Heterogeneous kinetics of AKT signaling in individual cells are accounted for by variable protein concentration

In most solid cancers, cells harboring oncogenic mutations represent only a sub-fraction of the entire population. Within this sub-fraction the expression level of mutated proteins can vary significantly due to cellular variability limiting the efficiency of targeted therapy. To address the causes of the heterogeneity, we performed a systematic analysis of one of the most frequently mutated pathways in cancer cells, the phosphatidylinositol 3 kinase (PI3K) signaling pathway. Among others PI3K signaling is activated by the hepatocyte growth factor (HGF) that regulates proliferation of hepatocytes during liver regeneration but also fosters tumor cell proliferation. HGF mediated responses of PI3K signaling were monitored both at the single cell and cell population level in primary mouse hepatocytes and in the hepatoma cell line Hepa1_6. Interestingly, we observed that the HGF mediated AKT responses at the level of individual cells is rather heterogeneous. However, the overall average behavior of the single cells strongly resembled the dynamics of AKT activation determined at the cell population level. To gain insights into the molecular cause for the observed heterogeneous behavior of individual cells, we employed dynamic mathematical modeling in a stochastic framework. Our analysis demonstrated that intrinsic noise was not sufficient to explain the observed kinetic behavior, but rather the importance of extrinsic noise has to be considered. Thus, distinct from gene expression in the examined signaling pathway fluctuations of the reaction rates has only a minor impact whereas variability in the concentration of the various signaling components even in a clonal cell population is a key determinant for the kinetic behavior

Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib.

IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines

Context-specific flow through the MEK/ERK module produces cell- and ligand-specific patterns of ERK single and double phosphorylation.

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of the MAPK kinase MEK and its targets extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK

T160‐phosphorylated CDK

Liver regeneration is a tightly controlled process mainly achieved by proliferation of usually quiescent hepatocytes. The specific molecular mechanisms ensuring cell division only in response to proliferative signals such as hepatocyte growth factor (HGF) are not fully understood. Here, we combined quantitative time-resolved analysis of primary mouse hepatocyte proliferation at the single cell and at the population level with mathematical modeling. We showed that numerous G1/S transition components are activated upon hepatocyte isolation whereas DNA replication only occurs upon additional HGF stimulation. In response to HGF, Cyclin:CDK complex formation was increased, p21 rather than p27 was regulated, and Rb expression was enhanced. Quantification of protein levels at the restriction point showed an excess of CDK2 over CDK4 and limiting amounts of the transcription factor E2F-1. Analysis with our mathematical model revealed that T160 phosphorylation of CDK2 correlated best with growth factor-dependent proliferation, which we validated experimentally on both the population and the single cell level. In conclusion, we identified CDK2 phosphorylation as a gate-keeping mechanism to maintain hepatocyte quiescence in the absence of HGF

Inpatient vs outpatient management and timing of delivery of uncomplicated monochorionic monoamniotic twin pregnancy: the MONOMONO study

Monoamniotic twins are at increased risk of perinatal complications. Perinatal mortality has been reported to be high, primarily related to cord entanglement. International guidelines made no recommendation regarding whether these women should be managed in the hospital or can be safely managed in outpatient settings. Moreover, timing of planned delivery in these women is also a subject of debate

Social status and cardiovascular disease: a Mediterranean case. Results from the Italian Progetto CUORE cohort study

<p>Abstract</p> <p>Background</p> <p>Social factors could offer useful information for planning prevention strategy for cardiovascular diseases. This analysis aims to explore the relationship between education, marital status and major cardiovascular risk factors and to evaluate the role of social status indicators in predicting cardiovascular events and deaths in several Italian cohorts.</p> <p>Methods</p> <p>The population is representative of Italy, where the incidence of the disease is low. Data from the Progetto CUORE, a prospective study of cohorts enrolled between 1983-1997, were used; 7520 men and 13127 women aged 35-69 years free of previous cardiovascular events and followed for an average of 11 years. Educational level and marital status were used as the main indicators of social status.</p> <p>Results</p> <p>About 70% of the studied population had a low or medium level of education (less than high school) and more than 80% was married or cohabitating. There was an inverse relationship between educational level and major cardiovascular risk factors in both genders. Significantly higher major cardiovascular risk factors were detected in married or cohabitating women, with the exception of smoking. Cardiovascular risk score was lower in married or cohabitating men. No relationship between incidence of cardiac events and the two social status indicators was observed. Cardiovascular case-fatality was significantly higher in men who were not married and not cohabitating (HR 3.20, 95%CI: 2.21-4.64). The higher cardiovascular risk observed in those with a low level of education deserves careful attention even if during the follow-up it did not seem to determine an increase of cardiac events.</p> <p>Conclusions</p> <p>Preventive interventions on cardiovascular risk should be addressed mostly to people with less education. Cardiovascular risk score and case-fatality resulted higher in men living alone while cardiovascular factors were higher in women married or cohabitating. Such gender differences seem peculiar of our population and require further research on unexpected cultural and behavioural influences.</p