The application of MANOVA to analyse <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> metabolomic data from factorially designed experiments

Metabolomic technologies produce complex multivariate datasets and researchers are faced with the daunting task of extracting information from these data. Principal component analysis (PCA) has been widely applied in the field of metabolomics to reduce data dimensionality and for visualising trends within the complex data. Although PCA is very useful, it cannot handle multi-factorial experimental designs and, often, clear trends of biological interest are not observed when plotting various PC combinations. Even if patterns are observed, PCA provides no measure of their significance. Multivariate analysis of variance (MANOVA) applied to these PCs enables the statistical evaluation of main treatments and, more importantly, their interactions within the experimental design. The power and scope of MANOVA is demonstrated through two different factorially designed metabolomic investigations using Arabidopsis ethylene signalling mutants and their wild-type. One investigation has multiple experimental factors including challenge with the economically important pathogen Botrytis cinerea and also replicate experiments, while the second has different sample preparation methods and one level of replication ‘nested’ within the design. In both investigations there are specific factors of biological interest and there are also factors incorporated within the experimental design, which affect the data. The versatility of MANOVA is displayed by using data from two different metabolomic techniques; profiling using direct injection mass spectroscopy (DIMS) and fingerprinting using fourier transform infra-red (FT-IR) spectroscopy. MANOVA found significant main effects and interactions in both experiments, allowing a more complete and comprehensive interpretation of the variation within each investigation, than with PCA alone. Canonical variate analysis (CVA) was applied to investigate these effects and their biological significance. In conclusion, the application of MANOVA followed by CVA provided extra information than PCA alone and proved to be a valuable statistical addition in the overwhelming task of analysing metabolomic data.     

Developing synthetic conical nanopores for biosensing applications

In this review we bring together recent results from our group focused towards the development of biosensors from single conically-shaped artificial nanopores. The nanopores, used in the work presented here, were prepared using the track-etch process. The fabrication of track-etched conical nanopores has been optimized to allow for single nanopores with reproducible dimensions to be prepared. We have also demonstrated techniques that allow for easy and controllable manipulation of nanopore geometry (e.g., cone angle). We will consider the ion transport properties of the conical nanopores and factors that affect these properties. Methods for introducing functions that mimic biological ion channels, such as voltage-gating, into these nanopores will also be addressed. Three prototype sensors developed from single conical nanopores will be presented. In the first two sensors, the single conical nanopores function as resistive-pulse sensors and detect the presence of analytes as current-blockade events in the ion current. The third sensor functions in an on/off mode, much like a ligand-gated ion channel. In the presence of a target analyte, the ion current permanently shuts off.     

The association of the ankle-brachial index with incident coronary heart disease: the Atherosclerosis Risk In Communities (ARIC) study, 1987–2001

Background

We have developed a rat cell model for studying collagen type I production in coronary artery adventitial fibroblasts. Increased deposition of adventitial collagen type I leads to stiffening of the blood vessel, increased blood pressure, arteriosclerosis and coronary heart disease. Although the source and mechanism of collagen deposition is yet unknown, the adventitia appears to play a significant role. To demonstrate the application of our cell model, cultured adventitial fibroblasts were treated with sex hormones and the effect on collagen production measured.

Methods

Hearts (10–12 weeks) were harvested and the left anterior descending coronary artery (LAD) was isolated and removed. Tissue explants were cultured and cells (passages 2–4) were confirmed as fibroblasts using immunohistochemistry. Optimal conditions were determined for cell tissue harvest, timing, proliferation and culture conditions. Fibroblasts were exposed to 10^-7 M testosterone or 10^-7 M estrogen for 24 hours and either immunostained for collagen type I or subjected to ELISA.

Results

Results showed increased collagen staining in fibroblasts treated with testosterone compared to control and decreased staining with estrogen. ELISA results showed that testosterone increased collagen I by 20% whereas estrogen decreased collagen I by 15%.

Conclusion

Data demonstrates the usefulness of our cell model in studying the specific role of the adventitia apart from other blood vessel tissue in rat coronary arteries. Results suggest opposite effects of testosterone and estrogen on collagen synthesis in the rat coronary artery adventitial fibroblasts.     

Elucidation of spheroid formation with and without the extrusion step

Spheroid formation mechanisms were investigated using extrusion-spheronization (ES) and rotary processing (RP). Using ES (cross-hatch), ES (teardrop), and RP (teardrop), spheroids with similar mass median diameter (MMD) and span were produced using equivalent formulation and spheronization conditions. During spheronization, the teardrop-studded rotating frictional surface, with increased peripheral tip speed and duration, produced spheroids of equivalent MMD and span to those produced by the cross-hatch rotating frictional plate surface. The roundness of these spheroids was also similar. RP required less water to produce spheroids of MMD similar to that of spheroids produced by ES. However, these RP spheroids were less spherical. Image analysis of 625 spheroids per batch indicated that the size distribution of RP spheroids had significantly greater SD, positive skewness, and kurtosis. Morphological examination of time-sampled spheroids produced by ES indicated that spheroid formation occurred predominatly by attrition and layering, while RP spheroids were formed by nucleation, agglomeration, layering, and coalescence. RP produced spheroids with higher crushing strength than that of ES-produced spheroids. The amount of moisture lost during spheronization for spheroids produced by ES had minimal influence on their eventual size. Differences in process and formulation parameters, in addition to size distribution and observed morphological changes, enabled a greater understanding of spheroid formation and methods to optimize spheroid production. Published: February 9, 2007     

Acidification effects on biofouling communities: winners and losers

How ocean acidification affects marine life is a major concern for science and society. However, its impacts on encrusting biofouling communities, that are both the initial colonizers of hard substrata and of great economic importance, are almost unknown. We showed that community composition changed significantly, from 92% spirorbids, 3% ascidians and 4% sponges initially to 47% spirorbids, 23% ascidians and 29% sponges after 100 days in acidified conditions (pH 7.7). In low pH, numbers of the spirorbid Neodexiospira pseudocorrugata were reduced ×5 compared to controls. The two ascidians present behaved differently with Aplidium sp. decreasing ×10 in pH 7.7, whereas Molgula sp. numbers were ×4 higher in low pH than controls. Calcareous sponge (Leucosolenia sp.) numbers increased ×2.5 in pH 7.7 over controls. The diatom and filamentous algal community was also more poorly developed in the low pH treatments compared to controls. Colonization of new surfaces likewise showed large decreases in spirorbid numbers, but numbers of sponges and Molgula sp. increased. Spirorbid losses appeared due to both recruitment failure and loss of existing tubes. Spirorbid tubes are comprised of a loose prismatic fabric of calcite crystals. Loss of tube materials appeared due to changes in the binding matrix and not crystal dissolution, as SEM analyses showed crystal surfaces were not pitted or dissolved in low pH conditions. Biofouling communities face dramatic future changes with reductions in groups with hard exposed exoskeletons and domination by soft‐bodied ascidians and sponges.     

C7L family of poxvirus host range genes inhibits antiviral activities induced by type I interferons and interferon regulatory factor 1

Vaccinia virus (VACV) K1L and C7L function equivalently in many mammalian cells to support VACV replication and antagonize antiviral activities induced by type I interferons (IFNs). While K1L is limited to orthopoxviruses, genes that are homologous to C7L are found in diverse mammalian poxviruses. In this study, we showed that the C7L homologues from sheeppox virus and swinepox virus could rescue the replication defect of a VACV mutant deleted of both K1L and C7L (vK1L(-)C7L(-)). Interestingly, the sheeppox virus C7L homologue could rescue the replication of vK1L(-)C7L(-) in human HeLa cells but not in murine 3T3 and LA-4 cells, in contrast to all other C7L homologues. Replacing amino acids 134 and 135 of the sheeppox virus C7L homologue, however, made it functional in the two murine cell lines, suggesting that these two residues are critical for antagonizing a putative host restriction factor which has some subtle sequence variation in human and murine cells. Furthermore, the C7L family of host range genes from diverse mammalian poxviruses were all capable of antagonizing type I IFN-induced antiviral activities against VACV. Screening of a library of more than 350 IFN-stimulated genes (ISGs) identified interferon-regulated factor 1 (IRF1) as an inhibitor of vK1L(-)C7L(-) but not wild-type VACV. Expression of either K1L or C7L, however, rendered vK1L(-)C7L(-) resistant to IRF1-induced antiviral activities. Altogether, our data show that K1L and C7L antagonize IRF1-induced antiviral activities and that the host modulation function of C7L is evolutionally conserved in all poxviruses that can readily replicate in tissue-cultured mammalian cells.     

Cutting edge: nitrogen bisphosphonate-induced inflammation is dependent upon mast cells and IL-1

Nitrogen-containing bisphosphonates (NBPs) are taken by millions for bone disorders but may cause serious inflammatory reactions. In this study, we used a murine peritonitis model to characterize the inflammatory mechanisms of these agents. At dosages comparable to those used in humans, injection of NBPs into the peritoneum caused recruitment of neutrophils, followed by an influx of monocytes. These cellular changes corresponded to an initial increase in IL-1α, which preceded a rise in multiple other proinflammatory cytokines. IL-1R, IL-1α, and IL-1β were required for neutrophil recruitment, whereas other MyD88-dependent signaling pathways were needed for the monocyte influx. Mice deficient in mast cells, but not mice lacking lymphocytes, were resistant to NBP-induced inflammation, and reconstitution of these mice with mast cells restored sensitivity to NBPs. These results document the critical role of mast cells and IL-1 in NBP-mediated inflammatory reactions.     

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A_27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8⁺ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.