Segmentation of left ventricle in short-axis echocardiographic sequences by weighted radial edge filtering and adaptive recovery of dropout regions

In this paper, we present a weighted radial edge filtering algorithm with adaptive recovery of dropout regions for the semi-automatic delineation of endocardial contours in short-axis echocardiographic image sequences. The proposed algorithm requires minimal user intervention at the end diastolic frame of the image sequence for specifying the candidate points of the contour. The region of interest is identified by fitting an ellipse in the region defined by the specified points. Subsequently, the ellipse centre is used for originating the radial lines for filtering. A weighted radial edge filter is employed for the detection of edge points. The outliers are corrected by global as well as local statistics. Dropout regions are recovered by incorporating the important temporal information from the previous frame by means of recursive least squares adaptive filter. This ensures fairly accurate segmentation of the cardiac structures for further determination of the functional cardiac parameters. The proposed algorithm was applied to 10 data-sets over a full cardiac cycle and the results were validated by comparing computer-generated boundaries to those manually outlined by two experts using Hausdorff distance (HD) measure, radial mean square error (rmse) and contour similarity index. The rmse was 1.83 mm with a HD of 5.12 ± 1.21 mm. We have also compared our results with two existing approaches, level set and optical flow. The results indicate an improvement when compared with ground truth due to incorporation of temporal clues. The weighted radial edge filtering algorithm in conjunction with adaptive dropout recovery offers semi-automatic segmentation of heart chambers in 2D echocardiography sequences for accurate assessment of global left ventricular function to guide therapy and staging of the cardiovascular diseases.     

Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity

We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different heights to yield substrates of different rigidities. The tips of the elastomeric microposts are functionalized with extracellular matrix through microcontact printing to promote cell adhesion. These substrates, therefore, present the same topographical cues to adherent cells while varying substrate rigidity only through manipulation of micropost height. This protocol describes how to fabricate the silicon micropost array masters (~2 weeks to complete) and elastomeric substrates (3 d), as well as how to perform cell culture experiments (1-14 d), immunofluorescence imaging (2 d), traction force analysis (2 d) and stem cell differentiation assays (1 d) on these substrates in order to examine the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization and differentiation.     

Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication

Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase that is critical for virus replication and spread. The extent of substrate conservation and the key common cell-signaling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach, we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched, and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective antiviral strategies.     

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.     

Acute drug treatment in the early C. elegans embryo

Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.     

Meeting report of the RNA Ontology Consortium January 8-9, 2011

This report summarizes the proceedings of the structure mapping working group meeting of the RNA Ontology Consortium (ROC), held in Kona, Hawaii on January 8-9, 2011. The ROC hosted this workshop to facilitate collaborations among those researchers formalizing concepts in RNA, those developing RNA-related software, and those performing genome annotation and standardization. The workshop included three software presentations, extended round-table discussions, and the constitution of two new working groups, the first to address the need for better software integration and the second to discuss standardization and benchmarking of existing RNA annotation pipelines. These working groups have subsequently pursued concrete implementation of actions suggested during the discussion. Further information about the ROC and its activities can be found at http://roc.bgsu.edu/.     

Efficacy of bacterial consortium-AIE2 for contemporaneous Cr(VI) and azo dye bioremediation in batch and continuous bioreactor systems, monitoring steady-state bacterial dynamics using qPCR assays

Bacterial consortium-AIE2 with a capability of contemporaneous Cr(VI) reduction and azo dye RV5 decolourization was developed from industrial wastewaters by enrichment culture technique. The 16S rRNA gene based molecular analyses revealed that the consortium bacterial community structure consisted of four bacterial strains namely, Alcaligenes sp. DMA, Bacillus sp. DMB, Stenotrophomonas sp. DMS and Enterococcus sp. DME. Cumulative mechanism of Cr(VI) reduction by the consortium was determined using in vitro Cr(VI) reduction assays. Similarly, the complete degradation of Reactive Violet 5 (RV5) dye was confirmed by FTIR spectroscopic analysis. Consortium-AIE2 exhibited simultaneous bioremediation efficiencies of (97.8 ± 1.4) % and (74.1 ± 1.2) % in treatment of both 50 mg l⁻¹ Cr(VI) and RV5 dye concentrations within 48 h of incubation at pH 7 and 37°C in batch systems. Continuous bioreactor systems achieved simultaneous bioremediation efficiencies of (98.4 ± 1.5) % and (97.5 ± 1.4) % after the onset of steady-state at 50 mg l⁻¹ input Cr(VI) and 25 mg l⁻¹ input RV5 concentrations, respectively, at medium dilution rate (D) of 0.014 h⁻¹. The 16S rRNA gene copy numbers in the continuous bioreactor as determined by real-time PCR assay indicated that Alcaligenes sp. DMA and Bacillus sp. DMB dominated consortium bacterial community during the active continuous bioremediation process.     

Complex inheritance of the plasmodial surface anion channel in a Plasmodium falciparum genetic cross

Human erythrocytes infected with the malaria parasite Plasmodium falciparum have increased permeabilities to many solutes. The plasmodial surface anion channel (PSAC) may mediate these changes. Despite good understanding of the biochemical and biophysical properties, the genetic basis of PSAC activity remains unknown. Functional polymorphisms in laboratory isolates and two mutants generated by in vitro selection implicate a parasite-encoded channel, although parasite-induced modifications of endogenous channels have not been formally excluded. Here, we identified stable differences in furosemide efficacy against PSAC activity induced by HB3 and 3D7A parasites. This difference was apparent in both single PSAC patch-clamp recordings and in sorbitol-mediated osmotic lysis measurements, confirming that Cl^- and sorbitol are transported by a single-channel type. Examination of 19 progeny from a genetic cross between HB3 and 3D7A revealed complex inheritance with some cloned progeny exhibiting furosemide affinities outside the range of parental values. Isolates generated by selfing of the 3D7A clone also exhibited altered furosemide affinities, implicating changes in one or more alleles during meiosis or passage through a primate host. PSAC may be encoded by multiple parasite genes (e.g. a multi-gene family or multiple genes that encode distinct channel subunits) or a single polymorphic gene under strong selective pressure.     

First steps in the direction of synthetic,allosteric, direct inhibitors of thrombin and factor Xa

Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that (i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; (ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and (iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes.