Development and characterization of novel microsatellite markers for Arion slug species

Seventeen polymorphic microsatellite markers were isolated and characterized in Arion vulgaris/lusitanicus, which belongs to the worst European slug pests with serious economic and ecological impact. These markers were tested on 23 individuals collected in a population in Switzerland. Numbers of alleles ranged from 2 to 14 per locus, observed and expected heterozygosities ranged from 0.174 to 0.87, and from 0.162 to 0.903, respectively. These loci were also successfully amplified and were polymorphic in the closely related species A. rufus and A. ater. These loci represent the first highly polymorphic nuclear markers described for A. vulgaris and pave the way for population genetics and molecular ecology research of the important Arion pest slugs.

Association of TNF-alpha genetic variants with HPV-associated oral cancer risk---a case-control study

The incidence of OSCC in younger population and in those who never smoked or drank has increased since the last decade. This increase may be attributable to increase of infection with HPV. The pro-inflammatory cytokine TNF-&agr; has the role in the pathogenesis of chronic inflammatory diseases and was found to control HPV infection in cervical cancer studies. Our study aimed to investigate the association between the four polymorphisms located in TNF-&agr; promoter region, -308(rs1800629), -857(rs1799724), -863(rs1800630) and -1031(rs1799964), and the risk of HPV-related OSCC. In this hospital-based case-control study, 325 cases and 335 controls were included. We found that HPV 16 seropositivity was associated with an increased risk of oral cancer (OR = 3.1, 95% CI, 2.1–4.6). Each of the polymorphism showed to increase the risk of HPV-related OSCC. And after combining the risk genotypes and using the low-risk group (0–1 combined risk genotypes) and HPV16 seronegativity as the reference group, only the high-risk groups (3–4 combined risk genotypes) and HPV16 seronegativity were associated with a low OR of 1.8 (95% CI, 1.1–2.8), while the low-risk and high-risk groups and HPV16 seropositivity were significantly associated with a higher OR of 2.7 (95% CI, 1.3–5.8) and 8.5 (95% CI, 3.7–19.4), respectively. In addition, the joint effects were greater among the young subjects (aged<50), males, never smokers or never drinkers, and patients with oropharyngeal cancer. Overall, the four TNF-&agr; polymorphisms, individually or collectively, would result in a significantly increased risk for HPV16-associated oral cancer in a non-Hispanic white population. More large sized studies are needed for future investigation.^

Expression of TNF-$\alpha$ protein and C -FOS and TNF mRNA during human peripheral blood monocyte maturation and activation

Human peripheral blood monocytes (HPBM) were isolated by centrifugal elutriation from mononuclear cell enriched fractions after routine plateletapheresis and the relationship between maturation of HPBM to macrophage-like cells and activation for tumoricidal activity determined. HPBM were cultured for various times in RPMI 1640 supplemented with 5% pooled human AB serum and cytotoxicity to $\sp{125}$IUDR labeled A375M, a human melanoma cell line, and TNF-$\alpha$ release determined by cytolysis of actinomycin D treated L929 cells. Freshly isolated HPBM or those exposed to recombinant IFN-$\gamma$(1.0 U/ml) were not cytolytic and did not release TNF-$\alpha$ into culture supernatants. Exposure to bacterial lipopolysaccharide (LPS, 1.0 $\upsilon$g/ml) stimulated cytolytic activity and release of TNF-$\alpha$. Maximal release of TNF-$\alpha$ protein occurred at 8 hrs and returned to baseline by 72 hrs. Expression of TNF-$\alpha$ protein was determined by Western blotting. Neither freshly isolated nor IFN-$\gamma$ treated HPBM expressed TNF protein at any time during in vitro culture. LPS treated HPBM maximally expressed the 17KD TNF-$\alpha$ protein at 8 hrs, and protein was not detected after 36 hrs of in vitro culture. Expression of TNF-$\alpha$ mRNA was determined by Northern blotting. Freshly isolated HPBM express TNF-$\alpha$ mRNA which decays to basal levels by 6 hrs of in vitro culture. IFN-$\gamma$ treatment maintains TNF-$\alpha$ mRNA expression for up to 48 hrs of culture, after which it is undetectable. LPS induces TNF-$\alpha$ mRNA after 30 minutes of exposure with maximal accumulation occurring between 4 to 8 hrs. TNF mRNA was not detected in control HPBM at any time after 6 hrs or IFN-$\gamma$ treated HPBM after 48 hrs of in vitro culture. A pulse of LPS the last 24 hrs of in vitro culture induces the accumulation of TNF-$\alpha$ mRNA in HPBM cultured for 3, 5, and 7 days, with the magnitude of induction decreasing approximately 10 fold between 3 and 7 days. Induction of TNF-$\alpha$ mRNA occurred in the absence of detectable TNF-$\alpha$ protein or supernatant activity. Maturation of HPBM to macrophage-like cells controls competence for activation, magnitude and duration of the activation response. ^

Microbiota and Microsatellite genotypes of Pacific oysters stemming from three oyster bed in the Northern Wadden Sea

Background: Studies of oyster microbiomes have revealed that a limited number of microbes, including pathogens, can dominate microbial communities in host tissues such as gills and gut. Much of the bacterial diversity however remains underexplored and unexplained, although environmental conditions and host genetics have been implicated. We used 454 next generation 16S rRNA amplicon sequencing of individually tagged PCR reactions to explore the diversity of bacterial communities in gill tissue of the invasive Pacific oyster Crassostrea gigas stemming from genetically differentiated beds under ambient outdoor conditions and after a multifaceted disturbance treatment imposing stress on the host. Results: While the gill associated microbial communities in oysters were dominated by few abundant taxa (i.e. Sphingomonas, Mycoplasma) the distribution of rare bacterial groups correlated to relatedness between the hosts under ambient conditions. Exposing the host to disturbance broke apart this relationship by removing rare phylotypes thereby reducing overall microbial diversity. Shifts in the microbiome composition in response to stress did not result in a net increase in genera known to contain potentially pathogenic strains. Conclusion: The decrease in microbial diversity and the disassociation between population genetic structure of the hosts and their associated microbiome suggest that disturbance (i.e. stress) may play a significant role for the assembly of the natural microbiome. Such community shifts may in turn also feed back on the course of disease and the occurrence of mass mortality events in oyster populations.

Assessing the identity of the variety 'Pedro Giménez' grown in Argentina through the use of microsatellite markers

Pedro Giménez' is a white criolla variety cropped in Argentina, mainly in Mendoza and San Juan, being the most planted white variety destined for wine making in the country. Its origin remains unknown, as well as its relationship with Spanish variety 'Pedro Ximénez', mostly grown in Jerez, Spain. Previous works have probed that most of Criollas varieties existing in America at the moment, are the offspring of 'Muscat of Alexandria' x 'Criolla Chica'. The aim of the present work was to compare 'Pedro Giménez' with the Spanish variety 'Pedro Ximénez', and to establish its degree of relatedness to 'Muscat of Alexandria' and 'Criolla Chica'. Therefore we used a set of 18 nuclear SSR loci and 3 chloroplast SSR loci. 'Pedro Giménez' shared only 38% of the alleles under analysis with 'Pedro Ximénez', indicating that they are indeed two different varieties. In all 18 polymorphic nuclear SSR loci 'Pedro Giménez' shared 50% of its alleles with 'Muscat of Alexandria', while the other 50% of the alleles present in 'Pedro Giménez' were also present in 'Criolla Chica'. This data, along with those from the chloroplast SSR analysis, strongly suggest that 'Pedro Giménez' is the progeny of 'Muscat of Alexandria' x 'Criolla Chica', being the latest one the most likely female progenitor.

Percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by doxorubicin and TNF-alpha co-treatments

The present dataset contains the source data for Figure 2B of Tentner et al. (2012). The data shows the percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by various doses of doxorubicin (0, 2 and 10 µmole/L) in the presence (100 ng/mL) or absence (0 ng/mL) of TNF-alpha co-treatment. For the six treatment conditions investigated, cell counts were made by flow cytometry at times 6, 12, 24, and 48 h following treatment; CULTURE DETAILS: U2OS cells were obtained from ATCC were maintained at 21% oxygen and 5% CO2 in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2mM L-glutamine, and used within 15-20 passages. The first thymidine block was released by washing the plates three times with PBS, and incubating them in fresh thymidine-free media for 12 h. A second thymidine block was then performed by re-addition of thymidine to 2.5 mM followed by incubation for an additional 18 h. Media was aspirated, plates were washed 3 with PBS, and replaced with fresh media in the presence or absence of 10 mM aphidicolin; ANALYSIS DETAILS: See supplementary journal publication; RESULT: The authors of the supplementary journal publication conclude that TNF enhances dose-dependent cell death following doxorubicin-induced DNA damage with minimal affect on dose-dependent cell-cycle arrest.

Genotypic data for nine microsatellite loci for the seagrass Zostera muelleri collections from Lake Macquarie, NSW, Australia

Seagrasses are ecosystem engineers that offer important habitat for a large number of species and provide a range of ecosystem services. Many seagrass ecosystems are dominated by a single species; with research showing that genotypic diversity at fine spatial scales plays an important role in maintaining a range of ecosystem functions. However, for most seagrass species, information on fine-scale patterns of genetic variation in natural populations is lacking. In this study we use a hierarchical sampling design to determine levels of genetic and genotypic diversity at different spatial scales (centimeters, meters, kilometers) in the Australian seagrass Zostera muelleri. Our analysis shows that at fine-spatial scales (< 1 m) levels of genotypic diversity are relatively low (R (Plots) = 0.37 ± 0.06 SE), although there is some intermingling of genotypes. At the site (10's m) and meadow location (km) scale we found higher levels of genotypic diversity (R (sites) = 0.79 ± 0.04 SE; R (Locations) = 0.78 ± 0.04 SE). We found some sharing of genotypes between sites within meadows, but no sharing of genotypes between meadow locations. We also detected a high level of genetic structuring between meadow locations (FST = 0.278). Taken together, our results indicate that both sexual and asexual reproduction are important in maintaining meadows of Z. muelleri. The dominant mechanism of asexual reproduction appears to occur via localised rhizome extension, although the sharing of a limited number of genotypes over the scale of 10's of metres could also result from the localised dispersal and recruitment of fragments. The large number of unique genotypes at the meadow scale indicates that sexual reproduction is important in maintaining these populations, while the high level of genetic structuring suggests little gene flow and connectivity between our study sites. These results imply that recovery from disturbances will occur through both sexual and asexual regeneration, but the limited connectivity at the landscape-scale implies that recovery at meadow-scale losses is likely to be limited.

Rapid sizing tool for a low cost university microsatellite structure subsystem

In recent decays university class small satellites are creating many opportunities for space research and professional trainings while at the same time responding to constrained budgets. In this work the main focus is on developing a simple and rapid structural sizing tool considering the main objectives of a low cost university class microsatellite project. In satellite projects, structure subsystem is one of the influential subsystems as a driver of the cost and acceptance of the final design. At the first steps of such projects there is no confirmed data regarding the launch vehicle or even in some cases there is no data for the satellite payload. Due to these facts, developing simple sizing tools at conceptual design phase for obtaining an over view of the effect of different variables is useful before entering complex calculations in detailed design phases. In this study, after developing a simple analytical model of satellite structure subsystem, a design space is evaluated with practical boundaries considering mass and dimensions constraints of such projects. The results are useful to give initial insight to establish the system level structural sizing

Validation of microsatellite markers for cytotype discrimination in the model grass Brachypodium distachyon

Brachypodium distachyon (2n = 2x = 10) is a small annual grass species where the existence of three different cytotypes (10, 20 and 30 chromosomes) has long been regarded as a case of autopolyploid series, with x = 5. However, it has been demonstrated that the cytotypes assumed to be polyploids represent two separate Brachypodium species recently named as B. stacei (2n = 2x = 20) and B. hybridum (2n = 4x = 30). The aim of this study was to find a PCR-based alternative approach that could replace standard cytotyping methods (i. e., chromosome counting and flow cytometry) to characterize each of the three Brachypodium species. We have analyzed with four microsatellite (SSR) markers eighty-three Brachypodium distachyon-type lines from varied locations in Spain, including the Balearic and Canary Islands. Within this set of lines, 64, 4 and 15 had 10, 20 and 30 chromosomes, respectively. The surveyed markers produced cytotype-specific SSR profiles. So, a single amplification product was generated in the diploid samples, with non-overlapping allelic ranges between the 2n = 10 and 2n = 20 cytotypes, whereas two bands, one in the size range of each of the diploid cytotypes, were amplified in the 2n = 30 lines. Furthermore, the remarkable size difference obtained with the SSR ALB165 allowed the identification of the Brachypodium species by simple agarose gel electrophoresis.