Acetaminophen (APAP) is a safe analgesic and antipyretic drug. However, APAP overdose leads to massive hepatocyte death. Cell death during APAP toxicity occurs by oncotic necrosis, in which the ...release of intracellular contents can elicit a reactive inflammatory response. We have previously demonstrated that an intravascular gradient of chemokines and mitochondria‐derived formyl peptides collaborate to guide neutrophils to sites of liver necrosis by CXC chemokine receptor 2 (CXCR2) and formyl peptide receptor 1 (FPR1), respectively. Here, we investigated the role of CXCR2 chemokines and mitochondrial products during APAP‐induced liver injury and in liver neutrophil influx and hepatotoxicity. During APAP overdose, neutrophils accumulated into the liver, and blockage of neutrophil infiltration by anti–granulocyte receptor 1 depletion or combined CXCR2‐FPR1 antagonism significantly prevented hepatotoxicity. In agreement with our in vivo data, isolated human neutrophils were cytotoxic to HepG2 cells when cocultured, and the mechanism of neutrophil killing was dependent on direct contact with HepG2 cells and the CXCR2‐FPR1–signaling pathway. Also, in mice and humans, serum levels of both mitochondrial DNA (mitDNA) and CXCR2 chemokines were higher during acute liver injury, suggesting that necrosis products may reach remote organs through the circulation, leading to a systemic inflammatory response. Accordingly, APAP‐treated mice exhibited marked systemic inflammation and lung injury, which was prevented by CXCR2‐FPR1 blockage and Toll‐like receptor 9 (TLR9) absence (TLR9−/− mice). Conclusion: Chemokines and mitochondrial products (e.g., formyl peptides and mitDNA) collaborate in neutrophil‐mediated injury and systemic inflammation during acute liver failure. Hepatocyte death is amplified by liver neutrophil infiltration, and the release of necrotic products into the circulation may trigger a systemic inflammatory response and remote lung injury. (HEPATOLOGY 2012;56:1971–1982)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
OBJECTIVES:We recently described how spontaneous effort during mechanical ventilation can cause “pendelluft,” that is, displacement of gas from nondependent (more recruited) lung to dependent (less ...recruited) lung during early inspiration. Such transfer depends on the coexistence of more recruited (source) liquid-like lung regions together with less recruited (target) solid-like lung regions. Pendelluft may improve gas exchange, but because of tidal recruitment, it may also contribute to injury. We hypothesize that higher positive end-expiratory pressure levels decrease the propensity to pendelluft and that with lower positive end-expiratory pressure levels, pendelluft is associated with improved gas exchange but increased tidal recruitment.
DESIGN:Crossover design.
SETTING:University animal research laboratory.
SUBJECTS:Anesthetized landrace pigs.
INTERVENTIONS:Surfactant depletion was achieved by saline lavage in anesthetized pigs, and ventilator-induced lung injury was produced by ventilation with high tidal volume and low positive end-expiratory pressure. Ventilation was continued in each of four conditionspositive end-expiratory pressure (low or optimized positive end-expiratory pressure after recruitment) and spontaneous breathing (present or absent). Tidal recruitment was assessed using dynamic CT and regional ventilation/perfusion using electric impedance tomography. Esophageal pressure was measured using an esophageal balloon manometer.
MEASUREMENTS AND RESULTS:Among the four conditions, spontaneous breathing at low positive end-expiratory pressure not only caused the largest degree of pendelluft, which was associated with improved ventilation/perfusion matching and oxygenation, but also generated the greatest tidal recruitment. At low positive end-expiratory pressure, paralysis worsened oxygenation but reduced tidal recruitment. Optimized positive end-expiratory pressure decreased the magnitude of spontaneous efforts (measured by esophageal pressure) despite using less sedation, from –5.6 ± 1.3 to –2.0 ± 0.7 cm H2O, while concomitantly reducing pendelluft and tidal recruitment. No pendelluft was observed in the absence of spontaneous effort.
CONCLUSIONS:Spontaneous effort at low positive end-expiratory pressure improved oxygenation but promoted tidal recruitment associated with pendelluft. Optimized positive end-expiratory pressure (set after lung recruitment) may reverse the harmful effects of spontaneous breathing by reducing inspiratory effort, pendelluft, and tidal recruitment.
Abstract
The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan ...sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-
O
or 6-
O
sulfate groups than on
N
-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the
Coronaviridae
.
Background and Purpose
Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID‐19. Heparin (delivered systemically) is currently used to treat anticoagulant ...anomalies in COVID‐19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID‐19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS‐CoV‐2, in vitro, is needed.
Experimental Approach
Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS‐CoV‐2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined.
Key Results
All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150‐fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor.
Conclusion and Implications
This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS‐CoV‐2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID‐19.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
To identify drugs that could potentially be used to treat infection with SARS-CoV-2, a high throughput 384-well assay was developed to measure the binding of the receptor binding domain (RBD) of the ...viral S1 protein to its main receptor, angiotensin converting enzyme 2 (ACE2). The RBD was fused to both a HiBIT tag and an IL6 secretion signal to enable facile collection from the cell culture media. The addition of culture media containing this protein, termed HiBIT-RBD, to cells expressing ACE2 led to binding that was specific to ACE2 and both time and concentration dependant, Binding could be inhibited by both RBD expressed in E. coli and by a full length S1 - Fc fusion protein (Fc-fused S1) expressed in eukaryotic cells. The mutation of residues that are known to play a role in the interaction of RBD with ACE2 also reduced binding. This assay may be used to identify drugs which inhibit the viral uptake into cells mediated by binding to ACE2.
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1.A high-throughput, 384-well plate assay was developed to measure the binding S1 RBD to ACE2;2.HiBIT-RBD binds to cells expressing ACE2 specifically and in a time dependant fashion;3.The binding of HiBIT-RBD to ACE2 can be inhibited using recombinantly expressed SARS-CoV-2 RBD and full-length, Spike S1;4.Site specific mutations within the RBD demonstrate the specificity of this assay.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be ...viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed.
Sulfur containing glycosides offer an exciting prospect for inclusion within noncanonical glycan sequences, particularly as enabling probes for chemical glycobiology and for carbohydrate-based ...therapeutic development. In this context, we required access to 4-thio-d-glucopyranose and sought its chemical synthesis. Unable to isolate this material in homogenous form, we observed instead a thermodynamic preference for interconversion of the pyranose to 4-thio-d-glucofuranose. Accordingly, we present an improved method to access both bis(4-thio-d-glucopyranoside)-4,4′-disulfide and 4-thio-d-glucofuranose from a single precursor, demonstrating that the latter compound can be accessed from the former using a dithiothreitol controlled reduction of the disulfide. The dithiothreitol-mediated interconversion between pyranose (monomer and disulfide) and furanose forms for this thiosugar is monitored by 1H NMR spectroscopy over a 24-h period. Access to these materials will support accessing sulfur-containing mimetics of glucose and derivatives therefrom, such as sugar nucleotides.
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•Access to 4-thio-glucopyranose disulfide.•DTT controlled release and interconversion to 4-thio-glucofuranose.•1H NMR time course experiments monitoring pyranose to furanose interconversion.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this paper we analyze the descriptive statistics of the Google search volume for the terms related to the National Reserve of Copper and Associates (RENCA), a Brazilian mineral reserve in the ...Amazon of 4.6 million hectares, before and after the government signed the decree releasing it for exploration. First, we analyze the volume of searches for expressions related to RENCA in Google Trends using descriptive statistics; second, we assess the cross-correlation coefficient rho.sub.DCCA, which measures the cross-correlation between two nonstationary time series across different time scales. After the government announced the release of the RENCA reserve, there was an increase in the average volume of Google searches for related terms, showing people's concern about the announcement. By using the cross-correlation coefficient rho.sub.DCCA, we identify strong cross-correlations between the different expressions related to RENCA in Google Trends. Our work shows the utility of Google Trends as an indicator of the perception of environmental policies. Additionally, we show that rho.sub.DCCA can be used as a tool to measure the cross-correlation between synonyms extracted from Google Trends for various time scales.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2 •–). O2 •– reacts at diffusional rates with nitric oxide to yield a potent ...nitrating species, peroxynitrite anion (ONOO–). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a k of ∼1.0 × 105 M–1 s–1 for the reaction of hMnSOD with ONOO– by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO–, including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of k was performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO– at a rate of ∼1 × 106 M–1 s–1 to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k of ∼1.0 × 105 M–1 s–1, fully consistent with the direct method. Proteomic analysis indicated that ONOO–, but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO– with MnIII to yield MnIV and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO– and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.
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IJS, KILJ, NUK, PNG, UL, UM
Cardiac rhythms are related to heart electrical activity, being an essential aspect of the cardiovascular physiology. Usually, these rhythms are represented by electrocardiograms (ECGs) that are ...useful to detect cardiac pathologies. This paper investigates the control of cardiac rhythms in order to induce normal rhythms from pathological responses. The strategy is based on the electrocardiograms and considers different pathologies. An intelligent controller is proposed considering the ECG as the observable variable. In order to allow the assessment of the control performance, synthetic ECGs are produced from a reduced-order mathematical model that presents close agreement with experimental measurements. The adopted model comprises a network of oscillators formed by sinoatrial node, atrioventricular node and His-Purkinje complex. Three nonlinear oscillators are employed to represent each one of these nodes that are connected by delayed couplings. The controller considers the control variable at the His-Purkinje complex. To evaluate the ability of the control law to deal with both intra- and interpatient variability, the heart model is assumed to be not available to the controller designer, being used only in the simulator to assess the control performance. The incorporation of artificial neural networks into a Lyapunov-based control scheme, however, allows the presented intelligent approach to compensate for unknown cardiac dynamics. Results show that abnormal rhythms can be avoided by applying the proposed control scheme, turning the electrocardiogram closer to the expected normal behavior and preventing critical cardiac responses.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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