Project description:Quercetin has been described to have a wide range of beneficial effects in humans, ranging from anti-carcinogenic properties to reduced risk of cardiovascular disease. We tested whether a daily supplementation of quercetin leads to reproducible changes in gene expression profiles of human monocytes. Experiment Overall Design: CD14+ monocytes were isolated from three healthy volunteers before and after a 2 wk supplementation period of 150mg quercetin per day. With this design every subject served as his or her own control (baseline sample = control)
Project description:Gene expression profiling of the classical (CD14++CD16-), intermediate (CD14++CD16+) and nonclassical (CD14+CD16+) human monocyte subsets
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
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:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs. One-condition experment, gene expression of 3A6
Project description:CD14+ monocytes, the predominant population in human blood, are primarily engaged in host defense and pro-inflammatory cytokine responses. Aberrant monocyte activity causes life-threatening cytokine storms, while dysfunctional monocytes lead to 'immunoparalysis.' Understanding the mechanisms controlling monocyte functions is therefore paramount. Here, we reveal platelets' vital role in human monocytes' pro-inflammatory responses. Natural low platelet counts in patients with immune thrombocytopenia (ITP) , platelet depletion in healthy human monocytes, or in vivo platelet depletion in mice, result in monocyte immunoparalysis, characterized by reduced pro-inflammatory gene expression and weakened cytokine responses to immune challenge. Remarkably, supplementation with fresh platelets reverses monocyte immunoparalysis. In mice, thrombocytopenia results in down-regulation of myeloid innate immune genes, and compromised host defense transcriptional programs in monocytes despite normal responses to LPS. Platelets control monocyte cytokines independently of traditional cross-talk pathways, acting as reservoirs of transcription factors like NF?B and MAPK p38. We pinpointed a vesicle-derived NF?B2 transfer to human monocytes by mass spectrometry-based proteomics. Functionally, platelets proportionally restored impaired cytokine secretion in human monocytes lacking MAPK p38a and NF?B p65 and NF?B2. We unveil the intercellular transfer of inflammatory regulators, positioning platelets as central checkpoints in monocyte-mediated inflammation.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes