Inhibition of mTORC2 promotes natriuresis in Dahl salt-sensitive rats via the decrease of NCC and ENaC activity.

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.

Replication efficiencies of human cytomegalovirus-infected epithelial cells are dependent on source of virus production.

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.

BioModME for building and simulating dynamic computational models of complex biological systems.

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.

Synaptic and mitochondrial mechanisms behind alcohol-induced imbalance of excitatory/inhibitory synaptic activity and associated cognitive and behavioral abnormalities.

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.

Physiologically-Based Pharmacokinetic Modeling of Blood Clearance of Liver Fluorescent Markers for the Assessment of the Degree of Hepatic Ischemia-Reperfusion Injury.

During liver transplantation, ischemia-reperfusion injury (IRI) is inevitable and decreases the overall success of the surgery. While guidelines exist, there is no reliable way to quantitatively assess the degree of IRI present in the liver. Our recent study has shown a correlation between the bile-to-plasma ratio of FDA-approved sodium fluorescein (SF) and the degree of hepatic IRI, presumably due to IRI-induced decrease in the activity of the hepatic multidrug resistance-associated protein 2 (MRP2); however, the contribution of SF blood clearance via the bile is still convoluted with other factors, such as renal clearance. In this work, we sought to computationally model SF blood clearance via the bile. First, we converted extant SF fluorescence data from rat whole blood, plasma, and bile to concentrations using calibration curves. Next, based on these SF concentration data, we generated a "liver-centric", physiologically-based pharmacokinetic (PBPK) model of SF liver uptake and clearance via the bile. Model simulations show that SF bile concentration is highly sensitive to change in the activity of hepatic MPR2. These simulations suggest that SF bile clearance along with the PBPK model can be used to quantify the effect of IRI on the activity of MRP2.Clinical Relevance- This study establishes the theory necessary to generate a model for predicting the degree of IRI during liver transplantation.

Simulating the Evolution of Signaling Signatures During CART-Cell and Tumor Cell Interactions.

Immunotherapies have been proven to have significant therapeutic efficacy in the treatment of cancer. The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for in silico based prediction of CART-cell functionality prior to experimentation.Clinical Relevance- Analysis of CART-cell signaling signatures can inform future CAR receptor design and combination therapy approaches aimed at improving therapy response.

Effects of N2 O elimination on the elimination of second gases in a two-step mathematical model of heterogeneous gas exchange.

We have investigated the elimination of inert gases in the lung during the elimination of nitrous oxide (N2 O) using a two-step mathematical model that allows the contribution from net gas volume expansion, which occurs in Step 2, to be separated from other factors. When a second inert gas is used in addition to N2 O, the effect on that gas appears as an extra volume of the gas eliminated in association with the dilution produced by N2 O washout in Step 2. We first considered the effect of elimination in a single gas-exchanging unit under steady-state conditions and then extended our analysis to a lung having a log-normal distribution of ventilation and perfusion. A further increase in inert gas elimination was demonstrated with gases of low solubility in the presence of the increased ventilation-perfusion mismatch that is known to occur during anesthesia. These effects are transient because N2 O elimination depletes the input of that gas from mixed venous blood to the lung, thereby rapidly reducing the magnitude of the diluting action.

Computational Modeling of Substrate-Dependent Mitochondrial Respiration and Bioenergetics in the Heart and Kidney Cortex and Outer Medulla.

Integrated computational modeling provides a mechanistic and quantitative framework to characterize alterations in mitochondrial respiration and bioenergetics in response to different metabolic substrates in-silico. These alterations play critical roles in the pathogenesis of diseases affecting metabolically active organs such as heart and kidney. Therefore, the present study aimed to develop and validate thermodynamically constrained integrated computational models of mitochondrial respiration and bioenergetics in the heart and kidney cortex and outer medulla (OM). The models incorporated the kinetics of major biochemical reactions and transport processes as well as regulatory mechanisms in the mitochondria of these tissues. Intrinsic model parameters such as Michaelis-Menten constants were fixed at previously estimated values, while extrinsic model parameters such as maximal reaction and transport velocities were estimated separately for each tissue. This was achieved by fitting the model solutions to our recently published respirometry data measured in isolated rat heart and kidney cortex and OM mitochondria utilizing various NADH- and FADH2-linked metabolic substrates. The models were validated by predicting additional respirometry and bioenergetics data, which were not used for estimating the extrinsic model parameters. The models were able to predict tissue-specific and substrate-dependent mitochondrial emergent metabolic system properties such as redox states, enzyme and transporter fluxes, metabolite concentrations, membrane potential, and respiratory control index under diverse physiological and pathological conditions. The models were also able to quantitatively characterize differential regulations of NADH- and FADH2-linked metabolic pathways, which contribute differently toward regulations of oxidative phosphorylation and ATP synthesis in the heart and kidney cortex and OM mitochondria.

Metabolic Responses of Normal Rat Kidneys to a High Salt Intake.

In this study, novel methods were developed, which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to 4.0% NaCl diet. In addition, targeted mRNA expression analysis of cortical segments was performed. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. A novel finding was the increased expression of glycolysis-related genes in Cx and isolated proximal tubular segments in response to an HS diet, consistent with increased release of pyruvate and lactate from the kidney to the renal venous blood. Data suggests that aerobic glycolysis (eg, Warburg effect) may contribute to energy production under these circumstances. The study provides evidence that kidney metabolism responds to an HS diet enabling enhanced energy production while protecting from oxidative stress and injury. Metabolomic and transcriptomic analysis of kidneys of Sprague-Dawley rats fed a high salt diet.

Effects of ROS pathway inhibitors and NADH and FADH2 linked substrates on mitochondrial bioenergetics and ROS emission in the heart and kidney cortex and outer medulla.

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.

Simulating the Evolution of Signaling Signatures during CART-Cell - Tumor Cell Interactions.

Immunotherapies have been proven to have significant therapeutic efficacy in the treatment of cancer. The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for in silico based prediction of CART-cell functionality prior to experimentation.

Metabolic responses of normal rat kidneys to a high salt intake.

In the present study, novel methods were developed which allowed continuous (24/7) measurement of blood pressure (BP) and renal blood flow (RBF) in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O 2 and metabolites. The study determined the effects of a high salt (HS) diet upon whole kidney O 2 consumption and the metabolomic profiles of normal Sprague Dawley (SD) rats. A separate group of rats was studied to determine changes in the cortex (Cx) and outer medulla (OM) tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to a 4.0% NaCl diet. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O 2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. Increased glycolysis was evident with the elevation of mRNA expression encoding key glycolytic enzymes and release of pyruvate and lactate from the kidney in the renal venous blood. Glycolytic production of NADH is used in either the production of lactate or oxidized via the malate aspartate shuttle. Aerobic glycolysis (e.g., Warburg-effect) may account for the needed increase in cellular energy. The study provides evidence that kidney metabolism responds to a HS diet enabling enhanced energy production while protecting from oxidate stress and injury.

Computational modeling of protracted HCMV replication using genome substrates and protein temporal profiles.

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 ∼96 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.

Fluorescein clearance kinetics in blood and bile indicates hepatic ischemia-reperfusion injury in rats.

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.

Depolarized mitochondrial membrane potential and protection with duroquinone in isolated perfused lungs from rats exposed to hyperoxia.

Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction. Our objective was to use a previously developed experimental-computational approach to estimate tissue Δψm in intact lungs of rats exposed to hyperoxia and to evaluate the ability of duroquinone (DQ) to reverse any hyperoxia-induced depolarization of lung Δψm. Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 h, after which lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6 G (R6G) during three single-pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. Computational modeling was used to estimate lung Δψm from R6G data. Rat exposure to hyperoxia resulted in partial depolarization (-33 mV) of lung Δψm and complex I inhibition depolarized lung Δψm by -83 mV. Results also demonstrate the efficacy of DQ to fully reverse both rotenone- and hyperoxia-induced lung Δψm depolarization. This study demonstrates hyperoxia-induced Δψm depolarization in intact lungs and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in intact lungs.NEW & NOTEWORTHY This study is the first to measure hyperoxia-induced Δψm depolarization in isolated perfused lungs. Hyperoxia resulted in a partial depolarization of Δψm, which was fully reversed with duroquinone, demonstrating the utility of this approach for assessing the impact of potential therapies that target mitochondria such as exogenous quinones.

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla.

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H2O2 emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H2O2 emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.

Crosstalk between Plk1, p53, cell cycle, and G2/M DNA damage checkpoint regulation in cancer: computational modeling and analysis.

Different cancer cell lines can have varying responses to the same perturbations or stressful conditions. Cancer cells that have DNA damage checkpoint-related mutations are often more sensitive to gene perturbations including altered Plk1 and p53 activities than cancer cells without these mutations. The perturbations often induce a cell cycle arrest in the former cancer, whereas they only delay the cell cycle progression in the latter cancer. To study crosstalk between Plk1, p53, and G2/M DNA damage checkpoint leading to differential cell cycle regulations, we developed a computational model by extending our recently developed model of mitotic cell cycle and including these key interactions. We have used the model to analyze the cancer cell cycle progression under various gene perturbations including Plk1-depletion conditions. We also analyzed mutations and perturbations in approximately 1800 different cell lines available in the Cancer Dependency Map and grouped lines by genes that are represented in our model. Our model successfully explained phenotypes of various cancer cell lines under different gene perturbations. Several sensitivity analysis approaches were used to identify the range of key parameter values that lead to the cell cycle arrest in cancer cells. Our resulting model can be used to predict the effect of potential treatments targeting key mitotic and DNA damage checkpoint regulators on cell cycle progression of different types of cancer cells.

Substrate- and Calcium-Dependent Differential Regulation of Mitochondrial Oxidative Phosphorylation and Energy Production in the Heart and Kidney.

Mitochondrial dehydrogenases are differentially stimulated by Ca2+. Ca2+ has also diverse regulatory effects on mitochondrial transporters and other enzymes. However, the consequences of these regulatory effects on mitochondrial oxidative phosphorylation (OxPhos) and ATP production, and the dependencies of these consequences on respiratory substrates, have not been investigated between the kidney and heart despite the fact that kidney energy requirements are second only to those of the heart. Our objective was, therefore, to elucidate these relationships in isolated mitochondria from the kidney outer medulla (OM) and heart. ADP-induced mitochondrial respiration was measured at different CaCl2 concentrations in the presence of various respiratory substrates, including pyruvate + malate (PM), glutamate + malate (GM), alpha-ketoglutarate + malate (AM), palmitoyl-carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). The results showed that, in both heart and OM mitochondria, and for most complex I substrates, Ca2+ effects are biphasic: small increases in Ca2+ concentration stimulated, while large increases inhibited mitochondrial respiration. Furthermore, significant differences in substrate- and Ca2+-dependent O2 utilization towards ATP production between heart and OM mitochondria were observed. With PM and PCM substrates, Ca2+ showed more prominent stimulatory effects in OM than in heart mitochondria, while with GM and AM substrates, Ca2+ had similar biphasic regulatory effects in both OM and heart mitochondria. In contrast, with complex II substrate SUC + ROT, only inhibitory effects on mitochondrial respiration was observed in both the heart and the OM. We conclude that the regulatory effects of Ca2+ on mitochondrial OxPhos and ATP synthesis are biphasic, substrate-dependent, and tissue-specific.

Mechanistic computational modeling of the kinetics and regulation of NADPH oxidase 2 assembly and activation facilitating superoxide production.

Reactive oxygen species (ROS) play a crucial role in many physiological processes. However, ROS overproduction leads to oxidative stress, which plays a critical role in cell injury/death and the pathogenesis of many diseases. Members of NADPH oxidase (NOX) family, most of which are comprised of membrane and cytosolic components, are known to be the major nonmitochondrial sources of ROS in many cells. NOX2 is a widely-expressed and well-studied NOX family member, which is activated upon assembly of its membrane subunits gp91 phox and p22 phox with its cytosolic subunits p40 phox , p47 phox , p67 phox , and Rac, facilitating ROS production. NOX2 activation is also enhanced by GTP and inhibited by GDP. However, there remains a lack of a mechanistic, quantitative, and integrated understanding of the kinetics and regulation of the assembly of these subunits and their relative contributions toward NOX2 activation and ROS production. Toward this end, we have developed a mechanistic computational model, which incorporates a generalized random rapid equilibrium binding mechanism for NOX2 assembly and activation as well as regulations by GTP (activation), GDP (inhibition), and individual subunits enhancing the binding of other subunits (mutual binding enhancement). The resulting model replicates diverse published kinetic data, including subunit concentration-dependent NOX2 activation and ROS production, under different assay conditions, with appropriate estimates of the unknown model parameters. The model provides a mechanistic, quantitative, and integrated framework for investigating the critical roles of NOX2 subunits in NOX2 assembly and activation facilitating ROS production in a variety of physiological and pathophysiological conditions. However, there is also a need for better quantitative kinetic data based on current understanding of NOX2 assembly and activation in order to test and further develop this model.

Network mechanisms and dysfunction within an integrated computational model of progression through mitosis in the human cell cycle.

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.