Petri nets for systems and synthetic biology

We give a description of a Petri net-based framework for modelling and analysing biochemical pathways, which uni¯es the qualita- tive, stochastic and continuous paradigms. Each perspective adds its con- tribution to the understanding of the system, thus the three approaches do not compete, but complement each other. We illustrate our approach by applying it to an extended model of the three stage cascade, which forms the core of the ERK signal transduction pathway. Consequently our focus is on transient behaviour analysis. We demonstrate how quali- tative descriptions are abstractions over stochastic or continuous descrip- tions, and show that the stochastic and continuous models approximate each other. Although our framework is based on Petri nets, it can be applied more widely to other formalisms which are used to model and analyse biochemical networks

Biochemical Testing

Clinical Correlation and Diagnosis highlights the improvements in methodological approaches for the purposes of disease diagnosis and health research. Chapters cover such topics as serum protein electrophoresis, urinary iodine measurement, blood collection tubes, semi-solid phase assay and advancement in analytical and bioanalytical techniques, and serological diagnostic tools for Zika virus, among other subjects. All these will not be possible without a proper laboratory management where this book also includes the Tissue Bank ATMP Production as a model. The chapters are expected to provide a new perspective in health science which may trigger a further exploration into the diagnostic and research field

Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding

Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ~30 Å apart, remains elusive. We construct an atomically detailed model of G protein activation by combining three powerful computational methods: metadynamics, Markov state models (MSMs), and CARDS analysis of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochemical data. Surprisingly, the rate-limiting step for GDP release correlates with tilting rather than translation of the GPCR-binding helix 5. β-Strands 1 - 3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotide-binding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examining unbinding processes with slow off-rates

Immuno-modulatory effects of intervertebral disc cells

Low back pain is a highly prevalent, chronic, and costly medical condition predominantly triggered by intervertebral disc degeneration (IDD). IDD is often caused by structural and biochemical changes in intervertebral discs (IVD) that prompt a pathologic shift from an anabolic to catabolic state, affecting extracellular matrix (ECM) production, enzyme generation, cytokine and chemokine production, neurotrophic and angiogenic factor production. The IVD is an immune-privileged organ. However, during degeneration immune cells and inflammatory factors can infiltrate through defects in the cartilage endplate and annulus fibrosus fissures, further accelerating the catabolic environment. Remarkably, though, catabolic ECM disruption also occurs in the absence of immune cell infiltration, largely due to native disc cell production of catabolic enzymes and cytokines. An unbalanced metabolism could be induced by many different factors, including a harsh microenvironment, biomechanical cues, genetics, and infection. The complex, multifactorial nature of IDD brings the challenge of identifying key factors which initiate the degenerative cascade, eventually leading to back pain. These factors are often investigated through methods including animal models, 3D cell culture, bioreactors, and computational models. However, the crosstalk between the IVD, immune system, and shifted metabolism is frequently misconstrued, often with the assumption that the presence of cytokines and chemokines is synonymous to inflammation or an immune response, which is not true for the intact disc. Therefore, this review will tackle immunomodulatory and IVD cell roles in IDD, clarifying the differences between cellular involvements and implications for therapeutic development and assessing models used to explore inflammatory or catabolic IVD environments

Identification of Biomarkers, Pathways, Immune Properties of Mitophagy Genes, and Prediction Models for Intervertebral Disc Degeneration

Yongxiong Huang,1,2,* Xianshuai Qiu,3,* Jinlian Liu,4 Jiangtao Wan,5 Cheng Yu,1 Chun Liu,1 Yang Duan,1 Chong Chen,2 Jingxing Dai,6 Jun Ouyang,6 Ming Liu,3 Shaoxiong Min,1,5 Sujun Qiu1 1Department of Spine Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510280, People’s Republic of China; 2Department of Spine Surgery, Guangdong Provincial People’s Hospital, Southern Medical University, Guangzhou, 510000, People’s Republic of China; 3Department of Orthopedics and Sports Medicine Center, Heyou Hospital, Foshan, 528333, People’s Republic of China; 4Department of Pathophysiology, Guangdong Provincial Key Laboratory of Shock and Microcirculation, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, People’s Republic of China; 5Shenzhen Key Laboratory of Spine Surgery, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, 518036, People’s Republic of China; 6Guangdong Provincial Key Laboratory of Medical Biomechanics & National Key Discipline of Human Anatomy, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, People’s Republic of China*These authors contributed equally to this workCorrespondence: Sujun Qiu, Department of Spine Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510280, People’s Republic of China, Email [email protected] Shaoxiong Min, Shenzhen Key Laboratory of Spine Surgery, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, 518036, People’s Republic of China, Email [email protected]: Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). The mechanism of IDD development and progression is not fully understood. Peripheral biomarkers are increasingly vital non-radioactive methods in early detection and diagnosis for IDD. Nevertheless, less attention has been paid to the role of mitophagy genes in the progress of IDD. This study aimed to identify the mitophagy disease-causing genes in the process of IDD and mitophagy diagnostic biomarkers for IDD.Methods: Mitophagy-related differentially expressed genes (MRDEGs) related to IDD were investigated by analyzing the microarray datasets of IDD cases from GEO, PathCards and Molecular Signatures Databases. We used R software, WGCNA, PPI, mRNA-miRNA, mRNA-TF, GO, KEGG, GSEA, GSVA and Cytoscape to analyze and visualize the data. We further used ssGSEA for immunoinfiltration analysis to obtain different immune cell infiltration. LASSO model was developed to screen for genes that met the diagnostic gene model requirements. Finally, qRT-PCR, Western blotting and HE were used to verify hub genes and their expression from clinical IDD samples.Results: We identified 14 MRDEGs and 12 hub genes. GO, KEGG, GSEA and GSVA analyses demonstrated that hub genes were critical for the development of IDD. LASSO diagnostic model consisted of six hub genes, among which SQSTM1, ATG7 and OPTN were significantly different between the two IDD disease subtypes. At the same time, SQSTM1 also had a high correlation with immune characteristic subtypes. The results of qRT-PCR and Western blotting also indicated that these genes were significantly differentially expressed in nucleus pulposus cells (NPCs) of the IDD group.Conclusion: We explored an association between MRDEGs-associated signature in IDD and validated that hub genes like SQSTM1 might serve as biomarkers for diagnostic and therapeutic targets for IDD. Meanwhile, this study can provide new insights into the functional characteristics and mechanism of mitophagy in the development of IDD. Keywords: intervertebral disc degeneration, mitophagy-related differentially expressed genes, immune infiltration, diagnostic model, SQSTM1, nucleus pulposus cell

Development of a Nucleus Pulposus Implant for Intervertebral Disc Repair

The intervertebral disc (IVD) is a fibrocartilaginous tissue connecting adjacent vertebrae in the spinal column. It comprises three distinct tissues: a gelatinous core known as the nucleus pulposus (NP), concentric fibrous rings encircling the NP known as the annulus fibrosus (AF), and the superior/inferior cartilaginous endplates (CEPs). The complex mechanical interplay of these tissues allows the IVD to withstand complex loading in the spine while maintaining trunk stability and flexibility. IVD pathologies, such as IVD degeneration (IDD) and herniation, are associated with cell-mediated inflammation in vivo. This inflammation creates a catabolic environment which degrades the extracellular matrix (ECM) of the IVD. Since ECM composition influences the mechanical properties of the tissue, this degradation compromises spine biomechanics, which may lead to low back pain, radiculopathy, and disability. Current treatment strategies for IVD pathologies are either palliative or are aggressive and only partly redress spine biomechanics. Furthermore, current interventions are not regenerative and may induce collateral pathologies in adjacent IVDs due to abnormal biomechanics post-repair. Mechanically robust scaffolds derived from decellularized tissues have the potential to re-establish spine biomechanics, alleviate the underlying pathology, and regenerate the native tissue. We have previously demonstrated the ability to decellularize bovine NP to form acellular bovine NP (ABNP), which exhibited a biomimetic ECM composition, supported cell seeding, and partially restored spine kinematics in an ex vivo model of IVD injury. Despite this, further refinement was necessary to improve its mechanical properties and characterize its regenerative potential. Furthermore, prior to translation, the ABNP must be evaluated using a well-characterized animal model that reproduces clinically relevant aspects of IDD. The goals of this research were to: i) fortify the mechanical properties of the ABNP, ii) determine the in vitro cytocompatibility and regenerative capacity of the ABNP, and iii) characterize a novel animal model of IVD degeneration for future in vivo testing of the implant

The disease mechanisms of skeletal dysplasia caused by two aggrecan mutations

PhD ThesisSkeletal dysplasias are a complex group of over 350 disorders of cartilage and bone with a combined incidence of 1/5000. Diagnosis is difficult and based on clinical and radiographic findings, with many cases having no known genetic cause. An allelic series of mutations has been identified in aggrecan that results in a broad phenotypic spectrum, including spondyloepimetaphyseal dysplasia (SEMD) and familial osteochondritis dissecans (OCD). Aggrecan, a large chondroitin sulfated proteoglycan, attracts ions and water molecules, allowing the cartilage to withstand the high mechanical load found in the skeletal joint. The SEMD (p.D2276N) and OCD (p.V2303M) causative mutations are found at highly conserved residues in the C-type lectin domain (CLD) which interacts with other extracellular matrix (ECM) molecules to provide stability. PolyGene Transgenetics introduced mutations into the C57Bl/6 mouse line using homologous gene targeting. The resultant mice were assessed with radiography, morphometry and growth measurements. Histological techniques, electron microscopy, RNA-sequencing and SDSPAGE Western blotting were used to analyse the underlying patho-molecular mechanisms. The homozygous V2019M Acan (OCD) mouse exhibits mild disproportionate short stature, whereas the homozygous D1983N Acan (SEMD) mouse has severe disproportionate short stature with associated skeletal abnormalities, including mid-face hypoplasia. OCD and SEMD mice also exhibit intervertebral disc degeneration, which has not previously been reported in human patients. OCD and SEMD growth plate cartilage is disorganised, with reduced aggrecan in the extracellular matrix, increased apoptosis and mis-expression of other extracellular matrix proteins. The unfolded protein response is not upregulated in either OCD or SEMD cartilage, although SEMD chondrocytes exhibit mild cellular stress. Comparative transcriptomic analysis indicates that autophagic flux, vesicular transport and regulation of chondrocyte differentiation are altered between these two models. The data presented in this thesis demonstrates that OCD and SEMD mice recapitulate the human short stature phenotypes and that altered matrix organisation may impair cellular differentiation, causing reduced bone growth. These mouse models could be used to further investigate the disease mechanisms of these two skeletal disorders.Financial support from the SYBIL project (funded by the European Commission’s Seventh Framework Programme for Research) and RUBICON network (funded by the European Union's Horizon 2020 research and innovation programme) is gratefully acknowledged

DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics

The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs. Machine learning methods predicted a 'hydration matrisome' that connects extracellular matrix with MRI intensity. Importantly, the static proteome used as point-references can be integrated with dynamic proteome (SILAC/degradome) and transcriptome data from multiple clinical samples, enhancing robustness and clinical relevance. The data, findings, and methodology, available on a web interface (http://www.sbms.hku.hk/dclab/DIPPER/), will be valuable references in the field of IVD biology and proteomic analytics

Biomodelkit - a framework for modular biomodel-engineering

Otto-von-Guericke-Universität Magdeburg, Fakultät für Naturwissenschaften, Dissertation, 2017von Dipl.-Ing. Mary-Ann BlätkeLiteraturverzeichnis: Seite [177]-18