Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Algorithmen zum Vergleich visueller Explorationsmuster

Our actions and intentions characterize the movement patterns of our eyes. Our visual exploration is driven by a mixture of cognitive processes and a conflict between the inspection of detail and the maintenance of an up-to-date overview. As a consequence, the determination of the influence of separate behavioral factors is challenging. The work at hand examines how eye movement sequences can be compared to each other. This process is at the core of almost every eye-tracking study as it answers questions such as: "Does gaze behavior of a patient group differ from his/her control group?," "How does experts’ visual exploration differ from novices’?," "How does the composition of a painting influence the observer’s gaze?" Therefore, several eye movement processing steps are revised: The issue of data quality is discussed with focus on methods and benchmarks to assure good quality during pupil detection, gaze mapping and eye movement identification in dynamic scenarios. Furthermore, eye-tracking data is integrated with physiological parameters such as ECG, galvanic skin conductivity and pupil dilation. The fusion of these complementary physiological sensors helps to disambiguate gaze and attention allocation. This thesis proposes a novel method for the comparison of visual scan patterns, which is based on the frequency of short snippets of the whole eye movement sequence. Combined with current techniques in machine learning, the method is adaptable to a multitude of applications. Visualization and aggregation procedures for frequently traversed gaze trails are demonstrated on the basis of the finding that these short patterns are highly characteristic to many applications. The proposed comparison technique is evaluated against state-of-the-art approaches on a new collection of data from a broad spectrum of eye-tracking experiments, ranging from static viewing tasks to highly dynamic outdoor scenarios. It effectively predicts the observer’s task in a conjunction search task and in the more complex Yarbus experiment significantly above chance level. Furthermore, it is possible to assess driving fitness and the driver’s secondary task, as well as to classify the expertise of neurosurgeons. This new approach is able to identify the influence of a single experimental factor upon eye movement sequences. In contrast to all competing methods, it generalizes well over a broad spectrum of experimental designs.Unsere Handlungen und Absichten spiegeln sich in den Bewegungen unserer Augen wieder. Die visuelle Exploration wird von einem Mix an kognitiven Prozessen und dem Konflikt sowohl Details erkennen zu können, als auch einen aktuellen Überblick zu bewahren, angetrieben. Deshalb ist es schwierig den Effekt einzelner Verhaltensfaktoren zu isolieren. Diese Arbeit untersucht, wie Augenbewegungssequenzen miteinander verglichen werden können. Dieser Vergleich ist Herzstück fast jeder Eye-Tracking Studie und beantwortet Fragen wie: "Unterscheidet sich das Blickverhalten einer Patientengruppe von der Kontrollgruppe?", "Wie unterscheidet sich das Explorationsverhalten von Experten und Anfängern?", "Wie wirkt sich die Komposition eines Bildes auf den Blick des Betrachters aus?" Dafür sind mehrere Verarbeitungsprozesse der Augenbewegungen notwendig: Die Frage der Datenqualität wird mit Schwerpunkt auf Benchmarks zur Sicherstellung guter Pupillenerkennung, Blickrichtungsbestimmung und Augenbewegungsklassifikation in dynamischen Szenarien behandelt. Außerdem werden Blickdaten mit anderen physiologischen Signalen, wie EKG, Hautleitwert und Pupillendurchmesser kombiniert. Die Fusion dieser sich ergänzenden Sensoren ermöglicht es Blick- und Aufmerksamkeitszuwendung voneinander zu trennen. In dieser Arbeit wird ein neuer Algorithmus für den Vergleich von visuellen Blicksequenzen vorgestellt, der auf Häufigkeitsverteilung kurzer Teilsequenzen in der Gesamtsequenz basiert. Kombiniert mit einem maschinellen Lernverfahren kann sich diese Methode selbstständig an eine Vielzahl von Applikationen anpassen. Visualisierungsformen und Aggregationsmethoden für häufig genutzte Blickpfade, die ebenfalls auf der Annahme basieren, dass diese kurzen Teilsequenzen charakteristisch für viele Anwendungen sind, werden demonstriert. Die vorgestellte Methode wird auf einem neuen Datensatz, der ein breites Spektrum an typischen Eye-Tracking Experimenten enthält, gegen den Stand der Technik evaluiert. Diese Daten enthalten sowohl statische, als auch hochdynamische Realszenarien. So kann die Aufgabenstellung des Betrachters während einer seriellen Suchaufgabe aber auch während der komplexeren Aufgabenstellung des Yarbus Experiments signifikant über der Ratewahrscheinlichkeit klassifiziert werden. Außerdem ist es möglich Fahrtüchtigkeit und durchgeführte Nebenaufgabe eines Fahrzeugführers, sowie den Erfahrungslevel von Chirurgen zu bestimmen. Dieser neuartige Ansatz ist in der Lage den Einfluss einzelner Faktoren auf eine Blicksequenz zu identifizieren. Im Gegensatz zu anderen Methoden generalisiert der Ansatz auf ein breites Spektrum an experimentellen Designs

Metabolic Fingerprinting of Murine L929 Fibroblasts as a Cell-Based Tumour Suppressor Model System for Methionine Restriction

Since Otto Warburg reported in 1924 that cancer cells address their increased energy requirement through a massive intake of glucose, the cellular energy level has offered a therapeutic anticancer strategy. Methionine restriction (MetR) is one of the most effective approaches for inducing low-energy metabolism (LEM) due to the central position in metabolism of this amino acid. However, no simple in vitro system for the rapid analysis of MetR is currently available, and this study establishes the murine cell line L929 as such a model system. L929 cells react rapidly and efficiently to MetR, and the analysis of more than 150 different metabolites belonging to different classes (amino acids, urea and tricarboxylic acid cycle (TCA) cycles, carbohydrates, etc.) by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. The system facilitates the rapid and efficient testing of potential cancer therapeutic metabolic targets. To date, MS studies of MetR have been performed using organisms and yeast, and the current LC/MS analysis of the intra- and extracellular metabolites in the murine cell line L929 over a period of 5 days thus provides new insights into the effects of MetR at the cellular metabolic level

Cysteine restriction in murine L929 fibroblasts as an alternative strategy to methionine restriction in cancer therapy

Methionine restriction (MetR) is an efficient method of amino acid restriction (AR) in cells and organisms that induces low energy metabolism (LEM) similar to caloric restriction (CR). The implementation of MetR as a therapy for cancer or other diseases is not simple since the elimination of a single amino acid in the diet is difficult. However, the in vivo turnover rate of cysteine is usually higher than the rate of intake through food. For this reason, every cell can enzymatically synthesize cysteine from methionine, which enables the use of specific enzymatic inhibitors. In this work, we analysed the potential of cysteine restriction (CysR) in the murine cell line L929. This study determined metabolic fingerprints using mass spectrometry (LC/MS). The profiles were compared with profiles created in an earlier work under MetR. The study was supplemented by proliferation studies using D-amino acid analogues and inhibitors of intracellular cysteine synthesis. CysR showed a proliferation inhibition potential comparable to that of MetR. However, the metabolic footprints differed significantly and showed that CysR does not induce classic LEM at the metabolic level. Nevertheless, CysR offers great potential as an alternative for decisive interventions in general and tumour metabolism at the metabolic level

Low energy status under methionine restriction is essentially independent of proliferation or cell contact inhibition

Nonlimited proliferation is one of the most striking features of neoplastic cells. The basis of cell division is the sufficient presence of mass (amino acids) and energy (ATP and NADH). A sophisticated intracellular network permanently measures the mass and energy levels. Thus, in vivo restrictions in the form of amino acid, protein, or caloric restrictions strongly affect absolute lifespan and age-associated diseases such as cancer. The induction of permanent low energy metabolism (LEM) is essential in this process. The murine cell line L929 responds to methionine restriction (MetR) for a short time period with LEM at the metabolic level defined by a characteristic fingerprint consisting of the molecules acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP. Here, we used mass spectrometry (LC/MS) to investigate the influence of proliferation and contact inhibition on the energy status of cells. Interestingly, the energy status was essentially independent of proliferation or contact inhibition. LC/MS analyses showed that in full medium, the cells maintain active and energetic metabolism for optional proliferation. In contrast, MetR induced LEM independently of proliferation or contact inhibition. These results are important for cell behaviour under MetR and for the optional application of restrictions in cancer therapy

Free Skin Grafting to Reconstruct Donor Sites after Radial Forearm Flap Harvesting: A Prospective Study with Platelet-Rich Fibrin (PRF)

Reconstruction of the donor site after radial forearm flap harvesting is a common procedure in maxillofacial plastic surgery. It is normally carried out with split-thickness or full-thickness free skin grafts. Unfortunately, free skin graft transplantation faces wound healing impairments such as necrosis, (partial) graft loss, or tendon exposure. Several studies have investigated methods to reduce these impairments and demonstrated improvements if the wound bed is optimised, for example, through negative-pressure wound therapy or vacuum-assisted closure. However, these methods are device-dependent, expansive, and time-consuming. Therefore, the application of platelet-rich fibrin (PRF) to the wound bed could be a simple, cost-effective, and device-independent method to optimise wound-bed conditions instead. In this study, PRF membranes were applied between the wound bed and skin graft. Results of this study indicate improvements in the PRF versus non-PRF group (93.44% versus 86.96% graft survival, p = 0.0292). PRF applied to the wound bed increases graft survival and reduces impairments. A possible explanation for this is the release of growth factors, which stimulate angiogenesis and fibroblast migration. Furthermore, the solid PRF membranes act as a mechanical barrier (&ldquo;lubrication&rdquo; layer) to protect the skin graft from tendon motion. The results of this study support the application of PRF in donor-site reconstruction with free skin grafts

Mandibular intraosseous pseudocarcinomatous hyperplasia: a case report

Background Mandibular pseudocarcinomatous hyperplasia is a rare and generally benign pathology. We report on one of these rare cases. Case presentation The case history of a 73-year-old white man stated that he had a carcinoma of the oropharynx, which was primarily treated with radiotherapy and chemotherapy 4 years prior. As a result of radiotherapy he developed an osteoradionecrosis of his mandible and a consecutive pathological fracture of his left mandibular angle. Subsequent osteosynthesis was performed with a reconstruction plate. When we first saw him, his reconstruction plate was partially exposed with intraoral and extraoral fistulation. The resected bone of his defect-bordering jaw showed the typical pathohistological findings of an intraosseous mandibular pseudocarcinomatous hyperplasia. After a first reconstruction attempt with an iliac crest graft failed, definitive reconstruction of his mandible with a microvascular anastomosed fibula graft was achieved. Conclusions Intraosseous pseudocarcinomatous hyperplasia of the mandible is a rare differential diagnosis in maxillofacial surgery. Besides other benign epithelial neoplasms, such as calcifying epithelial odontogenic tumor, squamous odontogenic tumor, or different forms of ameloblastoma, the far more frequent invasive squamous cell carcinoma needs to be excluded. A misinterpretation of pseudocarcinomatous hyperplasia as squamous cell carcinoma must be avoided because it can lead to a massive overtreatment