Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent

The epidermal compartment is complex and organized into several strata composed of keratinocytes (KCs), including basal, spinous, granular, and corniWed layers. The continuous process of self-renewal and barrier formation is dependent on a homeostatic balance achieved amongst KCs involving proliferation, diVerentiation, and cell death. To determine genes responsible for initiating and maintaining a corniWed epidermis, organotypic cultures comprised entirely of stratiWed KCs creating epidermal equivalents (EE) were raised from a submerged state to an air/liquid (A/L) interface. Compared to the array proWle of submerged cultures containing KCs predominantly in a proliferative (relatively undiVerentiated) state, EEs raised to an A/L interface displayed a remarkably consistent and distinct proWle of mRNAs. Cultures lifted to an A/L interface triggered the induction of gene groups that regulate proliferation, diVerentiation, and cell death. Next, diVerentially expressed microRNAs (miRNAs) and long noncoding (lncRNA) RNAs were identiWed in EEs. Several diVerentially expressed miRNAs were validated by qRT-PCR and Northern blots. miRNAs 203, 205 and Let-7b were up-regulated at early time points (6, 18 and 24 h) but downregulated by 120 h. To study the lncRNA regulation in EEs, we proWled lncRNA expression by microarray and validated the results by qRT-PCR. Although the diVerential expression of several lncRNAs is suggestive of a role in epidermal diVerentiation, their biological functions remain to be elucidated. The current studies lay the foundation for relevant model systems to address such fundamentally important biological aspects of epidermal structure and function in normal and diseased human skin

Characterization of Novel Paternal ncRNAs at the Plagl1 Locus, Including Hymai, Predicted to Interact with Regulators of Active Chromatin

Genomic imprinting is a complex epigenetic mechanism of transcriptional control that utilizes DNA methylation and histone modifications to bring about parent-of-origin specific monoallelic expression in mammals. Genes subject to imprinting are often organised in clusters associated with large non-coding RNAs (ncRNAs), some of which have cis-regulatory functions. Here we have undertaken a detailed allelic expression analysis of an imprinted domain on mouse proximal chromosome 10 comprising the paternally expressed Plagl1 gene. We identified three novel Plagl1 transcripts, only one of which contains protein-coding exons. In addition, we characterised two unspliced ncRNAs, Hymai, the mouse orthologue of HYMAI, and Plagl1it (Plagl1 intronic transcript), a transcript located in intron 5 of Plagl1. Imprinted expression of these novel ncRNAs requires DNMT3L-mediated maternal DNA methylation, which is also indispensable for establishing the correct chromatin profile at the Plagl1 DMR. Significantly, the two ncRNAs are retained in the nucleus, consistent with a potential regulatory function at the imprinted domain. Analysis with catRAPID, a protein-ncRNA association prediction algorithm, suggests that Hymai and Plagl1it RNAs both have potentially high affinity for Trithorax chromatin regulators. The two ncRNAs could therefore help to protect the paternal allele from DNA methylation by attracting Trithorax proteins that mediate H3 lysine-4 methylation

Reliability of Therapist Effects in Practice-Based Psychotherapy Research : A Guide for the Planning of Future Studies

This paper aims to provide researchers with practical information on sample sizes for accurate estimations of therapist effects (TEs). The investigations are based on an integrated sample of 48,648 patients treated by 1800 therapists. Multilevel modeling and resampling were used to realize varying sample size conditions to generate empirical estimates of TEs. Sample size tables, including varying sample size conditions, were constructed and study examples given. This study gives an insight into the potential size of the TE and provides researchers with a practical guide to aid the planning of future studies in this field

Psychometric properties of two physical activity questionnaires, the AQuAA and the PASE, in cancer patients

Background: This study aimed to evaluate the reliability and validity of two self-report physical activity (PA) questionnaires - the AQuAA (Activity Questionnaire for Adults and Adolescents) and PASE (Physical Activity Scale for the Elderly) - in cancer patients. Methods: Test-retest reliability was determined by administering the questionnaires twice within 5 days. Intraclass correlation coefficient (ICC), standard error of measurement (SEM) and smallest detectable difference (SDD) were calculated. Construct validity was determined by comparing the questionnaire results with ActiGraph accelerometer scores using Spearman correlation coefficients (r(s)) and ICCs. Content validity was examined using the Three-Step Test-Interview (TSTI). Results: Reliability for the AQuAA scores were fair to excellent (ICC = 0.57 to 0.78). Reliability for the PASE scores ranged from good to excellent (ICC = 0.67 to 0.90). Correlations between the ActiGraph and the AQuAA and the PASE were low (r(s) = 0.05 and 0.16 respectively, and ICC = -0.001 to 0.44). The TSTI showed that participants experienced difficulties with the examples provided with the questions, the perceptions of intensity level of PA, and with recalling the time spent on PA. Conclusions: Both questionnaires showed good to excellent test-retest reliability for most scores. Construct validity of both questionnaires was low, as indicated by the low correlations with the ActiGraph. Except for a few difficulties that participants perceived when filling out the questionnaires, the content validity of both questionnaires was goo

Differentiating Protein-Coding and Noncoding RNA: Challenges and Ambiguities

The assumption that RNA can be readily classified into either protein-coding or non-protein–coding categories has pervaded biology for close to 50 years. Until recently, discrimination between these two categories was relatively straightforward: most transcripts were clearly identifiable as protein-coding messenger RNAs (mRNAs), and readily distinguished from the small number of well-characterized non-protein–coding RNAs (ncRNAs), such as transfer, ribosomal, and spliceosomal RNAs. Recent genome-wide studies have revealed the existence of thousands of noncoding transcripts, whose function and significance are unclear. The discovery of this hidden transcriptome and the implicit challenge it presents to our understanding of the expression and regulation of genetic information has made the need to distinguish between mRNAs and ncRNAs both more pressing and more complicated. In this Review, we consider the diverse strategies employed to discriminate between protein-coding and noncoding transcripts and the fundamental difficulties that are inherent in what may superficially appear to be a simple problem. Misannotations can also run in both directions: some ncRNAs may actually encode peptides, and some of those currently thought to do so may not. Moreover, recent studies have shown that some RNAs can function both as mRNAs and intrinsically as functional ncRNAs, which may be a relatively widespread phenomenon. We conclude that it is difficult to annotate an RNA unequivocally as protein-coding or noncoding, with overlapping protein-coding and noncoding transcripts further confounding this distinction. In addition, the finding that some transcripts can function both intrinsically at the RNA level and to encode proteins suggests a false dichotomy between mRNAs and ncRNAs. Therefore, the functionality of any transcript at the RNA level should not be discounted

Genomic and Transcriptional Co-Localization of Protein-Coding and Long Non-Coding RNA Pairs in the Developing Brain

Besides protein-coding mRNAs, eukaryotic transcriptomes include many long non-protein-coding RNAs (ncRNAs) of unknown function that are transcribed away from protein-coding loci. Here, we have identified 659 intergenic long ncRNAs whose genomic sequences individually exhibit evolutionary constraint, a hallmark of functionality. Of this set, those expressed in the brain are more frequently conserved and are significantly enriched with predicted RNA secondary structures. Furthermore, brain-expressed long ncRNAs are preferentially located adjacent to protein-coding genes that are (1) also expressed in the brain and (2) involved in transcriptional regulation or in nervous system development. This led us to the hypothesis that spatiotemporal co-expression of ncRNAs and nearby protein-coding genes represents a general phenomenon, a prediction that was confirmed subsequently by in situ hybridisation in developing and adult mouse brain. We provide the full set of constrained long ncRNAs as an important experimental resource and present, for the first time, substantive and predictive criteria for prioritising long ncRNA and mRNA transcript pairs when investigating their biological functions and contributions to development and disease

The Genetic Signatures of Noncoding RNAs

The majority of the genome in animals and plants is transcribed in a developmentally regulated manner to produce large numbers of non–protein-coding RNAs (ncRNAs), whose incidence increases with developmental complexity. There is growing evidence that these transcripts are functional, particularly in the regulation of epigenetic processes, leading to the suggestion that they compose a hitherto hidden layer of genomic programming in humans and other complex organisms. However, to date, very few have been identified in genetic screens. Here I show that this is explicable by an historic emphasis, both phenotypically and technically, on mutations in protein-coding sequences, and by presumptions about the nature of regulatory mutations. Most variations in regulatory sequences produce relatively subtle phenotypic changes, in contrast to mutations in protein-coding sequences that frequently cause catastrophic component failure. Until recently, most mapping projects have focused on protein-coding sequences, and the limited number of identified regulatory mutations have been interpreted as affecting conventional cis-acting promoter and enhancer elements, although these regions are often themselves transcribed. Moreover, ncRNA-directed regulatory circuits underpin most, if not all, complex genetic phenomena in eukaryotes, including RNA interference-related processes such as transcriptional and post-transcriptional gene silencing, position effect variegation, hybrid dysgenesis, chromosome dosage compensation, parental imprinting and allelic exclusion, paramutation, and possibly transvection and transinduction. The next frontier is the identification and functional characterization of the myriad sequence variations that influence quantitative traits, disease susceptibility, and other complex characteristics, which are being shown by genome-wide association studies to lie mostly in noncoding, presumably regulatory, regions. There is every possibility that many of these variations will alter the interactions between regulatory RNAs and their targets, a prospect that should be borne in mind in future functional analyses

Banks’ liquidity buffers and the role of liquidity regulation

We assess the determinants of banks’ liquidity holdings using data for nearly 7000 banks from 25 OECD countries. We highlight the role of several bank-specific, institutional and policy variables in shaping banks’ liquidity risk management. Our main question is whether liquidity regulation neutralizes banks’ incentives to hold liquid assets. Without liquidity regulation, the determinants of banks’ liquidity buffers are a combination of bank-specific and country-specific variables. While most incentives are neutralized by liquidity regulation, a bank’s disclosure requirements remain important. The complementarity of disclosure and liquidity requirements provides a strong rationale for considering them jointly in the design of regulation