Magnetic resonance imaging, computed tomography, and conventional X-ray in 3 cases of symmelia

BACKGROUND: Symmelia is a rare birth defect, often combined with severe malformations of the urogenital system and the lower gastrointestinal tract. Additionally, a deformed pelvis and various degrees of separation of the lower limbs are present. CASES: We report the examination findings of 3 autopsy specimens of symmelia using magnetic resonance imaging (MRI) and computed tomography (CT) with 3-dimensional (3D) reconstructions, and conventional X-ray. CONCLUSIONS: MRI and CT with the addition of 3D visualization can be used additionally with autopsy and conventional X-ray images in the investigation of such complex anatomical abnormalities.     

Comparison of Amersham and Agilent microarray technologies through quantitative noise analysis

We carried out a series of replicate experiments on DNA microarrays using two cell lines and two technologies--the Agilent Human 1A Microarray and the GE Amersham Codelink Uniset Human 20K I Bioarray. We demonstrated that quantifying the noise level as a function of signal strength allows identification of the absolute and differential mRNA expression levels at which biological variability can be resolved above measurement noise. This represents a new formulation of a sensitivity threshold that can be used to compare platforms. It was found that the correlation in expression level between platforms is considerably worse than the correlation between replicate measurements taken using the same platform. In addition, we carried out replicate measurements at different stages of sample processing. This novel approach enables us to quantify the noise introduced into the measurements at each step of the experimental protocol. We demonstrated how this information can be used to determine the most efficient means of using replicates to reduce experimental uncertainty.     

The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.     

Calculating kinetics and pathways of protein-ligand association

While studies of protein-ligand association have mostly focused on the native complex and its stability (binding affinity), relatively little attention has been paid to the association process that precedes the formation of the complex. Here we review approaches to study the kinetics of association and association mechanisms, i.e. the probability distribution of association pathways. Selected methods are described that allow these properties to be calculated quantitatively from simulation models. We summarize some applications of these methods and finally propose a model mechanism by which proteins may efficiently screen potential ligands for those that can be natively bound.     

Phototrophic Phylotypes Dominate Mesothermal Microbial Mats Associated with Hot Springs in Yellowstone National Park

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.     

FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.     

Blur and disparity are complementary cues to depth

Estimating depth from binocular disparity is extremely precise, and the cue does not depend on statistical regularities in the environment. Thus, disparity is commonly regarded as the best visual cue for determining 3D layout. But depth from disparity is only precise near where one is looking; it is quite imprecise elsewhere. Away from fixation, vision resorts to using other depth cues-e.g., linear perspective, familiar size, aerial perspective. But those cues depend on statistical regularities in the environment and are therefore not always reliable. Depth from defocus blur relies on fewer assumptions and has the same geometric constraints as disparity but different physiological constraints. Blur could in principle fill in the parts of visual space where disparity is imprecise. We tested this possibility with a depth-discrimination experiment. Disparity was more precise near fixation and blur was indeed more precise away from fixation. When both cues were available, observers relied on the more informative one. Blur appears to play an important, previously unrecognized role in depth perception. Our findings lead to a new hypothesis about the evolution of slit-shaped pupils and have implications for the design and implementation of stereo 3D displays.     

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ～90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.     

Identification and characterization of histatin 1 salivary complexes by using mass spectrometry

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein's function is the identification of its interaction partners. We investigated interactions among and functions of histatin 1 and the other proteins that are present in saliva by using high‐throughput mass spectrometric techniques. This led to the identification of 43 proteins able to interact with histatin 1. In addition, we found that these protein–protein interactions protect complex partners from proteolysis and modulate their antifungal activity. Our data contribute significantly to characterization of the salivary interactome and to understanding the biology of salivary protein complexes.