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Abstract Chromothripsis is a frequent form of genome instability, whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or multiple chromosome(s). However, little is known about the heterogeneity of chromothripsis across different clones from the same tumour, as well as changes in response to treatment. Here we analyse single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma and neural stem cells (n = 9). We reconstruct the order of somatic events, identify early alterations likely linked to chromothripsis and depict the contribution of chromothripsis to malignancy. We characterise subclonal variation of chromothripsis and its effects on extrachromosomal circular DNA, cancer drivers and putatively druggable targets. Furthermore, we highlight the causative role and the fitness consequences of specific rearrangements in neural progenitors.

Abstract Stem cells are a hallmark of animal multicellularity. Sox and POU transcription factors are associated with stemness and were believed to be animal innovations, reported absent in their unicellular relatives. Here we describe unicellular Sox and POU factors. Choanoflagellate and filasterean Sox proteins have DNA-binding specificity similar to mammalian Sox2. Choanoflagellate—but not filasterean—Sox can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSCs) through interacting with the mouse POU member Oct4. In contrast, choanoflagellate POU has a distinct DNA-binding profile and cannot generate iPSCs. Ancestrally reconstructed Sox proteins indicate that iPSC formation capacity is pervasive among resurrected sequences, thus loss of Sox2-like properties fostered Sox family subfunctionalization. Our findings imply that the evolution of animal stem cells might have involved the exaptation of a pre-existing set of transcription factors, where pre-animal Sox was biochemically similar to extant Sox, whilst POU factors required evolutionary innovations.

Abstract Background Despite great advances in proliferative diabetic retinopathy (PDR) therapy over the last decades, one third of treated patients continue to lose vision. While resident vitreous macrophages called hyalocytes have been implicated in the pathophysiology of vitreoretinal proliferative disease previously, little is known about their exact role in PDR. In this study, we address molecular and cellular alterations in the vitreous of PDR patients as a means towards assessing the potential contribution of hyalocytes to disease pathogenesis. Results A total of 55 patients were included in this study encompassing RNA-Sequencing analysis of hyalocytes isolated from the vitreous of PDR and control patients, multiplex immunoassay and ELISA analyses of vitreous samples from PDR and control patients, as well as isolation and immunohistochemical staining of cultured porcine hyalocytes. Transcriptional analysis revealed an enhanced inflammatory response of hyalocytes contributing to the cytokine pool within the vitreous of PDR patients by expressing interleukin-6, among others. Further, increased angiopoietin-2 expression indicated that hyalocytes from PDR patients undergo a proangiogenic shift and may thus mediate the formation of retinal neovascularizations, the hallmark of PDR. Finally, RNA-Sequencing revealed an upregulation of factors known from hemoglobin catabolism in hyalocytes from PDR patients. By immunohistochemistry, cultured porcine hyalocytes exposed to red blood cells were shown to engulf and phagocytose these, which reveals hyalocytes’ potential to dispose of erythrocytes. Thus, our data suggest a potential role for vitreous macrophages in erythrophagocytosis and, thereby, clearance of vitreous hemorrhage, a severe complication of PDR. Conclusion Our results strongly indicate a critical role for vitreous hyalocytes in key pathophysiological processes of proliferative diabetic retinopathy: inflammation, angiomodulation and erythrophagocytosis. Immunomodulation of hyalocytes may thus prove an essential novel therapeutic approach in diabetic vitreoretinal disease.

Teaching in inclusive schools comes with a variety of ethical challenges, for which student teachers of general and special needs education should acquire ethical competence as part of their inclusion-oriented teacher training. Due to the lack of empirical studies on this area of teacher training in Germany to date, students in Masters degree programmes (N = 585; age: M = 25.35; SD = 3.28; 71.6% female) were asked to assess the need and relevance of acquiring ethical skills in an online survey. The prospective teachers considered ethical competencies to be necessary for everyday tasks in inclusive schools; nevertheless, only 18.9% stated that they had acquired the relevant skills in their previous studies. Accordingly, they wished for a higher priority of imparting ethical competencies in future teacher training. Here they preferred application-related competence goals, such as being able to reflect critically on ethical decisions or to justify ethical decisions in communication with others. In addition to poorly developed ethical skills, students reported a lack of acquisition of further competencies that are proven to be success factors for the implementation of inclusive education. Student teachers for special needs education held a more positive attitude towards school inclusion and placed a greater value on imparting ethical competence in future teacher training than student teachers for general education

Abstract Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a life-threatening autoimmune disease that often results in kidney failure caused by crescentic glomerulonephritis (GN). To date, treatment of most patients with ANCA-GN relies on non-specific immunosuppressive agents, which may have serious adverse effects and be only partially effective. Here, using spatial and single-cell transcriptome analysis, we characterize inflammatory niches in kidney samples from 34 patients with ANCA-GN and identify proinflammatory, cytokine-producing CD4+ and CD8+ T cells as a pathogenic signature. We then utilize these transcriptomic profiles for digital pharmacology and identify ustekinumab, a monoclonal antibody targeting IL-12 and IL-23, as the strongest therapeutic drug to use. Moreover, four patients with relapsing ANCA-GN are treated with ustekinumab in combination with low-dose cyclophosphamide and steroids, with ustekinumab given subcutaneously (90 mg) at weeks 0, 4, 12, and 24. Patients are followed up for 26 weeks to find this treatment well-tolerated and inducing clinical responses, including improved kidney function and Birmingham Vasculitis Activity Score, in all ANCA-GN patients. Our findings thus suggest that targeting of pathogenic T cells in ANCA-GN patients with ustekinumab might represent a potential approach and warrants further investigation in clinical trials.

Abstract Human development relies on the correct replication, maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages, and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells, we identify that several patterning signals—including WNT, BMP, and FGF—converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers, but re-emerges in neural progenitors. In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development.

Centrosomes are the major microtubule organizing centers of animal cells. Supernumerary centrosomes are a common feature of human tumors and associated with karyotype abnormalities and aggressive disease, but whether they are cause or consequence of cancer remains controversial. Here, we analyzed the consequences of centrosome amplification by generating transgenic mice in which centrosome numbers can be increased by overexpression of the structural centrosome protein STIL. We show that STIL overexpression induces centrosome amplification and aneuploidy, leading to senescence, apoptosis, and impaired proliferation in mouse embryonic fibroblasts, and microcephaly with increased perinatal lethality and shortened lifespan in mice. Importantly, both overall tumor formation in mice with constitutive, global STIL overexpression and chemical skin carcinogenesis in animals with inducible, skin-specific STIL overexpression were reduced, an effect that was not rescued by concomitant interference with p53 function. These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and delayed spontaneous as well as carcinogen-induced tumor formation.

IntroductionSubstance use disorders (SUDs) are common, and there is evidence of clinically significant benefit of cognitive behavioral therapy (CBT). The efficacy of CBT in SUDs has been confirmed, although relapse rates of 40%–60% have been reported. An enhancement of CBT-based therapy through an animal-assisted intervention (AAI) with sheep to normalize the occurrence of negative emotions and improve positive emotions as well as mindfulness and self-efficacy expectancy was investigated.MethodsA single-session AAI with sheep in a group setting was investigated against treatment as usual over time. N = 36 psychiatric inpatients with SUDs were examined by questionnaires before and 1 week after the intervention and additionally immediately after the intervention.ResultsPositive emotions improved significantly in the AAI group 1 week after the intervention with a medium effect size, but not in the control group. Similarly, mindfulness and self-efficacy expectancy improved over time in the AAI group. When exploratory results were evaluated immediately after the intervention while still on the farm, the effects in favor of AAI were even larger.ConclusionsAAI can thus be considered effective in improving positive emotions, mindfulness, and self-efficacy expectancy. The impressive effect sizes immediately after the intervention encourage us to consider what can be done to maintain these even greater effect sizes over time.Clinical Trial Registrationhttps://drks.de/search/de/trial/DRKS00027539, identifier DRKS 00027539.

Abstract Highly selective for K+ at neutral pH, the TWIK1 channel becomes permeable to Na+ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5–6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K+-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na+-permeable conformation of the channel.

Higher eukaryotes’ life is impossible without copper redox activity and, literally, every breath we take biochemically demonstrates this. However, this dependence comes at a considerable price to ensure target-oriented copper action. Thereto its uptake, distribution but also excretion are executed by specialized proteins with high affinity for the transition metal. Consequently, malfunction of copper enzymes/transporters, as is the case in hereditary Wilson disease that affects the intracellular copper transporter ATP7B, comes with serious cellular damage. One hallmark of this disease is the progressive copper accumulation, primarily in liver but also brain that becomes deadly if left untreated. Such excess copper toxicity may also result from accidental ingestion or attempted suicide. Recent research has shed new light into the cell-toxic mechanisms and primarily affected intracellular targets and processes of such excess copper that may even be exploited with respect to cancer therapy. Moreover, new therapies are currently under development to fight against deadly toxic copper.

IntroductionOsteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis.Methods and ResultsMurine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors.DiscussionOur findings define integrin α2β1 as an early marker of OC differentiation.

Abstract Background This controlled pilot study investigates the effect of the combined use of cognitive restructuring (CR) and imagery rescripting (IR) compared to treatment as usual among inpatients with moderate and severe depression. Alongside expert ratings and self-report tools, fitness wristbands were used as an assessment tool. Methods In addition to the standard inpatient care (SIC) program, 33 inpatients with moderate and severe depression were randomly assigned to an intervention group (two sessions of IR and CR) or an active treatment-as-usual (TAU) control group (two sessions of problem-solving and build-up of positive activity). Depression severity was assessed by the Hamilton Depression Rating Scale-21 (HDRS-21), the Beck Depression Inventory-II (BDI-II), and as a diagnostic adjunct daily step count via the Fitbit Charge 3™. We applied for analyses of HDRS-21 and BDI-II, 2 × 2 repeated-measures analysis of variance (ANOVA), and an asymptotic Wilcoxon test for step count. Results The main effect of time on both treatments was η2 = .402. Based on the data from the HDRS-21, patients in the intervention group achieved significantly greater improvements over time than the TAU group (η2 = .34). The BDI-II data did not demonstrate a significant interaction effect by group (η2 = .067). The daily hourly step count for participants of the intervention group was significantly higher (r = .67) than the step count for the control group. Conclusions The findings support the utilization of imagery-based interventions for treating depression. They also provide insights into using fitness trackers as psychopathological assessment tools for depressed patients. Trial registration The trial is registered at the German Clinical Trials Register (Deutsches Register Klinischer Studien) under the registration number: DRKS00030809.

Ripples are a typical form of neural activity in hippocampal neural networks associated with the replay of episodic memories during sleep as well as sleep-related plasticity and memory consolidation. The emergence of ripples has been observed both dependent as well as independent of input from other brain areas and often coincides with dendritic spikes. Yet, it is unclear how input-evoked and spontaneous ripples as well as dendritic excitability affect plasticity and consolidation. Here, we use mathematical modeling to compare these cases. We find that consolidation as well as the emergence of spontaneous ripples depends on a reliable propagation of activity in feed-forward structures which constitute memory representations. This propagation is facilitated by excitable dendrites, which entail that a few strong synapses are sufficient to trigger neuronal firing. In this situation, stimulation-evoked ripples lead to the potentiation of weak synapses within the feed-forward structure and, thus, to a consolidation of a more general sequence memory. However, spontaneous ripples that occur without stimulation, only consolidate a sparse backbone of the existing strong feed-forward structure. Based on this, we test a recently hypothesized scenario in which the excitability of dendrites is transiently enhanced after learning, and show that such a transient increase can strengthen, restructure and consolidate even weak hippocampal memories, which would be forgotten otherwise. Hence, a transient increase in dendritic excitability would indeed provide a mechanism for stabilizing memories.

BackgroundRichter transformation refers to the progression of an initially slow-growing small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) into an aggressive lymphoma, typically diffuse large B-cell lymphoma (DLBCL) or Hodgkin lymphoma.Case presentationThe patient presented with a rapid onset of localized cervical swelling, accompanied by monoclonal B-cell lymphocytosis displaying a CLL immunophenotype. The histopathological analysis identified a Burkitt lymphoma (BL) located in the submandibular gland and adjacent lymph node. The patient’s bone marrow displayed a minor infiltration of monoclonal B-cells with a CLL immunophenotype (< 10%). Molecular analysis demonstrated the presence of the same monoclonal rearrangement in the framework region (FR3 region) of the immunoglobulin heavy chain (IGH) locus. High-throughput sequencing of the immunoglobulin heavy and light chains also confirmed the presence of the same rearrangement in SLL/CLL and in the Burkitt lymphoma sample, but also highlighted the presence of a second rearrangement in the Burkitt lymphoma cells, not shared with the SLL/CLL cells in the bone marrow. The patient was treated with DA-EPOCH-R, which lead to a complete metabolic response.ConclusionThis report provides an exceptionally rare description of a CLL-type monoclonal B-cell lymphocytosis transforming into a very aggressive Burkitt lymphoma in a treatment naïve patient.