Comparison of extraction methods for intracellular metabolomics of human tissues.

Analyses of metabolic compounds inside cells or tissues provide high information content since they represent the endpoint of biological information flow and are a snapshot of the integration of many regulatory processes. However, quantification of the abundance of metabolites requires their careful extraction. We present a comprehensive study comparing ten extraction protocols in four human sample types (liver tissue, bone marrow, HL60, and HEK cells) aiming to detect and quantify up to 630 metabolites of different chemical classes. We show that the extraction efficiency and repeatability are highly variable across protocols, tissues, and chemical classes of metabolites. We used different quality metrics including the limit of detection and variability between replicates as well as the sum of concentrations as a global estimate of analytical repeatability of the extraction. The coverage of extracted metabolites depends on the used solvents, which has implications for the design of measurements of different sample types and metabolic compounds of interest. The benchmark dataset can be explored in an easy-to-use, interactive, and flexible online resource (R/shiny app MetaboExtract: http://www.metaboextract.shiny.dkfz.de) for context-specific selection of the optimal extraction method. Furthermore, data processing and conversion functionality underlying the shiny app are accessible as an R package: https://cran.r-project.org/package=MetAlyzer.

CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells.

Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3'-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.

Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells.

Hematopoietic stem cells (HSCs) and distinct multipotent progenitor (MPP) populations (MPP1-4) contained within the Lin-Sca-1+c-Kit+ (LSK) compartment have previously been identified using diverse surface-marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+CD135-CD48-CD150-). Upon transplantation, MPP5 supports initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. MPP5, capable of generating MPP1-4 but not HSCs, represents a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells, we performed RNA-sequencing (RNA-seq) analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with functional reconstitution activity, MPP5 is located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset.

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1.

Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.

Differential Alternative Polyadenylation Landscapes Mediate Hematopoietic Stem Cell Activation and Regulate Glutamine Metabolism.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.

Biglycan expression in the melanoma microenvironment promotes invasiveness via increased tissue stiffness inducing integrin-ß1 expression.

Novel targeted and immunotherapeutic approaches have revolutionized the treatment of metastatic melanoma. A better understanding of the melanoma-microenvironment, in particular the interaction of cells with extracellular matrix molecules, may help to further improve these new therapeutic strategies.We observed that the extracellular matrix molecule biglycan (Bgn) was expressed in certain human melanoma cells and primary fibroblasts when evaluated by microarray-based gene expression analysis. Bgn expression in the melanoma tissues correlated with low overall-survival and low progression-free-survival in patients. To understand the functional role of Bgn we used gene-targeted mice lacking functional Bgn. Here we observed that melanoma growth, metastasis-formation and tumor-related death were reduced in Bgn-/- mice compared to Bgn+/+ mice. In vitro invasion of melanoma cells into organotypic-matrices derived from Bgn-/- fibroblasts was reduced compared to melanoma invasion into Bgn-proficient matrices. Tissue stiffness as determined by atomic-force-microscopy was reduced in Bgn-/- matrices. Isolation of melanoma cells and fibroblasts from the stiffer Bgn+/+ matrices revealed an increase in integrin-ß1 expression compared to the Bgn-/- fibroblast matrices. Overexpression of integrin-ß1 in B16-melanoma cells abolished the survival benefit seen in Bgn-/- mice. Consistent with the studies performed in mice, the abundance of Bgn-expression in human melanoma samples positively correlated with the expression of integrin-ß1, which is in agreement with results from the organotypic invasion-assay and the in vivo mouse studies.This study describes a novel role for Bgn-related tissue stiffness in the melanoma-microenvironment via regulation of integrin-ß1 expression by melanoma cells in both mice and humans.

Synergistic antiviral activity of ribavirin and interferon-α against parrot bornaviruses in avian cells.

Avian bornaviruses are the causative agents of proventricular dilatation disease (PDD), a widely distributed and often fatal disease in captive psittacines. Because neither specific prevention measures nor therapies against PDD and bornavirus infections are currently available, new antiviral strategies are required to improve animal health. We show here that the nucleoside analogue ribavirin inhibited bornavirus activity in a polymerase reconstitution assay and reduced viral load in avian cell lines infected with two different parrot bornaviruses. Furthermore, we observed that ribavirin enhanced type I IFN signalling in avian cells. Combined treatment of avian bornavirus-infected cells with ribavirin and recombinant IFN-α strongly enhanced the antiviral efficiency compared to either drug alone. The combined use of ribavirin and type I IFN might represent a promising new strategy for therapeutic treatment of captive parrots persistently infected with avian bornaviruses.