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Acute optogenetic stimulation of serotonin neurons reduces cell proliferation in the dentate gyrus of mice
Get PDFThe dentate gyrus of the hippocampus is targeted by axons from serotonin raphe neurons, where the neurotransmitter modulates adult neurogenesis and antidepressant action, and mediates the neurogenic effect of running. Whether running-induced cell proliferation is directly mediated by serotonin remains unknown. Here, we took advantage of Tph2-ChR2-YFP transgenic mice in which the light-sensitive protein channelrhodopsin-2 (ChR2) is specifically expressed in tryptophan hydroxylase 2 (TPH2)-expressing neurons. We selectively activated serotonin neurons via optogenetics and determined the effect on cell proliferation in the dentate gyrus. Our data reveal a significant reduction in the number of newly generated cells upon overnight raphe stimulation. The decrease in cell proliferation was absent when serotonin neurons were light-activated for six consecutive nights. However, we observed an interhemispheric difference in BrdU-positive cell numbers. We conclude that acute network dynamics occur between serotonin raphe neurons and the hippocampus, directly affecting precursor cell proliferation
Leveraging large language models for data analysis automation
Get PDFData analysis is constrained by a shortage of skilled experts, particularly in biology, where detailed data analysis and subsequent interpretation is vital for understanding complex biological processes and developing new treatments and diagnostics. One possible solution to this shortage in experts would be making use of Large Language Models (LLMs) for generating data analysis pipelines. However, although LLMs have shown great potential when used for code generation tasks, questions regarding the accuracy of LLMs when prompted with domain expert questions such as omics related data analysis questions, remain unanswered. To address this, we developed mergen, an R package that leverages LLMs for data analysis code generation and execution. We evaluated the performance of this data analysis system using various data analysis tasks for genomics. Our primary goal is to enable researchers to conduct data analysis by simply describing their objectives and the desired analyses for specific datasets through clear text. Our approach improves code generation via specialized prompt engineering and error feedback mechanisms. In addition, our system can execute the data analysis workflows prescribed by the LLM providing the results of the data analysis workflow for human review. Our evaluation of this system reveals that while LLMs effectively generate code for some data analysis tasks, challenges remain in executable code generation, especially for complex data analysis tasks. The best performance was seen with the self-correction mechanism, in which self-correct was able to increase the percentage of executable code when compared to the simple strategy by 22.5% for tasks of complexity 2. For tasks for complexity 3, 4 and 5, this increase was 52.5%, 27.5% and 15% respectively. Using a chi-squared test, it was shown that significant differences could be found using the different prompting strategies. Our study contributes to a better understanding of LLM capabilities and limitations, providing software infrastructure and practical insights for their effective integration into data analysis workflow
Radiation retinopathy: microangiopathy-inflammation-neurodegeneration
Get PDFPURPOSE: Proton irradiation is used to treat choroidal melanoma of the eye. The impact on non-malignant retinal cells is currently understudied. Therefore, we here report a mouse model to investigate the impact of proton irradiation on the retina. METHODS: We performed a proton beam irradiation of 5–15 Cobalt-Gray-Equivalent (CGE) of the eyes of female C57Bl6/J (Cx3cr1(+/+)), Cx3cr1(gfp/+) and Cx3cr1(gfp/gfp) mice mimicking the clinical situation and evaluated the structure, function and cellular composition of the retina up to 24 weeks after irradiation. RESULTS: Proton beam irradiation of the eye with 15 CGE leads to cataract formation after 24 weeks without affecting the gross anatomy of the retinal vasculature as shown by Fundus imaging in all genotypes respectively. However, 10 and 15 CGE, lead to a significant decrease in NG2 positive cell numbers and all three dosages induced an increase in GFAP immunoreactivity. At 24 weeks a dosage of 15 CGE resulted in functional impairment and a decrease of NG2 positive cells in both WT and Cx3cr1 animals. Iba1 cell immunoreactivity was increased in all genotypes. However, in the Cx3cr1 animals the invasion of Iba1 cells into the deep vascular layer was partially prevented. This was accompanied by a less severe functional impairment in the irradiated Cx3cr1(gfp/gfp) vs. WT. CONCLUSIONS: Although the gross anatomy of the retina does not seem to be affected by proton beam irradiation, the cellular composition and retinal function changed significantly in both WT and Cx3cr1 mice reflecting the clinical situation. Moreover, cataract formation was one of the major long-term effects of irradiation. We conclude that the murine model (WT and Cx3cr1 genotype) can be used to investigate proton-beam associated side effects in vivo as well as to test prospective interventions. Moreover, the loss of Cx3cr1 seems to be partially protective
Short association fibres form topographic sheets in the human V1-V2 processing stream
Get PDFDespite the importance of short association fibres (SAF) for human brain function their structures remain understudied. It is not known how SAF are organised across the brain, and how consistent their geometries and locations are across individuals. To address this gap, we mapped the precise structures of SAF in the primary (V1) and secondary (V2) visual cortex in a group of participants in vivo and a post mortem specimen. We assessed the consistency of SAF geometries and their expected structural and functional topography using probabilistic tractography on sub-millimetre resolution diffusion-weighted MRI combined with functional MRI retinotopic maps in vivo. We found that dense SAF connected V1 and V2, forming sheet structures with retinotopic topography and bearing consistent geometries that resembled the local V1–V2 cortical folding. In vivo findings were corroborated by the robust and fine-grained post mortem reference. Our in vivo approach provides important insights into SAF organisation and could be applied to studies across species on cortical and SAF reorganisation and support neuronavigation
Pronounced impairment of B cell differentiation during bone regeneration in adult immune experienced mice
Get PDFINTRODUCTION: Alterations of the adaptive immune system have been shown to impact bone healing and may result in impaired healing in some patients. Apart from T cells, B cells are the key drivers of adaptive immunity. Therefore, their role in age-associated impairments of bone healing might be essential to understand delays during the healing process. B cells are essential for bone formation, and their dysfunction has been associated with aging or autoimmune diseases. But whether age-associated changes in B cell phenotypes are involved in bone regeneration is unknown. METHODS: Here, we aimed to characterize the role of immune aging in B cell phenotypes during the early inflammatory phase of bone healing. By comparing non-immune experienced with young and immune experienced mice we aimed to analyze the effect of gained immune experience on B cells. Our single cell proteo-genomics analysis quantified thousands of transcriptomes of cells that were isolated from post osteotomy hematoma and the proximal and distal bone marrow cavities, and enabled us to evaluate cell proportion, differential gene expression and cell trajectories. RESULTS: While the B cell proportion in young and non-immune experienced animals did not significantly change from 2 to 5 days post osteotomy in the hematoma, we found a significant decrease of the B cell proportion in the immune experienced mice, which was accompanied by the decreased expression of B cell specific genes, suggesting a specific response in immune experienced animals. Furthermore, we detected the most extensive B cell differentiation block in immune-experienced mice compared to non-immune experienced and young animals, predominantly in the transition from immature to mature B cells. DISCUSSION: Our results suggest that the pronounced impairment of B cell production found in immune experienced animals plays an important role in the initial phase leading to delayed bone healing. Therefore, novel therapeutic approaches may be able target the B cell differentiation defect to retain B cell functionality even in the immune experienced setting, which is prone to delayed healing
Neuromuscular dysfunction in patient-derived FUS(R244RR)-ALS iPSC model via axonal downregulation of neuromuscular junction proteins
Get PDFAmyotrophic lateral sclerosis (ALS) is a neurodegenerative condition characterized by the progressive degeneration of motor neurons, ultimately resulting in death due to respiratory failure. A common feature among ALS cases is the early loss of axons, pointing to defects in axonal transport and translation as initial disease indicators. ALS is associated with mutations in RNA-binding proteins, such as FUS (Fused in Sarcoma). Here, we established a FUSR244RR-ALS hiPSC-derived model that recapitulates the motor neuron survival and muscle contractility defects characteristic of ALS patients. Analysis of the protein and mRNA expression profiles in axonal and somatodendritic compartments of ALS-afflicted and isogenic control motor neurons revealed a selective downregulation of proteins essential for the neuromuscular junction function in FUS-ALS axons. Furthermore, analysis of FUS CLIP and RIP data showed that FUS binds mRNAs encoding these proteins. This work shed light on the pathogenic mechanisms of ALS and emphasized the importance of axonal gene expression analysis in elucidating the mechanisms of neurodegenerative disorders
Total bilirubin as a marker for hemolysis and outcome in patients with severe ARDS treated with veno-venous ECMO
Get PDFBACKGROUND: Hemolysis is a common complication in critically ill patients with sepsis, acute respiratory distress syndrome (ARDS) or therapy with extracorporeal membrane oxygenation (ECMO). Heme degradation product bilirubin might accumulate in conditions of significant hemolysis. In patients with ARDS and therapy with veno-venous ECMO (vvECMO), the prognostic potential of elevated initial total bilirubin (tBili) was investigated. METHODS: Retrospective analysis of patients with ARDS and vvECMO-therapy (n = 327) admitted to a tertiary ARDS center. A tBili cut-off value was determined by binary recursive partitioning. Baseline characteristics were compared and relevant variables were included in a multivariate logistic regression model with backward variable selection. Primary endpoint was survival within 28 days analyzed with Kaplan-Meier-curves and cox regression. Secondary endpoints included failure free composites for organ dysfunction, renal replacement therapy (RRT), vasopressor therapy and ECMO within 28 days and were compared using competing risk regression analysis. RESULTS: A cut-off value of 3.6mg/dl divided the cohort for ICU mortality (tBili ≤ 3.6mg/dl: 46% (n = 273) vs. tBili > 3.6mg/dl: 78% (n = 54), p 3.6mg/dl showed a higher 28-day mortality (HR 3.03 [95%CI 2.07-4.43], p 3.6mg/dl had a higher mortality and lower chances for recovery from organ dysfunction, RRT, and ECMO within 28 days. The tBili-cut-off value may be useful to identify patients at risk for unfavorable outcomes
Faecal cytolysin is associated with worse survival in patients with primary sclerosing cholangitis
No full textBACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an immune-related cholangiopathy without treatment options beyond liver transplantation. The gut–liver axis, especially the role of gut microbes, has emerged as a crucial pathway contributing to PSC pathogenesis. Recent research has revealed Enterococcus (E.) faecalis and its virulence factor cytolysin to increase mortality risk in patients with alcohol-associated hepatitis. Thus, we studied the role of enterococci, particularly E. faecalis and its virulence factor genes cytolysin and gelatinase, in faecal samples from patients with PSC. METHODS: To assess the relevance of Enterococcus species, we performed 16S rRNA gene amplicon analysis in faecal samples from 60 patients with PSC. We validated our findings by qPCR of faecal microbial DNA in an extended cohort of 105 patients with PSC, 104 patients with inflammatory bowel disease (IBD) and 68 healthy subjects. RESULTS: High-throughput 16S rRNA amplicon analysis revealed an increased relative abundance of enterococci in PSC patients compared with healthy controls and IBD patients, respectively, (p < 0.0001). PSC patients with high enterococci abundance had a decreased probability of transplant-free survival (p = 0.028). E. faecalis and its virulence factors cytolysin and gelatinase were more abundant in patients with PSC. Higher faecal cytolysin was associated with lower overall survival (p = 0.04), while survival was independent of gelatinase levels. CONCLUSION: Our data highlight the association of E. faecalis and faecal cytolysin with lower survival in patients with PSC. These data should prompt further research into the pathogenic role of cytolysin-positive E. faecalis, and to explore its role as a potential therapeutic target
TNAP expressing adventitial pericytes contribute to myogenesis during foetal development
Get PDFOBJECTIVE: During growth and differentiation of skeletal muscle, cell types other than canonical myoblasts can be recruited to a myogenic fate. Among these, TNAP(+) pericytes can differentiate into skeletal or smooth muscle cells during postnatal growth and contribute to muscle regeneration. However, their role in muscle development has not been investigated. This study aims to characterise pericyte fate choices during embryonic and foetal myogenesis, occurring in the second half of gestation. APPROACH AND RESULTS: Using Cre-loxP lineage tracing with multiple reporters including the multifluorescent Confetti, we labelled TNAP(+) precursors in vivo and assessed the smooth or skeletal muscle differentiation in their lineage at a perinatal stage. We found that TNAP(+) cells contribute in vivo to skeletal and smooth muscle cells, as well as other pericytes, also during pre-natal muscle development. The resulting clones showed that such fate choices are likely to depend on distinct unipotent progenitors rather than multipotent progenitors. In addition, we isolated and differentiated in vitro foetal cells derived from TNAP(+) precursors, which showed that they are not spontaneously myogenic unless co-cultured with other skeletal muscle cells. CONCLUSIONS: This work extends our understanding of the differentiative potency of these non- canonical skeletal muscle progenitors during prenatal life, with a view to a future application of this knowledge to optimise cell therapies for muscle wasting disorders
Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3
No full textRegulatory T (T(reg)) cells, which specifically express the master transcription factor FOXP3, have a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases. Although stimulation of naive CD4(+) T cells in the presence of TGFβ and IL-2 can induce FOXP3(+) T(reg) cells in vitro (iT(reg) cells), the resulting cells are often unstable and have thus far hampered translational efforts. A systematic approach towards understanding the regulatory networks that dictate T(reg) differentiation could lead to more effective iT(reg) cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalogue gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. We identify the RBPJ-NCOR repressor complex as a novel, context-specific negative regulator of FOXP3 expression. RBPJ-targeted knockout enhanced iT(reg) differentiation and function, independent of canonical Notch signalling. Repeated cytokine and T cell receptor signalling stimulation in vitro revealed that RBPJ-deficient iT(reg) cells exhibit increased phenotypic stability compared with control cells through DNA demethylation of the FOXP3 enhancer CNS2, reinforcing FOXP3 expression. Conversely, overexpression of RBPJ potently suppressed FOXP3 induction through direct modulation of FOXP3 histone acetylation by HDAC3. Finally, RBPJ-ablated human iT(reg) cells more effectively suppressed xenogeneic graft-versus-host disease than control iT(reg) cells in a humanized mouse model. Together, our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease