Network-based approaches to explore complex biological systems towards network medicine

Network medicine relies on different types of networks: from the molecular level of protein–protein interactions to gene regulatory network and correlation studies of gene expression. Among network approaches based on the analysis of the topological properties of protein–protein interaction (PPI) networks, we discuss the widespread DIAMOnD (disease module detection) algorithm. Starting from the assumption that PPI networks can be viewed as maps where diseases can be identified with localized perturbation within a specific neighborhood (i.e., disease modules), DIAMOnD performs a systematic analysis of the human PPI network to uncover new disease-associated genes by exploiting the connectivity significance instead of connection density. The past few years have witnessed the increasing interest in understanding the molecular mechanism of post-transcriptional regulation with a special emphasis on non-coding RNAs since they are emerging as key regulators of many cellular processes in both physiological and pathological states. Recent findings show that coding genes are not the only targets that microRNAs interact with. In fact, there is a pool of different RNAs—including long non-coding RNAs (lncRNAs) —competing with each other to attract microRNAs for interactions, thus acting as competing endogenous RNAs (ceRNAs). The framework of regulatory networks provides a powerful tool to gather new insights into ceRNA regulatory mechanisms. Here, we describe a data-driven model recently developed to explore the lncRNA-associated ceRNA activity in breast invasive carcinoma. On the other hand, a very promising example of the co-expression network is the one implemented by the software SWIM (switch miner), which combines topological properties of correlation networks with gene expression data in order to identify a small pool of genes—called switch genes—critically associated with drastic changes in cell phenotype. Here, we describe SWIM tool along with its applications to cancer research and compare its predictions with DIAMOnD disease genes

SWIM: A computational tool to unveiling crucial nodes in complex biological networks

SWItchMiner (SWIM) is a wizard-like software implementation of a procedure, previously described, able to extract information contained in complex networks. Specifically, SWIM allows unearthing the existence of a new class of hubs, called "fight-club hubs", characterized by a marked negative correlation with their first nearest neighbors. Among them, a special subset of genes, called "switch genes", appears to be characterized by an unusual pattern of intra- and inter-module connections that confers them a crucial topological role, interestingly mirrored by the evidence of their clinic-biological relevance. Here, we applied SWIM to a large panel of cancer datasets from The Cancer Genome Atlas, in order to highlight switch genes that could be critically associated with the drastic changes in the physiological state of cells or tissues induced by the cancer development. We discovered that switch genes are found in all cancers we studied and they encompass protein coding genes and non-coding RNAs, recovering many known key cancer players but also many new potential biomarkers not yet characterized in cancer context. Furthermore, SWIM is amenable to detect switch genes in different organisms and cell conditions, with the potential to uncover important players in biologically relevant scenarios, including but not limited to human cancer

Distinct transcriptome responses to water limitation in isohydric and anisohydric grapevine cultivars

Background: Grapevine (Vitis vinifera L.) is an economically important crop with a wide geographical distribution, reflecting its ability to grow successfully in a range of climates. However, many vineyards are located in regions with seasonal drought, and these are often predicted to be global climate change hotspots. Climate change affects the entire physiology of grapevine, with strong effects on yield, wine quality and typicity, making it difficult to produce berries of optimal enological quality and consistent stability over the forthcoming decades. Results: Here we investigated the reactions of two grapevine cultivars to water stress, the isohydric variety Montepulciano and the anisohydric variety Sangiovese, by examining physiological and molecular perturbations in the leaf and berry. A multidisciplinary approach was used to characterize the distinct stomatal behavior of the two cultivars and its impact on leaf and berry gene expression. Positive associations were found among the photosynthetic, physiological and transcriptional modifications, and candidate genes encoding master regulators of the water stress response were identified using an integrated approach based on the analysis of topological co-expression network properties. In particular, the genome-wide transcriptional study indicated that the isohydric behavior relies upon the following responses: i) faster transcriptome response after stress imposition; ii) faster abscisic acid-related gene modulation; iii) more rapid expression of heat shock protein (HSP) genes and iv) reversion of gene-expression profile at rewatering. Conversely, that reactive oxygen species (ROS)-scavenging enzymes, molecular chaperones and abiotic stress-related genes were induced earlier and more strongly in the anisohydric cultivar. Conclusions: Overall, the present work found original evidence of a molecular basis for the proposed classification between isohydric and anisohydric grapevine genotypes

A PheWAS Model Of Autism Spectrum Disorder

Children with Autism Spectrum Disorder (ASD) exhibit a wide diversity in type, number, and severity of social deficits as well as communicative and cognitive difficulties. It is a challenge to categorize the phenotypes of a particular ASD patient with their unique genetic variants. There is a need for a better understanding of the connections between genotype information and the phenotypes to sort out the heterogeneity of ASD. In this study, single nucleotide polymorphism (SNP) and phenotype data obtained from a simplex ASD sample are combined using a PheWAS-inspired approach to construct a phenotype-phenotype network. The network is clustered, yielding groups of etiologically related phenotypes. These clusters are analyzed to identify relevant genes associated with each set of phenotypes. The results identified multiple discriminant SNPs associated with varied phenotype clusters such as ASD aberrant behavior (self-injury, compulsiveness and hyperactivity), as well as IQ and language skills. Overall, these SNPs were linked to 22 significant genes. An extensive literature search revealed that eight of these are known to have strong evidence of association with ASD. The others have been linked to related disorders such as mental conditions, cognition, and social functioning, Clinical relevance - This study further informs on connections between certain groups of ASD phenotypes and their unique genetic variants. Such insight regarding the heterogeneity of ASD would support clinicians to advance more tailored interventions and improve outcomes for ASD patients

Integrated Network Analysis Identifies Fight-Club Nodes as a Class of Hubs Encompassing Key Putative Switch Genes That Induce Major Transcriptome Reprogramming during Grapevine Development

We developed an approach that integrates different network-based methods to analyze the correlation network arising from large-scale gene expression data. By studying grapevine (Vitis vinifera) and tomato (Solanum lycopersicum) gene expression atlases and a grapevine berry transcriptomic data set during the transition from immature to mature growth, we identified a category named “fight-club hubs” characterized by a marked negative correlation with the expression profiles of neighboring genes in the network. A special subset named “switch genes” was identified, with the additional property of many significant negative correlations outside their own group in the network. Switch genes are involved in multiple processes and include transcription factors that may be considered master regulators of the previously reported transcriptome remodeling that marks the developmental shift from immature to mature growth. All switch genes, expressed at low levels in vegetative/green tissues, showed a significant increase in mature/woody organs, suggesting a potential regulatory role during the developmental transition. Finally, our analysis of tomato gene expression data sets showed that wild-type switch genes are downregulated in ripening-deficient mutants. The identification of known master regulators of tomato fruit maturation suggests our method is suitable for the detection of key regulators of organ development in different fleshy fruit crops

Transcriptomic and genomic structural variation analyses on grape cultivars reveal new insights into the genotype-dependent responses to water stress

Grapevine (Vitis vinifera L.) is importantly cultivated worldwide for table grape and wine production. Its cultivation requires irrigation supply, especially in arid and semiarid areas. Water deficiency can affect berry and wine quality mostly depending on the extent of plant perceived stress, which is a cultivar-specific trait. We tested the physiological and molecular responses to water deficiency of two table grape cultivars, Italia and Autumn royal, and we highlighted their different adaptation. Microarray analyses revealed that Autumn royal reacts involving only 29 genes, related to plant stress response and ABA/hormone signal transduction, to modulate the response to water deficit. Instead, cultivar Italia orchestrates a very broad response (we found 1037 differentially expressed genes) that modifies the cell wall organization, carbohydrate metabolism, response to reactive oxygen species, hormones and osmotic stress. For the first time, we integrated transcriptomic data with cultivar-specific genomics and found that ABA-perception and –signalling are key factors mediating the varietal-specific behaviour of the early response to drought. We were thus able to isolate candidate genes for the genotype-dependent response to drought. These insights will allow the identification of reliable plant stress indicators and the definition of sustainable cultivar-specific protocols for water management

Berry transcriptome comparison of ten Italian grapevine varieties

Lo sviluppo della bacca di vite pu\uf2 essere descritto come una successione di cambiamenti fisiologici e biochimici che riflettono la modulazione trascrizionale di molti geni. Nello scorso decennio molti studi trascrittomici sono stati eseguiti per descrivere in modo pi\uf9 approfondito questo processo di sviluppo dinamico e complesso. Tuttavia, la maggior parte di questi studi trascrittomici si sono focalizzati solo su un\u2019unica variet\ue0 per volta e quindi vi \ue8 ancora una mancanza di risorse per poter effettuare comparazioni sullo sviluppo della bacca in differenti variet\ue0 di vite. Questa tesi riguarda la prima comparazione del trascrittoma della bacca di vite effettuato attraverso RNA sequencing di 120 campioni di RNA, corrispondenti alle bacche di dieci variet\ue0 raccolte a quattro stadi fenologici, due precedenti e due successivi all\u2019invaiatura, in triplicato biologico. Quest\u2019analisi RNA-seq ha mostrato un\u2019evidente e profonda transizione del trascrittoma dalla fase verde alla maturazione che avviene all\u2019invaiatura indipendentemente da colore della buccia e variet\ue0, che coinvolge la soppressione di diversi processi metabolici relativi alla crescita vegetativa, e l\u2019induzione di solo poche vie, come processi di metabolismo secondario e di risposta a stimoli biotici. Questo importante riprogramma del trascrittoma durante la maturazione \ue8 stato evidenziato da diversi approcci: correlazione con distanza di Pearson, analisi a componenti principali (PCA), O2PLS-DA, ricerca di biomarcatori, analisi clustering e network di correlazione. La creazione della prima via trascrittomica di sviluppo della bacca di vite, corrispondente a geni aventi un profilo di espressione simile durante tutto lo sviluppo indipendentemente dalla variet\ue0, ha permesso di identificare geni coinvolti nei maggiori processi biologici che avvengono durante la maturazione del frutto. Infine, l\u2019espressione dei geni appartenenti alla via biosintetica dei fenilpropanoidi/flavonoidi si sono mostrati insufficienti da soli nello spiegare le differenze trascrittomiche tra variet\ue0 rosse e bianche; tuttavia si presuppone che questi \u2013 probabilmente per effetto dell\u2019accumulo di antociani nella buccia della bacca dall\u2019inizio della maturazione \u2013 influenzino comunque il programma della fase di maturazione, determinando il coinvolgimento e reclutamento di geni appartenenti ad altri processi biologici.Grape berry development can be described as a succession of physiological and biochemical changes reflecting the transcriptional modulation of many genes. In the last decade, many transcriptomic studies have been carried out to deeper describe this dynamic and complex development. Nonetheless, most of those transcriptomic studies focused on one single variety at a time and then there is still a lack of resources for comparing berry development in different grape varieties. This thesis describes the first berry transcriptome comparison carried out by RNA sequencing of 120 RNA samples, corresponding to 10-variety berries collected at four phenological growth stages, two pre- and two post-v\ue9raison, in biological triplication. This RNA-Seq analysis showed an evident deep green-to-maturation transcriptome shift occurring at v\ue9raison independently on skin colour and variety, which involves the suppression of diverse metabolic processes related to vegetative growth, and the induction of only a few pathways, such as secondary metabolic processes and responses to biotic stimuli. This fundamental transcriptome reprogramming during ripening was highlighted by distinct approaches: Pearson\u2019s correlation distance, PCA, O2PLS-DA, biomarker discovery, clustering analysis and correlation network method. The establishment of the first grape berry development transcriptomic route, corresponding to the genes having similar patterns of expression during whole development independently on the variety, allowed identifying genes involved in the main biological processes occurring during berry development. Finally, the expression of phenylpropanoid/flavonoid biosynthetic pathway-related genes was found to be insufficient by itself to explain the differences between red- and white-grape transcriptomes, however it was supposed to influence \u2013 supposedly by the effect of anthocyanins accumulation in berry skin since the onset of ripening \u2013 maturation-phase transcriptional program, determining the recruitment of genes belonging to other biological processes

MOLECULAR ANALYSIS OF EPIGENETIC MEMORY OF STRESS ESTABLISHMENT AND LONG-TERM MAINTENANCE IN A PERENNIAL WOODY PLANT

Plants adapt to extreme environmental conditions through physiological adaptations, which are usually transient. Recent research has suggested that environmental conditions can activate a memory of stress that can result in a primed response to subsequent stress events. While the effect of priming has been observed in many plants, the underlying mechanisms are puzzling and seldom studied. A large body of research has been developed in the last decade linking response to stress, stress priming, and memory of stress with epigenetic mechanisms. This understanding of plant epigenetics has opened the door to the application of epigenetics to crop improvement, such as the use of epigenetic breeding for the generation of more resilient crops. Although well-studied in annual and model species, research on epigenetic memory of stress in perennials is still minimal. Viticulture, a perennial form of agriculture, is highly dependent on climatic conditions, not only for yield but also for fruit quality, which is the most important factor affecting produce value at the farm gate and would benefit from more in-depth knowledge on epigenetic memory of stress. Here we present the results of an experiment conducted over two growing seasons, which constitute the first comprehensive study providing insights into the memory of stress establishment and temporal maintenance, and its potential effect on priming in a perennial crop. Gene expression and DNA methylation data were obtained from 222 plants exposed to the most common forms of abiotic stress faced by vineyards (drought, heat, and combined drought and heat). Our results indicate that the effect of the combined stress on physiology and gene expression is more severe than that of individual stresses, but not simply additive. Common genes expressed under both individual and combined treatments included heat-shock proteins, mitogen-activated kinases, and sugar-metabolizing enzymes, while phenylpropanoid biosynthesis and histone-modifying genes were unique to the combined stress treatment. We also found evidence of the establishment of memory of stress after the heat and combined stress, but not after drought, and that epigenetic chromatin modifications may play an important role during this process. Additionally, we identified genes that are differentially expressed in primed plants one year after their initial exposure to environmental insult and in the absence of recurrent stress. Moreover, primed plants showed a stronger response in gene expression to recurrent stress than plants exposed for the first time to that same stress. Finally, we explored the effect that two types of vegetative propagation may have on the maintenance of epigenetic memory of stress in primed grapevines. Briefly, although primed propagules generated using callused cuttings presented more differentially expressed genes in response to a second stress than those propagated using layering, only primed layered propagules showed differentially expressed genes in the absence of a recurrent stress, suggesting that the established stress memory is, at least partially, lost during cutting propagation. Collectively, our results constitute the first molecular evidence of long-term stress memory in grapevine and lay the foundation for the development of a comprehensive model integrating plant response to stress, the establishment of epigenetic memory of stress, and its maintenance, over time and during vegetative propagation in perennial plants