Stocking may increase mitochondrial DNA diversity but fails to halt the decline of endangered Atlantic salmon populations

Over the last 50 years, Spanish Atlantic salmon (Salmo salar) populations have been in decline. In order to bolster these populations, rivers were stocked with fish of northern European origin during the period 1974–1996, probably also introducing the furunculosis-inducing pathogen, Aeromonas salmonicida. Here we assess the relative importance of processes influencing mitochondrial (mt)DNA variability in these populations from 1948 to 2002. Genetic material collected over this period from four rivers in northern Spain (Cantabria) was used to detect variability at the mtDNA ND1 gene. Before stocking, a single haplotype was found at high frequency (0.980). Following stocking, haplotype diversity (h) increased in all rivers (mean h before stocking was 0.041, and 0.245 afterwards). These increases were due principally to the dramatic increase in frequency of a previously very low frequency haplotype, reported at higher frequencies in northern European populations proximate to those used to stock Cantabrian rivers. Genetic structuring increased after stocking: among-river differentiation was low before stocking (1950s/1960s Φ _ST = –0.00296–0.00284), increasing considerably at the height of stocking (1980s Φ _ST = 0.18932) and decreasing post-stocking (1990s/2002 Φ _ST = 0.04934–0.03852). Gene flow from stocked fish therefore seems to have had a substantial role in increasing mtDNA variability. Additionally, we found significant differentiation between individuals that had probably died from infectious disease and apparently healthy, angled fish, suggesting a possible role for pathogen-driven selection of mtDNA variation. Our results suggest that stocking with non-native fish may increase genetic diversity in the short term, but may not reverse population declines.     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.     

Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.     

Independently evolving species in asexual bdelloid rotifers

Asexuals are an important test case for theories of why species exist. If asexual clades displayed the same pattern of discrete variation as sexual clades, this would challenge the traditional view that sex is necessary for diversification into species. However, critical evidence has been lacking: all putative examples have involved organisms with recent or ongoing histories of recombination and have relied on visual interpretation of patterns of genetic and phenotypic variation rather than on formal tests of alternative evolutionary scenarios. Here we show that a classic asexual clade, the bdelloid rotifers, has diversified into distinct evolutionary species. Intensive sampling of the genus Rotaria reveals the presence of well-separated genetic clusters indicative of independent evolution. Moreover, combined genetic and morphological analyses reveal divergent selection in feeding morphology, indicative of niche divergence. Some of the morphologically coherent groups experiencing divergent selection contain several genetic clusters, in common with findings of cryptic species in sexual organisms. Our results show that the main causes of speciation in sexual organisms, population isolation and divergent selection, have the same qualitative effects in an asexual clade. The study also demonstrates how combined molecular and morphological analyses can shed new light on the evolutionary nature of species.     

Retrovirus-polymer complexes: study of the factors affecting the dose response of transduction

We have previously shown that complexes of Polybrene (PB), chondroitin sulfate C (CSC), and retrovirus transduce cells more efficiently than uncomplexed virus because the complexes are large and sediment, reaching the cells more rapidly than by diffusion. Transduction reaches a peak at equal weight concentrations of CSC and PB and declines when the dose of PB is higher or lower than CSC. We hypothesized that the nonlinear dose response of transduction was a complex function of the molecular characteristics of the polymers, cell viability, and the number of viruses incorporated into the complexes. To test this hypothesis, we formed complexes using an amphotropic retrovirus and several pairs of oppositely charged polymers and used them to transduce murine fibroblasts. We examined the effect of the type and concentration of polymers used on cell viability, the size and charge of the complexes, the number of viruses incorporated into the complexes, and virus binding and transduction. Transduction was enhanced (2.5- to 5.5-fold) regardless of which polymers were used and was maximized when the number of positive charge groups was in slight excess (15-28%) of the number of negative charge groups. Higher doses of cationic polymer were cytotoxic, whereas complexes formed with lower doses were smaller, contained fewer viruses, and sedimented more slowly. These results show that the dose response of transduction by virus-polymer complexes is nonlinear because excess cationic polymer is cytotoxic, whereas excess anionic polymer reduces the number of active viruses that are delivered to the cells.     

A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells

Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of porcine bone-marrow-derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 x 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three-dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two-dimensional monolayer cultivation method.     

Early queen infection shapes developmental dynamics and induces long-term disease protection in incipient ant colonies

Infections early in life can have enduring effects on an organism's development and immunity. In this study, we show that this equally applies to developing ‘superorganisms’––incipient social insect colonies. When we exposed newly mated Lasius niger ant queens to a low pathogen dose, their colonies grew more slowly than controls before winter, but reached similar sizes afterwards. Independent of exposure, queen hibernation survival improved when the ratio of pupae to workers was small. Queens that reared fewer pupae before worker emergence exhibited lower pathogen levels, indicating that high brood rearing efforts interfere with the ability of the queen's immune system to suppress pathogen proliferation. Early-life queen pathogen exposure also improved the immunocompetence of her worker offspring, as demonstrated by challenging the workers to the same pathogen a year later. Transgenerational transfer of the queen's pathogen experience to her workforce can hence durably reduce the disease susceptibility of the whole superorganism.