Can adults learn L2 grammar after prolonged exposure under incidental conditions?

While late second language (L2) learning is assumed to be largely explicit, there is evidence that adults are able to acquire grammar under incidental exposure conditions, and that the acquisition of this knowledge may be implicit in nature. Here, we revisit the question of whether adults can learn grammar incidentally and investigate whether word order and morphology are susceptible to incidental learning to the same degree. In experiment 1, adult English monolinguals were exposed to an artificial language (Kepidalo) that had case marking and variable word order: a canonical Subject-Object-Verb order and a non-canonical Object-Subject-Verb. In a five-session online study, participants received vocabulary training while being incidentally exposed to grammar, and completed a series of picture-selection and grammaticality judgment tasks assessing grammatical knowledge. Despite extensive exposure to input, and although performance on vocabulary increased significantly across sessions, learners' grammatical comprehension showed little improvement over time, and this was limited to Subject-Object-Verb sentences only. Furthermore, participants were better at detecting word order than case marking violations in the grammaticality judgment tasks. Experiment 2 further increased the amount of incidental exposure whilst examining native speakers of German, which exhibits higher morphological richness. Testing was followed by a post-test metalinguistic awareness questionnaire. Although greater learning effects were observed, participants continued to have difficulties with case marking. The findings also demonstrated that language outcomes were modulated by learners' level of metalinguistic awareness. Taken together, the results of the two experiments underscore adult learners' difficulty with case marking and point towards the presence of a threshold in incidental L2 grammar learning, which appears to be tightly linked to prior first language experience. In addition, our findings continue to highlight the facilitative role of conscious awareness on L2 outcomes.

Long-term memory predictors of adult language learning at the interface between syntactic form and meaning.

Recent neurocognitive models of second language learning have posited specific roles for declarative and procedural memory in the processing of novel linguistic stimuli. Pursuing this line of investigation, the present exploratory study examined the role of declarative and procedural memory abilities in the early stages of adult comprehension of sentences in a miniature language with natural language characteristics (BrocantoJ). Thirty-six native Italian young adults were aurally exposed to BrocantoJ in the context of a computer game over three sessions on consecutive days. Following vocabulary training and passive exposure, participants were asked to perform game moves described by aural sentences in the language. Game trials differed with respect to the information the visual context offered. In part of the trials processing of relationships between grammatical properties of the language (word order and morphological case marking) and noun semantics (thematic role) was necessary in order reach an accurate outcome, whereas in others nongrammatical contextual cues were sufficient. Declarative and procedural learning abilities were respectively indexed by visual and verbal declarative memory measures and by a measure of visual implicit sequence learning. Overall, the results indicated a substantial role of declarative learning ability in the early stages of sentence comprehension, thus confirming theoretical predictions and the findings of previous similar studies in miniature artificial language paradigms. However, for trials that specifically probed the learning of relationships between morphosyntax and semantics, a positive interaction between declarative and procedural learning ability also emerged, indicating the cooperative engagement of both types of learning abilities in the processing of relationships between ruled-based grammar and interpretation in the early stages of exposure to a new language in adults.

CDK inhibitors reduce cell proliferation and reverse hypoxia-induced metastasis of neuroblastoma tumours in a chick embryo model.

Neuroblastoma is a paediatric cancer with a poor prognosis. This is in part due to widespread metastasis at time of presentation, which is refractory to current treatment modalities. New therapeutic agents that can control not only tumour growth but also metastasis are urgently needed. The differentiation therapy, retinoic acid, is currently used in clinic, leading to terminal differentiation of neuroblastoma cells thus reducing tumour growth in the primary tumour as well as at metastatic sites. However, retinoic acid only works in a subset of patients. We investigated the potential of CDK inhibitors, Palbociclib and RO-3306, on neuroblastoma cell differentiation, tumour progression and metastasis by utilising a 3R compliant cost effective preclinical chick embryo model. In both SK-N-AS and BE(2)C cell lines, when engrafted on the chorioallantoic membrane of chick embryos, we observed a reduction of tumour cell proliferation as well as a reduction in hypoxia preconditioning-driven metastasis by 60%. In addition, the expression of a panel of genes with known roles in metastasis, which increased upon hypoxia-preconditioning, was largely reduced by a CDK1 inhibitor. These results provide a promising alternative to currently existing therapies and might aid the development of new treatment protocols for retinoic acid-resistant patients.

Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery.

BACKGROUND: Neuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours. Retinoic acid (RA) promotes differentiation of some neuroblastoma tumours and cell lines, and is successfully used clinically, supporting the view that differentiation therapy is a promising strategy for treatment of neuroblastoma. To improve treatment of a wider range of tumour types, development and testing of novel differentiation agents is essential. New pre-clinical models are therefore required to test therapies in a rapid cost effective way in order to identify the most useful agents. METHODS: As a proof of principle, differentiation upon ATRA treatment of two MYCN-amplified neuroblastoma cell lines, IMR32 and BE2C, was measured both in cell cultures and in tumours formed on the chick chorioallantoic membrane (CAM). Differentiation was assessed by 1) change in cell morphology, 2) reduction in cell proliferation using Ki67 staining and 3) changes in differentiation markers (STMN4 and ROBO2) and stem cell marker (KLF4). Results were compared to MLN8237, a classical Aurora Kinase A inhibitor. For the in vivo experiments, cells were implanted on the CAM at embryonic day 7 (E7), ATRA treatment was between E11 and E13 and tumours were analysed at E14. RESULTS: Treatment of IMR32 and BE2C cells in vitro with 10 µM ATRA resulted in a change in cell morphology, a 65% decrease in cell proliferation, upregulation of STMN4 and ROBO2 and downregulation of KLF4. ATRA proved more effective than MLN8237 in these assays. In vivo, 100 µM ATRA repetitive treatment at E11, E12 and E13 promoted a change in expression of differentiation markers and reduced proliferation by 43% (p < 0.05). 40 µM ATRA treatment at E11 and E13 reduced proliferation by 37% (p < 0.05) and also changed cell morphology within the tumour. CONCLUSION: Differentiation of neuroblastoma tumours formed on the chick CAM can be analysed by changes in cell morphology, proliferation and gene expression. The well-described effects of ATRA on neuroblastoma differentiation were recapitulated within 3 days in the chick embryo model, which therefore offers a rapid, cost effective model compliant with the 3Rs to select promising drugs for further preclinical analysis.

Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide.

OBJECTIVE: Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models. METHODS: Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model. RESULTS: Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data. CONCLUSIONS: We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

The Chorioallantoic Membrane of the Chick Embryo to Assess Tumor Formation and Metastasis.

The chorioallantoic membrane (CAM) of the chick embryo is a suitable and convenient platform for the assessment of tumor formation and metastatic dissemination. Here, we describe tumor cell engraftment on the extraembryonic CAM and further monitoring of tumor growth and metastasis.

Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity.

Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo.

Evaluating the effectiveness of transferrin receptor-1 (TfR1) as a magnetic resonance reporter gene.

Magnetic resonance (MR) reporter genes have the potential for tracking the biodistribution and fate of cells in vivo, thus allowing the safety, efficacy and mechanisms of action of cell-based therapies to be comprehensively assessed. In this study, we evaluate the effectiveness of the iron importer transferrin receptor-1 (TfR1) as an MR reporter gene in the model cell line CHO-K1. Overexpression of the TfR1 transgene led to a reduction in the levels of endogenous TfR1 mRNA, but to a 60-fold increase in total TfR1 protein levels. Although the mRNA levels of ferritin heavy chain-1 (Fth1) did not change, Fth1 protein levels increased 13-fold. The concentration of intracellular iron increased significantly, even when cells were cultured in medium that was not supplemented with iron and the amount of iron in the extracellular environment was thus at physiological levels. However, we found that, by supplementing the cell culture medium with ferric citrate, a comparable degree of iron uptake and MR contrast could be achieved in control cells that did not express the TfR1 transgene. Sufficient MR contrast to enable the cells to be detected in vivo following their administration into the midbrain of chick embryos was obtained irrespective of the reporter gene. We conclude that TfR1 is not an effective reporter and that, to track the biodistribution of cells with MR imaging in the short term, it is sufficient to simply culture cells in the presence of ferric citrate. Copyright © 2016 The Authors Contrast Media & Molecular Imaging Published by John Wiley & Sons Ltd.

Overexpression of the MRI Reporter Genes Ferritin and Transferrin Receptor Affect Iron Homeostasis and Produce Limited Contrast in Mesenchymal Stem Cells.

Imaging technologies that allow the non-invasive monitoring of stem cells in vivo play a vital role in cell-based regenerative therapies. Recently, much interest has been generated in reporter genes that enable simultaneous monitoring of the anatomical location and viability of cells using magnetic resonance imaging (MRI). Here, we investigate the efficacy of ferritin heavy chain-1 (Fth1) and transferrin receptor-1 (TfR1) as reporters for tracking mesenchymal stem cells. The overexpression of TfR1 was well tolerated by the cells but Fth1 was found to affect the cell's iron homeostasis, leading to phenotypic changes in the absence of iron supplementation and an upregulation in transcript and protein levels of the cell's endogenous transferrin receptor. Neither the sole overexpression of Fth1 nor TfR1 resulted in significant increases in intracellular iron content, although significant differences were seen when the two reporter genes were used in combination, in the presence of high concentrations of iron. The supplementation of the culture medium with iron sources was a more efficient means to obtain contrast than the use of reporter genes, where high levels of intracellular iron were reflected in transverse (T2) relaxation. The feasibility of imaging iron-supplemented cells by MRI is shown using a 3R-compliant chick embryo model.

Characterisation of multiple regulatory domains spanning the major transcriptional start site of the FUS gene, a candidate gene for motor neurone disease.

Fused-In-Sarcoma (FUS) is a candidate gene for neurological disorders including motor neurone disease and Parkinson׳s disease in addition to various types of cancer. Recently it has been reported that over expression of FUS causes motor neurone disease in mouse models hence mutations leading to changes in gene expression may contribute to the development of neurodegenerative disease. Genome evolutionary conservation was used to predict important cis-acting DNA regulators of the FUS gene promoter that direct transcription. The putative regulators identified were analysed in reporter gene assays in cells and in chick embryos. Our analysis indicated in addition to regulatory domains 5' of the transcriptional start site an important regulatory domain resides in intron 1 of the gene itself. This intronic domain functioned both in cell lines and in vivo in the neural tube of the chick embryo including developing motor neurones. Our data suggest the interaction of multiple domains including intronic domains are involved in expression of FUS. A better understanding of the regulation of expression of FUS may give insight into how its stimulus inducible expression may be associated with neurological disorders.

Assessing the efficacy of nano- and micro-sized magnetic particles as contrast agents for MRI cell tracking.

Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of labelled cells in vivo. Currently, a wide range of such contrast agents is available with sizes varying from several nanometers up to a few micrometers and consisting of single or multiple magnetic cores. Here, we evaluate the effectiveness of these different particles for labelling and imaging stem cells, using a mouse mesenchymal stem cell line to investigate intracellular uptake, retention and processing of nano- and microsized contrast agents. The effect of intracellular confinement on transverse relaxivity was measured by MRI at 7 T and in compliance with the principles of the '3Rs', the suitability of the contrast agents for MR-based cell tracking in vivo was tested using a chick embryo model. We show that for all particles tested, relaxivity was markedly reduced following cellular internalisation, indicating that contrast agent relaxivity in colloidal suspension does not accurately predict performance in MR-based cell tracking studies. Using a bimodal imaging approach comprising fluorescence and MRI, we demonstrate that labelled MSC remain viable following in vivo transplantation and can be tracked effectively using MRI. Importantly, our data suggest that larger particles might confer advantages for longer-term imaging.

An evaluation of a SVA retrotransposon in the FUS promoter as a transcriptional regulator and its association to ALS.

Genetic mutations of FUS have been linked to many diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration. A primate specific and polymorphic retrotransposon of the SINE-VNTR-Alu (SVA) family is present upstream of the FUS gene. Here we have demonstrated that this retrotransposon can act as a classical transcriptional regulatory domain in the context of a reporter gene construct both in vitro in the human SK-N-AS neuroblastoma cell line and in vivo in a chick embryo model. We have also demonstrated that the SVA is composed of multiple distinct regulatory domains, one of which is a variable number tandem repeat (VNTR). The ability of the SVA and its component parts to direct reporter gene expression supported a hypothesis that this region could direct differential FUS expression in vivo. The SVA may therefore contribute to the modulation of FUS expression exhibited in and associated with neurological disorders including ALS where FUS regulation may be an important parameter in progression of the disease. As VNTRs are often clinical associates for disease progression we determined the extent of polymorphism within the SVA. In total 2 variants of the SVA were identified based within a central VNTR. Preliminary analysis addressed the association of these SVA variants within a small sporadic ALS cohort but did not reach statistical significance, although we did not include other parameters such as SNPs within the SVA or an environmental factor in this analysis. The latter may be particularly important as the transcriptional and epigenetic properties of the SVA are likely to be directed by the environment of the cell.

IgLONs form heterodimeric complexes on forebrain neurons.

IgLONs are a family of four GPI-anchored cell adhesion molecules that regulate neurite outgrowth and synaptogenesis and may act as tumour suppressor genes. Recently we have proposed that two members of the IgLON family act as a heterodimeric complex termed DIgLON. Neurons isolated from chick forebrain co-express all six combinations of IgLONs and the intensity of fluorescence for each pair of IgLONs was highly correlated. Antibody-patching experiments on forebrain neurons show complex formation for IgLON pairs but not between unrelated GPI-anchored glycoproteins. Thus IgLONs are the first GPI-anchored family of glycoproteins shown to form heterodimeric complexes in the plane of the membrane.

DIgLONs inhibit initiation of neurite outgrowth from forebrain neurons via an IgLON-containing receptor complex.

IgLONs are a family of four GPI-anchored cell adhesion molecules that regulate neurite outgrowth, synaptogenesis and may act as tumour suppressor genes. IgLONs are thought to function as monomers or homodimers and we have proposed that IgLONs also act as heterodimeric complexes termed Dimeric IgLONs or DIgLONs. Here we show that the initiation of neurite outgrowth is inhibited from a subset of chick embryonic day (E) 7 or 8 forebrain neurons when they are cultured on CHO cell lines expressing DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP but not on CHO cells that express single IgLONs CEPU-1 or OBCAM. Surprisingly at the younger age of E6 forebrain neurons do not respond to DIgLONs. Since there is little difference in expression of IgLONs on the surface of chick forebrain neurons at these two ages we suggest IgLONs alone are not the receptor on the responding forebrain neurons. A DIgLON heterodimeric recombinant protein DIgLON:CEPU-1-OBCAM-Fc also blocked neurite outgrowth from E8 chick forebrain neurons. However, when IgLONs were removed from the surface of these E8 neurons they no longer responded to DIgLON:CEPU-1-OBCAM-Fc substrate, indicating that IgLONs form at least a component of the neuronal cell receptor complex involved in this inhibition of neurite outgrowth. Inhibitors pertussis toxin and Y27632 reversed the inhibition of neurite outgrowth on a DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP substrate. This suggests the involvement of a G-protein coupled receptor and activation of Rho A. In summary we provide evidence that DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP complexes regulate initiation of neurite outgrowth on forebrain neurons via an IgLON-containing receptor complex.

Diglons are heterodimeric proteins composed of IgLON subunits, and Diglon-CO inhibits neurite outgrowth from cerebellar granule cells.

IgLONs are a family of four cell adhesion molecules belonging to the Ig superfamily that are thought to play a role in cell-cell recognition and growth-cone migration. One member of the family, opioid-binding cell-adhesion molecule (OBCAM), might act as a tumour suppressor. Previous work has shown that limbic-system-associated protein (LAMP), CEPU-1/Neurotrimin and OBCAM interact homophilically and heterophilically within the family. Here, we show that, based on their relative affinities, CEPU-1 might be both a homo- and a heterophilic cell adhesion molecule, whereas LAMP and OBCAM act only as heterophilic cell adhesion molecules. A binding assay using recombinant IgLONs fused to human Fc showed that IgLONs are organized in the plane of the membrane as heterodimers, and we propose that IgLONs function predominantly as subunits of heterodimeric proteins (Diglons). Thus, the four IgLONs can form six Diglons. Furthermore, although singly transfected cell lines have little effect on neurite outgrowth, CHO cell lines expressing both CEPU-1 and OBCAM (Diglon-CO) inhibit neurite outgrowth from cerebellar granule cells.

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth.

The IgLONs are a family of glycosyl phosphatidyl inositol-linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell-cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell-cell adhesion and recognition.