Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.     

Isolation and characterization of a mixotrophic sulfur-oxidizing Thermus scotoductus

Thermophilic, faculatatively mixotrophic sulfur-oxidizing bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland. The strain, IT-7254, used thiosulfate and elemental sulfur as electron donors, oxygen and nitrate as electron acceptors, and acetate and other organic compounds as carbon sources. After a few days of growth in the presence of thiosulfate, this strain formed sulfur globules. Comparison of intracellular enzymes and heme proteins of heterotrophically and mixotrophically grown cells showed some differences. The new isolate belonged to Thermus scotoductus because the small subunit (SSU) rRNA gene sequence analysis showed 98.6% sequence similarity and 84% DNA:DNA reassociation to Thermus scotoductus NMX2 A. 1. It is also close to Thermus antranikianii HN3-7, with 98.3% and 79% SSU rRNA sequence similarity and DNA:DNA reassociation, respectively. It was also found that both Thermus NMX2 A.1 and T. antranikianii HN3-7 were able to oxidize thiosulfate but that the T. scotoductus type strain SE-1 was not. This is the first report of Thermus strains that are capable of mixotrophic growth with sulfur oxidation.     

Examining genetic structure in a bogus yucca moth: a sequential approach to phylogeography

Understanding the phylogeography of a species requires not only elucidating patterns of genetic structure among populations, but also identifying the possible evolutionary events creating that structure. The use of a single phylogeographic test or analysis, however, usually provides a picture of genetic structure without revealing the possible underlying evolutionary causes. We used current analytical techniques in a sequential approach to examine genetic structure and its underlying causes in the bogus yucca moth Prodoxus decipiens (Lepidoptera: Prodoxidae). Both historical biogeography and recent human transplantations of the moth's host plants provided a priori expectations of the pattern of genetic structure and its underlying causes. We evaluated these expectations by using a progression of phylogenetic, demographic, and population genetic analyses of mtDNA sequence data from 476 individuals distributed across 25 populations that encompassed the range of P. decipiens. The combination of these analyses revealed that much of the genetic structure has evolved more recently than suggested by historical biogeography, has been influenced by changes in demography, and can be best explained by long distance dispersal and isolation by distance. We suggest that performing a suite of analyses that focus on different temporal scales may be an effective approach to investigating the patterns and causes of genetic structure within species.     

Enantiomeric separations of amino alcohols by packed-column SFC on Hypercarb with L-(+)-tartaric acid as chiral selector

The use of L-(+)-tartaric acid as a chiral mobile phase additive (CMPA) has been investigated in a packed-column SFC system. The CMPA, carbon dioxide, and methanol, containing a high concentration of aliphatic amine additive, were used as the mobile phase and Hypercarb as support [Gyllenhaal O., Karlsson A., SFC of metoprolol and other amino alcohols on Hypercarb (in preparation)]. Good enantioselectivities were obtained for tertiary amine homologues of 2-amino alcohols, used as beta-adrenoreceptor-blocking drugs. Moderate selectivities were observed for aromatic compounds having a second substituent in the ortho-position. The overall retention was influenced by the aromaticity of the analytes as well as the presence of free electron pairs in the molecule. Increased concentrations of CMPA gave higher retention and also increased the enantioselectivity. The practical utility of this present enantioselective system was demonstrated on one batch of (S)-metoprolol that was N-methylated with methyl iodide. The enantiomeric separation was accomplished within 10 min.     

Self-diffusion of nonfreezing water in porous carbohydrate polymer systems studied with nuclear magnetic resonance

Water is an integral part of the structure in biological porous materials such as wood and starch. A problem often encountered in the preparation of samples for, e.g., electron microscopy is that removal of water leads to a decreasing distance between supermolecular structural elements and a distortion of the structure. It is, therefore, of interest to find methods to investigate these materials in the native water-swollen state. We present a method to study water-swollen biological porous structures using NMR to determine the amount and self-diffusion of water within the porous objects. The contribution of bulk water to the NMR signal is eliminated by performing experiments below the bulk freezing temperature. Further decrease of the temperature leads to a gradual freezing of water within the porous objects. The contribution of the freezing water fraction to the migration of water through the porous network is, thus, estimated. The results are rationalized in terms of the ultrastructure of the samples studied, namely, wood pulp fibers and potato starch granules.     

Chloroplast movements in leaves: Influence on chlorophyll fluorescence and measurements of light-induced absorbance changes related to ΔpH and zeaxanthin formation

Light-induced chloroplast movements were found to cause changes in chlorophyll fluorescence emission, closely matching those in leaf absorptance, both in terms of the kinetics and the maximum extent of the changes observed in different species. The results demonstrate that chloroplast movements can have a significant effect on the efficiency of light utilization in photosynthesis. They further show that chloroplast movements need to be taken into account in measurements of fluorescence quenching and especially in measurements of light-induced optical changes used to monitor zeaxanthin formation and pH associated light scattering in leaves. Means of minimizing and of adjusting for the influences of chloroplast movements in such measurements are discussed.Abbreviations F fluorescence emission - PFD photon flux density - R reflectance - T transmittance - absorptance C.I.W.-D.P.B. Publication No. 1116.     

Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments

We examined the relationship between non-photochemical quenching (NPQ) and xanthophyll de-epoxidation in the unicellular algae Euglena gracilis, Ochromonas danica, Phaeodactylum tricornutum, and Dunaliella tertiolecta. Generally, low-light-grown algae had a smaller pool of xanthophyll-cycle pigments per chlorophyll than medium-light-grown grown cells, but they developed more NPQ during exposure to high light. Thus, lumen acidification was apparently lower in medium-light-grown cells in spite of the exposure to a photon flux density (PFD) three times the growth PFD. In darkness Dunaliella maintained a relatively large content of de-epoxidized xanthophylls, and NPQ developed without concomitant de-epoxidation in response to a 5-min exposure to high light. Violaxanthin de-epoxidation that occurred during longer exposures to light did not cause a further rise in NPQ in Dunaliella. In Ochromonas, NPQ and xanthophyll de-epoxidation increased simultaneously during a 15-min exposure to high light. A further rise in NPQ was not accompanied by xanthophyll de-epoxidation. In Phaeodactylum, the rise in NPQ and de-epoxidation were nearly linearly related during a 60-min exposure to high light. NPQ recovered quickly after darkening in these three algae and no significant photodamage occurred. In Euglena no xanthophyll-conversions and no quickly reversible NPQ occured in response to high light, suggesting that photodamage occurred. Dunaliella has similar light-harvesting and xanthophyll-cycle pigments as higher plants but the relationship between NPQ and DPS during the exposure to high light was different from the linear relationship that is commonly observed in plants. Conversely, Phaeodactylum, which has different light-harvesting and xanthophyll-cycle pigments, had a relationship similar to that in plants.     

How Can Inequalities in Mortality Be Reduced? A Quantitative Analysis of 6 Risk Factors in 21 European Populations

Background

Socioeconomic inequalities in mortality are one of the greatest challenges for health policy in all European countries, but the potential for reducing these inequalities is unclear. We therefore quantified the impact of equalizing the distribution of six risk factors for mortality: smoking, overweight, lack of physical exercise, lack of social participation, low income, and economic inactivity.

Methods

We collected and harmonized data on mortality and risk factors by educational level for 21 European populations in the early 2000s. The impact of the risk factors on mortality in each educational group was determined using Population Attributable Fractions. We estimated the impact on inequalities in mortality of two scenarios: a theoretical upward levelling scenario in which inequalities in the risk factor were completely eliminated, and a more realistic best practice scenario, in which inequalities in the risk factor were reduced to those seen in the country with the smallest inequalities for that risk factor.

Findings

In general, upward levelling of inequalities in smoking, low income and economic inactivity hold the greatest potential for reducing inequalities in mortality. While the importance of low income is similar across Europe, smoking is more important in the North and East, and overweight in the South. On the basis of best practice scenarios the potential for reducing inequalities in mortality is often smaller, but still substantial in many countries for smoking and physical inactivity.

Interpretation

Theoretically, there is a great potential for reducing inequalities in mortality in most European countries, for example by equity-oriented tobacco control policies, income redistribution and employment policies. Although it is necessary to achieve substantial degrees of upward levelling to make a notable difference for inequalities in mortality, the existence of best practice countries with more favourable distributions for some of these risk factors suggests that this is feasible.