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Abstract Single-cell sequencing datasets are key in biology and medicine for unraveling insights into heterogeneous cell populations with unprecedented resolution. Here, we construct a single-cell multi-omics map of human tissues through in-depth characterizations of datasets from five single-cell omics, spatial transcriptomics, and two bulk omics across 125 healthy adult and fetal tissues. We construct its complement web-based platform, the Single Cell Atlas (SCA, www.singlecellatlas.org ), to enable vast interactive data exploration of deep multi-omics signatures across human fetal and adult tissues. The atlas resources and database queries aspire to serve as a one-stop, comprehensive, and time-effective resource for various omics studies.

Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous, chronic inflammatory process of the lungs and, like other complex diseases, is caused by both genetic and environmental factors. Detailed understanding of the molecular mechanisms of complex diseases requires the study of the interplay among different biomolecular layers, and thus the integration of different omics data types. In this study, we investigated COPD-associated molecular mechanisms through a correlation-based network integration of lung tissue RNA-seq and DNA methylation data of COPD cases (n = 446) and controls (n = 346) derived from the Lung Tissue Research Consortium. First, we performed a SWIM-network based analysis to build separate correlation networks for RNA-seq and DNA methylation data for our case-control study population. Then, we developed a method to integrate the results into a coupled network of differentially expressed and differentially methylated genes to investigate their relationships across both molecular layers. The functional enrichment analysis of the nodes of the coupled network revealed a strikingly significant enrichment in Immune System components, both innate and adaptive, as well as immune-system component communication (interleukin and cytokine-cytokine signaling). Our analysis allowed us to reveal novel putative COPD-associated genes and to analyze their relationships, both at the transcriptomics and epigenomics levels, thus contributing to an improved understanding of COPD pathogenesis.

Abstract Obesity is the main risk factor for many non-communicable diseases. In clinical practice, unspecific markers are used for the determination of metabolic alterations and inflammation, without allowing the characterization of subjects at higher risk of complications. Circulating microRNAs represent an attractive approach for early screening to identify subjects affected by obesity more at risk of developing connected pathologies. The aim of this study was the identification of circulating free and extracellular vesicles (EVs)-embedded microRNAs able to identify obese patients at higher risk of type 2 diabetes (DM2). The expression data of circulating microRNAs derived from obese patients (OB), with DM2 (OBDM) and healthy donors were combined with clinical data, through network-based methodology implemented by weighted gene co-expression network analysis. The six circulating microRNAs overexpressed in OBDM patients were evaluated in a second group of patients, confirming the overexpression of miR-155-5p in OBDM patients. Interestingly, the combination of miR-155-5p with serum levels of IL-8, Leptin and RAGE was useful to identify OB patients most at risk of developing DM2. These results suggest that miR-155-5p is a potential circulating biomarker for DM2 and that the combination of this microRNA with other inflammatory markers in OB patients can predict the risk of developing DM2.

BackgroundAutism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder. Major interplays between the gastrointestinal (GI) tract and the central nervous system (CNS) seem to be driven by gut microbiota (GM). Herein, we provide a GM functional characterization, based on GM metabolomics, mapping of bacterial biochemical pathways, and anamnestic, clinical, and nutritional patient metadata.MethodsFecal samples collected from children with ASD and neurotypical children were analyzed by gas-chromatography mass spectrometry coupled with solid phase microextraction (GC–MS/SPME) to determine volatile organic compounds (VOCs) associated with the metataxonomic approach by 16S rRNA gene sequencing. Multivariate and univariate statistical analyses assessed differential VOC profiles and relationships with ASD anamnestic and clinical features for biomarker discovery. Multiple web-based and machine learning (ML) models identified metabolic predictors of disease and network analyses correlated GM ecological and metabolic patterns.ResultsThe GM core volatilome for all ASD patients was characterized by a high concentration of 1-pentanol, 1-butanol, phenyl ethyl alcohol; benzeneacetaldehyde, octadecanal, tetradecanal; methyl isobutyl ketone, 2-hexanone, acetone; acetic, propanoic, 3-methyl-butanoic and 2-methyl-propanoic acids; indole and skatole; and o-cymene. Patients were stratified based on age, GI symptoms, and ASD severity symptoms. Disease risk prediction allowed us to associate butanoic acid with subjects older than 5 years, indole with the absence of GI symptoms and low disease severity, propanoic acid with the ASD risk group, and p-cymene with ASD symptoms, all based on the predictive CBCL-EXT scale. The HistGradientBoostingClassifier model classified ASD patients vs. CTRLs by an accuracy of 89%, based on methyl isobutyl ketone, benzeneacetaldehyde, phenyl ethyl alcohol, ethanol, butanoic acid, octadecane, acetic acid, skatole, and tetradecanal features. LogisticRegression models corroborated methyl isobutyl ketone, benzeneacetaldehyde, phenyl ethyl alcohol, skatole, and acetic acid as ASD predictors.ConclusionOur results will aid the development of advanced clinical decision support systems (CDSSs), assisted by ML models, for advanced ASD-personalized medicine, based on omics data integrated into electronic health/medical records. Furthermore, new ASD screening strategies based on GM-related predictors could be used to improve ASD risk assessment by uncovering novel ASD onset and risk predictors.

The tumor immune microenvironment is an important prognostic determinant in colorectal cancer (CRC). Here the authors show that tumor infiltrating neutrophils expressing high levels of CD15 interact with CD8+ T effector memory skewing them to produce GZMK, associated with tumor progression in CRC patients.

Abstract Alzheimer’s disease (AD) is the most common neurodegenerative disease that currently lacks available effective therapy. Thus, identifying novel molecular biomarkers for diagnosis and treatment of AD is urgently demanded. In this study, we exploited tools and concepts of the emerging research area of Network Medicine to unveil a novel putative disease gene signature associated with AD. We proposed a new pipeline, which combines the strengths of two consolidated algorithms of the Network Medicine: DIseAse MOdule Detection (DIAMOnD), designed to predict new disease-associated genes within the human interactome network; and SWItch Miner (SWIM), designed to predict important (switch) genes within the co-expression network. Our integrated computational analysis allowed us to enlarge the set of the known disease genes associated to AD with additional 14 genes that may be proposed as new potential diagnostic biomarkers and therapeutic targets for AD phenotype.

Abstract Background Recently, we developed a mathematical model for identifying putative competing endogenous RNA (ceRNA) interactions. This methodology has aroused a broad acknowledgment within the scientific community thanks to the encouraging results achieved when applied to breast invasive carcinoma, leading to the identification of PVT1, a long non-coding RNA functioning as ceRNA for the miR-200 family. The main shortcoming of the model is that it is no freely available and implemented in MATLAB®, a proprietary programming platform requiring a paid license for installing, operating, manipulating, and running the software. Results Breaking through these model limitations demands to distribute it in an open-source, freely accessible environment, such as R, designed for an ordinary audience of users that are not able to afford a proprietary solution. Here, we present SPINNAKER (SPongeINteractionNetworkmAKER), the open-source version of our widely established mathematical model for predicting ceRNAs crosstalk, that is released as an exhaustive collection of R functions. SPINNAKER has been even designed for providing many additional features that facilitate its usability, make it more efficient in terms of further implementation and extension, and less intense in terms of computational execution time. Conclusions SPINNAKER source code is freely available at https://github.com/sportingCode/SPINNAKER.git together with a thoroughgoing PPT-based guideline. In order to help users get the key points more conveniently, also a practical R-styled plain-text guideline is provided. Finally, a short movie is available to help the user to set the own directory, properly.

Abstract Background Historically, the molecular classification of colorectal cancer (CRC) was based on the global genomic status, which identified microsatellite instability in mismatch repair (MMR) deficient CRC, and chromosomal instability in MMR proficient CRC. With the introduction of immune checkpoint inhibitors, the microsatellite and chromosomal instability classification regained momentum as the microsatellite instability condition predicted sensitivity to immune checkpoint inhibitors, possibly due to both high tumor mutation burden (TMB) and high levels of infiltrating lymphocytes. Conversely, proficient MMR CRC are mostly resistant to immunotherapy. To better understand the relationship between the microsatellite and chromosomal instability classification, and eventually discover additional CRC subgroups relevant for therapeutic decisions, we developed a computational pipeline that include molecular integrative analysis of genomic, epigenomic and transcriptomic data. Results The first step of the pipeline was based on unsupervised hierarchical clustering analysis of copy number variations (CNVs) versus hypermutation status that identified a first CRC cluster with few CNVs enriched in Hypermutated and microsatellite instability samples, a second CRC cluster with a high number of CNVs mostly including non-HM and microsatellite stable samples, and a third cluster (7.8% of the entire dataset) with low CNVs and low TMB, which shared clinical-pathological features with Hypermutated CRCs and thus defined Hypermutated-like CRCs. The mutational features, DNA methylation profile and base substitution fingerprints of these tumors revealed that Hypermutated-like patients are molecularly distinct from Hypermutated and non-Hypermutated tumors and are likely to develop and progress through different genetic events. Transcriptomic analysis highlighted further differences amongst the three groups and revealed an inflamed tumor microenvironment and modulation Immune Checkpoint Genes in Hypermutated-like CRCs. Conclusion Therefore, our work highlights Hypermutated-like tumors as a distinct and previously unidentified CRC subgroup possibly responsive to immune checkpoint inhibitors. If further validated, these findings can lead to expanding the fraction of patients eligible to immunotherapy.

Abstract Background Gene expression is the result of the balance between transcription and degradation. Recent experimental findings have shown fine and specific regulation of RNA degradation and the presence of various molecular machinery purposely devoted to this task, such as RNA binding proteins, non-coding RNAs, etc. A biological process can be studied by measuring time-courses of RNA abundance in response of internal and/or external stimuli, using recent technologies, such as the microarrays or the Next Generation Sequencing devices. Unfortunately, the picture provided by looking only at the transcriptome abundance may not gain insight into its dynamic regulation. By contrast, independent simultaneous measurement of RNA expression and half-lives could provide such valuable additional insight. A computational approach to the estimation of RNAs half-lives from RNA expression time profiles data, can be a low-cost alternative to its experimental measurement which may be also affected by various artifacts. Results Here we present a computational methodology, called StaRTrEK (STAbility Rates ThRough Expression Kinetics), able to estimate half-life values basing only on genome-wide gene expression time series without transcriptional inhibition. The StaRTrEK algorithm makes use of a simple first order kinetic model and of a $$l_1$$ l 1 -norm regularized least square optimization approach to find its parameter values. Estimates provided by StaRTrEK are validated using simulated data and three independent experimental datasets of two short (6 samples) and one long (48 samples) time-courses. Conclusions We believe that our algorithm can be used as a fast valuable computational complement to time-course experimental gene expression studies by adding a relevant kinetic property, i.e. the RNA half-life, with a strong biological interpretation, thus providing a dynamic picture of what is going in a cell during the biological process under study.

Abstract Despite advances in modern medicine that led to improvements in cardiovascular outcomes, cardiovascular disease (CVD) remains the leading cause of mortality and morbidity globally. Thus, there is an urgent need for new approaches to improve CVD drug treatments. As the development time and cost of drug discovery to clinical application are excessive, alternate strategies for drug development are warranted. Among these are included computational approaches based on omics data for drug repositioning, which have attracted increasing attention. In this work, we developed an adjusted similarity measure implemented by the algorithm SAveRUNNER to reposition drugs for cardiovascular diseases while, at the same time, considering the side effects of drug candidates. We analyzed nine cardiovascular disorders and two side effects. We formulated both disease disorders and side effects as network modules in the human interactome, and considered those drug candidates that are proximal to disease modules but far from side-effects modules as ideal. Our method provides a list of drug candidates for cardiovascular diseases that are unlikely to produce common, adverse side-effects. This approach incorporating side effects is applicable to other diseases, as well.

Abstract The outcome of patients affected by high-risk or metastatic neuroblastoma (NB) remains grim, with ≥ 50% of the children experiencing relapse or progression of the disease despite multimodal, intensive treatment. In order to identify new strategies to improve the overall survival and the quality of life of these children, we recently developed and optimized a third-generation GD2-specific chimeric antigen receptor (CAR) construct, which is currently under evaluation in our Institution in a phase I/II clinical trial (NCT03373097) enrolling patients with relapsed/refractory NB. We observed that our CAR T-cells are able to induce marked tumor reduction and even achieve complete remission with a higher efficiency than that of other CAR T-cells reported in previous studies. However, often responses are not sustained and relapses occur. Here, we demonstrate for the first time a mechanism of resistance to GD2.CAR T-cell treatment, showing how polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) increase in the peripheral blood (PB) of NB patients after GD2.CAR T-cell treatment in case of relapse and loss of response. In vitro, isolated PMN-MDSC demonstrate to inhibit the anti-tumor cytotoxicity of different generations of GD2.CAR T-cells. Gene-expression profiling of GD2.CAR T-cells “conditioned” with PMN-MDSC shows downregulation of genes involved in cell activation, signal transduction, inflammation and cytokine/chemokine secretion. Analysis of NB gene-expression dataset confirms a correlation between expression of these genes and patient outcome. Moreover, in patients treated with GD2.CAR T-cells, the frequency of circulating PMN-MDSC inversely correlates with the levels of GD2.CAR T-cells, resulting more elevated in patients who did not respond or lost response to the treatment. The presence and the frequency of PMN-MDSC in PB of high-risk and metastatic NB represents a useful prognostic marker to predict the response to GD2.CAR T-cells and other adoptive immunotherapy. This study underlines the importance of further optimization of both CAR T-cells and clinical trial in order to target elements of the tumor microenvironment.

Abstract Cancer stem-like cells (CSCs) have self-renewal abilities responsible for cancer progression, therapy resistance, and metastatic growth. The glioblastoma stem-like cells are the most studied among CSC populations. A recent study identified four transcription factors (SOX2, SALL2, OLIG2, and POU3F2) as the minimal core sufficient to reprogram differentiated glioblastoma (GBM) cells into stem-like cells. Transcriptomic data of GBM tissues and cell lines from two different datasets were then analyzed by the SWItch Miner (SWIM), a network-based software, and FOSL1 was identified as a putative regulator of the previously identified minimal core. Herein, we selected NTERA-2 and HEK293T cells to perform an in vitro study to investigate the role of FOSL1 in the reprogramming mechanisms. We transfected the two cell lines with a constitutive FOSL1 cDNA plasmid. We demonstrated that FOSL1 directly regulates the four transcription factors binding their promoter regions, is involved in the deregulation of several stemness markers, and reduces the cells’ ability to generate aggregates increasing the extracellular matrix component FN1. Although further experiments are necessary, our data suggest that FOSL1 reprograms the stemness by regulating the core of the four transcription factors.

Autism spectrum disorders (ASDs) is a multifactorial neurodevelopmental disorder. The communication between the gastrointestinal (GI) tract and the central nervous system seems driven by gut microbiota (GM). Herein, we provide GM profiling, considering GI functional symptoms, neurological impairment, and dietary habits. Forty-one and 35 fecal samples collected from ASD and neurotypical children (CTRLs), respectively, (age range, 3–15 years) were analyzed by 16S targeted-metagenomics (the V3–V4 region) and inflammation and permeability markers (i.e., sIgA, zonulin lysozyme), and then correlated with subjects’ metadata. Our ASD cohort was characterized as follows: 30/41 (73%) with GI functional symptoms; 24/41 (58%) picky eaters (PEs), with one or more dietary needs, including 10/41 (24%) with food selectivity (FS); 36/41 (88%) presenting high and medium autism severity symptoms (HMASSs). Among the cohort with GI symptoms, 28/30 (93%) showed HMASSs, 17/30 (57%) were picky eaters and only 8/30 (27%) with food selectivity. The remaining 11/41 (27%) ASDs without GI symptoms that were characterized by HMASS for 8/11 (72%) and 7/11 (63%) were picky eaters. GM ecology was investigated for the overall ASD cohort versus CTRLs; ASDs with GI and without GI, respectively, versus CTRLs; ASD with GI versus ASD without GI; ASDs with HMASS versus low ASSs; PEs versus no-PEs; and FS versus absence of FS. In particular, the GM of ASDs, compared to CTRLs, was characterized by the increase of Proteobacteria, Bacteroidetes, Rikenellaceae, Pasteurellaceae, Klebsiella, Bacteroides, Roseburia, Lactobacillus, Prevotella, Sutterella, Staphylococcus, and Haemophilus. Moreover, Sutterella, Roseburia and Fusobacterium were associated to ASD with GI symptoms compared to CTRLs. Interestingly, ASD with GI symptoms showed higher value of zonulin and lower levels of lysozyme, which were also characterized by differentially expressed predicted functional pathways. Multiple machine learning models classified correctly 80% overall ASDs, compared with CTRLs, based on Bacteroides, Lactobacillus, Prevotella, Staphylococcus, Sutterella, and Haemophilus features. In conclusion, in our patient cohort, regardless of the evaluation of many factors potentially modulating the GM profile, the major phenotypic determinant affecting the GM was represented by GI hallmarks and patients’ age.

Abstract Background Currently, no proven effective drugs for the novel coronavirus disease COVID-19 exist and despite widespread vaccination campaigns, we are far short from herd immunity. The number of people who are still vulnerable to the virus is too high to hamper new outbreaks, leading a compelling need to find new therapeutic options devoted to combat SARS-CoV-2 infection. Drug repurposing represents an effective drug discovery strategy from existing drugs that could shorten the time and reduce the cost compared to de novo drug discovery. Results We developed a network-based tool for drug repurposing provided as a freely available R-code, called SAveRUNNER (Searching off-lAbel dRUg aNd NEtwoRk), with the aim to offer a promising framework to efficiently detect putative novel indications for currently marketed drugs against diseases of interest. SAveRUNNER predicts drug–disease associations by quantifying the interplay between the drug targets and the disease-associated proteins in the human interactome through the computation of a novel network-based similarity measure, which prioritizes associations between drugs and diseases located in the same network neighborhoods. Conclusions The algorithm was successfully applied to predict off-label drugs to be repositioned against the new human coronavirus (2019-nCoV/SARS-CoV-2), and it achieved a high accuracy in the identification of well-known drug indications, thus revealing itself as a powerful tool to rapidly detect potential novel medical indications for various drugs that are worth of further investigation. SAveRUNNER source code is freely available at https://github.com/giuliafiscon/SAveRUNNER.git , along with a comprehensive user guide.

Abstract In this study, we integrate the outcomes of co-expression network analysis with the human interactome network to predict novel putative disease genes and modules. We first apply the SWItch Miner (SWIM) methodology, which predicts important (switch) genes within the co-expression network that regulate disease state transitions, then map them to the human protein–protein interaction network (PPI, or interactome) to predict novel disease–disease relationships (i.e., a SWIM-informed diseasome). Although the relevance of switch genes to an observed phenotype has been recently assessed, their performance at the system or network level constitutes a new, potentially fascinating territory yet to be explored. Quantifying the interplay between switch genes and human diseases in the interactome network, we found that switch genes associated with specific disorders are closer to each other than to other nodes in the network, and tend to form localized connected subnetworks. These subnetworks overlap between similar diseases and are situated in different neighborhoods for pathologically distinct phenotypes, consistent with the well-known topological proximity property of disease genes. These findings allow us to demonstrate how SWIM-based correlation network analysis can serve as a useful tool for efficient screening of potentially new disease gene associations. When integrated with an interactome-based network analysis, it not only identifies novel candidate disease genes, but also may offer testable hypotheses by which to elucidate the molecular underpinnings of human disease and reveal commonalities between seemingly unrelated diseases.

Abstract Background Rewiring of metabolism induced by oncogenic K-Ras in cancer cells involves both glucose and glutamine utilization sustaining enhanced, unrestricted growth. The development of effective anti-cancer treatments targeting metabolism may be facilitated by the identification and rational combinatorial targeting of metabolic pathways. Methods We performed mass spectrometric metabolomics analysis in vitro and in vivo experiments to evaluate the efficacy of drugs and identify metabolic connectivity. Results We show that K-Ras-mutant lung and colon cancer cells exhibit a distinct metabolic rewiring, the latter being more dependent on respiration. Combined treatment with the glutaminase inhibitor CB-839 and the PI3K/aldolase inhibitor NVP-BKM120 more consistently reduces cell growth of tumor xenografts. Maximal growth inhibition correlates with the disruption of redox homeostasis, involving loss of reduced glutathione regeneration, redox cofactors, and a decreased connectivity among metabolites primarily involved in nucleic acid metabolism. Conclusions Our findings open the way to develop metabolic connectivity profiling as a tool for a selective strategy of combined drug repositioning in precision oncology.

Tumor microenvironment (TME) includes a wide variety of cell types and soluble factors capable of suppressing immune-responses. While the role of NK cells in TME has been analyzed, limited information is available on the presence and the effect of polymorphonuclear (PMN) myeloid-derived suppressor cells, (MDSC). Among the immunomodulatory cells present in TME, MDSC are potentially efficient in counteracting the anti-tumor activity of several effector cells. We show that PMN-MDSC are present in high numbers in the PB of patients with primary or metastatic lung tumor. Their frequency correlated with the overall survival of patients. In addition, it inversely correlated with low frequencies of NK cells both in the PB and in tumor lesions. Moreover, such NK cells displayed an impaired anti-tumor activity, even those isolated from PB. The compromised function of NK cells was consequent to their interaction with PMN-MDSC. Indeed, we show that the expression of major activating NK receptors, the NK cytolytic activity and the cytokine production were inhibited upon co-culture with PMN-MDSC through both cell-to-cell contact and soluble factors. In this context, we show that exosomes derived from PMN-MDSC are responsible of a significant immunosuppressive effect on NK cell-mediated anti-tumor activity. Our data may provide a novel useful tool to implement the tumor immunoscore. Indeed, the detection of PMN-MDSC in the PB may be of prognostic value, providing clues on the presence and extension of both adult and pediatric tumors and information on the efficacy not only of immune response but also of immunotherapy and, possibly, on the clinical outcome.

Background Triple-negative breast cancers (TNBCs) display poor prognosis, have a high risk of tumour recurrence, and exhibit high resistance to drug treatments. Based on their gene expression profiles, the majority of TNBCs are classified as basal-like breast cancers. Currently, there are not available widely-accepted prognostic markers to predict outcomes in basal-like subtype, so the selection of new prognostic indicators for this BC phenotype represents an unmet clinical challenge. Results Here, we attempted to address this challenging issue by exploiting a bioinformatics pipeline able to integrate transcriptomic, genomic, epigenomic, and clinical data freely accessible from public repositories. This pipeline starts from the application of the well-established network-based SWIM methodology on the transcriptomic data to unveil important (switch) genes in relation with a complex disease of interest. Then, survival and linear regression analyses are performed to associate the gene expression profiles of the switch genes with both the patients’ clinical outcome and the disease aggressiveness. This allows us to identify a prognostic gene signature that in turn is fed to the last step of the pipeline consisting of an analysis at DNA level, to investigate whether variations in the expression of identified prognostic switch genes could be related to genetic (copy number variations) or epigenetic (DNA methylation differences) alterations in their gene loci, or to the activities of transcription factors binding to their promoter regions. Finally, changes in the protein expression levels corresponding to the so far identified prognostic switch genes are evaluated by immunohistochemical staining results taking advantage of the Human Protein Atlas. Conclusion The application of the proposed pipeline on the dataset of The Cancer Genome Atlas (TCGA)-Breast Invasive Carcinoma (BRCA) patients affected by basal-like subtype led to an in silico recognition of a basal-like specific gene signature composed of 11 potential prognostic biomarkers to be further investigated.

The SARS-CoV-2 pandemic is a worldwide public health emergency. Despite the beginning of a vaccination campaign, the search for new drugs to appropriately treat COVID-19 patients remains a priority. Drug repurposing represents a faster and cheaper method than de novo drug discovery. In this study, we examined three different network-based approaches to identify potentially repurposable drugs to treat COVID-19. We analyzed transcriptomic data from whole blood cells of patients with COVID-19 and 21 other related conditions, as compared with those of healthy subjects. In addition to conventionally used drugs (e.g., anticoagulants, antihistaminics, anti-TNFα antibodies, corticosteroids), unconventional candidate compounds, such as SCN5A inhibitors and drugs active in the central nervous system, were identified. Clinical judgment and validation through clinical trials are always mandatory before use of the identified drugs in a clinical setting.

Breast cancer (BC) is a heterogeneous and complex disease characterized by different subtypes with distinct morphologies and clinical implications and for which new and effective treatment options are urgently demanded. The computational approaches recently developed for drug repurposing provide a very promising opportunity to offer tools that efficiently screen potential novel medical indications for various drugs that are already approved and used in clinical practice. Here, we started with disease-associated genes that were identified through a transcriptome-based analysis, which we used to predict potential repurposable drugs for various breast cancer subtypes by using an algorithm that we developed for drug repurposing called SAveRUNNER. Our findings were also in silico validated by performing a gene set enrichment analysis, which confirmed that most of the predicted repurposable drugs may have a potential treatment effect against breast cancer pathophenotypes.

Abstract Background miRNAs regulate the expression of several genes with one miRNA able to target multiple genes and with one gene able to be simultaneously targeted by more than one miRNA. Therefore, it has become indispensable to shorten the long list of miRNA-target interactions to put in the spotlight in order to gain insight into understanding the regulatory mechanism orchestrated by miRNAs in various cellular processes. A reasonable solution is certainly to prioritize miRNA-target interactions to maximize the effectiveness of the downstream analysis. Results We propose a new and easy-to-use web tool MIENTURNET (MicroRNA ENrichment TURned NETwork) that receives in input a list of miRNAs or mRNAs and tackles the problem of prioritizing miRNA-target interactions by performing a statistical analysis followed by a fully featured network-based visualization and analysis. The statistics is used to assess the significance of an over-representation of miRNA-target interactions and then MIENTURNET filters based on the statistical significance associated with each miRNA-target interaction. In addition, the holistic approach of the network theory is used to infer possible evidences of miRNA regulation by capturing emergent properties of the miRNA-target regulatory network that would be not evident through a pairwise analysis of the individual components. Conclusion MIENTURNET offers the possibility to consistently perform both statistical and network-based analyses by using only a single tool leading to a more effective prioritization of the miRNA-target interactions. This has the potential to avoid researchers without computational and informatics skills to navigate multiple websites and thus to independently investigate miRNA activity in every cellular process of interest in an easy and at the same time exhaustive way thanks to the intuitive web interface. The web application along with a well-documented and comprehensive user guide are freely available at http://userver.bio.uniroma1.it/apps/mienturnet/ without any login requirement.

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide. They are typically characterized by a high incidence of local recurrence, which is the most common cause of death in HNSCC patients. TP53 is the most frequently mutated gene in HNSCC and patients carrying TP53 mutations are associated with a higher probability to develop local recurrence. MiRNAs, which are among the mediators of the oncogenic activity of mt-p53 protein, emerge as an appealing tool for screening, diagnosis and prognosis of cancer. We previously identified a signature of 12 miRNAs whose aberrant expression associated with TP53 mutations and was prognostic for HNSCC. Among them miR-96-5p emerges as an oncogenic miRNAs with prognostic significance in HNSCC. Methods To evaluate the oncogenic role of miR-96-5p in a tumoral context, we performed colony formation, cell migration and cell viability assays in two HNSCC cell lines transfected for miR-96-5p mimic or inhibitor and treated with or without radio/chemo-therapy. In addition, to identify genes positively and negatively correlated to miR-96-5p expression in HNSCC, we analyzed the correlation between gene expression and miR-96-5p level in the subset of TCGA HNSCC tumors carrying missense TP53 mutations by Spearman and Pearson correlation. To finally identify targets of miR-96-5p, we used in silico analysis and the luciferase reporter assay to confirm PTEN as direct target. Results Our data showed that overexpression of miR-96-5p led to increased cell migration and radio-resistance, chemotherapy resistance in HNSCC cells. In agreement with these results, among the most statistically significant pathways in which miR-96-5p is involved, are focal Adhesion, extracellular matrix organization and PI3K-Akt-mTOR-signaling pathway. As a direct target of miR-96-5p, we identified PTEN, the main negative regulator of PI3K-Akt signalling pathway activation. Conclusions These results highlight a new mechanism of chemo/radio-resistance insurgence in HNSCC cells and support the possibility that miR-96-5p expression could be used as a novel promising biomarker to predict radiotherapy response and local recurrence development in HNSCC patients. In addition, the identification of pathways in which miR-96-5p is involved could contribute to develop new therapeutic strategies to overcome radio-resistance.

The novelty of new human coronavirus COVID-19/SARS-CoV-2 and the lack of effective drugs and vaccines gave rise to a wide variety of strategies employed to fight this worldwide pandemic. Many of these strategies rely on the repositioning of existing drugs that could shorten the time and reduce the cost compared to de novo drug discovery. In this study, we presented a new network-based algorithm for drug repositioning, called SAveRUNNER (Searching off-lAbel dRUg aNd NEtwoRk), which predicts drug-disease associations by quantifying the interplay between the drug targets and the disease-specific proteins in the human interactome via a novel network-based similarity measure that prioritizes associations between drugs and diseases locating in the same network neighborhoods. Specifically, we applied SAveRUNNER on a panel of 14 selected diseases with a consolidated knowledge about their disease-causing genes and that have been found to be related to COVID-19 for genetic similarity (i.e., SARS), comorbidity (e.g., cardiovascular diseases), or for their association to drugs tentatively repurposed to treat COVID-19 (e.g., malaria, HIV, rheumatoid arthritis). Focusing specifically on SARS subnetwork, we identified 282 repurposable drugs, including some the most rumored off-label drugs for COVID-19 treatments (e.g., chloroquine, hydroxychloroquine, tocilizumab, heparin), as well as a new combination therapy of 5 drugs (hydroxychloroquine, chloroquine, lopinavir, ritonavir, remdesivir), actually used in clinical practice. Furthermore, to maximize the efficiency of putative downstream validation experiments, we prioritized 24 potential anti-SARS-CoV repurposable drugs based on their network-based similarity values. These top-ranked drugs include ACE-inhibitors, monoclonal antibodies (e.g., anti-IFNγ, anti-TNFα, anti-IL12, anti-IL1β, anti-IL6), and thrombin inhibitors. Finally, our findings were in-silico validated by performing a gene set enrichment analysis, which confirmed that most of the network-predicted repurposable drugs may have a potential treatment effect against human coronavirus infections.

Non-nonavalent vaccine (9v) Human papillomavirus (HPV) types have been shown to have high prevalence among HIV-positive women. Here, 1444 cervical samples were tested for HPV DNA positivity. Co-infections of the 9v HPV types with other HPV types were evaluated. The HPV81 L1 and L2 genes were used to investigate the genetic variability of antigenic epitopes. HPV-positive samples were genotyped using the HPVCLART2 assay. The L1 and L2 protein sequences were analyzed using a self-optimized prediction method to predict their secondary structure. Co-occurrence probabilities of the 9v HPV types were calculated. Non9v types represented 49% of the HPV infections; 31.2% of the non9v HPV types were among the low-grade squamous intraepithelial lesion samples, and 27.3% among the high-grade squamous intraepithelial lesion samples, and several genotypes were low risk. The co-occurrence of 9v HPV types with the other genotypes was not correlated with the filogenetic distance. HPV81 showed an amino-acid substitution within the BC loop (N75Q) and the FGb loop (T315N). In the L2 protein, all of the mutations were located outside antigenic sites. The weak cross-protection of the 9v types suggests the relevance of a sustainable and effective screening program, which should be implemented by HPV DNA testing that does not include only high-risk types.

Extensive molecular characterization of human colorectal cancer (CRC) via Next Generation Sequencing (NGS) indicated that genetic or epigenetic dysregulation of a relevant, but limited, number of molecular pathways typically occurs in this tumor. The molecular picture of the disease is significantly complicated by the frequent occurrence of individually rare genetic aberrations, which expand tumor heterogeneity. Inter- and intratumor molecular heterogeneity is very likely responsible for the remarkable individual variability in the response to conventional and target-driven first-line therapies, in metastatic CRC (mCRC) patients, whose median overall survival remains unsatisfactory. Implementation of an extensive molecular characterization of mCRC in the clinical routine does not yet appear feasible on a large scale, while multigene panel sequencing of most commonly mutated oncogene/oncosuppressor hotspots is more easily achievable. Here, we report that clinical multigene panel sequencing performed for anti-EGFR therapy predictive purposes in 639 formalin-fixed paraffin-embedded (FFPE) mCRC specimens revealed previously unknown pairwise mutation associations and a high proportion of cases carrying actionable gene mutations. Most importantly, a simple principal component analysis directed the delineation of a new molecular stratification of mCRC patients in eight groups characterized by non-random, specific mutational association patterns (MAPs), aggregating samples with similar biology. These data were validated on a The Cancer Genome Atlas (TCGA) CRC dataset. The proposed stratification may provide great opportunities to direct more informed therapeutic decisions in the majority of mCRC cases.

Abstract Background It is well-known that glioblastoma contains self-renewing, stem-like subpopulation with the ability to sustain tumor growth. These cells – called cancer stem-like cells – share certain phenotypic characteristics with untransformed stem cells and are resistant to many conventional cancer therapies, which might explain the limitations in curing human malignancies. Thus, the identification of genes controlling the differentiation of these stem-like cells is becoming a successful therapeutic strategy, owing to the promise of novel targets for treating malignancies. Methods Recently, we developed SWIM, a software able to unveil a small pool of genes – called switch genes – critically associated with drastic changes in cell phenotype. Here, we applied SWIM to the expression profiling of glioblastoma stem-like cells and conventional glioma cell lines, in order to identify switch genes related to stem-like phenotype. Results SWIM identifies 171 switch genes that are all down-regulated in glioblastoma stem-like cells. This list encompasses genes like CAV1, COL5A1, COL6A3, FLNB, HMMR, ITGA3, ITGA5, MET, SDC1, THBS1, and VEGFC, involved in “ECM-receptor interaction“ and “focal adhesion” pathways. The inhibition of switch genes highly correlates with the activation of genes related to neural development and differentiation, such as the 4-core OLIG2, POU3F2, SALL2, SOX2, whose induction has been shown to be sufficient to reprogram differentiated glioblastoma into stem-like cells. Among switch genes, the transcription factor FOSL1 appears as the brightest star since: it is down-regulated in stem-like cells; it highly negatively correlates with the 4-core genes that are all up-regulated in stem-like cells; the promoter regions of the 4-core genes harbor a consensus binding motif for FOSL1. Conclusions We suggest that the inhibition of switch genes in stem-like cells could induce the deregulation of cell communication pathways, contributing to neoplastic progression and tumor invasiveness. Conversely, their activation could restore the physiological equilibrium between cell adhesion and migration, hampering the progression of cancer. Moreover, we posit FOSL1 as promising candidate to orchestrate the differentiation of cancer stem-like cells by repressing the 4-core genes’ expression, which severely halts cancer growth and might affect the therapeutic outcome. We suggest FOSL1 as novel putative therapeutic and prognostic biomarker, worthy of further investigation.

Abstract Background Circular RNAs are a class of endogenous RNAs with various functions in eukaryotic cells. Worthy of note, circular RNAs play a critical role in cancer. Currently, nothing is known about their role in head and neck squamous cell carcinoma (HNSCC). The identification of circular RNAs in HNSCC might become useful for diagnostic and therapeutic strategies in HNSCC. Results Using samples from 115 HNSCC patients, we find that circPVT1 is over-expressed in tumors compared to matched non-tumoral tissues, with particular enrichment in patients with TP53 mutations. circPVT1 up- and down-regulation determine, respectively, an increase and a reduction of the malignant phenotype in HNSCC cell lines. We show that circPVT1 expression is transcriptionally enhanced by the mut-p53/YAP/TEAD complex. circPVT1 acts as an oncogene modulating the expression of miR-497-5p and genes involved in the control of cell proliferation. Conclusions This study shows the oncogenic role of circPVT1 in HNSCC, extending current knowledge about the role of circular RNAs in cancer.

The MRI of the prostate is the gold standard for the detection of clinically significant prostate cancer (csPCa). Nonetheless, MRI still misses around 11% of clinically significant disease. The aim was to comprehensively integrate tissue and circulating microRNA profiling, MRI biomarkers and clinical data to implement PCa early detection. In this prospective cohort study, 76 biopsy naïve patients underwent MRI and MRI directed biopsy. A sentinel sample of 15 patients was selected for a pilot molecular analysis. Weighted gene coexpression network analysis was applied to identify the microRNAs drivers of csPCa. MicroRNA–target gene interaction maps were constructed, and enrichment analysis performed. The ANOVA on ranks test and ROC analysis were performed for statistics. Disease status was associated with the underexpression of the miRNA profiled; a correlation was found with ADC (r = −0.51, p = 0.02) and normalized ADC values (r = −0.64, p = 0.002). The overexpression of miRNAs from plasma was associated with csPCa (r = 0.72; p = 0.02), and with PI-RADS assessment score (r = 0.73; p = 0.02); a linear correlation was found with biomarkers of diffusion and perfusion. Among the 800 profiled microRNA, eleven were identified as correlating with PCa, among which miR-548a-3p, miR-138-5p and miR-520d-3p were confirmed using the RT-qPCR approach on an additional cohort of ten subjects. ROC analysis showed an accuracy of >90%. Provided an additional validation set of the identified miRNAs on a larger cohort, we propose a diagnostic paradigm shift that sees molecular data and MRI biomarkers as the prebiopsy triage of patients at risk for PCa. This approach will allow for accurate patient allocation to biopsy, and for stratification into risk group categories, reducing overdiagnosis and overtreatment.

BackgroundCystic fibrosis (CF) is a disorder affecting the respiratory, digestive, reproductive systems and sweat glands. This lethal hereditary disease has known or suspected links to the dysbiosis gut microbiota. High-throughput meta-omics-based approaches may assist in unveiling this complex network of symbiosis modifications.ObjectivesThe aim of this study was to provide a predictive and functional model of the gut microbiota enterophenotype of pediatric patients affected by CF under clinical stability.MethodsThirty-one fecal samples were collected from CF patients and healthy children (HC) (age range, 1-6 years) and analysed using targeted-metagenomics and metabolomics to characterize the ecology and metabolism of CF-linked gut microbiota. The multidimensional data were low fused and processed by chemometric classification analysis.ResultsThe fused metagenomics and metabolomics based gut microbiota profile was characterized by a high abundance of Propionibacterium, Staphylococcus and Clostridiaceae, including Clostridium difficile, and a low abundance of Eggerthella, Eubacterium, Ruminococcus, Dorea, Faecalibacterium prausnitzii, and Lachnospiraceae, associated with overexpression of 4-aminobutyrate (GABA), choline, ethanol, propylbutyrate, and pyridine and low levels of sarcosine, 4-methylphenol, uracil, glucose, acetate, phenol, benzaldehyde, and methylacetate. The CF gut microbiota pattern revealed an enterophenotype intrinsically linked to disease, regardless of age, and with dysbiosis uninduced by reduced pancreatic function and only partially related to oral antibiotic administration or lung colonization/infection.ConclusionsAll together, the results obtained suggest that the gut microbiota enterophenotypes of CF, together with endogenous and bacterial CF biomarkers, are direct expression of functional alterations at the intestinal level. Hence, it's possible to infer that CFTR impairment causes the gut ecosystem imbalance.This new understanding of CF host-gut microbiota interactions may be helpful to rationalize novel clinical interventions to improve the affected children's nutritional status and intestinal function.

Recent findings have identified competing endogenous RNAs (ceRNAs) as the drivers in many disease conditions, including cancers. The ceRNAs indirectly regulate each other by reducing the amount of microRNAs (miRNAs) available to target messenger RNAs (mRNAs). The ceRNA interactions mediated by miRNAs are modulated by a titration mechanism, i.e. large changes in the ceRNA expression levels either overcome, or relieve, the miRNA repression on competing RNAs; similarly, a very large miRNA overexpression may abolish competition. The ceRNAs are also called miRNA "decoys" or miRNA "sponges" and encompass different RNAs competing with each other to attract miRNAs for interactions: mRNA, long non-coding RNAs (lncRNAs), pseudogenes, or circular RNAs. Recently, we developed a computational method for identifying ceRNA-ceRNA interactions in breast invasive carcinoma. We were interested in unveiling which lncRNAs could exert the ceRNA activity. We found a drastic rewiring in the cross-talks between ceRNAs from the physiological to the pathological condition. The main actor of this dysregulated lncRNA-associated ceRNA network was the lncRNA PVT1, which revealed a net biding preference towards the miR-200 family members in normal breast tissues. Despite its up-regulation in breast cancer tissues, mimicked by the miR-200 family members, PVT1 stops working as ceRNA in the cancerous state. The specific conditions required for a ceRNA landscape to occur are still far from being determined. Here, we emphasized the importance of the relative concentration of the ceRNAs, and their related miRNAs. In particular, we focused on the withdrawal in breast cancer tissues of the PVT1 ceRNA activity and performed a gene expression and sequence analysis of its multiple isoforms. We found that the PVT1 isoform harbouring the binding site for a representative miRNA of the miR-200 family shows a drastic decrease in its relative concentration with respect to the miRNA abundance in breast cancer tissues, providing a plausibility argument to the breakdown of the sponge program orchestrated by the oncogene PVT1.

OBJECTIVES:Identifying the predictive factors of Sustained Virological Response (SVR) represents an important challenge in new interferon-based DAA therapies. Here, we analyzed the kinetics of antiviral response associated with a triple drug regimen, and the association between negative residual viral load at different time points during treatment. METHODS:Twenty-three HCV genotype 1 (GT 1a n = 11; GT1b n = 12) infected patients were included in the study. Linear Discriminant Analysis (LDA) was used to establish possible association between HCV RNA values at days 1 and 4 from start of therapy and SVR. Principal component analysis (PCA) was applied to analyze the correlation between HCV RNA slope and SVR. A ultrasensitive (US) method was established to measure the residual HCV viral load in those samples which resulted "detected

The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. 1H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota "core" of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology.

Network analysis provides deep insight into real complex systems. Revealing the link between topological and functional role of network elements can be crucial to understand the mechanisms underlying the system. Here we propose a Cytoscape plugin (GIANT) to perform network clustering and characterize nodes at the light of a modified Guimerà-Amaral cartography. This approach results into a vivid picture of the a topological/functional relationship at both local and global level. The plugin has been already approved and uploaded on the Cytoscape APP store.

In this article we present evidence for a relationship between chromosome gene loci and the topological properties of the protein-protein interaction network corresponding to the set of genes under consideration. Specifically, for each chromosome of the Saccharomyces cerevisiae genome, the distribution of the intra-chromosome inter-gene distances was analyzed and a positive correlation with the distance among the corresponding proteins of the protein-protein interaction network was found. In order to study this relationship we used concepts based on non-parametric statistics and information theory. We provide statistical evidence that if two genes are closely located, then it is likely that their protein products are closely located in the protein-protein interaction network, or in other words, that they are involved in the same biological process.

The transcriptome in a cell is finely regulated by a large number of molecular mechanisms able to control the balance between mRNA production and degradation. Recent experimental findings have evidenced that fine and specific regulation of degradation is needed for proper orchestration of a global cell response to environmental conditions. We developed a computational technique based on stochastic modeling, to infer condition-specific individual mRNA half-lives directly from gene expression time-courses. Predictions from our method were validated by experimentally measured mRNA decay rates during the intraerythrocytic developmental cycle of Plasmodium falciparum. We then applied our methodology to publicly available data on the reproductive and metabolic cycle of budding yeast. Strikingly, our analysis revealed, in all cases, the presence of periodic changes in decay rates of sequentially induced genes and co-ordination strategies between transcription and degradation, thus suggesting a general principle for the proper coordination of transcription and degradation machinery in response to internal and/or external stimuli.

The time of initiation of antiretroviral therapy in HIV-1 infected patients has a determinant effect on the viral dynamics. The question is, how far can the therapy be delayed? Is sooner always better? We resort to clinical data and to microsimulations to forecast the dynamics of the viral load at therapy interruption after prolonged antiretroviral treatment. A computational model previously evaluated, produces results that are statistically adherent to clinical data. In addition, it allows a finer grain analysis of the impact of the therapy initiation point to the disease course. We find a swift increase of the viral density as a function of the time of initiation of the therapy measured when the therapy is stopped. In particular there is a critical time delay with respect to the infection instant beyond which the therapy does not affect the viral rebound. Initiation of the treatment is beneficial because it can down-regulate the immune activation, hence limiting viral replication and spread.