Project description:Microarrays revolutionized biological research by enabling gene expression comparisons on a transcriptome-wide scale. Microarrays, however, do not estimate absolute expression level accurately. At present, high throughput sequencing is emerging as an alternative methodology for transcriptome studies. Although free of many limitations imposed by microarray design, its potential to estimate absolute transcript levels is unknown. In this study, we evaluate relative accuracy of microarrays and transcriptome sequencing (RNA-Seq) using third methodology: proteomics. We find that RNA-Seq provides a better estimate of absolute expression levels.
Project description:Homo sapiens fresh whole blood was infected with Candida tropicalis. RNA-pool of both species extracted at 0min (control), 15, 30, 60, 120, 240 min. Samples are rRNA depleted. Measurement of Candida tropicalis gene expression.
Project description:Homo sapiens fresh whole blood was infected with Candida parapsilosis. RNA-pool of both species extracted at 0min (control), 15, 30, 60, 120, 240 min. Samples are rRNA depleted. Measurement of Homo sapiens gene expression.
Project description:Homo sapiens fresh whole blood was infected with Candida glabrata. RNA-pool of both species extracted at 0min (control), 15, 30, 60, 120, 240 min. Samples are rRNA depleted. Measurement of Candida glabrata gene expression.
Project description:Homo sapiens fresh whole blood was infected with Candida albicans SC5314. RNA-pool of both species extracted at 0min (control), 15, 30, 60, 120, 240 min. Samples are rRNA depleted. Expression measurement of Homo sapiens genes.
Project description:Microarrays revolutionized biological research by enabling gene expression comparisons on a transcriptome-wide scale. Microarrays, however, do not estimate absolute expression level accurately. At present, high throughput sequencing is emerging as an alternative methodology for transcriptome studies. Although free of many limitations imposed by microarray design, its potential to estimate absolute transcript levels is unknown. In this study, we evaluate relative accuracy of microarrays and transcriptome sequencing (RNA-Seq) using third methodology: proteomics. We find that RNA-Seq provides a better estimate of absolute expression levels. We first determined whether we could reproduce the agreement between mRNA expression estimates measured by microarrays and by RNA-Seq reported in other studies. For this purpose, we collected mRNA expression data in two independent cerebellar samples, each containing pooled mRNA from 5 adult human individuals, using both methodologies. Next, to test whether biological variation among samples would substantially reduce correlation strength, we compared expression levels determined by RNA-Seq in two pooled samples to the microarray data obtained from different individuals. For this purpose we used expression measurements obtained using Affymetrix Exon Arrays in 5 individual adult human cerebellar samples, none of which were included in the two pooled samples. Further, since technical and stochastic variation are extremely unlikely to result in better correlation between mRNA and protein expression measurements, we argue that the technology resulting in better correlation must provide more accurate measurements.
Project description:Transcriptional profiling of Homo sapiens inflammatory skin diseases (whole skin biospies): Psoriasis (Pso), vs Atopic Dermatitis (AD) vs Lichen planus (Li), vs Contact Eczema (KE), vs Healthy control (KO) In recent years, different genes and proteins have been highlighted as potential biomarkers for psoriasis, one of the most common inflammatory skin diseases worldwide. However, most of these markers are not psoriasis-specific but also found in other inflammatory disorders. We performed an unsupervised cluster analysis of gene expression profiles in 150 psoriasis patients and other inflammatory skin diseases (atopic dermatitis, lichen planus, contact eczema, and healthy controls). We identified a cluster of IL-17/TNFα-associated genes specifically expressed in psoriasis, among which IL-36γ was the most outstanding marker. In subsequent immunohistological analyses IL-36γ was confirmed to be expressed in psoriasis lesions only. IL-36γ peripheral blood serum levels were found to be closely associated with disease activity, and they decreased after anti-TNFα-treatment. Furthermore, IL-36γ immunohistochemistry was found to be a helpful marker in the histological differential diagnosis between psoriasis and eczema in diagnostically challenging cases. These features highlight IL-36γ as a valuable biomarker in psoriasis patients, both for diagnostic purposes and measurement of disease activity during the clinical course. Furthermore, IL-36γ might also provide a future drug target, due to its potential amplifier role in TNFα- and IL-17 pathways in psoriatic skin inflammation. In recent years, different genes and proteins have been highlighted as potential biomarkers for psoriasis, one of the most common inflammatory skin diseases worldwide. However, most of these markers are not psoriasis-specific but also found in other inflammatory disorders. We performed an unsupervised cluster analysis of gene expression profiles in 150 psoriasis patients and other inflammatory skin diseases (atopic dermatitis, lichen planus, contact eczema, and healthy controls). We identified a cluster of IL-17/TNFα-associated genes specifically expressed in psoriasis, among which IL-36γ was the most outstanding marker. In subsequent immunohistological analyses IL-36γ was confirmed to be expressed in psoriasis lesions only. IL-36γ peripheral blood serum levels were found to be closely associated with disease activity, and they decreased after anti-TNFα-treatment. Furthermore, IL-36γ immunohistochemistry was found to be a helpful marker in the histological differential diagnosis between psoriasis and eczema in diagnostically challenging cases. These features highlight IL-36γ as a valuable biomarker in psoriasis patients, both for diagnostic purposes and measurement of disease activity during the clinical course. Furthermore, IL-36γ might also provide a future drug target, due to its potential amplifier role in TNFα- and IL-17 pathways in psoriatic skin inflammation.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain.A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development.Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.