Quantitative analysis and phase assemblage of basic oxygen furnace slag hydration.

Basic oxygen furnace (BOF) slag from steelmaking could be applied as a binder in building materials, reducing the CO2 footprint and solid waste, which is relevant for industrial waste management and circular economy. However, its use is mostly restricted because its hydraulic activity is poorly understood. The BOF slag was hydrated in this study, and its reaction products were systematically characterized using XRD, QXRD, and SEM/EDX-based phase mapping. Internal consistency checks of the data were performed between the analytical techniques. The results revealed that the composition of the amorphous hydration products could be identified and quantified, and the main hydration products were hydrogarnets and C-S-H gel. An extended milling process significantly improved the reactivity, and all the major slag phases, including wüstite, participated in the reaction. Brownmillerite formed hydrogarnets during the first 7 days of hydration. The new hydration products contributed to the immobilization of vanadium and chromium. Particle size played an important role in the amount of C2S reacting, the composition of the hydrogarnets and C-S-H gel, their proportions, and the immobilization capacity. Based on the findings, an overall hydration reaction was developed.

Anaemia among intensive care unit survivors and association with days alive and at home: an observational study.

Anaemia is highly prevalent at the time of intensive care unit discharge and is persistent for a high proportion of intensive care unit survivors. Whether anaemia is a driver of impaired recovery after critical illness is uncertain. The aim of this study was to test the hypothesis that, in adult intensive care survivors, anaemia at the time of intensive care unit discharge independently predicts decreased days at home-90. This retrospective cohort study was conducted in a tertiary intensive care unit in Perth, Western Australia. All patients aged ≥ 16 years, discharged alive from their index intensive care unit admission and without documented treatment limitations were included. Median (IQR [range]) age of the 6358 participants was 61 (46-72 [16-95]) years and included 3385 (53.2%) unplanned admissions. Intensive care unit discharge with a haemoglobin concentration < 100 g.l-1 occurred in 2886 (45.4%) patients, a threshold that identified a cohort with significantly lower days at home-90 (median (IQR [range]) 80 (64-85 [0-90]) days vs. 85 (77-88 [0-90]) days (median difference 5 days, 95%CI 4.4-5.5, p < 0.0001). The association followed a severity-response relationship with more severe anaemia predicting lower days at home-90. When accounting for prespecified covariates including admission haemoglobin concentration and red blood cell transfusion, anaemia at intensive care unit discharge remained a significant predictor of decreased days at home-90, relative risk 0.96 (0.93-0.98), p < 0.002. These findings support the need for interventional trials investigating whether this risk is modifiable.

The hypoxia-sensor carbonic anhydrase IX affects macrophage metabolism, but is not a suitable biomarker for human cardiovascular disease.

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.

RNA editing blood biomarkers for predicting mood alterations in HCV patients.

Treatment-emergent depression is a common complication in patients with chronic hepatitis C virus (HCV) infection undergoing antiviral combination therapy with IFN-α and ribavirin. It has recently been shown that changes in A-to-I RNA editing rates are associated with various pathologies such as inflammatory disorders, depression and suicide. Interestingly, IFN-α induces gene expression of the RNA editing enzyme ADAR1-1 (ADAR1a-p150) and alters overall RNA editing activity. In this study, we took advantage of the high prevalence of pharmacologically induced depression in patients treated with IFN-α and ribavirin to test the interest of RNA editing-related biomarkers in white blood cells of patients. In this 16-week longitudinal study, a small cohort of patients was clinically evaluated using standard assessment methods prior to and during antiviral therapy and blood samples were collected to analyse RNA editing modifications. A-I RNA editing activity on the phosphodiesterase 8A (PDE8A) gene, a previously identified RNA editing hotspot in the context of lupus erythematosus, was quantified by using an ultra-deep next-generation sequencing approach. We also monitored gene expression levels of the ADAR enzymes and the PDE8A gene during treatment by qPCR. As expected, psychiatric evaluation could track treatment-emergent depression, which occurred in 30% of HCV patients. We show that PDE8A RNA editing is increased in all patients following interferon treatment, but differently in 30% of patients. This effect was mimicked in a cellular model using SHSY-5Y neuroblastoma cells. By combining the data of A-I RNA editing and gene expression, we generated an algorithm that allowed discrimination between the group of patients who developed a treatment-emergent depression and those who did not. The current model of drug-induced depression identified A-I RNA editing biomarkers as useful tools for the identification of individuals at risk of developing depression in an objective, quantifiable biological blood test.

Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression.

Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide.

Common variants associated with blood lipid levels do not affect carotid plaque composition.

INTRODUCTION: Although plasma lipid levels are known to influence the risk of cardiovascular disease (CVD), little is known about their effect on atherosclerotic plaque composition. To date, large-scale genome-wide association studies have identified 157 common single-nucleotide polymorphisms (SNPs) that influence plasma lipid levels, providing a powerful tool to investigate the effect of plasma lipid levels on atherosclerotic plaque composition. METHODS: In this study, we included 1443 carotid endarterectomy patients from the Athero-Express Biobank Study with genotype data. Plasma concentrations of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and triglycerides (TG) were determined at the time of endarterectomy. Atherosclerotic plaques, obtained during surgery, were histologically examined. For all patients, we calculated weighted genetic burden scores (GBS) for all lipid traits on the basis of the available genotype data. Plasma lipid levels and GBS were tested for association with 7 histological features using linear and logistic regression models. RESULTS: All GBS were associated with their respective plasma lipid concentrations (pHDL-C = 2.4 × 10(-14), pLDL-C = 0.003, pTC = 2.1 × 10(-6), pTG = 3.4 × 10(-8)). Neither the measured plasma lipids, nor the GBS, were associated with histological features of atherosclerotic plaque composition. In addition, neither the plasma lipids nor the GBS were associated with clinical endpoints within 3 years of follow-up, with the notable exception of a negative association between HDL-C and composite cardiovascular endpoints. CONCLUSION: This study found no evidence that plasma lipid levels or their genetic determinants influence carotid plaque composition.

Nuclear receptor coregulators differentially modulate induction and glucocorticoid receptor-mediated repression of the corticotropin-releasing hormone gene.

Nuclear receptor coregulators are proteins that modulate the transcriptional activity of steroid receptors and may explain cell-specific effects of glucocorticoid receptor action. Based on the uneven distribution of a number of coregulators in CRH-expressing cells in the hypothalamus of the rat brain, we tested the hypothesis that these proteins are involved as mediators in the glucocorticoid-induced repression of the CRH promoter. Therefore, we assessed the role of coregulator proteins on both induction and repression of CRH in the AtT-20 cell line, a model system for CRH repression by glucocorticoids. The steroid receptor coactivator 1a (SRC1a), SRC-1e, nuclear corepressor (N-CoR), and silencing mediator of the retinoid and thyroid hormone receptor (SMRT) were studied in this system. We show that the concentration of glucocorticoid receptor and the type of ligand, i.e. corticosterone or dexamethasone, determines the repression. Furthermore, overexpression of SRC1a, but not SRC1e, increased both efficacy and potency of the glucocorticoid receptor-mediated repression of the forskolin-induced CRH promoter. Unexpectedly, cotransfection of the corepressors N-CoR and SMRT did not affect the corticosterone-dependent repression but resulted in a marked decrease of the forskolin stimulation of the CRH gene. Altogether, our data demonstrate that 1) the concentration of the receptor, 2) the type of ligand, and 3) the coregulator recruited all determine the expression and the repression of the CRH gene. We conclude that modulation of coregulator activity may play a role in the control of the hypothalamus-pituitary-adrenal axis.

Steroid receptor coregulator diversity: what can it mean for the stressed brain?

Glucocorticoid hormones modulate brain function and as such are crucial for responding and adjusting to physical and psychological stressors. Their effects are mediated via mineralo- and glucocorticoid receptors, which in large measure act as transcription factors to modulate transcription of target genes, in a receptor-, cell-, and state-specific manner. The nature and magnitude of these transcriptional effects depend on the presence and activity of downstream proteins, such as steroid receptor coactivators and corepressors (together: coregulators), many of which are expressed in the brain. We address the role of coregulators for mineralo- and glucocorticoid receptor-mediated modulation of gene transcription. We first address evidence from cell lines for the importance of coregulator stoichiometry for steroid signaling. The in vivo importance of coregulators-when possible specifically for glucocorticoid signaling in the brain-is discussed based on knockout mice, transient knockdown of steroid receptor coactivators, and distribution and regulation of coactivator expression in the brain. We conclude that for a better understanding of modulation of brain function by glucocorticoids, it is necessary to take into account the role of coregulators, and to assess their importance relative to changes in hormone levels and receptor expression.

Neuroanatomical distribution and colocalisation of nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid receptors (SMRT) in rat brain.

The two structurally related nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid receptors (SMRT) proteins have been found to differentially affect the transcriptional activity of numerous nuclear receptors, such as thyroid hormone, retinoic acid and steroid receptors. Because of the numerous effects mediated by nuclear receptors in brain, it is of interest to extend these in vitro data and to explore the cellular distribution of both corepressors in brain tissue. We therefore examined, using in situ hybridisation, whether the relative abundance of these two functionally distinct corepressors differed in rat brain and pituitary. We find that although both N-CoR and SMRT transcripts are ubiquitously expressed in brain, striking differences in their respective levels of expression could be observed in discrete areas of brain stem, thalamus, hypothalamus and hippocampus. Using dual-label immunofluorescence, we examined in selected glucocorticoid sensitive areas involved in the regulation of the hypothalamus-pituitary-adrenal axis activity, the respective protein abundance of N-CoR and SMRT. Protein abundance was largely concurrent with the mRNA expression levels, with SMRT relatively more abundant in hypothalamus and brain stem areas. Colocalisation of N-CoR and SMRT was demonstrated by confocal microscopy in most areas studied. Taken together, these findings are consistent with the idea that the uneven neuroanatomical distribution of N-CoR and SMRT protein may contribute to the site-specific effects exerted by hormones, such as glucocorticoids, in the brain.

Steroid receptor coactivator-1 splice variants differentially affect corticosteroid receptor signaling.

The mechanisms of receptor- and cell-specific effects of the adrenal corticosteroid hormones via mineralo- (MRs) and glucocorticoid receptors (GRs) are still poorly understood. Because the expression levels of two splice variants of the steroid receptor coactivator-1 (SRC-1) 1a and 1e, can differ significantly in certain cell populations, we tested the hypothesis that their relative abundance could determine cell- and receptor-specific effects of corticosteroid receptor-mediated transcription. In transient transfections, we demonstrate three novel types of SRC-1a- and SRC-1e-specific effects for corticosteroid receptors. One is promoter dependence: SRC-1e much more potently coactivated transcription from several multiple response element-containing promoters. Mammalian 1-hydrid studies indicated that this likely does not involve promoter-specific coactivator recruitment. Endogenous phenylethanolamine-N-methyltransferase mRNA induction via GRs was also differentially affected by the splice variants. Another type is receptor specificity: responses mediated by the N-terminal part of the MR, but not the GR, were augmented by SRC-1e at synergizing response elements. SRC fragment SRC(988-1240) by the MR but not the GR N-terminal fragment in a 1-hybrid assay. The last type, for GRs, is ligand dependence. Due to effects on partial agonism of RU486-activated GRs, different ratios of SRC-1a and 1e can lead to large differences in the extent of antagonism of RU486 on GR-mediated transcription. Furthermore, we show that SRC-1e but not SRC-1a mRNA expression was regulated in the pituitary by corticosterone. We conclude that the cellular differences in SRC-1a to SRC-1e ratio demonstrated in vivo might be involved in cell-specific responses to corticosteroids in a promoter- and ligand-dependent way.

Expression profiling in laser-microdissected hippocampal subregions in rat brain reveals large subregion-specific differences in expression.

Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser-microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion-specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion-specific expression or regulation.

A short phosphodiester window is sufficient to direct RNase H-dependent RNA cleavage by antisense peptide nucleic acid.

The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H. The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions. It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay.

Standardization of allergenic extracts of Aspergillus fumigatus. Liberation of IgE-binding components during cultivation.

During cultivation of Aspergillus fumigatus a rapid liberation of IgE-binding components was found reaching maximum values during the logarithmic phase of growth (phase I). After a fall in IgE-binding titers during phase II, appearance of additional IgE-binding components was noted during the period of lysis of the microorganism (phase III). These latter allergenic components are different from the phase I IgE-binding components, as was shown by crossed-inhibition studies. The number of precipitating antigenic components was not related with the corresponding IgE-binding titers and showed an increase during all phases of growth. The rapid changes in both IgE- and IgG-binding properties and the discrepancies between precipitating properties and IgE binding are discussed in relation to standardization and quality control of aspergillus extracts.

Antibody determination against Aspergillus fumigatus by means of the enzyme-linked immunosorbent assay. I. Liberation of polystyrene binding components during cultivation.

The liberation of polystyrene binding antigenic components (PBC) of Aspergillus fumigatus involved in ELISA IgG measurements was studied during cultivation in the culture filtrates. PBC were rapidly liberated into the culture medium during the logarithmic phase of growth (phase I), were in part degraded or reconsumed during the stationary phase (phase II) and again secreted (other PBC) during the lytic phase (phase III). ELISA IgG titers and numbers of precipitating components are not always related with one another, may show an inverse relationship; maximal ELISA titers are reached during phase I when precipitating components are limited. The striking similarity between the liberation of PBC and IgE-binding components is discussed.

Antibody determination against Aspergillus fumigatus by means of the enzyme-linked immunosorbent assay. II. Physico- and immunochemical properties of the polystyrene-binding components.

Polystyrene-binding antigenic components (PBC) of Aspergillus fumigatus involved in ELISA IgG antibody determinations and the corresponding numbers of precipitates found by double immunodiffusion (DID) and/or crossed immunoelectrophoresis (CIE) were studied using culture filtrate antigenic preparations fractionated by Sephadex G-100 chromatography, concanavalin A (ConA) affinity chromatography, isoelectric focusing (IEF) and TCA precipitation. It is shown that PBC, immunologically active in ELISA, are characterized by high molecular weights and ConA-binding properties. The pI values of these components were 3.5-6 and above 8. The glycoprotein and/or mucopolysaccharide nature is suggested by the binding to ConA and their partial resistance to trichloroacetic acid treatment. By contrast, low-molecular weight components and antigenic components that are not absorbed by ConA being active in DID and/or CIE showed less or absence of binding to the polystyrene surface.

Cutaneous reactions to captopril. Predictive value of skin tests.

Cutaneous reactions associated with captopril treatment occurred in fifteen out of eighty-nine patients (17%). Dose reduction invariably led to improvement of the reaction but later recurrences were frequent (six patients). In four out of the fifteen patients captopril withdrawal ultimately was necessary. Skin tests and in vitro lymphocyte transformation tests with captopril were performed in these fifteen patients and also in nine captopril-treated control patients without adverse reactions. Positive epicutaneous skin tests were observed in five out of the fifteen patients including the four in whom captopril had to be withdrawn, but in none of the controls. Intracutaneous skin tests were positive in ten of the patients with cutaneous reactions and in two control patients. Captopril-induced in vitro lymphocyte transformation occurred in most patients with cutaneous reactions whereas in control patients captopril suppressed the in vitro lymphocyte proliferative response. Skin biopsies revealed histologic changes consistent with lymphocytic vasculitis. We conclude that epicutaneous skin tests with captopril are helpful in predicting the necessity of captopril withdrawal.

The glomerular polyanion (GPA) of the rat kidney. I. Concanavalin-A activated cells affect the glomerular polyanion in vitro.

Rat peritoneal exudate cells (PEC) or human peripheral blood cells stimulated with mitogens in vitro were cultured in tissue culture chambers mounted on rat or human cryostat kidney sections. After 20 h, the cultures were discontinued and the glomerular polyanion (GPA) of the glomeruli was studied using the colloidal iron stain. The results show that in contrast to PHA-stimulated cells, rat PEC or human blood cells stimulated with Con-A are able to reduce GPA stainability. It was further shown that rat PEC activated with Con-A for 20 or 48 h were able to suppress in vitro lymphocyte transformation of syngeneic blood lymphocytes in a co-culture system following mitogenic stimulation. In view of recent concepts according to which disturbance of T cell function is associated with in vivo loss of GPA in some forms of the nephrotic state, we conclude that investigation into the possible in vivo significance of the present observations is worthwhile.