Molecular models of human P-glycoprotein in two different catalytic states

P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Cellular pharmacokinetics of telavancin, a novel lipoglycopeptide antibiotic, and analysis of lysosomal changes in cultured eukaryotic cells (J774 mouse macrophages and rat embryonic fibroblasts)

Background Telavancin is a lipoglycopeptide with multiple mechanisms of action that include membrane-destabilizing effects towards bacterial cells. It shows bactericidal activity against forms of Staphylococcus aureus (phagolysosomal infection) with different resistance phenotypes [methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus]. We examine here the uptake, efflux and intracellular distribution of telavancin in eukaryotic cells as well as its potential to induce lysosomal changes (in comparison with vancomycin and oritavancin). Methods J774 macrophages and rat embryo fibroblasts were exposed for up to 24 and 72 h to telavancin (5-90 mg/L). The following studies were performed: measurement of (14)C-labelled telavancin cellular uptake and subcellular distribution (cell fractionation), determination of pericellular membrane integrity (lactate dehydrogenase release), electron microscopy with morphometric analysis of changes in lysosome size and determination of total phospholipid and cholesterol content. Results The uptake of telavancin proceeded linearly as a function of time and concentration in both cell types (clearance rate of approximately 10 mL/g of protein/h). Efflux (macrophages) was approximately 5.7-fold slower. Telavancin subcellular distribution was superimposable on that of a lysosomal marker (N-acetyl-beta-hexosaminidase). It did not cause an increase in the release of lactate dehydrogenase and did not induce significant increases in total phospholipid or cholesterol content. It caused only mild morphological lysosomal alterations (similar to vancomycin and much less than oritavancin by morphometric analysis). Conclusions Telavancin is taken up by eukaryotic cells and localizes in lysosomes, causing mild morphological alterations without evidence of lipid metabolism alterations. These data support our observations that telavancin is active against intracellular S. aureus

Penicillin-binding Proteins (PBP) and Lmo0441 (a PBP-like protein) play a role in Beta-lactam sensitivity of Listeria monocytogenes

While seven penicillin-binding proteins (PBPs) or PBP-like proteins have been identified either by radiolabelled penicillin binding studies or genomic analysis, only PBP3 has been considered of interest for Beta-lactams activity against Listeria monocytogenes. Herein we reveal that both PBP4 and Lmo0441 (a PBP-like protein) play a direct role in cephalosporin activity in L. monocytogenes while PBP4 additionally has a protective affect against both penicillin and carbapenem

Antiretroviral-induced adverse drug reactions in HIV-infected patients in Mali: a resource-limited setting experience

Background: There are few reports in the literature from sub-Saharan Africa (SSA) regarding antiretroviral-induced adverse drug reactions (ADRs). Antiretroviral therapy (ART) is now widely available in SSA, and ADRs during HIV infection are also frequent. In this study, we reported the frequency and risk factors of ART-induced ADRs in a Malian population.Methods: This prospective cohort study was performed in the HIV Care and Counseling Centre (CESAC) of Mali from 2011 to 2012. Adult patients infected with HIV and who had recently started ART were included and followed-up clinically Were included in this study, adult patients living with HIV and had recently started ART who were followed up for at least 6 months to determine the incidence of ADRs using Naranjo’s classification scale.Results: During this study, 357 (42.3%) patients presented ADRs (40.1% of our patients (n=338) experienced at least one ADR, and 2.2% (n=19) experienced at least two ADRs). The prevalence of ADRs by organ system was: 45.9% neurological (n=164); 29.4% metabolic (blood chemistry) (n=105); 15.4% hematological (n=55). High probable rate of ADR was observed as indicated by the Naranjo score in 83.7% of the cases. Zidovudine (AZT) and stavudine (d4T) use was identified as a risk factor for either anaemia or peripheral neuropathy whereas nevirapine (NVP) and female gender were risk factors for skin reactions. Patients with advance disease had the highest rate of ADRs compared to the others.Conclusions: Based on the Naranjo probability scale, our data show that ADRs such as peripheral neuropathy and anemia are very frequent. These ADR was linked to AZT and D4T. Our findings highlight the need for active monitoring, continuous pharmacovigilance of ART and change of some ART drug in this population

Correlation between cytotoxicity induced by Pseudomonas aeruginosa clinical isolates from acute infections and IL-1β secretion in a model of human THP-1 monocytes

Type III secretion system (T3SS) in Pseudomonas aeruginosa is associated with poor clinical outcome in acute infections. T3SS allows for injection of bacterial exotoxins (e.g. ExoU or ExoS) into the host cell, causing cytotoxicity. It also activates the cytosolic NLRC4 inflammasome, activating caspase-1, inducing cytotoxicity and release of mature IL-1β, which impairs bacterial clearance. In addition, flagellum-mediated motility has been suggested to also modulate inflammasome response and IL-1β release. Yet the capacity of clinical isolates to induce IL-1β release and its relation with cytotoxicity have never been investigated. Using 20 clinical isolates from acute infections with variable T3SS expression levels and human monocytes, our aim was to correlate IL-1β release with toxin expression, flagellar motility and cytotoxicity. ExoU-producing isolates caused massive cell death but minimal release of IL-1β, while those expressing T3SS but not ExoU (i.e. expressing ExoS or no toxins) induced caspase-1 activation and IL-1β release, the level of which was correlated with cytotoxicity. Both effects were prevented by a specific caspase-1 inhibitor. Flagellar motility was not correlated with cytotoxicity or IL-1β release. No apoptosis was detected. Thus, T3SS cytotoxicity is accompanied by a modification in cytokine balance for P. aeruginosa clinical isolates that do not express Exo

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Naert, T., Tulkens, D., Edwards, N. A., Carron, M., Shaidani, N. I., Wlizla, M., Boel, A., Demuynck, S., Horb, M. E., Coucke, P., Willaert, A., Zorn, A. M., & Vleminckx, K. Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos. Scientific Reports, 10(1), (2020): 14662, doi:10.1038/s41598-020-71412-0.CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.Research in the Vleminckx laboratory is supported by the Research Foundation—Flanders (FWO-Vlaanderen) (Grants G0A1515N and G029413N), by the Belgian Science Policy (Interuniversity Attraction Poles—IAP7/07) and by the Concerted Research Actions from Ghent University (BOF15/GOA/011). Further support was obtained by the Hercules Foundation, Flanders (Grant AUGE/11/14) and the Desmoid Tumor Research Foundation and the Desmoid Tumour Foundation Canada. T.N. is funded by “Kom op tegen Kanker” (Stand up to Cancer), the Flemish cancer society and previously held PhD fellowship with VLAIO-HERMES during the course of this work. D.T. and M. C. hold a PhD fellowship from the Research Foundation-Flanders (FWO-Vlaanderen). The Zorn Lab is supported by Funding from NIH National Institute of Child Health and Human Development (NICHD) P01 HD093363. A.W. and A.B. are supported by the Ghent University (Universiteit Gent) Methusalem grant BOFMET2015000401 to Anne De Paepe. The National Xenopus Resource and Horb lab is supported by funding from the National Institutes of Health (P40 OD010997 and R01 HD084409)

The Putative De-N-acetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones

Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1–3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5–7 fold) and mexX (3.6–5.4 fold) in the parental strain but to a lower extent (3–4-fold for gyrA and 1.8–2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target

Nonclinical and pharmacokinetic assessments to evaluate the potential of tedizolid and linezolid to affect mitochondrial function

Prolonged treatment with the oxazolidinone linezolid is associated with myelosuppression, lactic acidosis, and neuropathies, toxicities likely caused by impairment of mitochondrial protein synthesis (MPS). To evaluate the potential of the novel oxazolidinone tedizolid to cause similar side effects, nonclinical and pharmacokinetic assessments were conducted. In isolated rat heart mitochondria, tedizolid inhibited MPS more potently than did linezolid (average [± standard error of the mean] 50% inhibitory concentration [IC50] for MPS of 0.31 ± 0.02 μM versus 6.4 ± 1.2 μM). However, a rigorous 9-month rat study comparing placebo and high-dose tedizolid (resulting in steady-state area under the plasma concentration-time curve values about 8-fold greater than those with the standard therapeutic dose in humans) showed no evidence of neuropathy. Additional studies explored why prolonged, high-dose tedizolid did not cause these mitochondriopathic side effects despite potent MPS inhibition by tedizolid. Murine macrophage (J774) cell fractionation studies found no evidence of a stable association of tedizolid with eukaryotic mitochondria. Monte Carlo simulations based on population pharmacokinetic models showed that over the course of a dosing interval using standard therapeutic doses, free plasma concentrations fell below the respective MPS IC50 in 84% of tedizolid-treated patients (for a median duration of 7.94 h) and 38% of linezolid-treated patients (for a median duration of 0 h). Therapeutic doses of tedizolid, but not linezolid, may therefore allow for mitochondrial recovery during antibacterial therapy. The overall results suggest that tedizolid has less potential to cause myelosuppression and neuropathy than that of linezolid during prolonged treatment courses. This, however, remains a hypothesis that must be confirmed in clinical studies