Automated Detection and Analysis of Ca2+ Sparks in x-y Image Stacks Using a Thresholding Algorithm Implemented within the Open-Source Image Analysis Platform ImageJ

Previous studies have used analysis of Ca²⁺ sparks extensively to investigate both normal and pathological Ca²⁺ regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca²⁺ sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca²⁺ spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca²⁺ sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics.     

Genome-Wide Association Study of Celiac Disease in North America Confirms FRMD4B as New Celiac Locus

We performed a genome-wide association study (GWAS) of 1550 North American celiac disease cases and 3084 controls. Twelve SNPs, distributed across four regions (3p21.31, 4q27, 6q15, 6q25), were significantly associated with disease (p-value <1.0×10⁻⁷), and a further seven SNPs, across four additional regions (1q24.3, 10p15.1, 6q22.31, 17q21.32) had suggestive evidence (1.0×10⁻⁷ < p-value < 1.0×10⁻⁶). This study replicated a previous suggestive association within FRMD4B (3p14.1), confirming it as a celiac disease locus. All four regions with significant associations and two regions with suggestive results (1q24.3, 10p15.1) were known disease loci. The 6q22.31 and 10p11.23 regions were not replicated. A total of 410 SNPs distributed across the eight significant and suggestive regions were tested for association with dermatitis herpetiformis and microscopic colitis. Preliminary, suggestive statistical evidence for association with the two traits was found at chromosomes 3p21.31, 6q15, 6q25, 1q24.3 and 10p11.23, with future studies being required to validate the reported associations.     

Convergent Polishing: A Simple,Rapid, Full Aperture Polishing Process of High Quality Optical Flats & Spheres

Convergent Polishing is a novel polishing system and method for finishing flat and spherical glass optics in which a workpiece, independent of its initial shape (i.e., surface figure), will converge to final surface figure with excellent surface quality under a fixed, unchanging set of polishing parameters in a single polishing iteration. In contrast, conventional full aperture polishing methods require multiple, often long, iterative cycles involving polishing, metrology and process changes to achieve the desired surface figure. The Convergent Polishing process is based on the concept of workpiece-lap height mismatch resulting in pressure differential that decreases with removal and results in the workpiece converging to the shape of the lap. The successful implementation of the Convergent Polishing process is a result of the combination of a number of technologies to remove all sources of non-uniform spatial material removal (except for workpiece-lap mismatch) for surface figure convergence and to reduce the number of rogue particles in the system for low scratch densities and low roughness. The Convergent Polishing process has been demonstrated for the fabrication of both flats and spheres of various shapes, sizes, and aspect ratios on various glass materials. The practical impact is that high quality optical components can be fabricated more rapidly, more repeatedly, with less metrology, and with less labor, resulting in lower unit costs. In this study, the Convergent Polishing protocol is specifically described for fabricating 26.5 cm square fused silica flats from a fine ground surface to a polished ~λ/2 surface figure after polishing 4 hr per surface on a 81 cm diameter polisher.     

The Role of Galectin-1 and Galectin-3 in the Mucosal Immune Response to Citrobacter rodentium Infection

Despite their abundance at gastrointestinal sites, little is known about the role of galectins in gut immune responses. We have therefore investigated the Citrobacter rodentium model of colonic infection and inflammation in Galectin-1 or Galectin-3 null mice. Gal-3 null mice showed a slight delay in colonisation after inoculation with C. rodentium and a slight delay in resolution of infection, associated with delayed T cell, macrophage and dendritic cell infiltration into the gut mucosa. However, Gal-1 null mice also demonstrated reduced T cell and macrophage responses to infection. Despite the reduced T cell and macrophage response in Gal-1 null mice, there was no effect on C. rodentium infection kinetics and pathology. Overall, Gal-1 and Gal-3 play only a minor role in immunity to a gut bacterial pathogen.     

Succinate production and citrate catabolism by Cheddar cheese nonstarter lactobacilli

AIMS: To identify strains of Cheddar cheese nonstarter lactobacilli that synthesize succinate from common precursors and characterize the biochemical pathways utilized. METHODS AND RESULTS: Whole cell incubations of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus zeae and Lactobacillus rhamnosus, were used to identify strains that accumulated succinate from citrate, l-lactate, aspartic acid or isocitrate. In vivo 13C-nuclear magnetic resonance spectroscopy (13C-NMR) identified the biochemical pathway involved at pH 7.0, and under conditions more representative of the cheese ripening environment (pH 5.1/4% NaCl/13 degrees C). Enzyme assays on cell-free extracts were used to support the pathway suggested by 13C-NMR. CONCLUSIONS: The Lact. plantarum strains studied synthesize succinate from citrate by the reductive tricarboxylic acid (TCA) cycle at either pH 7.0 or pH 5.1/4% NaCl/13 degrees C. Lactobacillus casei, Lact. zeae and Lact. rhamnosus strains lack one or more enzymatic activities present in this pathway, and do not accumulate succinate from any of the four precursors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of Lact. plantarum strains to milk during cheese manufacture may increase the accumulation of the flavour enhancer succinate.     

The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge

Urea synthesis via the hepatic ornithine urea cycle (OUC) has been well described in elasmobranchs, but it is unknown whether OUC enzymes are also present in extrahepatic tissues. Muscle and liver urea, trimethylamine oxide (TMAO), and other organic osmolytes, as well as selected OUC enzymes (carbamoyl phosphate synthetase III, ornithine transcarbamoylase, arginase, and the accessory enzyme glutamine synthetase), were measured in adult little skates (Raja erinacea) exposed to 100% or 75% seawater for 5 d. Activities of all four OUC enzymes were detected in the muscle. There were no changes in muscle OUC activities in skates exposed to 75% seawater; however, arginase activity was significantly lower in the liver, compared to controls. Urea, TMAO, and several other osmolytes were significantly lower in the muscle of little skates exposed to 75% seawater, whereas only glycerophosphorylcholine was significantly lower in the liver. Urea excretion rates were twofold higher in skates exposed to 75% seawater. Taken together, these data suggest that a functional OUC may be present in the skeletal muscle tissues of R. erinacea. As well, enhanced urea excretion rates and the downregulation of the anchor OUC enzyme, arginase, in the liver may be critical in regulating tissue urea content under dilute-seawater stress.     

Correlation of in vitro chemopreventive efficacy data from the human epidermal cell assay with animal efficacy data and clinical trial plasma levels

The human epidermal cell (HEC) assay, which uses carcinogen exposed normal skin keratinocytes to screen for cancer prevention efficacy, was used to screen possible preventive agents. The endpoints measured were inhibition of carcinogen-induced growth and induction of involucrin, an early marker of differentiation. Sixteen of twenty agents (apigenin, apomine, budesonide, N-(2-carboxyphenyl)retinamide, ellagic acid, ibuprofen, indomethacin, melatonin, (-)-2-oxo-4-thiazolidine carboxylic acid, polyphenon E, resveratrol, beta-sitosterol, sulfasalazine, vitamin E acetate, and zileuton) were positive in at least one of the two assay endpoints. Four agents (4-methoxyphenol, naringenin, palmitoylcarnitine chloride, and silymarin) were negative in the assay. Nine of the sixteen agents were positive for both endpoints. Agents that showed the greatest response included: ellagic acid > budesonide, ibuprofen > apigenin, and quinicrine dihydrochloride. Fifty-eight of sixty-five agents that have been evaluated in the HEC assay have also been evaluated in one or more rodent bioassays for cancer prevention and several are in clinical trials for cancer prevention. The assay has an overall predictive accuracy of approximately 91.4% for efficacy in rodent cancer prevention irrespective of the species used, the tissue model, or the carcinogen used. Comparison of the efficacious concentrations in vitro to plasma levels in clinical trials show that concentrations that produced efficacy in the HEC assay were achieved in clinical studies for 31 of 33 agents for which plasma levels and/or C(max) levels were available. For two agents, 9-cis-retinoic acid (RA) and dehydroepiandrosterone (DHEA), the plasma levels greatly exceeded the highest concentration (HC) found to have efficacy in vitro. Thus, the HEC assay has an excellent predictive potential for animal efficacy and is responsive at clinically achievable concentrations.     

A high-density genetic map of hexaploid wheat (Triticum aestivum L.) from the cross Chinese Spring × SQ1 and its use to compare QTLs for grain yield across a range of environments

A population of 96 doubled haploid lines (DHLs) was prepared from F₁ plants of the hexaploid wheat cross Chinese Spring × SQ1 (a high abscisic acid-expressing breeding line) and was mapped with 567 RFLP, AFLP, SSR, morphological and biochemical markers covering all 21 chromosomes, with a total map length of 3,522 cM. Although the map lengths for each genome were very similar, the D genome had only half the markers of the other two genomes. The map was used to identify quantitative trait loci (QTLs) for yield and yield components from a combination of 24 site × treatment × year combinations, including nutrient stress, drought stress and salt stress treatments. Although yield QTLs were widely distributed around the genome, 17 clusters of yield QTLs from five or more trials were identified: two on group 1 chromosomes, one each on group 2 and group 3, five on group 4, four on group 5, one on group 6 and three on group 7. The strongest yield QTL effects were on chromosomes 7AL and 7BL, due mainly to variation in grain numbers per ear. Three of the yield QTL clusters were largely site-specific, while four clusters were largely associated with one or other of the stress treatments. Three of the yield QTL clusters were coincident with the dwarfing gene Rht-B1 on 4BS and with the vernalisation genes Vrn-A1 on 5AL and Vrn-D1 on 5DL. Yields of each DHL were calculated for trial mean yields of 6 g plant^–1 and 2 g plant^–1 (equivalent to about 8 t ha^–1 and 2.5 t ha^–1, respectively), representing optimum and moderately stressed conditions. Analyses of these yield estimates using interval mapping confirmed the group-7 effects on yield and, at 2 g plant^–1, identified two additional major yield QTLs on chromosomes 1D and 5A. Many of the yield QTL clusters corresponded with QTLs already reported in wheat and, on the basis of comparative genetics, also in rice. The implications of these results for improving wheat yield stability are discussed.