Syntactic Markovian Bisimulation for Chemical Reaction Networks

In chemical reaction networks (CRNs) with stochastic semantics based on continuous-time Markov chains (CTMCs), the typically large populations of species cause combinatorially large state spaces. This makes the analysis very difficult in practice and represents the major bottleneck for the applicability of minimization techniques based, for instance, on lumpability. In this paper we present syntactic Markovian bisimulation (SMB), a notion of bisimulation developed in the Larsen-Skou style of probabilistic bisimulation, defined over the structure of a CRN rather than over its underlying CTMC. SMB identifies a lumpable partition of the CTMC state space a priori, in the sense that it is an equivalence relation over species implying that two CTMC states are lumpable when they are invariant with respect to the total population of species within the same equivalence class. We develop an efficient partition-refinement algorithm which computes the largest SMB of a CRN in polynomial time in the number of species and reactions. We also provide an algorithm for obtaining a quotient network from an SMB that induces the lumped CTMC directly, thus avoiding the generation of the state space of the original CRN altogether. In practice, we show that SMB allows significant reductions in a number of models from the literature. Finally, we study SMB with respect to the deterministic semantics of CRNs based on ordinary differential equations (ODEs), where each equation gives the time-course evolution of the concentration of a species. SMB implies forward CRN bisimulation, a recently developed behavioral notion of equivalence for the ODE semantics, in an analogous sense: it yields a smaller ODE system that keeps track of the sums of the solutions for equivalent species.Comment: Extended version (with proofs), of the corresponding paper published at KimFest 2017 (http://kimfest.cs.aau.dk/

Reverse Engineering Gene Networks with ANN: Variability in Network Inference Algorithms

Motivation :Reconstructing the topology of a gene regulatory network is one of the key tasks in systems biology. Despite of the wide variety of proposed methods, very little work has been dedicated to the assessment of their stability properties. Here we present a methodical comparison of the performance of a novel method (RegnANN) for gene network inference based on multilayer perceptrons with three reference algorithms (ARACNE, CLR, KELLER), focussing our analysis on the prediction variability induced by both the network intrinsic structure and the available data. Results: The extensive evaluation on both synthetic data and a selection of gene modules of "Escherichia coli" indicates that all the algorithms suffer of instability and variability issues with regards to the reconstruction of the topology of the network. This instability makes objectively very hard the task of establishing which method performs best. Nevertheless, RegnANN shows MCC scores that compare very favorably with all the other inference methods tested. Availability: The software for the RegnANN inference algorithm is distributed under GPL3 and it is available at the corresponding author home page (http://mpba.fbk.eu/grimaldi/regnann-supmat

Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells

Inhibitory effects of retinoic acid metabolism blocking agents (RAMBAs) on the growth of human prostate cancer cells and LNCaP prostate tumour xenografts in SCID mice

In recent studies, we have identified several highly potent all-trans-retinoic acid (ATRA) metabolism blocking agents (RAMBAs). On the basis of previous effects of liarozole (a first-generation RAMBA) on the catabolism of ATRA and on growth of rat Dunning R3227G prostate tumours, we assessed the effects of our novel RAMBAs on human prostate tumour (PCA) cell lines. We examined three different PCA cell lines to determine their capacity to induce P450-mediated oxidation of ATRA. Among the three different cell lines, enhanced catabolism was detected in LNCaP, whereas it was not found in PC-3 and DU-145. This catabolism was strongly inhibited by our RAMBAs, the most potent being VN/14-1, VN/50-1, VN/66-1, and VN/69-1 with IC50 values of 6.5, 90.0, 62.5, and 90.0 nM, respectively. The RAMBAs inhibited the growth of LNCaP cells with IC50 values in the μM-range. In LNCaP cell proliferation assays, VN/14-1, VN/50-1, VN/66-1, and VN/69-1 also enhanced by 47-, 60-, 70-, and 65-fold, respectively, the ATRA-mediated antiproliferative activity. We then examined the molecular mechanism underlying the growth inhibitory properties of ATRA alone and in combination with RAMBAs. The mechanism appeared to involve the induction of differentiation, cell-cycle arrest, and induction of apoptosis (TUNEL), involving increase in Bad expression and decrease in Bcl-2 expression. Treatment of LNCaP tumours growing in SCID mice with VN/66-1 and VN/69-1 resulted in modest but statistically significant tumour growth inhibition of 44 and 47%, respectively, while treatment with VN/14-1 was unexpectedly ineffective. These results suggest that some of our novel RAMBAs may be useful agents for the treatment of prostate cancer

Automatic Design of Synthetic Gene Circuits through Mixed Integer Non-linear Programming

Automatic design of synthetic gene circuits poses a significant challenge to synthetic biology, primarily due to the complexity of biological systems, and the lack of rigorous optimization methods that can cope with the combinatorial explosion as the number of biological parts increases. Current optimization methods for synthetic gene design rely on heuristic algorithms that are usually not deterministic, deliver sub-optimal solutions, and provide no guaranties on convergence or error bounds. Here, we introduce an optimization framework for the problem of part selection in synthetic gene circuits that is based on mixed integer non-linear programming (MINLP), which is a deterministic method that finds the globally optimal solution and guarantees convergence in finite time. Given a synthetic gene circuit, a library of characterized parts, and user-defined constraints, our method can find the optimal selection of parts that satisfy the constraints and best approximates the objective function given by the user. We evaluated the proposed method in the design of three synthetic circuits (a toggle switch, a transcriptional cascade, and a band detector), with both experimentally constructed and synthetic promoter libraries. Scalability and robustness analysis shows that the proposed framework scales well with the library size and the solution space. The work described here is a step towards a unifying, realistic framework for the automated design of biological circuits

Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion.

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial

IMPORTANCE: Secretory phospholipase A2(sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measurewas a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95%CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95%CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246. Copyright 2014 American Medical Association. All rights reserved

A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells

A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4- Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies