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Cyclic di-nucleotides (CDN) are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA) is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8 + cytotoxic T lymphocytes (CTL) is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α) is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Perimortem caesarean section is very rare, mostly resulting in high mortality of mother and/or fetus. We report a case of successful resuscitation of both mother and newborn following maternal cardiac arrest prior to delivery. Postoperative outcome was complicated by severe bleeding and coagulopathy following fibrinolysis and subcapsular hepatic haematoma. We consider a fast reaction time based on a special in-hospital emergency team for immediate caesarean section and an aggressive management of coagulopathy as major factors that led to both patients recovery without neurological sequelae.

EndoNet is a new database that provides information about the components of endocrine networks and their relations. It focuses on the endocrine cell-to-cell communication and enables the analysis of intercellular regulatory pathways in humans. In the EndoNet data model, two classes of components span a bipartite directed graph. One class represents the hormones (in the broadest sense) secreted by defined donor cells. The other class consists of the acceptor or target cells expressing the corresponding hormone receptors. The identity and anatomical environment of cell types, tissues and organs is defined through references to the CYTOMER ontology. With the EndoNet user interface, it is possible to query the database for hormones, receptors or tissues and to combine several items from different search rounds in one complex result set, from which a network can be reconstructed and visualized. For each entity, a detailed characteristics page is available. Some well-established endocrine pathways are offered as showcases in the form of predefined result sets. These sets can be used as a starting point for a more complex query or for obtaining a quick overview. The EndoNet database is accessible at http://endonet.bioinf.med.uni-goettingen.de/.

Scaffold/matrix attachment regions (S/MARs) are essential regulatory DNA elements of eukaryotic cells. They are major determinants of locus control of gene expression and can shield gene expression from position effects. Experimental detection of S/MARs requires substantial effort and is not suitable for large-scale screening of genomic sequences. In silico prediction of S/MARs can provide a crucial first selection step to reduce the number of candidates. We used experimentally defined S/MAR sequences as the training set and generated a library of new S/MAR-associated, AT-rich patterns described as weight matrices. A new tool called SMARTest was developed that identifies potential S/MARs by performing a density analysis based on the S/MAR matrix library (http://www.genomatix.de/cgi-bin/smartest_pd/smartest.pl). S/MAR predictions were evaluated by using six genomic sequences from animal and plant for which S/MARs and non-S/MARs were experimentally mapped. SMARTest reached a sensitivity of 38% and a specificity of 68%. In contrast to previous algorithms, the SMARTest approach does not depend on the sequence context and is suitable to analyze long genomic sequences up to the size of whole chromosomes. To demonstrate the feasibility of large-scale S/MAR prediction, we analyzed the recently published chromosome 22 sequence and found 1198 S/MAR candidates.

A polyclonal antibody was raised against a recombinant Chlamydomonas 14-3-3-beta-galactosidase (beta-Gal) fusion protein and characterized for its epitope specificity towards the corresponding Chlamydomonas 14-3-3 protein by scan-peptide analysis. This antibody recognized four Chlamydomonas polypeptides with apparent molecular masses 32, 30, 27, and 24 kDa, which also reacted with the antiserum depleted of anti-(Escherichia coli beta-Gal) IgG, but not with the corresponding preimmune serum or the antiserum preincubated with purified 14-3-3 proteins. Western-blot analyses performed with the antibody depleted of anti-(beta-Gal) IgG revealed that more or less pronounced levels of 14-3-3 proteins were present in all subcellular fractions of Chlamydomonas reinhardtii except the nuclei. The highest levels of 14-3-3 protein were observed in the cytosol and microsomal fraction. The 30-kDa isoform was predominant in the cytosol, whereas the 27-kDa isoform was prevalent in the microsomes. When microsomal membranes were separated by sucrose-density-gradient centrifugation, Western-blot analysis revealed distinct patterns of 14-3-3 isoforms in the endoplasmic reticulum, dictyosome, and plasma membrane fractions identified by marker enzyme activities. These findings indicate that the four 14-3-3 proteins of C. reinhardtii differentially interact with endoplasmic reticulum, dictyosomes, and plasma membrane.

Members of the 14-3-3 protein family have been identified as regulatory elements in intracellular signalling pathways and cell cycle control. Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Chlamydomonas reinhardtii. In this communication, we describe the nucleotide sequence, the genomic organization and the cell-cycle-dependent expression of the corresponding gene. The coding sequence of this gene was found to be interrupted by four introns of 124, 116, 81, and 659 bp, respectively. Introns 2-4 were found in conserved positions as compared to the Arabidopsis 14-3-3 genes. A counterpart to intron 1 absent in the Arabidopsis 14-3-3 genes was found in the human 14-3-3 epsilon gene.

We have isolated and sequenced a 1464 bp cDNA from the unicellular green alga Chlamydomonas reinhardtii encoding an acidic polypeptide (259 aa) with considerable homologies to the 14-3-3 proteins of animals, yeasts and higher plants. Like the other members of this highly conserved protein kinase regulatory protein family, the deduced amino acid sequence of the Chlamydomonas 14-3-3 protein includes two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca(2+)-binding sites is located within the C-terminal part of this polypeptide (positions 208-219). EF hand motifs are also present in the 14-3-3 proteins of some higher plants but not in those of animals and yeasts.