TY - JOUR A1 - Akhoundi, Mohammad A1 - Downing, Tim A1 - Votýpka, Jan A1 - Kuhls, Katrin A1 - Lukeš, Julius A1 - Cannet, Arnaud A1 - Ravel, Christophe A1 - Marty, Pierre A1 - Delaunay, Pascal A1 - Kasbari, Mohamed A1 - Granouillac, Bruno A1 - Gradoni, Luigi A1 - Sereno, Denis T1 - Leishmania infections: Molecular targets and diagnosis JF - Molecular Aspects of Medicine N2 - Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs. KW - molecular marker KW - diagnostic method KW - hybrid strain KW - sympatric species KW - genome-wide map Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9908 SN - 0098-2997 VL - 57 SP - 1 EP - 29 ER - TY - JOUR A1 - Amro, Ahmad A1 - Al-Dwibe, Hamida A1 - Gashout, Aisha A1 - Moskalenko, Olga A1 - Galafin, Marlena A1 - Hamarsheh, Omar A1 - Frohme, Marcus A1 - Jaeschke, Anja A1 - Schönian, Gabriele A1 - Kuhls, Katrin T1 - Spatiotemporal and molecular epidemiology of cutaneous leishmaniasis in Libya JF - PLoS Neglected Tropical Diseases N2 - Cutaneous leishmaniasis (CL) is a skin infection caused by a single-celled parasite that is transmitted by the bite of a phlebotomine sandfly. CL is the most common form of leishmaniasis characterized by localized lesions in the skin and mucous membranes. The disease is prevalent in all countries around the Mediterranean Basin. In this paper, we describe spatiotemporal and eco-epidemiological parameters of CL in Libya. Moreover, we explored current spatiotemporal distributions of CL cases and explored the future projection of the disease. Our study indicates the presence of higher risk of CL in the coastal regions of Libya. Future projection until 2060 showed a trend of increasing incidence of CL in the north-western part of Libya, a spread along the coastal region and a possible emergence of new endemics in the north-eastern districts of Libya. These scenarios should be considered by health authorities in order to develop appropriate intervention strategies and plan effective control programs. Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-10054 SN - 1935-2735 VL - 11 IS - 9 ER - TY - JOUR A1 - Bay, Daniyah H. A1 - Busch, Annika A1 - Lisdat, Fred A1 - Iida, Keisuke A1 - Ikebukuro, Kazunori A1 - Nagasawa, Kazuo A1 - Karube, Isao A1 - Yoshida, Wataru T1 - Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands JF - BMC Molecular Biology N2 - G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay. KW - Cdc6 KW - Dele KW - G-quadruplex KW - transcriptional regulation KW - 7OTD Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9811 SN - 1471-2199 VL - 18 IS - 17 ER - TY - THES A1 - Fischbach, Jens T1 - Isothermale Amplifikationsmethoden für den DNA- und Pyrophosphat-abhängigen Pathogennachweis N2 - Hintergrund: Etablierte Protein- und Nukleinsäure-basierte Methoden für den spezifischen Pathogennachweis sind nur unter standardisierten Laborbedingungen von geschultem Personal durchführbar und daher mit einem hohen Zeit- und Kostenaufwand verbunden. In der Nukleinsäure-basierten Diagnostik kann durch die Einführung der isothermalen Amplifikation eine schnelle und kostengünstige Alternative zur Polymerase-Kettenreaktion (PCR) verwendet werden. Die Loop-mediated isothermal amplification (LAMP) bietet aufgrund der hohen Amplifikationseffizienz vielfältige Detektionsmöglichkeiten, die sowohl für Schnelltest- als auch für Monitoring-Anwendungen geeignet sind. Ein wesentliches Ziel dieser Arbeit war die Verbesserung der Anwendbarkeit der LAMP und die Entwicklung einer neuen Methode für den einfachen, schnellen und günstigen Nachweis von Pathogenen mittels alternativer DNA- oder Pyrophosphat-abhängiger Detektionsverfahren. Hier wurden zunächst direkte und indirekte Detektionsmethoden untersucht und darauf aufbauend ein Verfahren entwickelt, mit dem neue Metallionen-abhängige Fluoreszenzfarbstoffe für die selektive Detektion von Pyrophosphat in der LAMP und anderen enzymatischen Reaktionen identifiziert werden können. Als Alternative für die DNA-basierte Detektion in der digitalen LAMP sollten die zuvor etablierten Farbstoffe für den Pyrophosphatnachweis in einer Emulsion getestet werden. Abschließend wurde ein neuer Reaktionsmechanismus für die effiziente Generierung hochmolekularer DNA unter isothermalen Bedingungen als Alternative zur LAMP entwickelt. Ergebnisse: Für den Nachweis RNA- und DNA-basierter Phythopathogene konnte die Echtzeit- und Endpunktdetektion mit verschiedenen Farbstoffen in einem geschlossenen System etabliert werden. Hier wurde Berberin als DNA-interkalierender Fluoreszenzfarbstoff mit vergleichbarer Sensitivität zu SYBR Green und EvaGreen erfolgreich in der LAMP mit Echtzeitdetektion eingesetzt. Ein Vorteil von Berberin gegenüber den anderen Farbstoffen ist die Toleranz der DNA-Polymerase auch bei hohen Farbstoffkonzentrationen. Berberin kann daher auch in der geschlossenen LAMP-Reaktion ohne zusätzliche Anpassung der Reaktionsbedingungen für die Endpunktdetektion verwendet werden. Darüber hinaus konnte Hydroxynaphtholblau (HNB), das für den kolorimetrischen Endpunktnachweis bekannt ist, erstmals auch für die fluorimetrische Detektion der LAMP in Echtzeit eingesetzt werden. Zusätzlich konnten in der Arbeit weitere Metallionen-abhängige Farbstoffe zur indirekten Detektion der LAMP über das Pyrophosphat identifiziert werden. Dafür wurde eine iterative Methode entwickelt, mit der potenzielle Farbstoffe hinsichtlich ihrer Enzymkompatibilität und ihrer spektralen Eigenschaften bei An- oder Abwesenheit von Manganionen selektiert werden können. Mithilfe eines kombinatorischen Screenings im Mikrotiterplattenformat konnte die komplexe Konzentrationsabhängigkeit zwischen den einzelnen Komponenten für einen fluorimetrischen Verdrängungsnachweis untersucht werden. Durch die Visualisierung des Signal-Rausch-Verhältnis’ als Intensitätsmatrix (heatmap) konnten zunächst Alizarinrot S und Tetrazyklin unter simulierten Reaktionsbedingungen selektiert werden. In der anschließenden enzymatischen LAMP-Reaktion konnte insbesondere Alizarinrot S als günstiger, nicht-toxischer und robuster Fluoreszenzfarbstoff identifiziert werden und zeigte eine Pyrophosphat-abhängige Zunahme der Fluoreszenzintensität. Die zuvor etablierten Farbstoffe (HNB, Calcein und Alizarinrot S) konnten anschließend erfolgreich für die indirekte, fluorimetrische Detektion von Pyrophosphat in einer LAMP-optimierten Emulsion eingesetzt werden. Die Stabilität und Homogenität der generierten Emulsion wurde durch den Zusatz des Emulgators Poloxamer 188 verbessert. Durch die fluoreszenzmikroskopische Analyse der Emulsion war eine eindeutige Diskriminierung der positiven und negativen Tröpfchen vor allem bei Einsatz von Calcein und Alizarinrot S möglich. Aufgrund des komplexen Primer-Designs und der hohen Wahrscheinlichkeit unspezifischer Amplifikation in der LAMP wurde eine neue Bst DNA-Polymerase-abhängige isothermale Amplifikationsreaktion entwickelt. Durch die Integration einer spezifischen Linkerstruktur (abasische Stelle oder Hexaethylenglykol) zwischen zwei Primersequenzen konnte ein bifunktioneller Primer die effiziente Regenerierung der Primerbindungsstellen gewährleisten. Der neue Primer induziert nach der spezifischen Hybridisierung auf dem Templat die Rückfaltung zu einer Haarnadelstruktur und blockiert gleichzeitig die Polymeraseaktivität am Gegenstrang, wodurch eine autozyklische Amplifikation trotz konstanter Reaktionstemperatur möglich ist. Die Effizienz der „Hinge-initiated Primer dependent Amplification“ (HIP) konnte abschließend durch die Verkürzung der Distanz zwischen einem modifizierten Hinge-Primer und einem PCR-ähnlichen Primer verbessert werden. Schlussfolgerung: Die LAMP hat sich aufgrund der hohen Robustheit und Effizienz zu einer leistungsfähigen Alternative für die klassische PCR in der molekularbiologischen Diagnostik entwickelt. Unterschiedliche Detektionsverfahren verbessern die Leistungsfähigkeit der qualitativen und quantitativen LAMP für die Feldanwendungen und für die Diagnostik, da die neuen DNA- und Pyrophosphat-abhängigen Nachweismethoden in einer geschlossenen Reaktion eingesetzt werden können und so eine einfache Pathogendiagnostik ermöglichen. Die gezeigten Methoden können darüber hinaus zu einer Kostensenkung und Zeitersparnis gegenüber den herkömmlichen Methoden beitragen. Ein attraktives Ziel stellt die Weiterentwicklung der HIP für den Pathogennachweis als Alternative zur LAMP dar. Hierbei können die neuen LAMP-Detektionsverfahren ebenfalls Anwendung finden. Die Verwendung von Bst DNA-Polymerase-abhängigen Reaktionen ermöglicht darüber hinaus die Integration einer robusten isothermalen Amplifikation in mikrofluidische Systeme. Durch die Kombination der Probenvorbereitung, Amplifikation und Detektion sind zukünftige Anwendungen mit kurzer Analysezeit und geringem apparativen Aufwand insbesondere in der Pathogendiagnostik möglich. N2 - Background: Established protein- and nucleic acid-based methods for the specific pathogen detection are usually performed under standardized laboratory conditions by trained staff and are associated with long processing time and high costs. In nucleic acid-based pathogen diagnostics, the isothermal amplification can be used as a rapid and cost-effective alternative to the polymerase chain reaction (PCR). Among all isothermal techniques, the loop-mediated isothermal amplification (LAMP) offers a wide range of applications for the rapid endpoint and real-time detection. A major goal of this work, was to improve the applicability of LAMP and the development of a new method to get a simple, fast and cost-effective diagnostic tool that is based on the detection of DNA and pyrophosphate. For this purpose, direct and indirect detection methods were investigated as well as additional metal ion-dependent fluorescent dyes for the selective detection of pyrophosphate in LAMP or other enzymatic reactions identified. As an alternative to the DNA-based digital LAMP, the previously established dyes were tested for the detection of pyrophosphate in emulsion. Finally, a new reaction mechanism was developed that allows the efficient generation of high molecular weight DNA under isothermal reaction conditions. Results: The detection of RNA- and DNA-based phytopathogens in closed reactions was established successfully with different dyes for real-time and endpoint detection. Berberine as DNA-intercalating fluorescent dye was used in the real-time detection of LAMP with comparable sensitivity to SYBR Green and EvaGreen for the first time. Additionally, the results revealed adequate tolerance of the Bst DNA polymerase to higher concentrations of the dye. Thus, it could be used directly in a closed LAMP reaction without any optimization. Furthermore, the magnesium indicator hydroxynaphthol blue (HNB) was used for fluorometric real-time detection in LAMP for the first time. To extend the number of indirect detection methods for the accumulating pyrophosphate in LAMP and other enzymatic reactions, new metal-ion-dependent dyes were identified. The developed platform could support the iterative process of finding new fluorescent dyes with regard to enzyme compatibility and their spectral properties in the presence or absence of manganese ions. To obtain a selective fluorometric displacement assay, the complex concentration dependence between all components was investigated successfully by the establishment of a combinatorial screening in a microtiter plate. The visualization of the calculated signal-to-noise ratio was then used to identify alizarin red S and tetracycline as promising candidates under simulated reaction conditions. By testing both dyes in the enzymatic assay, alizarin red S was confirmed as low-cost, non-toxic and robust dye for the pyrophosphate dependent increase of the fluorescence intensity. The previously established dyes (HNB, calcein and alizarin red S) were applied successfully for the indirect and fluorometric detection of pyrophosphate in a LAMP-optimized emulsion. The stability and homogeneity of the generated emulsion was increased by adding the surfactant poloxamer 188. The fluorescence microscopic analysis showed a distinct discrimination between positive and negative droplets, in particular by using calcein, HNB and alizarin red S. Additionally, a new amplification reaction that is also based on the Bst DNA polymerase was developed to prevent the complicated primer design and likelihood of unspecific amplification in LAMP. The efficient regeneration of the single stranded priming site was achieved by the integration of a specific linker (abasic site or hexaethylenglycol) between two priming sites to create a bifunctional hinge-primer. After the hybridization on the template sequence, the hinge-primer was used to induce the refolding to a hairpin structure and for blocking the polymerase activity on the reverse strand. Thus, an autocyclic amplification can be achieved at isothermal reaction conditions. Finally, the efficiency of the hinge-initiated primer dependent amplification (HIP) was improved by decreasing the distance between the modified hinge-primer and the corresponding PCR-like primer. Conclusion: Due to its robustness and efficiency, LAMP has been developed to a powerful alternative for the standardized PCR-based diagnostics in molecular biology in the past years. Different detection methods improve the performance of the qualitative and quantitative LAMP in field applications as well as in diagnostics. The new DNA and pyrophosphate based assays can be used in closed reactions and contribute to a simple pathogen detection. Furthermore, the advancements can lead to a considerable reduction of costs and time compared to conventional methods. An attractive achievement is the further optimization of the HIP as sensitive pathogen assay by using LAMP-based detection methods. The use of Bst DNA polymerasedependent reactions will allow a robust integration of the isothermal amplification in microfluidic systems. By combining sample preparation, amplification and detection in one device, powerful applications with short analysis time and low instrumental requirements are a future perspective in pathogen diagnostics. KW - DNA KW - LAMP KW - Pathogen KW - Isothermale Amplifikation KW - Pyrophosphat Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-18314 ER - TY - JOUR A1 - Fischbach, Jens A1 - Frohme, Marcus A1 - Glökler, Jörn T1 - Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method JF - Scientific Reports N2 - The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel “hinge-primer” approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity. KW - DNA synthesis KW - pathogen Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9824 SN - 2045-2322 VL - 7 ER - TY - GEN A1 - Gering, Vanessa A1 - Kober, Liane A1 - Brödel, Oliver A1 - Koskei, Richard A1 - Mugendi, Beatrice A1 - Frohme, Marcus T1 - LC-MS/MS als Methode zur Detektion und Quantifizierung der Inhaltsstoffe in Kaffee T2 - Wissenschaftliche Beiträge 2017 N2 - Kaffee ist das wohl populärste Heißgetränk weltweit. Zwei botanische Arten sind für den Weltmarkt von Bedeutung und unterscheiden sich bezüglich ihres Anbaugebietes und der Inhaltsstoffe. Kaffee enthält über 850 bisher identifizierte Aromastoffe, welche den Geschmack bilden. Außerdem haben die Prozessierung der geernteten Kirsche bis zur Bohne, das Röstprofil und das Aufbrühen einen großen Einfluss darauf. In dieser Arbeit wurde die Auswirkung der Prozessierungsmethode auf ausgewählte Inhaltsstoffe mit Hilfe der Flüsssig-Chromatographie-Tandem-Massenspektroskopie (LC-MS/MS) untersucht. Dafür wurden drei Prozessierungsmethoden, die unterschiedlich viel Wasser für das Ablösen des Fruchtfleisches von den Kaffeekirschen benötigen, und zwei Kultursorten miteinander verglichen. Die Ergebnisse zeigten einen geringfügig höheren Gehalt der zwei wichtigsten Inhaltsstoffe Koffein und Chlorogensäure in den Bohnen, bei deren Prozessierung im Eco-Pulper-Verfahren weniger Wasser eingesetzt wurde. Bis auf einen verringerten Gehalt an Saccharose wurde für weitere wichtige Inhaltsstoffe (Acrylamid, Niacin, Zitronensäure und Vanillin) kein signifikanter Unterschied bei höherem Wassereinsatz gefunden. Insofern man die untersuchten Inhaltsstoffe als Qualitätsmarker betrachtet, ist im Vergleich zu den anderen Prozessierungsmethoden das Eco-Pulper-Verfahren die beste Wahl, insbesondere da es als wassersparende Methode einen entscheidenden Beitrag zur Nachhaltigkeit des Kaffeeanbaus leistet. N2 - Coffee is the most popular soft drink in the world. Two botanical species of coffee are important for the world market and differ not only in their area of cultivation but also their components. Coffee contains more than 850 aromatic compounds which constitute its taste and flavour. Also, the method selected for the processing from the harvested cherry to the bean, the roasting profile, and the method of brewing are relevant. In this work, liquid chromatography tandem-mass-spectroscopy (LC-MS/MS) is used to investigate how the processing method influences selected aromatic compounds. Thus, three different processing methods differing in the amount of water needed for separating the coffee beans from the cherries, and two cultivars were compared. The results showed slightly higher concentrations of the caffeine and chlorogenic acid components in coffee processed with less water (the eco-pulper method). Apart from a reduced sucrose concentration, we found no significant difference in other important compounds (acrylamide, niacin, citric acid and vanillin) when plenty of water was used. Taking the investigated compounds as quality markers, the eco-pulper method is the best choice among the processing methods, especially since it conserves water, which could therefore significantly contribute to a sustainable cultivation of coffee. Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9362 SN - 0949-8214 VL - 21 SP - 7 EP - 12 ER - TY - JOUR A1 - Gottschalk, Josefine Johanna A1 - Stollfuß, Carsten A1 - Lietzau, Kai-Henning A1 - Foitzik, Andreas A1 - Richetta, Maria T1 - Conceptual study for long-term monitoring of chemotherapeutic induced cell reactions by ESPI JF - tm - Technisches Messen N2 - During the last years, various approaches on an individualized drug therapy for benign cells have been researched. However, due to the complex topic a universal approach has not been found up until this point. Commonly, the effect of cytotoxic drugs on benign cells is in most cases the same compared to regular cells while the actual effect on patient still can't be predicted. In order to reduce unwanted side effects or unspecific drug reactions a test system for patients which allows to analyse the interaction between cytotoxic agents and the targeted cells is needed. Furthermore, this should also include an adequate measurement system which is capable to work in a natural environment and without any additional preparation. In terms of this work, a first proof of concept with different benign cells and cytotoxic agents is presented while monitoring the obtained displacement using electronic speckle pattern interferometry (ESPI). KW - interferometry KW - ESPI KW - anticancer drug KW - cellular displacement Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-10676 SN - 2196-7113 VL - 85 IS - 2 SP - 111 EP - 118 ER - TY - JOUR A1 - Hornemann, Andrea A1 - Sinning, Denise A1 - Cortes, Sofia A1 - Campino, Lenea A1 - Emmer, Peggy A1 - Kuhls, Katrin A1 - Ulm, Gerhard A1 - Frohme, Marcus A1 - Beckhoff, Burkhard T1 - A pilot study on fingerprinting Leishmania species from the Old World using Fourier transform infrared spectroscopy JF - Analytical and Bioanalytical Chemistry N2 - Leishmania species are protozoan parasites and the causative agents of leishmaniasis, a vector borne disease that imposes a large health burden on individuals living mainly in tropical and subtropical regions. Different Leishmania species are responsible for the distinct clinical patterns, such as cutaneous, mucocutaneous, and visceral leishmaniasis, with the latter being potentially fatal if left untreated. For this reason, it is important to perform correct species identification and differentiation. Fourier transform infrared spectroscopy (FTIR) is an analytical spectroscopic technique increasingly being used as a potential tool for identification of microorganisms for diagnostic purposes. By employing mid-infrared (MIR) spectral data, it is not only possible to assess the chemical structures but also to achieve differentiation supported by multivariate statistic analysis. This work comprises a pilot study on differentiation of Leishmania species of the Old World (L. major, L. tropica, L. infantum, and L. donovani) as well as hybrids of distinct species by using vibrational spectroscopic fingerprints. Films of intact Leishmania parasites and their deoxyribonucleic acid (DNA) were characterized comparatively with respect to their biochemical nature and MIR spectral patterns. The strains’ hyperspectral datasets were multivariately examined by means of variance-based principal components analysis (PCA) and distance-based hierarchical cluster analysis (HCA). With the implementation of MIR spectral datasets we show that a phenotypic differentiation of Leishmania at species and intra-species level is feasible. Thus, FTIR spectroscopy can be further exploited for building up spectral databases of Leishmania parasites in view of high-throughput analysis of clinical specimens. KW - Fourier transform infrared spectroscopy KW - hierarchical cluster analysis (HCA) KW - principal components analysis (PCA) KW - Leishmania KW - DNA KW - multivariate differentiation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-10080 SN - 1432-1130 ER - TY - JOUR A1 - Kaltdorf, Kristin Verena A1 - Schulze, Katja A1 - Helmprobst, Frederik A1 - Kollmannsberger, Philip A1 - Dandekar, Thomas A1 - Stigloher, Christian T1 - FIJI Macro 3D ART VeSElecT: 3D Automated Reconstruction Tool for Vesicle Structures of Electron Tomograms JF - PLoS Computational Biology N2 - Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i) in embryonic Danio rerio 4 and 8 days past fertilization (dpf) and (ii) to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ) wild-type and its septin mutant (unc-59(e261)). We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261) on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement). This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter). Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles) and specificity (true vesicles) as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual annotation. Both automatic and semi-automatic modes are explained including a tutorial. Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9218 SN - 1553-7358 VL - 13 IS - 1 ER - TY - JOUR A1 - Karakuş, Mehmet A1 - Nasereddin, Abed A1 - Onay, Hüseyin A1 - Karaca, Emin A1 - Özkeklikçi, Ahmet A1 - Jaffe, Charles L. A1 - Kuhls, Katrin A1 - Özbilgin, Ahmet A1 - Ertabaklar, Hatice A1 - Demir, Samiye A1 - Özbel, Yusuf A1 - Töz, Seray T1 - Epidemiological analysis of Leishmania tropica strains and giemsa-stained smears from Syrian and Turkish leishmaniasis patients using multilocus microsatellite typing (MLMT) JF - PLoS Neglected Tropical Diseases N2 - Turkey is located in an important geographical location, in terms of the epidemiology of vector-borne diseases, linking Asia and Europe. Cutaneous leishmaniasis (CL) is one of the endemic diseases in a Turkey and according to the Ministry Health of Turkey, 45% of CL patients originate from Şanlıurfa province located in southeastern Turkey. Herein, the epidemiological status of CL, caused by L. tropica, in Turkey was examined using multilocus microsatellite typing (MLMT) of strains obtained from Turkish and Syrian patients. A total of 38 cryopreserved strains and 20 Giemsa-stained smears were included in the present study. MLMT was performed using 12 highly specific microsatellite markers. Delta K (ΔK) calculation and Bayesian statistics were used to determine the population structure. Three main populations (POP A, B and C) were identified and further examination revealed the presence of three subpopulations for POP B and C. Combined analysis was performed using the data of previously typed L. tropica strains and Mediterranean and Şanlıurfa populations were identified. This finding suggests that the epidemiological status of L. tropica is more complicated than expected when compared to previous studies. A new population, comprised of Syrian L. tropica samples, was reported for the first time in Turkey, and the data presented here will provide new epidemiological information for further studies. Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9313 SN - 1935-2735 VL - 11 IS - 4 ER -