Internal soft-tissue anatomy of cambrian 'orsten' arthropods as revealed by synchrotron x-ray tomographic microscopy

The world-famous ‘Orsten’ Konservat-Lagerstätte has yielded detailed information about Cambrian arthropods and their morphology. Internal organs or soft tissues have, however, rarely been reported, an obvious palaeobiological drawback. In this study, we employed synchrotron radiation X-ray tomographic microscopy (SRXTM) to study microscopic ‘Orsten’ arthropods from the Cambrian of Sweden: Skara minuta and two phosphatocopine species, Hesslandona sp. and Hesslandona trituberculata. This exceptionally high-resolution technique reveals internal organs or soft tissues that allow detailed comparison with equivalent structures in extant crustaceans and functional inferences to be made. The S. minuta specimen shows the digestive system and muscles that extend to the extremities. The slanting anterior portion of the head and anterior position of the mouth with a straight oesophagus suggest a primarily brushing and scraping way of feeding. The prominent head appendage muscles indicate muscle strength and good capacity for food manipulation. In the phosphatocopines the bulbous labrum is one of the most prominent morphological structures of the body. All specimens analysed reveal pairs of muscle bundles within the labrum. Based on comparisons with extant crustacean relatives, these muscles would fulfil the function of moving the labrum up and down in order to open the buccal cavity. The results of this pilot study demonstrate that there is still much to be learned about the ‘Orsten’ taxa.     

Lactobacillus reuteri Prevents Diet-Induced Obesity,but not Atherosclerosis,in a Strain Dependent Fashion in Apoe?/? Mice

Objective

To investigate whether the specific strains of Lactobacillus reuteri modulates the metabolic syndrome in Apoe−/− mice.

Methods

8 week-old Apoe−/− mice were subdivided into four groups who received either L. reuteri ATCC PTA 4659 (ATCC), DSM 17938 (DSM), L6798, or no bacterial supplement in the drinking water for 12 weeks. The mice were fed a high-fat Western diet with 0.2% cholesterol and body weights were monitored weekly. At the end of the study, oral glucose and insulin tolerance tests were conducted. In addition, adipose and liver weights were recorded along with analyses of mRNA expression of ileal Angiopoietin-like protein 4 (Angptl4), the macrophage marker F4/80 encoded by the gene Emr1 and liver Acetyl-CoA carboxylase 1 (Acc1), Fatty acid synthase (Fas) and Carnitine palmitoyltransferase 1a (Cpt1a). Atherosclerosis was assessed in the aortic root region of the heart.

Results and Conclusions

Mice receiving L. reuteri ATCC gained significantly less body weight than the control mice, whereas the L6798 mice gained significantly more. Adipose and liver weights were also reduced in the ATCC group. Serum insulin levels were lower in the ATCC group, but no significant effects were observed in the glucose or insulin tolerance tests. Lipogenic genes in the liver were not altered by any of the bacterial treatments, however, increased expression of Cpt1a was found in the ATCC group, indicating increased β-oxidation. Correspondingly, the liver trended towards having lower fat content. There were no effects on inflammatory markers, blood cholesterol or atherosclerosis. In conclusion, the probiotic L. reuteri strain ATCC PTA 4659 partly prevented diet-induced obesity, possibly via a previously unknown mechanism of inducing liver expression of Cpt1a.     

Cellular effects of HER3-specific affibody molecules

Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.     

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.     

Micro and macro-habitat associations in saproxylic beetles: implications for biodiversity management

Restoration of habitats is critically important in preventing full realization of the extinction debt owed as a result of anthropogenic habitat destruction. Although much emphasis has been placed on macrohabitats, suitable microhabitats are also vital for the survival of most species. The aim of this large-scale field experiment was to evaluate the relative importance of manipulated microhabitats, i.e., dead wood substrates of spruce (snags, and logs that were burned, inoculated with wood fungi or shaded) and macrohabitats, i.e., stand types (clear-cuts, mature managed forests, and forest reserves) for species richness, abundance and assemblage composition of all saproxylic and red-listed saproxylic beetles. Beetles were collected in emergence traps in 30 forest stands in 2001, 2003, 2004 and 2006. More individuals emerged from snags and untreated logs than from burned and shaded logs, but species richness did not differ among substrates. Assemblage composition differed among substrates for both all saproxylics and red-listed saproxylic species, mainly attributed to different assemblage composition on snags. This suggests that the practise of leaving snags for conservation purposes should be complemented with log supplementation. Clear-cuts supported fewer species and different assemblages from mature managed forests and reserves. Neither abundance, nor species richness or assemblage composition differed between reserves and mature managed forests. This suggests that managed stands subjected to selective cutting, not clear-felling, maintain sufficient old growth characteristics and continuity to maintain more or less intact assemblages of saproxylic beetles. Thus, alternative management methods, e.g., continuity forestry should be considered for some of these stands to maintain continuity and conservation values. Furthermore, the significantly higher estimated abundance per ha of red-listed beetles in reserves underlines the importance of reserves for maintaining viable populations of rare red-listed species and as source areas for saproxylic species in boreal forest landscapes.     

Highly improved homopolymer aware nucleotide-protein alignments with 454 data

ABSTRACT: BACKGROUND: Roche 454 sequencing is the leading sequencing technology for producing long read high throughput sequence data. Unlike most methods where sequencing errors translate to base uncertainties, 454 sequencing inaccuracies create nucleotide gaps. These gaps are particularly troublesome for translated search tools such as BLASTx where they introduce frame-shifts and result in regions of decreased identity and/or terminated alignments, which affect further analysis. RESULTS: To address this issue, the Homopolymer Aware Cross Alignment Tool (HAXAT) was developed. HAXAT uses a novel dynamic programming algorithm for solving the optimal local alignment between a 454 nucleotide and a protein sequence by allowing frame-shifts, guided by 454 flowpeak values. The algorithm is an efficient minimal extension of the Smith-Waterman-Gotoh algorithm that easily fits in into other tools.Experiments using HAXAT demonstrate, through the introduction of 454 specific frame-shift penalties, significantly increased accuracy of alignments spanning homopolymer sequence errors. The full effect of the new parameters introduced with this novel alignment model is explored. Experimental results evaluating homopolymer inaccuracy through alignments show a two to five-fold increase in Matthews Correlation Coefficient over previous algorithms, for 454-derived data. CONCLUSIONS: This increased accuracy provided by HAXAT does not only result in improved homologue estimations, but also provides un-interrupted reading-frames, which greatly facilitate further analysis of protein space, for example phylogenetic analysis.The alignment tool is available at http://bioinfo.ifm.liu.se/454tools/haxat.     

Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K_d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K_d, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K_d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.     

The effect of anisotropic systematic errors in estimating helical angles

A common question in movement studies is how the results should be interpreted with respect to systematic and random errors. In this study, simulations are made in order to see how a rigid body's orientation in space (i.e. helical angle between two orientations) is affected by (1) a systematic error added to a single marker (2) a combination of this systematic error and Gaussian white noise. The orientation was estimated after adding a systematic error to one marker within the rigid body. This procedure was repeated with Gaussian noise added to each marker. In conclusion, results show that the systematic error's effect on estimated orientation depends on number of markers in the rigid body and also on which direction the systematic error is added. The systematic error has no effect if the error is added along the radial axis (i.e. the line connecting centre of mass and the affected marker).     

The correlation between cellular size and protein expression levels--normalization for global protein profiling

An automated image analysis system was used for protein quantification of 1862 human proteins in 47 cancer cell lines and 12 clinical cell samples using cell microarrays and immunohistochemistry. The analysis suggests that most proteins are expressed in a cell size dependent manner, and that normalization is required for comparative protein quantification in order to correct for the inherent bias of cell size and systematic ambiguities associated with immunohistochemistry. Two reference standards were evaluated, and normalized protein expression values were found to allow for protein profiling across a panel of morphologically diverse cells, revealing putative patterns of over- and underexpression. Using this approach, proteins with stable expression as well as cell-line specific expression were identified. The results demonstrate the value of large-scale, automated proteome analysis using immunohistochemistry, in revealing functional correlations and establishing methods to interpret and mine proteomic data.