Digitale alltagsintegrierte Förderdiagnostik mit DAF-L. Ein Ansatz für nachhaltige Diagnostik und Förderung?

Im BMBF-Verbundprojekt „Digitale alltagsintegrierte Förderdiagnostik – Lesen in der inklusiven Bildung“ (DaF-L) werden ein digitales adaptives Lesescreening und darauf abgestimmte Lesepakete zur Förderung für den inklusiven Unterricht in dritten Grundschulklassen entwickelt. Die Lesepakete enthalten unterrichtliche Fördereinheiten auf drei Niveaustufen und werden im Kontrollgruppendesign auf ihre Wirksamkeit hin überprüft. Das Lesescreening und die Lesepakete sollen als Open Educational Resource auf der Onlineplattform Levumi (https://www.levumi.de/) als Gesamtpaket für Lehrkräfte in inklusiven Grundschulen zur Verfügung stehen. In diesem Beitrag wird beschrieben, wie im Teilprojekt „Expertenbefragungen“ eine möglichst hohe Nachhaltigkeit des Lesescreenings und der Förderbausteine gesichert werden kann. Dazu werden erste Einblicke aus der Begleitstudie vorgestellt. (DIPF/Orig.

Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3 kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some mis-orientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, that gives rise to a non-lethal primary skeletal ciliopathy combined with defective motile cilia and PCD

Paralog-specific TTC30 regulation of Sonic hedgehog signaling

The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A–IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly

A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease

Intronic enhancers of the human SNCA gene predominantly regulate its expression in brain in vivo.

Evidence from patients with Parkinson's disease (PD) and our previously reported α-synuclein (SNCA) transgenic rat model support the idea that increased SNCA protein is a substantial risk factor of PD pathogenesis. However, little is known about the transcription control of the human SNCA gene in the brain in vivo. Here, we identified that the DYT6 gene product THAP1 (THAP domain-containing apoptosis-associated protein 1) and its interaction partner CTCF (CCCTC-binding factor) act as transcription regulators of SNCA. THAP1 controls SNCA intronic enhancers' activities, while CTCF regulates its enhancer-promoter loop formation. The SNCA intronic enhancers present neurodevelopment-dependent activities and form enhancer clusters similar to "super-enhancers" in the brain, in which the PD-associated single-nucleotide polymorphisms are enriched. Deletion of the SNCA intronic enhancer clusters prevents the release of paused RNA polymerase II from its promoter and subsequently reduces its expression drastically in the brain, which may provide new therapeutic approaches to prevent its accumulation and thus related neurodegenerative diseases defined as synucleinopathies

Multi-capillary column-ion mobility spectrometry (MCC-IMS) as a new method for the quantification of occupational exposure to sevoflurane in anaesthesia workplaces: an observational feasibility study

BACKGROUND: Occupational exposure to sevoflurane has the potential to cause health damage in hospital personnel. Workplace contamination with the substance mostly is assessed by using photoacoustic infrared spectrometry with detection limits of 10 ppbv. Multi-capillary column-ion mobility spectrometry (MCC-IMS) could be an alternative technology for the quantification of sevoflurane in the room air and could be even more accurate because of potentially lower detection limits. The aim of this study was to test the hypothesis that MCC-IMS is able to detect and monitor very low concentrations of sevoflurane (<10 ppbv) and to evaluate the exposure of hospital personnel to sevoflurane during paediatric anaesthesia and in the post anaesthesia care unit (PACU). METHODS: A MCC-IMS device was calibrated to several concentrations of sevoflurane and limits of detection (LOD) and quantification (LOQ) were calculated. Sevoflurane exposure of hospital personnel was measured at two anaesthesia workplaces and time-weighted average (TWA) values were calculated. RESULTS: The LOD was 0.0068 ppbv and the LOQ was 0.0189 ppbv. During paediatric anaesthesia the mean sevoflurane concentration was 46.9 ppbv (8.0 - 314.7 ppbv) with TWA values between 5.8 and 45.7 ppbv. In the PACU the mean sevoflurane concentration was 27.9 ppbv (8.0 – 170.2 ppbv) and TWA values reached from 8.3 to 45.1 ppbv. CONCLUSIONS: MCC-IMS shows a significantly lower LOD and LOQ than comparable methods. It is a reliable technology for monitoring sevoflurane concentrations at anaesthesia workplaces and has a particular strength in quantifying low-level contaminations of sevoflurane. The exposure of the personnel working in these areas did not exceed recommended limits and therefore adverse health effects are unlikely

Erprobung der Aufgabenpakete ILeA plus Deutsch: Bericht zum Teilprojekt 2: überarbeitete und ergänzte Version vom 29.11.2019

Ziel des Projektes ILeAplus Deutsch ist es, ein digitalisiertes Verfahren zur lernprozessbegleitenden Diagnostik und Förderung im Fach Deutsch in der Primarstufe in Brandenburg (Jahrgangstufen 1- 6) zu entwickeln, zu erproben und zu normieren (Liebers, Latzko, Reinhold & Ritter, 2016). Dies erfolgt im Auftrag des Landes Brandenburg (Ministerium für Bildung, Jugend und Sport (MBJS)). Die in Teilprojekt 1 entwickelten Aufgabenpakete umfassen Aufgaben zu schriftsprachlichen Voraussetzung (Jahrgangstufe 1) sowie Leseflüssigkeit, Leseverstehen und Rechtschreibung (Jahrgangsstufen 2-6). Die Aufgaben werden webbasiert durchgeführt und mit einer Schulverwaltungssoftware verknüpft, die die Auswertung und Rückmeldung der Ergebnisse und Förderempfehlungen ermöglicht. Die Erprobung der digitalen Aufgabenpakete (Teilprojekt 2) fand im Zeitraum von 5 Wochen im September und Oktober 2017 in ausgewählten brandenburgischen Schulen statt. Insgesamt nahmen 1101 Schüler*innen aus 11 Schulen mit insgesamt 49 Klassen der Jahrgangsstufen 1 bis 5 teil. Die Durchführung der digitalen Aufgabenpakete erfolgt unter Anleitung der Lehrkraft. Zudem wurden parallel in den Jahrgangsstufen 2 bis 6 normierte Schulleistungstests zur Sicherung der Konstruktvalidität durchgeführt. Der Bericht enthält detaillierte Ergebnisse zur psychometrischen Prüfung der Aufgabenpakete, den Empfehlungen zur Überarbeitung sowie zu den Testgütekriterien der finalen Testversionen. Zudem enthält der Bericht die Ergebnisse der Begleitevaluation bzgl. Handhabbarkeit und Verständlichkeit der Aufgaben für Lehrkräfte und Schüler*innen.:Inhaltsverzeichnis 1 Ausgangssituation 2 Ziele und Fragestellung der Erprobung 3 Methodisches Vorgehen 3.1 Design 3.2 Stichprobe 3.3 Instrumente der Datenerhebung 3.3.1 Webbasierter Aufgabensatz ILeAplus Deutsch, Aufgabenpakete A bis D 3.3.2 Dokumentationsbogen zur Durchführung der Erprobung 3.3.3 Feedbackbogen für Lehrpersonen 3.3.4 Schülerfragebogen 3.3.5 Schülerleistungstests Deutsch 3.4 Durchführung der Erprobung 3.4.1 Vorbereitung der Erprobung in den Schulen 3.4.2 Durchführung der Erprobung 3.5 Methoden der Datensammlung und Auswertung 3.5 Rückmeldungen an die Erprobungsschulen 4 Ergebnisse der Erprobung 4.1 Teilnehmer*innen an der Erprobung und Rücklaufquoten 4.2 Lehrerfeedback 4.2.1 Handhabbarkeit der Aufgabenpakete für die Schüler*innen 4.2.2 Verständlichkeit der Aufgabenformulierungen und Abbildungen für die Schüler*innen 4.2.3 Handhabbarkeit der Auswertungen 4.4 Testgütekriterien der Aufgabenpakete, Skalen und Items 4.4.1 Statistische Prüfverfahren (Testtheoretische Prüfung) 4.4.2 Ergebnisse zu Früher Literalität 4.4.3 Ergebnisse zu Leseflüssigkeit 4.4.4 Ergebnisse zum Leseverständnis 4.4.5 Ergebnisse zum Rechtschreiben 5 Schlussfolgerungen für die Erstellung der Normierungsfassung der Aufgabenpakete A bis C Deutsch 5.1 Schlussfolgerungen zu den Aufgaben zur Frühen Literalität 5.2 Schlussfolgerungen zu den Aufgaben zur Leseflüssigkeit 5.3 Schlussfolgerungen zu den Aufgaben zum Leseverständnis 5.4 Schlussfolgerungen zu den Aufgaben zur Rechtschreibung 6 Psychometrische Qualität der Normierungsfassung der Aufgabenpakete A bis C Deutsch [ergänzt 13.03.2019] 6.1 Normierungsfassung Frühe Literalität (Pakete AI & AII) 6.1.1 Reliabilität 6.1.2 Validität 6.2 Normierungsfassung Leseflüssigkeit 6.2.1 Reliabilität 6.2.2 Validität 6.3 Normierungsfassung Leseverständnis 6.3.1 Reliabilität 6.3.2 Validität 6.4 Normierungsfassung Rechtschreibung 6.4.1 Reliabilität 6.4.2 Validität Literatur Anlage