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Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin’s basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3′-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3′-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3′-RTMS6m repair molecule.

Human skin contains a population of memory T cells that supports tissue homeostasis and provides protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tissue environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rγnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES was generated from human keratinocytes and fibroblasts and was initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC, this reductionist system allowed us to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

Cross-language authorship attribution problems rely on either translation to enable the use of single-language features, or language-independent feature extraction methods. Until recently, the lack of datasets for this problem hindered the development of the latter, and single-language solutions were performed on machine-translated corpora. In this paper, we present a novel language-independent feature for authorship analysis based on dependency graphs and universal part of speech tags, called DT-grams (dependency tree grams), which are constructed by selecting specific sub-parts of the dependency graph of sentences. We evaluate DT-grams by performing cross-language authorship attribution on untranslated datasets of bilingual authors, showing that, on average, they achieve a macro-averaged F1 score of 0.081 higher than previous methods across five different language pairs. Additionally, by providing results for a diverse set of features for comparison, we provide a baseline on the previously undocumented task of untranslated cross-language authorship attribution., To be published in: "32. GI-Workshop Grundlagen von Datenbanken"

BackgroundCutaneous squamous cell carcinomas (cSCC) are the primary cause of premature deaths in patients suffering from the rare skin-fragility disorder recessive dystrophic epidermolysis bullosa, which is in marked contrast to the rarely metastasizing nature of these carcinomas in the general population. This remarkable difference is attributed to the frequent development of chronic wounds caused by an impaired skin integrity. However, the specific molecular and cellular changes to malignancy, and whether there are common players in different types of aggressive cSCCs, remain relatively undefined.MethodsMiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimized in situ hybridization protocol. Functional impact of overexpression of a dysregulated miRNA was assessed in migration and 3D spheroid assays. Sample-matched transcriptome data was generated to support the identification of disease relevant miRNA targets.ResultsSeveral miRNAs were identified as dysregulated in cSCCs as compared to controls. These included the metastasis-linked miR-10b, which was significantly upregulated in primary cell cultures and in archival biopsies. At the functional level, overexpression of miR-10b conferred the stem cell-characteristic of 3D-spheroid formation capacity to keratinocytes, and impaired their mobility. Analysis of miR-10b downstream effects identified a novel putative target of miR-10b, the actin- and tubulin cytoskeleton-associated protein DIAPH2.ConclusionThe discovery that miR-10b confers an aspect of cancer stemness – that of enhanced tumor cell adhesion, known to facilitate metastatic colonization - provides an important avenue for future development of novel therapies targeting this metastasis-linked miRNA.

Human skin contains a population of memory T cells that supports tissue homeostasis and provides protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tissue environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES was generated from human keratinocytes and fibroblasts and was initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC, this reductionist system allowed us to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases. (VLID)5327496

Marine species have evolved a variety of physical or chemical strategies to diminish damage from elevated environmental ultraviolet radiation. Mycosporine-like amino acids, a group of widely distributed small water soluble compounds, are biologically relevant because of their photo-protective potential. In addition, presumed antioxidant and skin protective strategies raise the interest for possible medicinal and cosmetic applications. In this study the first CE method for the quantification of mycosporine-like amino acids in marine species is presented. A borate buffer system consisting of 30 mM sodium tetraborate in water at a pH-value of 10.3 enabled the baseline separation of five MAAs, namely palythine, mycosporine-serinol, asterina-330, shinorine and porphyra-334, in 27 min. Separation voltage, temperature and detection wavelength were 25 kV, 25 °C and 320 nm, respectively. The optimized method was fully validated and applied for the quantitative determination of MAAs in the marine macroalgae Palmaria palmata, Porphyra umbilicalis, and Porphyra sp., as well as the lichen Lichina pygmaea.

As one of the most frequently occurring monomers in the biosphere, glucosamine is a valuable metabolite for several applications. Although microbial glucosamine production is still in its infancy, it offers the possibility to circumvent problems associated with traditional production by hydrolysis. Of particular interest is a study with Aspergillus niger, which reports for the first time high glucosamine excretion in the early phase of citric acid production. These results have relevance for both the commercial glucosamine production and deeper insight into the regulation of organic acid excretion in fungi. To investigate glucosamine excretion, we performed bioreactor batch cultivations with Penicillium ochrochloron CBS123.824 and A. niger CBS120.49 using cultivation conditions which are known to trigger the production of citric acid. Glucosamine detection in culture filtrates was achieved by two photometric methods, High performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and HPLC with mass spectrometry detection (HPLC-MS). Surprisingly, we detected no glucosamine at all. Based on a critical review of published data for A. niger, we conclude that the reported high levels of excreted glucosamine might be an experimental artifact. However, growth experiments with glucosamine as a combined or single source for carbon or nitrogen showed that both organisms are in principle able to transport glucosamine across their plasma membrane, which is a prerequisite for the excretion of glucosamine.

Introduction Multidisciplinary, complex rehabilitation interventions are an important part of the treatment of chronic diseases. However, little is known about the effectiveness of routine rehabilitation interventions within the German healthcare system. Due to the nature of the social insurance system in Germany, randomised controlled trials examining the effects of rehabilitation interventions are challenging to implement and scarcely accessible. Consequently, alternative pre-post designs can be employed to assess pre-post effects of medical rehabilitation programmes. We present a protocol of systematic review and meta-analysis methods to assess the pre-post effects of rehabilitation interventions in Germany. Methods and analysis The respective study will be conducted within the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. A systematic literature review will be conducted to identify studies reporting the pre-post effects (start of intervention vs end of intervention or later) in German healthcare. Studies investigating the following disease groups will be included: orthopaedics, rheumatology, oncology, pulmonology, cardiology, endocrinology, gastroenterology and psychosomatics. The primary outcomes of interest are physical/mental quality of life, physical functioning and social participation for all disease groups as well as pain (orthopaedic and rheumatologic patients only), blood pressure (cardiac patients only), asthma control (patients with asthma only), dyspnoea (patients with chronic obstructive pulmonary disease only) and depression/anxiety (psychosomatic patients only). We will invite the principal investigators of the identified studies to provide additional individual patient data. We aim to perform the meta-analyses using individual patient data as well as aggregate data. We will examine the effects of both study-level and patient-level moderators by using a meta-regression method. Ethics and dissemination Only studies that have received institutional approval from an ethics committee and present anonymised individual patient data will be included in the meta-analysis. The results will be presented in a peer-reviewed publication and at research conferences. A declaration of no objection by the ethics committee of the University of Würzburg is available (number 20180411 01). Trial registration number CRD42018080316.

Smart factories, in which processes are partially or completely automated and digitalized, are the future. Automobile production is also currently undergoing said change towards ‘Industry 4.0’. While the degree of automation is very high in car body construction due to the use of robots, the degree of automation in logistics varies depending on the process. By contrast, in transportation processes, such as the use of tugger trains, autonomous transport systems or forklifts, automation is at a very advanced stage of development. In picking processes the employee continues to be at the center of attention due to his flexibility in movement sequences and grasping. Assistance systems such as smart glasses, smart watches or scanner gloves can nevertheless support employees and provide process improvements. The present research project focuses on the use of Augmented Reality (AR) technologies, in particular the usage of smart glasses in manual man-to-goods order picking processes. The required information for order picking is displayed in smart glasses in the picker’s field of view. In scientific research, their usage has been tested primarily under laboratory conditions. I subsequently identified a research gap investigating a full shift usage of smart glasses under real industrial production conditions. For this reason, the present PhD thesis includes a full shift field study, which examines the impact of the smart glasses usage in order picking processes on the employee and the process. As a first step, a process for shop floor employee involvement in innovation projects based on Rogers’ ‘Diffusion of Innovations’ will be developed. Afterwards, the suitability of workstations for AR-usage will be evaluated using the Rasmussen skills-rules-knowledge-framework. In addition, an objective procedure for classifying individual activities into Rasmussen’s framework will be developed. After selecting the test workstation, which contains most of the process steps supportable by AR-technologies, I will compare scanning mechanisms for the interaction with the warehouse management system. The core of the research project is a full shift field study with 23 employees in automotive assembly supply, which examines the impact of a smart glasses usage on the number of errors, the error types, the task completion time, the attention, the subjective strain, the visual fatigue of the employees and the likelihood of symptomatic occurrence of simulator sickness. In contrast to most existing studies, the field study is conducted in real production in accordance to the rhythm of the assembly line. The test system is connected to the warehouse management system. Employees interact with the warehouse management system with the aid of a scanner. The age range of the participants represents the regular workforce. Besides that, I will select a smart glasses hardware, which enables the inclusion of corrective glasses wearers. All the above points contribute to a test under real industrial conditions. As a basis for comparison serves the previous process, which displays the picking orders on a monitor attached to the target shelf. In this system a scan glove is used as interaction mechanism. The evaluation of the study results determine a time saving of 22% and an error reduction of at least 33%, depending on the error type. Due to these promising results, I explain further steps for series introduction. In addition, I discuss further potentials of the usage of data glasses in order picking processes. Smarte Fabriken, in welchen Prozesse teilweise oder gänzlich automatisiert und digitalisiert sind, sind die Zukunft. Auch die Automobilproduktion befindet sich derzeit in diesem Wandel hin zur „Industrie 4.0“. Während beispielsweise im Karosseriebau der Automatisierungsgrad durch den Einsatz von Robotern sehr hoch ist, ist er in der Logistik prozessspezifisch unterschiedlich ausgeprägt. Bei Transportprozessen, wie dem Einsatz von Routenzügen, fahrerlosen Transportsystemen oder Gabelstaplern, ist der Entwicklungsstand der Automatisierung sehr weit fortgeschritten. In Kommissionierprozessen hingegen steht der Mitarbeiter durch seine Flexibilität in Bewegungsabläufen und im Greifen weiterhin im Mittelpunkt. Der Einsatz von Assistenzsystemen wie Datenbrillen, Smart Watches oder Scanner-Handschuhen kann dennoch Mitarbeiter unterstützen und Prozessverbesserungen hervorrufen. In dem vorliegenden Forschungsprojekt steht der Einsatz von Augmented Reality (AR)-Technologien, insbesondere der Einsatz von Smart Glasses, in der manuellen Mann-zur-Ware Kommissionierung im Fokus der Betrachtung. Dabei werden dem Mitarbeiter die zum Kommissionieren benötigten Informationen in einer Datenbrille im Sichtfeld angezeigt. In der Wissenschaft wurde ein Einsatz vor allem in Laborstudien getestet. Eine Studie über eine volle Schicht im industriellen Kontext wird als Forschungslücke angegeben. Aus diesem Grund beinhaltet die vorliegende Doktorarbeit eine Feldstudie im Vollschichtbetrieb, welche die Auswirkungen der Nutzung von Smart Glasses in der Kommissionierung auf den Mitarbeiter und den Prozess untersucht. Als ersten Schritt wird ein Prozess zur Mitarbeitereinbeziehung in Innovationsprojekten am Shop Floor basierend auf Rogers „Diffusion of Innovations“ erarbeitet. Im Anschluss wird ein für den AR-Einsatz geeigneter Arbeitsplatz mithilfe des Rasmussen-Modells menschlichen Verhaltens ausgewählt. Dazu wird ein objektives Verfahren zur Klassifizierung einzelner Aktivitäten in das Rasmussen-Modell entwickelt. Nach der Auswahl des Arbeitsplatzes, welcher die meisten durch AR-Technologien unterstützbare Prozessschritte enthält, werden Scan-Mechanismen zur Interaktion mit dem Warehouse Management System verglichen. Kern des Forschungsprojekts ist ein Vollschicht-Feldversuch mit 23 Mitarbeitern in der automobilen Montageversorgung, welcher die Auswirkungen des Einsatzes von Datenbrillen auf die Fehleranzahl, die Fehlerart, die Durchführungszeit, auf die Aufmerksamkeit, die subjektive Beanspruchung, die visuelle Ermüdung der Mitarbeiter und eine mögliche auftretende Simulatorkrankheit untersucht. Im Gegensatz zu den meisten bereits existierenden Studien erfolgt der Versuch im Realbetrieb unter Einhaltung des Produktionstakts. Dabei wird das Testsystem an das Warehouse Management System angebunden. Die Interaktion wird durch den Mitarbeiter selbst und nicht über einen Wizard-of-Oz-Ansatz durchgeführt. Die Altersspanne der Teilnehmer deckt im Versuch die gesamte Breite der Belegschaft ab. Darüber hinaus wird eine Datenbrillen-Hardware genutzt, welche die Einbeziehung von Brillenträgern ermöglicht. All die oben genannten Punkte führen zu einem Versuch unter Realbedingungen. Als Vergleichsbasis dient der jetzige Prozess, welcher die Kommissionieraufträge auf einem am Zielregal mitgeführten Monitor darstellt. Als Interaktionsmechanismus im bisherigen System dient ein Scan-Handschuh. Nach der Auswertung der Ergebnisse der Probandenstudie, welche u.a. eine Zeiteinsparung von 22% und eine Fehlerreduzierung von mindestens 33%, abhängig von der Fehlerart, nachweist, werden weitere Schritte zur Serieneinführung aufgezeigt. Darüber hinaus werden weitere Potenziale des Einsatzes von Datenbrillen in der Kommissionierung diskutiert.

Human skin contains a population of memory T cells that support tissue homeostasis and provide protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tisse environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rγnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES were generated from human keratinocytes and fibroblasts and is initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC this reductionist system allowed to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

RNA trans-splicing has become a versatile tool in the gene therapy of monogenetic diseases. This technique is especially valuable for the correction of mutations in large genes such as COL7A1, which underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa. Over 800 mutations spanning the entire length of the COL7A1 gene have been associated with defects in type VII collagen, leading to excessive fragility of epithelial tissues, the hallmark of dystrophic epidermolysis bullosa (DEB). In the present study, we designed an RNA trans-splicing molecule (RTM) that is capable of repairing any given mutation within a 4200 nucleotide region spanning the 3′ half of COL7A1. The selected RTM, RTM28, was able to induce accurate trans-splicing into endogenous COL7A1 pre-mRNA transcripts in a type VII collagen-deficient DEB patient-derived cell line. Correct trans-splicing was detected at the RNA level by semiquantitative RT-PCR and correction of full-length type VII collagen was confirmed at the protein level by immunofluorescence and western blot analyses. Our results demonstrate that RTM28, which covers >60% of all mutations reported in DEB and is thus the longest RTM described so far for the repair of COL7A1, represents a promising candidate for therapeutic applications.

In recent years, RNA trans-splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher trans-splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA trans-splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the trans-splicing efficiency. Thus, we assume that besides the RNA trans-splicing molecule, short antisense molecules can act as splicing modulators, thereby increasing the trans-splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases.

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA-based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA splicing modulation by antisense oligonucleotides and RNA trans-splicing, transcript replacement and genome editing by in vitro-transcribed mRNAs, and gene knockdown by small interfering RNA and anti-sense oligonucleotides. We present the current state of these therapeutic approaches and critically discuss their opportunities, limitations and the challenges that remain to be solved. The aim of this review was to set the stage for the development of new and better therapies to improve the lives of patients and families affected by a genodermatosis.

We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

(VLID)2704563

submitted by Michaela Murauer, BSc. Universität Linz, Masterarbeit, 2017