Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome

Two novel mitochondrial gene arrangements are identified in an agamid lizard and a ranid frog. Statistical tests incorporating phylogeny indicate a link between novel vertebrate mitochondrial gene orders and movement of the origin of light-strand replication. A mechanism involving errors in light-strand replication and tandem duplication of genes is proposed for rearrangement of vertebrate mitochondrial genes. A second mechanism involving small direct repeats also is identified. These mechanisms implicate gene order as a reliable phylogenetic character. Shifts in gene order define major lineages without evidence of parallelism or reversal. The loss of the origin of light-strand replication from its typical vertebrate position evolves in parallel and, therefore, is a less reliable phylogenetic character. Gene junctions also evolve in parallel. Sequencing across multigenic regions, in particular transfer RNA genes, should be a major focus of future systematic studies to locate novel gene orders and to provide a better understanding of the evolution of the vertebrate mitochondrial genome.      

Distribution patterns of Baltic herring larvae, Clupea harengus L., in the coastal waters off Helsinki, Finland

Changes in the spatial distribution of Baltic herring larvaethrough time were investigated in the waters off Helsinki, thenorthern Baltic Sea. The average larval densities were comparableto the relatively low levels (mean around 2 larvae m^–2)found in other studies in the Gulf of Finland. In shallow baysvirtually no herring larvae were found in early June. In lateJune and early July the densities of larvae longer than 10 mmincreased to 1–4 larvae m^–2 in shallow areas (depth<2 m), i.e. to levels higher than those recorded in deeperwaters. In combination with data on the size distributions ofthe larvae, these observations suggest that a significant proportionof the herring larvae are found in shallow water after the yolksac stage. We conclude that shallow coastal waters are potentiallyimportant nursery areas for herring larvae and that this featureof herring larval biology should be taken into account in planningsampling programs for larval herring.     

Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex

A novel form of mitochondrial DNA (mtDNA) inheritance has previously been documented for the blue mussel (Mytilus edulis). Female mussels inherit their mtDNA solely from their mother while males inherit mtDNA from both their mother and their father. In males, the paternal mtDNA is preferentially amplified so that the male gonad is highly enriched for the paternal mtDNA that is then transmitted from fathers to sons. We demonstrate that this mode of mtDNA inheritance also operates in the closely related species M. galloprovincialis and M. trossulus. The evolutionary relationship between the male and female mtDNA lineages is estimated by phylogenetic analysis of 455 nucleotides from the large subunit ribosomal RNA gene. We have found that the male and female lineages are highly divergent; the divergence of these lineages began prior to the speciation of the three species of blue mussels. Further, the separation between the male and female lineages is estimated to have occurred between 5.3 and 5.7 MYA.      

Characterization and crystallization of an active N-terminally truncated form of the Escherichia coli glycogen branching enzyme.

The prokaryotic glycogen branching enzymes (GBE) can be divided into two groups on the basis of their primary structures: the first group of enzymes, which includes GBE from Escherichia coli, is characterized by a long N-terminal extension that is absent in the enzymes of the second group. The extension consists of approximately 100 amino-acid residues with unknown function. In order to characterize the function of this region, the 728 amino-acid residue, full-length E. coli GBE, and a truncated form (nGBE) missing the first 107 amino-acid residues were overexpressed in E. coli. Both enzymes were purified to homogeneity by a simple purification procedure involving ammonium sulphate precipitation, ion-exchange chromatography, and a second ammonium sulphate precipitation. Purified full-length enzyme was poorly soluble and formed aggregates, which were inactive, at concentrations above 1 mg.mL-1. In contrast, the truncated form could be concentrated to 6 mg.mL-1 without any visible signs of aggregation or loss of activity on concentration. The ability to overexpress nGBE in a highly soluble form has allowed us to produce diffracting crystals of a branching enzyme for the first time. A comparison of the specific activities of purified GBE and nGBE in assays where amylose was used as substrate demonstrated that nGBE retained approximately half of the branching activity of full-length GBE and is therefore a suitable model for the study of the enzymes' catalytic mechanism.     

Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.     

Observer bias in randomized clinical trials with measurement scale outcomes: a systematic review of trials with both blinded and nonblinded assessors

Background:

Clinical trials are commonly done without blinded outcome assessors despite the risk of bias. We wanted to evaluate the effect of nonblinded outcome assessment on estimated effects in randomized clinical trials with outcomes that involved subjective measurement scales.

Methods:

We conducted a systematic review of randomized clinical trials with both blinded and nonblinded assessment of the same measurement scale outcome. We searched PubMed, EMBASE, PsycINFO, CINAHL, Cochrane Central Register of Controlled Trials, HighWire Press and Google Scholar for relevant studies. Two investigators agreed on the inclusion of trials and the outcome scale. For each trial, we calculated the difference in effect size (i.e., standardized mean difference between nonblinded and blinded assessments). A difference in effect size of less than 0 suggested that nonblinded assessors generated more optimistic estimates of effect. We pooled the differences in effect size using inverse variance random-effects meta-analysis and used metaregression to identify potential reasons for variation.

Results:

We included 24 trials in our review. The main meta-analysis included 16 trials (involving 2854 patients) with subjective outcomes. The estimated treatment effect was more beneficial when based on nonblinded assessors (pooled difference in effect size −0.23 [95% confidence interval (CI) −0.40 to −0.06]). In relative terms, nonblinded assessors exaggerated the pooled effect size by 68% (95% CI 14% to 230%). Heterogeneity was moderate (I² = 46%, p = 0.02) and unexplained by metaregression.

Interpretation:

We provide empirical evidence for observer bias in randomized clinical trials with subjective measurement scale outcomes. A failure to blind assessors of outcomes in such trials results in a high risk of substantial bias.A failure to blind assessors of outcomes in randomized clinical trials may result in bias. Observer bias, sometimes called “detection bias” or “ascertainment bias,” occurs when outcome assessments are systematically influenced by the assessors’ conscious or unconscious predispositions — for example, because of hope or expectations, often favouring the experimental intervention.¹Blinded outcome assessors are used in many trials to avoid such bias. However, the use of non-blinded assessors remains common,^2–4 especially in nonpharmacological trials; for example, non-blinded outcome assessment was used in 90% of trials involving orthopedic traumatology³ and 74% of trials involving strength training for muscles.⁴Unfortunately, the empirical evidence on observer bias in randomized clinical trials has been incomplete. Meta-epidemiological studies have compared double-blind trials with similar trials that were not double-blind.^5,6 However, such studies address blinding crudely because “double-blind” is an ambiguous term.^3,7 Furthermore, the risk of confounding is considerable in indirect between-trial analyses, as “double-blind” trials may have better overall methods and larger sample sizes than trials that are not reported as “double-blind.”A more reliable approach involves analyses of trials that use both blinded and nonblinded outcome assessors, because such a within-trial design provides a direct comparison between blinded and nonblinded assessments of the same outcome in the same patients. Our previous analysis of such trials with binary outcomes found substantial observer bias.⁸Although subjective measurement scales such as illness severity scores are popular, they may be susceptible to observer bias. They are frequently used as outcomes in clinical scenarios with no naturally distinct categories, and adjacent subcategories on a scale typically involve minor and vaguely defined differences.We decided to systematically review trials with both blinded and nonblinded assessment of outcomes using the same measurement scales. Our primary objective was to evaluate the impact of nonblinded outcome assessment on estimated treatment effects in randomized clinical trials. Our secondary objective was to examine reasons for variation in observer bias.