Restriction of access to the central cavity is a major contributor to substrate selectivity in plant ABCG transporters.

ABCG46 of the legume Medicago truncatula is an ABC-type transporter responsible for highly selective translocation of the phenylpropanoids, 4-coumarate, and liquiritigenin, over the plasma membrane. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of phylogenetic and biochemical analyses, AlphaFold2 structure prediction, molecular dynamics simulations, and mutagenesis. We discovered an unusually narrow transient access path to the central cavity of MtABCG46 that constitutes an initial filter responsible for the selective translocation of phenylpropanoids through a lipid bilayer. Furthermore, we identified remote residue F562 as pivotal for maintaining the stability of this filter. The determination of individual amino acids that impact the selective transport of specialized metabolites may provide new opportunities associated with ABCGs being of interest, in many biological scenarios.

Comparing Petri net-based models of biological systems using Holmes.

MOTIVATION: The first and necessary step in systems approach to study biological phenomena is building a formal model. One of the possibilities is to construct a model based on Petri nets. They have an intuitive graphical representation on one hand, and on the other, can be analyzed using formal mathematical methods. Finding homologies or conserved processes playing important roles in various biological systems can be done by comparing models. The ones expressed as Petri nets are especially well-suited for such a comparison, but there is a lack of software tools for this task. RESULTS: To resolve this problem, a new analytical tool has been implemented in Holmes application and described in this article. It offers four different comparison methods, i.e. the ones based on t-invariants, decomposition, graphlets and branching vertices. AVAILABILITY AND IMPLEMENTATION: Available at http://www.cs.put.poznan.pl/mradom/Holmes/holmes.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System.

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein-cargo and protein-protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

A Stochastic Petri Net-Based Model of the Involvement of Interleukin 18 in Atherosclerosis.

Interleukin 18 (IL-18) is a proinflammatory and proatherogenic cytokine with pleiotropic properties, which is involved in T and NK cell maturation and the synthesis of other inflammatory cytokines and cell adhesion molecules. It plays a significant role in orchestrating the cytokine cascade, accelerates atherosclerosis and influences plaque vulnerability. To investigate the influence of IL-18 cytokine on atherosclerosis development, a stochastic Petri net model was built and then analyzed. First, MCT-sets and t-clusters were generated, then knockout and simulation-based analysis was conducted. The application of systems approach that was used in this research enabled an in-depth analysis of the studied phenomenon. Our results gave us better insight into the studied phenomenon and allow revealing that activation of macrophages by the classical pathway and IL-18-MyD88 signaling axis is crucial for the modeled process.

A Role of Inflammation and Immunity in Essential Hypertension-Modeled and Analyzed Using Petri Nets.

Recent studies have shown that the innate and adaptive immune system, together with low-grade inflammation, may play an important role in essential hypertension. In this work, to verify the importance of selected factors for the development of essential hypertension, we created a Petri net-based model and analyzed it. The analysis was based mainly on t-invariants, knockouts of selected fragments of the net and its simulations. The blockade of the renin-angiotensin (RAA) system revealed that the most significant effect on the emergence of essential hypertension has RAA activation. This blockade affects: (1) the formation of angiotensin II, (2) inflammatory process (by influencing C-reactive protein (CRP)), (3) the initiation of blood coagulation, (4) bradykinin generation via the kallikrein-kinin system, (5) activation of lymphocytes in hypertension, (6) the participation of TNF alpha in the activation of the acute phase response, and (7) activation of NADPH oxidase-a key enzyme of oxidative stress. On the other hand, we found that the blockade of the activation of the RAA system may not eliminate hypertension that can occur due to disturbances associated with the osmotically independent binding of Na in the interstitium. Moreover, we revealed that inflammation alone is not enough to trigger primary hypertension, but it can coexist with it. We believe that our research may contribute to a better understanding of the pathology of hypertension. It can help identify potential subprocesses, which blocking will allow better control of essential hypertension.

Holmes: a graphical tool for development, simulation and analysis of Petri net based models of complex biological systems.

SUMMARY: Model development and its analysis is a fundamental step in systems biology. The theory of Petri nets offers a tool for such a task. Since the rapid development of computer science, a variety of tools for Petri nets emerged, offering various analytical algorithms. From this follows a problem of using different programs to analyse a single model. Many file formats and different representations of results make the analysis much harder. Especially for larger nets the ability to visualize the results in a proper form provides a huge help in the understanding of their significance. We present a new tool for Petri nets development and analysis called Holmes. Our program contains algorithms for model analysis based on different types of Petri nets, e.g. invariant generator, Maximum Common Transitions (MCT) sets and cluster modules, simulation algorithms or knockout analysis tools. A very important feature is the ability to visualize the results of almost all analytical modules. The integration of such modules into one graphical environment allows a researcher to fully devote his or her time to the model building and analysis. AVAILABILITY AND IMPLEMENTATION: Available at http://www.cs.put.poznan.pl/mradom/Holmes/holmes.html. CONTACT: piotr@cs.put.poznan.pl.

Hemojuvelin-hepcidin axis modeled and analyzed using Petri nets.

Systems biology approach to investigate biological phenomena seems to be very promising because it is capable to capture one of the fundamental properties of living organisms, i.e. their inherent complexity. It allows for analysis biological entities as complex systems of interacting objects. The first and necessary step of such an analysis is building a precise model of the studied biological system. This model is expressed in the language of some branch of mathematics, as for example, differential equations. During the last two decades the theory of Petri nets has appeared to be very well suited for building models of biological systems. The structure of these nets reflects the structure of interacting biological molecules and processes. Moreover, on one hand, Petri nets have intuitive graphical representation being very helpful in understanding the structure of the system and on the other hand, there is a lot of mathematical methods and software tools supporting an analysis of the properties of the nets. In this paper a Petri net based model of the hemojuvelin-hepcidin axis involved in the maintenance of the human body iron homeostasis is presented. The analysis based mainly on T-invariants of the model properties has been made and some biological conclusions have been drawn.

Control of Cholesterol Metabolism Using a Systems Approach.

Cholesterol is an essential component of mammalian cells and is involved in many fundamental physiological processes; hence, its homeostasis in the body is tightly controlled, and any disturbance has serious consequences. Disruption of the cellular metabolism of cholesterol, accompanied by inflammation and oxidative stress, promotes the formation of atherosclerotic plaques and, consequently, is one of the leading causes of death in the Western world. Therefore, new drugs to regulate disturbed cholesterol metabolism are used and developed, which help to control cholesterol homeostasis but still do not entirely cure atherosclerosis. In this study, a Petri net-based model of human cholesterol metabolism affected by a local inflammation and oxidative stress, has been created and analyzed. The use of knockout of selected pathways allowed us to observe and study the effect of various combinations of commonly used drugs on atherosclerosis. The analysis results led to the conclusion that combination therapy, targeting multiple pathways, may be a fundamental concept in the development of more effective strategies for the treatment and prevention of atherosclerosis.

Full-size ABC transporters from the ABCG subfamily in medicago truncatula.

Full-size ATP-binding cassette (ABC) transporters belonging to the ABCG subfamily are unique for plants and fungi. There is growing evidence that certain of these proteins play a role in plant defense or signaling systems. As yet, a complete set of full-size ABCG protein genes has been inventoried and classified in only two plants: Arabidopsis thaliana and Oryza sativa. Recently, a domain-based clustering analysis has predicted the presence of at least 12 genes encoding such proteins in the Lotus japonicus genome. Here, we identify and classify 19 genes coding full-size ABCG proteins in Medicago truncatula, a model legume plant. We have found that the majority of these genes are expressed in roots and flowers whereas only a few are expressed in leaves. Expression of several has been induced upon pathogenic infection in both roots and leaves. ABCG messenger RNAs have been detected in root nodules forming during symbiosis of legume plants and nitrogen-fixing bacteria. The data presented provide a scaffold for further studies of the physiological function of Medicago ABCG transporters and their possible role in modulating plant-microorganism interactions.

Petri net-based model of the human DNA base excision repair pathway.

Cellular DNA is daily exposed to several damaging agents causing a plethora of DNA lesions. As a first aid to restore DNA integrity, several enzymes got specialized in damage recognition and lesion removal during the process called base excision repair (BER). A large number of DNA damage types and several different readers of nucleic acids lesions during BER pathway as well as two sub-pathways were considered in the definition of a model using the Petri net framework. The intuitive graphical representation in combination with precise mathematical analysis methods are the strong advantages of the Petri net-based representation of biological processes and make Petri nets a promising approach for modeling and analysis of human BER. The reported results provide new information that will aid efforts to characterize in silico knockouts as well as help to predict the sensitivity of the cell with inactivated repair proteins to different types of DNA damage. The results can also help in identifying the by-passing pathways that may lead to lack of pronounced phenotypes associated with mutations in some of the proteins. This knowledge is very useful when DNA damage-inducing drugs are introduced for cancer therapy, and lack of DNA repair is desirable for tumor cell death.

An Algorithm for Sequencing by Hybridization Based on an Alternating DNA Chip.

Sequencing by hybridization allows the reconstruction of the DNA string of a given length from smaller fragments. These fragments are obtained in the hybridization experiment in which the DNA hybridizes to a DNA chip. In a classical approach, the chip consists of all oligonucleotides of a given length, with only one type of oligonucleotide for each probe of the chip. In this paper, we propose an algorithm solving the non-classical case of SBH, where the chip probes consist set of oligonucleotides described by some specific pattern. We will present the definition of such a non-classical DNA chip and the algorithm solving a sequencing problem related to such a chip. Unlike recent metaheuristic approaches to the classical SBH problem, the proposed algorithm tries to find an exact sequence, and even in the presence of all the hybridization errors in spectrum is very often able to do so in a short time. If only negative errors from repetitions are allowed, then the algorithm is able to reconstruct sequences having length of thousands nucleotides.

The role of Fenton reaction in ROS-induced toxicity underlying atherosclerosis - modeled and analyzed using a Petri net-based approach.

The superoxide-driven Fenton reaction plays an important role in the transformation of poorly reactive radicals into highly reactive ones. These highly reactive species (ROS), especially hydroxyl radicals can lead to many disturbances contributing to the endothelial dysfunction being a starting point for atherosclerosis. Although, iron has been identified as a possible culprit influencing formation of ROS, its significance in this process is still debatable. To better understand this phenomenon, the influence of blockade of Fenton reaction in a proposed Petri net-based model of the selected aspects of the iron ROS-induced toxicity in atherosclerosis has been evaluated. As a result of the blockade of iron ions formation in the model, even up to 70% of the paths leading to the progression of atherosclerosis in this model has been blocked. In addition, after adding to the model, the blockade of the lipids peroxidation paths, progression of atherosclerotic plaque has been not observed. This allowed to conclude that the superoxide-driven Fenton reaction plays a significant role in the atherosclerosis.

Petri net-based approach to modeling and analysis of selected aspects of the molecular regulation of angiogenesis.

The functioning of both normal and pathological tissues depends on an adequate supply of oxygen through the blood vessels. A process called angiogenesis, in which new endothelial cells and smooth muscles interact with each other, forming new blood vessels either from the existing ones or from a primary vascular plexus, is particularly important and interesting, due to new therapeutic possibilities it offers. This is a multi-step and very complex process, so an accurate understanding of the underlying mechanisms is a significant task, especially in recent years, with the constantly increasing amount of new data that must be taken into account. A systems approach is necessary for these studies because it is not sufficient to analyze the properties of the building blocks separately and an analysis of the whole network of interactions is essential. This approach is based on building a mathematical model of the system, while the model is expressed in the formal language of a mathematical theory. Recently, the theory of Petri nets was shown to be especially promising for the modeling and analysis of biological phenomena. This analysis, based mainly on t-invariants, has led to a particularly important finding that a direct link (close connection) exist between transforming growth factor ß1 (TGF-ß1), endothelial nitric oxide synthase (eNOS), nitric oxide (NO), and hypoxia-inducible factor 1, the molecules that play a crucial roles during angiogenesis. We have shown that TGF-ß1 may participate in the inhibition of angiogenesis through the upregulation of eNOS expression, which is responsible for catalyzing NO production. The results obtained in the previous studies, concerning the effects of NO on angiogenesis, have not been conclusive, and therefore, our study may contribute to a better understanding of this phenomenon.

A multilevel ant colony optimization algorithm for classical and isothermic DNA sequencing by hybridization with multiplicity information available.

The classical sequencing by hybridization takes into account a binary information about sequence composition. A given element from an oligonucleotide library is or is not a part of the target sequence. However, the DNA chip technology has been developed and it enables to receive a partial information about multiplicity of each oligonucleotide the analyzed sequence consist of. Currently, it is not possible to assess the exact data of such type but even partial information should be very useful. Two realistic multiplicity information models are taken into consideration in this paper. The first one, called "one and many" assumes that it is possible to obtain information if a given oligonucleotide occurs in a reconstructed sequence once or more than once. According to the second model, called "one, two and many", one is able to receive from biochemical experiment information if a given oligonucleotide is present in an analyzed sequence once, twice or at least three times. An ant colony optimization algorithm has been implemented to verify the above models and to compare with existing algorithms for sequencing by hybridization which utilize the additional information. The proposed algorithm solves the problem with any kind of hybridization errors. Computational experiment results confirm that using even the partial information about multiplicity leads to increased quality of reconstructed sequences. Moreover, they also show that the more precise model enables to obtain better solutions and the ant colony optimization algorithm outperforms the existing ones. Test data sets and the proposed ant colony optimization algorithm are available on: http://bioserver.cs.put.poznan.pl/download/ACO4mSBH.zip.

Usefulness of serum interleukin-18 in predicting cardiovascular mortality in patients with chronic kidney disease--systems and clinical approach.

The aim of this study was to check if serum interleukin-18 (IL-18) predicts 2-year cardiovascular mortality in patients at various stages of chronic kidney disease (CKD) and history of acute myocardial infarction (AMI) within the previous year. Diabetes mellitus was one of the key factors of exclusion. It was found that an increase in serum concentration of IL-18 above the cut-off point (1584.5 pg/mL) was characterized by 20.63-fold higher risk of cardiovascular deaths among studied patients. IL-18 serum concentration was found to be superior to the well-known cardiovascular risk parameters, like high sensitivity C-reactive protein (hsCRP), carotid intima media thickness (CIMT), glomerular filtration rate, albumins, ferritin, N-terminal prohormone of brain natriuretic peptide (NT-proBNP) in prognosis of cardiovascular mortality. The best predictive for IL-18 were 4 variables, such as CIMT, NT-proBNP, albumins and hsCRP, as they predicted its concentration at 89.5%. Concluding, IL-18 seems to be important indicator and predictor of cardiovascular death in two-year follow-up among non-diabetic patients suffering from CKD, with history of AMI in the previous year. The importance of IL-18 in the process of atherosclerotic plaque formation has been confirmed by systems analysis based on a formal model expressed in the language of Petri nets theory.