Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters

Breakfast partly restores the anti-inflammatory function of high-density lipoproteins from patients with type 2 diabetes mellitus

BACKGROUND AND AIMS: High-density lipoproteins (HDL) of patients with type 2 diabetes mellitus (T2DM) have impaired anti-inflammatory activities. The anti-inflammatory activity of HDL has been determined ex vivo after isolation by different methods from blood mostly obtained after overnight fasting. We first determined the effect of the HDL isolation method, and subsequently the effect of food intake on the anti-inflammatory function of HDL from T2DM patients. METHODS: Blood was collected from healthy controls and T2DM patients after an overnight fast, and from T2DM patients 3 h after breakfast (n = 17 each). HDL was isolated by a two-step density gradient ultracentrifugation in iodixanol (HDL(DGUC2)), by sequential salt density flotation (HDL(SEQ)) or by PEG precipitation (HDL(PEG)). The anti-inflammatory function of HDL was determined by the reduction of the TNFα-induced expression of VCAM-1 in human coronary artery endothelial cells (HCAEC) and retinal endothelial cells (REC). RESULTS: HDL isolated by the three different methods from healthy controls inhibited TNFα-induced VCAM-1 expression in HCAEC. With apoA-I at 0.7 μM, HDL(DGUC2) and HDL(SEQ) were similarly effective (16% versus 14% reduction; n = 3; p > 0.05) but less effective than HDL(PEG) (28%, p < 0.05). Since ultracentrifugation removes most of the unbound plasma proteins, we used HDL(DGUC2) for further experiments. With apoA-I at 3.2 μM, HDL from fasting healthy controls and T2DM patients reduced TNFα-induced VCAM-1 expression in HCAEC by 58 ± 13% and 51 ± 20%, respectively (p = 0.35), and in REC by 42 ± 13% and 25 ± 18%, respectively (p < 0.05). Compared to preprandial HDL, postprandial HDL from T2DM patients reduced VCAM-1 expression by 56 ± 16% (paired test: p < 0.001) in HCAEC and by 34 ± 13% (paired test: p < 0.05) in REC. CONCLUSIONS: The ex vivo anti-inflammatory activity of HDL is affected by the HDL isolation method. Two-step ultracentrifugation in an iodixanol gradient is a suitable method for HDL isolation when testing HDL anti-inflammatory function. The anti-inflammatory activity of HDL from overnight fasted T2DM patients is significantly impaired in REC but not in HCAEC. The anti-inflammatory function of HDL is partly restored by food intake

Excess maternal salt intake produces sex-specific hypertension in offspring: putative roles for kidney and gastrointestinal sodium handling.

Hypertension is common and contributes, via cardiovascular disease, towards a large proportion of adult deaths in the Western World. High salt intake leads to high blood pressure, even when occurring prior to birth - a mechanism purported to reside in altered kidney development and later function. Using a combination of in vitro and in vivo approaches we tested whether increased maternal salt intake influences fetal kidney development to render the adult individual more susceptible to salt retention and hypertension. We found that salt-loaded pregnant rat dams were hypernatraemic at day 20 gestation (147±5 vs. 128±5 mmoles/L). Increased extracellular salt impeded murine kidney development in vitro, but had little effect in vivo. Kidneys of the adult offspring had few structural or functional abnormalities, but male and female offspring were hypernatraemic (166±4 vs. 149±2 mmoles/L), with a marked increase in plasma corticosterone (e.g. male offspring; 11.9 [9.3-14.8] vs. 2.8 [2.0-8.3] nmol/L median [IQR]). Furthermore, adult male, but not female, offspring had higher mean arterial blood pressure (effect size, +16 [9-21] mm Hg; mean [95% C.I.]. With no clear indication that the kidneys of salt-exposed offspring retained more sodium per se, we conducted a preliminary investigation of their gastrointestinal electrolyte handling and found increased expression of proximal colon solute carrier family 9 (sodium/hydrogen exchanger), member 3 (SLC9A3) together with altered faecal characteristics and electrolyte handling, relative to control offspring. On the basis of these data we suggest that excess salt exposure, via maternal diet, at a vulnerable period of brain and gut development in the rat neonate lays the foundation for sustained increases in blood pressure later in life. Hence, our evidence further supports the argument that excess dietary salt should be avoided per se, particularly in the range of foods consumed by physiologically immature young

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Bulk enrichment of transplantable hemopoietic stem cell subsets from lipopolysaccharide-stimulated murine spleen

Counterflow centrifugal elutriation (CCE) in combination with density flotation centrifugation and fluorescence-activated cell sorting on wheat-germ agglutinin-FITC(WGA)-binding cells within the light-scatter 'blast window' were used consecutively to enrich pluripotent hemopoietic stem cells (HSC) in bulk from lipopolysaccharide-stimulated mouse spleen. The medium-to-strong WGA+ve fraction contained 3.106 cells isolated from 3-4 x 109 spleen cells, with an average of 126% day-12 CFU-S and 65% day-8 CFU-S as calculated on the basis of their seeding fraction, suggesting that virtually all cells represented in vivo macroscopic colony formers. In view of the large differences reported elsewhere between stem cell subsets differing in reconstitutive capacity and secondary stem cell generation ability, we also studied various isolated cell fractions with respect to spleen colony formation, radioprotective ability, and spleen- and marrow-repopulating ability. Day-8 and day-12 CFU-S copurified when isolated by CCE. Cells from a fraction with high affinity for WGA were most highly enriched for their radioprotective ability (RPA) and their ability to repopulate the cellularity of the spleen and femur of irradiated recipients. This fraction contained virtually pure day-12 CFU-S. However, the ability to generate secondary day-12 CFU-S and CFU-GM in irradiated organs was enriched most in the medium WGA+ve cell fraction. MRA and SRA, according to the latter criteria, could therefore be partly separated from day-12 CFU-S and RPA on the basis of affinity for WGA. The data strongly suggest that at least part of all day-12 CFU-S have a high potential to proliferate and differentiate into mature progeny, but a relatively low self-renewal ability, and may therefore not be representative of the genuine stem cell