Predictive Approach Identifies Molecular Targets and Interventions to Restore Angiogenesis in Wounds With Delayed Healing

Impaired angiogenesis is a hallmark of wounds with delayed healing, and currently used therapies to restore angiogenesis have limited efficacy. Here, we employ a computational simulation-based approach to identify influential molecular and cellular processes, as well as protein targets, whose modulation may stimulate angiogenesis in wounds. We developed a mathematical model that captures the time courses for platelets, 9 cell types, 29 proteins, and oxygen, which are involved in inflammation, proliferation, and angiogenesis during wound healing. We validated our model using previously published experimental data. By performing global sensitivity analysis on thousands of simulated wound-healing scenarios, we identified six processes (among the 133 modeled in total) whose modulation may improve angiogenesis in wounds. By simulating knockouts of 25 modeled proteins and by simulating different wound-oxygenation levels, we identified four proteins [namely, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and angiopoietin-2 (ANG-2)], as well as oxygen, as therapeutic targets for stimulating angiogenesis in wounds. Our modeling results indicated that simultaneous inhibition of TGF-β and supplementation of either FGF-2 or ANG-2 could be more effective in stimulating wound angiogenesis than the modulation of either protein alone. Our findings suggest experimentally testable intervention strategies to restore angiogenesis in wounds with delayed healing

Kinetic model facilitates analysis of fibrin generation and its modulation by clotting factors: implications for hemostasis-enhancing therapies

We developed a computational model that accounts for essential kinetic features of thrombin generation, fibrin formation, and fibrinolysis in diverse in vitro systems. We applied it to characterize strategies to improve hemostasis

Positive Autoregulation Shapes Response Timing and Intensity in Two-component Signal Transduction Systems

Positive feedback loops are regulatory elements that can modulate expression output, kinetics and noise in genetic circuits. Transcriptional regulators participating in such loops are often expressed from two promoters, one constitutive and one autoregulated. Here, we investigate the interplay of promoter strengths and the intensity of the stimulus activating the transcriptional regulator in defining the output of a positively autoregulated genetic circuit. Using a mathematical model of twocomponent regulatory systems, which are present in all domains of life, we establish that positive feedback strongly affects the steady-state output levels at both low and high levels of stimulus if the constitutive promoter of the regulator is weak. By contrast, the effect of positive feedback is negligible when the constitutive promoter is sufficiently strong, unless the stimulus intensity is very high. Furthermore, we determine that positive feedback can affect both transient and steady state output levels even in the simplest genetic regulatory systems. We tested our modeling predictions by abolishing the positive feedback loop in the two-component regulatory system PhoP/PhoQ of Salmonella enterica, which resulted in diminished induction of PhoP-activated genes

Evolution and Dynamics of Regulatory Architectures Controlling Polymyxin B Resistance in Enteric Bacteria

Complex genetic networks consist of structural modules that determine the levels and timing of a cellular response. While the functional properties of the regulatory architectures that make up these modules have been extensively studied, the evolutionary history of regulatory architectures has remained largely unexplored. Here, we investigate the transition between direct and indirect regulatory pathways governing inducible resistance to the antibiotic polymyxin B in enteric bacteria. We identify a novel regulatory architecture—designated feedforward connector loop—that relies on a regulatory protein that connects signal transduction systems post-translationally, allowing one system to respond to a signal activating another system. The feedforward connector loop is characterized by rapid activation, slow deactivation, and elevated mRNA expression levels in comparison with the direct regulation circuit. Our results suggest that, both functionally and evolutionarily, the feedforward connector loop is the transitional stage between direct transcriptional control and indirect regulation

Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants.

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS

Functional Assessment of EnvZ/OmpR Two-Component System in Shewanella oneidensis

EnvZ and OmpR constitute the bacterial two-component signal transduction system known to mediate osmotic stress response in a number of Gram-negative bacteria. In an effort to understand the mechanism through which Shewanella oneidensis senses and responds to environmental osmolarity changes, structure of the ompR-envZ operon was determined with Northern blotting assay and roles of the EnvZ/OmpR two-component system in response to various stresses were investigated with mutational analysis, quantitative reverse transcriptase PCR (qRT-PCR), and phenotype microarrays. Results from the mutational analysis and qRT-PCR suggested that the EnvZ/OmpR system contributed to osmotic stress response of S. oneidensis and very likely engaged a similar strategy employed by E. coli, which involved reciprocal regulation of two major porin coding genes. Additionally, the ompR-envZ system was also found related to cell motility. We further showed that the ompR-envZ dependent regulation of porin genes and motility resided almost completely on ompR and only partially on envZ, indicating additional mechanisms for OmpR phosphorylation. In contrast to E. coli lacking ompR-envZ, however, growth of S. oneidensis did not show a significant dependence on ompR-envZ even under osmotic stress. Further analysis with phenotype microarrays revealed that the S. oneidensis strains lacking a complete ompR-envZ system displayed hypersensitivities to a number of agents, especially in alkaline environment. Taken together, our results suggest that the function of the ompR-envZ system in S. oneidensis, although still connected with osmoregulation, has diverged considerably from that of E. coli. Additional mechanism must exist to support growth of S. oneidensis under osmotic stress

Signal integration in bacterial two-component regulatory systems

Two-component systems (TCSs) and phosphorelays are key mediators of bacterial signal transduction. The signals activating these systems promote the phosphorylated state of a response regulator, which is generally the form that carries out specific functions such as binding to DNA and catalysis of biochemical reactions. An emerging class of proteins—termed TCS connectors—modulate the output of TCSs by affecting the phosphorylation state of response regulators. TCS connectors use different mechanisms of action for signal integration, as well as in the coordination and fine-tuning of cellular processes. Present in both Gram-positive and Gram-negative bacteria, TCS connectors are critical for a variety of physiological functions including sporulation, competence, antibiotic resistance, and the transition to stationary phase

Computational Identification of Mechanistic Factors That Determine the Timing and Intensity of the Inflammatory Response

<div><p>Timely resolution of inflammation is critical for the restoration of homeostasis in injured or infected tissue. Chronic inflammation is often characterized by a persistent increase in the concentrations of inflammatory cells and molecular mediators, whose distinct amount and timing characteristics offer an opportunity to identify effective therapeutic regulatory targets. Here, we used our recently developed computational model of local inflammation to identify potential targets for molecular interventions and to investigate the effects of individual and combined inhibition of such targets. This was accomplished via the development and application of computational strategies involving the simulation and analysis of thousands of inflammatory scenarios. We found that modulation of macrophage influx and efflux is an effective potential strategy to regulate the amount of inflammatory cells and molecular mediators in both normal and chronic inflammatory scenarios. We identified three molecular mediators − tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and the chemokine CXCL8 − as potential molecular targets whose individual or combined inhibition may robustly regulate both the amount and timing properties of the kinetic trajectories for neutrophils and macrophages in chronic inflammation. Modulation of macrophage flux, as well as of the abundance of TNF-α, TGF-β, and CXCL8, may improve the resolution of chronic inflammation.</p></div

Inflammation index regulation by modifying the model parameters identified in the sensitivity and correlation analyses.

<p>Shown are the normalized total neutrophil (<b>a</b>) and total macrophage (<b>b</b>) concentrations during chronic (solid red) inflammation. We simulated chronic inflammation by increasing the macrophage influx rate parameter by 5 fold, a strategy we employed in our previous model [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004460#pcbi.1004460.ref011" target="_blank">11</a>]. These curves appeared in our previous model results (see Figure 5 in [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004460#pcbi.1004460.ref011" target="_blank">11</a>]) and are reproduced here for comparison purposes. Neutrophil restoration modifications included increasing the CXCL8 degradation rate by 5 fold (solid black), increasing the TGF-β degradation rate by 15 fold (dashed black), and increasing TNF-α inhibition by TGF-β by 5 fold (dotted black). Macrophage restoration modifications comprised increasing the TNF-α degradation rate by 20 fold (black solid), increasing the macrophage efflux rate by 2 fold (dashed black), and increasing IL-10 production rate by 5 fold (dotted black). These parameter-specific fold changes were chosen so as to reduce the respective parameter-specific target inflammation indices by at least ~10% during a chronic inflammatory scenario (the target indices are defined in the description of the six parameter modification strategies in the Results Section). All the model-predicted values were normalized to the respective maximum values from the chronic inflammation simulations. The tables in the figure show the quantitative index values calculated from the respective simulated kinetic trajectories.</p

Total neutrophil and total macrophage concentrations for inflammatory mediator inhibition at different time points.

<p>Green and red lines represent model predictions for acute and chronic inflammation, respectively. We simulated chronic inflammation by increasing the initial platelet concentration by 100 fold, increasing the macrophage influx rate parameter by 1.5 fold, and simultaneously decreasing the platelet degradation rate parameter by 1.5 fold. In all the subplots, the time points of inhibitor addition (represented by the black lines) are as follows: dotted, 24 h; dashed, 48 h; and solid, 72 h. Shown are normalized neutrophil (<b>a</b>-<b>c</b>) and normalized macrophage (<b>d</b>-<b>f</b>) kinetics for CXCL8, TNF-α, and combined CXCL8 + TNF-α inhibition. The concentration of each of the considered inhibitors was equal to 200 nM. All model-predicted values were normalized to the respective maximum values from the chronic inflammation simulations. In our simulations, the 72 h time point was chosen for mediator inhibitor addition based on an experimental study that evaluated the efficacy of several pro-resolution drugs in a mouse peritoneal infection model [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004460#pcbi.1004460.ref017" target="_blank">17</a>]. Furthermore, we chose the 24 h and 48 h time points for mediator inhibitor addition because they represent the times at which the neutrophil and macrophage concentrations peak, respectively, in our acute inflammation simulation [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004460#pcbi.1004460.ref011" target="_blank">11</a>].</p