Project description:Human bone marrow is a complex, diversified and well-organized hematopoietic network changing composition with age. The purpose of this study was to analyze variations in relative precursor B cell abundance in bone marrow with age by means of global gene expression profiling. RNA was isolated from composite bone marrow from 25 healthy children, adolescents and adults age 2 months to 28 years. As reference transcript for precursor B cells we used recombination activating gene RAG1 exploring the data for other transcripts showing the same profile as RAG1 with age. We identified 54 genes with correlated expression profiles to RAG1 (r ? 0.9, p = 0), characterized by high expression at 3 - 20 months followed by a fast decline to lower signal levels maintained until early adulthood. Immunophenotyping from a similar healthy age-matched cohort (n = 37) showed a comparable decrease of precursor B cells. Of the 54 genes 15 were characteristically B cell associated representing cell surface molecules (CD19, CD72, CD79A, CD79B, CD180, IGL@, IGLL1, VPREB1, VPREB3), a signal transduction molecule (BLNK) and transcription factors (DNTT, EBF1, PAX5, POU2AF1, RAG2). Of the remaining transcripts some may represent novel B cell transcripts or genes involved in control of B cells. Bone marrow was obtained from healthy children eligible for elective minor surgery and voluntary health care workers. The bone marrow samples (2.5ml) were immediately after aspiration transferred to PAXgene tubes for mRNA stabilization before RNA extraction and hybridization on Affymetrix microarrays. To that end, the study presents a picture of the total marrow activity with minimal manipulation that would otherwise influence gene expression results. We used microarrays to determine age-related changes in precursor B cell transcripts in bone marrow from 25 healthy children and adults and searched for other transcripts showing the same expression profile with age.
Project description:Human bone marrow is a complex, diversified and well-organized hematopoietic network changing composition with age. The purpose of this study was to analyze variations in relative precursor B cell abundance in bone marrow with age by means of global gene expression profiling. RNA was isolated from composite bone marrow from 25 healthy children, adolescents and adults age 2 months to 28 years. As reference transcript for precursor B cells we used recombination activating gene RAG1 exploring the data for other transcripts showing the same profile as RAG1 with age. We identified 54 genes with correlated expression profiles to RAG1 (r ≥ 0.9, p = 0), characterized by high expression at 3 - 20 months followed by a fast decline to lower signal levels maintained until early adulthood. Immunophenotyping from a similar healthy age-matched cohort (n = 37) showed a comparable decrease of precursor B cells. Of the 54 genes 15 were characteristically B cell associated representing cell surface molecules (CD19, CD72, CD79A, CD79B, CD180, IGL@, IGLL1, VPREB1, VPREB3), a signal transduction molecule (BLNK) and transcription factors (DNTT, EBF1, PAX5, POU2AF1, RAG2). Of the remaining transcripts some may represent novel B cell transcripts or genes involved in control of B cells. Bone marrow was obtained from healthy children eligible for elective minor surgery and voluntary health care workers. The bone marrow samples (2.5ml) were immediately after aspiration transferred to PAXgene tubes for mRNA stabilization before RNA extraction and hybridization on Affymetrix microarrays. To that end, the study presents a picture of the total marrow activity with minimal manipulation that would otherwise influence gene expression results.
Project description:1,322 morphologically unidentified fragmentary bone specimens were analyzed using MALDI-TOF and a subset of 341 bone specimens with LC-MS/MS in order to characterize their proteome for species identification and potential hominin specimens related to the LRJ transitional period derived from the site Ilsenhöhle Ranis, Germany (50°39.7563’N, 11°33.9139’E).
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:Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionallyin vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHB's anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demonstrate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.