Single cell transcriptome sequencing of stimulated and frozen human peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection, defending our bodies from harmful pathogens. In biomedical research, PBMCs are commonly used to study global immune response to disease outbreak and progression, pathogen infections, for vaccine development and a multitude of other clinical applications. Over the past few years, the revolution in single-cell RNA sequencing (scRNA-seq) has enabled an unbiased quantification of gene expression in thousands of individual cells, which provides a more efficient tool to decipher the immune system in human diseases. In this work, we generate scRNA-seq data from human PBMCs at high sequencing depth (>100,000 reads/cell) for more than 30,000 cells, in resting, stimulated, fresh and frozen conditions. The data generated can be used for benchmarking batch correction and data integration methods, and to study the effect of freezing-thawing cycles on the quality of immune cell populations and their transcriptomic profiles.

Whole genome: next-generation sequencing as a virus safety test for biotechnological products.

The availability of next-generation sequencing techniques is about to strongly modify the strategies of control of the viral safety of biologicals products. It is now possible to use the tools of metagenomics, which is the study of the microbial genetic sequences recovered directly from a given sample. In this setting, the sequence of all the nucleic acids species of the sample are determined and compared with those in databases. We describe the pipeline we are currently using and show comparison of its analytical sensitivity with that of polymerase chain reaction together with results of time course experiments in infected cells. We propose to test cell supernatants for viral genomes associated to capsids and testing cells for viral RNA transcripts as the hallmark for virus replication, and we suggest rules of interpretation of the results. We also discuss the applicability of next-generation sequencing for the control of raw materials.

Heparan sulfate mimetics can efficiently mobilize long-term hematopoietic stem cells.

BACKGROUND: Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important. DESIGN AND METHODS: Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation. RESULTS: Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice. CONCLUSIONS: This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.

Molecular model of human heparanase with proposed binding mode of a heparan sulfate oligosaccharide and catalytic amino acids.

Heparan sulfate is abundantly present in the extracellular matrix. As other glycosaminoglycans, it is synthesized in the Golgi apparatus and then exposed on the cell surface. The glucuronidase activity of human heparanase plays a major role in the structural remodeling of the extracellular matrix, which underlies cell migration, hence tumor invasion. Heparanase is therefore a major target for anti-cancer treatment. Several inhibitors of its enzymatic activity have been synthesized. However, their design is limited by the absence of experimental structure of the protein. Homology modeling is proposed based on the structure of the endoxylanase from Penicillium simplicissimum co-crystallized with a series of xylan oligosaccharide. The new heparanase model is consistent with the few experimental data suited for the validation of such work. Furthermore, the presence of natural substrates in the template structure allowed us to propose a binding model for a hydrolyzed heparin sulfate pentasaccharide. Several lysine residues have been identified to play a key role in binding to the anionic polysaccharide substrate. In addition, two phenylalanine residues are also potentially important for the interaction with the substrate. The enzymatic mechanism investigated in the light of this new model allows for the proposal of several amino acids that can influence the protonation state of the nucleophile and the proton donor.

Molecular modeling of the interaction between heparan sulfate and cellular growth factors: bringing pieces together.

Heparan sulfate is a polysaccharide belonging to the glycaminoglycan family. It interacts with numerous proteins of the extracellular matrix, in particular cellular growth factors. The number of experimental protein-heparin sulfate complexes obtained by crystallography or nuclear magnetic resonance is limited. Alternatively, computational approaches can be employed. Generally, they restrain the conformation of the glycosidic rings and linkages in order to reduce the complexity of the problem. Modeling the interaction between protein and heparan sulfate is indeed challenging because of the large size of the fragment needed for a strong binding, the flexibility brought by the glycosidic rings and linkages and the high density of negative charges. We propose a two-step method based on molecular docking and molecular dynamics simulation. Molecular docking allows exploring the positioning of a rigid heparin sulfate fragment on the protein surface. Molecular dynamics refine selected docking models by explicitly representing solvent molecules and not restraining the polysaccharide backbone. The interaction of a hexamer of heparin sulfate was studied in interaction with fibroblast growth factor 2 and stromal cell-derived factor 1α. This approach shed light on the plasticity of the growth factors interacting with heparan sulfate. This approach can be extended to the study of other protein/glycosaminoglycan complexes.

Evaluation of the gene-specific dye bias in cDNA microarray experiments.

MOTIVATION: In cDNA microarray experiments all samples are labeled with either Cy3 or Cy5. Systematic and gene-specific dye bias effects have been observed in dual-color experiments. In contrast to systematic effects which can be corrected by a normalization method, the gene-specific dye bias is not completely suppressed and may alter the conclusions about the differentially expressed genes. METHODS: The gene-specific dye bias is taken into account using an analysis of variance model. We propose an index, named label bias index, to measure the gene-specific dye bias. It requires at least two self-self hybridization cDNA microarrays. RESULTS: After lowess normalization we have found that the gene-specific dye bias is the major source of experimental variability between replicates. The ratio (R/G) may exceed 2. As a consequence false positive genes may be found in direct comparison without dye-swap. The stability of this artifact and its consequences on gene variance and on direct or indirect comparisons are addressed. AVAILABILITY: http://www.inapg.inra.fr/ens_rech/mathinfo/recherche/mathematique

Spotting effect in microarray experiments.

BACKGROUND: Microarray data must be normalized because they suffer from multiple biases. We have identified a source of spatial experimental variability that significantly affects data obtained with Cy3/Cy5 spotted glass arrays. It yields a periodic pattern altering both signal (Cy3/Cy5 ratio) and intensity across the array. RESULTS: Using the variogram, a geostatistical tool, we characterized the observed variability, called here the spotting effect because it most probably arises during steps in the array printing procedure. CONCLUSIONS: The spotting effect is not appropriately corrected by current normalization methods, even by those addressing spatial variability. Importantly, the spotting effect may alter differential and clustering analysis.

Opposing roles of C/EBPbeta and AP-1 in the control of fibroblast proliferation and growth arrest-specific gene expression.

Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C/EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had a proliferative advantage over cells with one or two functional copies of this gene. C/EBP inhibition enhanced the expression of the three major components of AP-1 in cycling CEF, namely c-Jun, JunD, and Fra-2, and stimulated AP-1 activity. In contrast, the over-expression of C/EBPbeta caused a dramatic reduction in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF over-expressing c-Jun, and to a lesser extent JunD and Fra-2, did not growth arrest at high cell density and did not express p20K. Therefore, AP-1 interfered with the action of C/EBPbeta at high cell density, indicating that these factors play opposing roles in the control of GAS gene expression and CEF proliferation.