Contrasting genetic diversity and population structure among three sympatric Madagascan shorebirds: parallels with rarity,endemism, and dispersal

Understanding the relative contributions of intrinsic and extrinsic factors to population structure and genetic diversity is a central goal of conservation and evolutionary genetics. One way to achieve this is through comparative population genetic analysis of sympatric sister taxa, which allows evaluation of intrinsic factors such as population demography and life history while controlling for phylogenetic relatedness and geography. We used ten conserved microsatellites to explore the population structure and genetic diversity of three sympatric and closely related plover species in southwestern Madagascar: Kittlitz's plover (Charadrius pecuarius), white‐fronted plover (C. marginatus), and Madagascar plover (C. thoracicus). Bayesian clustering revealed strong population structure in the rare and endemic Madagascar plover, intermediate population structure in the white‐fronted plover, and no detectable population structure in the geographically widespread Kittlitz's plover. In contrast, allelic richness and heterozygosity were highest for the Kittlitz's plover, intermediate for the white‐fronted plover and lowest for the Madagascar plover. No evidence was found in support of the “watershed mechanism” proposed to facilitate vicariant divergence of Madagascan lemurs and reptiles, which we attribute to the vagility of birds. However, we found a significant pattern of genetic isolation by distance among populations of the Madagascar plover, but not for the other two species. These findings suggest that interspecific variation in rarity, endemism, and dispersal propensity may influence genetic structure and diversity, even in highly vagile species.     

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10⁷ transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P_pampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ~91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.     

SWIMMING SPEEDS OF SINGING AND NON-SINGING HUMPBACK WHALES DURING MIGRATION

Limited data exist on swimming speeds of humpback whales ( Megaptera novaeangliae ) and none on swimming speeds of singing whales during migration. We tracked humpback whales visually and acoustically during migration from the breeding grounds past our study site on the east coast of Australia (latitude 26°28'S). The mean swimming speed for whales while singing was 2.5 km/h, significantly less than for non-singing whales with a mean of 4.0 km/h but significantly greater than the mean of 1.6 km/h observed for singing whales on the Hawaiian breeding grounds. Between song sessions, there was no significant difference in speeds between whales that had been singing and other whales. Migration speeds were less for whales while singing but increased during the season. Although humpback whales can swim rapidly while singing (maximum observed 15.6 km/h), they generally do not do so, even during migration. Slower migration by singers would delay their return to the polar feeding areas and may be costly, but may be a strategy to provide access to more females.     

Mitogen-activated protein kinase phosphatase 1/dual specificity phosphatase 1 mediates glucocorticoid inhibition of osteoblast proliferation

Steroid-induced osteoporosis is a common side effect of long-term treatment with glucocorticoid (GC) drugs. GCs have multiple systemic effects that may influence bone metabolism but also directly affect osteoblasts by decreasing proliferation. This may be beneficial at low concentrations, enhancing differentiation. However, high-dose treatment produces a severe deficit in the proliferative osteoblastic compartment. We provide causal evidence that this effect of GC is mediated by induction of the dual-specificity MAPK phosphatase, MKP-1/DUSP1. Excessive MKP-1 production is both necessary and sufficient to account for the impaired osteoblastic response to mitogens. Overexpression of MKP-1 after either GC treatment or transfection ablates the mitogenic response in osteoblasts. Knockdown of MKP-1 using either immunodepletion of MKP-1 before in vitro dephosphorylation assay or short interference RNA transfection prevents inactivation of ERK by GCs. Neither c-jun N-terminal kinase nor p38 MAPK is activated by the mitogenic cocktail in 20% fetal calf serum, but their activation by a DNA-damaging agent (UV irradiation) was inhibited by either GC treatment or overexpression of MKP-1, indicating regulation of all three MAPKs by MKP-1 in osteoblasts. However, an inhibitor of the MAPK/ERK kinase-ERK pathway inhibited osteoblast proliferation whereas inhibitors of c-jun N-terminal kinase or p38 MAPK had no effect, suggesting that ERK is the MAPK that controls osteoblast proliferation. Regulation of ERK by MKP-1 provides a novel mechanism for control of osteoblast proliferation by GCs.     

Development of injectable thermogelling chitosan-inorganic phosphate solutions for biomedical applications

Thermosetting polymers are attractive candidates for biomedical applications as noninvasive therapeutic delivery vehicles. In the present study, the feasibility of developing a neutral physiological temperature setting injectable formulation based on chitosan and an inorganic phosphate salt have been demonstrated. The in situ gelling system was developed by adding different concentrations of ammonium hydrogen phosphate (AHP) to chitosan solution. The resulting solutions have pH in the range of approximately 7-7.2. The gelling time of the chitosan-AHP solution was determined by incubating the solutions at 37 degrees C. Depending on the concentrations of AHP added, the gelling time varied from 5 min to 30 h at 37 degrees C. Addition of various diluents to chitosan-AHP solution did not significantly change the gelling time of the solutions. The gels were found to be cytocompatible as evidenced from in vitro cytocompatibility evaluation using MC3T3-E1 mouse osteoblast like cells. The feasibility of using the gels as a stem cell carrier vehicle as well as a macromolecular delivery vehicle has been demonstrated.     

Comparative genomics of <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> with emphasis on strains from meat

Lactobacillus sakei is a lactic acid bacterium important in food microbiology mainly due to its ability to ferment and preserve meat. The genome sequence of L. sakei strain 23K has revealed specialized metabolic capacities that reflect the bacterium’s adaption to meat products, and that differentiate it from other LAB. An extensive genomic diversity analysis was conducted to elucidate the core features of the species, and to provide a better comprehension of niche adaptation of the organism. Here, we describe the genomic comparison of 18 strains of L. sakei originating mainly from processed meat against the 23K strain by comparative genome hybridization. Pulsed field gel electrophoresis was used to estimate the genome sizes of the strains, which varied from 1.880 to 2.175 Mb, and the 23K genome was among the smallest. Consequently, a large part of the genome of this strain belongs to a common gene pool invariant in this species. The majority of genes important in adaption to meat products, the ability to flexibly use meat components, and robustness during meat processing and storage were conserved, such as genes involved in nucleoside scavenging, catabolism of arginine, and the ability to cope with changing redox and oxygen levels, which is indicative of the role these genes play in niche specialization within the L. sakei species. Moreover, an additional set of sequenced L. sakei genes beyond the 23K genome was present on the microarray used, and it was demonstrated that all the strains carry remnants of or complete bacteriocin operons. The genomic divergence corresponded mainly to five regions in the 23K genome, which showed features consistent with horizontal gene transfer. Carbohydrate-fermentation profiles of the strains were evaluated in light of the CGH data, and for most substrates, the genotypes were consistent with the phenotypes. We have demonstrated a highly conserved organization of the L. sakei genomes investigated, and the 23K strain is a suitable model organism to study core features of the L. sakei species.     

Radiation-induced free-radical transformation of phospholipids: MALDI-TOF MS study

Under the action of free-radical reaction initiators on membrane phospholipids, complex processes are taking place in both hydrophobic and hydrophilic parts of the phospholipids. Realization of these processes results in a mixture consisting of the initial lipids and their peroxidation and fragmentation products. Identification of compounds in such mixtures requires analytical methods of high sensitivity, reproducibility and accuracy to be applied. These properties are characteristic of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. In the studies of radiation-induced free-radical transformations of phosphatidylglycerol, the MALDI-TOF MS in combination with thin layer chromatography (TLC) has been shown to be able to detect and identify products of free-radical transformations taking place in both hydrophilic and hydrophobic parts of the phospholipid. Thus, the MALDI-TOF MS can serve as a suitable analytical tool to investigate free-radical transformations of lipids.     

Construction and Application of a luxABCDE Reporter System for Real-Time Monitoring of Enterococcus faecalis Gene Expression and Growth

The present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging of Enterococcus faecalis. To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains of E. faecalis were constructed. pSL101 harbors the luxABCDE operon from pPL2lux and the pREG696 broad-host-range replicon and axe-txe toxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly (R(2) > 0.98) with the viable-cell count. We employed lux-tagged E. faecalis strains to monitor growth in real time in milk and urine in vitro. Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in the Galleria mellonella model system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI of E. faecalis and an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.