Towards In Silico identification of genes contributing to similarity of patients’ multi-omics profiles: a case study of acute myeloid leukemia

We propose a computational framework for selecting biologically plausible genes identified by clustering of multi-omics data that reveal patients' similarity, thus giving researchers a more comprehensive view on any given disease. We employ spectral clustering of a similarity network created by fusion of three similarity networks, based on mRNA expression of immune genes, miRNA expression and DNA methylation data, using SNF_v2.1 software. For each cluster, we rank multi-omics features, ensuring the best separation between clusters, and select the top-ranked features that preserve clustering. To find genes targeted by DNA methylation and miRNAs found in the top-ranked features, we use chromosome-conformation capture data and miRNet 2.0 software, respectively. To identify informative genes, these combined sets of target genes are analyzed in terms of their enrichment in somatic/germline mutations, GO biological processes/pathways terms and known sets of genes considered to be important in relation to a given disease, as recorded in the Molecular Signature Database from GSEA. The protein-protein interaction (PPI) networks were analyzed to identify genes that are hubs of PPI networks. We used data recorded in The Cancer Genome Atlas for patients with acute myeloid leukemia to demonstrate our approach, and discuss our findings in the context of results in the literature

Palynomorphs of the Normapolles group and related plant mesofossils from the Iharkút vertebrate site, Bakony Mountains (Hungary)

Abstract Palynological and paleobotanical investigation of bonebeds and other strata of the Csehbánya Formation from the vertebrate locality at Iharkút (Bakony Mts, Hungary) reveals well-preserved Santonian palynological assemblages dominated by the Normapolles group, with a minor component consisting of other angiosperm pollen, some gymnosperm pollen, and spores. Eleven species of Normapolles-type pollen grains belonging to seven genera and fruit remains of a new taxon, Sphaeracostata barbackae gen. et sp. nov., are described. The new species is very abundant in the material, represented by ca. 1000 specimens. The genus Caryanthus Friis and an unnamed form previously reported from Haţeg by Lindfors et al. (2010) are also present. Plants producing Normapolles-type pollen grains diversified during the Late Cretaceous, with a bloom in the Santonian. The palynostratigraphy of the Upper Cretaceous terrestrial sediments in the studied region is based on Normapolles-related species. The studied assemblage is assigned to the Oculopollis zaklinskaiae-Tetracolporopollenites (Brecolpites) globosus Zone (or Zone C) indicating a late Santonian age. Comparison of the Iharkút palynoflora with other known Upper Cretaceous palynofloras of Central Europe shows diachronous occurrence of Normapolles taxa at different geographic localities and warrants further investigation. The ecological requirements of the amphibian fauna reflect azonal conditions controlled by the availability of water, which is in agreement with the inferred ecological conditions based on the paleobotanical investigations. The fauna is of entirely non-marine character, further supported by isotope studies, in line with our data showing that the palynological samples contain no marine forms

Restrained Th17 response and myeloid cell infiltration into the central nervous system by human decidua-derived mesenchymal stem cells during experimental autoimmune encephalomyelitis

Background: Multiple sclerosis is a widespread inflammatory demyelinating disease. Several immunomodulatory therapies are available, including interferon-β, glatiramer acetate, natalizumab, fingolimod, and mitoxantrone. Although useful to delay disease progression, they do not provide a definitive cure and are associated with some undesirable side-effects. Accordingly, the search for new therapeutic methods constitutes an active investigation field. The use of mesenchymal stem cells (MSCs) to modify the disease course is currently the subject of intense interest. Decidua-derived MSCs (DMSCs) are a cell population obtained from human placental extraembryonic membranes able to differentiate into the three germ layers. This study explores the therapeutic potential of DMSCs. Methods: We used the experimental autoimmune encephalomyelitis (EAE) animal model to evaluate the effect of DMSCs on clinical signs of the disease and on the presence of inflammatory infiltrates in the central nervous system. We also compared the inflammatory profile of spleen T cells from DMSC-treated mice with that of EAE control animals, and the influence of DMSCs on the in vitro definition of the Th17 phenotype. Furthermore, we analyzed the effects on the presence of some critical cell types in central nervous system infiltrates. Results: Preventive intraperitoneal injection of DMSCs resulted in a significant delay of external signs of EAE. In addition, treatment of animals already presenting with moderate symptoms resulted in mild EAE with reduced disease scores. Besides decreased inflammatory infiltration, diminished percentages of CD4+IL17+, CD11b+Ly6G+ and CD11b+Ly6C+ cells were found in infiltrates of treated animals. Early immune response was mitigated, with spleen cells of DMSC-treated mice displaying low proliferative response to antigen, decreased production of interleukin (IL)-17, and increased production of the anti-inflammatory cytokines IL-4 and IL-10. Moreover, lower RORγT and higher GATA-3 expression levels were detected in DMSC-treated mice. DMSCs also showed a detrimental influence on the in vitro definition of the Th17 phenotype. Conclusions: DMSCs modulated the clinical course of EAE, modified the frequency and cell composition of the central nervous system infiltrates during the disease, and mediated an impairment of Th17 phenotype establishment in favor of the Th2 subtype. These results suggest that DMSCs might provide a new cell-based therapy for the control of multiple sclerosis.This work was sponsored by grants from Acción Estratégica en Salud (PI13/00297 and PI11/00581), the Neurosciences and Aging Foundation, the Francisco Soria Melguizo Foundation, Octopharma, and Parkinson Madrid (PI2012/0032).S

A Concerted HIF-1α/MT1-MMP Signalling Axis Regulates the Expression of the 3BP2 Adaptor Protein in Hypoxic Mesenchymal Stromal Cells

Increased plasticity, migratory and immunosuppressive abilities characterize mesenchymal stromal cells (MSC) which enable them to be active participants in the development of hypoxic solid tumours. Our understanding of the oncogenic adaptation of MSC to hypoxia however lacks the identification and characterization of specific biomarkers. In this study, we assessed the hypoxic regulation of 3BP2/SH3BP2 (Abl SH3-binding protein 2), an immune response adaptor/scaffold protein which regulates leukocyte differentiation and motility. Gene silencing of 3BP2 abrogated MSC migration in response to hypoxic cues and generation of MSC stably expressing the transcription factor hypoxia inducible factor 1alpha (HIF-1α) resulted in increased endogenous 3BP2 expression as well as cell migration. Analysis of the 3BP2 promoter sequence revealed only one potential HIF-1α binding site within the human but none in the murine sequence. An alternate early signalling cascade that regulated 3BP2 expression was found to involve membrane type-1 matrix metalloproteinase (MT1-MMP) transcriptional regulation which gene silencing abrogated 3BP2 expression in response to hypoxia. Collectively, we provide evidence for a concerted HIF-1α/MT1-MMP signalling axis that explains the induction of adaptor protein 3BP2 and which may link protein binding partners together and stimulate oncogenic MSC migration. These mechanistic observations support the potential for malignant transformation of MSC within hypoxic tumour stroma and may contribute to evasion of the immune system by a tumour

Monocytes induce STAT3 activation in human mesenchymal stem cells to promote osteoblast formation

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair

IL-27 Regulates IL-18 Binding Protein in Skin Resident Cells

IL-18 is an important mediator involved in chronic inflammatory conditions such as cutaneous lupus erythematosus, psoriasis and chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. Here we provide evidence for a yet not described anti-inflammatory mode of action on skin resident cells. Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dose-dependent and highly inducible mRNA expression and secretion of IL-18BP upon IL-27 stimulation. Other IL-12 family members failed to induce IL-18BP. The production of IL-18BP peaked between 48–72 h after stimulation and was sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional activity of IL-27. The data are in support of a significant anti-inflammatory effect of IL-27 on skin resident cells. An important novel property of IL-27 in skin pathobiology may be to counter-regulate IL-18 activities by acting on keratinocytes and importantly also on dermal fibroblasts

BAFF Promotes Th17 Cells and Aggravates Experimental Autoimmune Encephalomyelitis

BAFF, in addition to promoting B cell survival and differentiation, may affect T cells. The objective of this study was to determine the effect of BAFF on Th17 cell generation and its ramifications for the Th17 cell-driven disease, EAE.Th17 cells were increased in BAFF-Tg B6 (B6.BTg) mice and decreased in B6.Baff(-/-) mice. Th17 cells in B6.Baff(-/-) mice bearing a BAFF Tg (B6.Baff(-/-).BTg mice) were identical to those in B6.BTg mice, indicating that membrane BAFF is dispensable for Th17 cell generation as long as soluble BAFF is plentiful. In T + non-T cell criss-cross co-cultures, Th17 cell generation was greatest in cultures containing B6.BTg T cells and lowest in cultures containing B6.Baff(-/-) T cells, regardless of the source of non-T cells. In cultures containing only T cells, Th17 cell generation followed an identical pattern. CD4(+) cell expression of CD126 (IL-6R α chain) was increased in B6.BTg mice and decreased in B6.Baff(-/-) mice, and activation of STAT3 following stimulation with IL-6 + TGF-β was also greatest in B6.BTg cells and lowest in B6.Baff(-/-) cells. EAE was clinically and pathologically most severe in B6.BTg mice and least severe in B6.Baff(-/-) mice and correlated with MOG(35-55) peptide-induced Th17 cell responses.Collectively, these findings document a contribution of BAFF to pathogenic Th17 cell responses and suggest that BAFF antagonism may be efficacious in Th17 cell-driven diseases

Development and evaluation of real time RT-PCR assays for detection and typing of Bluetongue virus

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures

Identification and Differentiation of the Twenty Six Bluetongue Virus Serotypes by RT–PCR Amplification of the Serotype-Specific Genome Segment 2

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT–PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT–PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm)

Thyroid and pituitary gland development from hatching through metamorphosis of a teleost flatfish, the Atlantic halibut

Fish larval development, not least the spectacular process of flatfish metamorphosis, appears to be under complex endocrine control, many aspects of which are still not fully elucidated. In order to obtain data on the functional development of two major endocrine glands, the pituitary and the thyroid, during flatfish metamorphosis, histology, immunohistochemistry and in situ hybridization techniques were applied on larvae of the Atlantic halibut (Hippoglossus hippoglossus), a large, marine flatfish species, from hatching through metamorphosis. The material was obtained from a commercial hatchery. Larval age is defined as day-degrees (D =accumulated daily temperature from hatching). Sporadic thyroid follicles are first detected in larvae at 142 D (27 days post-hatch), prior to the completion of yolk sack absorption. Both the number and activity of the follicles increase markedly after yolk sack absorption and continue to do so during subsequent development. The larval triiodothyronine (T3) and thyroxine (T4) content increases, subsequent to yolk absorption, and coincides with the proliferation of thyroid follicles. A second increase of both T3 and T4 occurs around the start of metamorphosis and the T3 content further increases at the metamorphic climax. Overall, the T3 content is lower than T4. The pituitary gland can first be distinguished as a separate organ at the yolk sack stage. During subsequent development, the gland becomes more elongated and differentiates into neurohypophysis (NH), pars distalis (PD) and pars intermedia (PI). The first sporadic endocrine pituitary cells are observed at the yolk sack stage, somatotrophs (growth hormone producing cells) and somatolactotrophs (somatolactin producing cells) are first observed at 121 D (23 days post-hatch), and lactotrophs (prolactin producing cells) at 134 D (25 days post-hatch). Scarce thyrotrophs are evident after detection of the first thyroid follicles (142 D ), but coincident with a phase in which follicle number and activity increase (260 D ). The somatotrophs are clustered in the medium ventral region of the PD, lactotrophs in the anterior part of the PD and somatolactotrophs are scattered in the mid and posterior region of the pituitary. At around 600 D , coinciding with the start of metamorphosis, somatolactotrophs are restricted to the interdigitating tissue of the NH. During larval development, the pituitary endocrine cells become more numerous. The present data on thyroid development support the notion that thyroid hormones may play a significant role in Atlantic halibut metamorphosis. The time of appearance and the subsequent proliferation of pituitary somatotrophs, lactotrophs, somatolactotrophs and thyrotrophs indicate at which stages of larval development and metamorphosis these endocrine cells may start to play active regulatory roles.This work has been carried out within the projects ‘‘Endocrine Control as a Determinant of Larval Quality in Fish Aquaculture’’ (CT-96-1422) and ‘‘Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis’’ (Q5RS-2002-01192), with financial support from the Commission of the European Communities. However, it does not necessarily reflect the Commission’s views and in no way anticipates its future policy in this area. This project was further supported by the Swedish Council for Agricultural and Forestry Research and Pluriannual funding to CCMAR by the Portuguese Science and Technology Council