Enhancement of essential cofactors for in vivo biocatalysis.

A scarcity of cofactors, necessary metabolites or substrates for in vivo enzymatic reactions, is among the major barriers for product synthesis in metabolically engineered cells. This work compares our recently developed cofactor-boosting strategy, which uses xylose reductase (XR) and lactose to increase the intracellular levels of reduced or oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), adenosine triphosphate (ATP) and acetyl coenzymeA (acetyl-CoA), with other previously reported methods. We demonstrated that the XR/lactose approach enhances levels of sugar alcohols and sugar phosphates, which leads to elevated levels of crucial cofactors required by specific metabolic pathways. The patterns of cofactor enhancement are not uniform and depend upon the specific pathway components that are overexpressed. We term this model the "user-pool" model. Here, we investigated metabolite alteration in the fatty-alcohol-producing system in the presence of XR/lactose within an early time frame (5 min after the bioconversion started). All metabolite data were analyzed using untargeted metabolomics. We found that the XR/lactose system could improve fatty-alcohol production as early as 5 min after the bioconversion started. The enhancement of key cofactors and intermediates, such as hexitol, NAD(P)H, ATP, 3-phosphoglycerate, acetyl-CoA, 6-phosphogluconate (6-PG) and glutathione, was consistent with those previously reported on a longer time scale (after 1 h). However, measurements performed at the early time reported here showed detectable differences in metabolite enhancement patterns, such as those of ATP, NADPH, acetyl-CoA and glutathione. These data could serve as a basis for future analysis of metabolic flux alteration by the XR/lactose system. Comparative analysis of the cofactor enhancement by XR and other methods suggests that XR/lactose can serve as a simple tool to increase levels of various cofactors for microbial cell factories.

Monomeric α-synuclein activates the plasma membrane calcium pump.

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

Modeling indicates degradation of mRNA and protein as a potential regulation mechanisms during cold acclimation.

Plants are constantly exposed to temperature fluctuations, which have direct effects on all cellular reactions because temperature influences reaction likelihood and speed. Chloroplasts are crucial to temperature acclimation responses of plants, due to their photosynthetic reactions whose products play a central role in plant metabolism. Consequently, chloroplasts serve as sensors of temperature changes and are simultaneously major targets of temperature acclimation. The core subunits of the complexes involved in the light reactions of photosynthesis are encoded in the chloroplast. As a result, it is assumed that temperature acclimation in plants requires regulatory responses in chloroplast gene expression and protein turnover. We conducted western blot experiments to assess changes in the accumulation of two photosynthetic complexes (PSII, and Cytb6f complex) and the ATP synthase in tobacco plants over two days of acclimation to low temperature. Surprisingly, the concentration of proteins within the chloroplast varied negligibly compared to controls. To explain this observation, we used a simplified Ordinary Differential Equation (ODE) model of transcription, translation, mRNA degradation and protein degradation to explain how the protein concentration can be kept constant. This model takes into account temperature effects on these processes. Through simulations of the ODE model, we show that mRNA and protein degradation are possible targets for control during temperature acclimation. Our model provides a basis for future directions in research and the analysis of future results.

Acclimation in plants - the Green Hub consortium.

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.

Deep Hidden Physics Modeling of Cell Signaling Networks.

According to the WHO, cancer is the second most common cause of death worldwide. The social and economic damage caused by cancer is high and rising. In Europe, the annual direct medical expenses alone amount to more than €129 billion. This results in an urgent need for new and sustainable therapeutics, which has currently not been met by the pharmaceutical industry; only 3.4% of cancer drugs entering Phase I clinical trials get to market. Phosphorylation sites are parts of the core machinery of kinase signaling networks, which are known to be dysfunctional in all types of cancer. Indeed, kinases are the second most common drug target yet. However, these inhibitors block all functions of a protein, and they commonly lead to the development of resistance and increased toxicity. To facilitate global and mechanistic modeling of cancer and clinically relevant cell signaling networks, the community will have to develop sophisticated data-driven deep-learning and mechanistic computational models that generate in silico probabilistic predictions of molecular signaling network rearrangements causally implicated in cancer.

Loss of hepatic Mboat7 leads to liver fibrosis.

OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.

An Antimicrobial Peptide Induces FIG1-Dependent Cell Death During Cell Cycle Arrest in Yeast.

Although most antibiotics act on cells that are actively dividing and non-dividing cells such as in microbe sporulation or cancer stem cells represent a new paradigm for the control of disease. In addition to their relevance to health, such antibiotics may promote our understanding of the relationship between the cell cycle and cell death. No antibiotic specifically acting on microbial cells arrested in their cell cycle has been identified until the present time. In this study we used an antimicrobial peptide derived from α-pheromone, IP-1, targeted against MATa Saccharomyces cerevisiae cells in order to assess its dependence on cell cycle arrest to kill cells. Analysis by flow cytometry and fluorescence microscopy of various null mutations of genes involved in biological processes activated by the pheromone pathway (the mitogen-activated protein kinase pathway, cell cycle arrest, cell proliferation, autophagy, calcium influx) showed that IP-1 requires arrest in G0/G1 in order to kill yeast cells. Isolating cells in different cell cycle phases by elutriation provided further evidence that entry into cell cycle arrest, and not into G1 phase, is necessary if our peptide is to kill yeast cells. We also describe a variant of IP-1 that does not activate the pheromone pathway and consequently does not kill yeast cells that express the pheromone's receptor; the use of this variant peptide in combination with different cell cycle inhibitors that induce cell cycle arrest independently of the pheromone pathway confirmed that it is cell cycle arrest that is required for the cell death induced by this peptide in yeast. We show that the cell death induced by IP-1 differs from that induced by α-pheromone and depends on FIG1 in a way independent of the cell cycle arrest induced by the pheromone. Thus, IP-1 is the first molecule described that specifically kills microbial cells during cell cycle arrest, a subject of interest beyond the process of mating in yeast cells. The experimental system described in this study should be useful in the study of the mechanisms at play in the communication between cell cycle arrest and cell death on other organisms, hence promoting the development of new antibiotics.

Alterations of mTOR signaling impact metabolic stress resistance in colorectal carcinomas with BRAF and KRAS mutations.

Metabolic reprogramming is as a hallmark of cancer, and several studies have reported that BRAF and KRAS tumors may be accompanied by a deregulation of cellular metabolism. We investigated how BRAFV600E and KRASG12V affect cell metabolism, stress resistance and signaling in colorectal carcinoma cells driven by these mutations. KRASG12V expressing cells are characterized by the induction of glycolysis, accumulation of lactic acid and sensitivity to glycolytic inhibition. Notably mathematical modelling confirmed the critical role of MCT1 designating the survival of KRASG12V cells. Carcinoma cells harboring BRAFV600E remain resistant towards alterations of glucose supply or application of signaling or metabolic inhibitors. Altogether these data demonstrate that an oncogene-specific decoupling of mTOR from AMPK or AKT signaling accounts for alterations of resistance mechanisms and metabolic phenotypes. Indeed the inhibition of mTOR in BRAFV600E cells counteracts the metabolic predisposition and demonstrates mTOR as a potential target in BRAFV600E-driven colorectal carcinomas.

Differential T cell response against BK virus regulatory and structural antigens: A viral dynamics modelling approach.

BK virus (BKV) associated nephropathy affects 1-10% of kidney transplant recipients, leading to graft failure in about 50% of cases. Immune responses against different BKV antigens have been shown to have a prognostic value for disease development. Data currently suggest that the structural antigens and regulatory antigens of BKV might each trigger a different mode of action of the immune response. To study the influence of different modes of action of the cellular immune response on BKV clearance dynamics, we have analysed the kinetics of BKV plasma load and anti-BKV T cell response (Elispot) in six patients with BKV associated nephropathy using ODE modelling. The results show that only a small number of hypotheses on the mode of action are compatible with the empirical data. The hypothesis with the highest empirical support is that structural antigens trigger blocking of virus production from infected cells, whereas regulatory antigens trigger an acceleration of death of infected cells. These differential modes of action could be important for our understanding of BKV resolution, as according to the hypothesis, only regulatory antigens would trigger a fast and continuous clearance of the viral load. Other hypotheses showed a lower degree of empirical support, but could potentially explain the clearing mechanisms of individual patients. Our results highlight the heterogeneity of the dynamics, including the delay between immune response against structural versus regulatory antigens, and its relevance for BKV clearance. Our modelling approach is the first that studies the process of BKV clearance by bringing together viral and immune kinetics and can provide a framework for personalised hypotheses generation on the interrelations between cellular immunity and viral dynamics.

Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation.

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.

Estimation of immune cell content in tumour tissue using single-cell RNA-seq data.

As interactions between the immune system and tumour cells are governed by a complex network of cell-cell interactions, knowing the specific immune cell composition of a solid tumour may be essential to predict a patient's response to immunotherapy. Here, we analyse in depth how to derive the cellular composition of a solid tumour from bulk gene expression data by mathematical deconvolution, using indication-specific and cell type-specific reference gene expression profiles (RGEPs) from tumour-derived single-cell RNA sequencing data. We demonstrate that tumour-derived RGEPs are essential for the successful deconvolution and that RGEPs from peripheral blood are insufficient. We distinguish nine major cell types, as well as three T cell subtypes. Using the tumour-derived RGEPs, we can estimate the content of many tumours associated immune and stromal cell types, their therapeutically relevant ratios, as well as an improved gene expression profile of the malignant cells.

Comprehensive mathematical model of oxidative phosphorylation valid for physiological and pathological conditions.

We developed a mathematical model of oxidative phosphorylation (OXPHOS) that allows for a precise description of mitochondrial function with respect to the respiratory flux and the ATP production. The model reproduced flux-force relationships under various experimental conditions (state 3 and 4, uncoupling, and shortage of respiratory substrate) as well as time courses, exhibiting correct P/O ratios. The model was able to reproduce experimental threshold curves for perturbations of the respiratory chain complexes, the F1 F0 -ATP synthase, the ADP/ATP carrier, the phosphate/OH carrier, and the proton leak. Thus, the model is well suited to study complex interactions within the OXPHOS system, especially with respect to physiological adaptations or pathological modifications, influencing substrate and product affinities or maximal catalytic rates. Moreover, it could be a useful tool to study the role of OXPHOS and its capacity to compensate or enhance physiopathologies of the mitochondrial and cellular energy metabolism.

Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a Dynamic In Vitro Model.

UNLABELLED: Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. In order to evaluate viral transfer between infected PBMC and EC, cocultivation assays were performed. Only EHV-1 was transferred from PBMC to EC, and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to that of cells infected with parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taking the results together, we conclude that systemic spread and EC infection by EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer. IMPORTANCE: EHV-1 is widespread throughout the world and causes substantial economic losses through outbreaks of respiratory disease, abortion, and myeloencephalopathy. Despite many years of research, no fully protective vaccines have been developed, and several aspects of viral pathogenesis still need to be uncovered. In the current study, we investigated the molecular mechanisms that facilitate the cell-associated viremia, which is arguably the most important aspect of EHV-1 pathogenesis. The newly discovered functions of gB and pUS3 add new facets to their previously reported roles. Due to the conserved nature of cell-associated viremia among numerous herpesviruses, these results are also very relevant for viruses such as varicella-zoster virus, pseudorabies virus, human cytomegalovirus, and others. In addition, the constructed mutant and recombinant viruses exhibit potent in vitro replication but have significant defects in certain stages of the disease course. These viruses therefore show much promise as candidates for future live vaccines.

Yeast mating and image-based quantification of spatial pattern formation.

Communication between cells is a ubiquitous feature of cell populations and is frequently realized by secretion and detection of signaling molecules. Direct visualization of the resulting complex gradients between secreting and receiving cells is often impossible due to the small size of diffusing molecules and because such visualization requires experimental perturbations such as attachment of fluorescent markers, which can change diffusion properties. We designed a method to estimate such extracellular concentration profiles in vivo by using spatiotemporal mathematical models derived from microscopic analysis. This method is applied to populations of thousands of haploid yeast cells during mating in order to quantify the extracellular distributions of the pheromone α-factor and the activity of the aspartyl protease Bar1. We demonstrate that Bar1 limits the range of the extracellular pheromone signal and is critical in establishing α-factor concentration gradients, which is crucial for effective mating. Moreover, haploid populations of wild type yeast cells, but not BAR1 deletion strains, create a pheromone pattern in which cells differentially grow and mate, with low pheromone regions where cells continue to bud and regions with higher pheromone levels and gradients where cells conjugate to form diploids. However, this effect seems to be exclusive to high-density cultures. Our results show a new role of Bar1 protease regulating the pheromone distribution within larger populations and not only locally inside an ascus or among few cells. As a consequence, wild type populations have not only higher mating efficiency, but also higher growth rates than mixed MATa bar1Δ/MATα cultures. We provide an explanation of how a rapidly diffusing molecule can be exploited by cells to provide spatial information that divides the population into different transcriptional programs and phenotypes.

Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin.

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.

A variable fork rate affects timing of origin firing and S phase dynamics in Saccharomyces cerevisiae.

Activation (in the following referred to as firing) of replication origins is a continuous and irreversible process regulated by availability of DNA replication molecules and cyclin-dependent kinase activities, which are often altered in human cancers. The temporal, progressive origin firing throughout S phase appears as a characteristic replication profile, and computational models have been developed to describe this process. Although evidence from yeast to human indicates that a range of replication fork rates is observed experimentally in order to complete a timely S phase, those models incorporate velocities that are uniform across the genome. Taking advantage of the availability of replication profiles, chromosomal position and replication timing, here we investigated how fork rate may affect origin firing in budding yeast. Our analysis suggested that patterns of origin firing can be observed from a modulation of the fork rate that strongly correlates with origin density. Replication profiles of chromosomes with a low origin density were fitted with a variable fork rate, whereas for the ones with a high origin density a constant fork rate was appropriate. This indeed supports the previously reported correlation between inter-origin distance and fork rate changes. Intriguingly, the calculated correlation between fork rate and timing of origin firing allowed the estimation of firing efficiencies for the replication origins. This approach correctly retrieved origin efficiencies previously determined for chromosome VI and provided testable prediction for other chromosomal origins. Our results gain deeper insights into the temporal coordination of genome duplication, indicating that control of the replication fork rate is required for the timely origin firing during S phase.

Molecular insights into reprogramming-initiation events mediated by the OSKM gene regulatory network.

Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal.

Triple-negative breast cancer: present challenges and new perspectives.

Triple-negative breast cancers (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2), are typically associated with poor prognosis, due to aggressive tumor phenotype(s), only partial response to chemotherapy and present lack of clinically established targeted therapies. Advances in the design of individualized strategies for treatment of TNBC patients require further elucidation, by combined 'omics' approaches, of the molecular mechanisms underlying TNBC phenotypic heterogeneity, and the still poorly understood association of TNBC with BRCA1 mutations. An overview is here presented on TNBC profiling in terms of expression signatures, within the functional genomic breast tumor classification, and ongoing efforts toward identification of new therapy targets and bioimaging markers. Due to the complexity of aberrant molecular patterns involved in expression, pathological progression and biological/clinical heterogeneity, the search for novel TNBC biomarkers and therapy targets requires collection of multi-dimensional data sets, use of robust multivariate data analysis techniques and development of innovative systems biology approaches.

A systems biology approach: modelling of Aquaporin-2 trafficking.

In healthy individuals, dehydration of the body leads to release of the hormone vasopressin from the pituitary. Via the bloodstream, vasopressin reaches the collecting duct cells in the kidney, where the water channel Aquaporin-2 (AQP2) is expressed. After stimulation of the vasopressin V2 receptor by vasopressin, intracellular AQP2-containing vesicles fuse with the apical plasma membrane of the collecting duct cells. This leads to increased water reabsorption from the pro-urine into the blood and therefore to enhanced retention of water within the body. Using existing biological data we propose a mathematical model of AQP-2 trafficking and regulation in collecting duct cells. Our model includes the vasopressin receptor, adenylate cyclase, protein kinase A, and intracellular as well as membrane located AQP2. To model the chemical reactions we used ordinary differential equations (ODEs) based on mass action kinetics. We employ known protein concentrations and time series data to estimate the kinetic parameters of our model and demonstrate its validity. Through generating, testing and ranking different versions of the model, we show that some model versions can describe the data well as soon as important regulatory parts such as the reduction of the signal by internalization of the vasopressin-receptor or the negative feedback loop representing phosphodiesterase activity are included. We perform time-dependent sensitivity analysis to identify the reactions that have the greatest influence on the cAMP and membrane located AQP2 levels over time. We predict the time courses for membrane located AQP2 at different vasopressin concentrations, compare them with newly generated data and discuss the competencies of the model.

Towards a systems biology approach to mammalian cell cycle: modeling the entrance into S phase of quiescent fibroblasts after serum stimulation.

BACKGROUND: The cell cycle is a complex process that allows eukaryotic cells to replicate chromosomal DNA and partition it into two daughter cells. A relevant regulatory step is in the G0/G1 phase, a point called the restriction (R) point where intracellular and extracellular signals are monitored and integrated.Subcellular localization of cell cycle proteins is increasingly recognized as a major factor that regulates cell cycle transitions. Nevertheless, current mathematical models of the G1/S networks of mammalian cells do not consider this aspect. Hence, there is a need for a computational model that incorporates this regulatory aspect that has a relevant role in cancer, since altered localization of key cell cycle players, notably of inhibitors of cyclin-dependent kinases, has been reported to occur in neoplastic cells and to be linked to cancer aggressiveness. RESULTS: The network of the model components involved in the G1 to S transition process was identified through a literature and web-based data mining and the corresponding wiring diagram of the G1 to S transition drawn with Cell Designer notation. The model has been implemented in Mathematica using Ordinary Differential Equations. Time-courses of level and of sub-cellular localization of key cell cycle players in mouse fibroblasts re-entering the cell cycle after serum starvation/re-feeding have been used to constrain network design and parameter determination. The model allows to recapitulate events from growth factor stimulation to the onset of S phase. The R point estimated by simulation is consistent with the R point experimentally determined. CONCLUSION: The major element of novelty of our model of the G1 to S transition is the explicit modeling of cytoplasmic/nuclear shuttling of cyclins, cyclin-dependent kinases, their inhibitor and complexes. Sensitivity analysis of the network performance newly reveals that the biological effect brought about by Cki overexpression is strictly dependent on whether the Cki is promoting nuclear translocation of cyclin/Cdk containing complexes.