Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms

Self-productivity and complementarities in human development : evidence from MARS

This paper investigates the role of self-productivity and home resources in capability formation from infancy to adolescence. In addition, we study the complementarities between basic cognitive, motor and noncognitive abilities and social as well as academic achievement. Our data are taken from the Mannheim Study of Children at Risk (MARS), an epidemiological cohort study following the long-term outcome of early risk factors. Results indicate that initial risk conditions cumulate and that differences in basic abilities increase during development. Self-productivity rises in the developmental process and complementarities are evident. Noncognitive abilities promote cognitive abilities and social achievement. There is remarkable stability in the distribution of the economic and socio-emotional home resources during the early life cycle. This is presumably a major reason for the evolution of inequality in human development

Convergent genetic linkage and associations to language, speech and reading measures in families of probands with Specific Language Impairment

We analyzed genetic linkage and association of measures of language, speech and reading phenotypes to candidate regions in a single set of families ascertained for SLI. Sib-pair and family-based analyses were carried out for candidate gene loci for Reading Disability (RD) on chromosomes 1p36, 3p12-q13, 6p22, and 15q21, and the speech-language candidate region on 7q31 in a sample of 322 participants ascertained for Specific Language Impairment (SLI). Replication or suggestive replication of linkage was obtained in all of these regions, but the evidence suggests that the genetic influences may not be identical for the three domains. In particular, linkage analysis replicated the influence of genes on chromosome 6p for all three domains, but association analysis indicated that only one of the candidate genes for reading disability, KIAA0319, had a strong effect on language phenotypes. The findings are consistent with a multiple gene model of the comorbidity between language impairments and reading disability and have implications for neurocognitive developmental models and maturational processes

Cerebellar-dependent delay eyeblink conditioning in adolescents with Specific Language Impairment

Cerebellar impairments have been hypothesized as part of the pathogenesis of Specific Language Impairment (SLI), although direct evidence of cerebellar involvement is sparse. Eyeblink Conditioning (EBC) is a learning task with well documented cerebellar pathways. This is the first study of EBC in affected adolescents and controls. 16 adolescent controls, 15 adolescents with SLI, and 12 adult controls participated in a delay EBC task. Affected children had low general language performance, grammatical deficits but no speech impairments. The affected group did not differ from the control adolescent or control adult group, showing intact cerebellar functioning on the EBC task. This study did not support cerebellar impairment at the level of basic learning pathways as part of the pathogenesis of SLI. Outcomes do not rule out cerebellar influences on speech impairment, or possible other forms of cerebellar functioning as contributing to SLI

Bishydrocotarnines - Stereochemical Aspects

The bishydrocotarnines 2a and 2b1)' **) were converted into the urethanes 9a and 9b and into the carbamates 10a and 10b, which in turn were split to yield the sec. amines 11a and 11b. Cyclisation with diethyl oxalate led to the diketopiperazines 12a and 12b. Contrary to 9b, compound 9a was resolved into enantiomers on a cellulose carbamate column. This indicates that 9a is the D, L- and 9b is the meso form. NMR spectra of 12a and 12b led to an analogous conclusion. Die Bishydrocotarnine 2a und 2b1) werden in die Urethane 9a und 9b bzw. in die Carbamate 10a und 10b umgewandelt. Spaltung von 10a bzw. 10b in die sek. Amine 11a und 11b und deren Cyclisierung führen zu den Diketopiperazinen 12a und 12b. 9a ließ sich im Gegensatz zu 9b an einer Cellulosecarbamat-Säule in Enantiomere spalten. Danach ist 9a die D, L-, 9b die meso-Form. NMR-Spektren von 12a und 12b führen zu derselben Schlußfolgerung