Addressing accuracy and precision issues in iTRAQ quantitation

iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.

Input from the amygdala to the rat nucleus accumbens : its relationship with tyrosine hydroxylase immunoreactivity and identified neurons.

Both tyrosine hydroxylase-positive fibres from the mesolimbic dopamine system and amygdala projection fibres from the basolateral nucleus are known to terminate heavily in the nucleus accumbens. Caudal amygdala fibres travelling dorsally via the stria terminalis project densely to the nucleus accumbens shell, especially in the dopamine rich septal hook. The amygdala has been associated with the recognition of emotionally relevant stimuli while the mesolimbic dopamine system is implicated with reward mechanisms. There is behavioural and electrophysiological evidence that the amygdala input to the nucleus accumbens is modulated by the mesolimbic dopamine input, but it is not known how these pathways interact anatomically within the nucleus accumbens. Using a variety of neuroanatomical techniques including anterograde and retrograde tracing, immunocytochemistry and intracellular filling, we have demonstrated convergence of these inputs on to medium-sized spiny neurons. The terminals of the basolateral amygdala projection make asymmetrical synapses predominantly on the heads of spines which also receive on their necks or adjacent dendrites, symmetrical synaptic input from the mesolimbic dopamine system. Some of these neurons have also been identified as projection neurons, possibly to the ventral pallidum. We have shown a synaptic level how dopamine is positioned to modulate excitatory limbic input in the nucleus accumbens.

Esophageal stricture in children: Fiber-optic endoscopy and dilatation under fluoroscopic control

We made a retrospective analysis of the efficacy and complication rate of 268 esophageal dilatation procedures performed under fluoroscopic control using the fiber-optic endoscope in 45 children with esophageal stricture. Antegrade and retrograde stricture dilatation was performed under general anesthetic, mainly as an outpatient procedure. Thirty-six children had an esophageal stricture following tracheoesophageal fistula and/or esophageal atresia repair, and nine children had severe corrosive stricture of the esophagus following lye ingestion. The procedure was well tolerated and effective. © 1992 Raven Press, Ltd., New York.

Ion-molecule reactions of O,S-Dimethyl methylphosphonothioate : Evidence for intramolecular sulfur oxidation during VX perhydrolysis

The alkaline perhydrolysis of the nerve agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) was investigated by studying the ion-molecule reactions of HOO(-) with O,S-dimethyl methylphosphonothioate in a modified linear ion-trap mass spectrometer. In addition to simple proton transfer, two other abundant product ions are observed at m/z 125 and 109 corresponding to the S-methyl methylphosphonothioate and methyl methylphosphonate anions, respectively. The structure of these product ions is demonstrated by a combination of collision-induced dissociation and isotope-labeling experiments that also provide evidence for their formation by nucleophilic reaction pathways, namely, (i) S(N)2 at carbon to yield the S-methyl methylphosphonothioate anion and (ii) nucleophilic addition at phosphorus affording a reactive pentavalent intermediate that readily undergoes internal sulfur oxidation and concomitant elimination of CH(3)SOH to yield the methyl methylphosphonate anion. Consistent with previous Solution phase observations of VX perhydrolysis, the toxic P-O cleavage product is not observed in this VX model system and theoretical calculations identify P-O cleavage to be energetically uncompetitive. Conversely, intramolecular sulfur oxidation is calculated to be extremely exothermic and kinetically accessible explaining its competitiveness with the facile gas phase proton transfer process. Elimination of a sulfur moiety deactivates the nerve agent VX and thus the intramolecular sulfur oxidation process reported here is also able to explain the selective perhydrolysis of the nerve agent to relatively nontoxic products.

An application of fuzzy DL-Based semantic perception to soil container classification

Semantic perception and object labeling are key requirements for robots interacting with objects on a higher level. Symbolic annotation of objects allows the usage of planning algorithms for object interaction, for instance in a typical fetchand-carry scenario. In current research, perception is usually based on 3D scene reconstruction and geometric model matching, where trained features are matched with a 3D sample point cloud. In this work we propose a semantic perception method which is based on spatio-semantic features. These features are defined in a natural, symbolic way, such as geometry and spatial relation. In contrast to point-based model matching methods, a spatial ontology is used where objects are rather described how they "look like", similar to how a human would described unknown objects to another person. A fuzzy based reasoning approach matches perceivable features with a spatial ontology of the objects. The approach provides a method which is able to deal with senor noise and occlusions. Another advantage is that no training phase is needed in order to learn object features. The use-case of the proposed method is the detection of soil sample containers in an outdoor environment which have to be collected by a mobile robot. The approach is verified using real world experiments.

Mutations in the gene encoding IFT dynein complex component WDR34 cause jeune asphyxiating thoracic dystrophy

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.

Semantic context and visual feature effects in object naming : an fMRI study using arterial spin labeling

Previous behavioral studies reported a robust effect of increased naming latencies when objects to be named were blocked within semantic category, compared to items blocked between category. This semantic context effect has been attributed to various mechanisms including inhibition or excitation of lexico-semantic representations and incremental learning of associations between semantic features and names, and is hypothesized to increase demands on verbal self-monitoring during speech production. Objects within categories also share many visual structural features, introducing a potential confound when interpreting the level at which the context effect might occur. Consistent with previous findings, we report a significant increase in response latencies when naming categorically related objects within blocks, an effect associated with increased perfusion fMRI signal bilaterally in the hippocampus and in the left middle to posterior superior temporal cortex. No perfusion changes were observed in the middle section of the left middle temporal cortex, a region associated with retrieval of lexical-semantic information in previous object naming studies. Although a manipulation of visual feature similarity did not influence naming latencies, we observed perfusion increases in the perirhinal cortex for naming objects with similar visual features that interacted with the semantic context in which objects were named. These results provide support for the view that the semantic context effect in object naming occurs due to an incremental learning mechanism, and involves increased demands on verbal self-monitoring.

Clustering and labeling a web scale document collection using Wikipedia clusters

Clustering is an important technique in organising and categorising web scale documents. The main challenges faced in clustering the billions of documents available on the web are the processing power required and the sheer size of the datasets available. More importantly, it is nigh impossible to generate the labels for a general web document collection containing billions of documents and a vast taxonomy of topics. However, document clusters are most commonly evaluated by comparison to a ground truth set of labels for documents. This paper presents a clustering and labeling solution where the Wikipedia is clustered and hundreds of millions of web documents in ClueWeb12 are mapped on to those clusters. This solution is based on the assumption that the Wikipedia contains such a wide range of diverse topics that it represents a small scale web. We found that it was possible to perform the web scale document clustering and labeling process on one desktop computer under a couple of days for the Wikipedia clustering solution containing about 1000 clusters. It takes longer to execute a solution with finer granularity clusters such as 10,000 or 50,000. These results were evaluated using a set of external data.

Creating healthy food environments in NSW: A knowledge and evidence synthesis to inform advocacy and action: Food labeling

Reviews and synthesizes evidence to produce evidence-based recommendations on policy actions to improve food labeling for NSW Health

Deeper and wider fully convolutional network coupled with conditional random fields for scene labeling

Deep convolutional neural networks (DCNNs) have been employed in many computer vision tasks with great success due to their robustness in feature learning. One of the advantages of DCNNs is their representation robustness to object locations, which is useful for object recognition tasks. However, this also discards spatial information, which is useful when dealing with topological information of the image (e.g. scene labeling, face recognition). In this paper, we propose a deeper and wider network architecture to tackle the scene labeling task. The depth is achieved by incorporating predictions from multiple early layers of the DCNN. The width is achieved by combining multiple outputs of the network. We then further refine the parsing task by adopting graphical models (GMs) as a post-processing step to incorporate spatial and contextual information into the network. The new strategy for a deeper, wider convolutional network coupled with graphical models has shown promising results on the PASCAL-Context dataset.

Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a Km of 46 μM and a kcat of 3.5 s−1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a Ki of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.

On the usage of labels and icons in business process modeling

The value of business process models is dependent not only on the choice of graphical elements in the model, but also on their annotation with additional textual and graphical information. This research discusses the use of text and icons for labeling the graphical constructs in a process model. We use two established verb classification schemes to examine the choice of activity labels in process modeling practice. Based on our findings, we synthesize a set of twenty-five activity label categories. We propose a systematic approach for graphically representing these label categories through the use of graphical icons, such that the resulting process models are easier and more readily understandable by end users. Our findings contribute to an ongoing stream of research investigating the practice of process modeling and thereby contribute to the body of knowledge about conceptual modeling quality overall.

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

Soil carbon saturation : evaluation and corroboration by long-term incubations

Although current assessments of agricultural management practices on soil organic C (SOC) dynamics are usually conducted without any explicit consideration of limits to soil C storage, it has been hypothesized that the SOC pool has an upper, or saturation limit with respect to C input levels at steady state. Agricultural management practices that increase C input levels over time produce a new equilibrium soil C content. However, multiple C input level treatments that produce no increase in SOC stocks at equilibrium show that soils have become saturated with respect to C inputs. SOC storage of added C input is a function of how far a soil is from saturation level (saturation deficit) as well as C input level. We tested experimentally if C saturation deficit and varying C input levels influenced soil C stabilization of added C-13 in soils varying in SOC content and physiochemical characteristics. We incubated for 2.5 years soil samples from seven agricultural sites that were closer to (i.e., A-horizon) or further from (i.e., C-horizon) their C saturation limit. At the initiation of the incubations, samples received low or high C input levels of 13 C-labeled wheat straw. We also tested the effect of Ca addition and residue quality on a subset of these soils. We hypothesized that the proportion of C stabilized would be greater in samples with larger C Saturation deficits (i.e., the C- versus A-horizon samples) and that the relative stabilization efficiency (i.e., Delta SCC/Delta C input) would decrease as C input level increased. We found that C saturation deficit influenced the stabilization of added residue at six out of the seven sites and C addition level affected the stabilization of added residue in four sites, corroborating both hypotheses. Increasing Ca availability or decreasing residue quality had no effect on the stabilization of added residue. The amount of new C stabilized was significantly related to C saturation deficit, supporting the hypothesis that C saturation influenced C stabilization at all our sites. Our results suggest that soils with low C contents and degraded lands may have the greatest potential and efficiency to store added C because they are further from their saturation level. (c) 2008 Elsevier Ltd. All rights reserved.