Proteomics and disease network associations evaluation of environmentally relevant Bisphenol A concentrations in a human 3D neural stem cell model.

Bisphenol A (BPA) exposure is associated with a plethora of neurodevelopmental abnormalities and brain disorders. Previous studies have demonstrated BPA-induced perturbations to critical neural stem cell (NSC) characteristics, such as proliferation and differentiation, although the underlying molecular mechanisms remain under debate. The present study evaluated the effects of a repeated-dose exposure of environmentally relevant BPA concentrations during the in vitro 3D neural induction of human induced pluripotent stem cells (hiPSCs), emulating a chronic exposure scenario. Firstly, we demonstrated that our model is suitable for NSC differentiation during the early stages of embryonic brain development. Our morphological image analysis showed that BPA exposure at 0.01, 0.1 and 1 µM decreased the average spheroid size by day 21 (D21) of the neural induction, while no effect on cell viability was detected. No alteration to the rate of the neural induction was observed based on the expression of key neural lineage and neuroectodermal transcripts. Quantitative proteomics at D21 revealed several differentially abundant proteins across all BPA-treated groups with important functions in NSC proliferation and maintenance (e.g., FABP7, GPC4, GAP43, Wnt-8B, TPPP3). Additionally, a network analysis demonstrated alterations to the glycolytic pathway, potentially implicating BPA-induced changes to glycolytic signalling in NSC proliferation impairments, as well as the pathophysiology of brain disorders including intellectual disability, autism spectrum disorders, and amyotrophic lateral sclerosis (ALS). This study enhances the current understanding of BPA-related NSC aberrations based mostly on acute, often high dose exposures of rodent in vivo and in vitro models and human GWAS data in a novel human 3D cell-based model with real-life scenario relevant prolonged and low-level exposures, offering further mechanistic insights into the ramifications of BPA exposure on the developing human brain and consequently, later life neurological disorders.

AutoCore: A network-based definition of the core module of human autoimmunity and autoinflammation.

Although research on rare autoimmune and autoinflammatory diseases has enabled definition of nonredundant regulators of homeostasis in human immunity, because of the single gene-single disease nature of many of these diseases, contributing factors were mostly unveiled in sequential and noncoordinated individual studies. We used a network-based approach for integrating a set of 186 inborn errors of immunity with predominant autoimmunity/autoinflammation into a comprehensive map of human immune dysregulation, which we termed "AutoCore." The AutoCore is located centrally within the interactome of all protein-protein interactions, connecting and pinpointing multidisease markers for a range of common, polygenic autoimmune/autoinflammatory diseases. The AutoCore can be subdivided into 19 endotypes that correspond to molecularly and phenotypically cohesive disease subgroups, providing a molecular mechanism-based disease classification and rationale toward systematic targeting for therapeutic purposes. Our study provides a proof of concept for using network-based methods to systematically investigate the molecular relationships between individual rare diseases and address a range of conceptual, diagnostic, and therapeutic challenges.

Low-dose Bisphenol A exposure alters the functionality and cellular environment in a human cardiomyocyte model.

Early embryonic development represents a sensitive time-window during which the foetus might be vulnerable to the exposure of environmental contaminants, potentially leading to heart diseases also later in life. Bisphenol A (BPA), a synthetic chemical widely used in plastics manufacturing, has been associated with heart developmental defects, even in low concentrations. This study aims to investigate the effects of environmentally relevant doses of BPA on developing cardiomyocytes using a human induced pluripotent stem cell (hiPSC)-derived model. Firstly, a 2D in vitro differentiation system to obtain cardiomyocytes from hiPSCs (hiPSC-CMs) have been established and characterised to provide a suitable model for the early stages of cardiac development. Then, the effects of a repeated BPA exposure, starting from the undifferentiated stage throughout the differentiation process, were evaluated. The chemical significantly decreased the beat rate of hiPSC-CMs, extending the contraction and relaxation time in a dose-dependent manner. Quantitative proteomics analysis revealed a high abundance of basement membrane (BM) components (e.g., COL4A1, COL4A2, LAMC1, NID2) and a significant increase in TNNC1 and SERBP1 proteins in hiPSC-CMs treated with BPA. Network analysis of proteomics data supported altered extracellular matrix remodelling and provided a disease-gene association with well-known pathological conditions of the heart. Furthermore, upon hypoxia-reoxygenation challenge, hiPSC-CMs treated with BPA showed higher rate of apoptotic events. Taken together, our results revealed that a long-term treatment, even with low doses of BPA, interferes with hiPSC-CMs functionality and alters the surrounding cellular environment, providing new insights about diseases that might arise upon the toxin exposure. Our study contributes to the current understanding of BPA effects on developing human foetal cardiomyocytes, in correlation with human clinical observations and animal studies, and it provides a suitable model for New Approach Methodologies (NAMs) for environmental chemical hazard and risk assessment.

Maternal hyperglycemia induces alterations in hepatic amino acid, glucose and lipid metabolism of neonatal offspring: Multi-omics insights from a diabetic pig model.

OBJECTIVE: To gain mechanistic insights into adverse effects of maternal hyperglycemia on the liver of neonates, we performed a multi-omics analysis of liver tissue from piglets developed in genetically diabetic (mutant INS gene induced diabetes of youth; MIDY) or wild-type (WT) pigs. METHODS: Proteome, metabolome and lipidome profiles of liver and clinical parameters of serum samples from 3-day-old WT piglets (n = 9) born to MIDY mothers (PHG) were compared with those of WT piglets (n = 10) born to normoglycemic mothers (PNG). Furthermore, protein-protein interaction network analysis was used to reveal highly interacting proteins that participate in the same molecular mechanisms and to relate these mechanisms with human pathology. RESULTS: Hepatocytes of PHG displayed pronounced lipid droplet accumulation, although the abundances of central lipogenic enzymes such as fatty acid-synthase (FASN) were decreased. Additionally, circulating triglyceride (TG) levels were reduced as a trend. Serum levels of non-esterified free fatty acids (NEFA) were elevated in PHG, potentially stimulating hepatic gluconeogenesis. This is supported by elevated hepatic phosphoenolpyruvate carboxykinase (PCK1) and circulating alanine transaminase (ALT) levels. Even though targeted metabolomics showed strongly elevated phosphatidylcholine (PC) levels, the abundances of multiple key enzymes involved in major PC synthesis pathways - most prominently those from the Kennedy pathway - were paradoxically reduced in PHG liver. Conversely, enzymes involved in PC excretion and breakdown such as PC-specific translocase ATP-binding cassette 4 (ABCB4) and phospholipase A2 were increased in abundance. CONCLUSIONS: Our study indicates that maternal hyperglycemia without confounding obesity induces profound molecular changes in the liver of neonatal offspring. In particular, we found evidence for stimulated gluconeogenesis and hepatic lipid accumulation independent of de novo lipogenesis. Reduced levels of PC biosynthesis enzymes and increased levels of proteins involved in PC translocation or breakdown may represent counter-regulatory mechanisms to maternally elevated PC levels. Our comprehensive multi-omics dataset provides a valuable resource for future meta-analysis studies focusing on liver metabolism in newborns from diabetic mothers.

Network Approaches for Charting the Transcriptomic and Epigenetic Landscape of the Developmental Origins of Health and Disease.

The early developmental phase is of critical importance for human health and disease later in life. To decipher the molecular mechanisms at play, current biomedical research is increasingly relying on large quantities of diverse omics data. The integration and interpretation of the different datasets pose a critical challenge towards the holistic understanding of the complex biological processes that are involved in early development. In this review, we outline the major transcriptomic and epigenetic processes and the respective datasets that are most relevant for studying the periconceptional period. We cover both basic data processing and analysis steps, as well as more advanced data integration methods. A particular focus is given to network-based methods. Finally, we review the medical applications of such integrative analyses.

Discovering common pathogenetic processes between COVID-19 and diabetes mellitus by differential gene expression pattern analysis.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the newly discovered coronavirus, SARS-CoV-2. Increased severity of COVID-19 has been observed in patients with diabetes mellitus (DM). This study aimed to identify common transcriptional signatures, regulators and pathways between COVID-19 and DM. We have integrated human whole-genome transcriptomic datasets from COVID-19 and DM, followed by functional assessment with gene ontology (GO) and pathway analyses. In peripheral blood mononuclear cells (PBMCs), among the upregulated differentially expressed genes (DEGs), 32 were found to be commonly modulated in COVID-19 and type 2 diabetes (T2D), while 10 DEGs were commonly downregulated. As regards type 1 diabetes (T1D), 21 DEGs were commonly upregulated, and 29 DEGs were commonly downregulated in COVID-19 and T1D. Moreover, 35 DEGs were commonly upregulated in SARS-CoV-2 infected pancreas organoids and T2D islets, while 14 were commonly downregulated. Several GO terms were found in common between COVID-19 and DM. Prediction of the putative transcription factors involved in the upregulation of genes in COVID-19 and DM identified RELA to be implicated in both PBMCs and pancreas. Here, for the first time, we have characterized the biological processes and pathways commonly dysregulated in COVID-19 and DM, which could be in the next future used for the design of personalized treatment of COVID-19 patients suffering from DM as comorbidity.

A Network Medicine Approach for Drug Repurposing in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive hereditary muscular disease caused by a lack of dystrophin, leading to membrane instability, cell damage, and inflammatory response. However, gene-editing alone is not enough to restore the healthy phenotype and additional treatments are required. In the present study, we have first conducted a meta-analysis of three microarray datasets, GSE38417, GSE3307, and GSE6011, to identify the differentially expressed genes (DEGs) between healthy donors and DMD patients. We have then integrated this analysis with the knowledge obtained from DisGeNET and DIAMOnD, a well-known algorithm for drug-gene association discoveries in the human interactome. The data obtained allowed us to identify novel possible target genes and were used to predict potential therapeutical options that could reverse the pathological condition.

Profiling of inhibitory immune checkpoints in glioblastoma: Potential pathogenetic players.

Glioblastoma (GBM) represents the most frequent glial tumor, with almost 3 new cases per 100,000 people per year. Despite treatment, the prognosis for GBM patients remains extremely poor, with a median survival of 14.6 months, and a 5-year survival less than 5%. It is generally believed that GBM creates a highly immunosuppressive microenvironment, sustained by the expression of immune-regulatory factors, including inhibitory immune checkpoints, on both infiltrating cells and tumor cells. However, the trials assessing the efficacy of current immune checkpoint inhibitors in GBM are still disappointing. In the present study, the expression levels of several inhibitory immune checkpoints in GBM (CD276, VTCN1, CD47, PVR, TNFRSF14, CD200, LGALS9, NECTIN2 and CD48) were characterized in order to evaluate their potential as prognostic and eventually, therapeutic targets. Among the investigated immune checkpoints, TNFRSF14 and NECTIN2 were identified as the most promising targets in GBM. In particular, a higher TNFRSF14 expression was associated with worse overall survival and disease-free survival, and with a lower Th1 response.

Transcriptional landscape of SARS-CoV-2 infection dismantles pathogenic pathways activated by the virus, proposes unique sex-specific differences and predicts tailored therapeutic strategies.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has posed a serious threat to global health. As no specific therapeutics are yet available to control disease evolution, more in-depth understanding of the pathogenic mechanisms induced by SARS-CoV-2 will help to characterize new targets for the management of COVID-19. The present study identified a specific set of biological pathways altered in primary human lung epithelium upon SARS-CoV-2 infection, and a comparison with SARS-CoV from the 2003 pandemic was studied. The transcriptomic profiles were also exploited as possible novel therapeutic targets, and anti-signature perturbation analysis predicted potential drugs to control disease progression. Among them, Mitogen-activated protein kinase kinase (MEK), serine-threonine kinase (AKT), mammalian target of rapamycin (mTOR) and I kappa B Kinase (IKK) inhibitors emerged as candidate drugs. Finally, sex-specific differences that may underlie the higher COVID-19 mortality in men are proposed.

Transcriptomic Analysis Reveals Abnormal Expression of Prion Disease Gene Pathway in Brains from Patients with Autism Spectrum Disorders.

The role of infections in the pathogenesis of autism spectrum disorder (ASD) is still controversial. In this study, we aimed to evaluate markers of infections and immune activation in ASD by performing a meta-analysis of publicly available whole-genome transcriptomic datasets of brain samples from autistic patients and otherwise normal people. Among the differentially expressed genes, no significant enrichment was observed for infectious diseases previously associated with ASD, including herpes simplex virus-1 (HSV-1), cytomegalovirus and Epstein-Barr virus in brain samples, nor was it found in peripheral blood from ASD patients. Interestingly, a significant number of genes belonging to the "prion diseases" pathway were found to be modulated in our ASD brain meta-analysis. Overall, our data do not support an association between infection and ASD. However, the data do provide support for the involvement of pathways related to other neurodegenerative diseases and give input to uncover novel pathogenetic mechanisms underlying ASD.

Exploratory Analysis of iPSCS-Derived Neuronal Cells as Predictors of Diagnosis and Treatment of Alzheimer Disease.

Alzheimer's disease (AD) represents the most common neurodegenerative disorder, with 47 million affected people worldwide. Current treatment strategies are aimed at reducing the symptoms and do slow down the progression of the disease, but inevitably fail in the long-term. Induced pluripotent stem cells (iPSCs)-derived neuronal cells from AD patients have proven to be a reliable model for AD pathogenesis. Here, we have conducted an in silico analysis aimed at identifying pathogenic gene-expression profiles and novel drug candidates. The GSE117589 microarray dataset was used for the identification of Differentially Expressed Genes (DEGs) between iPSC-derived neuronal progenitor (NP) cells and neurons from AD patients and healthy donors. The Discriminant Analysis Module (DAM) algorithm was used for the identification of biomarkers of disease. Drugs with anti-signature gene perturbation profiles were identified using the L1000FWD software. DAM analysis was used to identify a list of potential biomarkers among the DEGs, able to discriminate AD patients from healthy people. Finally, anti-signature perturbation analysis identified potential anti-AD drugs. This study set the basis for the investigation of potential novel pharmacological strategies for AD. Furthermore, a subset of genes for the early diagnosis of AD is proposed.

Impaired Expression of Tetraspanin 32 (TSPAN32) in Memory T Cells of Patients with Multiple Sclerosis.

Tetraspanins are a conserved family of proteins involved in a number of biological processes. We have previously shown that Tetraspanin-32 (TSPAN32) is significantly downregulated upon activation of T helper cells via anti-CD3/CD28 stimulation. On the other hand, TSPAN32 is marginally modulated in activated Treg cells. A role for TSPAN32 in controlling the development of autoimmune responses is consistent with our observation that encephalitogenic T cells from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mice exhibit significantly lower levels of TSPAN32 as compared to naïve T cells. In the present study, by making use of ex vivo and in silico analysis, we aimed to better characterize the pathophysiological and diagnostic/prognostic role of TSPAN32 in T cell immunity and in multiple sclerosis (MS). We first show that TSPAN32 is significantly downregulated in memory T cells as compared to naïve T cells, and that it is further diminished upon ex vivo restimulation. Accordingly, following antigenic stimulation, myelin-specific memory T cells from MS patients showed significantly lower expression of TSPAN32 as compared to memory T cells from healthy donors (HD). The expression levels of TSPAN32 was significantly downregulated in peripheral blood mononuclear cells (PBMCs) from drug-naïve MS patients as compared to HD, irrespective of the disease state. Finally, when comparing patients undergoing early relapses in comparison to patients with longer stable disease, moderate but significantly lower levels of TSPAN32 expression were observed in PBMCs from the former group. Our data suggest a role for TSPAN32 in the immune responses underlying the pathophysiology of MS and represent a proof-of-concept for additional studies aiming at dissecting the eventual contribution of TSPAN32 in other autoimmune diseases and its possible use of TSPAN32 as a diagnostic factor and therapeutic target.

The Role of Macrophage Migration Inhibitory Factor in Alzheimer's Disease: Conventionally Pathogenetic or Unconventionally Protective?

Recent preclinical and clinical observations have offered relevant insights on the etiopathogenesis of late onset Alzheimer's disease (AD) and upregulated immunoinflammatory events have been described as underlying mechanisms involved in the development of AD. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine produced by several cells of the innate and adaptive immune system, as well as non-immune cells. In the present review, we highlight experimental, genetic, and clinical studies on MIF in rodent models of AD and AD patients, and we discuss emerging therapeutic opportunities for tailored modulation of the activity of MIF, that may potentially be applied to AD patients. Dismantling the exact role of MIF and its receptors in AD may offer novel diagnostic and therapeutic opportunities in AD.

Transcriptomic Analysis Reveals Involvement of the Macrophage Migration Inhibitory Factor Gene Network in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive hereditary muscular disease with X-linked recessive inheritance, that leads patients to premature death. The loss of dystrophin determines membrane instability, causing cell damage and inflammatory response. Macrophage migration inhibitory factor (MIF) is a cytokine that exerts pleiotropic properties and is implicated in the pathogenesis of a variety of diseases. Recently, converging data from independent studies have pointed to a possible role of MIF in dystrophic muscle disorders, including DMD. In the present study, we have investigated the modulation of MIF and MIF-related genes in degenerative muscle disorders, by making use of publicly available whole-genome expression datasets. We show here a significant enrichment of MIF and related genes in muscle samples from DMD patients, as well as from patients suffering from Becker's disease and limb-girdle muscular dystrophy type 2B. On the other hand, transcriptomic analysis of in vitro differentiated myotubes from healthy controls and DMD patients revealed no significant alteration in the expression levels of MIF-related genes. Finally, by analyzing DMD samples as a time series, we show that the modulation of the genes belonging to the MIF network is an early event in the DMD muscle and does not change with the increasing age of the patients, Overall, our analysis suggests that MIF may play a role in vivo during muscle degeneration, likely promoting inflammation and local microenvironment reaction.

Overexpression of Macrophage Migration Inhibitory Factor and Its Homologue D-Dopachrome Tautomerase as Negative Prognostic Factor in Neuroblastoma.

Neuroblastoma (NB) represents one of the most frequent pediatric solid tumors. Macrophage migration inhibitory factor (MIF) is a cytokine exerting multiple biological functions. More recently, a second member of the MIF family of cytokine has been identified, the D-dopachrome tautomerase (DDT), that exerts several overlapping functions with MIF. Growing evidence suggests a key role for MIF and DDT in the development of cancer. The aim of this study is to characterize the prognostic value of MIF and DDT in NB. We show that higher expression levels of MIF and DDT in Stage 4 NB samples are associated with a poorer prognosis, independently of the presence of MYCN amplification. Moreover, higher levels of MIF are mostly enriched by Th1 cells, while lower levels of MIF are associated with an increased proportion of B cells, Cytotoxic T cells, Dendritic cells and Natural Killer T cells. We also show that treatment with the histone deacetylase (HDAC) inhibitor, vorinostat, of the NB cell line, SH-SY5Y, determines a significant reduction in the expression of both MIF and DDT. Finally, MIF and DDT inhibition by short interfering RNA is able to revert vincristine sensitivity in vitro. Overall, our data suggest that MIF exert pro-tumorigenic properties in NB, likely by dampening antigen presentation and cytotoxic immune responses, and we propose the HDAC inhibitors as a potential therapeutic strategy for NB patients.

Prediction of PD-L1 Expression in Neuroblastoma via Computational Modeling.

Immunotherapy is a promising new therapeutic approach for neuroblastoma (NBM): an anti-GD2 vaccine combined with orally administered soluble beta-glucan is undergoing a phase II clinical trial and nivolumab and ipilimumab are being tested in recurrent and refractory tumors. Unfortunately, predictive biomarkers of response to immunotherapy are currently not available for NBM patients. The aim of this study was to create a computational network model simulating the different intracellular pathways involved in NBM, in order to predict how the tumor phenotype may be influenced to increase the sensitivity to anti-programmed cell death-ligand-1 (PD-L1)/programmed cell death-1 (PD-1) immunotherapy. The model runs on COPASI software. In order to determine the influence of intracellular signaling pathways on the expression of PD-L1 in NBM, we first developed an integrated network of protein kinase cascades. Michaelis-Menten kinetics were associated to each reaction in order to tailor the different enzymes kinetics, creating a system of ordinary differential equations (ODEs). The data of this study offers a first tool to be considered in the therapeutic management of the NBM patient undergoing immunotherapeutic treatment.

Modulation of Tetraspanin 32 (TSPAN32) Expression in T Cell-Mediated Immune Responses and in Multiple Sclerosis.

Tetraspanins are a conserved family of proteins involved in a number of biological processes including, cell-cell interactions, fertility, cancer metastasis and immune responses. It has previously been shown that TSPAN32 knockout mice have normal hemopoiesis and B-cell responses, but hyperproliferative T cells. Here, we show that TSPAN32 is expressed at higher levels in the lymphoid lineage as compared to myeloid cells. In vitro activation of T helper cells via anti-CD3/CD28 is associated with a significant downregulation of TSPAN32. Interestingly, engagement of CD3 is sufficient to modulate TSPAN32 expression, and its effect is potentiated by costimulation with anti-CD28, but not anti-CTLA4, -ICOS nor -PD1. Accordingly, we measured the transcriptomic levels of TSPAN32 in polarized T cells under Th1 and Th2 conditions and TSPAN32 resulted significantly reduced as compared with unstimulated cells. On the other hand, in Treg cells, TSPAN32 underwent minor changes upon activation. The in vitro data were finally translated into the context of multiple sclerosis (MS). Encephalitogenic T cells from Myelin Oligodendrocyte Glycoprotein (MOG)-Induced Experimental Autoimmune Encephalomyelitis (EAE) mice showed significantly lower levels of TSPAN32 and increased levels of CD9, CD53, CD82 and CD151. Similarly, in vitro-activated circulating CD4 T cells from MS patients showed lower levels of TSPAN32 as compared with cells from healthy donors. Overall, these data suggest an immunoregulatory role for TSPAN32 in T helper immune response and may represent a target of future immunoregulatory therapies for T cell-mediated autoimmune diseases.

Upregulation of IL-1 Receptor Antagonist in a Mouse Model of Migraine.

Migraine is a disorder characterized by attacks of monolateral headaches, often accompanied by nausea, vomiting, and photophobia. Around 30% of patients also report aura symptoms. The cause of the aura is believed to be related to the cortical spreading depression (CSD), a wave of neuronal and glial depolarization originating in the occipital cortex, followed by temporary neuronal silencing. During a migraine attack, increased expression of inflammatory mediators, along with a decrease in the expression of anti-inflammatory genes, have been observed. The aim of this study was to evaluate the expression of inflammatory genes, in particular that of IL-1 receptor antagonist (IL-1RN), following CSD in a mouse model of familial hemiplegic migraine type 1 (FHM-1). We show here that the expression of IL-1RN was upregulated after the CSD, suggesting a possible attempt to modulate the inflammatory response. This study allows researchers to better understand the development of the disease and aids in the search for new therapeutic strategies in migraine.