Does bait type and bait container configuration influence the performance of remote underwater video systems in temperate freshwater lakes for assessing fish community structure?

Hydrobiologia - Methods for the use of baited remote underwater video stations (BRUVS) have been tested and refined such that they are now widely used in marine research for assessing fish...     

Comparisons between Geographically Diverse Samples of Carried Staphylococcus aureus

Approximately one-third of the human population is asymptomatically colonized by Staphylococcus aureus. However, much of the global diversity within the carriage populations remains uncharacterized, and it is unclear to what degree the variation is geographically partitioned. We isolated 300 carriage isolates from 1,531 adults contemporaneously in four countries: France, Algeria, Moldova, and Cambodia. All strains were characterized by multilocus sequence typing. Six clonal complexes (CCs) were present in all four samples (CC30, -45, -121, -15, -5, and -8). Analyses based on the genotype frequencies revealed the French and Algerian samples to be most similar and the Cambodian sample to be most distinct. While this pattern is consistent with likely rates of human migration and geographic distance, stochastic clonal expansion also contributes to regional differences. Phylogenetic analysis revealed a highly divergent and uncharacterized genotype (ST1223) within Cambodia. This lineage is related to CC75, which has previously been observed only in remote aboriginal populations in northern Australia.Although better known as an important human pathogen, Staphylococcus aureus is typically a commensal species and asymptomatically colonizes approximately one-third of the human population globally (18, 20, 29). This high carriage rate potentially represents a vast reservoir of as-yet-uncharacterized S. aureus diversity, an appreciation of which should shed light on the forces underpinning the diversification and dissemination of S. aureus. There are comparatively few studies examining spatial or temporal genotype distributions within carriage populations, and the extent of biogeographical structure is currently unclear, as is the level of discrimination which might be required to detect such structure.Multilocus sequence typing (MLST) has proved to be very successful as an epidemiological tool in that it delimits S. aureus in to a small number of widespread and discrete clonal complexes (CCs) (6, 8). These can be readily identified as clusters of related genotypes which have diversified radially from “founder” genotypes (9), and because this organism is largely clonal (8), assignments of isolates to these groups is broadly robust to the many different typing methods employed (4, 10, 27). The high level of divergence between these lineages suggests that they are relatively ancient and temporally stable (7), and it is possible that isolated host populations may have been colonized by different S. aureus lineages in the past. However, any footprints of geographical partitioning are likely to have been compromised by high rates of migration in recent times, due largely to the advent of air travel.Previous studies addressing the characterization of carried populations have tended to focus on samples from Western Europe or North America, and these have generally not provided strong evidence for geographical structuring. In a recent study using amplified fragment length polymorphism to compare the carried populations in Holland and North America, the authors noted considerable overlap between the samples, suggesting that they effectively constituted a single unstructured population (17). Similarly, independent MLST studies have revealed regional consistencies in Europe, such as the predominance of CC30 in the United Kingdom (8), Ireland (3), and Switzerland (25). Given the high rates of admixture within Europe and North America, the absence of obvious geographical structuring in the carried S. aureus population in these regions is perhaps not very surprising.Although they are currently scarce, current data from carriage populations outside of Europe or North America point to greater geographical structuring. For example, a sample of carried S. aureus recovered from Bamako, Mali, has recently been characterized, constituting the first such study of an African population (23). Although many of the previously characterized CCs were also present in this sample, the authors noted a high frequency (∼25%) of a single genotype, ST152, which is phylogenetically divergent and noted very rarely in Europe. The high frequency of ST152 in this population raises the possibility that this genotype is endemic to the Malian population and possibly elsewhere in sub-Saharan Africa. This in turn hints at greater geographical partitioning on a global scale, although more representative samples are clearly required. To address this, we generated MLST data from contemporaneous carriage samples recovered from four countries representing three continents: France (Western Europe), Moldova (Eastern Europe), Algeria (North Africa), and Cambodia (Southeast Asia). To our knowledge, this is the first time such a study has been carried out on samples from Eastern Europe, North Africa, or Southeast Asia. These data were therefore generated to uncover diversity within the global carriage population but also to understand further the extent to which geographical distance and host migration can explain regional differences.     

High-throughput protein purification and quality assessment for crystallization

The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. "Structural biology-grade" proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are discussed in this chapter.     

Demethylation of Circulating Estrogen Receptor Alpha Gene in Cerebral Ischemic Stroke

Background

Estrogen is involved in neuron plasticity and can promote neuronal survival in stroke. Its actions are mostly exerted via estrogen receptor alpha (ERα). Previous animal studies have shown that ERα is upregulated by DNA demethylation following ischemic injury. This study investigated the methylation levels in the ERα promoter in the peripheral blood of ischemic stroke patients.

Methods

The study included 201 ischemic stroke patients, and 217 age- and sex-comparable healthy controls. The quantitative methylation level in the 14 CpG sites of the ERα promoter was measured by pyrosequencing in each participant. Multivariate regression model was used to adjust for stroke traditional risk factors. Stroke subtypes and sex-specific analysis were also conducted.

Results

The results demonstrated that the stroke cases had a lower ERα methylation level than controls in all 14 CpG sites, and site13 and site14 had significant adjusted p-values of 0.035 and 0.026, respectively. Stroke subtypes analysis showed that large-artery atherosclerosis and cardio-embolic subtypes had significantly lower methylation levels than the healthy controls at CpG site5, site9, site12, site13 and site14 with adjusted p = 0.039, 0.009, 0.025, 0.046 and 0.027 respectively. However, the methylation level for the patients with small vessel subtype was not significant. We combined the methylation data from the above five sites for further sex-specific analysis. The results showed that the significant association only existed in women (adjusted p = 0.011), but not in men (adjusted p = 0.300).

Conclusions

Female stroke cases have lower ERα methylation levels than those in the controls, especially in large-artery and cardio-embolic stroke subtypes. The study implies that women suffering from ischemic stroke of specific subtype may undergo different protective mechanisms to reduce the brain injury.     

EsxB,a secreted protein from Bacillus anthracis forms two distinct helical bundles

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.     

Sensor Domain of Histidine Kinase KinB of Pseudomonas: A HELIX-SWAPPED DIMER*

The overproduction of polysaccharide alginate is responsible for the formation of mucus in the lungs of cystic fibrosis patients. Histidine kinase KinB of the KinB-AlgB two-component system in Pseudomonas aeruginosa acts as a negative regulator of alginate biosynthesis. The modular architecture of KinB is similar to other histidine kinases. However, its periplasmic signal sensor domain is unique and is found only in the Pseudomonas genus. Here, we present the first crystal structures of the KinB sensor domain. The domain is a dimer in solution, and in the crystal it shows an atypical dimer of a helix-swapped four-helix bundle. A positively charged cavity is formed on the dimer interface and involves several strictly conserved residues, including Arg-60. A phosphate anion is bound asymmetrically in one of the structures. In silico docking identified several monophosphorylated sugars, including β-d-fructose 6-phosphate and β-d-mannose 6-phosphate, a precursor and an intermediate of alginate synthesis, respectively, as potential KinB ligands. Ligand binding was confirmed experimentally. Conformational transition from a symmetric to an asymmetric structure and decreasing dimer stability caused by ligand binding may be a part of the signal transduction mechanism of the KinB-AlgB two-component system.