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1.
Exp Parasitol ; 218: 108003, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980317

RESUMO

Dermatophagoides farinae, an important pathogen, has multiple allergens. However, their expression under physiological conditions are not understood. Our previous RNA-seq showed that allergens of D. farinae were up-regulated under temperature stress, implying that they may be involved in stress response. Here, we performed a comprehensive study. qRT-PCR detection indicated that 26 of the 34 allergens showed differential expression. Der f1 had the most abundant basic expression quantity. Der f 28.0201 (HSP70) and Der f3 had the same regulation pattern in 9 highly expressed transcripts, which only up-regulated at 41 °C and 43 °C, but Der f 28.0201 showed stronger regulation than Der f 3 (19.88-fold vs 6.02-fold). Whereas Der f 1, 2, 7, 21, 22, 27, and 30 were up-regulated under both heat and cold stress, and Der f 27 showed the strongest regulation ability among them. Der f 27 showed more significant up-regulation than Der f 28.0201 under heat stress (23.59-fold vs 19.88-fold), and Der f27 had more obvious up-regulation under cold than heat stress (30.70-fold vs 23.59-fold). The expression of Der f 27, 28.0201 and 1, and D. farinae survival rates significantly decreased following RNAi, indicating the upregulation of these allergens under temperature stress conferred thermo-tolerance or cold-tolerance to D. farinae. In this study, we described for the first time that these allergens have temperature-stress response functions. This new scientific discovery has important clinical value for revealing the more frequent and serious allergic diseases caused by D. farinae during the change of seasons.


Assuntos
Antígenos de Dermatophagoides/fisiologia , Resposta ao Choque Frio/fisiologia , Dermatophagoides farinae/fisiologia , Resposta ao Choque Térmico/fisiologia , Estresse Fisiológico/fisiologia , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/metabolismo , Antígenos de Dermatophagoides/farmacologia , Sequência de Bases , Dermatophagoides farinae/genética , Feminino , Inativação Gênica , Anotação de Sequência Molecular , RNA/química , RNA/isolamento & purificação , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/química , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Estresse Fisiológico/genética , Regulação para Cima
2.
Nat Commun ; 11(1): 4774, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963224

RESUMO

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL-1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.


Assuntos
Líquidos Corporais/química , Técnicas de Diagnóstico Molecular/métodos , Ácidos Nucleicos/isolamento & purificação , Sepse/diagnóstico , Candida/genética , Candida/isolamento & purificação , Candidíase/diagnóstico , Contagem de Colônia Microbiana , Doenças Transmissíveis/diagnóstico , DNA/isolamento & purificação , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Reação em Cadeia da Polimerase/métodos , RNA/isolamento & purificação , Sensibilidade e Especificidade , Sepse/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Urina
3.
Sensors (Basel) ; 20(16)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824787

RESUMO

Pandemics require a fast and immediate response to contain potential infectious carriers. In the recent 2020 Covid-19 worldwide pandemic, authorities all around the world have failed to identify potential carriers and contain it on time. Hence, a rapid and very sensitive testing method is required. Current diagnostic tools, reverse transcription PCR (RT-PCR) and real-time PCR (qPCR), have its pitfalls for quick pandemic containment such as the requirement for specialized professionals and instrumentation. Versatile electrochemical DNA/RNA sensors are a promising technological alternative for PCR based diagnosis. In an electrochemical DNA sensor, a nucleic acid hybridization event is converted into a quantifiable electrochemical signal. A critical challenge of electrochemical DNA sensors is sensitive detection of a low copy number of DNA/RNA in samples such as is the case for early onset of a disease. Signal amplification approaches are an important tool to overcome this sensitivity issue. In this review, the authors discuss the most recent signal amplification strategies employed in the electrochemical DNA/RNA diagnosis of pathogens.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais , Infecções por Coronavirus/diagnóstico , Técnicas Eletroquímicas , Pneumonia Viral/diagnóstico , Betacoronavirus/patogenicidade , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , DNA/isolamento & purificação , Epidemias/prevenção & controle , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real
4.
PLoS One ; 15(8): e0236104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32776939

RESUMO

There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molecular endpoints. Despite the cross-linking effects of formalin, its impact on nucleic acid quality and concentration, amplification, and sequencing are not well described. While fresh-frozen tissue is optimal for working with nucleic acids, FFPE samples have been shown to be conducive for molecular studies. Laser capture microdissection (LCM) is one technology which allows for collection of specific regions or cell populations from fresh or preserved specimens with pathological alterations, pathogens, or parasites. In this study, smallmouth bass (Micropterus dolomieu) liver was preserved in three different fixatives, including 10% neutral buffered formalin (NBF), Z-Fix® (ZF), and PAXgene® (PG) for four time periods (24 hr, 48 hr, seven days, and 14 days). Controls consisted of pieces of liver preserved in RNALater® or 95% ethanol. Smallmouth bass were chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors. Small liver sections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality. Sanger sequencing and the NanoString nCounter® technology were used to assess the suitability of these samples in downstream molecular techniques. The results revealed that of the formalin fixatives, NBF samples fixed for 24 and 48 hr were superior to ZF samples for both Sanger sequencing and the Nanostring nCounter®. The non-formalin PAXgene® samples were equally successful and they showed greater stability in nucleic acid quality and concentration over longer fixation times. This study demonstrated that small quantities of preserved tissue from smallmouth bass can be utilized in downstream molecular techniques; however, future studies will need to optimize the methods presented here for different tissue types, fish species, and pathological conditions.


Assuntos
Bass/genética , DNA/efeitos dos fármacos , Monitoramento Ambiental/métodos , Fixadores/efeitos adversos , RNA/efeitos dos fármacos , Animais , Clivagem do DNA/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Formaldeído/efeitos adversos , Perfilação da Expressão Gênica/métodos , Fígado/efeitos dos fármacos , Fígado/patologia , Microdissecção , Desnaturação de Ácido Nucleico/efeitos dos fármacos , RNA/isolamento & purificação , Estabilidade de RNA/efeitos dos fármacos , Análise de Sequência de DNA , Fatores de Tempo , Fixação de Tecidos/métodos , West Virginia
5.
Nat Protoc ; 15(8): 2568-2588, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32651564

RESUMO

RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.


Assuntos
Fracionamento Químico/métodos , Proteoma/isolamento & purificação , Proteínas de Ligação a RNA/isolamento & purificação , RNA/isolamento & purificação , Transcriptoma , Linhagem Celular , Humanos , Proteoma/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fluxo de Trabalho
6.
Reumatol. clín. (Barc.) ; 16(3): 229-234, mayo-jun. 2020. ilus, tab, graf
Artigo em Inglês | IBECS | ID: ibc-194356

RESUMO

BACKGROUND: IL-6 mRNA expression is significantly high in many autoimmune diseases such as Behçet's disease; this is often related with more aggressive phenotypes. Nevertheless, the essential molecular process for its high expression has not been completely realized. The aim of this study was undertaken to estimate the gene copy number variation and promoter methylation to IL-6's high expression. METHODS: This study was performed on 51 patients and 61 healthy controls. Initially, DNA and RNA were extracted from all specimens. Promoter methylation levels of IL-6 were evaluated by MeDIP-qPCR technique. Also, IL-6 gene expression was measured by Real-time PCR. After that, we evaluated the relationship between gene expression and methylation, as well as their relationship with clinical specification. RESULTS: As we expected, the expression level of IL-6 gene increased significantly in the patient group compared to the healthy subjects. Also, the relative promoter methylation level of the IL-6 mRNA was significantly lower in patient group compared to healthy group (p < 0.001). DISCUSSION: We disclosed that the promoter hypomethylation may be considered as one of the main defects for IL-6 mRNA high expression in patients with Behçet's disease


ANTECEDENTES: La expresión de ARNm de IL-6 es significativamente elevada en muchas enfermedades autoinmunes, tales como el síndrome de Behçet, y ello se relaciona a menudo con fenotipos más agresivos. Sin embargo, no se ha comprendido plenamente el proceso molecular esencial para esta expresión elevada. El objetivo de este estudio fue la estimación de la variación del número de copias del gen, y la metilación del promotor de la expresión elevada de IL-6. MÉTODOS: Este estudio se realizó en 51 pacientes y 61 controles sanos. Al inicio, se extrajo ADN y ARN de todas las muestras. Se evaluaron los niveles de metilación del promotor de IL-6 mediante la técnica MeDIP-qPCR. También se midió la expresión del gen IL-6 mediante PCR a tiempo real. Tras ello, evaluamos la relación entre la expresión del gen y la metilación, así como su relación con la especificación clínica. RESULTADOS: Según lo previsto, el nivel de expresión del gen IL-6 se incrementó significativamente en el grupo de pacientes, con respecto a los sujetos sanos. También encontramos que el nivel relativo de metilación del promotor de ARNm de IL-6 fue considerablemente menor en el grupo de pacientes, con respecto al grupo sano (p < 0,001). DISCUSIÓN: Concluimos que la hipometilación del promotor puede considerarse uno de los defectos principales de la expresión elevada de ARNm de IL-6, en los pacientes con síndrome de Behçet


Assuntos
Humanos , Metilação/efeitos dos fármacos , Interleucina-6/sangue , Interleucina-6/imunologia , Síndrome de Behçet/sangue , Síndrome de Behçet/genética , Interleucina-6/genética , Anti-Inflamatórios/uso terapêutico , RNA/isolamento & purificação , DNA/isolamento & purificação , Reação em Cadeia da Polimerase
7.
PLoS One ; 15(6): e0235218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584890

RESUMO

Previous research regarding Holstein cows has mainly focused on increasing milk yield. However, in order to maximize the economical profits of Holstein cattle farming, it is necessary to fully take advantage of Holstein bulls to produce high-grade beef. The present study aims to investigate different transcriptomic profiling of Holstein bulls and steers, via high-throughput RNA-sequencing (RNA-seq). The growth and beef quality traits of Holstein steers and bulls were characterized via assessment of weight, rib eye area, marbling score, shear force and intramuscular fat percentage of the longissimus lumborum (LL) muscle. The results indicated that castration improved the meat quality, yet reduced the meat yield. Subsequently, RNA-seq of the LL muscle from Holstein steers and bulls revealed a total of 56 differentially expressed genes (DEGs). We performed the functional enrichment analysis in Gene Ontology (GO) annotations of the DEGs using GOseq R package software and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using KOBAS tool. Through the integrated analysis of DEGs with reported QTLs and SNPs, seven promising candidate genes potentially affecting the beef quality of LL muscle following castration were discovered, including muscle structural protein coding genes (MYH1, MYH4, MYH10) and functional protein coding genes (GADL1, CYP2R1, EEPD1, SHISA3). Among them, MYH10, GADL1, CYP2R1, EEPD1 and SHISA3 were novel candidate genes associated with beef quality traits. Notably, EEPD1 was associated with both meat quality and reproduction traits, thus indicating its overlapping role in responding to hormone change, and subsequently inducing beef quality improvement. Our findings provide a complete dataset of gene expression profile of LL in Holstein bulls and steers, and will aid in understanding how castration influence meat yield and quality.


Assuntos
Qualidade dos Alimentos , Carne/análise , Músculo Esquelético/metabolismo , Transcriptoma , Animais , Carboxiliases/genética , Bovinos , Endodesoxirribonucleases/genética , Feminino , Masculino , Cadeias Pesadas de Miosina/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Análise de Sequência de RNA
8.
Braz J Med Biol Res ; 53(4): e9220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267310

RESUMO

Rab7, an important member of the Rab family, is closely related to autophagy, endocytosis, apoptosis, and tumor suppression but few studies have described its association with renal fibrosis. In the early stage, our group studied the effects of Rab7 on production and degradation of extracellular matrix in hypoxic renal tubular epithelial cells. Because cell culture in vitro is different from the environment in vivo, it is urgent to understand the effects in vivo. In our current study, we established a renal fibrosis model in Rab7-knock-in mice (prepared by CRISPR/Cas9 technology) and wild type (WT) C57BL/6 mice using unilateral ureteral obstruction (UUO). Seven and 14 days after UUO, the expression of the Rab7 protein in WT mice, as well as the autophagic activity, renal function, and the degree of renal fibrosis in WT and Rab7-knock-in mice were examined by blood biochemical assay, hematoxylin-eosin and Masson staining, immunohistochemistry, and western blotting. We found that the Rab7 expression in WT mice increased over time. Furthermore, the autophagic activity constantly increased in both groups, although it was higher in the Rab7-knock-in mice than in the WT mice at the same time point. Seven days after UUO, the degree of renal fibrosis was milder in the Rab7-knock-in mice than in the WT mice, but it became more severe 14 days after surgery. Similar results were found for renal function. Therefore, Rab7 suppressed renal fibrosis in mice initially, but eventually it aggravated fibrosis with the activation of autophagy.


Assuntos
Autofagia/fisiologia , Nefropatias/etiologia , Rim/patologia , Obstrução Ureteral/complicações , Proteínas rab de Ligação ao GTP/genética , Animais , Feminino , Fibrose , Masculino , Camundongos , Camundongos Knockout , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima , Proteínas rab de Ligação ao GTP/metabolismo
9.
PLoS One ; 15(4): e0231898, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302353

RESUMO

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis.


Assuntos
Matriz Extracelular/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Podócitos/metabolismo , RNA/metabolismo , Proteínas WT1/genética , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Glomerulosclerose Segmentar e Focal/genética , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/química , RNA/isolamento & purificação , Receptor EphA6/genética , Análise de Sequência de RNA
10.
PLoS One ; 15(4): e0231400, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294110

RESUMO

Marine dinoflagellates produce a diversity of polyketide toxins that are accumulated in marine food webs and are responsible for a variety of seafood poisonings. Reef-associated dinoflagellates of the genus Gambierdiscus produce toxins responsible for ciguatera poisoning (CP), which causes over 50,000 cases of illness annually worldwide. The biosynthetic machinery for dinoflagellate polyketides remains poorly understood. Recent transcriptomic and genomic sequencing projects have revealed the presence of Type I modular polyketide synthases in dinoflagellates, as well as a plethora of single domain transcripts with Type I sequence homology. The current transcriptome analysis compares polyketide synthase (PKS) gene transcripts expressed in two species of Gambierdiscus from French Polynesia: a highly toxic ciguatoxin producer, G. polynesiensis, versus a non-ciguatoxic species G. pacificus, each assembled from approximately 180 million Illumina 125 nt reads using Trinity, and compares their PKS content with previously published data from other Gambierdiscus species and more distantly related dinoflagellates. Both modular and single-domain PKS transcripts were present. Single domain ß-ketoacyl synthase (KS) transcripts were highly amplified in both species (98 in G. polynesiensis, 99 in G. pacificus), with smaller numbers of standalone acyl transferase (AT), ketoacyl reductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) domains. G. polynesiensis expressed both a larger number of multidomain PKSs, and larger numbers of modules per transcript, than the non-ciguatoxic G. pacificus. The largest PKS transcript in G. polynesiensis encoded a 10,516 aa, 7 module protein, predicted to synthesize part of the polyether backbone. Transcripts and gene models representing portions of this PKS are present in other species, suggesting that its function may be performed in those species by multiple interacting proteins. This study contributes to the building consensus that dinoflagellates utilize a combination of Type I modular and single domain PKS proteins, in an as yet undefined manner, to synthesize polyketides.


Assuntos
Dinoflagelados/enzimologia , Policetídeo Sintases/genética , Transcriptoma , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Ciguatoxinas/metabolismo , Dinoflagelados/classificação , Dinoflagelados/isolamento & purificação , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Filogenia , Policetídeo Sintases/metabolismo , Polinésia , RNA/química , RNA/isolamento & purificação , RNA/metabolismo
11.
Gene ; 743: 144614, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32222532

RESUMO

Buffalo reproduction struggles with a high incidence of early embryonic mortality. Effective treatment and prevention strategies for this condition are not available due to lack of understanding of molecular pathways in early pregnancy of this species. In the present study, we have attempted to understand these molecular pathways by characterizing the endometrial transcriptomic profiles of pregnant buffalos during early pregnancy. For the transcriptome profiling, buffalo endometrial tissues of 29-36 days of pregnancy and of nonpregnant luteal phase were collected from the local slaughterhouse. We confirmed the status of pregnancy based on the crown vertebral length of the foetus. Total RNA was isolated and sequencing was performed using the Illumina nextseq platform. The raw reads were filtered and mapped to the Bos taurus UMD 3.1 reference genome assembly. An average of 24,597 genes was investigated for differential expression between the two groups. Transcriptome data identified a total of 450 differentially expressed genes (using a cut off value of log2 fold changes >2 and <-2) in early pregnancy in comparison to the nonpregnant group (Padj < 0.05). Among these, 270 genes were significantly upregulated and 180 genes were downregulated. The most impacted pathways were related to secretion, transport, ionic homeostasis, mitosis and negative regulation of viral processes. In conclusion, our study characterized a unique set of DEGs, during the early pregnancy of buffalo, which potentially modulate the endometrial environment to establish and maintain a successful pregnancy.


Assuntos
Búfalos/fisiologia , Endométrio/metabolismo , Prenhez/genética , RNA/metabolismo , Transcriptoma/fisiologia , Animais , Regulação para Baixo , Feminino , Gravidez , RNA/genética , RNA/isolamento & purificação , RNA-Seq , Regulação para Cima
12.
Gastroenterol. hepatol. (Ed. impr.) ; 43(3): 107-116, mar. 2020. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-190783

RESUMO

Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca2+ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca2+ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis


Frizzled-2 desempeña un papel importante en el mantenimiento de la funcionalidad normal de los hepatocitos. Este estudio tiene como objetivo analizar el papel de la inhibición de Frizzled-2 en la protección de los hepatocitos BRL-3A de rata de la hipoxia/reoxigenación (H/R). El modelo de hepatocitos H/R in vitro se demostró con el cultivo de células BRL-3A en condiciones de H/R. El ARNip de Frizzled-2 se transinfectó en células BRL-3A para inhibir la señalización de Frizzled-2. Wnt5a y Frizzled-2 aumentaron considerablemente en las células BRL-3A tras el tratamiento con H/R. El tratamiento con H/R provocó citotoxicidad celular, una tasa de apoptosis temprana y el nivel de Ca2+ intracelular en células BRL-3A mientras que el gen frizzled-2 silenciado redujo la citotoxicidad celular inducida por H/R, la apoptosis y el nivel de Ca2+ intracelular. El estudio in vivo con ratones mostró, además, la regulación al alza de la vía de Frizzled-2/Wnt 5 y la expresión de caspasa 3 escindida en tejidos hepáticos con lesión por isquemia y reperfusión (LIR). En resumen, la inhibición de Frizzled-2 por su ARNip puede proteger a las células BRL-3A al atenuar la citotoxicidad celular y la apoptosis inducida por H/R


Assuntos
Animais , Camundongos , Ratos , Receptores Frizzled/antagonistas & inibidores , RNA/isolamento & purificação , Hipóxia/metabolismo , Apoptose/fisiologia , Receptores Frizzled/genética , Reação em Cadeia da Polimerase , Western Blotting , Caspase 3
13.
PLoS One ; 15(3): e0229791, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32150588

RESUMO

Saliva represents an ideal matrix for diagnostic biomarker development as it is readily available and requires no invasive collection procedures. However, salivary RNA is labile and rapidly degrades. Previous attempts to isolate RNA from saliva have yielded poor quality and low concentrations. Here we compare collection and processing methods and propose an approach for future studies. The effects of RNA stabilisers, storage temperatures, length of storage and fasting windows were investigated on pooled saliva samples from healthy volunteers. Isolated RNA was assessed for concentration and quality. Bacterial growth was investigated through RT-PCR using bacterial and human primers. Optimal conditions were implemented and quality controlled in a clinical setting. The addition of RNAlater increased mean RNA yield from 4912 ng/µl to 15,473 ng and RNA Integrity Number (RIN) from 4.5 to 7.0. No significant changes to RNA yield were observed for storage at room temperature beyond 1 day or at -80 °C. Bacterial growth did not occur in samples stored at ambient temperature for up to a week. There was a trend towards higher RNA concentration when saliva was collected after overnight fasting but no effect on RIN. In the clinic, RNA yields of 6307 ng and RINs of 3.9 were achieved, improving on previous reports. The method we describe here is a robust, clinically feasible saliva collection method using preservative that gives high concentrations and improved RINs compared to saliva collected without preservative.


Assuntos
RNA/isolamento & purificação , Saliva/química , Saliva/microbiologia , Manejo de Espécimes/métodos , Pesquisa Médica Translacional/métodos , Adolescente , Adulto , Bactérias/isolamento & purificação , Feminino , Voluntários Saudáveis , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Adulto Jovem
14.
BMC Res Notes ; 13(1): 77, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070402

RESUMO

OBJECTIVE: Peripheral blood is the most promising source of RNA biomarkers for diagnostic and epidemiological studies, because the presence of disease and prognostic information is reflected in the gene expression pattern. Quality RNA is used by a number of different downstream applications, so the selection of the most appropriate RNA stabilization and purification method is important. We have analyzed the RNA purified from 300 blood samples from 25 donors processed by two technicians using three methodologies with Tempus and PaxGene tubes. RESULTS: The best quality sample results were obtained with the Tempus Spin RNA Isolation Kit and the PaxGene Blood miRNA Kit, although larger amounts of RNA were obtained with the Tempus Spin RNA Isolation Kit. Lower Cq values were observed for RNA and miRNA genes in samples that were tested with PaxGene Blood miRNA Kit and Tempus Spin RNA Isolation Kit respectively. We identify the Tempus Spin RNA Isolation Kit as the most robust methodology, whilst the MagMax for Stabilized Blood Tubes RNA Isolation Kit showed the most instability. For biobanks, which process a large cohort and conduct epidemiological studies, the Tempus Spin RNA Isolation Kit is the most appropriate methodology. The study demonstrates the robustness of real-life procedures.


Assuntos
Coleta de Amostras Sanguíneas/instrumentação , Coleta de Amostras Sanguíneas/métodos , Estudos Epidemiológicos , RNA/sangue , RNA/isolamento & purificação , Humanos , RNA/genética , Estabilidade de RNA , Reprodutibilidade dos Testes
15.
PLoS One ; 15(2): e0229423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084228

RESUMO

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.


Assuntos
Leucócitos Mononucleares/metabolismo , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/métodos , Actinas/genética , Humanos , RNA/análise , RNA/genética , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Reprodutibilidade dos Testes
16.
Cancer Res ; 80(7): 1371-1373, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32075797

RESUMO

Blood-based liquid biopsies are considered a screening approach for early cancer detection. Sequencing technologies enable in-depth analyses of nucleic acids, including mutant cell-free (cf) DNA in the plasma. However, in the blood of patients with early-stage cancer the detection level of mutant cfDNA is relatively low, and complicated by the natural presence of noncancer cfDNA mutants attributed to aging-related processes. Consequently, analysis of methylated cfDNA patterns and alternative approaches such as tumor-educated platelets are gaining traction for the detection of early-stage tumors. Here, we dissect the use of platelet RNA as a potential biomarker for the development of early-stage, pan-cancer blood tests.


Assuntos
Biomarcadores Tumorais/sangue , Plaquetas , Detecção Precoce de Câncer/métodos , Neoplasias/diagnóstico , RNA/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Humanos , Biópsia Líquida , Mutação , Neoplasias/sangue , Neoplasias/genética , RNA/genética , RNA/isolamento & purificação , RNA-Seq
17.
Sci Rep ; 10(1): 1148, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980690

RESUMO

To improve the understanding of the complex biological processes underlying the development of non-alcoholic steatohepatitis (NASH), a multi-omics approach combining bulk RNA-sequencing based transcriptomics, quantitative proteomics and single-cell RNA-sequencing was used to characterize tissue biopsies from histologically validated diet-induced obese (DIO) NASH mice compared to chow-fed controls. Bulk RNA-sequencing and proteomics showed a clear distinction between phenotypes and a good correspondence between mRNA and protein level regulations, apart from specific regulatory events discovered by each technology. Transcriptomics-based gene set enrichment analysis revealed changes associated with key clinical manifestations of NASH, including impaired lipid metabolism, increased extracellular matrix formation/remodeling and pro-inflammatory responses, whereas proteomics-based gene set enrichment analysis pinpointed metabolic pathway perturbations. Integration with single-cell RNA-sequencing data identified key regulated cell types involved in development of NASH demonstrating the cellular heterogeneity and complexity of NASH pathogenesis.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/etiologia , Proteômica/métodos , Transcriptoma , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/genética , RNA/genética , RNA/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de RNA , Análise de Célula Única , Espectrometria de Massas em Tandem
18.
Int J Biol Macromol ; 146: 422-430, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31904458

RESUMO

Chitosan has emerged as a useful biomaterial employed in tissue engineering and drug delivery applications due to its tunable and interesting properties. However, chitosan is protonated at biological pH and thus carries positive charges, which renders chitosan incompatible with conventional methods of RNA extraction. RNA extraction is an important step in investigating cell responses and behavior through studying their gene expression transcriptional profiles. While some researchers have tried different techniques to improve the yield and purity of RNA extracted from cells encapsulated in chitosan-based biomaterials, no single study has investigated the effects of manipulating pH of the homogenate during RNA extraction on the yield and quality of total RNA. This study confirms the release and binding of RNA from chitosan to be pH dependent while analyzing the impact of pH changes during the tissue disruption and homogenization step of extraction on the resulting yield and quality of isolated RNA. This concept was applied to three commonly used methods of RNA extraction, using adult neural stem/progenitor cells (aNSPCs) encapsulated within methacrylamide chitosan (MAC) as a model chitosan-based bioscaffold. High pH conditions resulted in high yields with good quality using both TRIzol and CTAB. pH of the homogenate did not affect RNeasy spin columns, which worked best in neutral conditions with good quality, however, the overall yield was low. Results in total show that pH affected RNA interaction with a chitosan-based bioscaffold, and thus altered the concentration, purity, and integrity of isolated RNA, dependent on the method used.


Assuntos
Materiais Biocompatíveis/química , Quitosana/química , RNA/isolamento & purificação , Acrilamidas/química , Células-Tronco Adultas/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Células-Tronco Neurais/metabolismo , Ratos Endogâmicos F344 , Eletricidade Estática
19.
J Chromatogr A ; 1618: 460875, 2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31982098

RESUMO

Large RNAs including messenger RNAs (mRNAs) are promising candidates for development of new drug products and vaccines. Development of high resolution methods for direct analysis of large RNAs, especially for purity in general and size or length in particular, is critical to support new drug development and manufacture. However, resolution based on size or length for large RNAs is limited even by capillary electrophoresis (CE), which is one of the most efficient separation methods for nucleic acids in general. This paper presents a capillary gel electrophoresis (CGE) method for separating large RNA molecules by size or length under strongly denaturing, non-aqueous conditions. We believe that our work constitutes the first time that a gel suitable for CGE prepared with high molecular weight polymers and using only formamide as solvent has been successfully employed to analyze large RNAs on the basis of their size or length with high resolution. With an eye toward application for mRNAs in particular, separation conditions in this work were optimized for RNAs approximately 2000 nucleotides (nt) in length. As compared to a standard CGE method using an aqueous gel, resolution for commercially-available RNA ladder components at 1500 and 2000 nt is increased approximately 6-fold. The impacts of polymer type, molecular weight of the polymer, and polymer concentration on the separation were studied and optimized. Analysis of the results presented here also provides guidance for optimization of separation conditions for RNAs with different sizes as needed for particular applications in the future.


Assuntos
Química Farmacêutica/métodos , Eletroforese Capilar , RNA/isolamento & purificação , Peso Molecular , Polímeros/química , RNA/análise , Solventes/química
20.
Methods Mol Biol ; 2102: 17-34, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989548

RESUMO

Next generation sequencing (NGS) is routinely used in gene expression analyses. In particular, RNA-seq has been the method of choice for highly sensitive genome-wide quantification of RNA expression. The method can be used in a wide variety of model systems, including studies to gain insight into underlying mechanisms of toxicologic processes and disease development induced by environmental toxicants. RNA-seq has also been coupled to many other molecular biology protocols to monitor specific aspects of the gene expression process. Here, we describe two such coupling-(a) global run-on sequencing (GRO-seq) that coupled it to the nuclear run-on (NRO), and (b) polysome profiling that coupled it to sucrose-gradient-based polysome isolation. Simultaneous RNA-seq, GRO-seq, and polysome profiling analyses enabled genome-wide analysis of the mode of stability control of individual RNA molecules.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA-Seq/métodos , Técnicas de Cultura de Células/métodos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Desoxirribonucleases , Humanos , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA/isolamento & purificação , Estabilidade de RNA , Transcriptoma , Fluxo de Trabalho
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