Comprehensive molecular interaction map of TGFß induced epithelial to mesenchymal transition in breast cancer.

Breast cancer is one of the prevailing cancers globally, with a high mortality rate. Metastatic breast cancer (MBC) is an advanced stage of cancer, characterised by a highly nonlinear, heterogeneous process involving numerous singling pathways and regulatory interactions. Epithelial-mesenchymal transition (EMT) emerges as a key mechanism exploited by cancer cells. Transforming Growth Factor-ß (TGFß)-dependent signalling is attributed to promote EMT in advanced stages of breast cancer. A comprehensive regulatory map of TGFß induced EMT was developed through an extensive literature survey. The network assembled comprises of 312 distinct species (proteins, genes, RNAs, complexes), and 426 reactions (state transitions, nuclear translocations, complex associations, and dissociations). The map was developed by following Systems Biology Graphical Notation (SBGN) using Cell Designer and made publicly available using MINERVA ( http://35.174.227.105:8080/minerva/?id=Metastatic_Breast_Cancer_1 ). While the complete molecular mechanism of MBC is still not known, the map captures the elaborate signalling interplay of TGFß induced EMT-promoting MBC. Subsequently, the disease map assembled was translated into a Boolean model utilising CaSQ and analysed using Cell Collective. Simulations of these have captured the known experimental outcomes of TGFß induced EMT in MBC. Hub regulators of the assembled map were identified, and their transcriptome-based analysis confirmed their role in cancer metastasis. Elaborate analysis of this map may help in gaining additional insights into the development and progression of metastatic breast cancer.

Systems biology dissection of PTSD and MDD across brain regions, cell types, and blood.

The molecular pathology of stress-related disorders remains elusive. Our brain multiregion, multiomic study of posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) included the central nucleus of the amygdala, hippocampal dentate gyrus, and medial prefrontal cortex (mPFC). Genes and exons within the mPFC carried most disease signals replicated across two independent cohorts. Pathways pointed to immune function, neuronal and synaptic regulation, and stress hormones. Multiomic factor and gene network analyses provided the underlying genomic structure. Single nucleus RNA sequencing in dorsolateral PFC revealed dysregulated (stress-related) signals in neuronal and non-neuronal cell types. Analyses of brain-blood intersections in >50,000 UK Biobank participants were conducted along with fine-mapping of the results of PTSD and MDD genome-wide association studies to distinguish risk from disease processes. Our data suggest shared and distinct molecular pathology in both disorders and propose potential therapeutic targets and biomarkers.

Molecular complexity of quantitative immunity in plants: from QTL mapping to functional and systems biology.

In nature, plants defend themselves against pathogen attack by activating an arsenal of defense mechanisms. During the last decades, work mainly focused on the understanding of qualitative disease resistance mediated by a few genes conferring an almost complete resistance, while quantitative disease resistance (QDR) remains poorly understood despite the fact that it represents the predominant and more durable form of resistance in natural populations and crops. Here, we review our past and present work on the dissection of the complex mechanisms underlying QDR in Arabidopsis thaliana. The strategies, main steps and challenges of our studies related to one atypical QDR gene, RKS1 (Resistance related KinaSe 1), are presented. First, from genetic analyses by QTL (Quantitative Trait Locus) mapping and GWAs (Genome Wide Association studies), the identification, cloning and functional analysis of this gene have been used as a starting point for the exploration of the multiple and coordinated pathways acting together to mount the QDR response dependent on RKS1. Identification of RKS1 protein interactors and complexes was a first step, systems biology and reconstruction of protein networks were then used to decipher the molecular roadmap to the immune responses controlled by RKS1. Finally, exploration of the potential impact of key components of the RKS1-dependent gene network on leaf microbiota offers interesting and challenging perspectives to decipher how the plant immune systems interact with the microbial communities' systems.

Dans la nature, les plantes se défendent contre les attaques pathogènes en activant tout un arsenal de mécanismes de défense. Au cours des décennies passées, la recherche s'est principalement focalisée sur la compréhension de la résistance qualitative médiée par quelques gènes majeurs conférant une résistance quasi complète, alors que la résistance quantitative (QDR) demeure peu comprise bien qu'elle représente la forme de résistance prédominante et la plus durable dans les populations naturelles ou les cultures. Nous donnons ici une revue de nos travaux passés et présents sur la dissection des mécanismes complexes qui sous-tendent la QDR chez Arabidopsis thaliana. Les stratégies, étapes clés et défis de nos études concernant un gène QDR atypique, RKS1 (Resistance related KinaSe 1), sont rapportés. En premier lieu, à partir d'analyses génétiques par cartographie de QTL et GWA, l'identification, le clonage et l'analyse fonctionnelle de ce gène ont été utilisés comme point de départ à l'exploration des voies multiples et coordonnées agissant ensemble pour le développement de la réponse QDR dépendante de RKS1. L'identification des interacteurs et complexes protéiques impliquant RKS1 a été une première étape, la biologie des systèmes et la reconstruction de réseaux d'interactions protéines-protéines ont ensuite été mises en œuvre pour décoder les voies moléculaires conduisant aux réponses immunitaires contrôlées par RKS1. Finalement, l'exploration de l'impact potentiel de composantes clés du réseau de gènes dépendant de RKS1 sur le microbiote, offre des perspectives intéressantes et ambitieuses pour comprendre comment le système immunitaire de la plante interagit avec le système des communautés microbiennes.

A gray box framework that optimizes a white box logical model using a black box optimizer for simulating cellular responses to perturbations.

Predicting cellular responses to perturbations requires interpretable insights into molecular regulatory dynamics to perform reliable cell fate control, despite the confounding non-linearity of the underlying interactions. There is a growing interest in developing machine learning-based perturbation response prediction models to handle the non-linearity of perturbation data, but their interpretation in terms of molecular regulatory dynamics remains a challenge. Alternatively, for meaningful biological interpretation, logical network models such as Boolean networks are widely used in systems biology to represent intracellular molecular regulation. However, determining the appropriate regulatory logic of large-scale networks remains an obstacle due to the high-dimensional and discontinuous search space. To tackle these challenges, we present a scalable derivative-free optimizer trained by meta-reinforcement learning for Boolean network models. The logical network model optimized by the trained optimizer successfully predicts anti-cancer drug responses of cancer cell lines, while simultaneously providing insight into their underlying molecular regulatory mechanisms.

Systems biology of plant metabolic interactions.

Metabolism is the key cellular process of plant physiology. Understanding metabolism and its dynamical behavior under different conditions may help plant biotechnologists to design new cultivars with desired goals. Computational systems biochemistry and incorporation of different omics data unravelled active metabolism and its variations in plants. In this review, we mainly focus on the basics of flux balance analysis (FBA), elementary flux mode analysis (EFMA), and some advanced computational tools. We describe some important results that were obtained using these tools. Limitations and challenges are also discussed.

Morphological Stability for in silico Models of Avascular Tumors.

The landscape of computational modeling in cancer systems biology is diverse, offering a spectrum of models and frameworks, each with its own trade-offs and advantages. Ideally, models are meant to be useful in refining hypotheses, to sharpen experimental procedures and, in the longer run, even for applications in personalized medicine. One of the greatest challenges is to balance model realism and detail with experimental data to eventually produce useful data-driven models. We contribute to this quest by developing a transparent, highly parsimonious, first principle in silico model of a growing avascular tumor. We initially formulate the physiological considerations and the specific model within a stochastic cell-based framework. We next formulate a corresponding mean-field model using partial differential equations which is amenable to mathematical analysis. Despite a few notable differences between the two models, we are in this way able to successfully detail the impact of all parameters in the stability of the growth process and on the eventual tumor fate of the stochastic model. This facilitates the deduction of Bayesian priors for a given situation, but also provides important insights into the underlying mechanism of tumor growth and progression. Although the resulting model framework is relatively simple and transparent, it can still reproduce the full range of known emergent behavior. We identify a novel model instability arising from nutrient starvation and we also discuss additional insight concerning possible model additions and the effects of those. Thanks to the framework's flexibility, such additions can be readily included whenever the relevant data become available.

Systems genetics analysis of human body fat distribution genes identifies adipocyte processes.

Excess abdominal fat is a sexually dimorphic risk factor for cardio-metabolic disease and is approximated by the waist-to-hip ratio adjusted for body mass index (WHRadjBMI). Whereas this trait is highly heritable, few causal genes are known. We aimed to identify novel drivers of WHRadjBMI using systems genetics. We used two independent cohorts of adipose tissue gene expression and constructed sex- and depot-specific Bayesian networks to model gene-gene interactions from 8,492 genes. Using key driver analysis, we identified genes that, in silico and putatively in vitro, regulate many others. 51-119 key drivers in each network were replicated in both cohorts. In other cell types, 23 of these genes are found in crucial adipocyte pathways: Wnt signaling or mitochondrial function. We overexpressed or down-regulated seven key driver genes in human subcutaneous pre-adipocytes. Key driver genes ANAPC2 and RSPO1 inhibited adipogenesis, whereas PSME3 increased adipogenesis. RSPO1 increased Wnt signaling activity. In differentiated adipocytes, MIGA1 and UBR1 down-regulation led to mitochondrial dysfunction. These five genes regulate adipocyte function, and we hypothesize that they regulate fat distribution.

Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).

DeepFGRN: inference of gene regulatory network with regulation type based on directed graph embedding.

The inference of gene regulatory networks (GRNs) from gene expression profiles has been a key issue in systems biology, prompting many researchers to develop diverse computational methods. However, most of these methods do not reconstruct directed GRNs with regulatory types because of the lack of benchmark datasets or defects in the computational methods. Here, we collect benchmark datasets and propose a deep learning-based model, DeepFGRN, for reconstructing fine gene regulatory networks (FGRNs) with both regulation types and directions. In addition, the GRNs of real species are always large graphs with direction and high sparsity, which impede the advancement of GRN inference. Therefore, DeepFGRN builds a node bidirectional representation module to capture the directed graph embedding representation of the GRN. Specifically, the source and target generators are designed to learn the low-dimensional dense embedding of the source and target neighbors of a gene, respectively. An adversarial learning strategy is applied to iteratively learn the real neighbors of each gene. In addition, because the expression profiles of genes with regulatory associations are correlative, a correlation analysis module is designed. Specifically, this module not only fully extracts gene expression features, but also captures the correlation between regulators and target genes. Experimental results show that DeepFGRN has a competitive capability for both GRN and FGRN inference. Potential biomarkers and therapeutic drugs for breast cancer, liver cancer, lung cancer and coronavirus disease 2019 are identified based on the candidate FGRNs, providing a possible opportunity to advance our knowledge of disease treatments.

Biology System Description Language (BiSDL): a modeling language for the design of multicellular synthetic biological systems.

BACKGROUND: The Biology System Description Language (BiSDL) is an accessible, easy-to-use computational language for multicellular synthetic biology. It allows synthetic biologists to represent spatiality and multi-level cellular dynamics inherent to multicellular designs, filling a gap in the state of the art. Developed for designing and simulating spatial, multicellular synthetic biological systems, BiSDL integrates high-level conceptual design with detailed low-level modeling, fostering collaboration in the Design-Build-Test-Learn cycle. BiSDL descriptions directly compile into Nets-Within-Nets (NWNs) models, offering a unique approach to spatial and hierarchical modeling in biological systems. RESULTS: BiSDL's effectiveness is showcased through three case studies on complex multicellular systems: a bacterial consortium, a synthetic morphogen system and a conjugative plasmid transfer process. These studies highlight the BiSDL proficiency in representing spatial interactions and multi-level cellular dynamics. The language facilitates the compilation of conceptual designs into detailed, simulatable models, leveraging the NWNs formalism. This enables intuitive modeling of complex biological systems, making advanced computational tools more accessible to a broader range of researchers. CONCLUSIONS: BiSDL represents a significant step forward in computational languages for synthetic biology, providing a sophisticated yet user-friendly tool for designing and simulating complex biological systems with an emphasis on spatiality and cellular dynamics. Its introduction has the potential to transform research and development in synthetic biology, allowing for deeper insights and novel applications in understanding and manipulating multicellular systems.

A systems biology-based mathematical model demonstrates the potential anti-stress effectiveness of a multi-nutrient botanical formulation.

Stress is an adaptive response to the stressors that adversely affects physiological and psychological health. Stress elicits HPA axis activation, resulting in cortisol release, ultimately contributing to oxidative, inflammatory, physiological and mental stress. Nutritional supplementations with antioxidant, anti-inflammatory, and stress-relieving properties are among widely preferred complementary approaches for the stress management. However, there is limited research on the potential combined impact of vitamins, minerals and natural ingredients on stress. In the present study, we have investigated the effect of a multi-nutrient botanical formulation, Nutrilite® Daily Plus, on clinical stress parameters. The stress-modulatory effects were quantified at population level using a customized sub-clinical inflammation mathematical model. The model suggested that combined intervention of botanical and micronutrients lead to significant decline in physical stress (75% decline), mental stress (70% decline), oxidative stress (55% decline) and inflammatory stress (75% decline) as evident from reduction in key stress parameters such as ROS, TNF-α, blood pressure, cortisol levels and PSS scores at both individual and population levels. Further, at the population level, the intervention relieved stress in 85% of individuals who moved towards a healthy state. The in silico studies strongly predicts the use of Gotukola based Nutrilite® Daily Plus as promising anti-stress formulation.

Deciphering the similarities and disparities of molecular mechanisms behind respiratory epithelium response to HCoV-229E and SARS-CoV-2 and drug repurposing, a systems biology approach.

PURPOSE: Identifying the molecular mechanisms behind SARS-CoV-2 disparities and similarities will help find new treatments. The present study determines networks' shared and non-shared (specific) crucial elements in response to HCoV-229E and SARS-CoV-2 viruses to recommend candidate medications. METHODS: We retrieved the omics data on respiratory cells infected with HCoV-229E and SARS-CoV-2, constructed PPIN and GRN, and detected clusters and motifs. Using a drug-gene interaction network, we determined the similarities and disparities of mechanisms behind their host response and drug-repurposed. RESULTS: CXCL1, KLHL21, SMAD3, HIF1A, and STAT1 were the shared DEGs between both viruses' protein-protein interaction network (PPIN) and gene regulatory network (GRN). The NPM1 was a specific critical node for HCoV-229E and was a Hub-Bottleneck shared between PPI and GRN in HCoV-229E. The HLA-F, ADCY5, TRIM14, RPF1, and FGA were the seed proteins in subnetworks of the SARS-CoV-2 PPI network, and HSPA1A and RPL26 proteins were the seed in subnetworks of the PPI network of HCOV-229E. TRIM14, STAT2, and HLA-F played the same role for SARS-CoV-2. Top enriched KEGG pathways included cell cycle and proteasome in HCoV-229E and RIG-I-like receptor, Chemokine, Cytokine-cytokine, NOD-like receptor, and TNF signaling pathways in SARS-CoV-2. We suggest some candidate medications for COVID-19 patient lungs, including Noscapine, Isoetharine mesylate, Cycloserine, Ethamsylate, Cetylpyridinium, Tretinoin, Ixazomib, Vorinostat, Venetoclax, Vorinostat, Ixazomib, Venetoclax, and epoetin alfa for further in-vitro and in-vivo investigations. CONCLUSION: We suggested CXCL1, KLHL21, SMAD3, HIF1A, and STAT1, ADCY5, TRIM14, RPF1, and FGA, STAT2, and HLA-F as critical genes and Cetylpyridinium, Cycloserine, Noscapine, Ethamsylate, Epoetin alfa, Isoetharine mesylate, Ribavirin, and Tretinoin drugs to study further their importance in treating COVID-19 lung complications.

Cynomolgus macaques as a translational model of human immune responses to yellow fever 17D vaccination.

The non-human primate (NHP) model (specifically rhesus and cynomolgus macaques) has facilitated our understanding of the pathogenic mechanisms of yellow fever (YF) disease and allowed the evaluation of the safety and efficacy of YF-17D vaccines. However, the accuracy of this model in mimicking vaccine-induced immunity in humans remains to be fully determined. We used a systems biology approach to compare hematological, biochemical, transcriptomic, and innate and antibody-mediated immune responses in cynomolgus macaques and human participants following YF-17D vaccination. Immune response progression in cynomolgus macaques followed a similar course as in adult humans but with a slightly earlier onset. Yellow fever virus neutralizing antibody responses occurred earlier in cynomolgus macaques [by Day 7[(D7)], but titers > 10 were reached in both species by D14 post-vaccination and were not significantly different by D28 [plaque reduction neutralization assay (PRNT)50 titers 3.6 Log vs 3.5 Log in cynomolgus macaques and human participants, respectively; P = 0.821]. Changes in neutrophils, NK cells, monocytes, and T- and B-cell frequencies were higher in cynomolgus macaques and persisted for 4 weeks versus less than 2 weeks in humans. Low levels of systemic inflammatory cytokines (IL-1RA, IL-8, MIP-1α, IP-10, MCP-1, or VEGF) were detected in either or both species but with no or only slight changes versus baseline. Similar changes in gene expression profiles were elicited in both species. These included enriched and up-regulated type I IFN-associated viral sensing, antiviral innate response, and dendritic cell activation pathways D3-D7 post-vaccination in both species. Hematological and blood biochemical parameters remained relatively unchanged versus baseline in both species. Low-level YF-17D viremia (RNAemia) was transiently detected in some cynomolgus macaques [28% (5/18)] but generally absent in humans [except one participant (5%; 1/20)].IMPORTANCECynomolgus macaques were confirmed as a valid surrogate model for replicating YF-17D vaccine-induced responses in humans and suggest a key role for type I IFN.

Bioinformatics and system biology approaches to determine the connection of SARS-CoV-2 infection and intrahepatic cholangiocarcinoma.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), has infected millions of individuals worldwide, which poses a severe threat to human health. COVID-19 is a systemic ailment affecting various tissues and organs, including the lungs and liver. Intrahepatic cholangiocarcinoma (ICC) is one of the most common liver cancer, and cancer patients are particularly at high risk of SARS-CoV-2 infection. Nonetheless, few studies have investigated the impact of COVID-19 on ICC patients. METHODS: With the methods of systems biology and bioinformatics, this study explored the link between COVID-19 and ICC, and searched for potential therapeutic drugs. RESULTS: This study identified a total of 70 common differentially expressed genes (DEGs) shared by both diseases, shedding light on their shared functionalities. Enrichment analysis pinpointed metabolism and immunity as the primary areas influenced by these common genes. Subsequently, through protein-protein interaction (PPI) network analysis, we identified SCD, ACSL5, ACAT2, HSD17B4, ALDOA, ACSS1, ACADSB, CYP51A1, PSAT1, and HKDC1 as hub genes. Additionally, 44 transcription factors (TFs) and 112 microRNAs (miRNAs) were forecasted to regulate the hub genes. Most importantly, several drug candidates (Periodate-oxidized adenosine, Desipramine, Quercetin, Perfluoroheptanoic acid, Tetrandrine, Pentadecafluorooctanoic acid, Benzo[a]pyrene, SARIN, Dorzolamide, 8-Bromo-cAMP) may prove effective in treating ICC and COVID-19. CONCLUSION: This study is expected to provide valuable references and potential drugs for future research and treatment of COVID-19 and ICC.

Exploring the immune-inflammatory mechanism of Maxing Shigan Decoction in treating influenza virus A-induced pneumonia based on an integrated strategy of single-cell transcriptomics and systems biology.

BACKGROUND: Influenza is an acute respiratory infection caused by influenza virus. Maxing Shigan Decoction (MXSGD) is a commonly used traditional Chinese medicine prescription for the prevention and treatment of influenza. However, its mechanism remains unclear. METHOD: The mice model of influenza A virus pneumonia was established by nasal inoculation. After 3 days of intervention, the lung index was calculated, and the pathological changes of lung tissue were detected by HE staining. Firstly, transcriptomics technology was used to analyze the differential genes and important pathways in mouse lung tissue regulated by MXSGD. Then, real-time fluorescent quantitative PCR (RT-PCR) was used to verify the changes in mRNA expression in lung tissues. Finally, intestinal microbiome and intestinal metabolomics were performed to explore the effect of MXSGD on gut microbiota. RESULTS: The lung inflammatory cell infiltration in the MXSGD group was significantly reduced (p < 0.05). The results of bioinformatics analysis for transcriptomics results show that these genes are mainly involved in inflammatory factors and inflammation-related signal pathways mediated inflammation biological modules, etc. Intestinal microbiome showed that the intestinal flora Actinobacteriota level and Desulfobacterota level increased in MXSGD group, while Planctomycetota in MXSGD group decreased. Metabolites were mainly involved in primary bile acid biosynthesis, thiamine metabolism, etc. This suggests that MXSGD has a microbial-gut-lung axis regulation effect on mice with influenza A virus pneumonia. CONCLUSION: MXSGD may play an anti-inflammatory and immunoregulatory role by regulating intestinal microbiome and intestinal metabolic small molecules, and ultimately play a role in the treatment of influenza A virus pneumonia.

Gene networks and systems biology in Alzheimer's disease: Insights from multi-omics approaches.

Despite numerous studies in the field of dementia and Alzheimer's disease (AD), a comprehensive understanding of this devastating disease remains elusive. Bulk transcriptomics have provided insights into the underlying genetic factors at a high level. Subsequent technological advancements have focused on single-cell omics, encompassing techniques such as single-cell RNA sequencing and epigenomics, enabling the capture of RNA transcripts and chromatin states at a single cell or nucleus resolution. Furthermore, the emergence of spatial omics has allowed the study of gene responses in the vicinity of amyloid beta plaques or across various brain regions. With the vast amount of data generated, utilizing gene regulatory networks to comprehensively study this disease has become essential. This review delves into some techniques employed in the field of AD, explores the discoveries made using these techniques, and provides insights into the future of the field.

BioKC: a collaborative platform for curation and annotation of molecular interactions.

Curation of biomedical knowledge into systems biology diagrammatic or computational models is essential for studying complex biological processes. However, systems-level curation is a laborious manual process, especially when facing ever-increasing growth of domain literature. New findings demonstrating elaborate relationships between multiple molecules, pathways and cells have to be represented in a format suitable for systems biology applications. Importantly, curation should capture the complexity of molecular interactions in such a format together with annotations of the involved elements and support stable identifiers and versioning. This challenge calls for novel collaborative tools and platforms allowing to improve the quality and the output of the curation process. In particular, community-based curation, an important source of curated knowledge, requires support in role management, reviewing features and versioning. Here, we present Biological Knowledge Curation (BioKC), a web-based collaborative platform for the curation and annotation of biomedical knowledge following the standard data model from Systems Biology Markup Language (SBML). BioKC offers a graphical user interface for curation of complex molecular interactions and their annotation with stable identifiers and supporting sentences. With the support of collaborative curation and review, it allows to construct building blocks for systems biology diagrams and computational models. These building blocks can be published under stable identifiers and versioned and used as annotations, supporting knowledge building for modelling activities.

A Practical Guide to the Representation of Protein Regulation in the Web Application ChloroKB.

ChloroKB ( http://chlorokb.fr ) is a knowledge base providing synoptic representations of the metabolism of the model plant Arabidopsis thaliana and its regulation. Initially focused on plastid metabolism, ChloroKB now accounts for the metabolism throughout the cell. ChloroKB is based on the CellDesigner formalism. CellDesigner supports graphical notation and listing of the corresponding symbols based on the Systems Biology Graphical Notation. Thus, this formalism allows biologists to represent detailed biochemical processes in a way that can be easily understood and shared, facilitating communication between researchers. In this chapter, we will focus on a specificity of ChloroKB, the representation of multilayered regulation of protein activity. Information on regulation of protein activity is indeed central to understanding the plant response to fluctuating environmental conditions. However, the intrinsic diversity of the regulatory modes and the abundance of detail may hamper comprehension of the regulatory processes described in ChloroKB. With this chapter, ChloroKB users will be guided through the representation of these sophisticated biological processes of prime importance to understanding metabolism or for applied purposes. The descriptions provided, which summarize years of work and a broad bibliography in a few pages, can help speed up the integration of regulatory processes in kinetic models of plant metabolism.