Anti-mycobacterial treatment reduces high plasma levels of CXC-chemokines detected in active tuberculosis by cytometric bead array

Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometric bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Affymetrix Whole-Transcript Human Gene 1.0 ST array is highly concordant with standard 3' expression arrays.

The recently released Affymetrix Human Gene 1.0 ST array has two major differences compared with standard 3' based arrays: (i) it interrogates the entire mRNA transcript, and (ii) it uses DNA targets. To assess the impact of these differences on array performance, we performed a series of comparative hybridizations between the Human Gene 1.0 ST and the Affymetrix HG-U133 Plus 2.0 and the Illumina HumanRef-8 BeadChip arrays. Additionally, both RNA and DNA targets were hybridized on HG-U133 Plus 2.0 arrays. The results show that the overall reproducibility of the Gene 1.0 ST array is best. When looking only at the high intensity probes, the reproducibility of the Gene 1.0 ST array and the Illumina BeadChip array is equally good. Concordance of array results was assessed using different inter-platform mappings. Agreements are best between the two labeling protocols using HG-U133 Plus 2.0 array. The Gene 1.0 ST array is most concordant with the HG-U133 array hybridized with cDNA targets. This may reflect the impact of the target type. Overall, the high degree of correspondence provides strong evidence for the reliability of the Gene 1.0 ST array.

Effect of chronic hypoglycaemia on glucose concentration and glycogen content in rat brain: A localized 13C NMR study.

While chronic hypoglycaemia has been reported to increase unidirectional glucose transport across the blood-brain barrier (BBB) and to increase GLUT1 expression at the endothelium, the effect on steady-state brain d-glucose and brain glycogen content is currently unknown. Brain glucose and glycogen concentrations were directly measured in vivo using localized 13C magnetic resonance spectroscopy (MRS) following 12-14 days of hypoglycaemia. Brain glucose content was significantly increased by 48%, which is consistent with an increase in the maximal glucose transport rate, Tmax, by 58% compared with the sham-treated animals. The localized 13C NMR measurements of brain glucose were directly validated by comparison with biochemically determined brain glucose content after rapid focused microwave fixation (1.4 s at 4 kW). Both in vivo MRS and biochemical measurements implied that brain glycogen content was not affected by chronic hypoglycaemia, consistent with brain glucose being a major factor controlling brain glycogen content. We conclude that the increased glucose transporter expression in chronic hypoglycaemia leads to increased brain glucose content at a given level of glycaemia. Such increased brain glucose concentrations can result in a lowered glycaemic threshold of counter-regulation observed in chronic hypoglycaemia.

X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation

Background: Aproximately 5–10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects. Results: Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%). Conclusion:This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients.

Molecular role of Cx37 in advanced atherosclerosis: a micro-array study.

Recently, we showed that connexin37 (Cx37) protects against early atherosclerotic lesion development by regulating monocyte adhesion. The expression of this gap junction protein is altered in mouse and human atherosclerotic lesions; it is increased in macrophages newly recruited to the lesions and disappears from the endothelium of advanced plaques. To obtain more insight into the molecular role of Cx37 in advanced atherosclerosis, we used micro-array analysis for gene expression profiling in aortas of ApoE(-/-) and Cx37(-/-)ApoE(-/-) mice before and after 18 weeks of cholesterol-rich diet. Out of >15,000 genes, 106 genes were significantly differentially expressed in young mice before diet (P-value of <0.05, fold change of >0.7 or <-0.7, and intensity value >2.2 times background). Ingenuity pathway analysis (IPA) revealed differences in genes involved in cell-to-cell signaling and interaction, cellular compromise and nutritional disease. In addition, we identified 100 genes that were significantly perturbed after the cholesterol-rich diet. Similar to the analysis on 10-week-old mice, IPA revealed differences in genes involved in cell-to-cell signaling and interaction as well as to immuno-inflammatory disease. Furthermore, we found important changes in genes involved in vascular calcification and matrix degradation, some of which were confirmed at protein level by (immuno-)histochemistry. In conclusion, we suggest that Cx37 deficiency alters the global differential gene expression profiles in young mice towards a pro-inflammatory phenotype, which are then further influenced in advanced atherosclerosis. The results provide new insights into the significance of Cx37 in plaque calcification.

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

A systolic array for linear MIMO detection based on an all-swap lattice reduction algorithm

A systolic array to implement lattice-reduction-aided lineardetection is proposed for a MIMO receiver. The lattice reductionalgorithm and the ensuing linear detections are operated in the same array, which can be hardware-efficient. All-swap lattice reduction algorithm (ASLR) is considered for the systolic design.ASLR is a variant of the LLL algorithm, which processes all lattice basis vectors within one iteration. Lattice-reduction-aided linear detection based on ASLR and LLL algorithms have very similarbit-error-rate performance, while ASLR is more time efficient inthe systolic array, especially for systems with a large number ofantennas.

A developmental in vivo 1H NMR study in mice with genetic redox dysregulation: an animal model with relevance to schizophrenia

Glutathione (GSH), a major redox regulator and anti-oxidant, is decreased in cerebrospinal fluid and prefrontal cortex of schizophrenia patients. The gene of the key GSH-synthesizing enzyme, glutamate-cysteine ligase, modifier (GCLM) subunit, is associated with schizophrenia, suggesting that the deficit in the GSH system is of genetic origin. Using the GCLM knock-out (KO) mouse model with 60% decreased brain GSH levels, we have shown that redox dysregulation results in abnormal brain morphology and function. Current theory holds that schizophrenia is a developmental disease involving progressive anatomical and functional brain pathology. Here, we used GCLM KO mice to investigate the impact of a genetically dysregulated redox system on the neurochemical profile of the developing brain. The anterior and posterior cortical neurochemical profile of male and female GCLM KO, heterozygous and wildtype mice was determined by localised in vivo 1H NMR spectroscopy at 14.1 T (Varian/Magnex spectrometer) on post-natal days 10, 20, 30, 60 and 90. We show, for the first time, (1) that high quality 1H NMR spectra can be acquired from early developing mouse brains and (2) that recurrent anaesthesia by itself when administered at the same developmental days has no adverse effects on brain metabolites nor on adult behaviour. (3) Most importantly, our results reveal genotype and age specific changes for a number of metabolites revealing insight into normal brain development and about the impact of genetic GSH dysregulation.

Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

16q24.1 microdeletion in a premature newborn: Usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn.

OBJECTIVE:: Report of a 16q24.1 deletion in a premature newborn, demonstrating the usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn and multiple congenital malformations. DESIGN:: Descriptive case report. SETTING:: Genetic department and neonatal intensive care unit of a tertiary care children's hospital. INTERVENTIONS:: None. PATIENT:: We report the case of a preterm male infant, born at 26 wks of gestation. A cardiac malformation and bilateral hydronephrosis were diagnosed at 19 wks of gestation. Karyotype analysis was normal, and a 22q11.2 microdeletion was excluded by fluorescence in situ hybridization analysis. A cesarean section was performed due to fetal distress. The patient developed persistent pulmonary hypertension unresponsive to mechanical ventilation and nitric oxide treatment and expired at 16 hrs of life. MEASUREMENTS AND MAIN RESULTS:: An autopsy revealed partial atrioventricular canal malformation and showed bilateral dilation of the renal pelvocaliceal system with bilateral ureteral stenosis and annular pancreas. Array-based comparative genomic hybridization analysis (Agilent oligoNT 44K, Agilent Technologies, Santa Clara, CA) showed an interstitial microdeletion encompassing the forkhead box gene cluster in 16q24.1. Review of the pulmonary microscopic examination showed the characteristic features of alveolar capillary dysplasia with misalignment of pulmonary veins. Some features were less prominent due to the gestational age. CONCLUSIONS:: Our review of the literature shows that alveolar capillary dysplasia with misalignment of pulmonary veins is rare but probably underreported. Prematurity is not a usual presentation, and histologic features are difficult to interpret. In our case, array-based comparative genomic hybridization revealed a 16q24.1 deletion, leading to the final diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins. It emphasizes the usefulness of array-based comparative genomic hybridization analysis as a diagnostic tool with implications for both prognosis and management decisions in newborns with refractory persistent pulmonary hypertension and multiple congenital malformations.

The application of NMR and MS methods for detection of adulteration of wine, fruit juices, and olive oil. A review

This review covers two important techniques, high resolution nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), used to characterize food products and detect possible adulteration of wine, fruit juices, and olive oil, all important products of the Mediterranean Basin. Emphasis is placed on the complementary use of SNIF-NMR (site-specific natural isotopic fractionation nuclear magnetic resonance) and IRMS (isotope-ratio mass spectrometry) in association with chemometric methods for detecting the adulteration.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase.

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37-1.73]; combined P=2.58 · 10(-13)). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25-1.44; P=1.032 · 10(-14)). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16-3.66) for systolic and 1.40 (95% CI: 0.25-2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.