A 9 angstroms single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscope

Sub-nanometer resolution structure determination is becoming a common practice in electron cryomicroscopy of macromolecular assemblies. The data for these studies have until now been collected on photographic film. Using cytoplasmic polyhedrosis virus (CPV), a previously determined structure, as a test specimen, we show the feasibility of obtaining a 9 angstroms structure from images acquired from a 4 k x 4 k Gatan CCD on a 200 kV electron cryomicroscope. The match of the alpha-helices in the protein components of the CPV with the previous structure of the same virus validates the suitability of this type of camera as the recording media targeted for single particle reconstructions at sub-nanometer resolution.     

Sciurid phylogeny and the paraphyly of Holarctic ground squirrels (Spermophilus)

The squirrel family, Sciuridae, is one of the largest and most widely dispersed families of mammals. In spite of the wide distribution and conspicuousness of this group, phylogenetic relationships remain poorly understood. We used DNA sequence data from the mitochondrial cytochrome b gene of 114 species in 21 genera to infer phylogenetic relationships among sciurids based on maximum parsimony and Bayesian phylogenetic methods. Although we evaluated more complex alternative models of nucleotide substitution to reconstruct Bayesian phylogenies, none provided a better fit to the data than the GTR+G+I model. We used the reconstructed phylogenies to evaluate the current taxonomy of the Sciuridae. At essentially all levels of relationships, we found the phylogeny of squirrels to be in substantial conflict with the current taxonomy. At the highest level, the flying squirrels do not represent a basal divergence, and the current division of Sciuridae into two subfamilies is therefore not phylogenetically informative. At the tribal level, the Neotropical pygmy squirrel, Sciurillus, represents a basal divergence and is not closely related to the other members of the tribe Sciurini. At the genus level, the sciurine genus Sciurus is paraphyletic with respect to the dwarf squirrels (Microsciurus), and the Holarctic ground squirrels (Spermophilus) are paraphyletic with respect to antelope squirrels (Ammospermophilus), prairie dogs (Cynomys), and marmots (Marmota). Finally, several species of chipmunks and Holarctic ground squirrels do not appear monophyletic, indicating a need for reevaluation of alpha taxonomy.     

Turtle phylogeny: insights from a novel nuclear intron

Introns have gained considerable popularity as markers for molecular phylogenetics. However, no primers exist for a nuclear intron that amplifies across all turtles. Available data from morphology and mitochondrial DNA have not unambiguously resolved relationships within the superfamily Trionychoidea and the family Chelidae, which together form a large portion of extant turtle diversity. We tested the phylogenetic utility of a novel intron from the RNA fingerprint protein 35 (R35) as applied to these two areas of turtle systematics. We found the intron to be a single-copy locus that provides excellent resolving power for lineages among turtles, though problems with alignment made it impossible to infer deeper amniote relationships. Maximum parsimony and maximum likelihood both demonstrated the polyphyly of Trionychoidea and the reciprocal monophyly of Australian/New Guinea and South American chelid turtles. This is the first study to resolve such relationships with strong statistical support, and we suggest that R35 holds great promise for resolving additional persistent problems in the phylogeny of living turtles.     

Linking phylogenetics with population genetics to reconstruct the geographic origin of a species

Reconstructing ancestral geographic origins is critical for understanding the long-term evolution of a species. Bayesian methods have been proposed to test biogeographic hypotheses while accommodating uncertainty in phylogenetic reconstruction. However, the problem that certain taxa may have a disproportionate influence on conclusions has not been addressed. Here, we infer the geographic origin of Drosophila simulans using 2,014 bp of the period locus from 63 lines collected from 18 countries. We also analyze two previously published datasets, alcohol dehydrogenase related and NADH:ubiquinone reductase 75 kDa subunit precursor. Phylogenetic inferences of all three loci support Madagascar as the geographic origin of D. simulans. Our phylogenetic conclusions are robust to taxon resampling and to the potentially confounding effects of recombination. To test our phylogenetically derived hypothesis we develop a randomization test of the population genetics prediction that sequences from the geographic origin should contain more genetic polymorphism than those from derived populations. We find that the Madagascar population has elevated genetic polymorphism relative to non-Madagascar sequences. These data are corroborated by mitochondrial DNA sequence data.     

Synthesizing strength and conditioning research: the meta-analysis

As the body of research examining strength and conditioning topics grows, it is vital that researchers be able to effectively synthesize research findings. The meta-analysis has been shown to be a more advanced form of research synthesis than the conventional narrative review. Meta-analytic procedures are discussed and applications to strength related research are presented.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates the recently completed P. falciparum genome sequence and annotation, as well as draft sequence and annotation emerging from other Plasmodium sequencing projects. PlasmoDB currently houses information from five parasite species and provides tools for intra- and inter-species comparisons. Sequence information is integrated with other genomic-scale data emerging from the Plasmodium research community, including gene expression analysis from EST, SAGE and microarray projects and proteomics studies. The relational schema used to build PlasmoDB, GUS (Genomics Unified Schema) employs a highly structured format to accommodate the diverse data types generated by sequence and expression projects. A variety of tools allow researchers to formulate complex, biologically-based, queries of the database. A stand-alone version of the database is also available on CD-ROM (P. falciparum GenePlot), facilitating access to the data in situations where internet access is difficult (e.g. by malaria researchers working in the field). The goal of PlasmoDB is to facilitate utilization of the vast quantities of genomic-scale data produced by the global malaria research community. The software used to develop PlasmoDB has been used to create a second Apicomplexan parasite genome database, ToxoDB (http://ToxoDB.org).     

Chemical identification of<Emphasis Type="Italic"> N</Emphasis>-acyl homoserine lactone quorum-sensing signals produced by<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> strains in defined medium

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.