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.     

Bronchiectasis-Associated Hospitalizations in Germany, 2005–2011: A Population-Based Study of Disease Burden and Trends

Background

Representative population-based data on the epidemiology of bronchiectasis in Europe are limited. The aim of the present study was to investigate the current burden and the trends of bronchiectasis-associated hospitalizations and associated conditions in Germany in order to inform focused patient care and to facilitate the allocation of healthcare resources.

Methods

The nationwide diagnosis-related groups hospital statistics for the years 2005–2011 were used in order to identify hospitalizations with bronchiectasis as any hospital discharge diagnosis according to the International Classification of Diseases, 10th revision, code J47, (acquired) bronchiectasis. Poisson log-linear regression analysis was used to assess the significance of trends. In addition, the overall length of hospital stay (LOS) and the in-hospital mortality in comparison to the nationwide overall mortality due to bronchiectasis as the primary diagnosis was assessed.

Results

Overall, 61,838 records with bronchiectasis were extracted from more than 125 million hospitalizations. The average annual age-adjusted rate for bronchiectasis as any diagnosis was 9.4 hospitalizations per 100,000 population. Hospitalization rates increased significantly during the study period, with the highest rate of 39.4 hospitalizations per 100,000 population among men aged 75–84 years and the most pronounced average annual increases among females. Besides numerous bronchiectasis-associated conditions, chronic obstructive pulmonary disease (COPD) was most frequently found in up to 39.2% of hospitalizations with bronchiectasis as the primary diagnosis. The mean LOS was comparable to that for COPD. Overall, only 40% of bronchiectasis-associated deaths occurred inside the hospital.

Conclusions

The present study provides evidence of a changing epidemiology and a steadily increasing prevalence of bronchiectasis-associated hospitalizations. Moreover, it confirms the diversity of bronchiectasis-associated conditions and the possible association between bronchiectasis and COPD. As the major burden of disease may be managed out-of-hospital, prospective patient registries are needed to establish the exact prevalence of bronchiectasis according to the specific underlying condition.     

Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies

Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (iv, ip, sc), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000–300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.     

The Tyrosine Kinase Btk Regulates the Macrophage Response to Listeria monocytogenes Infection

In this study we investigated the role of Bruton''s tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk ^−/− BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk ^−/− BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk ^−/− BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk ^−/− mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk ^−/− mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.     

The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of β-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost- and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.     

Origin of the S‐Shaped JV Curve and the Light‐Soaking Issue in Inverted Organic Solar Cells

Inverted organic solar cells generally exhibit a strong s‐shaped kink in the current–voltage characteristics (JV curve) that may be removed by exposure to UV light (light‐soaking) leading to a drastically improved performance. Using in‐device characterization methods the origin of the light‐soaking issue in inverted solar cells employing titanium dioxide (TiO₂) as an electron selective layer is clarified. An injected hole reservoir accumulated at the TiO₂/organic interface of the pristine device is observed from extraction current transients; the hole reservoir increases the recombination and results in an s‐shape in the JV curve of pristine devices. The hole reservoir and the s‐shape is a result of the energetics at the selective contact in the pristine device; the effect of UV exposure is to decrease the work function of the indium tin oxide/TiO₂‐contact, increasing the built‐in potential. This hinders the build‐up of the hole reservoir and the s‐shape is removed. The proposed model is in excellent agreement with drift‐diffusion simulations.     

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS‐PAGE and LC‐MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane‐associated hypothetical proteins that might be involved in adhesion or host–pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose‐6‐phosphate‐transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 ( http://proteomecentral.proteomexchange.org/dataset/PXD002294 ).     

eGender—from e-Learning to e-Research: a web-based interactive knowledge-sharing platform for sex- and gender-specific medical education

Background

Sex and Gender Medicine is a novel discipline that provides equitable medical care for society and improves outcomes for both male and female patients. The integration of sex- and gender-specific knowledge into medical curricula is limited due to adequate learning material, systematic teacher training and an innovative communication strategy. We aimed at initiating an e-learning and knowledge-sharing platform for Sex and Gender Medicine, the eGender platform (http://egender.charite.de), to ensure that future doctors and health professionals will have adequate knowledge and communication skills on sex and gender differences in order to make informed decisions for their patients.

Methods

The web-based eGender knowledge-sharing platform was designed to support the blended learning pedagogical teaching concept and follows the didactic concept of constructivism. Learning materials developed by Sex and Gender Medicine experts of seven universities have been used as the basis for the new learning tools. The content of these tools is patient-centered and provides add-on information on gender-sensitive aspects of diseases. The structural part of eGender was designed and developed using the open source e-learning platform Moodle. The eGender platform comprises an English and a German version of e-learning modules: one focusing on basic knowledge and seven on specific medical disciplines. Each module consists of several courses corresponding to a disease or symptom complex. Self-organized learning has to be managed by using different learning tools, e.g., texts and audiovisual material, tools for online communication and collaborative work.

Results

More than 90 users from Europe registered for the eGender Medicine learning modules. The most frequently accessed module was “Gender Medicine—Basics” and the users favored discussion forums. These e-learning modules fulfill the quality criteria for higher education and are used within the elective Master Module “Gender Medicine—Basics” implemented into the accredited Master of Public Health at Charité—Berlin.

Conclusions

The eGender platform is a flexible and user-friendly electronical knowledge-sharing platform providing evidence-based high-quality learning material used by a growing number of registered users. The eGender Medicine learning modules could be key in the reform of medical curricula to integrate Sex and Gender Medicine into the education of health professionals.

     

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.