Phylogeographic evidence for two mesic refugia in a biodiversity hotspot

Phylogeographic studies of flora in species-rich south-western Australia point to complex evolutionary histories, reflecting patterns of persistence and resilience to climatic changes during the Pleistocene. We asked whether coastal areas of the mid-west and south, as well as granite outcrops and inland ranges, have acted as major refugia within this region during Pleistocene climatic fluctuations by analysing phylogeographic patterns in the shrub Calothamnus quadrifidus R.Br. (Myrtaceae). We determined variation in chloroplast DNA data for 41 populations across the geographic range. Relationships and major clades were resolved using parsimony and Bayesian analyses. We tested for demographic and spatial expansion of the major clades and estimated clade divergence dates using an uncorrelated, lognormal relaxed clock based on two conservative chloroplast mutation rates. Two distinct phylogeographic clades were identified showing divergence during the Pleistocene, consistent with other phylogeographic studies of south-west Australian flora, emphasising the impact of climatic oscillations in driving divergence in this landscape. The southern clade was more diverse, having higher haplotype diversity and greater genetic structure, while the northern clade showed evidence of fluctuation in population size. Regions of high haplotype diversity with adjacent areas of low diversity observed in each clade indicated the locations of two coastal refugia: one on the south coast and another along the mid-west coast. This is the first evidence for major Pleistocene refugia using chloroplast genetic data in a common, widespread species from this region.     

SearchDOGS Bacteria,Software That Provides Automated Identification of Potentially Missed Genes in Annotated Bacterial Genomes

We report the development of SearchDOGS Bacteria, software to automatically detect missing genes in annotated bacterial genomes by combining BLAST searches with comparative genomics. Having successfully applied the approach to yeast genomes, we redeveloped SearchDOGS to function as a standalone, downloadable package, requiring only a set of GenBank annotation files as input. The software automatically generates a homology structure using reciprocal BLAST and a synteny-based method; this is followed by a scan of the entire genome of each species for unannotated genes. Results are provided in a HTML interface, providing coordinates, BLAST results, syntenic location, omega values (Ka/Ks, where Ks is the number of synonymous substitutions per synonymous site and Ka is the number of nonsynonymous substitutions per nonsynonymous site) for protein conservation estimates, and other information for each candidate gene. Using SearchDOGS Bacteria, we identified 155 gene candidates in the Shigella boydii sb227 genome, including 56 candidates of length < 60 codons. SearchDOGS Bacteria has two major advantages over currently available annotation software. First, it outperforms current methods in terms of sensitivity and is highly effective at identifying small or highly diverged genes. Second, as a freely downloadable package, it can be used with unpublished or confidential data.     

Mathematical Model of Hyperbaric Oxygen Therapy Applied to Chronic Diabetic Wounds

The failure of certain wounds to heal (including diabetic foot ulcers) is a significant socioeconomic issue for countries worldwide. There is much debate about the best way to treat these wounds and one approach that is shrouded with controversy is hyperbaric oxygen therapy (HBOT), a technique that can reduce the risk of amputation in diabetic patients.     

Genetic barcoding of commercial Bêche-de-mer species (Echinodermata: Holothuroidea)

There are more than 47 species of holothurians used for bêche-de-mer production, many of which are locally overfished. With three exceptions, all bêche-de-mer species are Aspidochirotida and species identification of many of these is difficult. We analysed available genetic information and newly generated sequences to determine if genetic barcoding with the mitochondrial COI gene can be used to identify bêche-de-mer species. Although genetic data were available for ～50% of bêche-de-mer species, sufficient information and within-species replication were only available for six species. We generated 96 new COI sequences extending the existing database to cover most common species. COI unambiguously identified most bêche-de-mer species providing a genetic barcode for the identification of known species. In addition, conspecific (1.3%) variation and congeneric (16.9%) divergence were well separated ('barcoding-gap') albeit with a small overlap, which may lead to some error if genetic sampling alone was applied for species discovery. In addition to identification of adults, COI sequences were useful to identify juveniles that are often morphologically different. Sequence data showed that large (deep) and small (shallow) morphotypes of Holothuria atra are the same species, but suggested potential cryptic species within this taxon. For bêche-de-mer, the COI barcode proved useful in species clarification and discovery, but further genetic and taxonomic work is essential for several species. Some bêche-de-mer clades were problematic with morphologically disparate specimens sharing the same barcode. Our study indicated the presence of undescribed species (Bohadschia sp.) and species that constitute separate species in the Indian and Pacific Ocean (e.g. Holothuria fuscogilva).     

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.     

Campylobacter insulaenigrae isolates from northern elephant seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42 degrees C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: lack of evidence for other than a two state thermal denaturation

It is unclear whether the thermal denaturation of staphylococcal nuclease is a two state, three state, or variable two state process. The thermal denaturation of wild-type staphylococcal nuclease was followed by tryptophan fluorescence and circular dichroism signal at 222 nm, forty-two and fourteen times, respectively. Analysis of this data using a simple two state model gave melting temperatures of 53.0+/-0.4 degrees C (fluorescence) and 52.7+/-0.6 degrees C (CD) and van't Hoff enthalpies of 82.4+/-2.6 kcal/mol and 88.6+/-4.2 kcal/mol. Ninety-seven mutants also had these parameters determined by both fluorescence and CD. The average difference between the melting temperatures was 1.05+/-0.75 degrees and the average difference between van't Hoff enthalpies was 1.6+/-4.8 kcal/mol. These very similar results for the two spectroscopic probes of structure are discussed in the context of the different models that have been proposed for nuclease denaturation. It is concluded, for most nuclease variants, that the errors introduced by a two state assumption are negligible and either virtually all helical structure is lost in any initial unfolding event or any intermediate must have low stability.