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Background: Antigen (Ag)/IgE-mediated mast cell (MC) responses play detrimental roles in allergic diseases. MC activation via the high-affinity receptor for IgE (FceRI) is controlled by the Src family kinase Lyn. Lyn-deficient (-/-) bone marrow-derived MCs (BMMCs) have been shown by various laboratories to exert stronger activation of the PI3K pathway, degranulation, and production of pro-inflammatory cytokines compared to wild-type (wt) cells. This mimics the phenotype of BMMCs deficient for the SH2-containing inositol-5'-phosphatase 1 (SHIP1). In this line, Lyn has been demonstrated to tyrosine-phosphorylate and activate SHIP1, thereby constituting a negative feedback control of PI3K-mediated signals. However, several groups have also reported on Lyn-/- BMMCs degranulating weaker than wt BMMCs. Results: Lyn-/- BMMCs, which show a suppressed degranulation response, were found to exhibit abrogated tyrosine phosphorylation of SHIP1 as well. This indicated that even in the presence of reduced SHIP1 function MC degranulation is dependent on Lyn function. In contrast to the reduced immediate secretory response, pro-inflammatory cytokine production was augmented in Lyn-/- BMMCs. For closer analysis, Lyn/SHIP1-double-deficient (dko) BMMCs were generated. In support of the dominance of Lyn deficiency, dko BMMCs degranulated significantly weaker than SHIP1-/- BMMCs. This coincided with reduced LAT1 and PLC-γ1 phosphorylation as well as Ca2+ mobilization in those cells. Interestingly, activation of the NFκB pathway followed the same pattern as measured by IκBα phosphorylation/degradation as well as induction of NFκB target genes. This suggested that Ag-triggered NFκB activation involves a Ca2+-dependent step. Indeed, IκBα phosphorylation/degradation and NFκB target gene induction were controlled by the Ca2+-dependent phosphatase calcineurin. Conclusions: Lyn deficiency is dominant over SHIP1 deficiency in MCs with respect to Ag-triggered degranulation and preceding signaling events. Moreover, the NFκB pathway and respective targets are activated in a Lyn- and Ca2+-dependent manner, reinforcing the importance of Lyn for MC activation. [ABSTRACT FROM AUTHOR]

Background DNA methylation (DNAm) has important regulatory roles in many biological processes and diseases. It is the only epigenetic mark with a clear mechanism of mitotic inheritance and the only one easily available on a genome scale. Aberrant cytosine-phosphate-guanine (CpG) methylation has been discussed in the context of disease aetiology, especially cancer. CpG hypermethylation of promoter regions is often associated with silencing of tumour suppressor genes and hypomethylation with activation of oncogenes. Supervised principal component analysis (SPCA) is a popular machine learning method. However, in a recent application to phenotype prediction from DNAm data SPCA was inferior to the specific method EVORA. Results We present Model-Selection-SPCA (MS-SPCA), an enhanced version of SPCA. MS-SPCA applies several models that perform well in the training data to the test data and selects the very best models for final prediction based on parameters of the test data. We have applied MS-SPCA for phenotype prediction from genome-wide DNAm data. CpGs used for prediction are selected based on the quantification of three features of their methylation (average methylation difference, methylation variation difference and methylation-age-correlation). We analysed four independent case-control datasets that correspond to different stages of cervical cancer: (i) cases currently cytologically normal, but will later develop neoplastic transformations, (ii, iii) cases showing neoplastic transformations and (iv) cases with confirmed cancer. The first dataset was split into several smaller case-control datasets (samples either Human Papilloma Virus (HPV) positive or negative). We demonstrate that cytology normal HPV+ and HPV- samples contain DNAm patterns which are associated with later neoplastic transformations. We present evidence that DNAm patterns exist in cytology normal HPV- samples that (i) predispose to neoplastic transformations after HPV infection and (ii) predispose to HPV infection itself. MS-SPCA performs significantly better than EVORA. Conclusions MS-SPCA can be applied to many classification problems. Additional improvements could include usage of more than one principal component (PC), with automatic selection of the optimal number of PCs. We expect that MS-SPCA will be useful for analysing recent larger DNAm data to predict future neoplastic transformations. [ABSTRACT FROM AUTHOR]

Background: Gene reshuffling, point mutations and horizontal gene transfer contribute to bacterial genome variation, but require the genome to rewire its transcriptional circuitry to ensure that inserted, mutated or reshuffled genes are transcribed at appropriate levels. The genomes of Epsilonproteobacteria display very low synteny, due to high levels of reshuffling and reorganisation of gene order, but still share a significant number of gene orthologs allowing comparison. Here we present the primary transcriptome of the pathogenic Epsilonproteobacterium Campylobacter jejuni, and have used this for comparative and predictive transcriptomics in the Epsilonproteobacteria. Results: Differential RNA-sequencing using 454 sequencing technology was used to determine the primary transcriptome of C. jejuni NCTC 11168, which consists of 992 transcription start sites (TSS), which included 29 putative non-coding and stable RNAs, 266 intragenic (internal) TSS, and 206 antisense TSS. Several previously unknown features were identified in the C. jejuni transcriptional landscape, like leaderless mRNAs and potential leader peptides upstream of amino acid biosynthesis genes. A cross-species comparison of the primary transcriptomes of C. jejuni and the related Epsilonproteobacterium Helicobacter pylori highlighted a lack of conservation of operon organisation, position of intragenic and antisense promoters or leaderless mRNAs. Predictive comparisons using 40 other Epsilonproteobacterial genomes suggests that this lack of conservation of transcriptional features is common to all Epsilonproteobacterial genomes, and is associated with the absence of genome synteny in this subdivision of the Proteobacteria. Conclusions: Both the genomes and transcriptomes of Epsilonproteobacteria are highly variable, both at the genome level by combining and division of multicistronic operons, but also on the gene level by generation or deletion of promoter sequences and 5' untranslated regions. Regulatory features may have evolved after these species split from a common ancestor, with transcriptome rewiring compensating for changes introduced by genomic reshuffling and horizontal gene transfer [ABSTRACT FROM AUTHOR]

Activation of mast cells (MCs) can be achieved by the high-affinity receptor for IgE (FceRI) as well as by additional receptors such as the lipopolysaccharide (LPS) receptor and the receptor tyrosine kinase Kit (stem cell factor [SCF] receptor). Thus, pharmacological interventions which stabilize MCs in response to different receptors would be preferable in diseases with pathological systemic MC activation such as systemic mastocytosis. 1,4-Benzodiazepines (BDZs) have been reported to suppress MC effector functions. In the present study, our aim was to analyze molecularly the effects of BDZs on MC activation by comparison of the effects of the two BDZs Ro5-4864 and clonazepam, which markedly differ in their affinities for the archetypical BDZ recognition sites, i.e., the GABAA receptor and TSPO (previously termed peripheral-type BDZ receptor). Ro5-4864 is a selective agonist at TSPO, whereas clonazepam is a selective agonist at the GABAA receptor. Ro5-4864 suppressed pro-inflammatory MC effector functions in response to antigen (Ag) (degranulation/cytokine production) and LPS and SCF (cytokine production), whereas clonazepam was inactive. Signaling pathway analyses revealed inhibitory effects of Ro5-4864 on Ag-triggered production of reactive oxygen species, calcium mobilization and activation of different downstream kinases. The initial activation of Src family kinases was attenuated by Ro5-4864 offering a molecular explanation for the observed impacts on various downstream signaling elements. In conclusion, BDZs structurally related to Ro5-4864 might serve as multifunctional MC stabilizers without the sedative effect of GABAA receptorinteracting BDZs. [ABSTRACT FROM AUTHOR]

Background: Expansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation. Results: Here, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities. Conclusions: The ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny. [ABSTRACT FROM AUTHOR]

Activation of mast cells (MCs) can be achieved by the high-affinity receptor for IgE (FcεRI) as well as by additional receptors such as the lipopolysaccharide (LPS) receptor and the receptor tyrosine kinase Kit (stem cell factor [SCF] receptor). Thus, pharmacological interventions which stabilize MCs in response to different receptors would be preferable in diseases with pathological systemic MC activation such as systemic mastocytosis. 1,4-Benzodiazepines (BDZs) have been reported to suppress MC effector functions. In the present study, our aim was to analyze molecularly the effects of BDZs on MC activation by comparison of the effects of the two BDZs Ro5-4864 and clonazepam, which markedly differ in their affinities for the archetypical BDZ recognition sites, i.e., the GABAA receptor and TSPO (previously termed peripheral-type BDZ receptor). Ro5-4864 is a selective agonist at TSPO, whereas clonazepam is a selective agonist at the GABAA receptor. Ro5-4864 suppressed pro-inflammatory MC effector functions in response to antigen (Ag) (degranulation/cytokine production) and LPS and SCF (cytokine production), whereas clonazepam was inactive. Signaling pathway analyses revealed inhibitory effects of Ro5-4864 on Ag-triggered production of reactive oxygen species, calcium mobilization and activation of different downstream kinases. The initial activation of Src family kinases was attenuated by Ro5-4864 offering a molecular explanation for the observed impacts on various downstream signaling elements. In conclusion, BDZs structurally related to Ro5-4864 might serve as multifunctional MC stabilizers without the sedative effect of GABAA receptor-interacting BDZs.

Background: Bistability underlies basic biological phenomena, such as cell division, differentiation, cancer onset, and apoptosis. So far biologists identified two necessary conditions for bistability: positive feedback and ultrasensitivity., Results: Biological systems are based upon elementary mono- and bimolecular chemical reactions. In order to definitely clarify all necessary conditions for bistability we here present the corresponding minimal system. According to our definition, it contains the minimal number of (i) reactants, (ii) reactions, and (iii) terms in the corresponding ordinary differential equations (decreasing importance from i-iii). The minimal bistable system contains two reactants and four irreversible reactions (three bimolecular, one monomolecular).We discuss the roles of the reactions with respect to the necessary conditions for bistability: two reactions comprise the positive feedback loop, a third reaction filters out small stimuli thus enabling a stable 'off' state, and the fourth reaction prevents explosions. We argue that prevention of explosion is a third general necessary condition for bistability, which is so far lacking discussion in the literature.Moreover, in addition to proving that in two-component systems three steady states are necessary for bistability (five for tristability, etc.), we also present a simple general method to design such systems: one just needs one production and three different degradation mechanisms (one production, five degradations for tristability, etc.). This helps modelling multistable systems and it is important for corresponding synthetic biology projects., Conclusion: The presented minimal bistable system finally clarifies the often discussed question for the necessary conditions for bistability. The three necessary conditions are: positive feedback, a mechanism to filter out small stimuli and a mechanism to prevent explosions. This is important for modelling bistability with simple systems and for synthetically designing new bistable systems. Our simple model system is also well suited for corresponding teaching purposes.

Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive upregulation of components of the transforming growth factor (TGF)-beta pathway in RA SFBs, with 2 hits in the top 30 regulated pathways. The growth factor TGF-beta1, its receptor TGFBR1, the TGF-beta binding proteins LTBP1/2, the TGF-beta-releasing thrombospondin 1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs, whereas TGF-beta2 was downregulated. Upregulation of TGF-beta1 and THBS1 mRNA (both positively correlated with clinical markers of disease activity/severity) and downregulation of TGF-beta2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-beta1 showed a slightly higher expression, and the signal-transducing TGFBR1 and the TGF-beta-activating THBS1 a significantly higher expression in RA SFBs than in OA SFBs. Consistent with the upregulated TGF-beta pathway in RA SFBs, stimulation with TGF-beta1 resulted in a significantly enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA SFBs show broad, constitutive alterations of the TGF-beta pathway. The abundance of TGF-beta, in conjunction with an augmented mRNA and/or protein expression of TGF-beta-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-beta-induced effects on SFBs in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling.