Biology subjects

Green, K.

Publications and source records attributed to Green, K..

24 records · Page 2Linked to original sources

bioRxiv2019

Donor monocyte-derived macrophages promote human acute graft versus host disease

Myelopoiesis is invariably present, and contributes to pathology, in animal models of graft versus host disease (GVHD). In humans, a rich inflammatory infiltrate bearing macrophage markers has also been described in histological studies. In order to determine the origin, functional properties and role in pathogenesis of these cells, we isolated single cell suspensions from acute cutaneous GVHD and subjected them to genotype, transcriptome and in vitro functional analysis. A donor-derived population of CD11c+CD14+ cells was the dominant population of all leukocytes in GVHD. Surface phenotype and nanostring gene expression profiling indicated the closest steady-state counterpart of these cells to be monocyte-derived macrophages. In GVHD, however, there was upregulation of monocyte antigens SIRP and S100A8/9, and transcripts associated with leukocyte trafficking, pattern recognition, antigen presentation, and co-stimulation. Isolated GVHD macrophages stimulated greater proliferation and activation of allogeneic T cells, and secreted higher levels of inflammatory cytokines than their steady-state counterparts. In HLA-matched mixed leukocyte reactions, we also observed differentiation of activated macrophages with a similar phenotype. These exhibited cytopathicity to a cell line and mediated pathological damage to skin explants, independently of T cells. Together, these results define the origin, functional properties and potential pathogenic roles of human GVHD macrophages.

immunology↗

bioRxiv2019

PK-DB: PharmacoKinetics DataBase for Individualized and Stratified Computational Modeling

A multitude of pharmacokinetics studies have been published. However, due to the lack of an open database, pharmacokinetics data, as well as the corresponding meta-information, have been difficult to access. We present PK-DB (https://pk-db.com), an open database for pharmacokinetics information from clinical trials including pre-clinical research. PK-DB provides curated information on (i) characteristics of studied patient cohorts and subjects (e.g. age, bodyweight, smoking status); (ii) applied interventions (e.g. dosing, substance, route of application); (iii) measured pharmacokinetic time-courses; (iv) pharmacokinetic parameters (e.g. clearance, half-life, area under the curve). Key features are the representation of experimental errors, the normalization of measurement units, annotation of information to biological ontologies, calculation of pharmacokinetic parameters from concentration-time profiles, a workflow for collaborative data curation, strong validation rules on the data, computational access via a REST API as well as human access via a web interface. PK-DB enables meta-analysis based on data from multiple studies and data integration with computational models. A special focus lies on meta-data relevant for individualized and stratified computational modeling with methods like physiologically based pharmacokinetic (PBPK), pharmacokinetic/pharmacodynamic (PK/DB), or population pharmacokinetic (pop PK) modeling.

bioinformatics↗

bioRxiv2019

Decoding the development of the blood and immune systems during human fetal liver haematopoiesis

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs), yet remains poorly defined in humans. Using single cell transcriptome profiling of ~133,000 fetal liver and ~65,000 fetal skin and kidney cells, we identify the repertoire of blood and immune cells in first and early second trimesters of development. From this data, we infer differentiation trajectories from HSC/MPPs, and evaluate the impact of tissue microenvironment on blood and immune cell development. We predict coupling of mast cell differentiation with erythro-megakaryopoiesis and identify physiological erythropoiesis in fetal skin. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for understanding and harnessing the therapeutic potential of HSC/MPPs.

developmental biology↗

bioRxiv2019

A low-cost and high-precision scanning electrochemical microscope built with open source tools

A low-cost Scanning Electrochemical Microscope (SECM) was built with a 0.6 pA current measurement capability potentiostat and submicron resolution motorized stage, using open source software and hardware tools. The high performance potentiostat with a Python graphical user interface was built based on an open source project. Arduino boards, stepper motors, a manual XY micromanipulator stage, 3D printed couplers and gears were used in building the motorized stage. An open source motor control software was used for moving the motorized stage with high precision. An inverted microscope was utilized for viewing a standard microelectrode while scanning. The setup was tested in the formation of a map of electrochemical signals from an array of pores on a parafilm membrane. As the setup will be used in future biosensing experiments, DNA hybridization detection experiments were also performed with the setup.

biochemistry↗

bioRxiv2017

Alleviation of Al toxicity by Si is associated with the formation of Al-Si complexes in root tissues of sorghum

Silicon is reported to reduce the toxic effects of Al on root elongation but the in planta mechanism by which this occurs remains unclear. Using seedlings of soybean (Glycine max) and sorghum (Sorghum bicolor), we examined the effect of up to 2 mM Si on root elongation rate (RER) in Al-toxic nutrient solutions. Synchrotron-based low energy X-ray fluorescence (LEXRF) was then used for the in situ examination of the distribution of Al and Si within cross-sections cut from the apical tissues of sorghum roots. The addition of Si potentially increased RER in Al-toxic solutions, with RER being up to ca. 0.3 mm h-1 (14 %) higher for soybean and ca. 0.2 mm h-1 (17 %) higher for sorghum relative to solutions without added Si. This improvement in RER could not be attributed to a change in Al-chemistry of the bulk nutrient solution, nor was it due to a change in the concentration of Al within the apical (0-10 mm) root tissues. Using LEXRF to examine sorghum, it was demonstrated that in roots exposed to both Al and Si, much of the Al was co-located with Si in the mucigel and outer apoplast. These observations suggest that Si reduces the toxicity of Al in planta through formation of Al-Si complexes in mucigel and outer cellular tissues, thereby decreasing the binding of Al to the cell wall where it is known to inhibit wall loosening as required for cell elongation.

plant biology↗

bioRxiv2017

An improved method for the expression screening of membrane protein-GFP fusions in yeast

The expression and purification of membrane proteins is an extremely challenging area of work within Protein Science. Membrane proteins are required for compound screening and structure determination in industry. Here we describe some new and innovative methodology in developing the membrane protein GFP fusion primary expression screening in yeast. This methodology enables the expression of membrane proteins fused to GFP in both Saccharomyces cerevisiae and Pichia pastoris systems. This capability helps facilitate screening of constructs to establish which are suitable for membrane protein production for compound screening and structure determination\n\nIn terms of the primary screening work, we have developed both agar plate and liquid plate expression methodology in yeast. The two approaches correspond well, but the agar plate method is more rapid and we have shown it to have the advantage of allowing cells to be taken directly into confocal microscopy for immediate cell localisation data. Innovative work to extend the methanol induction time in the Pichia agar plate method established good differentiation from the background. A novel agar plate method was also developed for S.cerevisiae which is also presented. These screening methods allow triaging of constructs for either membrane protein preps for biochemical assays or progression to fluorescence size exclusion chromatography; where various detergents can be screened to determine the most appropriate for membrane protein solubilisation, the starting point for purification, crystallisation and structure determination.\n\nMembrane targets depicted to demonstrate the improved primary screening methodology are a copper transporter Ctr1p from S.cerevisiae and a water transporter Aqp4 from human origin.\n\nHighlightsAn improved method for the production of recombinant MP-GFP fusions in yeast is presented using agar plates.\n\nAn agar plate method for MP-GFP expression screening is described for Pichia pastoris, with improved induction methodology by the simple addition of methanol, allowing longer induction times for expression clarity.\n\nA new simple rapid agar plate method for MP-GFP expression screening is described for Saccharomyces cerevisiae.\n\nCells can be taken directly from agar plates into confocal microscopy studies for immediate cell localisation data and triaging.\n\nLiquid plate based screening methods are also described for both yeasts in comparison, to show there is corresponding data, helping validate the new agar plate methods.

biochemistry↗