Error, reproducibility and sensitivity: a pipeline for data processing of Agilent oligonucleotide expression arrays

Background: Expression microarrays are increasingly used to obtain large scale transcriptomic information on a wide range of biological samples. Nevertheless, there is still much debate on the best ways to process data, to design experiments and analyse the output. Furthermore, many of the more sophisticated mathematical approaches to data analysis in the literature remain inaccessible to much of the biological research community. In this study we examine ways of extracting and analysing a large data set obtained using the Agilent long oligonucleotide transcriptomics platform, applied to a set of human macrophage and dendritic cell samples. Results: We describe and validate a series of data extraction, transformation and normalisation steps which are implemented via a new R function. Analysis of replicate normalised reference data demonstrate that intrarray variability is small (only around 2 of the mean log signal), while interarray variability from replicate array measurements has a standard deviation (SD) of around 0.5 log(2) units (6 of mean). The common practise of working with ratios of Cy5/Cy3 signal offers little further improvement in terms of reducing error. Comparison to expression data obtained using Arabidopsis samples demonstrates that the large number of genes in each sample showing a low level of transcription reflect the real complexity of the cellular transcriptome. Multidimensional scaling is used to show that the processed data identifies an underlying structure which reflect some of the key biological variables which define the data set. This structure is robust, allowing reliable comparison of samples collected over a number of years and collected by a variety of operators. Conclusions: This study outlines a robust and easily implemented pipeline for extracting, transforming normalising and visualising transcriptomic array data from Agilent expression platform. The analysis is used to obtain quantitative estimates of the SD arising from experimental (non biological) intra- and interarray variability, and for a lower threshold for determining whether an individual gene is expressed. The study provides a reliable basis for further more extensive studies of the systems biology of eukaryotic cells.

Sex differences in reward-related decision processing under stress

Recent research indicates gender differences in the impact of stress on decision behavior, but little is known about the brain mechanisms involved in these gender-specific stress effects. The current study used functional magnetic resonance imaging (fMRI) to determine whether induced stress resulted in gender-specific patterns of brain activation during a decision task involving monetary reward. Specifically, we manipulated physiological stress levels using a cold pressor task, prior to a risky decision making task. Healthy men (n = 24, 12 stressed) and women (n = 23, 11 stressed) completed the decision task after either cold pressor stress or a control task during the period of cortisol response to the cold pressor. Gender differences in behavior were present in stressed participants but not controls, such that stress led to greater reward collection and faster decision speed in males but less reward collection and slower decision speed in females. A gender-by-stress interaction was observed for the dorsal striatum and anterior insula. With cold stress, activation in these regions was increased in males but decreased in females. The findings of this study indicate that the impact of stress on reward-related decision processing differs depending on gender.

Pronoun processing in Broca's aphasia: discourse-syntax effects in ambiguous anaphora resolution

Background: The interpretation of ambiguous subject pronouns in a null subject language, like Greek, requires that one possesses grammatical knowledge of the two subject pronominal forms, i.e., null and overt, and that discourse constraints regulating the distribution of the two pronouns in context are respected. Aims: We investigated whether the topic-shift feature encoded in overt subject pronouns would exert similar interpretive effects in a group of seven participants with Broca’s aphasia and a group of language-unimpaired adults during online processing of null and overt subject pronouns in referentially ambiguous contexts. Method & Procedures: An offline picture–sentence matching task was initially administered to investigate whether the participants with Broca’s aphasia had access to the gender and number features of clitic pronouns. An online self-paced listening picture-verification task was subsequently administered to examine how the aphasic individuals resolve pronoun ambiguities in contexts with either null or overt subject pronouns and how their performance compares to that of language-unimpaired adults. Outcomes & Results: Results demonstrate that the Broca group, along with controls, had intact access to the morphosyntactic features of clitic pronouns. However, the aphasic individuals showed decreased preference for non-salient antecedents in object position during the online resolution of ambiguous overt subject pronouns and preferred to pick the subject antecedent instead. Conclusions: Broca’s aphasic participants’ parsing decisions in the online task reflect their difficulty with establishing topic-shifted interpretations of the ambiguous overt subject pronouns. The presence of a local topic-shift effect in the immediate temporal vicinity of the overt pronoun suggests that sensitivity to the marked informational status of overt pronouns is preserved in the aphasic individuals, yet, it is blocked under conditions of global sentential processing.

Grey matter volume in the cerebellum is related to processing of grammatical rules in a second language: a structural Voxel-Based Morphometry study

The experience of learning and using a second language (L2) has been shown to affect the grey matter (GM) structure of the brain. Importantly, GM density in several cortical and subcortical areas has been shown to be related to performance in L2 tasks. Here we show that bilingualism can lead to increased GM volume in the cerebellum, a structure that has been related to the processing of grammatical rules. Additionally, the cerebellar GM volume of highly proficient L2 speakers is correlated to their performance in a task tapping on grammatical processing in a L2, demonstrating the importance of the cerebellum for the establishment and use of grammatical rules in a L2.

Constraints on L2 learners’ processing of wh- dependencies: evidence from eye-movements

Using the eye movement monitoring technique, the present study examined whether wh-dependency formation is sensitive to island constraints in second language (L2) sentence comprehension, and whether the presence of an intervening relative clause island has any effects on learners’ ability to ultimately resolve long wh-dependencies. Participants included proficient learners of L2 English from typologically different language backgrounds (German, Chinese), as well as a group of native English-speaking controls. Our results indicate that both the learners and the native speakers were sensitive to relative clause islands during processing, irrespective of typological differences between the learners’ L1s, but that the learners had more difficulty than native speakers linking distant wh-fillers to their lexical subcategorizers during processing. We provide a unified processing-based account for our findings.

The timing of island effects in nonnative sentence processing

Using the eye-movement monitoring technique in two reading comprehension experiments, we investigated the timing of constraints on wh-dependencies (so-called ‘island’ constraints) in native and nonnative sentence processing. Our results show that both native and nonnative speakers of English are sensitive to extraction islands during processing, suggesting that memory storage limitations affect native and nonnative comprehenders in essentially the same way. Furthermore, our results show that the timing of island effects in native compared to nonnative sentence comprehension is affected differently by the type of cue (semantic fit versus filled gaps) signalling whether dependency formation is possible at a potential gap site. Whereas English native speakers showed immediate sensitivity to filled gaps but not to lack of semantic fit, proficient German-speaking learners of L2 English showed the opposite sensitivity pattern. This indicates that initial wh-dependency formation in nonnative processing is based on semantic feature-matching rather than being structurally mediated as in native comprehension.

Processing reflexives in a second language: the timing of structural and discourse-level information

We report the results from two eye-movement monitoring experiments examining the processing of reflexive pronouns by proficient German-speaking learners of second language (L2) English. Our results show that the nonnative speakers initially tried to link English argument reflexives to a discourse-prominent but structurally inaccessible antecedent, thereby violating binding condition A. Our native speaker controls, in contrast, showed evidence of applying condition A immediately during processing. Together, our findings show that L2 learners’ initial focusing on a structurally inaccessible antecedent cannot be due to first language influence and is also independent of whether the inaccessible antecedent c-commands the reflexive. This suggests that unlike native speakers, nonnative speakers of English initially attempt to interpret reflexives through discourse-based coreference assignment rather than syntactic binding.

The role of working memory in the processing of reflexives

We report results from two eye-movement experiments that examined how differences in working memory (WM) capacity affect readers' application of structural constraints on reflexive anaphor resolution during sentence comprehension. We examined whether binding Principle A, a syntactic constraint on the interpretation of reflexives, is reducible to a memory friendly “recency” strategy, and whether WM capacity influences the degree to which readers create anaphoric dependencies ruled out by binding theory. Our results indicate that low and high WM span readers applied Principle A early during processing. However, contrary to previous findings, low span readers also showed immediate intrusion effects of a linearly closer but structurally inaccessible competitor antecedent. We interpret these findings as indicating that although the relative prominence of potential antecedents in WM can affect online anaphor resolution, Principle A is not reducible to a processing or linear distance based “least effort” constraint.

The time-course of morphological processing in a second language

We report findings from psycholinguistic experiments investigating the detailed timing of processing morphologically complex words by proficient adult second (L2) language learners of English in comparison to adult native (L1) speakers of English. The first study employed the masked priming technique to investigate -ed forms with a group of advanced Arabic-speaking learners of English. The results replicate previously found L1/L2 differences in morphological priming, even though in the present experiment an extra temporal delay was offered after the presentation of the prime words. The second study examined the timing of constraints against inflected forms inside derived words in English using the eye-movement monitoring technique and an additional acceptability judgment task with highly advanced Dutch L2 learners of English in comparison to adult L1 English controls. Whilst offline the L2 learners performed native-like, the eye-movement data showed that their online processing was not affected by the morphological constraint against regular plurals inside derived words in the same way as in native speakers. Taken together, these findings indicate that L2 learners are not just slower than native speakers in processing morphologically complex words, but that the L2 comprehension system employs real-time grammatical analysis (in this case, morphological information) less than the L1 system.

The processing of number and gender agreement in Spanish: an event related potential investigation of the effects of structural distance

Previous research suggests that the processing of agreement is affected by the distance between the agreeing elements. However, the unique contribution of structural distance (number of intervening syntactic phrases) to the processing of agreement remains an open question, since previous investigations do not tease apart structural and linear distance (number of intervening words). We used event related potentials (ERPs) to examine the extent to which structural distance impacts the processing of Spanish number and gender agreement. Violations were realized both within the phrase and across the phrase. Across both levels of structural distance, linear distance was kept constant, as was the syntactic category of the agreeing elements. Number and gender agreement violations elicited a robust P600 between 400 and 900ms, a component associated with morphosyntactic processing. No amplitude differences were observed between number and gender violations, suggesting that the two features are processed similarly at the brain level. Within-phrase agreement yielded more positive waveforms than across-phrase agreement, both for agreement violations and for grammatical sentences (no agreement by distance interaction). These effects can be interpreted as evidence that structural distance impacts the establishment of agreement overall, consistent with sentence processing models which predict that hierarchical structure impacts the processing of syntactic dependencies. However, due to the lack of an agreement by distance interaction, the possibility cannot be ruled out that these effects are driven by differences in syntactic predictability between the within-phrase and across-phrase configurations, notably the fact that the syntactic category of the critical word was more predictable in the within-phrase conditions.

Examining second language development using event-related potentials: a cross-sectional study on the processing of gender and number agreement

This cross-sectional study examines the role of L1-L2 differences and structural distance in the processing of gender and number agreement by English-speaking learners of Spanish at three different levels of proficiency. Preliminary results show that differences between the L1 and L2 impact L2 development, as sensitivity to gender agreement violations, as opposed to number agreement violations, emerges only in learners at advanced levels of proficiency. Results also show that the establishment of agreement dependencies is impacted by the structural distance between the agreeing elements for native speakers and for learners at intermediate and advanced levels of proficiency but not for low proficiency. The overall pattern of results suggests that the linguistic factors examined here impact development but do not constrain ultimate attainment; for advanced learners, results suggest that second language processing is qualitatively similar to native processing.

Individual differences and the role of the L1 in L2 processing: an ERP investigation

This study used ERP (event-related potentials) to examine both the role of the L1 and the role of individual differences in the processing of agreement violations. Theories of L2 acquisition differ with regard to whether or not native-like acquisition of L2 features is possible (Schwartz and Sprouse, 1994, 1996; Tsimpli and Mastropavlou, 2007), and the results of previous ERP studies are inconsistent when it comes to whether or not native-like processing is observed in response to L2 agreement violations (e.g., Sabourin, 2003; Tokowicz and MacWhinney, 2005). Furthermore, studies of learners in early stages of L2 acquisition have found variability in the emergence of native-like responses (e.g., McLaughlin et al., 2010; Tanner et al., 2009), but sources of variability have not been investigated. The current study examines responses to gender and number agreement violations in English-speaking learners of Spanish (n=24). Stimuli targeted agreement in three conditions: subject-verb agreement (el barco flota/*flotan), which is similar in Spanish and English; number agreement on adjectival predicates (la isla rocosa/*rocosas), a context in which agreement is not instantiated in English; and gender agreement on adjectival predicates (la isla rocosa/*rocoso), which is unique to Spanish. Grammaticality judgments and ERP responses were also tested for correlations with aptitude scores on the Modern Languages Aptitude Test (MLAT; Carroll and Sapon, 1959) and the Raven Advanced Progressive Matrices (Raven, 1965). Results are in line with theories that claim native-like processing is acquirable, since learners demonstrated similar ERP responses to a control group of native Spanish-speakers (n=8) with regard to all three agreement types. Additionally, the MLAT (but not the Raven) was significantly correlated with sensitivity to number violations, both in terms of grammaticality judgments and ERP amplitudes, indicating a role for verbal but not nonverbal aptitude in L2 processing.

Continuous-flow processing of gaseous ammonia using a Teflon AF-2400 tube-in-tube reactor: synthesis of thioureas and in-line titrations

A simple tube-in-tube reactor based on the gas-permeable membrane Teflon AF-2400 was used in the continuous flow reaction of gaseous ammonia with isothiocyanates and one isocyanate. A colourimetric in-line titration technique is also reported as a simple method to quantify the amount of ammonia taken up by the solvent in the system.

Morphosyntactic processing in advanced second language (L2) learners: An event-related potential investigation of the effects of L1-L2 similarity and structural distance

Different theoretical accounts of second language (L2) acquisition differ with respect to whether or not advanced learners are predicted to show native like processing for features not instantiated in the native language (L1). We examined how native speakers of English, a language with number but not gender agreement, process number and gender agreement in Spanish. We compare agreement within a determiner phrase (órgano muy complejo “[DP organ-MASC-SG very complex-MASC-SG]”) and across a verb phrase (cuadro es auténtico “painting-MASC-SG [VP is authentic-MASC-SG]”) in order to investigate whether native like processing is limited to local domains (e.g. within the phrase), in line with Clahsen and Felser (2006). We also examine whether morphological differences in how the L1 and L2 realize a shared feature impact processing by comparing number agreement between nouns and adjectives, where only Spanish instantiates agreement, and between demonstratives and nouns, where English also instantiates agreement. Similar to Spanish natives, advanced learners showed a P600 for both number and gender violations overall, in line with the Full Transfer/Full Access Hypothesis (Schwartz and Sprouse, 1996), which predicts that learners can show native-like processing for novel features. Results also show that learners can establish syntactic dependencies outside of local domains, as suggested by the presence of a P600 for both within and across phrase violations. Moreover, similar to native speakers, learners were impacted by the structural distance (number of intervening phrases) between the agreeing elements, as suggested by the more positive waveforms for within than across-phrase agreement overall. These results are consistent with the proposal that learners are sensitive to hierarchical structure.