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1.
Biochemistry (Mosc) ; 84(9): 1093-1106, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31693469

RESUMO

Differential expression of 30,003 genes was studied in the liver of female Wistar rats fed with isocaloric diets with the excess of fat, fructose, or cholesterol, or their combinations for 62 days using the method of whole-transcriptome profiling on a microchip. Relative mRNA expression levels of the Asah2, Crot, Crtc2, Fmo3, GSTA2, LOC1009122026, LOC102551184, NpY, NqO1, Prom1, Retsat, RGD1305464, Tmem104, and Whsc1 genes were also determined by RT-qPCR. All the tested diets affected differently the key metabolic pathways (KEGGs). Significant changes in the expression of steroid metabolism gene were observed in the liver of animals fed with the tested diets (except the high-fat high fructose diet). Both high-fat and high-fructose diets caused a significant decrease in the expression of squalene synthase (FDFT1 gene) responsible for the initial stage of cholesterol synthesis. On the contrary, in animals fed with the high-cholesterol diet (0.5% cholesterol), expression of the FDFT1 gene did not differ from the control group; however, these animals were characterized by changes in the expression of glucose and glycogen synthesis genes, which could lead to the suppression of glycogen synthesis and gluconeogenesis. At the same time, this group demonstrated different liver tissue morphology in comparison with the animals fed with the high-fructose high-fat diet, manifested as the presence of lipid vacuoles of a smaller size in hepatocytes. The high-fructose and high-fructose high-fat diets affected the metabolic pathways associated with intracellular protein catabolism (endocytosis, phagocytosis, proteasomal degradation, protein processing in the endoplasmic reticulum), tight junctions and intercellular contacts, adhesion molecules, and intracellular RNA transport. Rats fed with the high-fructose high-fat or high-cholesterol diets demonstrated consistent changes in the expression of the Crot, Prom1, and RGD1305464 genes, which reflected a coordinated shift in the regulation of lipid and carbohydrate metabolisms.


Assuntos
Colesterol/farmacologia , Gorduras na Dieta/farmacologia , Açúcares da Dieta/farmacologia , Frutose/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , RNA/genética , Transcriptoma/efeitos dos fármacos , Animais , Colesterol/administração & dosagem , Colesterol/metabolismo , Biologia Computacional , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/metabolismo , Açúcares da Dieta/administração & dosagem , Açúcares da Dieta/metabolismo , Feminino , Frutose/administração & dosagem , Frutose/metabolismo , Perfilação da Expressão Gênica , Fígado/citologia , RNA/análise , RNA/isolamento & purificação , Ratos , Ratos Wistar , Transcriptoma/genética
2.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura Ambiente , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
3.
Chem Biol Interact ; 309: 108675, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31150632

RESUMO

Liver fibrosis is a progression of chronic liver disease with lacks effective therapies at present. Schisandrin B (Sch B), a bioactive compound extracted from the traditional Chinese medicine Schisandra chinensis, was reported to benefit liver diseases. This study aimed to investigate the therapeutic effects and molecular mechanisms of Sch B against CCl4-induced liver fibrosis in rats. RNA sequencing and transcriptome analysis were performed collaboratively, including analysis of differential gene expression, gene ontology (GO) analysis, pathway analysis and pathway-act-network analysis. The results demonstrated that Sch B effectively alleviated CCl4-induced liver damage and fibrosis in rats, as evidenced by improved liver function and decreased extracellular matrix deposition. Furthermore, 4440 (1878 up-regulated, 2562 down-regulated) genes in the model group versus (vs) normal group, 4243 (2584 up-regulated, 1659 down-regulated) genes in Sch B-treated group vs model group were identified as differentially expressed genes (DEGs). Subsequently, GO analysis revealed that DEGs were mainly enriched in metabolism, oxidation-reduction, endoplasmic reticulum stress and apoptosis-related biological processes. Pathway analysis suggested that Sch B up-regulated cytochrome P450 drug metabolism, PPAR signaling pathways, and down-regulated glutathione metabolism pathways. In addition, the regulatory patterns of Sch B on key genes and pathways were also confirmed. In conclusion, our study demonstrated Sch B alleviated CCl4-induced liver fibrosis by multiple modulatory mechanisms, which provide new clues for further pharmacological study of Sch B.


Assuntos
Lignanas/farmacologia , Cirrose Hepática/patologia , Fígado/metabolismo , Compostos Policíclicos/farmacologia , Transcriptoma , Animais , Apoptose/efeitos dos fármacos , Tetracloreto de Carbono/toxicidade , Ciclo-Octanos/química , Ciclo-Octanos/farmacologia , Ciclo-Octanos/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Lignanas/química , Lignanas/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Masculino , Medicina Tradicional Chinesa , Compostos Policíclicos/química , Compostos Policíclicos/uso terapêutico , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Ratos , Ratos Wistar , Schisandra/química , Schisandra/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos
4.
Nat Commun ; 10(1): 2300, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127091

RESUMO

Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.


Assuntos
Transformação Celular Neoplásica/patologia , Interações Hospedeiro-Patógeno/genética , RNA Viral/metabolismo , RNA/metabolismo , Neoplasias do Colo do Útero/virologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Conjuntos de Dados como Assunto , Feminino , Técnicas de Silenciamento de Genes , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas E7 de Papillomavirus/genética , Polirribossomos/genética , Polirribossomos/metabolismo , RNA/genética , RNA/isolamento & purificação , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/isolamento & purificação , Análise de Sequência de RNA , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Environ Sci Pollut Res Int ; 26(17): 17204-17213, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012072

RESUMO

As transcriptomic studies are becoming more and more common, it is important to ensure that the RNA used in the analyses is of good quality. The RNA integrity may be compromised by storage temperature or freeze-thaw cycles, but these have not been well studied in poikilothermic fishes. This work studied the effects of tissue storage time and temperature, and freeze-thaw cycles of tissue and extracted RNA on RNA integrity in brown trout (Salmo trutta L.) liver. The storage time and temperature had an effect on RNA integrity, but RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (RIN > 7) was still obtained from samples preserved at - 20 °C for 6 months. Freeze-thaw cycles of tissue or RNA did not compromise the integrity of RNA. RNA degradation had an effect on RT-qPCR results, and the effect depended on gene. The RT-qPCR analysis of historical samples from a bleached kraft pulp mill effluent exposure in 1984 revealed no significant cyp1a induction. Recommendations are given for the preservation and handling procedures of samples designated for transcriptomic analyses.


Assuntos
Criopreservação/métodos , Congelamento , Fígado/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Truta , Animais , Masculino , RNA/isolamento & purificação , Manejo de Espécimes , Transcriptoma , Truta/genética
6.
Methods Mol Biol ; 1979: 9-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028629

RESUMO

The starting material for all single-cell protocols is a cell suspension. The particular functions and spatial distribution of immune cells generally make them easy to isolate them from the tissues where they dwell. Here we describe tissue dissociation protocols that have been used to obtain human immune cells from lymphoid and nonlymphoid tissues to be then used as input to single-cell methods. We highlight the main factors that can influence the final quality of single-cell data, namely the stress signatures that can bias its interpretation.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Manejo de Espécimes/métodos , Fracionamento Celular/métodos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Linfonodos/citologia , Linfonodos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , RNA/sangue , RNA/isolamento & purificação , Pele/citologia , Pele/metabolismo , Baço/citologia , Baço/metabolismo
7.
Nat Commun ; 10(1): 1605, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962428

RESUMO

Colonies of the bumblebee Bombus terrestris are characterized by wide phenotypic variability among genetically similar full-sister workers, suggesting a major role for epigenetic processes. Here, we report a high level of ADAR-mediated RNA editing in the bumblebee, despite the lack of an ADAR1-homolog. We identify 1.15 million unique genomic sites, and 164 recoding sites residing in 100 protein coding genes, including ion channels, transporters, and receptors predicted to affect brain function and behavior. Some edited sites are similarly edited in other insects, cephalopods and even mammals. The global editing level of protein coding and non-coding transcripts weakly correlates with task performance (brood care vs. foraging), but not affected by dominance rank or juvenile hormone known to influence physiology and behavior. Taken together, our findings show that brain editing levels are high in naturally behaving bees, and may be regulated by relatively short-term effects associated with brood care or foraging activities.


Assuntos
Abelhas/fisiologia , Comportamento Animal/fisiologia , Edição de RNA/fisiologia , RNA/genética , Comportamento Social , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Encéfalo/metabolismo , Epigênese Genética/fisiologia , Feminino , Variação Genética/genética , Variação Genética/fisiologia , Masculino , RNA/isolamento & purificação , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA
8.
J Fish Biol ; 95(2): 393-400, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31017661

RESUMO

The partial cDNA sequences of eight reference genes (actb, tuba1, gapdh58, gapdh59, eef1a1, RNA 18 s, pabpc1, ube2I) were cloned from largemouth bass Micropterus salmoides. The expression levels of these eight genes were compared in the various tissues (eye, spleen, kidney, gill, muscle, brain, liver, heart, gut and gonad) of M. salmoides fed on forage fish. The results showed that the candidate genes exhibited tissue-specific expression to various degrees and the stability ranking order was eef1a1 > tuba1 > RNA 18 s > pabpc1 > ube2I > actb > gapdh58 > gapdh59 among tissue types. Four candidate genes eef1a1, tuba1, RNA 18 s and actb were used to analyse the stability in liver tissues of largemouth bass between the forage-fish group and the formulated-feed group. The candidate genes also showed some changes in expression levels in the livers, while eef1a1 and tuba1 had the most stable expression in livers of fish fed on alternative diets within 10 candidates. So eef1a1 and tuba1 were recommended as optimal reference gene in quantitative real-time PCR analysis to normalise the expression levels of target genes in tissues and lives of the M. salmoides fed on alternative diets. In livers, the expression levels of gck normalised by eef1a1 and tuba1 showed the significant up-regulation in formulated feed group (P < 0.05) than those in forage-fish group. While sex difference has no significant effects on the expression levels of gck in both groups.


Assuntos
Bass/genética , Dieta/veterinária , Animais , Bass/anatomia & histologia , Bass/fisiologia , DNA Complementar/biossíntese , DNA Complementar/química , Bases de Dados de Ácidos Nucleicos , Expressão Gênica , Instabilidade Genômica , Glucoquinase/genética , Fígado/metabolismo , RNA/análise , RNA/isolamento & purificação , RNA/normas , Reação em Cadeia da Polimerase em Tempo Real
9.
Biomed Res Int ; 2019: 2756516, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834258

RESUMO

Circular RNAs (circRNAs) are newly discovered incipient non-coding RNAs with potential roles in disease progression in living organisms. Significant reports, since their inception, highlight the abundance and putative functional roles of circRNAs in every organism checked for, like O. sativa, Arabidopsis, human, and mouse. CircRNA expression is generally less than their linear mRNA counterparts which fairly explains the competitive edge of canonical splicing over non-canonical splicing. However, existing methods may not be sensitive enough for the discovery of low-level expressed circRNAs. By combining template-dependent multiple displacement amplification (tdMDA), Illumina sequencing, and bioinformatics tools, we have developed an experimental protocol that is able to detect 1,875 novel and known circRNAs from O. sativa. The same method also revealed 9,242 putative circRNAs in less than 40 million reads for the first time from the Nicotiana benthamiana whose genome has not been fully annotated. Supported by the PCR-based validation and Sanger sequencing of selective circRNAs, our method represents a valuable tool in profiling circRNAs from the organisms with or without genome annotation.


Assuntos
Biologia Computacional , Perfilação da Expressão Gênica/métodos , Processamento de RNA/genética , RNA/genética , Animais , Arabidopsis/genética , Genoma/genética , Humanos , Camundongos , MicroRNAs/genética , Anotação de Sequência Molecular , Oryza/genética , RNA/isolamento & purificação
11.
Nano Lett ; 19(4): 2641-2646, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30864449

RESUMO

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.


Assuntos
DNA/química , Microscopia de Fluorescência/métodos , Proteínas/isolamento & purificação , RNA/isolamento & purificação , Simulação por Computador , Cinética , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Proteínas/química , RNA/química
12.
BMC Genomics ; 20(1): 228, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894119

RESUMO

BACKGROUND: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. RESULTS: We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. CONCLUSIONS: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.


Assuntos
Citometria de Fluxo , RNA/genética , RNA/isolamento & purificação , Análise de Sequência de RNA , Peixe-Zebra/genética , Animais , Contagem de Células , Poli A/genética , Controle de Qualidade
13.
Photochem Photobiol Sci ; 18(5): 1280-1289, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30907896

RESUMO

Softening processes after ripening are a major factor contributing to the perishability of fleshy fruit and, together with mechanical damage, represent the onset of physiological decay. Softening involves multiple co-ordinated events leading to modifications of the cell wall architecture. Several studies described that UV-B radiation positively affects both the nutraceutical and aesthetical qualities of fruit. However, very few studies investigated the effect of UV-B irradiation on the activity of cell wall-related enzymes. This research aimed at studying how different UV-B treatments (10 min and 60 min) affect the activity of cell wall-modifying enzymes (pectin methylesterase, polygalacturonase and ß-galactosidase) together with the expression of some of their isoforms up to 36 h after UV-B treatment of peach (cv. Fairtime, melting phenotype) fruit. Results revealed that UV-B radiation did not affect the soluble solid content and the titratable acidity, two important parameters influencing consumers' choice and taste. In contrast, UV-B was effective at reducing the loss of firmness 24 h after the 60 min irradiation. Generally, a lower activity of the hydrolytic enzymes compared to untreated fruit was observed, regardless of the UV-B dose. However, gene expression did not reflect the corresponding enzymatic activity. Based on these results, UV-B irradiation might be a successful tool in reducing the loss of firmness of peach fruit during post-harvest, thus improving their quality and shelf-life.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/enzimologia , Frutas/metabolismo , Poligalacturonase/metabolismo , Prunus persica/metabolismo , beta-Galactosidase/metabolismo , Hidrolases de Éster Carboxílico/genética , Frutas/genética , Oxirredução , Fenótipo , Poligalacturonase/genética , Prunus persica/genética , RNA/genética , RNA/isolamento & purificação , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Raios Ultravioleta , beta-Galactosidase/genética
14.
Exp Parasitol ; 200: 67-72, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30904695

RESUMO

Demodex is a type of parasitic mite which could cause serious dermatoses in 11 orders of mammals. However, due to the tiny body with thick chitin hard to be ruptured as well as the difficulty in obtaining a large number of mites, the quantity and quality of extracted RNA could hardly satisfied for transcriptome sequencing. This has hampered the research on functional genes and molecular pathogenesis of Demodex for a long time. To solve the problems above, the present study established a new RNA extraction method in combination Azanno method with liquid nitrogen grinding using 16 human and canine Demodex mite samples. The RNA quality detection results of Agilent 2100 Bioanalyzer showed that 8 of 16 RNA samples met the requirements for trace RNA-Seq, with RIN of 5.0-6.5 and RNA quantity of 1.1-16.0 ng. RNA quality was affected by grinding process and parasitic position of Demodex. Enough grinding number (≥2000) in moderate time (≤20 min) was significant for mites' complete rupture and RNA degradation prevention. D. brevis (100%, 3/3) parasitizing in human sebaceous glands had significantly higher RNA qualification rate than D. folliculorum (57.14%, 4/7) parasitizing in human hair follicles. Yet D. canis parasitizing in dog had lower RNA qualification rate (16.67%, 1/6) as mites were embedded in skin tissues and blood clots. It should be pointed out that microplate reader had defects with a lower RNA qualification rate of 6.25% (1/16) unmatched with 2100 Bioanalyzer, reminding that it could be only used as reference in RNA quality evaluation.


Assuntos
Ácaros/genética , RNA/isolamento & purificação , Transcriptoma , Animais , Cães , Folículo Piloso/parasitologia , Humanos , Ácaros/classificação , RNA/química , RNA/normas , RNA Ribossômico 18S/isolamento & purificação , Glândulas Sebáceas/parasitologia , Análise de Sequência de RNA , Trombose/parasitologia
15.
Prep Biochem Biotechnol ; 49(3): 279-285, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30767702

RESUMO

In this study, we report a novel member of the attacin family from Hermetia illucens. The cDNA clone encoding the attacin-like protein was isolated by screening a cDNA library prepared from immunized fat body. The complete 510 bp cDNA of HI-attacin was predicted to encode a protein of 169 amino acids with a molecular weight of 17.7 kDa. The putative mature protein of H. illucens attacin (HI-attacin) had 50% identity with that of Bactrocera dorsalis attacin B. Phylogenetic analysis revealed that the HI-attacin was separated from the other dipteran attacins with a high bootstrap percentage. Compared to that in the other dipteran attacins, the G1 domain of HI-attacin was shorter and the sequence of the G2 domain of HI-attacin was more conserved than that of the G1 domain. We produced the recombinant attacin (rHI-attacin) protein using a prokaryotic expression system to confirm its antibacterial character. The rHI-attacin was produced as inclusion bodies and refolded by on-column refolding. rHI-attacin exhibited antibacterial activity against both Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Using real-time PCR, the expression of HI-attacin was was barely detected before the immunization, but was mostly evident in the fat body after immunization.


Assuntos
Antibacterianos/farmacologia , Dípteros/química , Proteínas de Insetos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Corpo Adiposo/química , Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Filogenia , RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência
16.
Methods Mol Biol ; 1956: 305-319, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30779041

RESUMO

A major hurdle for the treatment of cancer is the incomplete understanding of its evolution through the course of its emergence, dispersal, and relapse. Genetic and epigenetic changes in combination with external cues and selective forces are the driving factors behind tumor heterogeneity. Understanding this variability within and across patients may partly explain the unpredictable outcomes of cancer treatments. Measuring the variation of gene expression levels within cells of the same tumor is a crucial part of this endeavor. Hence, the recently developed single-cell RNA-sequencing (scRNA-seq) technologies have become a valuable tool for cancer research. In practice, however, this is still challenging, especially for clinical samples. Here, we describe mcSCRB-seq (molecular crowding single-cell RNA barcoding and sequencing), a highly sensitive and powerful plate-based scRNA-seq method, which shows great capability to generate transcriptome data for cancer cells. mcSCRB-seq is not only characterized by high sensitivity due to molecular crowding and the use of unique molecular identifiers (UMIs) but also features an easy workflow and a low per-cell cost and does not require specialized equipment.


Assuntos
Neoplasias/genética , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , DNA Complementar/genética , Citometria de Fluxo/métodos , Biblioteca Gênica , Humanos , RNA/isolamento & purificação , Transcrição Reversa , Software , Fluxo de Trabalho
17.
Nat Commun ; 10(1): 37, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30604742

RESUMO

The respiratory system undergoes a diversity of structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify the heterogeneity of pulmonary cell types and dynamic changes in gene expression mediating adaptation to respiration, here we perform single cell RNA analyses of mouse lung on postnatal day 1. Using an iterative cell type identification strategy we unbiasedly identify the heterogeneity of murine pulmonary cell types. We identify distinct populations of epithelial, endothelial, mesenchymal, and immune cells, each containing distinct subpopulations. Furthermore we compare temporal changes in RNA expression patterns before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, including activation of cell stress and the unfolded protein response during perinatal adaptation of the lung. The present data provide a single cell view of the adaptation to air breathing after birth.


Assuntos
Adaptação Fisiológica/genética , Pulmão/citologia , RNA/metabolismo , Fenômenos Fisiológicos Respiratórios , Análise de Célula Única/métodos , Animais , Animais Recém-Nascidos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , RNA/isolamento & purificação , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Análise de Sequência de RNA , Resposta a Proteínas não Dobradas/fisiologia
18.
PLoS Biol ; 17(1): e3000107, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629605

RESUMO

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ácidos Nucleicos/isolamento & purificação , Animais , DNA/isolamento & purificação , Humanos , Campos Magnéticos , Microesferas , RNA/isolamento & purificação
19.
Nucleic Acids Res ; 47(5): 2306-2321, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30605520

RESUMO

RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including super-enhancers and repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.


Assuntos
DNA/química , DNA/isolamento & purificação , Conformação de Ácido Nucleico , RNA/química , RNA/isolamento & purificação , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , DNA/genética , Elementos Facilitadores Genéticos/genética , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Purinas/química , Purinas/metabolismo , RNA/genética , RNA Longo não Codificante/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
20.
Biosens Bioelectron ; 128: 68-75, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30634076

RESUMO

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (~8 nL capture chamber) coupled with a thin-film photodiode (200 × 200 µm area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 µL of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.


Assuntos
Técnicas Biossensoriais , Ebolavirus/isolamento & purificação , RNA/isolamento & purificação , Sequência de Bases , Ebolavirus/química , Ebolavirus/genética , Fluorescência , Dispositivos Lab-On-A-Chip , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , RNA/genética , Dióxido de Silício/química
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