Placodus (Placodontia, Sauropterygia) dentaries from Winterswijk, The Netherlands (middle Anisian) and Hünfeld, Hesse, Germany (late Anisian) with comments on ontogenetic changes.

Two recently found dentaries from the Lower Muschelkalk of Winterswijk (The Netherlands) and from the Upper Muschelkalk of an outcrop in the vicinity of Hünfeld (Hesse, Germany) are studied and compared to lower jaws of placodonts. As a result, the here described specimens can be assigned to Placodus cf. gigas. However, this assignment should be regarded as preliminary due to the isolated nature of the material. More diagnostic material is necessary to validate this affiliation. A certain morphological variability in P. gigas dentaries that had been pointed out before is also obvious among the new material. Placodus gigas has a wide paleogeography and stratigraphic range and a revision of the material assigned to P. gigas with new methods is overdue but beyond the scope of the current paper. The dentary from Hünfeld is with about 4 cm preserved length the smallest so far known dentary of a Placodus. It provides interesting insights in morphological changes during ontogeny and reveals differences in trajectories when compared to dentaries of different ontogenetic stages of Cyamodus hildegardis.

Using systems medicine to identify a therapeutic agent with potential for repurposing in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) cause significant morbidity and mortality. Aberrant NF-κB signalling is strongly associated with these conditions, and several established drugs influence the NF-κB signalling network to exert their effect. This study aimed to identify drugs that alter NF-κB signalling and could be repositioned for use in IBD. The SysmedIBD Consortium established a novel drug-repurposing pipeline based on a combination of in silico drug discovery and biological assays targeted at demonstrating an impact on NF-κB signalling, and a murine model of IBD. The drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. The effects of clarithromycin effects were validated in several experiments: it influenced NF-κB-mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-κB protein shuttling in murine reporter enteroids; it suppressed NF-κB (p65) DNA binding in the small intestine of mice exposed to lipopolysaccharide; and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-κB (p65) nuclear translocation in human intestinal enteroids. These findings demonstrate that in silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of IBD, and that further clinical assessment of clarithromycin in the management of IBD is required.This article has an associated First Person interview with the joint first authors of the paper.

The Epigenetic Signature of Colonizing New Environments in Anolis Lizards.

Founder populations often show rapid divergence from source populations after colonizing new environments. Epigenetic modifications can mediate phenotypic responses to environmental change and may be an important mechanism promoting rapid differentiation in founder populations. Whereas many long-term studies have explored the extent to which divergence between source and founder populations is genetically heritable versus plastic, the role of epigenetic processes during colonization remains unclear. To investigate epigenetic modifications in founding populations, we experimentally colonized eight small Caribbean islands with brown anole lizards (Anolis sagrei) from a common source population. We then quantitatively measured genome-wide DNA methylation in liver tissue using reduced representation bisulfite sequencing of individuals transplanted onto islands with high- versus low-habitat quality. We found that lizard sex and habitat quality explained a significant proportion of epigenetic variation. Differentially methylated cytosines mapped to genes that encode proteins with functions likely to be relevant to habitat change (e.g., signal transduction, immune response, circadian rhythm). This study provides experimental evidence of a relationship between epigenetic responses and the earliest stages of colonization of novel environments in nature and suggests that habitat quality influences the nature of these epigenetic modifications.

Cell cycle phase regulates glucocorticoid receptor function.

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1). Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G(1). This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase. The impact of cell cycle-driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle. In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.

Distinct roles for Caf1, Ccr4, Edc3 and CutA in the co-ordination of transcript deadenylation, decapping and P-body formation in Aspergillus nidulans.

Transcript degradation is a key step in gene regulation. In eukaryotes, mRNA decay is generally initiated by removal of the poly(A) tail mediated by the Ccr4-Caf1-Not complex. Deadenylated transcripts are then rapidly degraded, primarily via the decapping-dependent pathway. Components of this pathway can be localized into highly dynamic cytoplasmic foci, the mRNA processing (P)-bodies. We have undertaken confocal fluorescence microscopy to monitor P-bodies in Aspergillus nidulans. As in other organisms a dynamic shift in P-body formation occurs in response to diverse physiological signals. Significantly, both this cellular response and the signalled degradation of specific transcripts are dependent on the nuclease activity of Caf1 but not Ccr4. P-body formation is disrupted in A. nidulans strains deleted for Edc3, an enhancer of decapping, or CutA, which encodes a nucleotidyltransferase that triggers mRNA decapping by the addition of a CUCU tag to the poly(A) tail. As with DeltacutA, Deltaedc3 led to reduced rates of transcript degradation. These data link P-bodies to both the optimization and regulation of transcript degradation.

P53 apoptosis mediator PERP: localization, function and caspase activation in uveal melanoma.

p53 apoptosis effector related to PMP-22 (PERP) is a transcriptional target gene of p53 tumour suppressor that is specifically induced during apoptosis and not during cell cycle arrest. In primary uveal melanoma (UM), the most common intraocular malignancy in adults that has a reportedly unaffected signalling pathway upstream of and including p53, PERP expression is down-regulated in the metastatic monosomy 3-type tumours, compared with the less aggressive disomy 3-type tumours. Here, we demonstrate experimentally, by the use of full-length PERP-green fluorescent protein (GFP) fusions and real-time confocal microscopy, the intracellular targeting and plasma membrane localization of PERP in living UM cells and show that expression of PERP induces caspase-mediated apoptosis in UM cells. Induction of PERP expression in GFP-PERP-transfected UM cells leads to increased levels of cleaved caspase-8 forms, as well as to reduction of its full-length substrate Bid, but not to detectable processing of caspase-9. The levels of mature caspase-8, -9 and -3 proteins significantly correlate with PERP expression levels in primary UMs. Transcriptional profiling of PERP and caspase-8 in tumour specimens indicates that the positive association of PERP and caspase-8 proteins is a consequence of post-translational processing, most likely at the level of caspase-8 cleavage, and not of increased transcription of pro-caspase-8. We conclude that PERP expression leads to activation of an extrinsic receptor-mediated apoptotic pathway, with a possible subsequent engagement of the intrinsic apoptotic pathway. The findings underline the apoptotic pathway mediated by PERP as a critical mechanism employed by UM tumours to modulate susceptibility to apoptosis.

Spatio-temporal protein dynamics in single living cells.

The development and application of single cell optical imaging has identified dynamic and oscillatory signalling processes in individual cells. This requires single cell analyses since the processes may otherwise be masked by the population average. These oscillations range in timing from seconds/minutes (e.g. calcium) to minutes/hours (e.g. NF-kappaB, Notch/Wnt and p53) and hours/days (e.g. circadian clock and cell cycle). Quantitative live cell measurement of the protein processes underlying these complex networks will allow characterisation of the core mechanisms that drive these signalling pathways and control cell function. Ultimately, such studies can be applied to develop predictive models of whole tissues and organisms.

HP1alpha guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation.

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

A dual Golgi- and mitochondria-localised Ala25Ser precursor cystatin C: an additional tool for characterising intracellular mis-localisation leading to increased AMD susceptibility.

An artificial mutant Ala25Ser precursor cystatin C was created to help elucidate the cause of intracellular mis-localisation of the biochemically related variant B (Ala25Thr) precursor cystatin C to the mitochondria. Homozygotes of variant B precursor cystatin C were reported to carry an increased susceptibility to developing the exudative form of AMD. Ala25Ser precursor cystatin C shows a dual distribution to the Golgi apparatus and to the mitochondria. This localisation is thus intermediary between that of wild-type cystatin C (targeted to ER/Golgi compartment) and that of variant B precursor cystatin C. Furthermore, the level of secretion of Ala25Ser cystatin C by RPE cells is intermediary between wild type and variant B cystatin C. Ala25Ser precursor cystatin C thus represents a biochemical intermediate between the wild type and the AMD-associated cystatin C and as such, is a novel tool for the investigation of the mechanism of intracellular mis-localisation of variant B cystatin C. Our findings further support the hypothesis that substitution of the alanine residue in the penultimate position of precursor cystatin C signal sequence with a less hydrophobic amino acid residue, such as threonine (as in variant B cystatin C) or serine is sufficient to impair the intracellular trafficking and processing of the protein.

Trafficking of osteonectin by retinal pigment epithelial cells: evidence for basolateral secretion.

Osteonectin is a glycoprotein that modulates several aspects of cellular behaviour including proliferation and adhesion. The retinal pigment epithelium forms a continuous monolayer of polarised cells immediately bellow the neuroretina, and is integral to the homeostasis of photoreceptor cells. While osteonectin is expressed by normal retinal pigment epithelium in situ, its expression is significantly increased in retinal pigment epithelial cells associated with several common retinal diseases. This pattern of expression implies an important role for osteonectin in the biology of retinal pigment epithelial cells. However, the trafficking, processing, and eventual fate of osteonectin in these cells is not clear at present. Although the theoretical report of a leader sequence within the osteonectin open reading frame and its extracellular presence in some tissues indirectly support secretion of the protein, there is no direct experimental demonstration of the secretion route to date. As a first step towards understanding the role of osteonectin in retinal pigment epithelium, we studied the intracellular distribution and trafficking of the protein in living cells. Here, we present experimental evidence that a precursor osteonectin fusion protein is targeted to the endoplasmic reticulum/Golgi pathway, with a likely basal secretion in retinal pigment epithelial cells. In addition, we show that the precursor osteonectin protein having the leader sequence masked fails to undergo secretion leading to cell death, a phenotype which may be of relevance not only for retinal pathology, but also for other diseases such as the bone disorder known as pseudoachondroplasia that is associated with a lack of osteonectin secretion.

Tumor necrosis factor-alpha activates the human prolactin gene promoter via nuclear factor-kappaB signaling.

Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-alpha activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-alpha in GH3 cells was confirmed using real-time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-alpha, Western blot analysis showed rapid inhibitory protein kappaB (IkappaBalpha) degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence-labeled p65 and IkappaBalpha fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IkappaBalpha degradation. This was associated with increased nuclear factor kappaB (NF-kappaB)-mediated transcription from an NF-kappaB-responsive luciferase reporter construct. Disruption of NF-kappaB signaling by expression of dominant-negative variants of IkappaB kinases or truncated IkappaBalpha abolished TNF-alpha activation of the prolactin promoter, suggesting that this effect was mediated by NF-kappaB. TNF-alpha signaling was found to interact with other endocrine signals to regulate prolactin gene expression and is likely to be a major paracrine modulator of lactotroph function.

Developing rat lung has a sided pacemaker region for morphogenesis-related airway peristalsis.

Prenatal airways from diverse species are capable of spontaneous peristaltic contractions in each trimester. The function of this smooth muscle activity is unknown. We demonstrate that peristalsis of the embryonic airway originates from a sided pacemaker focus, is stimulated in a calcium-dependent fashion by the pulmonary morphogen fibroblast growth factor-10 (FGF-10), and appears coupled to lung growth. Airway peristalsis may be crucial for lung development (thereby providing a physiologic role for airway smooth muscle) and play a hitherto unanticipated role in reported transgenic mutant lung phenotypes.

Unexpected intracellular localization of the AMD-associated cystatin C variant.

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.