Nucleic acid capture assay,a new method for direct quantitation of nucleic acids

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.     

Depolarized resonance light scattering by porphyrin and chlorophyll a aggregates.

A quantum mechanical model is developed for the observed resonance enhancement of light scattering by aggregates of electronically interacting chromophores. Aggregate size, monomer oscillator strength, extent of electronic coupling, and aggregate geometry are all important determinants of intensity in resonance light scattering (RLS) spectra. The theory also predicts the value of the depolarization ratio (rho(v)(90)) of RLS for a given aggregate geometry. These results are used to interpret the RLS depolarization ratios of four aggregates: tetrakis(4-sulfonatophenyl)porphine aggregated at low pH (rho(v)(90) = 0.17 at 488 nm), trans-bis(N-methylpyridinium-4-yl)-diphenylporphinato copper(II) aggregated in 0.2 M NaCl solution (rho(v)(90) = 0.13 at 450 nm) and on calf thymus DNA (rho(v)(90) = 0.20 at 454 nm), and chlorophyll a aggregates in formamide/water (rho(v)(90) = 0.23 and 0.32 at 469 and 699 nm, respectively). The analysis is consistent with a J-aggregate geometry for all four systems. Furthermore, the specific values of rho(v)(90) allow us to estimate the orientation of the monomer transition dipoles with respect to the long axis of the aggregate. We conclude that depolarized resonance light scattering spectroscopy is a powerful probe of the geometric and electronic structures of extended aggregates of strong chromophores.     

DESICCATION AND STARVATION TOLERANCE OF ADULT DROSOPHILA: OPPOSITE LATITUDINAL CLINES IN NATURAL POPULATIONS OF THREE DIFFERENT SPECIES

Desiccation and starvation tolerance were measured along latitudinal transects in three Drosophilid species (Drosophila ananassae, D. melanogaster, and Zaprionus indianus) of the Indian subcontinent. In each case, significant latitudinal clines were observed; desiccation tolerance increased with latitude while starvation tolerance decreased. Such field observations suggest that desiccation and starvation tolerance are fitness related traits that are independently selected in nature and genetically independent. It was, however, difficult to relate these genetic changes with precise climatic variables, except winter temperature. The overall negative correlation between the two traits, which was evidenced in natural populations, contrasts with a positive correlation generally observed in various laboratory selection experiments and that also seems to exist between different species. These observations point to the difficulty of interpreting correlations among fitness-related traits when different evolutionary levels are compared, and also different sets of data, that is, field versus laboratory studies.     

Impact of body melanisation on contrasting levels of desiccation resistance in a circumtropical and a generalist <Emphasis Type="Italic">Drosophila</Emphasis> species

We investigated the role of cuticular lipids, body melanisation and body size in conferring contrasting levels of desiccation resistance in latitudinal populations of Drosophila melanogaster and Drosophila ananassae on the Indian subcontinent. Contrary to the well known role of cuticular lipids in water proofing in diverse insect taxa, there is lack of geographical variations in the amount of cuticular lipids per fly in both the species. In D. ananassae, quite low levels of body melanisation are correlated with lower desiccation resistance. By contrast, increased levels of desiccation resistance are correlated with quite high melanisation in D. melanogaster. Thus, species specific cuticular melanisation patterns are significantly correlated with varying levels of desiccation resistance within as well as between populations and across species. Role of body melanisation in desiccation resistance is further supported by the fact that assorted dark and light flies differ significantly in cuticular water loss, hemolymph and dehydration tolerance. However, similar patterns of body size variation do not account for contrasting levels of desiccation resistance in these two Drosophila species. Climatic selection is evidenced by multiple regression analysis with seasonal amplitude of thermal and humidity changes (Tcv and RHcv) along latitude on the Indian subcontinent. Finally, the contrasting levels of species specific distribution patterns are negatively correlated with RHcv of sites of origin of populations i.e. a steeper negative slope for D. ananassae corresponds with its desiccation sensitivity as compared with D. melanogaster. Thus, evolutionary changes in body melanisation impact desiccation resistance potential as well as distribution patterns of these two Drosophila species on the Indian subcontinent.     

Correlated changes in thermotolerance traits and body color phenotypes in montane populations of Drosophila melanogaster: analysis of within- and between-population variations

Thermotolerance traits vary across geographical gradients but there is a lack of clinal variation in some Drosophila species. Thus, it is not clear whether thermotolerance or other correlated traits are the target of natural selection. In order to test selection responses, we investigated body melanization and thermotolerance traits in six altitudinal populations of Drosophila melanogaster . Based on rearing different geographical populations under uniform growth conditions at 21 °C (common garden experiments), clinal variations for cold resistance are in the direction opposite to heat resistance along an altitudinal gradient, that is darker flies from highland populations evidenced higher levels of cold resistance while lowland populations showed higher heat resistance. Phenotypic plastic responses for body melanization at 17–28 °C showed significant correlations with thermotolerance traits. At 17 °C, regression coefficients as a function of altitude are highly significant and positive for cold resistance but negative for heat knockdown. However, for flies reared at 28 °C, there is no elevational change in melanization as well as thermotolerance traits. Thus, both genetic and plastic changes of body melanization and thermotolerance traits suggest a correlated selection response. Further, within-population analyses of body melanization (based on dark, intermediate and light color phenotypes) showed significant associations with thermotolerance traits. Correlated variations in body melanization and thermal tolerances are associated with climatic thermal variability ( T _cv) but not with T _min. or T _max. along an altitudinal gradient.     

Cardiac resynchronization therapy: a meta-analysis of randomized controlled trials

Background

Studies of cardiac resynchronization therapy in addition to an implantable cardioverter defibrillator in patients with mild to moderate congestive heart failure had not been shown to reduce mortality until the recent RAFT trial (Resynchronization/Defibrillation for Ambulatory Heart Failure Trial). We performed a meta-analysis including the RAFT trial to determine the effect of cardiac resynchronization therapy with or without an implantable defibrillator on mortality.

Methods

We searched electronic databases and other sources for reports of randomized trials using a parallel or crossover design. We included studies involving patients with heart failure receiving optimal medical therapy that compared cardiac resynchronization therapy with optimal medical therapy alone, or cardiac resynchronization therapy plus an implantable defibrillator with a standard implantable defibrillator. The primary outcome was mortality. The optimum information size was considered to assess the minimum amount of information required in the literature to reach reliable conclusions about cardiac resynchronization therapy.

Results

Of 3071 reports identified, 12 studies (n = 7538) were included in our meta-analysis. Compared with optimal medical therapy alone, cardiac resynchronization therapy plus optimal medical therapy significantly reduced mortality (relative risk [RR] 0.73, 95% confidence interval [CI] 0.62–0.85). Compared with an implantable defibrillator alone, cardiac resynchronization therapy plus an implantable defibrillator significantly reduced mortality (RR 0.83, 95% CI 0.72–0.96). This last finding remained significant among patients with New York Heart Association (NYHA) class I or II disease (RR 0.80, 95% CI 0.67–0.96) but not among those with class III or IV disease (RR 0.84, 95% CI 0.69–1.07). Analysis of the optimum information size showed that the sequential monitoring boundary was crossed, which suggests no need for further clinical trials.

Interpretation

The cumulative evidence is now conclusive that the addition of cardiac resynchronization to optimal medical therapy or defibrillator therapy significantly reduces mortality among patients with heart failure.Congestive heart failure is currently reaching epidemic proportions in Canada, with 500 000 Canadians affected and 50 000 new patients identified each year.¹ It accounts for more than 100 000 hospital admissions per year and has a one-year mortality ranging from 15% to 50%, depending on the severity of heart failure.² By 2050, the number of patients with heart failure is projected to increase threefold.²Advances in medical therapies have resulted in substantial reductions in mortality associated with congestive heart failure.^3–7 The use of devices has recently become an important adjuvant therapy.⁸ Cardiac resynchronization therapy involves pacing from both the right and left ventricles simultaneously to improve myocardial efficiency (see radiographs in Appendix 1, at www.cmaj.ca/cgi/content/full/cmaj.101685/DC1). Cardiac resynchronization therapy has been shown to reduce morbidity and, when compared with medical therapy alone, to reduce mortality.^9–13 Until recently, it was not shown to reduce mortality among patients who also received an implantable cardioverter defibrillator. Among patients receiving optimal medical therapy, the Resynchronization/Defibrillation for Ambulatory Heart Failure Trial (RAFT) showed the superiority of cardiac resynchronization therapy in addition to an implantable defibrillator over a standard implantable defibrillator in reducing mortality and the combined outcome of death from any cause or hospital admission related to heart failure.¹⁴We performed a meta-analysis to further assess the effect on mortality of cardiac resynchronization therapy with and without an implantable defibrillator among patients with mildly symptomatic and advanced heart failure.     

Divergence of water balance mechanisms in two melanic Drosophila species from the western Himalayas

Drosophila busckii is more abundant under colder and drier montane habitats in the western Himalayas as compared to Drosophila melanogaster but the mechanistic basis of such climatic adaptations is largely unknown. We tested the hypothesis whether genetic variation or phenotypic plasticity of cuticular traits confer adaptive protection against desiccation stress in two melanic Drosophila species living under drier montane localities. For D. melanogaster, changes in melanisation are known to be associated with reduced water loss but there are no data on D. busckii. We investigated changes in body melanisation, cuticular lipids, desiccation resistance, water loss, extractable hemolymph volume (%), and dehydration tolerance in six sympatric populations of D. busckii and D. melanogaster over an altitudinal range of 640-2236 m. D. busckii is a melanic species but changes in cuticular water loss are negatively correlated with cuticular lipid mass and not with body melanisation. In D. melanogaster, there are no plastic effects (14-28 °C) for cuticular lipid mass but variation in body melanisation is associated with desiccation-related traits. Effects of organic solvents (hexane or chloroform: methanol), developmental plasticity and seasonal variation in cuticular lipids affect body water loss in D. busckii but no such changes occur in D. melanogaster. Thus, sympatric populations of D. busckii and D. melanogaster have evolved different water balance mechanisms under shared environmental conditions in the western Himalayas. Multiple measures of desiccation resistance in these species show clinal variation with altitude, consistent with adaptation to increased desiccation stress.     

Low-dosage inhibition of Dll4 signaling promotes wound healing by inducing functional neo-angiogenesis

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing.     

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.