Small volume laboratory on a chip measurements incorporating the quartz crystal microbalance to measure the viscosity-density product of room temperature ionic liquids

A microfluidic glass chip system incorporating a quartz crystal microbalance (QCM) to measure the square root of the viscosity-density product of room temperature ionic liquids (RTILs) is presented. The QCM covers a central recess on a glass chip, with a seal formed by tightly clamping from above outside the sensing region. The change in resonant frequency of the QCM allows for the determination of the square root viscosity-density product of RTILs to a limit of similar to 10 kg m(-2) s(-0.5). This method has reduced the sample size needed for characterization from 1.5 ml to only 30 mu l and allows the measurement to be made in an enclosed system.

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.

Toxic effects of cadmium and zinc on ectomycorrhizal colonization of scots pine (Pinus sylvestris L.) from soil inoculum

Scots pine seedlings colonized by ectomycorrhizal (ECM) fungi from natural soil inoculum were exposed to a range of Cd or Zn concentrations to investigate the effects of metals on ECM fungi-Scots pine associations in a realistic soil environment. Experiments focused on the relationship between the sensitivity of ECM fungi and their host plants, the influence of metals on ECM community dynamics on Scots pine roots, and the effects of metal exposure on ECM colonization from soil-borne propagules. Ectomycorrhizal colonization was inhibited by Cd and Zn, with a decrease in the proportion of ECM-colonized root tips. Shoot and root biomass, total root length, and total root-tip density, however, were unaffected by Cd or Zn. A decrease in the diversity of ECM morphotypes also occurred, which could have a negative effect on tree vigor. Overall, colonization by ECM fungi was more sensitive than seedling growth to Cd and Zn, and this could have serious implications for successful tree establishment on metal-contaminated soils.

Upregulation of osteopontin and alpha B crystallin in the normal-appearing white matter of multiple sclerosis: an immunohistochemical study utilizing tissue microarrays

Tissue microarrays assembled from control and multiple sclerosis (MS) brain tissue have been used to assess the expression patterns and cellular distribution of two antigens, the proinflammatory cytokine osteopontin and the inducible heat shock protein alpha B -crystallin, which have previously been implicated in MS pathogenesis. Tissue cores were taken from paraffin-embedded donor blocks containing chronic active or chronic inactive plaques and normal-appearing white matter (NAWM) in seven MS cases, and white matter (WM) in five control cases. Expression patterns of both proteins were assessed against myelin density and microglial activation in the different tissue categories. Both proteins showed increased expression in all categories of MS tissue compared with control WM. The results indicate progressive up-regulation of expression of osteopontin with increased plaque activity, while elevation of alpha B-crystallin expression in MS tissue was independent of demyelination. In MS NAWM a significant correlation was observed between high levels of expression of osteopontin and alpha B -crystallin. Osteopontin expression was predominantly confined to astrocytes throughout MS tissues. alpha B -crystallin was expressed on astrocytes, oligodendrocytes and occasionally on demyelinated axons. Taken together, these data indicate a wider distribution of osteopontin and alpha B -crystallin in MS tissues than previously described and support their proposed role in MS pathogenesis.

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Prediction of adipose tissue composition using Raman spectroscopy: average properties and individual fatty acids.

Abstract: Raman spectroscopy has been used for the first time to predict the FA composition of unextracted adipose tissue of pork, beef, lamb, and chicken. It was found that the bulk unsaturation parameters could be predicted successfully [R-2 = 0.97, root mean square error of prediction (RMSEP) = 4.6% of 4 sigma], with cis unsaturation, which accounted for the majority of the unsaturation, giving similar correlations. The combined abundance of all measured PUFA (>= 2 double bonds per chain) was also well predicted with R-2 = 0.97 and RMSEP = 4.0% of 4 sigma. Trans unsaturation was not as well modeled (R-2 = 0.52, RMSEP = 18% of 4 sigma); this reduced prediction ability can be attributed to the low levels of trans FA found in adipose tissue (0.035 times the cis unsaturation level). For the individual FA, the average partial least squares (PLS) regression coefficient of the 18 most abundant FA (relative abundances ranging from 0.1 to 38.6% of the total FA content) was R-2 = 0.73; the average RMSEP = 11.9% of 4 sigma. Regression coefficients and prediction errors for the five most abundant FA were all better than the average value (in some cases as low as RMSEP = 4.7% of 4 sigma). Cross-correlation between the abundances of the minor FA and more abundant acids could be determined by principal component analysis methods, and the resulting groups of correlated compounds were also well-predicted using PLS. The accuracy of the prediction of individual FA was at least as good as other spectroscopic methods, and the extremely straightforward sampling method meant that very rapid analysis of samples at ambient temperature was easily achieved. This work shows that Raman profiling of hundreds of samples per day is easily achievable with an automated sampling system.

Density-viscosity product of small-volume ionic liquid samples using quartz crystal impedance analysis

Quartz crystal impedance analysis has been developed as a technique to assess whether room-temperature ionic liquids are Newtonian fluids and as a small-volume method for determining the values of their viscosity-density product, rho eta. Changes in the impedance spectrum of a 5-MHz fundamental frequency quartz crystal induced by a water-miscible room-temperature ionic liquid, 1-butyl-3-methylimidazolium. trifluoromethylsulfonate ([C(4)mim][OTf]), were measured. From coupled frequency shift and bandwidth changes as the concentration was varied from 0 to 100% ionic liquid, it was determined that this liquid provided a Newtonian response. A second water-immiscible ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C(4)mim][NTf2], with concentration varied using methanol, was tested and also found to provide a Newtonian response. In both cases, the values of the square root of the viscosity-density product deduced from the small-volume quartz crystal technique were consistent with those measured using a viscometer and density meter. The third harmonic of the crystal was found to provide the closest agreement between the two measurement methods; the pure ionic liquids had the largest difference of similar to 10%. In addition, 18 pure ionic liquids were tested, and for 11 of these, good-quality frequency shift and bandwidth data were obtained; these 12 all had a Newtonian response. The frequency shift of the third harmonic was found to vary linearly with square root of viscosity-density product of the pure ionic liquids up to a value of root(rho eta) approximate to 18 kg m(-2) s(-1/2), but with a slope 10% smaller than that predicted by the Kanazawa and Gordon equation. It is envisaged that the quartz crystal technique could be used in a high-throughput microfluidic system for characterizing ionic liquids.

A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6

Lung T lymphocytes are important in pulmonary immunity and inflammation. it has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5% +/- 1.9% (n = 11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8 +/- 2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells. (C) 2008 Elsevier B.V. All rights reserved.

AKT1 Is Associated with Schizophrenia Across Multiple Symptom Dimensions in the Irish Study of High Density Schizophrenia Families

Background: The phosphatidylinositol 3-kinase (PI3K)-AKT signal transduction pathway is critical to cell growth and survival. In vitro functional studies indicate that the candidate schizophrenia susceptibility gene DTNBP1 influences AKT signaling to promote neuronal viability. The AKT1 gene has also been implicated in schizophrenia by association studies and decreased protein expression in the brains of schizophrenic patients. 
 Methods: The association of DTNBP1 in the Irish Study of High Density Schizophrenia Families (ISHDSF) prompted our investigation of AKT1 for association with disease in this sample. Eight single nucleotide polymorphisms spanning AKT1 were analyzed for association with schizophrenia across four definitions of affection and according to Operational Criteria Checklist of Psychotic Illness (OPCRIT) symptom scales. We examined expression of AKT1 messenger RNA from postmortem brain tissue of schizophrenic, bipolar, and control individuals. 
 Results: No single marker showed significant association, but the risk haplotype previously found over-transmitted to Caucasian schizophrenic patients was significantly under-transmitted in the ISHDSF (.01 < p < .05), across all OPCRIT symptom dimensions. Exploratory haplotype analysis confirmed association with schizophrenia toward the 5’ end of AKT1 (.008 < p < .049, uncorrected). We found significantly decreased RNA levels in prefrontal cortex of schizophrenic individuals, consistent with reduced AKT1 protein levels reported in schizophrenic brain. 
 Conclusions: The replication of association of AKT1 gene variants in a further Caucasian family sample adds support for involvement of AKT signaling in schizophrenia, perhaps encompassing a broader clinical phenotype that includes mood dysregulation. We show that AKT signaling might be compromised in schizophrenic and bipolar patients via reduced RNA expression of specific AKT isoforms.

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a (CO2)-C-13 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of C-13-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded C-13 compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from C-12 and C-13 RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.

Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

No evidence for linkage or association of neuregulin-1 (NRG1) with disease in the Irish study of high-density schizophrenia families (ISHDSF)

The neuregulin-1 gene (NRG1) at chromosome 8p21-22 has been implicated as a schizophrenia susceptibility gene in Icelandic, Scottish, Irish and mixed UK populations. The shared ancestry between these populations led us to investigate the NRG1 polymorphisms and appropriate marker haplotypes for linkage and/or association to schizophrenia in the Irish study of high-density schizophrenia families (ISHDSF). Neither single-point nor multi-point linkage analysis of NRG1 markers gave evidence for linkage independent of our pre-existing findings telomeric on 8p. Analysis of linkage disequilibrium (LD) across the 252 kb interval encompassing the 7 marker core Icelandic/Scottish NRG1 haplotype revealed two separate regions of modest LD, comprising markers SNP8NRG255133, SNP8NRG249130 and SNP8NRG243177 (telomeric) and microsatellites 478B14-428, 420M9-1395, D8S1810 and 420M9-116I12 (centromeric). From single marker analysis by TRANSMIT and FBAT we found no evidence for association with schizophrenia for any marker. Haplotype analysis for the three SNPs in LD region 1 and, separately, the four microsatellites in LD region 2 (analyzed in overlapping 2-marker windows), showed no evidence for overtransmission of specific haplotypes to affected individuals. We therefore conclude that if NRG1 does contain susceptibility alleles for schizophrenia, they impact quite weakly on risk in the ISHDSF.

Wnt signaling in age-related macular degeneration: human macular tissue and mouse model

BACKGROUND: The wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly.

METHODS: We examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD.

RESULTS: We found higher levels of LRP6 (a key Wnt signaling receptor) protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-β-catenin and phosphorylated-LRP6 were higher in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 retinas.

CONCLUSIONS: Wnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.