Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Fully automated software solution for protein quantitation by global metabolic labeling with stable isotopes

Metabolic stable isotope labeling is increasingly employed for accurate protein (and metabolite) quantitation using mass spectrometry (MS). It provides sample-specific isotopologues that can be used to facilitate comparative analysis of two or more samples. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has been used for almost a decade in proteomic research and analytical software solutions have been established that provide an easy and integrated workflow for elucidating sample abundance ratios for most MS data formats. While SILAC is a discrete labeling method using specific amino acids, global metabolic stable isotope labeling using isotopes such as (15)N labels the entire element content of the sample, i.e. for (15)N the entire peptide backbone in addition to all nitrogen-containing side chains. Although global metabolic labeling can deliver advantages with regard to isotope incorporation and costs, the requirements for data analysis are more demanding because, for instance for polypeptides, the mass difference introduced by the label depends on the amino acid composition. Consequently, there has been less progress on the automation of the data processing and mining steps for this type of protein quantitation. Here, we present a new integrated software solution for the quantitative analysis of protein expression in differential samples and show the benefits of high-resolution MS data in quantitative proteomic analyses.

Manual response preparation and saccade programming are linked to attention shifts: ERP evidence for covert attentional orienting and spatially specific modulations of visual processing

The premotor theory of attention claims that attentional shifts are triggered during response programming, regardless of which response modality is involved. To investigate this claim, event-related brain potentials (ERPs) were recorded while participants covertly prepared a left or right response, as indicated by a precue presented at the beginning of each trial. Cues signalled a left or right eye movement in the saccade task, and a left or right manual response in the manual task. The cued response had to be executed or withheld following the presentation of a Go/Nogo stimulus. Although there were systematic differences between ERPs triggered during covert manual and saccade preparation, lateralised ERP components sensitive to the direction of a cued response were very similar for both tasks, and also similar to the components previously found during cued shifts of endogenous spatial attention. This is consistent with the claim that the control of attention and of covert response preparation are closely linked. N1 components triggered by task-irrelevant visual probes presented during the covert response preparation interval were enhanced when these probes were presented close to cued response hand in the manual task, and at the saccade target location in the saccade task. This demonstrates that both manual and saccade preparation result in spatially specific modulations of visual processing, in line with the predictions of the premotor theory.

Preliminary observations on the presence of sustained tendon strain and eccentric contractions of the wrist extensors during a common manual task: implications for lateral epicondylitis

Lateral epicondylitis (LE) is hypothesized to occur as a result of repetitive, strenuous and abnormal postural activities of the elbow and wrist. There is still a lack of understanding of how wrist and forearm positions contribute to this condition during common manual tasks. In this study the wrist kinematics and the wrist extensors’ musculotendon patterns were investigated during a manual task believed to elicit LE symptoms in susceptible subjects. A 42-year-old right-handed male, with no history of LE, performed a repetitive movement involving pushing and turning a spring-loaded mechanism. Motion capture data were acquired for the upper limb and an inverse kinematic and dynamic analysis was subsequently carried out. Results illustrated the presence of eccentric contractions sustained by the extensor carpi radialis longus (ECRL), together with an almost constant level of tendon strain of both extensor carpi radialis brevis (ECRB) and extensor digitorum communis lateral (EDCL) branch. It is believed that these factors may partly contribute to the onset of LE as they are both responsible for the creation of microtears at the tendons’ origins. The methodology of this study can be used to explore muscle actions during movements that might cause or exacerbate LE.

Eco-labeling, rents, sales prices and occupancy rates: do LEED and Energy Star labeled offices obtain multiple premiums?

Drawing upon an updated and expanded dataset of Energy Star and LEED labeled commercial offices, this paper investigates the effect of eco-labeling on rental rates, sale prices and occupancy rates. Using OLS and robust regression procedures, hedonic modeling is used to test whether the presence of an eco-label has a significant positive effect on rental rates, sale prices and occupancy rates. The study suggests that estimated coefficients can be sensitive to outlier treatment. For sale prices and occupancy rates, there are notable differences between estimated coefficients for OLS and robust regressions. The results suggest that both Energy Star and LEED offices obtain rental premiums of approximately 3%. A 17% sale price premium is estimated for Energy Star labeled offices but no significant sale price premium is estimated for LEED labeled offices. Surprisingly, no significant occupancy premium is estimated for Energy Star labeled offices and a negative occupancy premium is estimated for LEED labeled offices.

An investigation of the effect of eco-labeling on office occupancy rates

This paper investigates the extent to which clients were able to influence performance measurement appraisals during the downturn in commercial property markets that began in the UK during the second half of 2007. The sharp change in market sentiment produced speculation that different client categories were attempting to influence their appraisers in different ways. In particular, it was recognised that the requirement for open-ended funds to meet redemptions gave them strong incentives to ensure that their asset values were marked down to market. Using data supplied by Investment Property Databank, we demonstrate that, indeed, unlisted open ended funds experienced sharper drops in capital values than other fund types in the second half of 2007, after the market turning point. These differences are statistically significant and cannot simply be explained by differences in portfolio composition. Client influence on appraisal forms one possible explanation of the results observed: the different pressures on fund managers resulting in different appraisal outcomes.

Field manual 3-24 and the heritage of counterinsurgency theory

The Counterinsurgency Manual FM 3-24 has been accused of being over-dependent on the counterinsurgency 'classics' Galula and Thompson. But comparison reveals that it is different in spirit. Galula and Thompson seek practical control; the Manual seeks to build 'legitimacy'. Its concept of legitimacy is superficially Weberian, but owes more to the writings of the American Max Manwaring. The Manual presupposes that a rights-based legal order can (other things being equal) be made to be cross-culturally attractive; 'effective governance' by itself can build legitimacy. The fusion of its methods with an ideology creates unrealistic criteria for success. Its weaknesses suggest a level of incapacity to think politically that will, in time, result in further failures.

Assessment of inter-examiner agreement and variability in the manual classification of auditory brainstem response

Abstract Background: The analysis of the Auditory Brainstem Response (ABR) is of fundamental importance to the investigation of the auditory system behaviour, though its interpretation has a subjective nature because of the manual process employed in its study and the clinical experience required for its analysis. When analysing the ABR, clinicians are often interested in the identification of ABR signal components referred to as Jewett waves. In particular, the detection and study of the time when these waves occur (i.e., the wave latency) is a practical tool for the diagnosis of disorders affecting the auditory system. Significant differences in inter-examiner results may lead to completely distinct clinical interpretations of the state of the auditory system. In this context, the aim of this research was to evaluate the inter-examiner agreement and variability in the manual classification of ABR. Methods: A total of 160 ABR data samples were collected, for four different stimulus intensity (80dBHL, 60dBHL, 40dBHL and 20dBHL), from 10 normal-hearing subjects (5 men and 5 women, from 20 to 52 years). Four examiners with expertise in the manual classification of ABR components participated in the study. The Bland-Altman statistical method was employed for the assessment of inter-examiner agreement and variability. The mean, standard deviation and error for the bias, which is the difference between examiners’ annotations, were estimated for each pair of examiners. Scatter plots and histograms were employed for data visualization and analysis. Results: In most comparisons the differences between examiner’s annotations were below 0.1 ms, which is clinically acceptable. In four cases, it was found a large error and standard deviation (>0.1 ms) that indicate the presence of outliers and thus, discrepancies between examiners. Conclusions: Our results quantify the inter-examiner agreement and variability of the manual analysis of ABR data, and they also allows for the determination of different patterns of manual ABR analysis.

Neuromantic : from semi-manual to semi-automatic reconstruction of neuron morphology

The ability to create accurate geometric models of neuronal morphology is important for understanding the role of shape in information processing. Despite a significant amount of research on automating neuron reconstructions from image stacks obtained via microscopy, in practice most data are still collected manually. This paper describes Neuromantic, an open source system for three dimensional digital tracing of neurites. Neuromantic reconstructions are comparable in quality to those of existing commercial and freeware systems while balancing speed and accuracy of manual reconstruction. The combination of semi-automatic tracing, intuitive editing, and ability of visualizing large image stacks on standard computing platforms provides a versatile tool that can help address the reconstructions availability bottleneck. Practical considerations for reducing the computational time and space requirements of the extended algorithm are also discussed.

Updated empirical model of genetically-modified maize grain production practices to achieve European Union labeling thresholds

An updated empirical approach is proposed for specifying coexistence requirements for genetically modified (GM) maize (Zea mays L.) production to ensure compliance with the 0.9% labeling threshold for food and feed in the European Union. The model improves on a previously published (Gustafson et al., 2006) empirical model by adding recent data sources to supplement the original database and including the following additional cases: (i) more than one GM maize source field adjacent to the conventional or organic field, (ii) the possibility of so-called “stacked” varieties with more than one GM trait, and (iii) lower pollen shed in the non-GM receptor field. These additional factors lead to the possibility for somewhat wider combinations of isolation distance and border rows than required in the original version of the empirical model. For instance, in the very conservative case of a 1-ha square non-GM maize field surrounded on all four sides by homozygous GM maize with 12 m isolation (the effective isolation distance for a single GM field), non-GM border rows of 12 m are required to be 95% confident of gene flow less than 0.9% in the non-GM field (with adventitious presence of 0.3%). Stacked traits of higher GM mass fraction and receptor fields of lower pollen shed would require a greater number of border rows to comply with the 0.9% threshold, and an updated extension to the model is provided to quantify these effects.

User's manual

'Typeform dialogues', as originally planned for publication, was an interactive CD-based screen interface presenting a comparative survey of typeform history and description. The CD was to be held inside a printed book that included two essays and a User’s Manual. Although 'Typeform dialogues' (CD+book) was not completed as planned, a PDF file was published containing the User’s Manual, together with background information about the interface and the project it was part of. The User's Manual presents all interface sections and features (using screen grabs) and explains their editorial rationale, design, and function.

Overcoming your child’s fears and worries: guided self-help manual for therapists working with groups

This manual for therapists accompanies ‘Overcoming your child’s fears and worries’ (Creswell & Willetts, 2007), a guide for parents to help their children overcome difficulties with anxiety