ARID3B Directly Regulates Ovarian Cancer Promoting Genes

The DNA-binding protein AT-Rich Interactive Domain 3B (ARID3B) is elevated in ovarian cancer and increases tumor growth in a xenograft model of ovarian cancer. However, relatively little is known about ARID3B''s function. In this study we perform the first genome wide screen for ARID3B direct target genes and ARID3B regulated pathways. We identified and confirmed numerous ARID3B target genes by chromatin immunoprecipitation (ChIP) followed by microarray and quantitative RT-PCR. Using motif-finding algorithms, we characterized a binding site for ARID3B, which is similar to the previously known site for the ARID3B paralogue ARID3A. Functionality of this predicted site was demonstrated by ChIP analysis. We next demonstrated that ARID3B induces expression of its targets in ovarian cancer cell lines. We validated that ARID3B binds to an epidermal growth factor receptor (EGFR) enhancer and increases mRNA expression. ARID3B also binds to the promoter of Wnt5A and its receptor FZD5. FZD5 is highly expressed in ovarian cancer cell lines, and is upregulated by exogenous ARID3B. Both ARID3B and FZD5 expression increase adhesion to extracellular matrix (ECM) components including collagen IV, fibronectin and vitronectin. ARID3B-increased adhesion to collagens II and IV require FZD5. This study directly demonstrates that ARID3B binds target genes in a sequence-specific manner, resulting in increased gene expression. Furthermore, our data indicate that ARID3B regulation of direct target genes in the Wnt pathway promotes adhesion of ovarian cancer cells.     

Dose-Dependent Effects of a Soluble Dietary Fibre (Pectin) on Food Intake,Adiposity, Gut Hypertrophy and Gut Satiety Hormone Secretion in Rats

Soluble fermentable dietary fibre elicits gut adaptations, increases satiety and potentially offers a natural sustainable means of body weight regulation. Here we aimed to quantify physiological responses to graded intakes of a specific dietary fibre (pectin) in an animal model. Four isocaloric semi-purified diets containing 0, 3.3%, 6.7% or 10% w/w apple pectin were offered ad libitum for 8 or 28 days to young adult male rats (n = 8/group). Measurements were made of voluntary food intake, body weight, initial and final body composition by magnetic resonance imaging, final gut regional weights and histology, and final plasma satiety hormone concentrations. In both 8- and 28-day cohorts, dietary pectin inclusion rate was negatively correlated with food intake, body weight gain and the change in body fat mass, with no effect on lean mass gain. In both cohorts, pectin had no effect on stomach weight but pectin inclusion rate was positively correlated with weights and lengths of small intestine and caecum, jejunum villus height and crypt depth, ileum crypt depth, and plasma total glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) concentrations, and at 8 days was correlated with weight and length of colon and with caecal mucosal depth. Therefore, the gut’s morphological and endocrine adaptations were dose-dependent, occurred within 8 days and were largely sustained for 28 days during continued dietary intervention. Increasing amounts of the soluble fermentable fibre pectin in the diet proportionately decreased food intake, body weight gain and body fat content, associated with proportionately increased satiety hormones GLP-1 and PYY and intestinal hypertrophy, supporting a role for soluble dietary fibre-induced satiety in healthy body weight regulation.     

High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc -) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells

The cystine-glutamate antiporter (system x_c ^-) is a Na⁺-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system x_c ^- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system x_c ^- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system x_c ^- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system x_c ^- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system x_c ^-. Human glioma cells were chosen based on their high system x_c ^- activity. Using these cells, [¹⁴C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well ¹⁴C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.     

Short-Term Treatment with Diminazene Aceturate Ameliorates the Reduction in Kidney ACE2 Activity in Rats with Subtotal Nephrectomy

Angiotensin converting enzyme (ACE) 2 is an important modulator of the renin angiotensin system (RAS) through its role to degrade angiotensin (Ang) II. Depletion of kidney ACE2 occurs following kidney injury due to renal mass reduction and may contribute to progressive kidney disease. This study assessed the effect of diminazine aceturate (DIZE), which has been described as an ACE2 activator, on kidney ACE2 mRNA and activity in rats with kidney injury due to subtotal nephrectomy (STNx). Sprague Dawley rats were divided into Control groups or underwent STNx; rats then received vehicle or the DIZE (s.c. 15 mg/kg/day) for 2 weeks. STNx led to hypertension (P<0.01), kidney hypertrophy (P<0.001) and impaired kidney function (P<0.001) compared to Control rats. STNx was associated with increased kidney cortical ACE activity, and reduced ACE2 mRNA in the cortex (P<0.01), with reduced cortical and medullary ACE2 activity (P<0.05), and increased urinary ACE2 excretion (P<0.05) compared to Control rats. Urinary ACE2 activity correlated positively with urinary protein excretion (P<0.001), and negatively with creatinine clearance (P=0.04). In STNx rats, DIZE had no effect on blood pressure or kidney function, but was associated with reduced cortical ACE activity (P<0.01), increased cortical ACE2 mRNA (P<0.05) and increased cortical and medullary ACE2 activity (P<0.05). The precise in vivo mechanism of action of DIZE is not clear, and its effects to increase ACE2 activity may be secondary to an increase in ACE2 mRNA abundance. In ex vivo studies, DIZE did not increase ACE2 activity in either Control or STNx kidney cortical membranes. It is not yet known if chronic administration of DIZE has long-term benefits to slow the progression of kidney disease.     

Rescue of Fructose-Induced Metabolic Syndrome by Antibiotics or Faecal Transplantation in a Rat Model of Obesity

A fructose-rich diet can induce metabolic syndrome, a combination of health disorders that increases the risk of diabetes and cardiovascular diseases. Diet is also known to alter the microbial composition of the gut, although it is not clear whether such alteration contributes to the development of metabolic syndrome. The aim of this work was to assess the possible link between the gut microbiota and the development of diet-induced metabolic syndrome in a rat model of obesity. Rats were fed either a standard or high-fructose diet. Groups of fructose-fed rats were treated with either antibiotics or faecal samples from control rats by oral gavage. Body composition, plasma metabolic parameters and markers of tissue oxidative stress were measured in all groups. A 16S DNA-sequencing approach was used to evaluate the bacterial composition of the gut of animals under different diets. The fructose-rich diet induced markers of metabolic syndrome, inflammation and oxidative stress, that were all significantly reduced when the animals were treated with antibiotic or faecal samples. The number of members of two bacterial genera, Coprococcus and Ruminococcus, was increased by the fructose-rich diet and reduced by both antibiotic and faecal treatments, pointing to a correlation between their abundance and the development of the metabolic syndrome. Our data indicate that in rats fed a fructose-rich diet the development of metabolic syndrome is directly correlated with variations of the gut content of specific bacterial taxa.     

Maternal Serum Meteorin Levels and the Risk of Preeclampsia

Background

Meteorin (METRN) is a recently described neutrophic factor with angiogenic properties. This is a nested case-control study in a longitudinal cohort study that describes the serum profile of METRN during different periods of gestation in healthy and preeclamptic pregnant women. Moreover, we explore the possible application of METRN as a biomarker.

Methods and Findings

Serum METRN was measured by ELISA in a longitudinal prospective cohort study in 37 healthy pregnant women, 16 mild preeclamptic women, and 20 healthy non-pregnant women during the menstrual cycle with the aim of assessing serum METRN levels and its correlations with other metabolic parameters. Immunostaining for METRN protein was performed in placenta. A multivariate logistic regression model was proposed and a classifier model was formulated for predicting preeclampsia in early and middle pregnancy. The performance in classification was evaluated using measures such as sensitivity, specificity, and the receiver operating characteristic (ROC) curve. In healthy pregnant women, serum METRN levels were significantly elevated in early pregnancy compared to middle and late pregnancy. METRN levels are significantly lower only in early pregnancy in preeclamptic women when compared to healthy pregnant women. Decision trees that did not include METRN levels in the first trimester had a reduced sensitivity of 56% in the detection of preeclamptic women, compared to a sensitivity of 69% when METRN was included.

Conclusions

The joint measurements of circulating METRN levels in the first trimester and systolic blood pressure and weight in the second trimester significantly increase the probabilities of predicting preeclampsia.     

Cellular Changes during Renal Failure-Induced Inflammatory Aortic Valve Disease

Background

Aortic valve calcification (AVC) secondary to renal failure (RF) is an inflammation-regulated process, but its pathogenesis remains unknown. We sought to assess the cellular processes that are involved in the early phases of aortic valve disease using a unique animal model of RF-associated AVC.

Methods

Aortic valves were obtained from rats that were fed a uremia-inducing diet exclusively for 2, 3, 4, 5, and 6 weeks as well as from controls. Pathological examination of the valves included histological characterization, von Kossa staining, and antigen expression analyses.

Results

After 2 weeks, we noted a significant increase in urea and creatinine levels, reflecting RF. RF parameters exacerbated until the Week 5 and plateaued. Whereas no histological changes or calcification was observed in the valves of any study group, macrophage accumulation became apparent as early as 2 weeks after the diet was started and rose after 3 weeks. By western blot, osteoblast markers were expressed after 2 weeks on the diet and decreased after 6 weeks. Collagen 3 was up-regulated after 3 weeks, plateauing at 4 weeks, whereas collagen 1 levels peaked at 2 and 4 weeks. Fibronectin levels increased gradually until Week 5 and decreased at 6 weeks. We observed early activation of the ERK pathway, whereas other pathways remained unchanged.

Conclusions

We concluded that RF induces dramatic changes at the cellular level, including macrophage accumulation, activation of cell signaling pathway and extracellular matrix modification. These changes precede valve calcification and may increase propensity for calcification, and have to be investigated further.