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Interferons (IFNs) are key cytokines involved in alerting the immune system to viral infection. After IFN stimulation, cellular transcriptional profile critically changes, leading to the expression of several IFN stimulated genes (ISGs) that exert a wide variety of antiviral activities. Despite many ISGs have been already identified, a comprehensive network of coding and non-coding genes with a central role in IFN-response still needs to be elucidated. We performed a global RNA-Seq transcriptome profile of the HCV permissive human hepatoma cell line Huh7.5 and its parental cell line Huh7, upon IFN treatment, to define a network of genes whose coordinated modulation plays a central role in IFN-response. Our study adds molecular actors, coding and non-coding genes, to the complex molecular network underlying IFN-response and shows how systems biology approaches, such as correlation networks, network’s topology and gene ontology analyses can be leveraged to this aim.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1–targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy. Human primary and metastatic tumors harbor CD4+ Treg cells that can suppress antitumor immune responses. Bonnal et al. identify an intratumoral type 1 Treg-like CD4+ T cell subset that expresses the transcription factor EOMES, granzyme K and CHI3L2. This EOMES+ T cell subset correlates with disease progression but is responsive to PD-1 checkpoint blockade immunotherapy.

Aim The review was conducted to collate the evidence on the current status of "Reproductive, Maternal, Newborn, Child and Adolescent Health" (RMNCAH) and related Behaviour Change Communication (BCC) interventions in RMNCAH in South Asia, particularly Bangladesh, Nepal and India. Methods Literature search was conducted on multiple databases and websites in March 2019. The search was limited to English language publications. The data on health indicators for the eight South Asian countries are compared in the review. This review narratively summarises the key RMNCAH and related BCC initiatives undertaken in Bangladesh, Nepal and India. Results The South Asian countries have achieved significant progress in socio-economic indicators related to health aligned with the Millennium Development Goals but face a number of challenges. There has been a significant development in financial incentives, programmes and policies framed for improving indicators of maternal and child health. Various RMNCAH initiatives which impact positively on maternal and child health indicators were identified. Conclusion Bangladesh, Nepal and India have made substantial improvement in maternal and child health indicators but at a slower pace than other countries of South Asia, in relation to which consistent disparities still exist. There are a number of BCC initiatives targeting RMNCAH. The improvement in RMNCAH indicators can be attributed to multiple initiatives wherein the contribution of BCC is relevant, because of their wide community outreach and effectiveness among all age-groups.

Interferons (IFNs) are key cytokines involved in alerting the immune system to viral infection. After IFN stimulation, cellular transcriptional profile critically changes, leading to the expression of several IFN stimulated genes (ISGs) that exert a wide variety of antiviral activities. Despite many ISGs have been already identified, a comprehensive network of coding and non-coding genes with a central role in IFN-response still needs to be elucidated. We performed a global RNA-Seq transcriptome profile of the HCV permissive human hepatoma cell line Huh7.5 and its parental cell line Huh7, upon IFN treatment, to define a network of genes whose coordinated modulation plays a central role in IFN-response. Our study adds molecular actors, coding and non-coding genes, to the complex molecular network underlying IFN-response and shows how systems biology approaches, such as correlation networks, network's topology and gene ontology analyses can be leveraged to this aim.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2. They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linkedwith susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+T lymphocytes, while CD4+T cells were less expanded and skewed toward TCMand TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+GZMB+effector cells were clonally expanded both during the infection and post-infection, while CD8+GZMK+lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+GZMB+and GZMK+subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+T cell population with memory precursor-like features.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID‐19, which negatively affects T‐cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T‐cell responses limit disease severity. Understanding how cDCs cope with SARS‐CoV‐2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection‐induced gene signatures, with the upregulation of interferon‐stimulated genes and interleukin (IL)‐6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti‐apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS‐CoV‐2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T‐cell activation. This article is protected by copyright. All rights reserved

Regulatory T (T

Fluorescence in situ hybridization (FISH) is a powerful single-cell technique that harnesses nucleic acid base pairing to detect the abundance and positioning of cellular RNA and DNA molecules in fixed samples. Recent technology development has paved the way to the construction of FISH probes entirely from synthetic oligonucleotides (oligos), allowing the optimization of thermodynamic properties together with the opportunity to design probes against any sequenced genome. However, comparatively little progress has been made in the development of computational tools to facilitate the oligos design, and even less has been done to extend their accessibility. OligoMiner is an open-source and modular pipeline written in Python that introduces a novel method of assessing probe specificity that employs supervised machine learning to predict probe binding specificity from genome-scale sequence alignment information. However, its use is restricted to only those people who are confident with command line interfaces because it lacks a Graphical User Interface (GUI), potentially cutting out many researchers from this technology. Here, we present OligoMinerApp (http://oligominerapp.org), a web-based application that aims to extend the OligoMiner framework through the implementation of a smart and easy-to-use GUI and the introduction of new functionalities specially designed to make effective probe mining available to everyone.

Glioblastoma (GBM) is the most aggressive human brain tumor. The high growth potential and decreased susceptibility to apoptosis of the glioma cells is mainly dependent on genetic amplifications or mutations of oncogenic or pro-apoptotic genes, respectively. We have previously shown that the activation of the M2 acetylcholine muscarinic receptors inhibited cell proliferation and induced apoptosis in two GBM cell lines and cancer stem cells. The aim of this study was to delve into the molecular mechanisms underlying the M2-mediated cell proliferation arrest. Exploiting U87MG and U251MG cell lines as model systems, we evaluated the ability of M2 receptors to interfere with Notch-1 and EGFR pathways, whose activation promotes GBM proliferation. We demonstrated that the activation of M2 receptors, by agonist treatment, counteracted Notch and EGFR signaling, through different regulatory cascades depending, at least in part, on p53 status. Only in U87MG cells, which mimic p53-wild type GBMs, did M2 activation trigger a molecular circuitry involving p53, Notch-1, and the tumor suppressor mir-34a-5p. This regulatory module negatively controls Notch-1, which affects cell proliferation mainly through the Notch-1/EGFR axis. Our data highlighted, for the first time, a molecular circuitry that is deregulated in the p53 wild type GBM, based on the cross-talk between M2 receptor and the Notch-1/EGFR pathways, mediated by mir-34a-5p.

// Pietro Laneve 1 , Agnese Po 2 , Annarita Favia 3 , Ivano Legnini 4 , Vincenzo Alfano 1, 2 , Jessica Rea 4 , Valerio Di Carlo 4, 11 , Valeria Bevilacqua 4, 12 , Evelina Miele 1, 13 , Angela Mastronuzzi 5 , Andrea Carai 6 , Franco Locatelli 5, 7 , Irene Bozzoni 1, 3, 4, 8 , Elisabetta Ferretti 9, 10 , Elisa Caffarelli 1, 3 1 Center for Life NanoScience@Sapienza, Istituto Italiano di Tecnologia, 00161 Rome, Italy 2 Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy 3 Institute of Molecular Biology and Pathology, National Research Council, 00185 Rome, Italy 4 Department of Biology and Biotechnology, Sapienza University of Rome, 00185 Rome, Italy 5 Department of Hematology/Oncology and Stem Cell Transplantation, Bambino Gesu Children’s Hospital, IRCCS, 00165 Rome, Italy 6 Department of Neuroscience and Neurorehabilitation, Neurosurgery Unit, Bambino Gesu Children’s Hospital, IRCCS, 00165 Rome, Italy 7 University of Pavia, Corso Strada Nuova, 27100 Pavia, Italy 8 Institute Pasteur Fondazione Cenci-Bolognetti, Sapienza University of Rome, 00185 Rome, Italy 9 Department of Experimental Medicine Sapienza University of Rome, 00161 Rome, Italy 10 Neuromed Institute, 86077 Pozzilli, Italy 11 Present addresses: Center for Genomic Regulation, 08003 Barcelona, Spain 12 Present addresses: Virology Program, INGM-Istituto Nazionale di Genetica Molecolare, 20122 Milan, Italy 13 Present addresses: Department of Hematology/Oncology and Stem Cell Transplantation, Bambino Gesu Children’s Hospital, IRCCS, 00165 Rome, Italy Correspondence to: Elisa Caffarelli, email: elisa.caffarelli@uniroma1.it Elisabetta Ferretti, email: elisabetta.ferretti@uniroma1.it Keywords: long noncoding RNAs, competing endogenous RNAs, microRNAs, cancer driver genes, Group 4 medulloblastoma Received: September 14, 2016 Accepted: February 27, 2017 Published: March 09, 2017 ABSTRACT Long noncoding RNAs (lncRNAs) are major regulators of physiological and disease-related gene expression, particularly in the central nervous system. Dysregulated lncRNA expression has been documented in several human cancers, and their tissue-specificity makes them attractive candidates as diagnostic/prognostic biomarkers and/or therapeutic agents. Here we show that linc-NeD125, which we previously characterized as a neuronal-induced lncRNA, is significantly overexpressed in Group 4 medulloblastomas (G4 MBs), the largest and least well characterized molecular MB subgroup. Mechanistically, linc-NeD125 is able to recruit the miRNA-induced silencing complex (miRISC) and to directly bind the microRNAs miR-19a-3p, miR-19b-3p and miR-106a-5p. Functionally, linc-NeD125 acts as a competing endogenous RNA (ceRNA) that, sequestering the three miRNAs, leads to de-repression of their targets CDK6, MYCN, SNCAIP, and KDM6A, which are major driver genes of G4 MB. Accordingly, linc-NeD125 downregulation reduces G4 cell proliferation. Moreover, we also provide evidence that linc-NeD125 ectopic expression in the aggressive Group 3 MB cells attenuates their proliferation, migration and invasion. This study unveils the first lncRNA-based ceRNA network in central nervous system tumours and provides a novel molecular circuit underlying the enigmatic Group 4 medulloblastoma.

The human genome contains some thousands of long non coding RNAs (lncRNAs). Many of these transcripts are presently considered crucial regulators of gene expression and functionally implicated in developmental processes in Eukaryotes. Notably, despite a huge number of lncRNAs are expressed in the Central Nervous System (CNS), only a few of them have been characterized in terms of molecular structure, gene expression regulation and function. In the present study, we identify linc-NeD125 as a novel cytoplasmic, neuronal-induced long intergenic non coding RNA (lincRNA). Linc-NeD125 represents the host gene for miR-125b-1, a microRNA with an established role as negative regulator of human neuroblastoma cell proliferation. Here, we demonstrate that these two overlapping non coding RNAs are coordinately induced during in vitro neuronal differentiation, and that their expression is regulated by different mechanisms. While the production of miR-125b-1 relies on transcriptional regulation, linc-NeD125 is controlled at the post-transcriptional level, through modulation of its stability. We also demonstrate that linc-NeD125 functions independently of the hosted microRNA, by reducing cell proliferation and activating the antiapoptotic factor BCL-2.

SBASE is a database of annotated protein domain sequences representing various structural, functional, ligand binding and topogenic segments of proteins. The current release of SBASE contains 27,211 entries which are provided with standardized names in order to facilitate retrieval. SBASE is cross-referenced to the major protein and nucleic acid databanks as well as to the PROSITE catalog of protein sequence patterns [Bairoch, A. (1992) Nucleic Acids Res., 20, Suppl., 2013-2118]. SBASE can be used to establish domain homologies through database search using programs such as FASTA [Lipman and Pearson (1985) Science, 227, 1436-1441], FASTDB [Brutlag et al. (1990) Comp. Appl. Biosci., 6, 237-245] or BLAST3 [Altschul and Lipman (1990) Proc. Natl. Acad. Sci. USA, 87, 5509-5513], which is especially useful in the case of loosely defined domain types for which efficient consensus patterns cannot be established. The use of SBASE is illustrated on the DNA binding protein Brain-4. The database and a set of search and retrieval tools are freely available on request to the authors or by anonymous 'ftp' file transfer fromftp.icgeb.trieste.it.