Detailed investigations of proximal tubular function in Imerslund-Grasbeck syndrome

BACKGROUND: Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B(12) receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. METHODS: Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. RESULTS: Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B(12) binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. CONCLUSIONS: In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria

Intelligente Vernetzung zur autonomen Fräsbearbeitung von Strukturbauteilen - Ergebnisbericht des BMBF Verbundprojektes TensorMill

Digitalisierte Prozesse können zukünftig zu einer intelligenten Fertigung beitragen, um den Herausforderungen einer intelligent vernetzten, autonomen Fertigung von sicherheitsrelevanten Integralbauteilen zu begegnen. Die Herausforderungen hierbei liegen insbesondere in der Aufzeichnung und Extraktion von nutzerrelevanten Daten zur Steigerung der Produktivität bei der Fertigung von sicherheitsrelevanten Integralbauteilen für die Luft- und Raumfahrtbranche. An diesem Punkt hat das Verbundforschungsprojekt „TensorMill“ angesetzt. Ziel des Projekts war es, die Produktivität in der spanenden Fertigung sicherheitsrelevanter Integralbauteile durch die Entwicklung und den Aufbau einer intelligent, vernetzten, autonomen Fertigung zu erhöhen und die Prozesssicherheit zu verbessern. Die intelligente Fertigung soll dabei in der Lage sein, auf möglichst viele Situationen im Fertigungsprozess mit Hilfe von künstlicher Intelligenz (KI) zu reagieren. Für die Implementierung der KI-basierten Lösungen sind im Projekt fortschrittliche Methoden und Vorgehensweisen entstanden, welche es ermöglichen, die Daten von Produktionsmitteln in einer einfachen Form nutzbar zu machen, damit diese einen Mehrwert für Hersteller und Anwender bringen. Die aufbereiteten Daten dienten schließlich der Umsetzung von KI-basierten Lösungen zur prozessparallelen Qualitätsprognose und Werkzeugzustandserkennung. Darüber hinaus wurde ein entwickeltes cyber-physisches Spannsystem entwickelt, um neuartige Ansätze zur Abdrängungskompensation und Echtzeitbewertung der Prozessstabilität zu erforschen

Characterization of Chromosomal Instability in Murine Colitis-Associated Colorectal Cancer

Patients suffering from ulcerative colitis (UC) bear an increased risk for colorectal cancer. Due to the sparsity of colitis-associated cancer (CAC) and the long duration between UC initiation and overt carcinoma, elucidating mechanisms of inflammation-associated carcinogenesis in the gut is particularly challenging. Adequate murine models are thus highly desirable. For human CACs a high frequency of chromosomal instability (CIN) reflected by aneuploidy could be shown, exceeding that of sporadic carcinomas. The aim of this study was to analyze mouse models of CAC with regard to CIN. Additionally, protein expression of p53, beta-catenin and Ki67 was measured to further characterize murine tumor development in comparison to UC-associated carcinogenesis in men.The AOM/DSS model (n = 23) and IL-10(-/-) mice (n = 8) were applied to monitor malignancy development via endoscopy and to analyze premalignant and malignant stages of CACs. CIN was assessed using DNA-image cytometry. Protein expression of p53, beta-catenin and Ki67 was evaluated by immunohistochemistry. The degree of inflammation was analyzed by histology and paralleled to local interferon-γ release.CIN was detected in 81.25% of all murine CACs induced by AOM/DSS, while all carcinomas that arose in IL-10(-/-) mice were chromosomally stable. Beta-catenin expression was strongly membranous in IL-10(-/-) mice, while 87.50% of AOM/DSS-induced tumors showed cytoplasmatic and/or nuclear translocation of beta-catenin. p53 expression was high in both models and Ki67 staining revealed higher proliferation of IL-10(-/-)-induced CACs.AOM/DSS-colitis, but not IL-10(-/-) mice, could provide a powerful murine model to mechanistically investigate CIN in colitis-associated carcinogenesis

Murine models of inflammation-driven carcinogenesis.

<p>(A) Endoscopic images of inflamed mucosa and tumor of AOM/DSS-colitis as well as the IL10^−/− model. Tumorigenesis was assessed by high resolution endoscopy <i>in vivo</i>. Methylene blue was used to enhance dysplasia detection as shown exemplarily for AOM/DSS colitis (B) Tumor size as rated by the tumor size score described in the methods section, IFNγ -levels, and inflammation score as assessed in H.E. staining for untreated controls, AOM/DSS-mice, and IL10^−/− mice. Bars represent means, whishkers represent standard error of the mean (SEM). Colonic mucosa samples of 10 healthy untreated C57BL/6J-mice were applied to assess histologic parameters and IFNγ -expression.</p

Immunohistochemistry for beta-catenin, p53 and Ki67 of murine premalignant tissue and CACs, details of 100x magnification.

<p>Immunohistochemistry for beta-catenin, p53 and Ki67 of murine premalignant tissue and CACs, details of 100x magnification.</p

Ploidy assessment according to Auer's classification, p53-, beta-catenin-, and Ki67-immunohistochemistry for murine tissue analyzed.

<p>AOM-CA_n: carcinomas of AOM/DSS-colitis. IL10CA_n: carcinomas of IL10^−/−-mice. CNTRL_n: premalignant tissue.</p><p>DNA-ploidy was assessed according to Auer (please refer to the text). p53 was assessed semiquantitatively, 0: no expression, 1 1–20% positive mucosa cells, 2: 21–50%, 3>50%</p><p>Beta-catenin: 1: membranous, 2: membranous-cytoplasmatic, 3: cytoplasmatic, 4: cytoplasmatic-nulcear, to 5, nuclear.</p

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor {kappa}B (NF-{kappa}B)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-{kappa}B, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-{gamma}, IL-2, and tumor necrosis factor-{alpha}, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis