Contributions of keratins to epithelial cell architecture and signaling

Keratin intermediate filament proteins form the major cytoskeleton in all epithelia. Increasing evidence suggests that keratins act as cytoskeletal scaffolds which locally regulate cell growth and survival. Many of these functions, however, are not understood in full, owing to keratin redundancy. Previous gene function studies have been impeded by gain of toxic function phenotypes. Therefore, transgenic mice lacking the entire keratin multiprotein family were analyzed and a corresponding keratinocyte cell line was generated. The deletion of keratins resulted in prenatal death at E9.5 due to severe growth retardation. Within the scope of this thesis it was demonstrated that this was in part caused by repressed protein biosynthesis, due to mislocalized GLUTs mediated by the mTOR pathway. The analysis of extraembryonic tissue further revealed an influence of keratins on the adhesion between endodermal and mesodermal cell layers. As a consequence, keratin depleted embryos suffered from reduced yolk sac haematopoiesis and vasculogenesis, due to altered hormone and growth factor gradients. Similar effects were reported for mislocalized trophoblast giant cells in the absence of keratins which altered signaling and hormone secretion leading to increased vascularization of the maternal decidua. Hyperoxia in the decidua entailed defective Hif1α and VEGF signaling, resulting in impaired placental vasculogenesis and concomitant impairment of maternal and embryonic gas and nutrient exchange. To overcome the limitation of an in vivo model in determining functions at the molecular level, a cell culture model was generated, the CK minus keratinocytes. First data indicated drastic changes in morphology of the ER and the Golgi complex, possibly ER stress, a hypothesis emerging form the acquired in vivo data of decreased glucose levels and increased hypoxia. Furthermore, similar to the mouse model desmosomes were altered, as demonstrated by DP and plectin relocalization and a misorganization of the actin cytoskeleton was observed. This suggests that the amount of keratin and eventually specific isotypes regulate formation and turnover of other multiprotein complexes, as cell junctions and other cytoskeletal systems, influencing cell attachment and migration in the epithelial tissue environment. Translational control, restructuring of the tissue environment and cell migration are implications for stem cell turnover during development and tissue repair, as well as tumorigenesis. This thesis provides for the first time the tools, the CK minus cells, to examine the influences of keratin free or keratin isotype specific cells on tumor development and tissue regeneration by reinjection. Furthermore, with this easily accessible cell culture system, isotype-specific functions can be examined by re-expression of individual keratin pairs. This will help to understand the function of keratins in vivo including their associated diseases and will have implications for therapy approaches

Genetic variants including markers from the exome chip and metabolite traits of type 2 diabetes

Diabetes-associated metabolites may aid the identification of new risk variants for type 2 diabetes. Using targeted metabolomics within a subsample of the German EPIC-Potsdam study (n = 2500), we tested previously published SNPs for their association with diabetes-associated metabolites and conducted an additional exploratory analysis using data from the exome chip including replication within 2,692 individuals from the German KORA F4 study. We identified a total of 16 loci associated with diabetes-related metabolite traits, including one novel association between rs499974 (MOGAT2) and a diacyl-phosphatidylcholine ratio (PC aa C40:5/PC aa C38:5). Gene-based tests on all exome chip variants revealed associations between GFRAL and PC aa C42:1/PC aa C42:0, BIN1 and SM (OH) C22:2/SM C18:0 and TFRC and SM (OH) C22:2/SM C16:1). Selecting variants for gene-based tests based on functional annotation identified one additional association between OR51Q1 and hexoses. Among single genetic variants consistently associated with diabetes-related metabolites, two (rs174550 (FADS1), rs3204953 (REV3L)) were significantly associated with type 2 diabetes in large-scale meta-analysis for type 2 diabetes. In conclusion, we identified a novel metabolite locus in single variant analyses and four genes within gene-based tests and confirmed two previously known mGWAS loci which might be relevant for the risk of type 2 diabetes

Markierungsfreie Online-Detektion der Hybridisierung auf einem DNA-Chip : Detektionsmethode für den Gensensor

Die Genomforschung hat sich in den letzten Jahren verstärkt in Richtung neuer analytischer Methoden orientiert, um der großen Anzahl an notwendigen Experimenten durch parallele Ansätze zu begegnen. Der größte Entwicklungsschritt ist hierbei die Verwendung von sogenannten DNA-Chips, womit flächige Arrays aus immobilisierten DNA-Fragmenten beschrieben werden, die zu Anwendungen von der Expressionsanalytik (high density arrays) bis hin zu diagnostischen Chips (low density arrays) herangezogen werden. Die Detektion auf Arrays erfolgt in kommerziellen Geräten in der Regel fast ausschließlich durch Fluoreszenzemission von farbstoffmarkierten Oligonukleotiden an der Oberfläche. In mehreren Beispielen wurde bereits die Detektion der Hybridisierung auf markierungsfreien Biosensoren, jedoch nur in Einkanalmessungen, gezeigt, wie z.B. auf SPR (Biacore), Gitterkoppler oder RIfS (Reflektometrische Interferenzspektroskopie). Diese oberflächenbasierten Detektionsmethoden erfordern keinerlei Markierung der zu detektierenden Komponenten, d.h. es kann im Einzelfall direkt mit Extrakten oder Amplifikaten aus komplexen Matrices heraus gearbeitet werden. Der Schritt der Farbstoffmarkierung entfällt und damit entfällt auch die mögliche Beeinflussung der Wechselwirkung durch die Markierung, genauso wie die Beeinflussung der Fluoreszenzintensität an der Oberfläche durch unspezifische Quenchingefffekte oder auch Photobleaching. Im Bremer Forschungs- und Entwicklungsverbund Gensensorik wird mit dem Ziel einer industriellen Umsetzung des Gensensorik Konzeptes an einer Integration der DNA-Chiptechnologie in ein Gesamtkonzept aus Bioinformatik, biochemischer Fragestellung, Oberflächenchemie, Chipproduktion, Detektion und Datenauswertung/-bewertung gearbeitet. Im Rahmen dieses FuE Verbundeses wird die parallele und zeitaufgelöste Detektion von Hybridisierungsreaktionen an der Oberfläche mittels Reflektometrischer Interferenzspektroskopie auf das Chipformat übertragen

Prevention and Treatment of Chemotherapy-Induced Peripheral Neuropathy (CIPN) with Non-Pharmacological Interventions: Clinical Recommendations from a Systematic Scoping Review and an Expert Consensus Process

Background: Most individuals affected by cancer who are treated with certain chemotherapies suffer of CIPN. Therefore, there is a high patient and provider interest in complementary non-pharmacological therapies, but its evidence base has not yet been clearly pointed out in the context of CIPN. Methods: The results of a scoping review overviewing the published clinical evidence on the application of complementary therapies for improving the complex CIPN symptomatology are synthesized with the recommendations of an expert consensus process aiming to draw attention to supportive strategies for CIPN. The scoping review, registered at PROSPERO 2020 (CRD 42020165851), followed the PRISMA-ScR and JBI guidelines. Relevant studies published in Pubmed/MEDLINE, PsycINFO, PEDro, Cochrane CENTRAL, and CINAHL between 2000 and 2021 were included. CASP was used to evaluate the methodologic quality of the studies. Results: Seventy-five studies with mixed study quality met the inclusion criteria. Manipulative therapies (including massage, reflexology, therapeutic touch), rhythmical embrocations, movement and mind–body therapies, acupuncture/acupressure, and TENS/Scrambler therapy were the most frequently analyzed in research and may be effective treatment options for CIPN. The expert panel approved 17 supportive interventions, most of them were phytotherapeutic interventions including external applications and cryotherapy, hydrotherapy, and tactile stimulation. More than two-thirds of the consented interventions were rated with moderate to high perceived clinical effectiveness in therapeutic use. Conclusions: The evidence of both the review and the expert panel supports a variety of complementary procedures regarding the supportive treatment of CIPN; however, the application on patients should be individually weighed in each case. Based on this meta-synthesis, interprofessional healthcare teams may open up a dialogue with patients interested in non-pharmacological treatment options to tailor complementary counselling and treatments to their needs

Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor

Removal of the entire keratin family of intermediate filament proteins from embryonic epithelia has surprising implications for mTOR signaling

Prevention and treatment of chemotherapy-induced peripheral neuropathy (CIPN) with non-pharmacological interventions : clinical recommendations from a systematic scoping review and an expert consensus process

Background: Most individuals affected by cancer who are treated with certain chemotherapies suffer of CIPN. Therefore, there is a high patient and provider interest in complementary non-pharmacological therapies, but its evidence base has not yet been clearly pointed out in the context of CIPN. Methods: The results of a scoping review overviewing the published clinical evidence on the application of complementary therapies for improving the complex CIPN symptomatology are synthesized with the recommendations of an expert consensus process aiming to draw attention to supportive strategies for CIPN. The scoping review, registered at PROSPERO 2020 (CRD 42020165851), followed the PRISMA-ScR and JBI guidelines. Relevant studies published in Pubmed/MEDLINE, PsycINFO, PEDro, Cochrane CENTRAL, and CINAHL between 2000 and 2021 were included. CASP was used to evaluate the methodologic quality of the studies. Results: Seventy-five studies with mixed study quality met the inclusion criteria. Manipulative therapies (including massage, reflexology, therapeutic touch), rhythmical embrocations, movement and mind-body therapies, acupuncture/acupressure, and TENS/Scrambler therapy were the most frequently analyzed in research and may be effective treatment options for CIPN. The expert panel approved 17 supportive interventions, most of them were phytotherapeutic interventions including external applications and cryotherapy, hydrotherapy, and tactile stimulation. More than two-thirds of the consented interventions were rated with moderate to high perceived clinical effectiveness in therapeutic use. Conclusions: The evidence of both the review and the expert panel supports a variety of complementary procedures regarding the supportive treatment of CIPN; however, the application on patients should be individually weighed in each case. Based on this meta-synthesis, interprofessional healthcare teams may open up a dialogue with patients interested in non-pharmacological treatment options to tailor complementary counselling and treatments to their needs