Characterization of candidate genes in inflammatory bowel disease-associated risk loci

GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn\u27s disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis

Phosphorylation controls autoinhibition of cytoplasmic linker protein-170

Author Posting. © American Society for Cell Biology, 2010. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 21 (2010): 2661-2673, doi:10.1091/mbc.E09-12-1036.Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.This work was supported by National Institutes of Health grant GM-25062 (to G.G.B.); Netherlands Organization for Scientific Research grants (to A. A. and N. G.); a Cancer Genomics Centre grant (to J.v.H.); and Presidential Program of Russian Academy of Sciences and RFBP grant 05-04-4915 (to E.S.N.)

Evaluation of high-dose rifampin in patients with new, smear-positive tuberculosis (HIRIF): study protocol for a randomized controlled trial.

BACKGROUND: Evidence has existed for decades that higher doses of rifampin may be more effective, but potentially more toxic, than standard doses used in tuberculosis treatment. Whether increased doses of rifampin could safely shorten treatment remains an open question. METHODS/DESIGN: The HIRIF study is a phase II randomized trial comparing rifampin doses of 20 and 15 mg/kg/day to the standard 10 mg/kg/day for the first 2 months of tuberculosis treatment. All participants receive standard doses of companion drugs and a standard continuation-phase treatment (4 months, 2 drugs). They are followed for 6 months post treatment. Study participants are adults with newly diagnosed, previously untreated, smear positive (≥2+) pulmonary tuberculosis. The primary outcome is rifampin area under the plasma concentration-time curve (AUC0-24) after at least 14 days of study treatment/minimum inhibitory concentration. 180 randomized participants affords 90 % statistical power to detect a difference of at least 14 mcg/mL*hr between the 20 mg/kg group and the 10 mg/kg group, assuming a loss to follow-up of up to 17 %. DISCUSSION: Extant evidence suggests the potential for increased doses of rifampin to shorten tuberculosis treatment duration. Early studies that explored this potential using intermittent, higher dosing were derailed by toxicity. Given the continued large, global burden of tuberculosis with nearly 10 million new cases annually, shortened regimens with existing drugs would offer an important advantage to patients and health systems. TRIAL REGISTRATION: This trial was registered with clinicaltrials.gov (registration number: NCT01408914 ) on 2 August 2011

The transcription factor STAT6 mediates direct repression of inflammatory enhancers and limits activation of alternatively polarized macrophages

The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli

Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2

The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1fnob2 mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1fnob2 mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts

The SOLAS air-sea gas exchange experiment (SAGE) 2004

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Deep Sea Research Part II: Topical Studies in Oceanography 58 (2011): 753-763, doi:10.1016/j.dsr2.2010.10.015.The SOLAS air-sea gas exchange experiment (SAGE) was a multiple-objective study investigating gas-transfer processes and the influence of iron fertilisation on biologically driven gas exchange in high-nitrate low-silicic acid low-chlorophyll (HNLSiLC) Sub-Antarctic waters characteristic of the expansive Subpolar Zone of the southern oceans. This paper provides a general introduction and summary of the main experimental findings. The release site was selected from a pre-voyage desktop study of environmental parameters to be in the south-west Bounty Trough (46.5°S 172.5°E) to the south-east of New Zealand and the experiment conducted between mid-March and mid-April 2004. In common with other mesoscale iron addition experiments (FeAX’s), SAGE was designed as a Lagrangian study quantifying key biological and physical drivers influencing the air-sea gas exchange processes of CO2, DMS and other biogenic gases associated with an iron-induced phytoplankton bloom. A dual tracer SF6/3He release enabled quantification of both the lateral evolution of a labelled volume (patch) of ocean and the air-sea tracer exchange at the 10’s of km’s scale, in conjunction with the iron fertilisation. Estimates from the dual-tracer experiment found a quadratic dependency of the gas exchange coefficient on windspeed that is widely applicable and describes air-sea gas exchange in strong wind regimes. Within the patch, local and micrometeorological gas exchange process studies (100 m scale) and physical variables such as near-surface turbulence, temperature microstructure at the interface, wave properties, and wind speed were quantified to further assist the development of gas exchange models for high-wind environments. There was a significant increase in the photosynthetic competence (Fv/Fm) of resident phytoplankton within the first day following iron addition, but in contrast to other FeAX’s, rates of net primary production and column-integrated chlorophyll a concentrations had only doubled relative to the unfertilised surrounding waters by the end of the experiment. After 15 days and four iron additions totalling 1.1 tonne Fe2+, this was a very modest response compared to the other mesoscale iron enrichment experiments. An investigation of the factors limiting bloom development considered co- limitation by light and other nutrients, the phytoplankton seed-stock and grazing regulation. Whilst incident light levels and the initial Si:N ratio were the lowest recorded in all FeAX’s to date, there was only a small seed-stock of diatoms (less than 1% of biomass) and the main response to iron addition was by the picophytoplankton. A high rate of dilution of the fertilised patch relative to phytoplankton growth rate, the greater than expected depth of the surface mixed layer and microzooplankton grazing were all considered as factors that prevented significant biomass accumulation. In line with the limited response, the enhanced biological draw-down of pCO2 was small and masked by a general increase in pCO2 due to mixing with higher pCO2 waters. The DMS precursor DMSP was kept in check through grazing activity and in contrast to most FeAX’s dissolved dimethylsulfide (DMS) concentration declined through the experiment. SAGE is an important low-end member in the range of responses to iron addition in FeAX’s. In the context of iron fertilisation as a geoengineering tool for atmospheric CO2 removal, SAGE has clearly demonstrated that a significant proportion of the low iron ocean may not produce a phytoplankton bloom in response to iron addition.SAGE was jointly funded through the New Zealand Foundation for Research, Science and Technology (FRST) programs (C01X0204) "Drivers and Mitigation of Global Change" and (C01X0223) "Ocean Ecosystems: Their Contribution to NZ Marine Productivity." Funding was also provided for specific collaborations by the US National Science Foundation from grants OCE-0326814 (Ward), OCE-0327779 (Ho), and OCE 0327188 OCE-0326814 (Minnett) and the UK Natural Environment Research Council NER/B/S/2003/00282 (Archer). The New Zealand International Science and Technology (ISAT) linkages fund provided additional funding (Archer and Ziolkowski), and the many collaborator institutions also provided valuable support