Études in vivo de l'épissage alternatif de l'exon EDIIIA du gène de la fibronectine

Résumé: Les évidences que l'exon EDIIIA varie dans son inclusion au niveau de l’ARN messager dans différentes lignées cellulaires suggèrent que l'épissage alternatif de cet exon est régulé. D'autres études ont démontré qu'il y avait une séquence "splicing enhancer (SE)" à l'intérieur de l'exon EDIIIA qui était nécessaire pour son inclusion dans l'ARN messager. Des études effectuées in vitro ont identifié une séquence riche en purines de 9 nt qui stimule l'utilisation du site d'épissage 3' en amont de EDIIIA. Cette séquence riche en purines lie les protéines SR. Nous avons construit deux mini-gènes dont l'un contient l'unité d'épissage alternatif EDIIIA et l'autre possède une délétion de la séquence riche en purines. Ces mini-gènes ont été transfectés dans des cellules WI38VA13 et MRC5, deux lignées dans lesquelles le pré-ARNm de la fibronectine est épissé de façon différente. Le patron d'épissage des mini-gènes a été analysé par RT-PCR et a démontré que la séquence riche en purines est nécessaire pour l'inclusion de l'exon EDIIIA dans l'ARN messager in vivo. Étant donné que l'élément "splicing enhancer" est requis pour l'inclusion de l'exon EDIIIA dans les deux lignées cellulaires, nos résultats suggèrent que la séquence riche en purines pourrait jouer un rôle dans la régulation de l'épissage alternatif. Nous avons étudié l'effet de l'expression des protéines SR sur la régulation de l'épissage alternatif de l'exon EDIIIA. Sachant que le niveau d'expression des protéines SRp40, SF2, et SC35 est moins abondant dans les cellules MRC5 que dans les cellules WI38VA13, nous avons tenté de surexprimer ces protéines SR dans les cellules MRC5. La surexpression des protéines SC35 et SF2 dans les cellules MRC5 inhibe la croissance des cellules. Nous avons obtenu des cellules stables transfectées par le cDNA de SRp40 mais le niveau de protéine SRp40 phosphorylée dans ces cellules était réduit. Cette diminution de la protéine SRp40 phosphorylée a été accompagnée par une réduction dans le niveau de produits incluent l'exon EDIIIA. Ces résultats suggèrent que la séquence riche en purines et la protéine SRp40 jouent un rôle dans la régulation de l'épissage alternatif. || Abstract : Cell-specific differences in the level of inclusion of the fibronectin EDIIIA exon suggest that the alternative splicing of this exon is regulated. Previous studies have revealed that the EDIIIA exon contains a splicing enhancer which stimulates its inclusion into the mRNA. Further investigation identified a 9 nt purine-rich sequence which stimulates the use of the upstream 3' splice site of EDIIIA in vitro. This sequence is hound by SR proteins. To study the possible role of the purine-rich sequence in the regulation of alternative splicing of the EDIIIA exon, a mini-gene construct was made containing the EDIIIA alternative splicing unit. Another mini-gene construct carried a deletion of the purine-rich sequence. These mini-genes were transfected into two cell lines (WI38VA13 and MRC5) which splice EDIIIA differently. The splicing pattern of these mini-genes was analyzed by RT-PCR and the results indicate that the purine-rich sequence is necessary for the inclusion of the EDIIIA exon in vivo. Because the splicing enhancer was required for EDIIIA inclusion in both cell lines, our results suggest that the purine-rich sequence may play a role in the regulation of alternative splicing. We also studied the effect of SR protein expression in the regulation of the alternative splicing of this exon. Because the level of SRp40, SC35, and SF2 are reduced in MRC5 cells which skip EDHIA more often than WI38VA13 cells, we attempted to overexpress these SR proteins in MRC5 cells. Overexpression of SC35 and SF2 in the MRC5 cells was probably toxic and inhibited the growth of the cells. Growing cells were obtained only by stable transfection of the SRp40 cDNA. Surprisingly, the amount of phosphorylated SRp40 protein expressed in the MRC5 cells was reduced. This decrease in phosphorylated SRp40 protein was accompanied by a reduction in the level of inclusion of the EDIII-A exon. These results suggest a role for the purine-rich sequence and SRp40 in the regulation of alternative splicing of the fibronectin EDIII-A exon

Prognosis of hyponatremia in elderly patients with fragility fractures

Funding This work is supported by an NHS Research Scotland (NRS) Career Research Fellowship to Dr Soiza.Peer reviewedPublisher PD

Anne Brontë and Geology : A Study of her Collection of Stones

Open access via T&F agreement Acknowledgements The authors would like to thank the staff of the Bront€e Parsonage Museum, especially Ann Dinsdale, Sarah Laycock, Emma Littlejohns, Charlotte Craig and Amy Rowbottom (formerly of the Bront€e Parsonage Museum); Ralph O’Connor, University of Aberdeen; Walter Ritchie, University of Aberdeen; William Zachs; Edward Chitham; Allan M. Bicket; Clare Flaherty; Jim Middleton, Scarborough Museums Trust; Jennifer Dunne (formerly of the Scarborough Museums Trust); Dave Horsley, Scarborough Royal National Lifeboat Institution; UK Hydrographic Office; The British Library, Boston Spa; the Department of English in the School of Language, Literature, Music and Visual Culture, and the Department Geology and Geophysics in the School of Geosciences at the University of Aberdeen for their financial assistance for fieldwork; M.J., A.J., and H.JPeer reviewedPublisher PD

Novel picornavirus in turkey poults with hepatitis, California, USA

To identify a candidate etiologic agent for turkey viral hepatitis, we analyzed samples from diseased turkey poults from 8 commercial flocks in California, USA, that were collected during 2008–2010. High-throughput pyrosequencing of RNA from livers of poults with turkey viral hepatitis (TVH) revealed picornavirus sequences. Subsequent cloning of the ≈9-kb genome showed an organization similar to that of picornaviruses with conservation of motifs within the P1, P2, and P3 genome regions, but also unique features, including a 1.2-kb sequence of unknown function at the junction of P1 and P2 regions. Real-time PCR confirmed viral RNA in liver, bile, intestine, serum, and cloacal swab specimens from diseased poults. Analysis of liver by in situ hybridization with viral probes and immunohistochemical testing of serum demonstrated viral nucleic acid and protein in livers of diseased poults. Molecular, anatomic, and immunologic evidence suggests that TVH is caused by a novel picornavirus, tentatively named turkey hepatitis virus

Geographic disparity in premature mortality in Ontario, 1992–1996

BACKGROUND: Standardized mortality ratios are used to identify geographic areas with higher or lower mortality than expected. This article examines geographic disparity in premature mortality in Ontario, Canada, at three geographic levels of population and considers factors that may underlie variations in premature mortality across geographic areas. All-cause, sex and disease chapter specific premature mortality were analyzed at the regional, district and public health unit level to determine the extent of geographic variation. Standardized mortality ratios for persons aged 0–74 years were calculated to identify geographic areas with significantly higher or lower premature mortality than expected, using Ontario death rates as the basis for the calculation of expected deaths in the local population. Data are also presented from the household component of the 1996/97 National Population Health Survey and from the 1996 Statistics Canada Census. RESULTS: Results showed approximately 20% higher than expected all-cause premature mortality for males and females in the North region. However, disparity in all-cause premature mortality in Ontario was most pronounced at the public health unit level, ranging from 20% lower than expected to 30% higher than expected. Premature mortality disparities were largely influenced by neoplasms, circulatory diseases, injuries and poisoning, respiratory diseases and digestive diseases, which accounted for more than 80% of all premature deaths. Premature mortality disparities were also more pronounced for disease chapter specific mortality. CONCLUSION: Geographic disparities in premature mortality are clearly greater at the small area level. Geographic disparities in premature mortality undoubtedly reflect the underlying distribution of population health determinants such as health related behaviours, social, economic and environmental influences

The mouse globin pseudogene beta h3 is descended from a premammalian delta-globin gene.

The beta h3 pseudogene of the BALB/c mouse contains sequence defects which prevent transcription and translation to produce a beta-globin. Comparison with other globin gene sequences indicates that beta h3 arose by recombination between an adult beta-globin gene and some significantly diverged globin sequence. Analysis of noncoding sequences shows that the 3' end of mouse beta h3 and the human delta-globin gene are both descended from an ancestral gene, which we call proto-delta. The origin of proto-delta must predate the mammalian radiation. A member of the L1 family of interspersed repetitive elements is inserted into the 3' untranslated delta-homologous sequence in beta h3 from BALB/c. beta h3 is a widespread feature of the rodent beta-globin complex, which has been fixed in the genome for 35 million years. Independent inactivation events produced pseudogenes located between the adult and nonadult beta-globin genes in the rodent, primate, rabbit, and goat lineages. One model to explain the abundance and evolutionary persistence of pseudogenes postulates that the mammalian genome simply has no efficient mechanism for deleting nonessential sequences. Consequently, the genomes of higher eukaryotes have been growing, by the accumulation of duplications, with doubling times of 200 +/- 100 million years

A randomised controlled trial and cost-effectiveness evaluation of 'booster' interventions to sustain increases in physical activity in middle-aged adults in deprived urban neighbourhoods

Background: More evidence is needed on the potential role of 'booster' interventions in the maintenance of increases in physical activity levels after a brief intervention in relatively sedentary populations. Objectives: To determine whether objectively measured physical activity, 6 months after a brief intervention, is increased in those receiving physical activity 'booster' consultations delivered in a motivational interviewing (MI) style, either face to face or by telephone. Design: Three-arm, parallel-group, pragmatic, superiority randomised controlled trial with nested qualitative research fidelity and geographical information systems and health economic substudies. Treatment allocation was carried out using a web-based simple randomisation procedure with equal allocation probabilities. Principal investigators and study statisticians were blinded to treatment allocation until after the final analysis only. Setting: Deprived areas of Sheffield, UK. Participants: Previously sedentary people, aged 40-64 years, living in deprived areas of Sheffield, UK, who had increased their physical activity levels after receiving a brief intervention. Interventions: Participants were randomised to the control group (no further intervention) or to two sessions of MI, either face to face ('full booster') or by telephone ('mini booster'). Sessions were delivered 1 and 2 months post-randomisation. Main outcome measures: The primary outcome was total energy expenditure (TEE) per day in kcal from 7-day accelerometry, measured using an Actiheart device (CamNtech Ltd, Cambridge, UK). Independent evaluation of practitioner competence was carried out using the Motivational Interviewing Treatment Integrity assessment. An estimate of the per-participant intervention costs, resource use data collected by questionnaire and health-related quality of life data were analysed to produce a range of economic models from a short-term NHS perspective. An additional series of models were developed that used TEE values to estimate the long-term cost-effectiveness. Results: In total, 282 people were randomised (control = 96; mini booster = 92, full booster = 94) of whom 160 had a minimum of 4 out of 7 days' accelerometry data at 3 months (control = 61, mini booster = 47, full booster = 52). The mean difference in TEE per day between baseline and 3 months favoured the control arm over the combined booster arm but this was not statistically significant (-39 kcal, 95% confidence interval -173 to 95, p = 0.57). The autonomy-enabled MI communication style was generally acceptable, although some participants wanted a more paternalistic approach and most expressed enthusiasm for monitoring and feedback components of the intervention and research. Full boosters were more popular than mini boosters. Practitioners achieved and maintained a consistent level of MI competence. Walking distance to the nearest municipal green space or leisure facilities was not associated with physical activity levels. Two alternative modelling approaches both suggested that neither intervention was likely to be cost-effective. Conclusions: Although some individuals do find a community-based, brief MI 'booster' intervention supportive, the low levels of recruitment and retention and the lack of impact on objectively measured physical activity levels in those with adequate outcome data suggest that it is unlikely to represent a clinically effective or cost-effective intervention for the maintenance of recently acquired physical activity increases in deprived middle-aged urban populations. Future research with middle-aged and relatively deprived populations should explore interventions to promote physical activity that require less proactive engagement from individuals, including environmental interventions

Concert recording 2019-04-09a

[Track 1]. Fanfare / James Barnes -- [Track 2]. Contrapunctus IX / J.S. Bach ; arranged by Michael Forbes -- [Track 3]. Celestial suite. I. Eclipse ; [Track 4]. II. Canzone lunaire. Pt. 1 ; [Track 5]. Pt. 2 ; [Track 6]. III. Solar plexus / Stephen Bulla -- [Track 7]. Misty Mountain / Howard ; arranged by Cody Hutchison -- [Track 8]. Passage to Bangkok / Rush ; arranged by Cody Hutchison -- [Track 9]. Power ; [Track 10]. Ballade / John Stevens -- [Track 11]. Cherokee / John Schooley -- [Track 12]. Go / Michael Forbes -- [Track 13]. Canzona Bergamasca / Samuel Scheidt ; arranged by Cody Hutchison -- [Track 14]. Shenandoah / traditional ; arranged by Erb. ; translated by Hutchison -- [Track 15]. War Machine / Anthony O\u27Toole

The diagonal-traverse homology search algorithm for locating similarities between two sequences

We present a fast computer algorithm for finding homology between two DNA sequences. It generates a two-dimensional display in which a diagonal string of dots represents a stretch of homology between the two sequences. Our algorithm performs the search very rapidly, and has no internal data storage requirement except for the sequences themselves. These characteristics make it particularly well suited for execution on microcomputers. Without slowing execution, the matching criterion can be that a specified fraction of contiguous bases must be identical. Even with gapped sequences, we have found large search windows to be surprisingly good for detecting poor homologies with nearly complete background suppression. A diagonal search pattern is used that reports the finds in a compact and logically ordered form. A simple and rapid plotting algorithm for unsophisticated printers is also reported