Search

Murine embryonic stem (ES) cells can be differentiated into functional neurons and glia in culture. This system has been shown to provide a valuable resource for gene discovery and expression pattern analysis. We have isolated 604 nonredundant clones from lan early ES neural differentiation subtraction library. Of these, 279 were novel or uncharacterized and 325 were known. The sequences have been submitted to the GenBank EST database (Accession Nos. AW244216-AW244819). To manage and analyze these sequences, we have developed a database (linked to the Rancourt lab web page: http://www.ucalgary.ca/~rancourt/) using the open integrated software package MYSQL. One EST (BR_END07F05; Accession No. AW244659.1) has been selected for follow-up based on bio-informatic analyses suggesting that it may encode a zinc finger transcription factor. Using Clontech multiple tissue Northern (MTNr) blot membranes, it has been demonstrated that a 2.3-kb transcript of the gene is expressed during mouse embryogenesis with a peak of expression at embryonic day 11. Expression is also observed using Northern blotting in adult tissues including brain and heart. RNase protection assays reveal similar patterns of expression during embryonic development and in adult tissues. Preliminary in situ hybridization studies show staining in the nervous system, limb buds, and craniofacial regions in 11- and 12.5-day-old mouse embryos. Bioinformatic analysis maps the human homologue to 19p13.3, a gene rich region to which a number of developmental/neurological disorders have been mapped. A knockout targeting vector is currently under construction, making use of a novel combination of bioinformatics and retrorecombination to streamline vector construction.

Prior utilization of a subtractive hybridization strategy has allowed us to isolate 604 nonredundant ESTs from ES cells undergoing neural differentiation. Of these, 279 were novel or uncharacterized, and 325 matched previously characterized genes, a high proportion of which were developmentally and/or neurally expressed. Novel tools, such as the use of microarrays and/or gene chips, promise to yield valuable information with regard to gene expression patterns in systems such as ours. In collaboration with the Ontario Cancer Institute (OCI), our ESTs were therefore arrayed on glass slides. Arrays were subsequently probed with Cy3-and Cy5-1abeled RNAs from various ES cell and neural stem cell sources. Using QuantArray(GSI Lumonics) software, scatter plots were generated, and tabulated data were exported to Microsoft Excel for subsequent analysis. After appropriate normalization and background subtraction, significantly up- or down-regulated clones from the arrays were selected for further analysis. We have also expanded the number of genes we are studying utilizing the Research Genetics GF400 mouse array. RNAs from various ES cell and neural stem cell systems have been studied using these arrays in conjunction with Pathways 3 software (Research Genetics). Several clones resulting from these screens have been investigated further using a combination of bench (RNase protection assay, Northern blot, in situ hybridization) and bioinformatic analyses (UniGene clustering, ORF finding, functional domain/motif identification). Clones of interest, in particular transcription factors containing Zinc finger domains, have been selected for further research.

Background: Current literature reports a gap between development of effective interventions to promote physical activity and the systematic uptake into real-world settings. Factors relating to implementation and scale-up of physical activity interventions have been examined, however the perspectives of multiple stakeholders from different domains are not well researched. The purpose of this study was to examine the perceived factors related to physical activity intervention implementation and scale-up in different domains from different stakeholders on the island of Ireland., Methods: Practitioners, researchers, funders and policy makers in Ireland were invited to take part in a semi-structured interview exploring factors related to the implementation and scale-up of eleven different physical activity interventions. A thematic analysis was conducted to identify factors related to the implementation and scale-up of the included interventions. The data collection and analysis were guided by the Consolidated Framework for Implementation Research., Results: Thirty-eight participants took part in the interviews which identified factors related to 1) intervention planning and practical considerations; 2) organisational structures, staffing and resources related to delivery; 3) reflection, evaluation and updating of the intervention; and 4) practical consideration related to scale-up. Furthermore, participants referred to the ongoing commitment, engagement, and support needed throughout the implementation process., Conclusions: Future research and practice needs to consider how different factors are experienced at different implementation stages and by the different stakeholder groups involved. The findings highlight multiple inter-related factors that influence the implementation and scale-up of physical activity interventions, but also identifies many strategies that can be utilised to aid future successes., (© 2023. The Author(s).)

Introduction: Physical activity among children and adolescents remains insufficient, despite the substantial efforts made by researchers and policymakers. Identifying and furthering our understanding of potential modifiable determinants of physical activity behaviour (PAB) and sedentary behaviour (SB) is crucial for the development of interventions that promote a shift from SB to PAB. The current protocol details the process through which a series of systematic literature reviews and meta-analyses (MAs) will be conducted to produce a best-evidence statement (BESt) and inform policymakers. The overall aim is to identify modifiable determinants that are associated with changes in PAB and SB in children and adolescents (aged 5-19 years) and to quantify their effect on, or association with, PAB/SB., Methods and Analysis: A search will be performed in MEDLINE, SportDiscus, Web of Science, PsychINFO and Cochrane Central Register of Controlled Trials. Randomised controlled trials (RCTs) and controlled trials (CTs) that investigate the effect of interventions on PAB/SB and longitudinal studies that investigate the associations between modifiable determinants and PAB/SB at multiple time points will be sought. Risk of bias assessments will be performed using adapted versions of Cochrane's RoB V.2.0 and ROBINS-I tools for RCTs and CTs, respectively, and an adapted version of the National Institute of Health's tool for longitudinal studies. Data will be synthesised narratively and, where possible, MAs will be performed using frequentist and Bayesian statistics. Modifiable determinants will be discussed considering the settings in which they were investigated and the PAB/SB measurement methods used., Ethics and Dissemination: No ethical approval is needed as no primary data will be collected. The findings will be disseminated in peer-reviewed publications and academic conferences where possible. The BESt will also be shared with policy makers within the DE-PASS consortium in the first instance., Systematic Review Registration: CRD42021282874., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Physical activity (PA) promotion is a complex challenge, with the Global Action Plan on Physical Activity (GAPPA) endorsing a systems approach and recommending countries assess existing areas of progress which can be strengthened. This paper reports a process facilitating a systems approach for identifying current good practice and gaps for promoting PA in Ireland. Elements of participatory action research were enabled through 3 stages: (1) aligning examples of actions from Irish policy documents (n = 3) to the GAPPA, (2) workshop with stakeholders across multiple sectors, and (3) review of outputs. Data collected through the workshop were analyzed using a deductive thematic analysis guided by the GAPPA. The policy context in Ireland aligns closely to the GAPPA with the creation of Active Systems the most common strategic objective across policy documents. Forty participants (50% male) took part in the systems approach workshop, which after revision resulted in 80 examples of good practice and 121 actions for greater impact. A pragmatic and replicable process facilitating a systems approach was adopted and showed current Irish policy and practices align with the GAPPA "good practices." The process provides existing areas of progress which can be strengthened, as well as the policy opportunities and practice gaps.

Background: Public policy is increasingly recognized as an important component of physical activity promotion. This paper reports on the current status of physical activity policy development and implementation in four European countries based on the Health-Enhancing Physical Activity Policy Audit Tool (HEPA PAT) developed by WHO. It compares the findings to previous studies and discusses the general utility of this tool and its unique features in relation to other instruments., Methods: The study was conducted as part of the Policy Evaluation Network ( www.jpi-pen.eu ) in Germany, Ireland, the Netherlands and Poland. Data collection built upon information obtained via the EU Physical Activity Monitoring Framework survey, additional desk research and expert opinion. Data analysis employed Howlett's policy cycle framework to map and compare national physical activity policies in the four countries., Results: In all countries under study, policy agenda-setting is influenced by prevalence data from national health monitoring systems, and the sport and/or health sector takes the lead in policy formulation. Key policy documents were located mainly in the health sector but also in sport, urban design and transport. Physical activity programmes implemented to meet policy objectives usually cover a broad range of target groups, but currently only a small selection of major policies are evaluated for effectiveness. National experts made several suggestions to other countries wishing to establish physical activity policies, e.g. regarding cross-sectoral support and coordination, comprehensive national action plans, and monitoring/surveillance., Conclusions: This study provides a detailed overview of physical activity policies in the four countries. Results show that national governments are already very active in the field but that there is room for improvement in a number of areas, e.g. regarding the contribution of sectors beyond sport and health. Using the HEPA PAT simultaneously in four countries also showed that procedures and timelines have to be adapted to national contexts. Overall, the instrument can make an important contribution to understanding and informing physical activity policy, especially when used as an add-on to regular monitoring tools like the EU HEPA Monitoring Framework.

In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08-0.85 MPa) and times (5-60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine., (Copyright © 2011 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Retinal neural transmission represents a key function of the eye. Identifying the molecular components of this vital process is helped by studies of selected human genetic eye disorders. For example, mutations in the calcium channel subunit gene CACNA1F cause incomplete X-linked congenital stationary night blindness (CSNB2 or iCSNB), a human retinal disorder with abnormal electrophysiological response and visual impairments consistent with a retinal neurotransmission defect. To understand the subcellular basis of this retinal disorder, we generated a mouse with a loss-of-function mutation by inserting a self-excising Cre-lox-neo cassette into exon 7 of the murine orthologue, Cacna1f. Electroretinography of the mutant mouse revealed a scotopic a-wave of marginally reduced amplitude compared with the wild-type mouse and absence of the post-receptoral b-wave and oscillatory potentials. Cone ERG responses together with visual evoked potentials and multi-unit activity in the superior colliculus were also absent. Calcium imaging in Fluo-4 loaded retinal slices depolarized with KCl showed 90% less peak signal in the photoreceptor synapses of the Cacna1f mutant than in wild-type mice. The absence of post-receptoral ERG responses and the diminished photoreceptor calcium signals are consistent with a loss of Ca((2+)) channel function in photoreceptors. Immunocytochemistry showed no detectable Ca(v)1.4 protein in the outer plexiform layer of Cacna1f-mutant mice, profound loss of photoreceptor synapses, and abnormal dendritic sprouting of second-order neurons in the photoreceptor layer. Together, these findings in the Cacna1f-mutant mouse reveal that the Ca(v)1.4 calcium channel is vital for the functional assembly and/or maintenance and synaptic functions of photoreceptor ribbon synapses. Moreover, the outcome of this study provides critical clues to the pathophysiology of the human retinal channelopathy of X-linked incomplete CSNB.

We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease.

Neurodegenerative diseases cost the economies of the developed world billions of dollars per annum. Given ageing population profiles and the increasing extent of this problem, there has been a surge of interest in neural stem cells and in neural differentiation protocols that yield neural cells for therapeutic transplantation. Due to the oncogenic potential of stem cells a better characterisation of neural differentiation, including the identification of new neurotrophic factors, is required. Stem cell cultures undergoing synchronous in vitro neural differentiation provide a valuable resource for gene discovery. Novel tools such as microarrays promise to yield information regarding gene expression in stem cells. With the completion of the yeast, C. elegans, Drosophila, human, and mouse genome projects, the functional characterisation of genes using genetic and bioinformatic tools will aid in the identification of important regulators of neural differentiation.

Family ZMK is a large Irish kindred that segregates progressive sensorineural hearing loss and retinitis pigmentosa. The symptoms in the family are almost identical to those observed in Usher syndrome type III. Unlike that in Usher syndrome type III, the inheritance pattern in this family is compatible with dominant, X-linked dominant, or maternal inheritance. Prior linkage studies had resulted in exclusion of most candidate loci and >90% of the genome. A tentative location for a causative nuclear gene had been established on 9q; however, it is notable that no markers were found at zero recombination with respect to the disease gene. The marked variability in symptoms, together with the observation of subclinical muscle abnormalities in a single muscle biopsy, stimulated sequencing of the entire mtDNA in affected and unaffected individuals. This revealed a number of previously reported polymorphisms and/or silent substitutions. However, a C-->A transversion at position 12258 in the gene encoding the second mitochondrial serine tRNA, MTTS2, was heteroplasmic and was found in family members only. This sequence change was not present in 270 normal individuals from the same ethnic background. The consensus C at this position is highly conserved and is present in species as divergent from Homo sapiens as vulture and platypus. The mutation probably disrupts the amino acid-acceptor stem of the tRNA molecule, affecting aminoacylation of the tRNA and thereby reducing the efficiency and accuracy of mitochondrial translation. In summary, the data presented provide substantial evidence that the C12258A mtDNA mutation is causative of the disease phenotype in family ZMK.

A gene for juvenile onset, open angle glaucoma (JOAG) has been localized to chromosome 1q21-31 in several families. Mutations in the trabecular meshwork-induced glucocorticoid response protein (TIGR) gene, which maps to this region, recently have been found in families segregating both JOAG and a later onset form of primary open angle glaucoma (POAG). We have analysed the TIGR gene in two families; one Spanish family segregating autosomal dominant JOAG and an Irish family with a later onset form of autosomal dominant POAG. We have found a G-T transversion in the first base of codon 426 in all affected members of the Spanish family, which results in a valine to phenylalanine amino acid substitution. We have also found a G-A transition at the first base of codon 367 that segregates through all but one branch of the Irish family and results in a glycine to arginine amino acid substitution. Members of this family that carry the Gly367Arg change also share a common haplotype that is neither present in any of the unaffected members of the family, nor in the branch that does not segregate the mutation. Identification of further mutations in the TIGR gene increases its importance in the etiology of open angle glaucoma.

A major difficulty associated with the design of gene therapies for autosomal dominant diseases is the immense intragenic heterogeneity often encountered in such conditions. In order to overcome such difficulties we have designed, and evaluated in vitro, three strategies which avoid a requirement to target individual mutations for genetic suppression. In the first, normal and mutant alleles are suppressed by targeting sequences in transcribed but untranslated regions of transcript (UTRs), enabling introduction of a replacement gene with the correct coding sequencing but altered UTRs to prevent suppression. A second approach involves suppression in coding sequence and concurrent introduction of a replacement gene by exploiting the degeneracy of the genetic code. A third strategy utilises intragenic polymorphism to suppress the disease allele specifically, the advantage being that a proportion of patients with different disease mutations have the same polymorphism. These approaches provide a wider choice of target sequence than those directed to single disease mutations and are appropriate for many mutations within a given gene. General methods for suppression may be directed towards the primary defect or a secondary effect associated with the disease process, such as apoptosis. Three general methods targeting the primary defect which circumvent problems of allelic genetic heterogeneity are explored in vitro using hammerhead ribozymes designed to target transcripts from the rhodopsin, peripherin and collagen 1A1 and 1A2 genes, extensive genetic heterogeneity being a feature of associated disease pathologies.

We have located three extended families in Ireland (population 3.5 million) with autosomal dominant simplex forms of Epidermolysis Bullosa (EBS). A mutation within the keratin type I (K14) gene (Met-->272-->Arg) in one family suffering from the generalized simplex (Koebner) form of the disease has been previously described (Humphries et al., Hum Mutat 2:37-42, 1993). Here we report on the identification of mutations within the remaining two families, both of whom suffer from the Weber-Cockayne form of the disease. These mutations, within the type II keratin (K5) gene, are Asn-->193-->Lys and Met-->327-->Thr. They have been shown in each case to co-segregate with the disease and are not present in the normal population. Within the three families, a total of 44 living persons with such mutations have been identified, providing a minimum prevalence estimate for the disease in the Irish population of approximately 1 in 80,000, compared to an overall estimated global incidence at birth for all forms of EB of 1 in 50,000. Therefore, these three mutations probably account for the majority of cases of EBS within this population.

Retinitis Pigmentosa (RP) is the most prevalent degenerative retinal disease of mendelian origin, currently affecting approximately 1.5 million people worldwide. To date it has been established that a minimum of five different genes maybe involved in the pathogenesis of autosomal dominant forms of RP (adRP). The genes encoding two retinal specific proteins, rhodopsin and peripherin/RDS, have been implicated in causing adRP due to the observation of many different mutations in these genes in patients suffering from RP. The three remaining adRP genes have been mapped to specific regions of human chromosomes but as yet are uncharacterized. We have investigated if there is evidence for the presence of another locus in the genome which when mutated causes adRP. We have utilised polymorphic genetic markers which have previously been mapped to each of the regions known to harbour adRP genes, to test for the exclusion or linkage of the disease gene segregating in a pedigree of Irish origin and find no evidence for linkage. Hence we provide definitive evidence for the involvement of yet another locus. The implications of high levels of genetic heterogeneity inherent in adRP are discussed in relation to diagnosis, prognosis and future therapies.

We have identified a single base change in exon 4 of the type I keratin gene which results in the replacement of a methionine for an arginine residue at codon 272 in an Irish family displaying an autosomal dominant simplex (Koebner) form of epidermolysis bullosa (EB). This family had previously provided tentative evidence for linkage to genetic markers on chromosome 1q. The mutation cosegregates with the disease, producing a lod score of 4.8 at theta = 0.