Integrative Analysis of DNA Methylation and Gene Expression Data Identifies EPAS1 as a Key Regulator of COPD

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a ‘causal’ role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.     

HLA-E allelic variants. Correlating differential expression,peptide affinities,crystal structures,and thermal stabilities

Previous studies of HLA-E allelic polymorphism have indicated that balancing selection may be acting to maintain two major alleles in most populations, indicating that a functional difference may exist between the alleles. The alleles differ at only one amino acid position, where an arginine at position 107 in HLA-E*0101 (E(R)) is replaced by a glycine in HLA-E*0103 (E(G)). To investigate possible functional differences, we have undertaken a study of the physical and biochemical properties of these two proteins. By comparing expression levels, we found that whereas steady-state protein levels were similar, the two alleles did in fact differ with respect to cell surface levels. To help explain this difference, we undertook studies of the relative differences in peptide affinity, complex stability, and three-dimensional structure between the alleles. The crystal structures for HLA-E(G) complexed with two distinct peptides were determined, and both were compared with the HLA-E(R) structure. No significant differences in the structure of HLA-E were induced as a result of binding different peptides or by the allelic substitution at position 107. However, there were clear differences in the relative affinity for peptide of each heavy chain, which correlated with and may be explained by differences between their thermal stabilities. These differences were completely consistent with the relative levels of the HLA-E alleles on the cell surface and may indeed correlate with functional differences. This in turn may help explain the apparent balancing selection acting on this locus.     

Origin of jurassic ammonite concretions assemblages at Alfeld,Germany: a biogenic alternative

The ecology of macrofossils in ammonite-rich concretions and in coeval host sediments from Lower Bajocian mudstones in N.W. Germany indicates a predominantly epifaunal suspensionfeeding community living at maximally 50–90 m on a firm mud bottom during a hiatus or reduced sedimentation. Concretions are lined below with shell debris and contain numerous dimorphicStephanoceras [Ammonitina]. The dimorphs, however, are not a corresponding pair, showing instead extreme and uniquely inverse dimorphic ratios. Distribution of fossils and other debris within the concretions and the host sediments suggests the ammonite shells were swept by currents into subcircular depressions, lined with shell debris. We propose a biogenic alternative to the current/wave scour hypothesis for the origin of the depressions, which are essential to the accumulation of concretion assemblages: the pits were produced by rays feeding on the benthic fauna. Extant rays (Elasmobranchia) excavate such subcircular pits by jetting water through the gills (or mouth) for winnowing of the fauna from the sediment. The ammonite shells were transported post-mortem into the pits by moderate bottom currents. Rapid burial occurred by either a mudflow or renewal of strong sedimentation. Concretion growth was initiated by decomposition of organic matter within the mud.     

Molecular cytogenetic evaluation in a patient with a translocation (3;21) associated with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES)

Blepharophimosis, ptosis, epicanthus inversus syndrome type I (BPES; OMIM 110100) is an autosomal dominant disorder affecting craniofacial development and ovarian function. We have identified a patient with BPES who carried a de novo reciprocal translocation [46, XX,t(3;21)(q23;q22.1)]. Fluorescence in situ hybridization analysis at band 3q23 using probes derived from BAC 175G20 (Research Genetics), PACs 108L15 and 169C10 (RPCI1), and cosmids AC174D4, AC68D3, AC44F5, and AC125C5 (Lawrence Livermore National Laboratory) was performed. The patient's breakpoint was found to lie within the overlapping region of the BAC and PACs but centromeric to all the cosmids. However, a 10.5-kb BamHI-digested fragment, common to the BAC and PAC clones, was shown to cross the breakpoint. The results have placed our patient's breakpoint proximal to that of the previously reported patient [46,XY,t(3;4)(q23;p15.2)] and within a 10.5-kb interval. This is the second patient in which a breakpoint was refined by molecular cytogenetics. Our findings emphasize the significance of this region for BPES.     

Interactions between NKG2x immunoreceptors and HLA-E ligands display overlapping affinities and thermodynamics

The NKG2x/CD94 family of C-type lectin-like immunoreceptors (x = A, B, C, E, and H) mediates surveillance of MHC class Ia cell surface expression, often dysregulated during infection or tumorigenesis, by recognizing the MHC class Ib protein HLA-E that specifically presents peptides derived from class Ia leader sequences. In this study, we determine the affinities and interaction thermodynamics between three NKG2x/CD94 receptors (NKG2A, NKG2C, and NKG2E) and complexes of HLA-E with four representative peptides. Inhibitory NKG2A/CD94 and activating NKG2E/CD94 receptors bind HLA-E with indistinguishable affinities, but with significantly higher affinities than the activating NKG2C/CD94 receptor. Despite minor sequence differences, the peptide presented by HLA-E significantly influenced the affinities; HLA-E allelic differences had no effect. These results reveal important constraints on the integration of opposing activating and inhibitory signals driving NK cell effector functions.     

Visualization and biochemical analyses of the emerging mammalian 14-3-3-phosphoproteome

Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.     

Evaluation of a web-based intervention to reduce antibiotic prescribing for LRTI in six European countries: quantitative process analysis of the GRACE/INTRO randomised controlled trial

Background

To reduce the spread of antibiotic resistance, there is a pressing need for worldwide implementation of effective interventions to promote more prudent prescribing of antibiotics for acute LRTI. This study is a process analysis of the GRACE/INTRO trial of a multifactorial intervention that reduced antibiotic prescribing for acute LRTI in six European countries. The aim was to understand how the interventions were implemented and to examine effects of the interventions on general practitioners’ (GPs’) and patients’ attitudes.

Methods

GPs were cluster randomised to one of three intervention groups or a control group. The intervention groups received web-based training in either use of the C-reactive protein (CRP) test, communication skills and use of a patient booklet, or training in both. GP attitudes were measured before and after the intervention using constructs from the Theory of Planned Behaviour and a Website Satisfaction Questionnaire. Effects of the interventions on patients were assessed by a post-intervention questionnaire assessing patient enablement, satisfaction with the consultation, and beliefs about the risks and need for antibiotics.

Results

GPs in all countries and intervention groups had very positive perceptions of the intervention and the web-based training, and felt that taking part had helped them to reduce prescribing. All GPs perceived reducing prescribing as more important and less risky following the intervention, and GPs in the communication groups reported increased confidence to reduce prescribing. Patients in the communication groups who received the booklet reported the highest levels of enablement and satisfaction and had greater awareness that antibiotics could be unnecessary and harmful.

Conclusions

Our findings suggest that the interventions should be broadly acceptable to both GPs and patients, as well as feasible to roll out more widely across Europe. There are also some indications that they could help to engender changes in GP and patient attitudes that will be helpful in the longer-term, such as increased awareness of the potential disadvantages of antibiotics and increased confidence to manage LRTI without them. Given the positive effects of the booklet on patient beliefs and attitudes, it seems logical to extend the use of the patient booklet to all patients.

     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.     

Age and growth of the tiger shark Galeocerdo cuvier off the east coast of Australia

Total lengths (L_T) at age and growth rates for south‐west Pacific Galeocerdo cuvier were estimated from vertebral growth‐band counts of 202 sagitally sectioned centra from 112 females (71–430 cm L_T), 79 males (72–351 cm L_T) and 11 of unknown sex. Captive growth data were also examined to complement vertebral age estimations. The sexes combined modelled growth coefficient (k = 0·08) was smaller than previously reported for G. cuvier populations elsewhere. Split‐band and narrow banding patterns were identified as potential sources of age underestimation in this species.