Timescale analysis of a mathematical model of acetaminophen metabolism and toxicity

Acetaminophen is a widespread and commonly used painkiller all over the world. However, it can cause liver damage when taken in large doses or at repeated chronic doses. Current models of acetaminophen metabolism are complex, and limited to numerical investigation though provide results that represent clinical investigation well. We derive a mathematical model based on mass action laws aimed at capturing the main dynamics of acetaminophen metabolism, in particular the contrast between normal and overdose cases, whilst remaining simple enough for detailed mathematical analysis that can identify key parameters and quantify their role in liver toxicity. We use singular perturbation analysis to separate the different timescales describing the sequence of events in acetaminophen metabolism, systematically identifying which parameters dominate during each of the successive stages. Using this approach we determined, in terms of the model parameters, the critical dose between safe and overdose cases, timescales for exhaustion and regeneration of important cofactors for acetaminophen metabolism and total toxin accumulation as a fraction of initial dose

Functionality of primary hepatic non-parenchymal cells in a 3D spheroid model and contribution to acetaminophen hepatotoxicity.

In addition to hepatocytes, the liver comprises a host of specialised non-parenchymal cells which are important to consider in the development of in vitro models which are both physiologically and toxicologically relevant. We have characterized a 3D co-culture system comprising primary human hepatocytes (PHH) and non-parenchymal cells (NPC) and applied it to the investigation of acetaminophen-induced toxicity. Firstly, we titrated ratios of PHH:NPC and confirmed the presence of functional NPCs via both immunohistochemistry and activation with both LPS and TGF-β. Based on these data we selected a ratio of 2:1 PHH:NPC for further studies. We observed that spheroids supplemented with NPCs were protected against acetaminophen (APAP) toxicity as determined by ATP (up to threefold difference in EC50 at day 14 compared to hepatocytes alone) and glutathione depletion, as well as miR-122 release. APAP metabolism was also altered in the presence of NPCs, with significantly lower levels of APAP-GSH detected. Expression of several CYP450 enzymes involved in the bioactivation of APAP was also lower in NPC-containing spheroids. Spheroids containing NPCs also expressed higher levels of miRNAs which have been implicated in APAP-induced hepatotoxicity, including miR-382 and miR-155 which have potential roles in liver regeneration and inflammation, respectively. These data indicate that the interaction between hepatocytes and NPCs can have significant metabolic and toxicological consequences important for the correct elucidation of hepatic safety mechanisms

Neural changes following cognitive behaviour therapy for psychosis: a longitudinal study

A growing body of evidence demonstrates that persistent positive symptoms, particularly delusions, can be improved by cognitive behaviour therapy for psychosis. Heightened perception and processing of threat are believed to constitute the genesis of delusions. The present study aimed to examine functional brain changes following cognitive behaviour therapy for psychosis. The study involved 56 outpatients with one or more persistent positive distressing symptoms of schizophrenia. Twenty-eight patients receiving cognitive behaviour therapy for psychosis for 6–8 months in addition to their usual treatment were matched with 28 patients receiving treatment as usual. Patients’ symptoms were assessed by a rater blind to treatment group, and they underwent functional magnetic resonance imaging during an affect processing task at baseline and end of treatment follow-up. The two groups were comparable at baseline in terms of clinical and demographic parameters and neural and behavioural responses to facial and control stimuli. The cognitive behaviour therapy for psychosis with treatment-as-usual group (22 subjects) showed significant clinical improvement compared with the treatment-as-usual group (16 subjects), which showed no change at follow-up. The cognitive behaviour therapy for psychosis with treatment-as-usual group, but not the treatment-as-usual group, showed decreased activation of the inferior frontal, insula, thalamus, putamen and occipital areas to fearful and angry expressions at treatment follow-up compared with baseline. Reduction of functional magnetic resonance imaging response during angry expressions correlated directly with symptom improvement. This study provides the first evidence that cognitive behaviour therapy for psychosis attenuates brain responses to threatening stimuli and suggests that cognitive behaviour therapy for psychosis may mediate symptom reduction by promoting processing of threats in a less distressing way

On the Nature of the X-ray Emission from the Ultraluminous X-ray Source, M33 X-8: New Constraints from NuSTAR and XMM-Newton

We present nearly simultaneous NuSTAR and XMM-Newton observations of the nearby (832 kpc) ultraluminous X-ray source (ULX) M33 X-8. M33 X-8 has a 0.3-10 keV luminosity of LX ~ 1.4 x 10^39 erg/s, near the boundary of the "ultraluminous" classification, making it an important source for understanding the link between typical Galactic X-ray binaries and ULXs. Past studies have shown that the 0.3-10 keV spectrum of X-8 can be characterized using an advection-dominated accretion disk model. We find that when fitting to our NuSTAR and XMM-Newton observations, an additional high-energy (>10 keV) Comptonization component is required, which allows us to rule out single advection-dominated disk and classical sub-Eddington models. With our new constraints, we analyze XMM-Newton data taken over the last 17 years to show that small (~30%) variations in the 0.3-10 keV flux of M33 X-8 result in spectral changes similar to those observed for other ULXs. The two most likely phenomenological scenarios suggested by the data are degenerate in terms of constraining the nature of the accreting compact object (i.e., black hole versus neutron star). We further present a search for pulsations using our suite of data; however, no clear pulsations are detected. Future observations designed to observe M33 X-8 at different flux levels across the full 0.3-30 keV range would significantly improve our constraints on the nature of this important source.Comment: Accepted for publication in ApJ (15 pages, 4 tables, 6 figures

Acute Metabolic Switch Assay Using Glucose/Galactose Medium in HepaRG Cells to Detect Mitochondrial Toxicity.

Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantages of working with a cell line, therefore, are considered an alternative for HepG2 cells in drug toxicity screening. A method is described here using HepaRG cells in an acute metabolic switch assay utilizing specific glucose/galactose media, a combined ATP-protein-LDH assay measuring three endpoints from one 96-well plate, and a criteria to label a compound as a mitochondrial toxin. © 2019 by John Wiley & Sons, Inc

Novel in vitro and mathematical models for the prediction of chemical toxicity

The focus of much scientific and medical research is directed towards understanding the disease process and defining therapeutic intervention strategies. Whilst the scientific basis of drug safety has received relatively little attention, despite the fact that adverse drug reactions (ADRs) are a major health concern and a serious impediment to development of new medicines. Toxicity issues account for ~21% drug attrition during drug development and safety testing strategies require considerable animal use. Mechanistic relationships between drug plasma levels and molecular/cellular events that culminate in whole organ toxicity underpins development of novel safety assessment strategies. Current in vitro test systems are poorly predictive of toxicity of chemicals entering the systemic circulation, particularly to the liver. Such systems fall short because of 1) the physiological gap between cells currently used & human hepatocytes existing in their native state, 2) the lack of physiological integration with other cells/systems within organs, required to amplify the initial toxicological lesion into overt toxicity, 3) the inability to assess how low level cell damage induced by chemicals may develop into overt organ toxicity in a minority of patients, 4) lack of consideration of systemic effects. Reproduction of centrilobular & periportal hepatocyte phenotypes in in vitro culture is crucial for sensitive detection of cellular stress. Hepatocyte metabolism/phenotype is dependent on cell position along the liver lobule, with corresponding differences in exposure to substrate, oxygen & hormone gradients. Application of bioartificial liver (BAL) technology can encompass in vitro predictive toxicity testing with enhanced sensitivity and improved mechanistic understanding. Combining this technology with mechanistic mathematical models describing intracellular metabolism, fluid-­‐flow, substrate, hormone and nutrient distribution provides the opportunity to design the BAL specifically to mimic the in vivo scenario. Such mathematical models enable theoretical hypothesis testing, will inform the design of in vitro experiments, and will enable both refinement and reduction of in vivo animal trials. In this way, development of novel mathematical modelling tools will help to focus and direct in vitro and in vivo research, and can be used as a framework for other areas of drug safety science

Multiscale modelling of drug transport and metabolism in liver spheroids

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo. While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery

Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study

Background: Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms—many related to the structure or function of red blood cells—and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. Methods: We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. Findings: Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11–0·20; p=2·61 × 10−58), blood group O (0·74, 0·66–0·82; p=6·26 × 10−8), and –α3·7-thalassaemia (0·83, 0·76–0·90; p=2·06 × 10−6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63–0·92; p=0·001) and FREM3 (0·64, 0·53–0·79; p=3·18 × 10−14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49–0·68; p=3·22 × 10−11), as was homozygosity (0·26, 0·11–0·62; p=0·002). Interpretation: Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. Funding: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative

AN RE/RM APPROACH TO THE DESIGN AND MANUFACTURE OF REMOVABLE PARTIAL DENTURES WITH A BIOCOMPATIBILITY ANALYSIS OF THE F75 Co-Cr SLM ALLOY RE/RM-PRIBLI@EK, NA^RTOVANJE IN IZDELAVA SNEMLJIVIH DELOV ZOBOVJA Z ANALIZO BIOKOMPATIBILNOSTI ZLITINE F75 Co-Cr SLM

The implementation of computer-aided technologies and systems has paved the way towards a significant advancement of the conventional modelling and manufacture of dental replacements. In this research the focus is on approach that combines reverse engineering as a modelling technique and rapid manufacture, i.e., selective laser melting, as the manufacturing technology, with special emphases on material selection in the fabrication of removable partial dentures. The paper presents the results of a biocompatibility analysis of the F75 Co-Cr dental alloy using the MTT eluate test. Keywords: reverse engineering, rapid manufacturing, selective laser melting, removable partial dentures, biocompatibility, Co-Cr alloys, MTT eluate test Uporaba ra~unalni{kih tehnologij in sistemov je odprla pot do pomembnega napredka konvencionalnega modeliranja in izdelave snemljivih zobnih nadomestkov. Te`i{~e te raziskave je pribli`ek, ki kombinira obratno in`enirstvo kot tehniko modeliranja in hitre izdelave z laserskim taljenjem kot izdelavno tehnologijo s posebnim poudarkom na izbiri materiala za snemljive dele zobovja. V~lanku so predstavljeni rezultati analize biokompatibilnosti zobne zlitine F75 Co-Cr z MTT-preizkusom eluiranja. Klju~ne besede: obratno in`enirstvo, hitra izdelava, selektivno lasersko taljenje, snemljivi deli zobovja, biokompatibilnost, Co-Cr zlitina, MTT-preizkus eluiranj

Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress.

BACKGROUND: Tissue hypoxia is a key feature of several endemic hepatic diseases, including alcoholic and non-alcoholic fatty liver disease, and organ failure. Hypoxia imposes a severe metabolic challenge on the liver, potentially disrupting its capacity to carry out essential functions including fuel storage and the integration of lipid metabolism at the whole-body level. Mitochondrial respiratory function is understood to be critical in mediating the hepatic hypoxic response, yet the time-dependent nature of this response and the role of the respiratory chain in this remain unclear. RESULTS: Here, we report that hepatic respiratory capacity is enhanced following short-term exposure to hypoxia (2 days, 10% O2) and is associated with increased abundance of the respiratory chain supercomplex III2+IV and increased cardiolipin levels. Suppression of this enhanced respiratory capacity, achieved via mild inhibition of mitochondrial complex III, disrupted metabolic homeostasis. Hypoxic exposure for 2 days led to accumulation of plasma and hepatic long chain acyl-carnitines. This was observed alongside depletion of hepatic triacylglycerol species with total chain lengths of 39-53 carbons, containing palmitic, palmitoleic, stearic, and oleic acids, which are associated with de novo lipogenesis. The changes to hepatic respiratory capacity and lipid metabolism following 2 days hypoxic exposure were transient, becoming resolved after 14 days in line with systemic acclimation to hypoxia and elevated circulating haemoglobin concentrations. CONCLUSIONS: The liver maintains metabolic homeostasis in response to shorter term hypoxic exposure through transient enhancement of respiratory chain capacity and alterations to lipid metabolism. These findings may have implications in understanding and treating hepatic pathologies associated with hypoxia.GlaxoSmithKlin