SNPflow: A Lightweight Application for the Processing,Storing and Automatic Quality Checking of Genotyping Assays

Single nucleotide polymorphisms (SNPs) play a prominent role in modern genetics. Current genotyping technologies such as Sequenom iPLEX, ABI TaqMan and KBioscience KASPar made the genotyping of huge SNP sets in large populations straightforward and allow the generation of hundreds of thousands of genotypes even in medium sized labs. While data generation is straightforward, the subsequent data conversion, storage and quality control steps are time-consuming, error-prone and require extensive bioinformatic support. In order to ease this tedious process, we developed SNPflow. SNPflow is a lightweight, intuitive and easily deployable application, which processes genotype data from Sequenom MassARRAY (iPLEX) and ABI 7900HT (TaqMan, KASPar) systems and is extendible to other genotyping methods as well. SNPflow automatically converts the raw output files to ready-to-use genotype lists, calculates all standard quality control values such as call rate, expected and real amount of replicates, minor allele frequency, absolute number of discordant replicates, discordance rate and the p-value of the HWE test, checks the plausibility of the observed genotype frequencies by comparing them to HapMap/1000-Genomes, provides a module for the processing of SNPs, which allow sex determination for DNA quality control purposes and, finally, stores all data in a relational database. SNPflow runs on all common operating systems and comes as both stand-alone version and multi-user version for laboratory-wide use. The software, a user manual, screenshots and a screencast illustrating the main features are available at http://genepi-snpflow.i-med.ac.at.     

Turning Up the Heat on a Hotspot: DNA Barcodes Reveal 80% More Species of Geometrid Moths along an Andean Elevational Gradient

We sampled 14,603 geometrid moths along a forested elevational gradient from 1020–3021 m in the southern Ecuadorian Andes, and then employed DNA barcoding to refine decisions on species boundaries initially made by morphology. We compared the results with those from an earlier study on the same but slightly shorter gradient that relied solely on morphological criteria to discriminate species. The present analysis revealed 1857 putative species, an 80% increase in species richness from the earlier study that detected only 1010 species. Measures of species richness and diversity that are less dependent on sample size were more than twice as high as in the earlier study, even when analysis was restricted to an identical elevational range. The estimated total number of geometrid species (new dataset) in the sampled area is 2350. Species richness at single sites was 32–43% higher, and the beta diversity component rose by 43–51%. These impacts of DNA barcoding on measures of richness reflect its capacity to reveal cryptic species that were overlooked in the first study. The overall results confirmed unique diversity patterns reported in the first investigation. Species diversity was uniformly high along the gradient, declining only slightly above 2800 m. Species turnover also showed little variation along the gradient, reinforcing the lack of evidence for discrete faunal zones. By confirming these major biodiversity patterns, the present study establishes that incomplete species delineation does not necessarily conceal trends of biodiversity along ecological gradients, but it impedes determination of the true magnitude of diversity and species turnover.     

Interactions of donor sources and media influence the histo‐morphological quality of full‐thickness skin models

Human artificial skin models are increasingly employed as non‐animal test platforms for research and medical purposes. However, the overall histopathological quality of such models may vary significantly. Therefore, the effects of manufacturing protocols and donor sources on the quality of skin models built‐up from fibroblasts and keratinocytes derived from juvenile foreskins is studied. Histo‐morphological parameters such as epidermal thickness, number of epidermal cell layers, dermal thickness, dermo‐epidermal adhesion and absence of cellular nuclei in the corneal layer are obtained and scored accordingly. In total, 144 full‐thickness skin models derived from 16 different donors, built‐up in triplicates using three different culture conditions were successfully generated. In univariate analysis both media and donor age affected the quality of skin models significantly. Both parameters remained statistically significant in multivariate analyses. Performing general linear model analyses we could show that individual medium‐donor‐interactions influence the quality. These observations suggest that the optimal choice of media may differ from donor to donor and coincides with findings where significant inter‐individual variations of growth rates in keratinocytes and fibroblasts have been described. Thus, the consideration of individual medium‐donor‐interactions may improve the overall quality of human organ models thereby forming a reproducible test platform for sophisticated clinical research.     

Spatially heterogeneous pressure raises risk of catastrophic shifts

Ecosystems may exhibit catastrophic shifts, i.e. abrupt and irreversible responses of ecosystem functions and services to continuous changes in external conditions. The search for early warning signs of approaching shifts has so far mainly been conducted on theoretical models assuming spatially-homogeneous external pressures (e.g. climatic). Here, we investigate how a spatially explicit pressure may affect ecosystems’ risk of catastrophic shifts and the associated spatial early-warning signs. As a case study, we studied a dryland vegetation model assuming ‘associational resistance’, i.e. the mutual reduction of local grazing impact by neighboring plants sharing the investment in defensive traits. Consequently, grazing pressure depends on the local density of plants and is thus spatially-explicit. We focus on the distribution of vegetation patch sizes, which can be assessed using remote sensing and are candidate early warning signs of catastrophic shifts in drylands. We found that spatially explicit grazing affected both the resilience and the spatial patterns of the landscape. Grazing impact became self-enhancing in more fragmented landscapes, disrupted patch growth and put apparently ‘healthy’ drylands under high risks of catastrophic shifts. Our study highlights that a spatially explicit pressure may affect the nature of the spatial pattern observed and thereby change the interpretation of the early warning signs. This may generalize to other ecosystems exhibiting self-organized spatial patterns, where a spatially-explicit pressure may interfere with pattern formation.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

Hyaluronic Acid Suppresses the Expression of Metalloproteinases in Osteoarthritic Cartilage Stimulated Simultaneously by Interleukin 1β and Mechanical Load

Purpose

In patients with osteoarthritis (OA), intraarticular injection of hyaluronic acid (HA) frequently results in reduced pain and improved function for prolonged periods of time, i.e. more than 6 months. However, the mechanisms underlying these effects are not fully understood. Our underlying hypothesis is that HA modifies the enzymatic breakdown of joint tissues.

Methods

To test this hypothesis, we examined osteochondral cylinders from 12 OA patients. In a bioreactor, these samples were stimulated by interleukin 1β (Il1ß) (2 ng/ml) plus mechanical load (2.0 Mpa at 0.5 Hz horizontal and 0.1 Hz vertical rotation), thus the experimental setup recapitulated both catabolic and anabolic clues of the OA joint.

Results

Upon addition of HA at either 1 or 3 mg/ml, we observed a significant suppression of expression of metalloproteinase (MMP)-13. A more detailed analysis based on the Kellgren and Lawrence (K&L) OA grade, showed a much greater degree of suppression of MMP-13 expression in grade IV as compared to grade II OA. In contrast to the observed MMP-13 suppression, treatment with HA resulted in a suppression of MMP-1 expression only at 1 mg/ml HA, while MMP-2 expression was not significantly affected by either HA concentration.

Conclusion

Together, these data suggest that under concurrent catabolic and anabolic stimulation, HA exhibits a pronounced suppressive effect on MMP-13. In the long-run these findings may benefit the development of treatment strategies aimed at blocking tissue degradation in OA patients.     

Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension

Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.     

Ciliate community structure and interactions within the planktonic food web in two alpine lakes of contrasting transparency

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