Evaluation of temperature gradient gel electrophoresis for the analysis of prey DNA within the guts of invertebrate predators

The utility of temperature gradient gel electrophoresis (TGGE) as a means of analysing the gut contents of predators was evaluated. Generalist predators consume multiple prey species and a species-specific primer approach may not always be a practical means of analysing predator responses to prey diversity in complex and biodiverse ecosystems. General invertebrate primers were used to amplify the gut contents of predators, generating banding patterns that identified component prey remains. There was no evidence of dominance of the polymerase chain reaction (PCR) by predator DNA. When applied to field samples of the carabid predator Pterostichus melanarius (Illiger) nine banding patterns were detected, including one for aphids. To further distinguish between species, group-specific primers were designed to separate species of earthworm and aphid. TGGE of the earthworm PCR products generated banding patterns that varied with haplotype in some species. Aphid and earthworm DNA could be detected in the guts of carabids for up to 24 h using TGGE. In P. melanarius, with low numbers of prey per insect gut (mean<3), interpretation of banding patterns proved to be tractable. Potential problems of interpretation of TGGE gels caused by multiple prey bands, cryptic bands, haplotype variation, taxonomic uncertainties (especially with regard to earthworms), secondary predation, scavenging and presence of parasites and parasitoids in the prey or the predators, are discussed. The results suggest that PCR, using combinations of general invertebrate and group-specific primers followed by TGGE, provides a potentially useful approach to the analysis of multiple uncharacterized prey in predators.     

Effects of altered estuarine submerged macrophyte bed cover on the omnivorous Cape stumpnose Rhabdosargus holubi

The ecological importance of submerged macrophyte beds to fishes within estuaries was investigated through the example of the ubiquitous Cape stumpnose Rhabdosargus holubi, an omnivorous, vegetation and estuary-dependent species, using stable-isotope techniques and long-term abundance (catch-per-unit-effort) data from the East Kleinemonde Estuary, South Africa. Outputs from a Bayesian mixing model using δ(13) C and δ(15) N signatures indicated that the submerged macrophytes Ruppia cirrhosa and Potamogeton pectinatus were not a primary source of nutrition for R. holubi, confirming previous work that revealed that macrophytes are consumed but not digested. Long-term seine netting data showed reduced abundance of R. holubi during a prolonged period of macrophyte senescence, suggesting that submerged macrophyte habitats provide shelter that reduces mortality (predation risk) and a food-rich foraging area.     

Additional analysis of dendrochemical data of Fallon, Nevada

Previously reported dendrochemical data showed temporal variability in concentration of tungsten (W) and cobalt (Co) in tree rings of Fallon, Nevada, US. Criticism of this work questioned the use of the Mann-Whitney test for determining change in element concentrations. Here, we demonstrate that Mann-Whitney is appropriate for comparing background element concentrations to possibly elevated concentrations in environmental media. Given that Mann-Whitney tests for differences in shapes of distributions, inter-tree variability (e.g., "coefficient of median variation") was calculated for each measured element across trees within subsites and time periods. For W and Co, the metals of highest interest in Fallon, inter-tree variability was always higher within versus outside of Fallon. For calibration purposes, this entire analysis was repeated at a different town, Sweet Home, Oregon, which has a known tungsten-powder facility, and inter-tree variability of W in tree rings confirmed the establishment date of that facility. Mann-Whitney testing of simulated data also confirmed its appropriateness for analysis of data affected by point-source contamination. This research adds important new dimensions to dendrochemistry of point-source contamination by adding analysis of inter-tree variability to analysis of central tendency. Fallon remains distinctive by a temporal increase in W beginning by the mid 1990s and by elevated Co since at least the early 1990s, as well as by high inter-tree variability for W and Co relative to comparison towns.     

Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness

The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when analyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ≥5 d and was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1 demonstrated that CD45(+)CD11c(+) cells (macrophages and dendritic cells), as well as collagen-1-producing CD45(-) cells (fibroblasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma.     

P65 truncation impacts P30 dynamics during Mycoplasma pneumoniae gliding

The cell wall-less prokaryote Mycoplasma pneumoniae is a major cause of community-acquired bronchitis and pneumonia in humans. Colonization is mediated largely by a differentiated terminal organelle, which is also the leading end in gliding motility. Cytadherence-associated proteins P30 and P65 appear to traffic concurrently to the distal end of developing terminal organelles. Here, truncation of P65 due to transposon insertion in the corresponding gene resulted in lower gliding velocity, reduced cytadherence, and decreased steady-state levels of several terminal organelle proteins, including P30. Utilizing fluorescent protein fusions, we followed terminal organelle development over time. New P30 foci appeared at nascent terminal organelles in P65 mutants, as in the wild type. However, with forward cell motility, P30 in the P65 mutants appeared to drag toward the trailing cell pole, where it was released, yielding a fluorescent trail to which truncated P65 colocalized. In contrast, P30 was only rarely observed at the trailing end of gliding wild-type cells. Complementation with the recombinant wild-type P65 allele by transposon delivery restored P65 levels and stabilized P30 localization to the terminal organelle.     

Phenotypic diversity and epigenomic variation - The utility of mass spectrometric analysis of DNA methylation

Epigenomic variation may underlie phenotypic diversity that is not attributable to differences in genomic sequence. Such processes provide an organism the flexibility to respond to changing environmental cues within its lifetime, and perhaps its offspring's lifetime, and would therefore be expected to confer a selective advantage in evolutionary terms. Analysis of epigenomic variation within a population may be both a useful measure of developmental exposures and an indicator of future phenotype. A key molecular indicator of epigenomic variation in organisms is the chemical modification of DNA by methylation at specific nucleotide residues in the genome. Here we discuss how mass spectrometry can be utilised to provide quantitative analysis of DNA methylation patterns across populations. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

You look familiar: how Malaysian Chinese recognize faces

East Asian and white Western observers employ different eye movement strategies for a variety of visual processing tasks, including face processing. Recent eye tracking studies on face recognition found that East Asians tend to integrate information holistically by focusing on the nose while white Westerners perceive faces featurally by moving between the eyes and mouth. The current study examines the eye movement strategy that Malaysian Chinese participants employ when recognizing East Asian, white Western, and African faces. Rather than adopting the Eastern or Western fixation pattern, Malaysian Chinese participants use a mixed strategy by focusing on the eyes and nose more than the mouth. The combination of Eastern and Western strategies proved advantageous in participants' ability to recognize East Asian and white Western faces, suggesting that individuals learn to use fixation patterns that are optimized for recognizing the faces with which they are more familiar.     

IQGAP1 is necessary for pulmonary vascular barrier protection in murine acute lung injury and pneumonia

We recently reported that integrin α(v)β(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel β(3)-integrin binding partner IQGAP1. Like integrin β(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin α(v)β(3) to the cell-cell junction. Similar to β(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of (125)I-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin α(v)β(3).     

Notch receptor-ligand binding and activation: insights from molecular studies

The Notch receptor is part of a core signalling pathway which is highly conserved in all metazoan species. It is required for various cell fate decisions at multiple stages of development and in the adult organism, with dysregulation of the pathway associated with genetic and acquired diseases including cancer. Although cellular and in vivo studies have provided considerable insight into the downstream consequences of Notch signalling, relatively little is known about the molecular basis of the receptor/ligand interaction and initial stages of activation. Recent advances in structure determination of the extracellular regions of human Notch-1 and one of its ligands Jagged-1 have given new insights into docking events occurring at the cell surface which may facilitate the development of new highly specific therapies. We review the structural data available for receptor and ligands and identify the challenges ahead.