Dgp71WD is required for the assembly of the acentrosomal meiosis I spindle, and is not a general targeting factor for the γ-TuRC

Dgp71WD/Nedd1 proteins are essential for mitotic spindle formation. In human cells, Nedd1 targets γ-tubulin to both centrosomes and spindles, but in other organisms the function of Dgp71WD/Nedd1 is less clear. In Drosophila cells, Dgp71WD plays a major part in targeting γ-tubulin to spindles, but not centrosomes, while in Xenopus egg extracts, Nedd1 acts as a more general microtubule (MT) organiser that can function independently of γ-tubulin. The interpretation of these studies, however, is complicated by the fact that some residual Dgp71WD/Nedd1 is likely present in the cells/extracts analysed. Here we generate a Dgp71WD null mutant lacking all but the last 12 nucleotides of coding sequence. The complete loss of Dgp71WD has no quantifiable effect on γ-tubulin or Centrosomin recruitment to the centrosome in larval brain cells. The recruitment of γ-tubulin to spindle MTs, however, is severely impaired, and spindle MT density is reduced in a manner that is indistinguishable from cells lacking Augmin or γ-TuRC function. In contrast, the absence of Dgp71WD leads to defects in the assembly of the acentrosomal female Meiosis I spindle that are more severe than those seen in Augmin or γ-TuRC mutants, indicating that Dgp71WD has additional functions that are independent of these complexes in oocytes. Moreover, the localisation of bicoid RNA during oogenesis, which requires γ-TuRC function, is unperturbed in Dgp71WD(120) mutants. Thus, Dgp71WD is not simply a general cofactor required for γ-TuRC and/or Augmin targeting, and it appears to have a crucial role independent of these complexes in the acentrosomal Meiosis I spindle

Genetic and environmental risk factors for atherosclerosis regulate transcription of phosphatase and actin regulating gene PHACTR1.

BACKGROUND AND AIMS: Coronary artery disease (CAD) risk is associated with non-coding genetic variants at the phosphatase and actin regulating protein 1(PHACTR1) gene locus. The PHACTR1 gene encodes an actin-binding protein with phosphatase regulating activity. The mechanism whereby PHACTR1 influences CAD risk is unknown. We hypothesized that PHACTR1 would be expressed in human cell types relevant to CAD and regulated by atherogenic or genetic factors. METHODS AND RESULTS: Using immunohistochemistry, we demonstrate that PHACTR1 protein is expressed strongly in human atherosclerotic plaque macrophages, lipid-laden foam cells, adventitial lymphocytes and endothelial cells. Using a combination of genomic analysis and molecular techniques, we demonstrate that PHACTR1 is expressed as multiple previously uncharacterized transcripts in macrophages, foam cells, lymphocytes and endothelial cells. Immunoblotting confirmed a total absence of PHACTR1 in vascular smooth muscle cells. Real-time quantitative PCR showed that PHACTR1 is regulated by atherogenic and inflammatory stimuli. In aortic endothelial cells, oxLDL and TNF-alpha both upregulated an intermediate length transcript. A short transcript expressed only in immune cells was upregulated in macrophages by oxidized low-density lipoprotein, and oxidized phospholipids but suppressed by lipopolysaccharide or TNF-alpha. In primary human macrophages, we identified a novel expression quantitative trait locus (eQTL) specific for this short transcript, whereby the risk allele at CAD risk SNP rs9349379 is associated with reduced PHACTR1 expression, similar to the effect of an inflammatory stimulus. CONCLUSIONS: Our data demonstrate that PHACTR1 is a key atherosclerosis candidate gene since it is regulated by atherogenic stimuli in macrophages and endothelial cells and we identify an effect of the genetic risk variant on PHACTR1 expression in macrophages that is similar to that of an inflammatory stimulus

Lipid-Induced Epigenomic Changes in Human Macrophages Identify a Coronary Artery Disease-Associated Variant that Regulates \u3cem\u3ePPAP2B\u3c/em\u3e Expression through Altered C/EBP-Beta Binding

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipid-laden foam cells. We used a combination of open chromatin mapping with formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and enhancer and transcription factor mapping using chromatin immuno-precipitation (ChIP-seq) in primary human macrophages before and after exposure to atherogenic oxidized low-density lipoprotein (oxLDL), with resultant foam cell formation. OxLDL-induced foam cell formation was associated with changes in a subset of open chromatin and active enhancer sites that strongly correlated with expression changes of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors including C/EBP-beta, which has no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased genomic binding events, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were significantly and specifically enriched in the subset of chromatin sites altered by oxLDL exposure, including rs72664324 in an oxLDL-induced enhancer at the PPAP2B locus. OxLDL increased C/EBP beta binding to this site and C/EBP beta binding and enhancer activity were stronger with the protective A allele of rs72664324. In addition, expression of the PPAP2B protein product LPP3 was present in foam cells in human atherosclerotic plaques and oxLDL exposure up-regulated LPP3 in macrophages resulting in increased degradation of pro-inflammatory mediators. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of studying epigenetic changes in disease processes involving pathogenic environmental stimuli

Microtubule organization: A complex solution

Microtubule nucleation within cells is catalyzed by γ-tubulin ring complexes localized at specific microtubule-organizing centers. In this issue, Muroyama et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601099) reveal heterogeneity in the composition and function of these complexes, with wide implications for how cells organize their microtubule arrays.P.T. Conduit was funded by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship (105653/Z/14/Z)

Evaluation of a simple low-cost intervention to empower people with chronic kidney disease to reduce their dietary salt intake : OxCKD1, a multi-center randomized controlled trial

Background To evaluate the efficacy of a simple low-cost intervention to empower people with CKD to reduce their dietary salt intake. Methods A randomized controlled trial in primary and secondary care comparing the OxSalt care bundle intervention versus standard care for 1 month. Participants were people with CKD and an eGFR >20 ml/min per 1.73 m2 and were recruited from primary and secondary care. The primary outcome was a reduction in dietary salt intake, as assessed by 24-hour urinary sodium excretion, after 1 month of the intervention. Results Two hundred and one participants were recruited. Dietary salt intake, as assessed from 24-hour urine sodium excretion, fell by 1.9 (±2.9) g/d in the intervention group compared with 0.4 (±2.7) g/d in the control group (P < 0.001). Salt intake was still reduced to a lesser extent over the following year in the intervention group. Conclusions A short, low-cost, easily delivered intervention empowers people with CKD to reduce their dietary salt intake

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle