifying miR-21-5p-positive ones in the subpopulation, which show significantly higher counts in the plasma of cancer patients than healthy controls. The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and opens up the possibility of multiplexed single EV analysis for protein and RNA markers.
Assuntos
Vesículas Extracelulares , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Vesículas Extracelulares/metabolismoRESUMO
Label-free detecting multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. Herein, we first thoroughly characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles is required.
RESUMO
Label-free detecting multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. Herein, we first thoroughly characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles is required.
RESUMO
ANALYSIS: of rare circulating extracellular vesicles (EV) from early cancers or different types of host cells requires extremely sensitive EV sensing technologies. Nanoplasmonic EV sensing technologies have demonstrated good analytical performances, but their sensitivity is often limited by EVs' diffusion to the active sensor surface for specific target EV capture. Here, we developed an advanced plasmonic EV platform with electrokinetically enhanced yields (KeyPLEX). The KeyPLEX system effectively overcomes diffusion-limited reactions with applied electroosmosis and dielectrophoresis forces. These forces bring EVs toward the sensor surface and concentrate them in specific areas. Using the keyPLEX, we showed significant improvements in detection sensitivity by â¼100-fold, leading to the sensitive detection of rare cancer EVs from human plasma samples in 10 min. The keyPLEX system could become a valuable tool for point-of-care rapid EV analysis.
Assuntos
Técnicas Biossensoriais , Vesículas Extracelulares , Neoplasias , Humanos , Neoplasias/diagnóstico , Eletro-OsmoseRESUMO
Peroxisome proliferator-activated receptor γ coactivator 1 α (PPARGC1A) is a transcriptional coactivator that is involved in a variety of biological processes including muscle fiber type composition. Here, we identified two single nucleotide polymorphisms (SNPs; *2690T>C and *2864T>C) and one insertion/deletion in the 3' untranslated region of porcine PPARGC1A. These SNPs were genotyped by direct sequencing in a total of 439 pigs representing three different pig breeds (Berkshire, n = 156; Yorkshire, n = 163; Landrace, n = 120). We evaluated the effects of diplotypes of individual PPARGC1A 3'UTR SNPs on muscle fiber characteristics and meat quality traits. The *2690T>C polymorphism was significantly associated with the percentage of type I and IIb fibers for both muscle fiber number and area composition (P < 0.05), and also showed a significant association with muscle pH, a parameter of meat quality (P = 0.0188). The *2864T>C polymorphism was also associated with meat quality traits including muscle pH (P = 0.0071), drip loss (P = 0.0006), and lightness (P = 0.0702), but showed no significant association with muscle fiber characteristics. Interestingly, each SNP affected PPARGC1A expression significantly at the protein level but not at the mRNA level, thereby accounting for phenotypic variability among genotypes. Taken together, our data suggest that the *2690T>C and *2864T>C polymorphisms can be used as genetic markers for selection toward improved meat quality.
Assuntos
Regiões 3' não Traduzidas/genética , Carne/normas , Fibras Musculares Esqueléticas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa Herdável , Sus scrofa/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Feminino , Regulação da Expressão Gênica , Técnicas de Genotipagem , Masculino , Dados de Sequência Molecular , Fatores de Transcrição/metabolismoRESUMO
Plasmonic biosensors are increasingly being used for the analysis of extracellular vesicles (EVs) originating from disease areas. However, the high non-specific binding of EVs to a gold-sensing surface has been a critical problem and hindered the true translational potential. Here, we report that direct antibody immobilization on the plasmonic gold surface via physisorption shows excellent capture of cancer-derived EVs with ultralow non-specific binding even at very high concentrations. Contrary to commonly used methods that involve thiol-based linker attachment and an EDC/sulfo-NHS reaction, we show a higher specific capture rate and >50-fold lower non-specific on citrate-capped plain and nanopatterned gold surfaces. The method provides a simple, fast, and reproducible means to functionalize plasmonic gold surfaces with antibodies for robust EV biosensing.
RESUMO
A simple and sensitive AuNP-coated magnetic beads (AMB)-based electrochemical biosensor platform was fabricated for bioassay. In this study, AuNP-conjugated magnetic particles were successfully prepared using biotin-streptavidin conjugation. The morphology and structure of the nanocomplex were characterized by scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX) and UV-visible spectroscopy. Moreover, cyclic voltammetry (CV) was used to investigate the effect of AuNP-MB on alkaline phosphatase (ALP) for electrochemical signal enhancement. An ALP-based electrochemical (EC) immunoassay was performed on the developed AuNP-MB complex with indium tin oxide (ITO) electrodes. Subsequently, the concentration of capture antibodies was well-optimized on the AMB complex via biotin-avidin conjugation. Lastly, the developed AuNP-MB immunoassay platform was verified with extracellular vesicle (EV) detection via immune response by showing the existence of EGFR proteins on glioblastoma multiforme (GBM)-derived EVs (108 particle/mL) spiked in human plasma. Therefore, the signal-enhanced ALP-based EC biosensor on AuNP-MB was favorably utilized as an immunoassay platform, revealing the potential application of biosensors in immunoassays in biological environments.
RESUMO
MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.
Assuntos
Tecido Adiposo/metabolismo , MicroRNAs/genética , Músculo Esquelético/metabolismo , Sus scrofa/genética , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma/genética , MicroRNAs/química , MicroRNAs/metabolismo , Dados de Sequência Molecular , Família Multigênica/genética , Conformação de Ácido Nucleico , Precursores de RNA/química , Precursores de RNA/genética , Análise de Sequência de RNARESUMO
The coast of the Korean peninsula experiences a range of human impacts, including pollution, shipping, reclamation, and aquaculture, that have motivated numerous local studies of macrobenthic organisms. In this paper, 1,492 subtidal stations were compiled from 23 studies (areas) to evaluate environmental quality on a broader scale. A common index in biomonitoring, Shannon-Wiener evenness proportion (SEP), could not incorporate azoic or single-species samples. This shortcoming was overcome by developing an inverse function of SEP (ISEP), which was positively correlated with independent measures of water quality available for nine sites and was not biased by the size of the sampling unit. Additionally, at Shihwa Dike, where samples were collected before and after reinstating a tidal connection with the ocean, ISEP values improved over time, as expected. Thus, it is now possible to assign Korean subtidal sites to seven ISEP "grades" and to use their values and trends to guide coastal management.
Assuntos
Organismos Aquáticos/classificação , Biodiversidade , Monitoramento Ambiental/métodos , Biomassa , República da Coreia , Água do Mar/química , Poluentes Químicos da Água/análise , Poluição da Água/estatística & dados numéricosRESUMO
Cap1 2'-O-ribose methyltransferase (CMTR1) modifies RNA transcripts containing the 7-methylguanosine cap via 2'-O-ribose methylation of the first transcribed nucleotide, yielding cap1 structures. However, the role of CMTR1 in small RNA-mediated gene silencing remains unknown. Here, we identified and characterized a Drosophila CMTR1 gene (dCMTR1) mutation. We found that the catalytic activity of dCMTR1 was involved in the biogenesis of small interfering RNAs (siRNAs) but not microRNAs. Additionally, dCMTR1 interacted with R2D2, a key component for the assembly of the RNA-induced silencing complex (RISC) containing Argonaute 2 (Ago2). Consistent with this finding, loss of dCMTR1 function impaired RISC assembly by inhibiting the unwinding of Ago2-bound siRNA duplexes, thus preventing the retention of the guide strand. Moreover, dCMTR1 is unlikely to modify siRNAs during RISC assembly. Collectively, our data indicate that dCMTR1 is a positive regulator of the small RNA pathway associated with Ago2 with roles in both siRNA biogenesis and RISC assembly.
Assuntos
Drosophila/metabolismo , Metiltransferases , RNA Interferente Pequeno , Complexo de Inativação Induzido por RNA , Animais , Proteínas Argonautas/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Mutação , Interferência de RNA , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/biossíntese , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
Extracellular vesicles (EVs)-nanoscale phospholipid vesicles secreted by cells-present new opportunities for molecular diagnosis from non-invasive liquid biopsies. Single EV protein analysis can be extremely valuable in studying EVs as circulating cancer biomarkers, but it is technically challenging due to weak detection signals associated with limited amounts of epitopes and small surface areas for antibody labeling. Here, a new, simple method that enables multiplexed analyses of EV markers with improved sensitivities is reported. Specifically, plasmon-enhanced fluorescence detection is implemented that amplifies fluorescence signals using surface plasmon resonances excited by periodic gold nanohole structures. It is shown that fluorescence signals in multiple channels are amplified by one order of magnitude, and both transmembrane and intravesicular markers can be detected at the single EV level. This approach can offer additional insight into understanding subtypes, heterogeneity, and production dynamics of EVs during disease development and progression.