TY - JOUR A1 - Grebinyk, Anna A1 - Grebinyk, Sergii A1 - Prylutska, Svitlana A1 - Ritter, Uwe A1 - Matyshevska, Olga A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - C60 fullerene accumulation in human leukemic cells and perspectives of LED-mediated photodynamic therapy JF - Free Radical Biology and Medicine N2 - Recent progress in nanobiotechnology has attracted interest to a biomedical application of the carbon nanostructure C60 fullerene since it possesses a unique structure and versatile biological activity. C60 fullerene potential application in the frame of cancer photodynamic therapy (PDT) relies on rapid development of new light sources as well as on better understanding of the fullerene interaction with cells. The aim of this study was to analyze C60 fullerene effects on human leukemic cells (CCRF-CEM) in combination with high power single chip light-emitting diodes (LEDs) light irradiation of different wavelengths: ultraviolet (UV, 365 nm), violet (405 nm), green (515 nm) and red (632 nm). The time-dependent accumulation of fullerene C60 in CCRF-CEM cells up to 250 ng/106 cells at 24 h with predominant localization within mitochondria was demonstrated with immunocytochemical staining and liquid chromatography mass spectrometry. In a cell viability assay we studied photoexcitation of the accumulated C60 nanostructures with ultraviolet or violet LEDs and could prove that significant phototoxic effects did arise. A less pronounced C60 fullerene phototoxic effect was observed after irradiation with green, and no effect was detected with red light. A C60 fullerene photoactivation with violet light induced substantial ROS generation and apoptotic cell death, confirmed by caspase3/7 activation and plasma membrane phosphatidylserine externalization. Our work proved C60 fullerene ability to induce apoptosis of leukemic cells after photoexcitation with high power single chip 405 nm LED as a light source. This underlined the potential for application of C60 nanostructure as a photosensitizer for anticancer therapy. Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-10588 SN - 1873-4596 VL - 124 SP - 319 EP - 327 ER - TY - JOUR A1 - Kinuthia, Chuaga A1 - Frohme, Marcus A1 - Brödel, Oliver A1 - Mugendi, Beatrice T1 - LCMS Analysis of Biochemical Composition in Different Kenyan Coffee Classifications JF - Food Science and Quality Management N2 - Kenyan coffee is classified by defects after grading by the ‘Devonshire method.’ The method involves classification of the coffee beans into different classes based on the raw and roasted coffees and cup quality, with class one being the best and ten the poorest. In this study, the relationship between classification of the coffee and the content of sucrose, trigonelline, caffeine and chlorogenic acids was determined by simultaneous LC-MS analysis. By using the sensory variables the class 3 coffee portrayed the best quality, followed by class 4 and 5 which were placed further distinctively from the other classes (6, 7, 8 and 9). The class 3 coffee had a high percent of non-defective beans with 94.31% and class 9 had a high defect count at 79.53% hence the defective beans increased with decrease in coffee class. The caffeine concentration in green coffee for class 3 coffee was significantly different from the rest of the coffees at 1.23 ± 0.00g/100g. A higher content of trigonelline levels was observed as the quality of the green coffee decreased in classes 6, 7 and 8. The highest level of sucrose in green coffee beans was observed in class 3 coffee and the lowest in the class 9. Lower content of chlorogenic acids were observed among the high quality coffees i.e. class 3, 4 and 5 with class 3 having the lowest while a higher content was observed among the lower quality coffees i.e. classes 6, 7 and 9 with class 6 having a higher content was it significantly different in class 6 compared to 7 and 9?. Quantities of different chemical components among the classes are clear indicators that the classification method used on the Kenyan coffees brings out the differences in coffee quality based on the analysis of the green and the roasted coffees. KW - biochemical composition KW - coffee classification KW - LCMS Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-10185 UR - https://iiste.org/Journals/index.php/FSQM/article/view/39645/40765 SN - 2225-0557 VL - 69 SP - 44 EP - 52 ER - TY - JOUR A1 - Grebinyk, Anna A1 - Prylutska, Svitlana A1 - Grebinyk, Sergii A1 - Evstigneev, Maxim A1 - Krysiuk, Iryna A1 - Skaterna, Tetiana A1 - Horak, Iryna A1 - Sun, Yanfang A1 - Drobot, Liudmyla A1 - Matyshevska, Olga A1 - Prylutskyy, Yuriy A1 - Ritter, Uwe A1 - Frohme, Marcus T1 - Antitumor efficiency of the natural alkaloid berberine complexed with C60 fullerene in Lewis lung carcinoma in vitro and in vivo JF - Cancer Nanotechnology N2 - Background Berberine (Ber) is a herbal alkaloid with pharmacological activity in general and a high anticancer potency in particular. However, due to its low bioavailability, the difficulty in reaching a target and choosing the right dose, there is a need to improve approaches of Ber use in anticancer therapy. In this study, Ber, noncovalently bound to a carbon nanostructure C60 fullerene (C60) at various molar ratios of the components, was explored against Lewis lung carcinoma (LLC). Methods C60–Ber noncovalent nanocomplexes were synthesized in 1:2, 1:1 and 2:1 molar ratios. Ber release from the nanocomplexes was studied after prolonged incubation at different pH with the liquid chromatography–mass spectrometry analysis of free Ber content. Biological effects of the free and C60-complaxated Ber were studied in vitro towards LLC cells with phase-contrast and fluorescence microscopy, flow cytometry, MTT reduction, caspase activity and wound closure assays. The treatment with C60–Ber nanocomplex was evaluated in vivo with the LLC-tumored C57Bl mice. The mice body weight, tumor size, tumor weight and tumor weight index were assessed for four groups, treated with saline, 15 mg C60/kg, 7.5 mg Ber/kg or 2:1 C60-Ber nanocomplex (15 mg C60/kg, 7.5 mg Ber/kg). Results Ber release from C60–Ber nanocomplexes was promoted with medium acidification. LLC cells treatment with C60–Ber nanocomplexes was followed by enhanced Ber intracellular uptake as compared to free Ber. The cytotoxicity of the studied agents followed the order: free Ber < 1:2 < 1:1 < 2:1 C60–Ber nanocomplex. The potency of cytotoxic effect of 2:1 C60–Ber nanocomplex was confirmed by 21.3-fold decrease of IC50 value (0.8 ± 0.3 µM) compared to IC50 for free Ber (17 ± 2 µM). C60–Ber nanocomplexes induced caspase 3/7 activation and suppressed the migration activity of LLC cells. The therapeutic potency of 2:1 C60–Ber nanocomplex was confirmed in a mouse model of LLC. The tumor growth in the group treated with 2:1 C60–Ber nanocomplex is suppressed by approximately 50% at the end of experiment, while in the tumor-bearing group treated with free Ber no therapeutic effect was detected. Conclusions This study indicates that complexation of natural alkaloid Ber with C60 may be a novel therapeutic strategy against lung carcinoma. Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-14749 SN - 1868-6966 VL - 12 SP - 24 PB - BioMed Central ER - TY - JOUR A1 - Pajčin, Ivana A1 - Vlajkov, Vanja A1 - Frohme, Marcus A1 - Grebinyk, Sergii A1 - Grahovac, Mila A1 - Mojićević, Marija A1 - Grahovac, Jovana T1 - Pepper Bacterial Spot Control by Bacillus velezensis: Bioprocess Solution JF - Microorganisms N2 - Pepper bacterial spot is one of the most severe plant diseases in terms of infection persistence and economic losses when it comes to fresh pepper fruits used in nutrition and industrial processing. In this study, Bacillus velezensis IP22 isolated from fresh cheese was used as a biocontrol agent of pepper bacterial spot, whose main causal agent is the cosmopolitan pathogen Xanthomonas euvesicatoria. After optimization of the cultivation medium composition aimed at maximizing of the antimicrobial activity against X. euvesicatoria and validation of the optimized medium at the scale of a laboratory bioreactor, in planta tests were performed. The results have showed significant suppression of bacterial spot symptoms in pepper plants by the produced biocontrol agent, as well as reduction of disease spreading on the healthy (uninoculated) pepper leaves. Furthermore, HPLC-MS (high pressure liquid chromatography–mass spectrometry) analysis was employed to examine antimicrobial metabolites produced by B. velezensis IP22, where lipopeptides were found with similar m/z values compared to lipopeptides from fengycin and locillomycin families. The bioprocess solution developed at the laboratory scale investigated in this study represents a promising strategy for production of pepper bacterial spot biocontrol agent based on B. velezensis IP22, a food isolate with a great perspective for application in plant protection. KW - Xanthomonas euvesicatoria KW - biological control KW - biocontrol agent KW - medium optimization KW - lipopeptide KW - fengycin KW - locillomycin KW - HPLC-MS KW - bioreactor KW - laboratory scale Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-13510 SN - 2076-2607 VL - 8 IS - 10 PB - MDPI ER - TY - GEN A1 - Schulze, Katja A1 - Glökler, Jörn A1 - Kadolsky, Marianne A1 - Frohme, Marcus T1 - Detektion des Kartoffelspindelknollen Viroids mit Hilfe der Loop Mediated Isothermal Amplification T2 - Wissenschaftliche Beiträge 2011 N2 - PSTV ist ein hoch infektiöses Viroid, das in Kartoffeln verkleinerte und spindelähnliche Knollen verursacht. Um Ernteverlusten vorzubeugen, ist eine Detektion in frühen Infektionsstadien von großer Bedeutung. Aufgrund ihrer hohen Sensitivität und Spezifität wird die PCR als Standardnachweisverfahren für PSTV verwendet. Nachteilig an dieser Methode sind der apparative Aufwand und die zeitaufwändige Durchführung. Als viel versprechende Alternative konnte der Nachweis von PSTV mit Hilfe der Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) gezeigt werden. Hierbei handelt es sich um eine einfache und schnelle Methode, für die wenig aufwändige Laborausrüstung benötigt wird. Dabei ermöglichte eine an den Amplifikationsprozess gekoppelte Fluoreszenzreaktion die Detektion von Produkten direkt nach der Nachweisreaktion mit dem bloßen Auge (bzw. unter UV-Licht). Die Ergebnisse konnten mit Hilfe einer Real Time Detektion des auftretenden Fluoreszenzsignals bestätigt werden. N2 - PSTV is a highly infectious viroid which leads to small and spindle shaped tubers in potatoes. The detection at an early stage of infection is important to minimize loss of harvest. Normally PCR is used for the detection of PSTV, because of its high sensitivity and specificity. Disadvantages of this method are the requirement of sophisticated equipment and the time consuming process. As a promising alternative, the detection of PSTV with the reverse transcription loop mediated isothermal amplification (RT-LAMP) was shown. LAMP is a very fast and simple detection method requiring only standard laboratory equipment. A fluorescence reaction, coupled to the amplification process, allowed the detection of amplification products directly after the reaction with the naked eye (or under UV light, respectively). Real time detection of the occurring fluorescence signal was possible and confirmed the obtained results. Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus-1025 SN - 0949-8214 VL - 15 SP - 7 EP - 12 ER - TY - JOUR A1 - Kober, Liane A1 - Meyer-Alert, Henriette A1 - Grienitz, Desirée A1 - Hollert, Henner A1 - Frohme, Marcus T1 - Intact cell mass spectrometry as a rapid and specific tool for the differentiation of toxic effects in cell-based ecotoxicological test systems JF - Analytical and Bioanalytical Chemistry N2 - In the last few decades, MALDI-TOF MS has become a useful technique not only in proteomics, but also as a fast and specific tool for whole cell analysis through intact cell mass spectrometry (IC-MS). The present study evaluated IC-MS as a novel tool for the detection of distinct patterns that can be observed after exposure to a certain toxin or concentration by utilizing the eukaryotic fish cell line RTL-W1. Two different viability assays were performed to define the range for IC-MS investigations, each of which employing copper sulfate, acridine, and β-naphthoflavone (BNF) as model compounds for several classes of environmental toxins. The IC-MS of RTL-W1 cells revealed not only specific spectral patterns for the various toxins, but also that the concentration used had an effect on RTL-W1 profiles. After the exposure with copper sulfate and acridine, the spectra of RTL-W1 showed a significant increase of certain peaks in the higher mass range (m/z >7000), which is probably attributed to the apoptosis of RTL-W1. On the contrary, exposure to BNF showed a distinct change of ion abundances only in the lower mass range (m/z <7000). Furthermore, a set of mass peaks could be identified as a specific biomarker for a single toxin treatment, so IC-MS demonstrates a new method for the distinction of toxic effects in fish cells. Due to fast sample preparation and high throughput, IC-MS offers great potential for ecotoxicological studies to investigate cellular effects of different substances and complex environmental samples. KW - RTL-W1 KW - intact cell mass spectrometry KW - MALDI KW - acridine KW - β-Naphthoflavone KW - copper sulfate Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-5900 SN - 1618-2650 VL - 407 IS - 25 SP - 7721 EP - 7731 ER - TY - JOUR A1 - Schulze, Katja A1 - Lang, Imke A1 - Enke, Heike A1 - Grohme, Diana A1 - Frohme, Marcus T1 - The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 JF - BMC Research Notes N2 - Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. KW - PCC6803 KW - PCC7002 KW - genetic instability KW - ethanol producer KW - 3D fluorescence scan KW - phycocyanin KW - absorption spectra KW - fluorescence microscopy KW - image analysis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-5911 SN - 1756-0500 VL - 8 IS - 160 ER - TY - JOUR A1 - Bilous, Nataliya A1 - Malko, Vladyslav A1 - Frohme, Marcus A1 - Nechyporenko, Alina T1 - Comparison of CNN-Based Architectures for Detection of Different Object Classes JF - AI N2 - (1) Background: Detecting people and technical objects in various situations, such as natural disasters and warfare, is critical to search and rescue operations and the safety of civilians. A fast and accurate detection of people and equipment can significantly increase the effectiveness of search and rescue missions and provide timely assistance to people. Computer vision and deep learning technologies play a key role in detecting the required objects due to their ability to analyze big volumes of visual data in real-time. (2) Methods: The performance of the neural networks such as You Only Look Once (YOLO) v4-v8, Faster R-CNN, Single Shot MultiBox Detector (SSD), and EfficientDet has been analyzed using COCO2017, SARD, SeaDronesSee, and VisDrone2019 datasets. The main metrics for comparison were mAP, Precision, Recall, F1-Score, and the ability of the neural network to work in real-time. (3) Results: The most important metrics for evaluating the efficiency and performance of models for a given task are accuracy (mAP), F1-Score, and processing speed (FPS). These metrics allow us to evaluate both the accuracy of object recognition and the ability to use the models in real-world environments where high processing speed is important. (4) Conclusion: Although different neural networks perform better on certain types of metrics, YOLO outperforms them on all metrics, showing the best results of mAP-0.88, F1-0.88, and FPS-48, so the focus was on these models. KW - EfficientDet KW - SSD KW - YOLO KW - deep learning KW - neural network KW - object detection Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-19634 VL - 5 IS - 4 SP - 2300 EP - 2320 PB - MDPI ER - TY - JOUR A1 - Przymus, Piotr A1 - Rykaczewski, Krzysztof A1 - Martín-Segura, Adrián A1 - Truu, Jaak A1 - De Santa Pau, Enrique Carrillo A1 - Kolev, Mikhail A1 - Naskinova, Irina A1 - Gruca, Aleksandra A1 - Sampri, Alexia A1 - Frohme, Marcus A1 - Nechyporenko, Alina T1 - Deep Learning in Microbiome Analysis: A Comprehensive Review of Neural Network Models JF - Frontiers in Microbiology N2 - Microbiome research, the study of microbial communities in diverse environments, has seen significant advances due to the integration of deep learning (DL) methods. These computational techniques have become essential for addressing the inherent complexity and high-dimensionality of microbiome data, which consist of different types of omics datasets. Deep learning algorithms have shown remarkable capabilities in pattern recognition, feature extraction, and predictive modeling, enabling researchers to uncover hidden relationships within microbial ecosystems. By automating the detection of functional genes, microbial interactions, and host-microbiome dynamics, DL methods offer unprecedented precision in understanding microbiome composition and its impact on health, disease, and the environment. However, despite their potential, deep learning approaches face significant challenges in microbiome research. Additionally, the biological variability in microbiome datasets requires tailored approaches to ensure robust and generalizable outcomes. As microbiome research continues to generate vast and complex datasets, addressing these challenges will be crucial for advancing microbiological insights and translating them into practical applications with DL. This review provides an overview of different deep learning models in microbiome research, discussing their strengths, practical uses, and implications for future studies. We examine how these models are being applied to solve key problems and highlight potential pathways to overcome current limitations, emphasizing the transformative impact DL could have on the field moving forward. KW - clasiffication KW - clustering KW - deep learning KW - natural language processing KW - microbiome Y1 - 2025 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-19889 SN - 1664-302X VL - 15 PB - Frontiers ER - TY - JOUR A1 - Narvekar, Prachiti A1 - Berriel Diaz, Mauricio A1 - Krones-Herzig, Anja A1 - Hardeland, Ulrike A1 - Strzoda, Daniela A1 - Stöhr, Sigrid A1 - Frohme, Marcus A1 - Herzig, Stephan T1 - Liver-specific loss of lipolysis-stimulated lipoprotein receptor triggers systemic hyperlipidemia in mice JF - Diabetes N2 - In mammals, proper storage and distribution of lipids in and between tissues is essential for the maintenance of energy homeostasis. In contrast, aberrantly high levels of triglycerides in the blood ("hypertriglyceridemia") represent a hallmark of the metabolic syndrome and type 2 diabetes. As hypertriglyceridemia has been identified as an important risk factor for cardiovascular complications, in this study we aimed to identify molecular mechanisms in aberrant triglyceride elevation under these conditions. To determine the importance of hepatic lipid handling for systemic dyslipidemia, we profiled the expression patterns of various hepatic lipid transporters and receptors under healthy and type 2 diabetic conditions. A differentially expressed lipoprotein receptor was functionally characterized by generating acute, liver-specific loss- and gain-of-function animal models. We show that the hepatic expression of lipid transporter lipolysis-stimulated lipoprotein receptor (LSR) is specifically impaired in mouse models of obesity and type 2 diabetes and can be restored by leptin replacement. Experimental imitation of this pathophysiological situation by liver-specific knockdown of LSR promotes hypertriglyceridemia and elevated apolipoprotein (Apo)B and E serum levels in lean wild-type and ApoE knockout mice. In contrast, genetic restoration of LSR expression in obese animals to wild-type levels improves serum triglyceride levels and serum profiles in these mice. The dysregulation of hepatic LSR under obese and diabetic conditions may provide a molecular rationale for systemic dyslipidemia in type 2 diabetes and the metabolic syndrome and represent a novel target for alternative treatment strategies in these patients. Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-19954 VL - 58 IS - 5 SP - 1040 EP - 1049 PB - American Diabetes Association ER - TY - JOUR A1 - Reuner, Andy A1 - Hengherr, Steffen A1 - Mali, Brahim A1 - Förster, Frank A1 - Arndt, Detlev A1 - Reinhardt, Richard A1 - Dandekar, Thomas A1 - Frohme, Marcus A1 - Brümmer, Franz A1 - Schill, Ralph O. T1 - Stress response in tardigrades: differential gene expression of molecular chaperones JF - Cell Stress and Chaperones N2 - Semi-terrestrial tardigrades exhibit a remarkable tolerance to desiccation by entering a state called anhydrobiosis. In this state, they show a strong resistance against several kinds of physical extremes. Because of the probable importance of stress proteins during the phases of dehydration and rehydration, the relative abundance of transcripts coding for two α-crystallin heat-shock proteins (Mt-sHsp17.2 and Mt-sHsp19.5), as well for the heat-shock proteins Mt-sHsp10, Mt-Hsp60, Mt-Hsp70 and Mt-Hsp90, were analysed in active and anhydrobiotic tardigrades of the species Milnesium tardigradum. They were also analysed in the transitional stage (I) of dehydration, the transitional stage (II) of rehydration and in heat-shocked specimens. A variable pattern of expression was detected, with most candidates being downregulated. Gene transcripts of one Mt-hsp70 isoform in the transitional stage I and Mt-hsp90 in the anhydrobiotic stage were significantly upregulated. A high gene expression (778.6-fold) was found for the small α-crystallin heat-shock protein gene Mt-sHsp17.2 after heat shock. We discuss the limited role of the stress-gene expression in the transitional stages between the active and anhydrobiotic tardigrades and other mechanisms which allow tardigrades to survive desiccation. KW - alpha-crystallin protein KW - anhydrobiosis KW - cryptobiosis KW - heat-shock protein KW - tardigrada KW - Milnesium tardigradum Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-19994 VL - 15 IS - 4 SP - 423 EP - 430 PB - Springer ER - TY - JOUR A1 - Tillich, Ulrich M. A1 - Lehmann, Sandra A1 - Schulze, Katja A1 - Dühring, Ulf A1 - Frohme, Marcus T1 - The Optimal Mutagen Dosage to Induce Point-Mutations in Synechocystis sp. PCC6803 and Its Application to Promote Temperature Tolerance JF - PLoS ONE N2 - Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria Synechocystis sp. PCC6803. Based on these results an optimal dosage of 10–50 J/m2 for UV and either 0.1 or 1 v% for MMS was determined. A Synechocystis wildtype culture was then mutagenized and selected for increased temperature tolerance in vivo. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate in vivo screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for in vivo, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods. Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6186 SN - 1932-6203 VL - 7 IS - 11 ER -