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Find video protocols related to scientific articles indexed in Pubmed.
Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses.
Gene
PUBLISHED: 07-17-2014
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Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is a powerful tool for the measurement of gene expression; however, the accuracy of this approach depends on the stability of reference genes. The objective of the present study was to identify the stable reference genes in orchardgrass (Dactylis glomerata L.), a principal cool-season forage grass in the world. Ten candidate reference genes were selected in this study including ATP-binding [ABC], actin [ACTIN], cyclophilin [CYP2], glyceraldehyde 3-phosphate dehydrogenase [GAPDH], beta-amylase 4 [BAM4], zeitlupe [ZTL], MAP Kinase 4 [MPK4], ubiquitin-conjugating enzyme [UBC], S-adenosylmethionine decarboxylase [SAMDC], and translationally controlled tumor protein [TCTP]. The candidate genes were assessed in orchardgrass leaves and roots under conditions of drought, high salinity, heat, waterlogging, and abscisic acid (ABA) treatments. We used GeNorm, BestKeeper, NormFinder, and RefFinder for qRT-PCR normalization and validation to determine that the expression of these reference genes was stress-dependent. ACTIN, CYP2, and ABC were found to be the most stably expressed genes for drought stress while ACTIN, TCTP, and ABC were the most stable under salt stress. ACTIN, CYP2, and ABC were all found to be good reference genes for studying heat stress. Likewise, CYP2, MPK4, and ABC were most suitable to study waterlogging, and ACTIN, CYP2, and MPK4 were determined as the three best reference genes for ABA studies. Our study identified and validated the possible reference genes in orchardgrass that may be used for quantification of target gene expression under various abiotic stresses.
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Biological characteristics and gene expression pattern of bone marrow mesenchymal stem cells in patients with psoriasis.
Exp. Dermatol.
PUBLISHED: 05-09-2014
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Mesenchymal stem cells (MSCs) have immunoregulatory and proangiogenic effects and are suggested to be involved in the pathological processes of immune-related diseases, including psoriasis. Biological characteristics of bone marrow MSCs (BMSCs) from patients with autoimmune diseases, such as systemic lupus erythematosus or rheumatoid arthritis, but not psoriasis, have been characterized. We compared the gene expression profile and biological characteristics of BMSCs from patients with psoriasis and healthy controls. Although the phenotype, differentiation potential and ability to support CD34(+) cell proliferation were similar to those of normal BMSCs, psoriatic BMSCs showed aberrant proliferative activity, increased apoptosis rate and a characteristic gene expression profile. These aberrations may develop after the abnormal immune response in psoriasis and result in BMSC dysfunction. The functionally deficient BMSCs may then fail to suppress overactive immune cells, thereby contributing to the pathogenesis of psoriasis.
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Complete genome of Hainan papaya ringspot virus using small RNA deep sequencing.
Virus Genes
PUBLISHED: 01-17-2014
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Small RNA deep sequencing allows for virus identification, virus genome assembly, and strain differentiation. In this study, papaya plants with virus-like symptoms collected in Hainan province were used for deep sequencing and small RNA library construction. After in silicon subtraction of the papaya sRNAs, small RNA reads were used to in the viral genome assembly using a reference-guided, iterative assembly approach. A nearly complete genome was assembled for a Hainan isolate of papaya ringspot virus (PRSV-HN-2). The complete PRSV-HN-2 genome (accession no.: KF734962) was obtained after a 15-nucleotide gap was filled by direct sequencing of the amplified genomic region. Direct sequencing of several random genomic regions of the PRSV isolate did not find any sequence discrepancy with the sRNA-assembled genome. The newly sequenced PRSV-HN-2 genome shared a nucleotide identity of 96 and 94 % to that of the PRSV-HN (EF183499) and PRSV-HN-1 (HQ424465) isolates, and together with these two isolates formed a new PRSV clade. These data demonstrate that the small RNA deep sequencing technology provides a viable and rapid mean to assemble complete viral genomes in plants.
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A Novel Mutation in the RPE65 Gene Causing Leber Congenital Amaurosis and Its Transcriptional Expression In Vitro.
PLoS ONE
PUBLISHED: 01-01-2014
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The retinal pigment epithelium-specific 65 kDa protein is an isomerase encoded by the RPE65 gene (MIM 180069) that is responsible for an essential enzymatic step required for the function of the visual cycle. Mutations in the RPE65 gene cause not only subtype II of Leber congenital amaurosis (LCA) but also early-onset severe retinal dystrophy (EOSRD). This study aims to investigate a Chinese case diagnosed as EOSRD and to characterize the polymorphisms of the RPE65 gene. A seven-year-old girl with clinical symptoms of EOSRD and her parents were recruited into this study. Ophthalmologic examinations, including best-corrected visual acuity, slit-lamp, Optical coherence tomography (OCT), and fundus examination with dilated pupils, were performed to determine the clinical characteristics of the whole family. We amplified and sequenced the entire coding region and adjacent intronic sequences of the coding regions of the RPE65 gene for the whole family to explore the possible mutation. Our results demonstrate that the patient exhibited the typical clinically features of EOSRD. Her bilateral decimal visual acuity was 0.3 and 0.4 in the left and right eyes, respectively. Spectral-domain optical coherence tomography (SD-OCT) was used to assess the retinal stratification for the whole family. All together, we identified four mutations within the RPE65 gene (c.1056G>A, c.1243+2T>A, c.1338+20A>C and c.1590C>A) in the patient. Among the four mutations, c.1056G>A and c.1338+20A>C had been reported previously and another two were found for the first time in this study. Her mother also carried the novel mutation (c.1243+2T>A). Either a single or a compound heterozygous or a homozygous one mutation is expected to cause EOSRD because mutations of RPE65 gene usually cause an autosomal recessive disease. Therefore, we speculate that the c.1590C>A mutation together with the c.1243+2T>A mutation may cause the patient's phenotype.
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Identification of candidate reference genes in perennial ryegrass for quantitative RT-PCR under various abiotic stress conditions.
PLoS ONE
PUBLISHED: 01-01-2014
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Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied.
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Inhibitory effect of sanguinarine on PKC-CPI-17 pathway mediating by muscarinic receptors in dispersed intestinal smooth muscle cells.
Res. Vet. Sci.
PUBLISHED: 07-15-2013
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This study investigated the inhibitory effects of sanguinarine (SA) on PKC-CPI-17 pathway in rat intestinal smooth muscle cells (ISMC). Previous studies indicate that the inhibitory effects of SA on ISMC contraction are possibly mediated by the Ca(2+) influx. ISMC was treated with 1?M SA for 24h remarkably inhibited the mRNA expression of m2 and m3 receptors. ISMC treated with 1 or 3?M SA for 30min significantly decreased the mRNA expression of PKC-?, PKC-?, PKC-?, and CPI-17. 1?M SA could markedly inhibit carbachol (CCh)-mediated increase PKC-?, PKC-?, and CPI-17 mRNA but had no effect in PKC-?.Treatment of ISMC with SA (1?M, 30min) caused a decrease in protein expression of PKC-?. However, the expression of CPI-17 was significantly inhibited in a time-dependent manner. These results demonstrate that the inhibitory effect of SA is coupled with alteration of PKC-mediated signal transduction and intracellular Ca(2+) concentration.
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Narrowband ultraviolet B interferes with gene expression in the peripheral blood T cells of patients with psoriasis.
Dermatology (Basel)
PUBLISHED: 04-25-2013
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Psoriasis pathogenesis and development are closely related to abnormal T cell activity. Narrowband ultraviolet B (NB-UVB) treatment markedly improves skin lesions in psoriasis.
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DNA methylation of dermal MSCs in psoriasis: identification of epigenetically dysregulated genes.
J. Dermatol. Sci.
PUBLISHED: 01-15-2013
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Mesenchymal stem cells (MSCs) are likely involved in pathological processes of immune-related diseases, including psoriasis, because of their immunoregulatory and pro-angiogenic effects. DNA methylation plays an essential role in regulating gene expression and maintaining cell function.
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Differential gene expression in peripheral blood T cells from patients with psoriasis, lichen planus, and atopic dermatitis.
J. Am. Acad. Dermatol.
PUBLISHED: 01-09-2013
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Psoriasis, lichen planus (LP), and atopic dermatitis (AD) are common chronic inflammatory skin diseases mediated by immune responses.
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Screening of differentially expressed genes and predominant expression of ? variable region of T cell receptor in peripheral T cells of psoriatic patients.
Eur J Dermatol
PUBLISHED: 09-29-2011
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Psoriasis is a skin disease featuring epithelial cell hyper-proliferation and T cell infiltration. Abnormal T cell immune responses play an important role in psoriatic pathogenesis. To screen differentially expressed genes in T cells of patients with plaque psoriasis, analyze the predominant expression of the ? variable region of T cell receptors and discuss the role of T cells in the pathogenesis of psoriasis. High throughput RNA sequencing and Real-time PCR were used. Results: A total of 907 genes were differentially expressed in peripheral T cells of patients with psoriasis. Among them, 695 genes were mapped to the Gene Ontology database, 14 gene terms were significantly enriched, and 418 genes were involved in signaling pathways such as apoptosis, B cell receptor signaling and T cell receptor signaling. TRBV2, TRBV5-7, TRBV6-6/6-9, TRBV12, TRBV24 and TRBV29 were significantly up-regulated in psoriatic patients compared to healthy subjects, among which, TRBV6-6/6-9, TRBV12 and TRBV29 are predominantly expressed in psoriatic patients. Many genes were differentially expressed in T cells of psoriatic patients, especially the TRBV gene family, which were predominantly expressed in T cells and might play an important role in abnormal immune responses of T cells in psoriatic patients.
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Differential cytokine secretion of cultured bone marrow stromal cells from patients with psoriasis and healthy volunteers.
Eur J Dermatol
PUBLISHED: 11-04-2009
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Psoriasis is a chronic inflammatory skin disorder associated with a host of immune abnormalities. We have recently proposed that psoriasis is related to aberrant hematopoiesis and hypothesized that aberrant hematopoiesis in psoriasis is due to the differential hematopoietic microenvironment. To investigate whether the hematopoietic microenvironment is altered in patients with psoriasis by comparing the levels of cytokines secreted from BMSCs in patients with psoriasis and those in healthy volunteers, flow cytometry analysis was used to examine the proportion of the positive cells and direct ELISA was used to measure the concentrations of cytokines secreted from BMSCs in patients with psoriasis and healthy volunteers. In comparison with normal controls, BMSCs from patients with psoriasis showed increased secretions of SCF, G-CSF, and IL-6, decreased secretions of IL-1alpha, IL-1beta, IL-3, IL-8, EGF, VEGF, TNF-alpha, LIF, HGF, PDGF and no alteration in the levels of GM-CSF, IL-11, IL-7 and the cell surface markers CD29, CD34, CD45 and HLA-DR. Our data demonstrate for the first time that the hematopoietic microenvironment is altered in patients with psoriasis, suggesting that an aberrant hematopoietic microenvironment may be one mechanism for the pathogenesis of the disease.
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Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system.
Appl. Microbiol. Biotechnol.
PUBLISHED: 03-14-2009
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In many species, the introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference (RNAi). RNAi is the process of sequence-specific, posttranscriptional gene silencing (PTGS) in animals and plants, mediated by dsRNA homologous to the silenced genes. In plants, PTGS is part of a defense mechanism against virus infection, and dsRNA is the pivotal factor that induces gene silencing. Here, we report an efficient method that can produce dsRNA using a bacterial prokaryotic expression system. Using the bacteriophage lambda-dependent Red recombination system, we knocked out the rnc genes of two different Escherichia coli strains and constructed three different vectors that could produce dsRNAs. This work explores the best vector/host combinations for high output of dsRNA. In the end, we found that strain M-JM109 or the M-JM109lacY mutant strain and the vector pGEM-CP480 are the best choices for producing great quantities of dsRNA. Resistance analyses and Northern blot showed that Tobacco mosaic virus infection could be inhibited by dsRNA, and the resistance was an RNA-mediated virus resistance. Our findings indicate that exogenous dsRNA could form the basis for an effective and environmentally friendly biotechnological tool that protects plants from virus infections.
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Promoter methylation status of p15 and p21 genes in HPP-CFCs of bone marrow of patients with psoriasis.
Eur J Dermatol
PUBLISHED: 01-20-2009
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Psoriasis is an inflammatory disease related to dysfunctional immunity. The dysfunctional immunity may influence the haematopoietic microenvironment or haematopoiesis in psoriasis. However, direct evidence is lacking. Our objective was to investigate the proliferation of hematopoietic cells from psoriatic patients and any link between the promoter methylation status of p15 and p21 genes and the colony formation ability of high proliferative potential colony-forming cells (HPP-CFCs). Marrow mononuclear cells were isolated from the bone marrow of psoriatic patients and normal controls by density gradient centrifugalization. Colony forming assays of HPP-CFCs were performed in vitro in methylcellulose semi-solid culture medium. mRNA expression and the promoter methylation status of p15 and p21 genes in HPP-CFCs were studied by semi-quantitative RT-PCR and methylation-specific PCR respectively. In methycellulose semi-solid culture system, the colony count of HPP-CFCs in bone marrow of psoriatic patients was significantly less than that of normal controls. Moreover, significantly lower positive frequencies of promoter methylation and higher transcription levels for p15 and p21 genes were observed in psoriasis in comparison to normal volunteers. The lower promoter methylation of p15 and p21 genes may be an important mechanism for the dysfunctional growth regulation pathways in HPP-CFCs in psoriasis.
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A novel mutation in the CYP4V2 gene in a Chinese patient with Biettis crystalline dystrophy.
Int Ophthalmol
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Bietti crystalline corneoretinal dystrophy (BCD, MIM 210370) is a type of hereditary retinal disorder which commonly occurs in China. It is known that mutations in the CYP4V2 gene result in BCD. The purpose of this study was to investigate the case of a Chinese family and characterize the polymorphisms of the CYP4V2 gene. A 29-year-old male (the son of a Chinese family) with typical clinical symptoms of BCD and his family were recruited into this study. Ophthalmologic examination, including best-corrected visual acuity, slit-lamp, and fundus examination with dilated pupils, was conducted to determine the clinical characteristics of the whole family. The entire coding region and adjacent intronic sequences of 11 coding regions of the CYP4V2 gene of the whole family were then amplified by polymerase chain reaction and sequenced. Our results show that the son had typical clinical features of BCD. His bilateral decimal visual acuity was 0.06 (left eye) and 0.01 (right eye). Bilateral crystal-like deposits were found in the posterior pole of his fundus, and differing extent of atrophy of the retinal pigment epithelium, and carpet-like retinal degeneration along with numerous tiny glittering crystals were also clearly observed. However, such characteristics were not found on the fundus of his parents eyes. Five mutations within the CYP4V2 gene (c.64C>G, c.775C>A, c.810T>G, c.1091-2A>G, and c.1399T>C) were identified in the son. Among the five mutations, four had previously been reported and the c.1399T>C was discovered for the first time. This novel mutation causes an amino acid substitution (C467R) in the CYP4V2 protein, but it was not detected in the parents. As there is no apparent relationship in genotype-phenotype correlation between the CYP4V2 gene and the occurrence of BCD, this novel mutation may be a possible cause that could induce the clinical phenotype of BCD.
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Expression of Notch receptor and its target gene Hes-1 in bone marrow CD34+ cells from patients with psoriasis.
Dermatology (Basel)
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Psoriasis is an autoimmune disease mediated mainly by dysfunctional peripheral blood T cells. Both CD4+/CD8+ T cells and CD4+CD25+ regulatory T cells derived from psoriatic CD34+ bone marrow cells in vitro have been found to be functionally similar to those psoriatic circulating and lesional T cells. Notch signaling participates in diverse cell fate decisions during T cell development and has been reported to influence the proliferation of hematopoietic stem cells and the differentiation of T cells. The purpose of this study was to investigate the expression levels of Notch receptor 1, 2 and its target gene Hes-1 in CD34+ cells from patients with psoriasis. The total RNA and protein of CD34+ cells were extracted, and the mRNA as well as protein expression of Notch1, Notch2 and Hes-1 were investigated using real-time PCR and Western blot assays. We found that the mRNA and protein expression levels of Notch1 and Hes-1 in psoriasis patients were higher compared to normal controls, while the Notch2 mRNA and protein expression levels in psoriasis patients were similar to those of normal controls. The elevated Notch1 and Hes-1 expression levels in psoriatic CD34+ cells might be one reason for the immune disorders which are mainly mediated by T cells.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.