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Lrrk2 alleles modulate inflammation during microbial infection of mice in a sex-dependent manner
No full textVariants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson's disease, leprosy, and Crohn's disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson's disease-linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen
Bile acid analogues are activators of pyrin inflammasome
No full textthe work was first published Jan 2019 in JBC - doi: 10.1074/jbc.RA118.005103. (PMID: 30647128)
Since publication a represenative from the Lewis-Sigler Institute for Integrative Genomics reached out to inquire if we would publish the full CRISPR data from the paper including the gene-level scores and relevant significance thresholds for their CRISPR ORCS database (orcs.thebiogrid.org/). ORCS houses the most comprehensive collection of gene-level scores from published CRISPR screens and makes them freely available to the research community.. For more information about BioGRID ORCS, please consult our recent paper (PMID: 30476227)
Target (MexB) and efflux based mechanisms decreasing the effectiveness of the efflux pump inhibitor D13-9001 in P. aeruginosa PAO1: uncovering a new role for MexMN-OprM in efflux of β-lactams and a novel regulatory circuit controlling MexMN expression
No full textEfflux pumps contribute to antibiotic resistance in Gram-negative pathogens.
Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-
9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased
susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these
fell into two categories: those with alterations in the target MexB (F628L and ΔV177)
and those with an alteration in a putative sensor kinase of unknown function, PA1438
(L172P). The alterations in MexB were consistent with reported structural studies of the
D13-9001 interaction with MexB. The PA1438L172P alteration mediated a �150-fold upregulation
of MexMN pump gene expression and a �50-fold upregulation of PA1438
and the neighboring response regulator gene, PA1437. We propose that these be renamed
mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN
was shown to partner with the outer membrane channel protein OprM and to pump
several �-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced
MexAB-OprM to efflux these compounds but was insusceptible to inhibition by
D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem
that was independent of MexMN-OprM. Expression of oprD, encoding the uptake
channel for these compounds, was downregulated, suggesting that this channel is also
part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding
MmnSL172P revealed, among other things, an interrelationship between the regulation of
mexMN and genes involved in heavy metal resistance
Discovery of a ZIP7 Inhibitor from a Notch Pathway Screen
No full textThe identification of activating mutations in NOTCH1 in over 50% of T-cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the Notch signaling pathway in this disease. The receptors and ligands of the Notch signaling pathway must be trafficked to the cell surface where they interact and activate signaling. A phenotypic screen for inhibitors of the Notch signaling pathway identified compounds that interfere with trafficking of Notch to the cell surface, and induce apoptosis in T-ALL via an ER stress mechanism. Comprehensive target identification approaches revealed a role for SLC39A7 / ZIP7, a Zrt-, Irt-like protein zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, but not the cytosol, suggesting ER zinc homeostasis is critical to both Notch signaling and ER stress. A diazirine-containing analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. The ZIP and the Zn transporter (ZnT) family of zinc transporters are found in all aspects of life, and both families of transporters play critical roles in cellular and physiological functions. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and to further investigate ZIP7 as a novel druggable node in the Notch pathway
Total synthesis of diaportheone A
No full textDiaportheone A (1), a chromone natural product was previously isolated from the endophytic fungi Diaporthe sp. P 133. Its structure was established by spectroscopic methods, however, its absolute configuration remained undefined. This study dealt on the total synthesis of diaportheone A (1) utilizing the cyclization and in situ thermal syn-elimination of a β-ketosulfoxide. The C-1R absolute configuration of the natural product was established by X-ray crystallography of the synthetic diaportheone A (1) (>99% ee) and comparison with the optical rotation
Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab fragment of NISTmAb
No full textHydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A) from fifteen laboratories. Laboratories reported ≈ 89,800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (≈ 78,900 centroids), giving ≈ 100 % coverage, and ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87 %) exhibited centroid mass laboratory repeatability precisions of 〈 sLab 〉 ≤ (0.15 ± 0.01) Da (1σx ̅ ), and all laboratories achieved 〈 sLab 〉 ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) oC and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Labs ( tHDX ) = (9.0 ± 0.9) % (1σ). A 9 laboratory cohort that immersed samples at THDX = 25 oC exhibited reproducibility of σreproducibility25C cohort ( tHDX ) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements
The BACE inhibitor NB-360 in preclinical models: From β-amyloid reduction to downstream disease-relevant effects
No full textInhibition of Beta-site- APP Cleaving Enzyme-1 (BACE-1) is a current approach to fight the β-amyloid (Aβ) deposition in the brains of patients with Alzheimer’s disease, and a number of BACE-1 inhibitors are currently being tested in clinical trials. The BACE-1 inhibitor NB-360, although not a clinical compound, turned out as a valuable pharmacological tool to investigate the effects of BACE-1 inhibition on the deposition of different Aβ species in APP transgenic mice. Furthermore, chronic animal studies with NB-360 discovered relationships between BACE-1 inhibition, Aβ deposition, and Aβ-related downstream effects on neuroinflammation, neuronal function and markers of neurodegeneration. NB-360 effects on the processing of physiological BACE-1 substrates as well as on non-enzymatic BACE-1 functions have been investigated and complement studies performed with BACE-1 knock-out mice. Since NB-360 is also an inhibitor for BACE-2, non-clinical studies revealed physiological effects of BACE-2 inhibition
Ontology mapping for semantically enabled applications
No full textIn this review, we provide a summary of recent progress in ontology mapping (OM) at a crucial time when biomedical research is under a deluge of an increasing amount and variety of data. This is particularly important for realising the full potential of semantically enabled or enriched applications and for meaningful insights, such as drug discovery, using machine-learning technologies. We discuss challenges and solutions for better ontology mappings, as well as how to select ontologies before their application. In addition, we describe tools and algorithms for ontology mapping, including evaluation of tool capability and quality of mappings. Finally, we outline the requirements for an ontology mapping service (OMS) and the progress being made towards implementation of such sustainable services
Oncostatin M reduces atherosclerosis development in APOE3Leiden.CETP mice and is associated with increased survival probability in humans
No full textObjective Previous studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied. Approach and results Gene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457). Conclusions Chronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect
Examining P-gp efflux kinetics guided by the BDDCS – Rational selection of in vitro assay designs and mathematical models
No full textThe generation of reliable kinetic parameters to describe P-glycoprotein (P-gp) activity is essential for predicting the impact of efflux transport on gastrointestinal drug absorption. The compound-specific selection of in vitro assay designs and ensuing data analysis methods is explored in this manuscript. We measured transcellular permeability and cellular uptake of five P-gp substrates in Caco-2 and LLC-PK1 MDR1 cells. Kinetic parameters of P-gp-mediated efflux transport (Km, Vmax) were derived from conventional and mechanistic compartmental models. The estimated apparent Km values based on medium concentrations in the conventional permeability model indicated significant differences between the cell lines. The respective intrinsic Km values based on unbound intracellular concentrations in the mechanistic compartmental models were significantly lower and comparable between cell lines and assay formats. Non-specific binding or lysosomal trapping were shown to cause discrepancies in the kinetic parameters obtained from different assay formats. A guidance for the selection of in vitro assays and kinetic assessment methods is proposed in line with the Biopharmaceutics Drug Disposition Classification System (BDDCS). The recommendations are expected to aid the acquisition of robust and reproducible kinetic parameters of P-gp-mediated efflux transport