Molecular Signatures Identify a Candidate Target of Balancing Selection in an arcD-Like Gene of Staphylococcus epidermidis

A comparative population genetics study revealed high levels of nucleotide polymorphism and intermediate-frequency alleles in an arcC gene of Staphylococcus epidermidis, but not in a homologous gene of the more aggressive human pathogen, Staphylococcus aureus. Further investigation showed that the arcC genes used in the multilocus sequence typing schemes of these two species were paralogs. Phylogenetic analyses of arcC-containing loci, including the arginine catabolic mobile element, from both species, suggested that these loci had an eventful history involving gene duplications, rearrangements, deletions, and horizontal transfers. The peak signatures in the polymorphic S. epidermidis locus were traced to an arcD-like gene adjacent to arcC; these signatures consisted of unusually elevated Tajima’s D and π/K ratios, which were robust to assumptions about recombination and species divergence time and among the most elevated in the S. epidermidis genome. Amino acid polymorphisms, including one that differed in polarity and hydropathy, were located in the peak signatures and defined two allelic lineages. Recombination events were detected between these allelic lineages and potential donors and recipients of S. epidermidis were identified in each case. By comparison, the orthologous gene of S. aureus showed no unusual signatures. The ArcD-like protein belonged to the unknown ion transporter 3 family and appeared to be unrelated to ArcD from the arginine deiminase pathway. These studies report the first comparative population genetics results for staphylococci and the first statistical evidence for a candidate target of balancing selection in S. epidermidis.     

A comparison of two approaches to extracting Cryptosporidium DNA from human stools as measured by a real-time PCR assay

Direct extraction of Cryptosporidium DNA from 46 stools by bead-beating, guanidine thiocyanate and silica purification provided slightly lower PCR positivity (93.5% vs. 100%) and higher threshold cycle values (mean 34.93 vs. 28.03; P=0.00) than spin-column extraction from boiled, semi-purified oocyst suspensions. However, direct extraction is cheaper, and amenable to automation.     

Reductions in primate abundance and diversity in a multiuse protected area: synergistic impacts of hunting and logging in a congo basin forest

This article explores spatial and temporal changes in diurnal primate abundance and behavior in response to hunting, logging, and conservation at the Dzanga Sangha Dense Forest Reserve (RDS), Central African Republic over time. We use a combination of line-transect surveys in 2002 and 2009 (N = 540 km) and ethnographic interviews (N = 210) to investigate changes in the status of cercopithecines and colobines at RDS, with additional comparisons to earlier work. This protected area was lightly logged in the 1970s and the park was gazetted in 1990, with multiple-use reserve sectors allocated. Since the park's inception, hunting and the trade of primates have increased, along with human migration, greater accessibility of arms, and reduction of preferred ungulate prey. Primates have declined in both the park and reserve sectors. Our data further suggest that at RDS hunting has had a greater impact on primate diversity and abundance than logging. We have identified changes in species-specific vulnerability to hunting over time, with Cercopithecus nictitans and Lophocebus albigena initially having appeared to be relatively resistant to hunting pressure in 2002. However, subsequently as gun hunting has increased at RDS, these species have become vulnerable. Although monkeys at RDS have been responding behaviorally to increased gun hunting, they are not able to keep pace with changing hunting practices. This study allows us to begin to understand synergistic impacts of hunting and logging, necessary if we are to recommend strategies to better secure the future of primates in multiuse protected areas.     

Tissue-specific effects of allergic rhinitis in mouse nasal epithelia

Allergic rhinitis (AR) can cause significant olfactory loss, but few studies have specifically investigated AR effects on olfactory and nasal respiratory tissues per se. To address this, we used a murine AR protocol employing nasal allergen infusion for both sensitization and challenges. Seven- to 11-week BALB/c mice were bilaterally infused with 1% ovalbumin (OVA) in phosphate-buffered saline (PBS) or PBS alone for 6 or 11 weeks, given single bilateral PBS or OVA infusions 24 h before sacrifice, or left untreated. High OVA-specific IgE serum levels and eosinophil infiltration confirmed AR induction. Olfactory (OE) and respiratory (RE) epithelia showed distinctly different responses, most conspicuously, massive eosinophil infiltration of immediately RE-subjacent lamina propria. In OE, such infiltration was minimal. Significant RE hypertrophy and hyperplasia also occurred, although OE organization was generally maintained and extensive disruption localized despite a 20% reduction in sensory neurons and globose basal cells after 11 weeks OVA. Pronounced Bowman's gland hypertrophy crowded both OE and olfactory nerve bundles. Cellular proliferation was widely distributed in RE but in OE was localized to normally thinner OE and RE-proximal OE, suggesting possible indirect RE influences. Terminal deoxynucleotide transferase (TdT) nick end labeling was greater in OE than RE and, in contrast to other effects, occurred with acute infusions and chronic PBS alone, often unilaterally. Following chronic OVA, AR-related bilateral increases appeared superimposed on those. These findings indicate AR effects on olfactory function may be complex, reflecting various levels of RE/OE responses and interactions.     

Cellular iron distribution in Bacillus anthracis

Although successful iron acquisition by pathogens within a host is a prerequisite for the establishment of infection, surprisingly little is known about the intracellular distribution of iron within bacterial pathogens. We have used a combination of anaerobic native liquid chromatography, inductively coupled plasma mass spectrometry, principal-component analysis, and peptide mass fingerprinting to investigate the cytosolic iron distribution in the pathogen Bacillus anthracis. Our studies identified three of the major iron pools as being associated with the electron transfer protein ferredoxin, the miniferritin Dps2, and the superoxide dismutase (SOD) enzymes SodA1 and SodA2. Although both SOD isozymes were predicted to utilize manganese cofactors, quantification of the metal ions associated with SodA1 and SodA2 in cell extracts established that SodA1 is associated with both manganese and iron, whereas SodA2 is bound exclusively to iron in vivo. These data were confirmed by in vitro assays using recombinant protein preparations, showing that SodA2 is active with an iron cofactor, while SodA1 is cambialistic, i.e., active with manganese or iron. Furthermore, we observe that B. anthracis cells exposed to superoxide stress increase their total iron content more than 2-fold over 60 min, while the manganese and zinc contents are unaffected. Notably, the acquired iron is not localized to the three identified cytosolic iron pools.     

Draft genome sequences of Staphylococcus aureus sequence type 34 (ST34) and ST42 hybrids

Staphylococcus aureus is a major cause of antimicrobial-resistant infections of humans. Hybrids of S. aureus, which originate from large-scale chromosomal recombinations between parents of distinct genetic backgrounds, are of interest from clinical and evolutionary perspectives. We present draft genome sequences of two S. aureus hybrids of sequence type 34 (ST34) and ST42.     

Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.     

Linking structural change with functional regulation-insights from mass spectrometry

A wide range of biophysical approaches has been applied to structural biology, all with the same overall goal-to understand the molecular machines that allow cells to function. While knowledge of the identity and composition of component protein subunits is an important foundation for understanding these macromolecular complexes it has become increasingly clear that knowledge of the exact composition alone is insufficient for understanding dynamic interactions and regulatory mechanisms. In this review we focus on recent developments of mass spectrometry (MS) that allow us to unravel the functional 'secrets' of non-covalent molecular machines.