Magnetic resonance imaging as a tool for quality control in extrusion-based bioprinting

Bioprinting is gaining importance for the manufacturing of tailor-made hydrogel scaffolds in tissue engineering, pharmaceutical research and cell therapy. However, structure fidelity and geometric deviations of printed objects heavily influence mass transport and process reproducibility. Fast, three-dimensional and nondestructive quality control methods will be decisive for the approval in larger studies or industry. Magnetic resonance imaging (MRI) meets these requirements for characterizing heterogeneous soft materials with different properties. Complementary to the idea of decentralized 3D printing, magnetic resonance tomography is common in medicine, and image data processing tools can be transferred system-independently. In this study, a MRI measurement and image analysis protocol was evaluated to jointly assess the reproducibility of three different hydrogels and a reference material. Critical parameters for object quality, namely porosity, hole areas and deviations along the height of the scaffolds are discussed. Geometric deviations could be correlated to specific process parameters, anomalies of the ink or changes of ambient conditions. This strategy allows the systematic investigation of complex 3D objects as well as an implementation as a process control tool. Combined with the monitoring of metadata this approach might pave the way for future industrial applications of 3D printing in the field of biopharmaceutics

A minisandwich experiment with blended ca-bentonite and pearson water—Hydration, swelling, solute transport and cation exchange

Shaft seals are geotechnical barriers in nuclear waste deposits and underground mines. The Sandwich sealing system consists of alternating sealing segments (DS) of bentonite and equipotential segments (ES). MiniSandwich experiments were performed with blended Ca-bentonite (90 mm diameter and 125 mm height) to study hydration, swelling, solute transport and cation exchange during hydration with A3 Pearson water, which resembles pore water of Opalinus Clay Formation at sandy facies. Two experiments were run in parallel with DS installed either in one-layer hydrate state (1W) or in air-dry two-layer hydrate (2W) state. Breakthrough at 0.3 MPa injection pressure occurred after 20 days and the fluid inlet was closed after 543 days, where 4289 mL and 2984 mL, respectively, passed both cells. Final hydraulic permeability was 2.0–2.7 × 10$^{-17}$ m$^{2}$. Cells were kept for another 142 days before dismantling. Swelling of DS resulted in slight compaction of ES. No changes in the mineralogy of the DS and ES material despite precipitated halite and sulfates occurred. Overall cation exchange capacity of the DS does not change, maintaining an overall value of 72 ± 2 cmol(+)/kg. Exchangeable Na$^{+}$ strongly increased while exchangeable Ca$^{2+}$ decreased. Exchangeable Mg$^{2+}$ and K$^{+}$ remained nearly constant. Sodium concentration in the outflow indicated two different exchange processes while the concentration of calcium and magnesium decreased potentially. Concentration of sulfate increased in the outflow, until it reached a constant value and chloride concentration decreased to a minimum before it slightly increased to a constant value. The available data set will be used to adapt numerical models for a mechanism-based description of the observed physical and geochemical processes

Neurosurgical morbidity in pediatric supratentorial midline low‐grade glioma: results from the German LGG studies

Surgical resection is a mainstay of treatment for pediatric low-grade glioma (LGG) within all current therapy algorithms, yet associated morbidity is scarcely reported. As supratentorial midline (SML) interventions are particularly challenging, we investigated the frequency of neurosurgical complications/new impairments aiming to identify their risk factors. Records were retrospectively analyzed from 318 patients with SML-LGG from successive German multicenter LGG studies, undergoing surgery between May 1998 and June 2020. Exactly 537 operations (230 resections, 167 biopsies, 140 nontumor procedures) were performed in 318 patients (54% male, median age: 7.6 years at diagnosis, 9.5 years at operation, 11% NF1, 42.5% optic pathway glioma). Surgical mortality rate was 0.93%. Applying the Drake classification, postoperative surgical morbidity was observed following 254/537 (47.3%) and medical morbidity following 97/537 (18.1%) patients with a 40.1% 30-day persistence rate for newly developed neurological deficits (65/162). Neuroendocrine impairment affected 53/318 patients (16.7%), visual deterioration 34/318 (10.7%). Postsurgical morbidity was associated with patient age <3 years at operation, tumor volume ≥80 cm3, presence of hydrocephalus, complete resection, surgery in centers with less than median reported tumor-related procedures and during the earlier study period between 1998 and 2006, while the neurosurgical approach, tumor location, NF1 status or previous nonsurgical treatment were not. Neurosurgery-associated morbidity was frequent in pediatric patients with SML-LGG undergoing surgery in the German LGG-studies. We identified patient- and institution-associated factors that may increase the risk for complications. We advocate that local multidisciplinary teams consider the planned extent of resection and surgical skills

ETMR-05: Single-cell transcriptomics of ETMR reveals developmental cellular programs and tumor-pericyte communications in the microenvironment [Abstract]

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are pediatric brain tumors bearing a grim prognosis, despite intensive multimodal therapeutic approaches. Insights into cellular heterogeneity and cellular communication of tumor cells with cells of the tumor microenvironment (TME), by applying single-cell (sc) techniques, potentially identify mechanisms of therapy resistance and target-directed treatment approaches. MATERIAL AND METHODS: To explore ETMR cell diversity, we used single-cell RNA sequencing (scRNA-seq) in human (n=2) and murine ETMR (transgenic mode; n=4) samples, spatial transcriptomics, 2D and 3D cultures (including co-cultures with TME cells), multiplex immunohistochemistry and drug screens. RESULTS: ETMR microenvironment is composed of tumor and non-tumor cell types. The ETMR malignant compartment harbour cells representing distinct transcriptional metaprograms, (NSC-like, NProg-like and Neuroblast-like), mirroring embryonic neurogenic cell states and fuelled by neurogenic pathways (WNT, SHH, Hippo). The ETMR TME is composed of oligodendrocyte and neuronal progenitor cells, neuroblasts, microglia, and pericytes. Tumor-specific ligand-receptor interaction analysis showed enrichment of intercellular communication between NProg-like ETMR cells and pericytes (PC). Functional network analyses reveal ETMR-PC interactions related to stem-cell signalling and extracellular matrix (ECM) organization, involving factors of the WNT, BMP, and CxCl12 networks. Results from ETMR-PC co-culture and spatial transcriptomics pointed to a pivotal role of pericytes in keeping ETMR in a germinal neurogenic state, enriched in stem-cell signalling. Drug screening considering cellular heterogeneity and cellular communication suggested novel therapeutic approaches. CONCLUSION: ETMR demonstrated diversity in the microenvironment, with enrichment of cell-cell communications with pericytes, supporting stem-cell signalling and interfering in the organization of the tumor extracellular matrix. Targeting ETMR-PC interactions might bring new opportunities for target-directed therapy

The Astropy Problem

The Astropy Project (http://astropy.org) is, in its own words, "a community effort to develop a single core package for Astronomy in Python and foster interoperability between Python astronomy packages." For five years this project has been managed, written, and operated as a grassroots, self-organized, almost entirely volunteer effort while the software is used by the majority of the astronomical community. Despite this, the project has always been and remains to this day effectively unfunded. Further, contributors receive little or no formal recognition for creating and supporting what is now critical software. This paper explores the problem in detail, outlines possible solutions to correct this, and presents a few suggestions on how to address the sustainability of general purpose astronomical software

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, we and others identified de novo FBXO11 variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, we and others identified de novo FBXO11 variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs

Identification and characterization of genetic causes of growth disorders

Hintergrund und Ziele Wachstumsstörungen werden durch das Abweichen des Höhenwachstums eines heranwachsenden Menschen von der Norm charakterisiert. Von einer Wachstumsverzögerung spricht man, wenn die Körperhöhe, bezogen auf die jeweilige Population, unterhalb der 3. Perzentile der Norm oder mehr als zwei Standardabweichungen unter dem Mittelwert des genetischen Zielbereichs einer Population liegt. Daher stellt Kleinwuchs eine häufige Fragestellung in der Pädiatrie und der humangenetischen Beratung dar. Obgleich Wachstum und Körperhöhe mit einer hohen Heretabilität von 80 bis 90 % einhergehen, kann die genetische Ursache des Kleinwuchses bei einem Großteil der Betroffenen bislang nicht aufgedeckt werden. Das Ziel dieser Arbeit war daher die Identifizierung ursächlicher genetischer Veränderungen idiopathischer Wachstumsstörungen bei Patienten mittels Exomsequenzierung. Methoden (Patienten, Material und Untersuchungsmethoden) Im Rahmen dieser Arbeit wurden zwölf nicht-konsanguine Familien mit betroffenen Kindern auf seltene Varianten als mögliche krankheitsverursachende Mutationen untersucht. Dazu wurde das Exom aller gesunden Elternpaare und Patienten mittels Next Generation Sequencing auf genetische Veränderungen durchsucht. Durch Sanger-Sequenzierung validierte Varianten wurden durch computergestütze Analysen und anhand ihrer Proteinfunktion bewertet. Für die vielversprechenden Kandidatengene wurden ein alternatives Splicen durch reverse Transkriptase Polymerase Kettenreaktion (rt-PCR) und die Expression der betroffenen Gene in den Patienten mittels quantitativer Realtime Polymerase Kettenreaktion untersucht. Anschließend wurde ein bezüglich Wachstumsstörungen gesundes Patientenkollektiv von 382 Individuen auf die identifizierten Varianten durchsucht. Für zwei Kandidatengene erfolgte zudem eine Mutationssuche bei weiteren Patienten mit bislang idiopathischer Wachstumsstörung aus einem internen Patientenkollektiv. Ergebnisse und Beobachtungen Durch Exomsequenzierung und anschließende Sanger-Bestätigung konnten bei den zwölf untersuchten Familien 30 Varianten ermittelt werden. Nach der Bewertung der Varianten durch computergestützte Analysen und Charakterisierung der Proteinfunktion der entsprechenden Gene konnten 14 potentiell krankheitsverursachende Varianten in neunGenen und acht Familien identifiziert werden. Bei drei Patienten konnten seltene, mit Kleinwuchs einhergehende Erkrankungen anhand der identifizierten Mutationen festgestellt werden (Kniest-Dysplasie, Mulibrey-Nanism, Hypertensions- und Brachydaktylie-Syndrom). Bei drei weiteren Patienten konnten Varianten in Kandidatengenen identifiziert werden. Das Gen MCF2L kodiert für ein überwiegend neuronal exprimiertes Onkogen und zeigte bei der Patientin eine erhöhte Expression auf mRNA-Ebene. Das Gen ARID4A ist in die Regulation der Zellproliferation involviert. Das Gen EIF3G wurde aufgrund der Ergebnisse der funktionellen Analysen und der Genfunktion mit Beteiligung an der Translationsinitiation von Proteinen als besonders interessantes Kandidatengen eingestuft. Eine Beeinträchtigung dieser Funktionen könnte zu einem gestörten Zellwachstum oder einer Signalwegstörung führen. Um eine Assoziation zwischen den Wachstumsphänotypen der Patienten und den Varianten in den Kandidatengenen zu bestätigen, müssten weitere Patienten mit Varianten in den entsprechenden Genen identifiziert werden oder weiterführende funktionelle Analysen durchgeführt werden. Schlussfolgerungen Zusammenfassend konnte durch diese Arbeit gezeigt werden, dass die Exomsequenzierung eine geeignete Methode zur Identifizierung genetischer Ursachen für Wachstumsstörungen und zur Detektion potentiell wachstumsrelevanter Gene darstellt. Weiterhin kann die Identifikation eines in der Regel seltenen genetischen Krankheitsbildes bei Patienten mit idiopathischer Wachstumsstörung therapeutische Konsequenzen im Sinne von Vorsorgeuntersuchungen oder spezifischen Therapien implizieren. Dies konnte am Beispiel des Mulibrey-Nanism mit einem erhöhtem Risiko für Neoplasien und kardiale Erkrankungen und des Hypertonie- und Brachydaktylie- Syndroms mit unbehandelt schwerer arterieller Hypertonie dargestellt werden.Objectives Growth disorders are characterized as deviation of body height of an individual from age related standards. Growth retardation is specified by body height under the 3rd percentile or more than two standard deviations below the average of the genetic target range of a defined population. Therefore growth retardation and disorders present a common medical issue in pediatric and human genetic counselling. Although growth and body height are associated with a high heritability of 80 to 90 %, the underlying genetic cause of short stature cannot be detected in the majority of affected individuals. The objective of this thesis was the identification of causal mutations in individuals with idiopathic growth disorders by exome sequencing. Design and Methods Twelve non-consanguineous families with affected children were analyzed in order to identify rare variants as possibly underlying genetic cause. Therefore exome sequencing of all affected individuals and their parents was performed. All variants confirmed by Sanger sequencing were further categorized by computer-based prediction analysis and evaluation of the protein function of the associated gene. For the resulting candidate genes I performed reverse transcription polymerase chain reaction (rt-PCR) in order to determine alternative splicing and expression analysis in the patients by quantitative Real-Time PCR (qPCR). Thereafter a control group of 382 individuals was screened for the presence of the identified variants. For two candidate genes a patient cohort with idiopathic growth disorders was examined in order to identify further mutations. Observations and Results By performing exome sequencing and Sanger confirmation I was able to identify 30 variants in twelve families. After characterization of the variants by computer-based prediction analysis and protein function of the corresponding genes 14 potentially pathogenic variants in nine genes and eight families remained. In three individuals I was able to determine rare growth disorders associated with the affected genes (Kniest dysplasia, Mulibrey nanism, hypertension and brachydaktyly syndrome). In three further patients we identified variants in candidate genes. MCF2L encodes an oncogene, that is predominantly expressed in neuronal tissue and showed elevated expression levels in the patient. ARID4A is a gene involved in the regulation of cell proliferation. EIF3G gene was further pursued due to the results of the functional analyses and its role in translation initiation of proteins.Impairment of this function could lead to alteration of cell growth or signaling pathways. In order to determine the pathogenicity of the identified variants and associated genes more patients with variants in these genes need to be identified and further functional experiments have to be performed. Conclusions I was able to underline the importance of exome sequencing as an appropriate method to identify causative and possibly pathogenic variants in individuals with idiopathic growth disorders. Furthermore the identification of rare genetic growth disorders can have therapeutic consequences like surveillance or specific therapy. This could be demonstrated for Mulibrey nanism where affected individuals have an elevated risk for neoplasia and cardiac disease as well as for hypertension and brachydaktyly syndrome which is associated with severe hypertension

Correlation of the absorbed dose to the blood and DNA damage in leukocytes after internal ex-vivo irradiation of blood samples with Ra-224

Background: Irradiation with α-particles creates densely packed damage tracks along particle trajectories in exposed cells, including complex DNA damage and closely spaced double-strand breaks (DSBs) in hit nuclei. Here, we investigated the correlation of the absorbed dose to the blood and the number of α-induced DNA damage tracks elicited in human blood leukocytes after ex-vivo in-solution exposure with Ra-224. The aim was to compare the data to previously published data on Ra-223 and to investigate differences in DNA damage induction between the two radium isotopes. Results: Blood samples from three healthy volunteers were exposed ex-vivo to six different concentrations of Ra-224 dichloride. Absorbed doses to the blood were calculated assuming local energy deposition of all α- and β-particles of the Ra-224 decay chain, ranging from 0 to 127 mGy. γ-H2AX + 53BP1 DNA damage co-staining and analysis was performed on ethanol-fixed leukocytes isolated from the irradiated blood samples. For damage quantification, α-induced DNA damage tracks and small γ-H2AX + 53BP1 DSB foci were enumerated in the exposed leukocytes. This revealed a linear relationship between the frequency of α-induced γ-H2AX damage tracks and the absorbed dose to the blood, while the frequency of small γ-H2AX + 53BP1 DSB foci indicative of β-irradiation was similar to baseline values. Conclusions: Our data provide a first estimation of the DNA damage induced by Ra-224 in peripheral blood mononuclear cells. A comparison with our previously published Ra-223 data suggests that there is no difference in the induction of radiation-induced DNA damage between the two radium isotopes due to their similar decay properties