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Human cytomegalovirus (HCMV) is a major cause of illness in immunocompromised individuals. The HCMV lytic cycle contributes to the clinical manifestations of infection. The lytic cycle occurs over â¼96 h in diverse cell types and consists of viral DNA (vDNA) genome replication and temporally distinct expression of hundreds of viral proteins. Given its complexity, understanding this elaborate system can be facilitated by the introduction of mechanistic computational modeling of temporal relationships. Therefore, we developed a multiplicity of infection (MOI)-dependent mechanistic computational model that simulates vDNA kinetics and late lytic replication based on in-house experimental data. The predictive capabilities were established by comparison to post hoc experimental data. Computational analysis of combinatorial regulatory mechanisms suggests increasing rates of protein degradation in association with increasing vDNA levels. The model framework also allows expansion to account for additional mechanisms regulating the processes. Simulating vDNA kinetics and the late lytic cycle for a wide range of MOIs yielded several unique observations. These include the presence of saturation behavior at high MOIs, inefficient replication at low MOIs, and a precise range of MOIs in which virus is maximized within a cell type, being 0.382 IU to 0.688 IU per fibroblast. The predicted saturation kinetics at high MOIs are likely related to the physical limitations of cellular machinery, while inefficient replication at low MOIs may indicate a minimum input material required to facilitate infection. In summary, we have developed and demonstrated the utility of a data-driven and expandable computational model simulating lytic HCMV infection.
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Simulación por Computador , Citomegalovirus , Genoma Viral , Proteínas Virales , Replicación Viral , Citomegalovirus/genética , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidad , ADN Viral/genética , ADN Viral/metabolismo , Fibroblastos/virología , Genoma Viral/genética , Humanos , Cinética , Factores de Tiempo , Proteínas Virales/análisis , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin 1 (mTORC1), partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (NS) or 4.0% NaCl (HS) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide (inhibitors of ENaC, NKCC2, or NCC, respectively) either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K+ associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects.
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Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically-based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multi-drug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; Vmax) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a 6-parameter version of this model to control time course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only 3 unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT and found the mean estimated value for each parameter and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of Vmax for MRP2 at 30 min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP.
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Mitochondria are major sources of reactive oxygen species (ROS), which play important roles in both physiological and pathological processes. However, the specific contributions of different ROS production and scavenging components in the mitochondria of metabolically active tissues such as heart and kidney cortex and outer medulla (OM) are not well understood. Therefore, the goal of this study was to determine contributions of different ROS production and scavenging components and provide detailed comparisons of mitochondrial respiration, bioenergetics, ROS emission between the heart and kidney cortex and OM using tissues obtained from the same Sprague-Dawley rat under identical conditions and perturbations. Specifically, data were obtained using both NADH-linked substrate pyruvate + malate and FADH2-linked substrate succinate followed by additions of inhibitors of different components of the electron transport chain (ETC) and oxidative phosphorylation (OxPhos) and other ROS production and scavenging systems. Currently, there is limited data available for the mitochondria of kidney cortex and OM, the two major energy-consuming tissues in the body only next to the heart, and scarce quantitative information on the interplay between mitochondrial ROS production and scavenging systems in the three tissues. The findings from this study demonstrate significant differences in mitochondrial respiratory and bioenergetic functions and ROS emission among the three tissues. The results quantify the rates of ROS production from different complexes of the ETC, identify the complexes responsible for variations in mitochondrial membrane depolarization and regulations of ROS production, and quantify the contributions of ROS scavenging enzymes towards overall mitochondrial ROS emission. These findings advance our fundamental knowledge of tissue-specific and substrate-dependent mitochondrial respiratory and bioenergetic functions and ROS emission. This is important given the critical role that excess ROS production, oxidative stress, and mitochondrial dysfunction in the heart and kidney cortex and OM play in the pathogenesis of cardiovascular and renal diseases, including salt-sensitive hypertension.
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Mitocondrias , NAD , Ratas , Animales , Especies Reactivas de Oxígeno/metabolismo , NAD/metabolismo , Ratas Sprague-Dawley , Mitocondrias/metabolismo , Metabolismo Energético , Corteza Renal/metabolismoRESUMEN
Quantitative measurement of the degree of hepatic ischemia-reperfusion injury (IRI) is crucial for developing therapeutic strategies for its treatment. We hypothesized that clearance of fluorescent dye through bile metabolism may reflect the degree of hepatic IRI. In this study, we investigated sodium fluorescein clearance kinetics in blood and bile for quantifying the degree of hepatic IRI. Warm ischemia times (WITs) of 0, 30, or 60 min followed by 1 h or 4 h of reperfusion, were applied to the median and lateral lobes of the liver in Sprague-Dawley rats. Subsequently, 2 mg/kg of sodium fluorescein was injected intravenously, and blood and bile samples were collected over 60 min to measure fluorescence intensities. The bile-to-plasma fluorescence ratios demonstrated an inverse correlation with WIT and were distinctly lower in the 60-min WIT group than in the control or 30-min WIT groups. Bile-to-plasma fluorescence ratios displayed superior discriminability for short versus long WITs when measured 1 h after reperfusion versus 4 h. We conclude that the bile-to-blood ratio of fluorescence after sodium fluorescein injection has the potential to enable the quantification of hepatic IRI severity.NEW & NOTEWORTHY Previous attempts to use fluorophore clearance to test liver function have relied on a single source of data. However, the kinetics of substrate processing via bile metabolism include decreasing levels in blood and increasing levels in bile. Thus, we analyzed data from blood and bile to better reflect fluorescein clearance kinetics.
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Bilis , Daño por Reperfusión , Animales , Bilis/metabolismo , Fluoresceína/metabolismo , Fluoresceína/uso terapéutico , Cinética , Hígado/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismoRESUMEN
The cellular protein-protein interaction network that governs cellular proliferation (cell cycle) is highly complex. Here, we have developed a novel computational model of human mitotic cell cycle, integrating diverse cellular mechanisms, for the purpose of generating new hypotheses and predicting new experiments designed to help understand complex diseases. The pathogenic state investigated is infection by a human herpesvirus. The model starts at mitotic entry initiated by the activities of Cyclin-dependent kinase 1 (CDK1) and Polo-like kinase 1 (PLK1), transitions through Anaphase-promoting complex (APC/C) bound to Cell division cycle protein 20 (CDC20), and ends upon mitotic exit mediated by APC/C bound to CDC20 homolog 1 (CDH1). It includes syntheses and multiple mechanisms of degradations of the mitotic proteins. Prior to this work, no such comprehensive model of the human mitotic cell cycle existed. The new model is based on a hybrid framework combining Michaelis-Menten and mass action kinetics for the mitotic interacting reactions. It simulates temporal changes in 12 different mitotic proteins and associated protein complexes in multiple states using 15 interacting reactions and 26 ordinary differential equations. We have defined model parameter values using both quantitative and qualitative data and using parameter values from relevant published models, and we have tested the model to reproduce the cardinal features of human mitosis determined experimentally by numerous laboratories. Like cancer, viruses create dysfunction to support infection. By simulating infection of the human herpesvirus, cytomegalovirus, we hypothesize that virus-mediated disruption of APC/C is necessary to establish a unique mitotic collapse with sustained CDK1 activity, consistent with known mechanisms of virus egress. With the rapid discovery of cellular protein-protein interaction networks and regulatory mechanisms, we anticipate that this model will be highly valuable in helping us to understand the network dynamics and identify potential points of therapeutic interventions.
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Biología Computacional/métodos , Mitosis/fisiología , Mapas de Interacción de Proteínas/fisiología , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Antígenos CD/metabolismo , Proteína Quinasa CDC2/metabolismo , Cadherinas/metabolismo , Proteínas Cdc20/metabolismo , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Humanos , Cinética , Modelos Teóricos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Quinasa Tipo Polo 1RESUMEN
We present a new size-modified Poisson-Boltzmann ion channel (SMPBIC) model and use it to calculate the electrostatic potential, ionic concentrations, and electrostatic solvation free energy for a voltage-dependent anion channel (VDAC) on a biological membrane in a solution mixture of multiple ionic species. In particular, the new SMPBIC model adopts a membrane surface charge density and a natural Neumann boundary condition to reflect the charge effect of the membrane on the electrostatics of VDAC. To avoid the singularity difficulties caused by the atomic charges of VDAC, the new SMPBIC model is split into three submodels such that the solution of one of the submodels is obtained analytically and contains all the singularity points of the SMPBIC model. The other two submodels are then solved numerically much more efficiently than the original SMPBIC model. As an application of this SMPBIC submodel partitioning scheme, we derive a new formula for computing the electrostatic solvation free energy. Numerical results for a human VDAC isoform 1 (hVDAC1) in three different salt solutions, each with up to five different ionic species, confirm the significant effects of membrane surface charges on both the electrostatics and ionic concentrations. The results also show that the new SMPBIC model can describe well the anion selectivity property of hVDAC1, and that the new electrostatic solvation free energy formula can significantly improve the accuracy of the currently used formula. © 2019 Wiley Periodicals, Inc.
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Canales Aniónicos Dependientes del Voltaje/química , Humanos , Iones/química , Modelos Moleculares , Solventes/química , Electricidad Estática , TermodinámicaRESUMEN
Cardiovascular-related pathologies are the single leading cause of death in patients with chronic kidney disease (CKD). Previously, we found that a 5/6th nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle, targeting peroxisome proliferator-activated receptor-α and altering the expression of numerous transcripts involved with fatty acid oxidation and glycolysis. In the present study, we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content, lipid peroxidation, and mitochondrial respiration in H9C2 cells. Cells were transfected with anti-miR-21-5p (40 nM), pre-miR-21-5p (20 nM), or the appropriate scrambled oligonucleotide controls before lipid treatment in culture or as part of the Agilent Seahorse XF fatty acid oxidation assay. Overexpression of miR-21-5p attenuated the lipid-induced increase in cellular lipid content, whereas suppression of miR-21-5p augmented it. The abundance of malondialdehyde, a product of lipid peroxidation, was significantly increased with lipid treatment in control cells but attenuated in pre-miR-21-5p-transfected cells. This suggests that miR-21-5p reduces oxidative stress. The cellular oxygen consumption rate (OCR) was increased in both pre-miR-21-5p- and anti-miR-21-5p-transfected cells. Levels of intracellular ATP were significantly higher in anti-mR-21-5p-transfected cells. Pre-miR-21-5p blocked additional increases in OCR in response to etomoxir and palmitic acid. Conversely, anti-miR-21-5p-transfected cells exhibited reduced OCR with both etomoxir and palmitic acid, and the glycolytic capacity was concomitantly reduced. Together, these results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. This likely occurs by reducing cellular lipid uptake and utilization, shifting cellular metabolism toward reliance on the glycolytic pathway. NEW & NOTEWORTHY Both overexpression and suppression of miR-21-5p augment basal and maximal mitochondrial respiration. Our data suggest that reliance on glycolytic and fatty acid oxidation pathways can be modulated by the abundance of miR-21-5p within the cell. miR-21-5p regulation of mitochondrial respiration can be modulated by extracellular lipids.
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Metabolismo de los Lípidos/genética , MicroARNs/genética , MicroARNs/fisiología , Mitocondrias/genética , Mitocondrias/metabolismo , Consumo de Oxígeno/genética , Animales , Línea Celular , Ácidos Grasos/metabolismo , Glucólisis , Peroxidación de Lípido/genética , Malondialdehído/metabolismo , Mioblastos/metabolismo , Oxidación-Reducción , Estrés Oxidativo/genética , RatasRESUMEN
BACKGROUND: Recent clinical studies suggest that the magnitude of the second gas effect is considerably greater on arterial blood partial pressures of volatile agents than on end-expired partial pressures, and a significant second gas effect on blood partial pressures of oxygen and volatile agents occurs even at relatively low rates of nitrous oxide uptake. We set out to further investigate the mechanism of this phenomenon with the help of mathematical modeling. METHODS: Log-normal distributions of ventilation and blood flow were generated representing the range of ventilation-perfusion scatter seen in patients during general anesthesia. Mixtures of nominal delivered concentrations of volatile agents (desflurane, isoflurane and diethyl ether) with and without 70% nitrous oxide were mathematically modeled using steady state mass-balance principles, and the magnitude of the second gas effect calculated as an augmentation ratio for the volatile agent, defined as the partial pressure in the presence to that in the absence of nitrous oxide. RESULTS: Increasing the degree of mismatch increased the second gas effect in blood. Simultaneously, the second gas effect decreased in the gas phase. The increase in blood was greatest for the least soluble gas, desflurane, and least for the most soluble gas, diethyl ether, while opposite results applied in the gas phase. CONCLUSIONS: Modeling of ventilation-perfusion inhomogeneity confirms that the second gas effect is greater in blood than in expired gas. Gas-based minimum alveolar concentration readings may therefore underestimate the depth of anesthesia during nitrous oxide anesthesia with volatile agents. The effect on minimum alveolar concentration is likely to be most pronounced for the less soluble volatile agents in current use.
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Anestesia por Inhalación/métodos , Anestésicos por Inhalación/administración & dosificación , Anestésicos por Inhalación/sangre , Modelos Teóricos , Relación Ventilacion-Perfusión/efectos de los fármacos , Humanos , Relación Ventilacion-Perfusión/fisiologíaRESUMEN
The manner in which mitochondria take up and store Ca(2+) remains highly debated. Recent experimental and computational evidence has suggested the presence of at least two modes of Ca(2+) uptake and a complex Ca(2+) sequestration mechanism in mitochondria. But how Mg(2+) regulates these different modes of Ca(2+) uptake as well as mitochondrial Ca(2+) sequestration is not known. In this study, we investigated two different ways by which mitochondria take up and sequester Ca(2+) by using two different protocols. Isolated guinea pig cardiac mitochondria were exposed to varying concentrations of CaCl2 in the presence or absence of MgCl2. In the first protocol, A, CaCl2 was added to the respiration buffer containing isolated mitochondria, whereas in the second protocol, B, mitochondria were added to the respiration buffer with CaCl2 already present. Protocol A resulted first in a fast transitory uptake followed by a slow gradual uptake. In contrast, protocol B only revealed a slow and gradual Ca(2+) uptake, which was approximately 40 % of the slow uptake rate observed in protocol A. These two types of Ca(2+) uptake modes were differentially modulated by extra-matrix Mg(2+). That is, Mg(2+) markedly inhibited the slow mode of Ca(2+) uptake in both protocols in a concentration-dependent manner, but not the fast mode of uptake exhibited in protocol A. Mg(2+) also inhibited Na(+)-dependent Ca(2+) extrusion. The general Ca(2+) binding properties of the mitochondrial Ca(2+) sequestration system were reaffirmed and shown to be independent of the mode of Ca(2+) uptake, i.e. through the fast or slow mode of uptake. In addition, extra-matrix Mg(2+) hindered Ca(2+) sequestration. Our results indicate that mitochondria exhibit different modes of Ca(2+) uptake depending on the nature of exposure to extra-matrix Ca(2+), which are differentially sensitive to Mg(2+). The implications of these findings in cardiomyocytes are discussed.
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Calcio/metabolismo , Magnesio/fisiología , Mitocondrias Cardíacas/metabolismo , Animales , Transporte Biológico , Calcio/farmacocinética , Cobayas , Cinética , Miocitos Cardíacos/metabolismoRESUMEN
PURPOSE: Equations for blood oxyhemoglobin (HbO2) and carbaminohemoglobin (HbCO2) dissociation curves that incorporate nonlinear biochemical interactions of oxygen and carbon dioxide with hemoglobin (Hb), covering a wide range of physiological conditions, are crucial for a number of practical applications. These include the development of physiologically-based computational models of alveolar-blood and blood-tissue O2CO2 transport, exchange, and metabolism, and the analysis of clinical and in vitro data. METHODS AND RESULTS: To this end, we have revisited, simplified, and extended our previous models of blood HbO2 and HbCO2 dissociation curves (Dash and Bassingthwaighte, Ann Biomed Eng 38:16831701, 2010), validated wherever possible by available experimental data, so that the models now accurately fit the low HbO2 saturation (SHbO2) range over a wide range of values of PCO2, pH, 2,3-DPG, and temperature. Our new equations incorporate a novel PO2-dependent variable cooperativity hypothesis for the binding of O2 to Hb, and a new equation for P50 of O2 that provides accurate shifts in the HbO2 and HbCO2 dissociation curves over a wide range of physiological conditions. The accuracy and efficiency of these equations in computing PO2 and PCO2 from the SHbO2 and SHbCO2 levels using simple iterative numerical schemes that give rapid convergence is a significant advantage over alternative SHbO2 and SHbCO2 models. CONCLUSION: The new SHbO2 and SHbCO2 models have significant computational modeling implications as they provide high accuracy under non-physiological conditions, such as ischemia and reperfusion, extremes in gas concentrations, high altitudes, and extreme temperatures.
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Dióxido de Carbono/sangre , Hemoglobinas/metabolismo , Oxígeno/sangre , Oxihemoglobinas/metabolismo , Temperatura , Hemoglobina A/análogos & derivados , Hemoglobina A/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Modelos BiológicosRESUMEN
The 'Pathway for Oxygen' is captured in a set of models describing quantitative relationships between fluxes and driving forces for the flux of oxygen from the external air source to the mitochondrial sink at cytochrome oxidase. The intervening processes involve convection, membrane permeation, diffusion of free and heme-bound O2 and enzymatic reactions. While this system's basic elements are simple: ventilation, alveolar gas exchange with blood, circulation of the blood, perfusion of an organ, uptake by tissue, and consumption by chemical reaction, integration of these pieces quickly becomes complex. This complexity led us to construct a tutorial on the ideas and principles; these first PathwayO2 models are simple but quantitative and cover: (1) a 'one-alveolus lung' with airway resistance, lung volume compliance, (2) bidirectional transport of solute gasses like O2 and CO2, (3) gas exchange between alveolar air and lung capillary blood, (4) gas solubility in blood, and circulation of blood through the capillary syncytium and back to the lung, and (5) blood-tissue gas exchange in capillaries. These open-source models are at Physiome.org and provide background for the many respiratory models there.
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Mitocondrias/metabolismo , Oxígeno/metabolismo , Transporte Biológico , Humanos , Pulmón/metabolismo , Modelos BiológicosRESUMEN
Mitochondrial dysfunction contributes to cardiac ischemia-reperfusion (IR) injury but volatile anesthetics (VA) may alter mitochondrial function to trigger cardioprotection. We hypothesized that the VA isoflurane (ISO) mediates cardioprotection in part by altering the function of several respiratory and transport proteins involved in oxidative phosphorylation (OxPhos). To test this we used fluorescence spectrophotometry to measure the effects of ISO (0, 0.5, 1, 2mM) on the time-course of interlinked mitochondrial bioenergetic variables during states 2, 3 and 4 respiration in the presence of either complex I substrate K(+)-pyruvate/malate (PM) or complex II substrate K(+)-succinate (SUC) at physiological levels of extra-matrix free Ca(2+) (~200nM) and Na(+) (10mM). To mimic ISO effects on mitochondrial functions and to clearly delineate the possible ISO targets, the observed actions of ISO were interpreted by comparing effects of ISO to those elicited by low concentrations of inhibitors that act at each respiratory complex, e.g. rotenone (ROT) at complex I or antimycin A (AA) at complex III. Our conclusions are based primarily on the similar responses of ISO and titrated concentrations of ETC. inhibitors during state 3. We found that with the substrate PM, ISO and ROT similarly decreased the magnitude of state 3 NADH oxidation and increased the duration of state 3 NADH oxidation, ΔΨm depolarization, and respiration in a concentration-dependent manner, whereas with substrate SUC, ISO and ROT decreased the duration of state 3 NADH oxidation, ΔΨm depolarization and respiration. Unlike AA, ISO reduced the magnitude of state 3 NADH oxidation with PM or SUC as substrate. With substrate SUC, after complete block of complex I with ROT, ISO and AA similarly increased the duration of state 3 ΔΨm depolarization and respiration. This study provides a mechanistic understanding in how ISO alters mitochondrial function in a way that may lead to cardioprotection.
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Complejo III de Transporte de Electrones/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Metabolismo Energético/efectos de los fármacos , Isoflurano/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Animales , Antimicina A/farmacología , Transporte de Electrón/efectos de los fármacos , Malatos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/fisiología , Modelos Biológicos , NAD/metabolismo , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ácido Pirúvico/metabolismo , Ratas , Ratas Wistar , Rotenona/farmacología , Espectrometría de Fluorescencia , Ácido Succínico/metabolismo , Desacopladores/farmacologíaRESUMEN
Cardiac mitochondria can act as a significant Ca(2+) sink and shape cytosolic Ca(2+) signals affecting various cellular processes, such as energy metabolism and excitation-contraction coupling. However, different mitochondrial Ca(2+) uptake mechanisms are still not well understood. In this study, we analysed recently published Ca(2+) uptake experiments performed on isolated guinea pig cardiac mitochondria using a computer model of mitochondrial bioenergetics and cation handling. The model analyses of the data suggest that the majority of mitochondrial Ca(2+) uptake, at physiological levels of cytosolic Ca(2+) and Mg(2+), occurs through a fast Ca(2+) uptake pathway, which is neither the Ca(2+) uniporter nor the rapid mode of Ca(2+) uptake. This fast Ca(2+) uptake component was explained by including a biophysical model of the ryanodine receptor (RyR) in the computer model. However, the Mg(2+)-dependent enhancement of the RyR adaptation was not evident in this RyR-type channel, in contrast to that of cardiac sarcoplasmic reticulum RyR. The extended computer model is corroborated by simulating an independent experimental dataset, featuring mitochondrial Ca(2+) uptake, egress and sequestration. The model analyses of the two datasets validate the existence of two classes of Ca(2+) buffers that comprise the mitochondrial Ca(2+) sequestration system. The modelling study further indicates that the Ca(2+) buffers respond differentially depending on the source of Ca(2+) uptake. In particular, it suggests that the Class 1 Ca(2+) buffering capacity is auto-regulated by the rate at which Ca(2+) is taken up by mitochondria.
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Calcio/metabolismo , Biología Computacional/métodos , Metabolismo Energético/fisiología , Mitocondrias Cardíacas/metabolismo , Modelos Teóricos , Animales , Predicción , CobayasRESUMEN
Alcohol consumption during pregnancy can significantly impact the brain development of the fetus, leading to long-term cognitive and behavioral problems. However, the underlying mechanisms are not well understood. In this study, we investigated the acute and chronic effects of binge-like alcohol exposure during the third trimester equivalent in postnatal day 7 (P7) mice on brain cell viability, synapse activity, cognitive and behavioral performance, and gene expression profiles at P60. Our results showed that alcohol exposure caused neuroapoptosis in P7 mouse brains immediately after a 6-hour exposure. In addition, P60 mice exposed to alcohol during P7 displayed impaired learning and memory abilities and anxiety-like behaviors. Electrophysiological analysis of hippocampal neurons revealed an excitatory/inhibitory imbalance in alcohol-treated P60 mice compared to controls, with decreased excitation and increased inhibition. Furthermore, our bioinformatic analysis of 376 dysregulated genes in P60 mouse brains following alcohol exposure identified 50 synapse-related and 23 mitochondria-related genes. These genes encoded proteins located in various parts of the synapse, synaptic cleft, extra-synaptic space, synaptic membranes, or mitochondria, and were associated with different biological processes and functions, including the regulation of synaptic transmission, transport, synaptic vesicle cycle, metabolism, synaptogenesis, mitochondrial activity, cognition, and behavior. The dysregulated synapse and mitochondrial genes were predicted to interact in overlapping networks. Our findings suggest that altered synaptic activities and signaling networks may contribute to alcohol-induced long-term cognitive and behavioral impairments in mice, providing new insights into the underlying synaptic and mitochondrial molecular mechanisms and potential neuroprotective strategies.
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Problema de Conducta , Femenino , Embarazo , Animales , Ratones , Etanol , Mitocondrias , Consumo de Bebidas Alcohólicas , ADN Mitocondrial , CogniciónRESUMEN
Summary: Molecular mechanisms of biological functions and disease processes are exceptionally complex, and our ability to interrogate and understand relationships is becoming increasingly dependent on the use of computational modeling. We have developed "BioModME," a standalone R-based web application package, providing an intuitive and comprehensive graphical user interface to help investigators build, solve, visualize, and analyze computational models of complex biological systems. Some important features of the application package include multi-region system modeling, custom reaction rate laws and equations, unit conversion, model parameter estimation utilizing experimental data, and import and export of model information in the Systems Biology Matkup Language format. The users can also export models to MATLAB, R, and Python languages and the equations to LaTeX and Mathematical Markup Language formats. Other important features include an online model development platform, multi-modality visualization tool, and efficient numerical solvers for differential-algebraic equations and optimization. Availability and implementation: All relevant software information including documentation and tutorials can be found at https://mcw.marquette.edu/biomedical-engineering/computational-systems-biology-lab/biomodme.php. Deployed software can be accessed at https://biomodme.ctsi.mcw.edu/. Source code is freely available for download at https://github.com/MCWComputationalBiologyLab/BioModME.
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Since the FDA's approval of chimeric antigen receptor (CAR) T cells in 2017, significant improvements have been made in the design of chimeric antigen receptor constructs and in the manufacturing of CAR T cell therapies resulting in increased in vivo CAR T cell persistence and improved clinical outcome in certain hematological malignancies. Despite the remarkable clinical response seen in some patients, challenges remain in achieving durable long-term tumor-free survival, reducing therapy associated malignancies and toxicities, and expanding on the types of cancers that can be treated with this therapeutic modality. Careful analysis of the biological factors demarcating efficacious from suboptimal CAR T cell responses will be of paramount importance to address these shortcomings. With the ever-expanding toolbox of experimental approaches, single-cell technologies, and computational resources, there is renowned interest in discovering new ways to streamline the development and validation of new CAR T cell products. Better and more accurate prognostic and predictive models can be developed to help guide and inform clinical decision making by incorporating these approaches into translational and clinical workflows. In this review, we provide a brief overview of recent advancements in CAR T cell manufacturing and describe the strategies used to selectively expand specific phenotypic subsets. Additionally, we review experimental approaches to assess CAR T cell functionality and summarize current in silico methods which have the potential to improve CAR T cell manufacturing and predict clinical outcomes.
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Adult rats exposed to hyperoxia (>95% O2) die from respiratory failure in 60-72 h. However, rats preconditioned with >95% O2 for 48 h followed by 24 h in room air acquire tolerance of hyperoxia (H-T), whereas rats preconditioned with 60% O2 for 7 days become more susceptible (H-S). Our objective was to evaluate lung tissue mitochondrial bioenergetics in H-T and H-S rats. Bioenergetics was assessed in mitochondria isolated from lung tissue of H-T, H-S, and control rats. Expressions of complexes involved in oxidative phosphorylation (OxPhos) were measured in lung tissue homogenate. Pulmonary endothelial filtration coefficient (Kf) and tissue mitochondrial membrane potential (Δψm) were evaluated in isolated perfused lungs (IPLs). Results show that ADP-induced state 3 OxPhos capacity (Vmax) decreased in H-S mitochondria but increased in H-T. Δψm repolarization time following ADP-stimulated depolarization increased in H-S mitochondria. Complex I expression decreased in H-T (38%) and H-S (43%) lung homogenate, whereas complex V expression increased (70%) in H-T lung homogenate. Δψm is unchanged in H-S and H-T lungs, but complex II has a larger contribution to Δψm in H-S than H-T lungs. Kf increased in H-S, but not in H-T lungs. For H-T, increased complex V expression and Vmax counter the effect of the decrease in complex I expression on Δψm. A larger complex II contribution to Δψm along with decreased Vmax and increased Kf could make H-S rats more hyperoxia susceptible. Results are clinically relevant since ventilation with ≥60% O2 is often required for extended periods in patients with acute respiratory distress syndrome (ARDS).NEW & NOTEWORTHY We assessed lung tissue mitochondrial bioenergetics in rats with tolerance (H-T) or susceptibility (H-S) to hyperoxia-induced ARDS. Results from studies in isolated mitochondria, tissue homogenate, and isolated perfused lungs show that mitochondrial bioenergetics are differentially altered in H-T and H-S lungs suggesting a potential role for mitochondrial bioenergetics in hyperoxia-induced ARDS. Results are clinically relevant since hyperoxia exposure is a primary therapy for patients with ARDS, and differential sensitivity to hyperoxia surely occurs in humans.
Asunto(s)
Lesión Pulmonar Aguda , Hiperoxia , Pulmón , Mitocondrias , Fosforilación Oxidativa , Ratas Sprague-Dawley , Animales , Hiperoxia/metabolismo , Hiperoxia/fisiopatología , Hiperoxia/complicaciones , Pulmón/metabolismo , Pulmón/fisiopatología , Ratas , Mitocondrias/metabolismo , Masculino , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/fisiopatología , Potencial de la Membrana Mitocondrial/fisiología , Metabolismo EnergéticoRESUMEN
Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus, and infection can lead to a range of symptomatology from mononucleosis to sepsis in immunocompromised individuals. HCMV is also the leading viral cause of congenital birth defects. Lytic replication is supported by many cell types with different kinetics and efficiencies leading to a plethora of pathologies. The goal of these studies was to elucidate HCMV replication efficiencies for viruses produced on different cell types upon infection of epithelial cells by combining experimental approaches with data-driven computational modeling. HCMV was generated from a common genetic background of TB40-BAC4, propagated on fibroblasts (TB40Fb) or epithelial cells (TB40Epi), and used to infect epithelial cells. We quantified cell-associated viral genomes (vDNA), protein levels (pUL44, pp28), and cell-free titers over time for each virus at different multiplicities of infection. We combined experimental quantification with data-driven simulations and determined that parameters describing vDNA synthesis were similar between sources. We found that pUL44 accumulation was higher in TB40Fb than TB40Epi. In contrast, pp28 accumulation was higher in TB40Epi which coincided with a significant increase in titer for TB40Epi over TB40Fb. These differences were most evident during live-cell imaging, which revealed syncytia-like formation during infection by TB40Epi. Simulations of the late lytic replication cycle yielded a larger synthesis constant for pp28 in TB40Epi along with increase in virus output despite similar rates of genome synthesis. By combining experimental and computational modeling approaches, our studies demonstrate that the cellular source of propagated virus impacts viral replication efficiency in target cell types.
RESUMEN
Modulation of mitochondrial free Ca(2+) ([Ca(2+)](m)) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia-reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca(2+)](m) and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2mM) on the magnitudes and time-courses of [Ca(2+)](m) and mitochondrial redox state (NADH), membrane potential (ΔΨ(m)), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10mM Na(+)- or K(+)-pyruvate/malate (NaPM or KPM) or Na(+)-succinate (NaSuc) followed by additions of isoflurane, 0.5mM CaCl(2) (≈200nM free Ca(2+) with 1mM EGTA buffer), and 250µM ADP. Isoflurane stepwise: (a) increased [Ca(2+)](m) in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ(m) and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ(m), and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ(m), and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca(2+) uptake by inhibiting the Na(+)/Ca(2+) exchanger (NCE), independent of changes in ΔΨ(m) and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca(2+)](m), while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.