A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material

This is the final version. Available from Springer via the DOI in this record.The fraction of intact monomer in a sample (moles/moles), the monomeric purity, is measured as a quality control in therapeutic monoclonal antibodies but is often unknown in research samples and remains a major source of variation in quantitative antibody-based techniques such as immunoassay development. Here, we describe a novel multiplex technique for estimating the monomeric purity and antigen affinity of research grade antibody samples. Light scattering was used to simultaneously observe the mass of antibody binding to biosensor surfaces functionalised with antigen (revealing Fab binding kinetics) or protein A/G (PAG). Initial estimates of monomeric purity in 7 antibody samples including a therapeutic infliximab biosimilar were estimated by observing a mass deficit on the PAG surface compared to the NISTmAb standard of high monomeric purity. Monomeric purity estimates were improved in a second step by observing the mass of antigen binding to the mass of antibody on the PAG surface. The NISTmAb and infliximab biosimilar displayed tightly controlled stoichiometries for antigen binding of 1.31 ± 0.57 and 1.71 ± 0.16 (95% confidence interval)—within the theoretical limit of 1–2 antigens per antibody depending on avidity. The other antibodies in the panel displayed antigen binding stoichiometries in the range 0.06–1.15, attributed to lower monomeric purity. The monomeric purity estimates were verified by electrospray ionization mass spectrometry (ESI), the gold standard technique for structural characterization of antibodies. ESI data indicated that the NISTmAb and infliximab biosimilar samples had monomeric purity values of 93.5% and 94.7%, respectively, whilst the research grade samples were significantly lower (54–89%). Our results demonstrate rapid quality control testing for monomeric purity of antibody samples (< 15 min) which could improve the reproducibility of antibody-based experiments.EPSR

Direct activation of NADPH oxidase 2 by 2-deoxyribose-1-phosphate triggers nuclear factor kappa B-dependent angiogenesis.

AbstractAims: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells.Results: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2−/− mice.Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex.Conclusions: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110–130

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine-type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine

Deep sequencing of the Mexican avocado transcriptome, an ancient angiosperm with a high content of fatty acids

Background: Avocado (Persea americana) is an economically important tropical fruit considered to be a good source of fatty acids. Despite its importance, the molecular and cellular characterization of biochemical and developmental processes in avocado is limited due to the lack of transcriptome and genomic information. Results: The transcriptomes of seeds, roots, stems, leaves, aerial buds and flowers were determined using different sequencing platforms. Additionally, the transcriptomes of three different stages of fruit ripening (pre-climacteric, climacteric and post-climacteric) were also analyzed. The analysis of the RNAseqatlas presented here reveals strong differences in gene expression patterns between different organs, especially between root and flower, but also reveals similarities among the gene expression patterns in other organs, such as stem, leaves and aerial buds (vegetative organs) or seed and fruit (storage organs). Important regulators, functional categories, and differentially expressed genes involved in avocado fruit ripening were identified. Additionally, to demonstrate the utility of the avocado gene expression atlas, we investigated the expression patterns of genes implicated in fatty acid metabolism and fruit ripening. Conclusions: A description of transcriptomic changes occurring during fruit ripening was obtained in Mexican avocado, contributing to a dynamic view of the expression patterns of genes involved in fatty acid biosynthesis and the fruit ripening process

Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors

BACKGROUND: Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce. RESULTS: We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features. CONCLUSIONS: These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.This work was supported by the Gatsby Charitable Foundation (RG62472), by the Royal Society (RG69135) and by the European Research Council (ERC-2014-STG, H2020, 637537)

Ekonomiskt utsatta barn : - en kvalitativ studie om fattigdomens påverkan på barns vardag

Syftet med detta lärdomsprov är att granska hur fattigdom kan påverka barns vardag. De mest centrala frågeställningarna i detta arbete är: Hur påverkas barn av fattigdom? Vilka följder medför fattigdom för barn och vilka tecken finns det på att barn kommer från familjer med olika ekonomiska resurser? Teoridelen behandlar socialpolitiken i Finland, familjens situation, samt vad fattigdom kan innebära för barnet. Som undersökningsmetod använde jag mig av kvalitativa intervjuer. I intervjuerna deltog dagvårdspersonal sammanlagt fyra stycken från två olika daghem i samma kommun. Intervjuerna gjordes både i grupp och som individuella intervjuer. Undersökningen visade att fattigdomen har en påverkan på barnet, men hur mycket ett barn påverkas av fattigdomens effekter kan dock vara individuellt. Barnets upplevelser av fattigdomens effekter kan bero på barnets ålder eller på graden av fattigdom.The aim with this thesis is to examine how poverty can affect children´s daily life. The most central questions of this thesis are: How does poverty affect children? What are the consequences of poverty? What are the signs which reflect if a child comes from a family with limited economical resources? The theoretical part describes social politics in Finland, family life and what poverty can mean for children. The study was carried out through qualitative interviews. Four people working in two different kindergartens in the same municipality participated. The interviews were conducted both in a group and individually. The study showed that poverty affects children, but that degree of influence is individual, depending on the degree of poverty and the age of the children

Exploring inhibition of Pdx1, a component of the PLP synthase complex of the human malaria parasite Plasmodium falciparum

Malaria tropica is a devastating infectious disease caused by Plasmodium falciparum. This parasite synthesizes vitamin B6 de novo via the pyridoxal 5-phosphate (PLP) synthase enzymatic complex consisting of PfPdx1 and PfPdx2 proteins. Biosynthesis of PLP is largely performed by PfPdx1, ammonia provided by PfPdx2 subunits, is condensed together with Dribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P). PfPdx1 accommodates both the R5P and G3P substrates and intricately coordinates the reaction mechanism, which is composed of a series of imine bond formations, leading to the production of PLP. We demonstrate that D-erythrose 4-phosphate (E4P) inhibits PfPdx1 in a dose dependent manner. We propose that the acyclic phospho-sugar E4P, with a C1 aldehyde group similar to acyclic R5P, could interfere with R5P imine bond formations in the PfPdx1 reaction mechanism. Molecular docking and subsequent screening identified the E4P hydrazide analogue, 4- phospho-D-erythronhydrazide (4PEHz), which selectively inhibited PfPdx1 with an IC50 of 43 μM. PfPdx1 contained in the heteromeric PLP synthase complex was shown to be more sensitive to 4PEHz and was inhibited with an IC50 of 16 μM. Moreover, the compound had an IC50 value of 10 μM against cultured P. falciparum intraerythrocytic parasites. To further analyse the selectivity of 4PEHz, transgenic cell lines over-expressing PfPdx1 and PfPdx2 showed that additional copies of the protein complex conferred protection against 4PEHz, implicating that the PLP synthase is directly affected by 4EPHz in vivo. These PfPdx1 inhibitors represent novel lead scaffolds which are capable of targeting PLP biosynthesis, and we propose this as a viable strategy for the development of newer therapeutics against malaria.This work was funded by the grants [WR 124/2] and [WR 124/3] from the Deutsche Forschungsgemeinschaft (DFG) to CW as well as the National Research Foundation (NRF) of South Africa [65876] and NRF-DFG Scientific exchange grants to LMB, IM and CW. SBR was supported by the PhD sandwich programme of the German Academic Exchange Programme (DAAD) [A/08/99008]. CW is supported by grant [2009/54325-2] from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) within the “Jovem Pesquisador” programme.http://www.biochemj.org/bj/default.ht

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

International audienceSubstrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the $Arabidopsis\ thaliana$ enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I$_{320}$ intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand $\beta$6 of the Pdx1 ($\beta \alpha$)$_8$-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 ${\AA}$ between substrate- and product-binding sites