Isolation and functional characterization of cold-regulated promoters, by digitally identifying peach fruit cold-induced genes from a large EST dataset

<p>Abstract</p> <p>Background</p> <p>Cold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset.</p> <p>Results</p> <p>Digital expression analyses of EST datasets, revealed 164 cold-induced peach genes, several of which show similarities to genes associated with cold acclimation and cold stress responses. The promoters of three of these cold-inducible genes (<it>Ppbec1</it>, <it>Ppxero2 </it>and <it>Pptha1</it>) were fused to the GUS reporter gene and characterized for cold-inducibility using both transient transformation assays in peach fruits (<it>in fruta</it>) and stable transformation in <it>Arabidopsis thaliana</it>. These assays demonstrate that the promoter <it>Pptha1 </it>is not cold-inducible, whereas the <it>Ppbec1 and Ppxero2 </it>promoter constructs are cold-inducible.</p> <p>Conclusion</p> <p>This work demonstrates that during cold storage, peach fruits differentially express genes that are associated with cold acclimation. Functional characterization of these promoters in transient transformation assays <it>in fruta </it>as well as stable transformation in Arabidopsis, demonstrate that the isolated <it>Ppbec1 </it>and <it>Ppxero2 </it>promoters are cold-inducible promoters, whereas the isolated <it>Pptha1 </it>promoter is not cold-inducible. Additionally, the cold-inducible activity of the <it>Ppbec1 </it>and <it>Ppxero2 </it>promoters suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. These results reveal that digital expression analyses may be used in non-model species to identify candidate genes whose promoters are differentially expressed in response to exogenous stimuli.</p

Duplication of the dystroglycan gene in most branches of teleost fish

<p>Abstract</p> <p>Background</p> <p>The dystroglycan (DG) complex is a major non-integrin cell adhesion system whose multiple biological roles involve, among others, skeletal muscle stability, embryonic development and synapse maturation. DG is composed of two subunits: α-DG, extracellular and highly glycosylated, and the transmembrane β-DG, linking the cytoskeleton to the surrounding basement membrane in a wide variety of tissues. A single copy of the DG gene (<it>DAG1</it>) has been identified so far in humans and other mammals, encoding for a precursor protein which is post-translationally cleaved to liberate the two DG subunits. Similarly, <it>D. rerio </it>(zebrafish) seems to have a single copy of <it>DAG1</it>, whose removal was shown to cause a severe dystrophic phenotype in adult animals, although it is known that during evolution, due to a whole genome duplication (WGD) event, many teleost fish acquired multiple copies of several genes (paralogues).</p> <p>Results</p> <p>Data mining of pufferfish (<it>T. nigroviridis </it>and <it>T. rubripes</it>) and other teleost fish (<it>O. latipes </it>and <it>G. aculeatus</it>) available nucleotide sequences revealed the presence of two functional paralogous DG sequences. RT-PCR analysis proved that both the DG sequences are transcribed in <it>T. nigroviridis</it>. One of the two DG sequences harbours an additional mini-intronic sequence, 137 bp long, interrupting the uncomplicated exon-intron-exon pattern displayed by <it>DAG1 </it>in mammals and <it>D. rerio</it>. A similar scenario emerged also in <it>D. labrax </it>(sea bass), from whose genome we have cloned and sequenced a new DG sequence that also harbours a shorter additional intronic sequence of 116 bp. Western blot analysis confirmed the presence of DG protein products in all the species analysed including two teleost Antarctic species (<it>T. bernacchii </it>and <it>C. hamatus</it>).</p> <p>Conclusion</p> <p>Our evolutionary analysis has shown that the whole-genome duplication event in the Class Actinopterygii (ray-finned fish) involved also <it>DAG1</it>. We unravelled new important molecular genetic details about fish orthologous DGs, which might help to increase the current knowledge on DG expression, maturation and targeting and on its physiopathological role in higher organisms.</p

Duplication of the dystroglycan gene in most branches of teleost fish

background: the dystroglycan (DG) complex is a major non-integrin cell adhesion system whose multiple biological roles involve, among others, skeletal muscle stability, embryonic development and synapse maturation. DG is composed of two subunits: α-DG, extracellular and highly glycosylated, and the transmembrane β-DG, linking the cytoskeleton to the surrounding basement membrane in a wide variety of tissues. a single copy of the DG gene (DAG1) has been identified so far in humans and other mammals, encoding for a precursor protein which is post-translationally cleaved to liberate the two DG subunits. similarly, D. rerio (zebrafish) seems to have a single copy of DAG1, whose removal was shown to cause a severe dystrophic phenotype in adult animals, although it is known that during evolution, due to a whole genome duplication (WGD) event, many teleost fish acquired multiple copies of several genes (paralogues). results: data mining of pufferfish (T. nigroviridis and T. rubripes) and other teleost fish (O. latipes and G. aculeatus) available nucleotide sequences revealed the presence of two functional paralogous DG sequences. RT-PCR analysis proved that both the DG sequences are transcribed in T. nigroviridis. One of the two DG sequences harbours an additional mini-intronic sequence, 137 bp long, interrupting the uncomplicated exon-intron-exon pattern displayed by DAG1 in mammals and D. rerio. a similar scenario emerged also in D. labrax (sea bass), from whose genome we have cloned and sequenced a new DG sequence that also harbours a shorter additional intronic sequence of 116 bp. western blot analysis confirmed the presence of DG protein products in all the species analysed including two teleost antarctic species (T. bernacchii and C. hamatus). conclusion: our evolutionary analysis has shown that the whole-genome duplication event in the class actinopterygii (ray-finned fish) involved also DAG1. we unravelled new important molecular genetic details about fish orthologous DGs, which might help to increase the current knowledge on DG expression, maturation and targeting and on its physiopathological role in higher organisms

An Immunological Analysis of Dystroglycan Subunits: Lessons Learned from a Small Cohort of Non-Congenital Dystrophic Patients

The dystroglycan (DG) expression pattern can be altered in severe muscular dystrophies. In fact, some congenital muscular dystrophies (CMDs) and limb-girdle muscular dystrophies (LGMDs) are caused by point mutations identified in six glycosyltransferase genes which are likely to target different steps along the posttranslational “O-glycosylation route” leading to a fully decorated and functional α-DG subunit. Indeed, hypoglycosylation of α-DG is thought to represent a major pathological event, in that it could reduce the DG’s ability to bind the basement membrane components, thus leading to sarcolemmal instability and necrosis. In order to set up an efficient standard immunological protocol, taking advantage of a wide panel of antibodies, we have analyzed the two DG subunits in a small cohort of adult dystrophic patients, whom an extensive medical examination had already clinically classified as affected by LGMD (5), Miyoshi (1) or distal (1) myopathy. Immunofluorescence analysis of skeletal muscle tissue sections revealed a proper sarcolemmal localization of the DG subunits in all the patients analyzed. However, Western blot analysis of lectin enriched skeletal muscle samples revealed an abnormal glycosylation of α-DG in two patients. Our work reinforces the notion that a careful immunological and biochemical analysis of the two DG subunits should be always considered as a prerequisite for the identification of new putative cases of dystroglycanopathy

The EuroLifeNet Study: How Different Microenvironments Influence Personal Exposure to PM 2.5 among High-School Students in Milan A. Borgini et al

Abstract Background: Epidemiological studies show that long-term exposure to PM is associated with an increased risk of cancer. The EuroLifeNet study measured the personal exposure to PM 2.5 in 90 pupils attending three schools in Milan, using a portable nephelometer, over a three-week period spanning November and December 2006. Background levels explained 40% of the variability of the exposure. Methods: As a second part of that study we analyzed the role of different microenvironments as determinants of personal exposure to PM 2.5 . Results: Exposure was influenced by the time of day, zone of the city and different microenvironments. Exposure was higher indoors than out, and indoors it was higher in the kitchen, particularly during cooking. In outdoor environments exposure was higher at bus stops where road traffic was heavy. Conclusions: Even though background concentration can be a good predictor of personal exposure to PM, students&apos; personal exposure is strongly influenced by different microenvironments and should be considered in population studies. The EuroLifeNet experience gives a contribution to personal exposure measure methodology

Measurements of Particulate Matter from Electronic and Conventional Cigarettes: A Comparative Analysis of Methods

Due to the growing popularity of electronic cigarettes (ECs) and heated tobacco products (HTPs) as alternatives to conventional cigarettes (CCs), there is an increasing need to monitor the emissions of these new devices. ECs generate significant concentrations of second-hand aerosol (ECSHA), which is visible in dense clouds and can be smelled. Particulate matter (PM) is an important component of CC, HTP and EC aerosols, and Optical Particle Counters (OPCs) enable its real-time measurement, which is expressed either as the number of particles or as mass. This study specifically addresses the limitations associated with EC mass measurement using OPC technology and identifies the strict necessity of the measurement of a corresponding density (k factor) not only for each specific PM source but also for the desired PM size. Therefore, a standard measurement requires the simultaneous operation of the OPC equipment and a certified reference instrument. Four different OPCs were used. Crucially, this study also proves that this setup may be inapplicable because the extreme volatility of EC-generated aerosols makes it impractical to gauge the correct EC k factor

Measuring for change: A multi-centre pre-post trial of an air quality feedback intervention to promote smoke-free homes

Introduction Second-hand smoke exposure in the home is a serious cause of ill-health for children. Behaviour change interventions have been developed to encourage parents to keep homes smoke-free. This study evaluates a novel air quality feedback intervention using remote air quality monitoring with SMS and email messaging to promote smoke-free homes among families from deprived areas. Methods This paper presents a pre-post study of this intervention. Using internet connected monitors developed with the Dylos DC1700, daily SMS and weekly email feedback provided for 16 days to participants recruited in four European countries. Participants were recruited based on their stage of change, in order to target those most able to achieve smoke-free homes. The primary outcome measure was median change in mean fine particulate matter (PM2.5) concentration between baseline and follow-up periods, while secondary outcome measures included change in time over the World Health Organisation (WHO) guideline limit for PM2.5 exposure over 24 h (25 µg/m3) in those periods and the number of homes where PM2.5 concentrations reduced. Telephone interviews were conducted with participants in Scotland post-intervention to explore intervention experience and perceived effectiveness. Results Of 86 homes that completed the intervention study, 57 (66%) experienced pre-post reductions in measured PM2.5. The median reduction experienced was 4.1 µg/m3 (a reduction of 19% from baseline, p = 0.008). Eight homes where concentrations were higher than the WHO guideline limit at baseline fell below that level at follow-up. In follow-up interviews, participants expressed positive views on the usefulness of air quality feedback. Discussion Household air quality monitoring with SMS and email feedback can lead to behaviour change and consequent reductions in SHS in homes, but within the context of our study few homes became totally smoke-free

Measuring for change: A multi-centre pre-post trial of an air quality feedback intervention to promote smoke-free homes

Introduction: Second-hand smoke exposure in the home is a serious cause of ill-health for children. Behaviour change interventions have been developed to encourage parents to keep homes smoke-free. This study evaluates a novel air quality feedback intervention using remote air quality monitoring with SMS and email messaging to promote smoke-free homes among families from deprived areas. Methods: This paper presents a pre-post study of this intervention. Using internet connected monitors developed with the Dylos DC1700, daily SMS and weekly email feedback provided for 16 days to participants recruited in four European countries. Participants were recruited based on their stage of change, in order to target those most able to achieve smoke-free homes. The primary outcome measure was median change in mean fine particulate matter (PM2.5) concentration between baseline and follow-up periods, while secondary outcome measures included change in time over the World Health Organisation (WHO) guideline limit for PM2.5 exposure over 24 h (25 mu g/m(3)) in those periods and the number of homes where PM2.5 concentrations reduced. Telephone interviews were conducted with participants in Scotland post-intervention to explore intervention experience and perceived effectiveness. Results: Of 86 homes that completed the intervention study, 57 (66%) experienced pre-post reductions in measured PM2.5. The median reduction experienced was 4.1 mu g/m(3) (a reduction of 19% from baseline, p = 0.008). Eight homes where concentrations were higher than the WHO guideline limit at baseline fell below that level at follow-up. In follow-up interviews, participants expressed positive views on the usefulness of air quality feedback. Discussion: Household air quality monitoring with SMS and email feedback can lead to behaviour change and consequent reductions in SHS in homes, but within the context of our study few homes became totally smoke-free