Biology subjects

Fischer, S.

Publications and source records attributed to Fischer, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

mTOR controls growth and internal architecture of human breast cancer spheroids

mTOR is a serine/threonine kinase participating in two distinct functional complexes: mTORC1 and mTORC2. mTORC1 regulates protein translation, cell cycle progression and autophagy. mTORC2 controls cell survival, proliferation and actin cytoskeleton reorganization. mTOR signaling is often deregulated during breast cancer. We investigated how mTOR and its downstream process autophagy determine the inner architecture of solid tumor aggregates at multiple levels. We focused on structures, which are morphologically altered during progression of cancer, including: nuclei number and volume, cell density, number, shape and volume of polarized structures. We developed a breast cancer spheroid model, in which the effects of mTOR and autophagy modulation were investigated by pharmacological inhibition mTOR signaling and induction autophagy, as well as using ATG7 KO spheroids. Spheroids were imaged with light sheet-based fluorescence microscopy and data were quantitatively analyzed. Our data suggest that mTORC1 regulates the spheroid volume, cell number and size. mTORC2 controls nuclei volume and determines growth and elongation of polarized acini inside a spheroid.

cell biology↗

bioRxiv2023

Kinetochore and ionomic adaptation to whole genome duplication

Transforming genomic and cellular landscapes in a single generation, whole genome duplication (WGD) brings fundamental challenges, but is also associated with diversification. How is WGD tolerated, and what processes commonly evolve to stabilize the resulting polyploid? Here we study this in Cochlearia spp., which have experienced multiple WGDs in the last 300,000 years. We first generate a chromosome-scale genome and sequence 113 individuals from 33 diploid, tetraploid, hexaploid, and outgroup populations. We detect the clearest post-WGD selection signatures in functionally interacting kinetochore components and ion transporters. We structurally model these derived selected alleles, identifying striking WGD-relevant functional variation, and then compare these results to independent recent post-WGD selection in Arabidopsis arenosa and Cardamine amara. Most prominent in these results is genetic evidence of at least four functionally interacting kinetochore complex subunits in adaptation to WGD at the centromere among our very top selective sweep outliers. In addition, some of the same biological processes evolve in all three WGDs, but specific genes recruited are flexible. This points to a polygenic basis for modifying systems that control the kinetochore, meiotic crossover number, DNA repair, ion homeostasis, and cell cycle. Given that DNA management (especially repair) is the most salient category with the strongest selection signal, we speculate that the generation rate of structural genomic variants may be altered by WGD in young polyploids, contributing to their occasionally spectacular adaptability observed across kingdoms. Significance StatementWhole-genome duplication (WGD) occurs in all kingdoms and is linked to adaptation, speciation, domestication, and even cancer outcome. But WGD is a shock to the system, and commonly disrupts cell division due to increased DNA management burden and transformed cell physiology. Nevertheless, the hopeful monster that survives WGD is special, occasionally experiencing runaway success. Why do some thrive but others die? Here we introduce a powerful new model, Cochlearia, which has benefitted from multiple WGDs, and we provide the first genetic evidence of rapid adaptation of functionally interacting components of the cell division machinery, the kinetochore. We also compare which processes and genes evolve to stabilize the new polyploid in three independent cases and highlight common mechanisms.

evolutionary biology↗

bioRxiv2023

Genetic Variants That Modify the Neuroendocrine Regulation of Foraging Behavior in C. elegans

The molecular mechanisms underlying diversity in animal behavior are not well understood. A major experimental challenge is determining the contribution of genetic variants that affect neuronal gene expression to differences in behavioral traits. The neuroendocrine TGF-beta ligand, DAF-7, regulates diverse behavioral responses of Caenorhabditis elegans to bacterial food and pathogens. The dynamic neuron-specific expression of daf-7 is modulated by environmental and endogenous bacteria-derived cues. Here, we investigated natural variation in the expression of daf-7 from the ASJ pair of chemosensory neurons and identified common variants in gap-2, encoding a GTPase-Activating Protein homologous to mammalian SynGAP proteins, which modify daf-7 expression cell-non-autonomously and promote exploratory foraging behavior in a DAF-7-dependent manner. Our data connect natural variation in neuron-specific gene expression to differences in behavior and suggest that genetic variation in neuroendocrine signaling pathways mediating host-microbe interactions may give rise to diversity in animal behavior.

neuroscience↗

bioRxiv2023

The role of the stress axis in mediating behavioural flexibility in a social cichlid, Neolamprologus pulcher

Behavioural flexibility plays a major role in the way animals cope with novel situations, and physiological stress responses are adaptive and highly efficient mechanisms to cope with unpredictable events. Previous studies investigating the role of stress responses in mediating behavioural flexibility were mostly done in laboratory rodents using stressors and cognitive challenges unrelated to the ecology of the species. To better understand how stress mediates behavioural flexibility in a natural context, direct manipulations of the stress response and cognitive tests in ecologically relevant contexts are needed. To this aim, we pharmacologically blocked glucocorticoid receptors (GR) in adult Neolamprologus pulcher using a minimally invasive application of a GR antagonist. GR blockade prevents the recovery after a stressful event, which we predicted to impair behavioural flexibility. After the application of the GR antagonist, we repeatedly exposed fish to a predator and tested their behavioural flexibility using a detour task, i.e. fish had to find a new, longer route to the shelter when the shortest route was blocked. While the latencies to find the shelter were not different between treatments, GR blocked fish showed more failed attempts during the detour tasks than control fish. Furthermore, weak performance during the detour tasks was accompanied by an increase of fear related behaviours. This suggests that blocking GR changed the perception of fear and resulted in an impaired behavioural flexibility. Therefore, our results support a causal link between the capacity to recover from stressors and behavioural flexibility in N. pulcher with potential consequences for an effective and adaptive coping with changing environments.

animal behavior and cognition↗

bioRxiv2023

Risk of urban Mayaro virus (Alphavirus: Togaviridae) transmission: is Aedes aegypti a competent vector?

Mayaro virus (MAYV) is an emerging pathogen endemic in Latin America and is the causative agent of fever and polyarthritis. Urban transmission depends on its ability to be transmitted by Aedes aegypti and to be amplified by humans. The aim of this study was to evaluate the susceptibility to infection and transmission and the presence of barriers to infection in different populations of Ae. aegypti for MAYV. Ae. aegypti eggs were collected from Cordoba, Buenos Aires and Rosario Cities (Argentina). Females were infected with five viral loads of MAYV strain (1 to 6 log10 PFU/ml) and maintained for 8 days. The presence of infectious viral particles in body, legs, and saliva was detected by plaquing assay in Vero cell monolayers. Through a bibliographic search, Ae. aegypti population data from Peru were incorporated and tested with different viral doses of MAYV. We build dose-response curves for Ae. aegypti populations to estimate infection (IR), dissemination (DR) and transmission (TR) based on MAYV viral loads detected in humans to estimate transmission risk occurring in an urban environment. The overall IR and DR were significantly associated with the viral doses and were not significantly affected by population origin. We found IR ranging for 3 to 84% (ID50% were higher than 5.5 log10 PFU/ml) and a DR reached 78% (DD50% higher than 6.0 log10 PFU/ml). The percentage of dissemination based on the infected mosquitoes ranged from 60 to 86% while the percentage of transmission based on disseminated mosquitoes ranged from 11 to 60%. Our results indicate that Ae. aegypti populations are not competent vectors for MAYV because they need higher viral doses than those developed by humans (3.9 - 4.5 log10 PFU/ml) to become infected. Only a very low proportion of infected mosquitoes with high 5 log10 PFU/ml are capable of transmitting it.

microbiology↗

bioRxiv2023

SAMD1 suppresses epithelial-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma

SAMD1 (SAM-domain containing protein 1), a CpG island-binding protein, plays a pivotal role in the repression of its target genes. Despite its significant correlation with outcomes in various tumor types, the role of SAMD1 in cancer has remained largely unexplored. In this study we focused on pancreatic ductal adenocarcinoma (PDAC) and revealed that SAMD1 acts as a repressor of genes associated with epithelial-mesenchymal transition (EMT). Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor of SAMD1. FBXO11 ubiquitinates SAMD1 within its DNA-binding winged helix domain and inhibits SAMD1 chromatin binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide new insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.

molecular biology↗

bioRxiv2023

IRF2BP2 counteracts the ATF7/JDP2 AP-1 heterodimer to prevent inflammatory overactivation in acute myeloid leukemia (AML) cells

Acute myeloid leukemia (AML) is a hematological malignancy characterized by abnormal proliferation and accumulation of immature myeloid cells in the bone marrow. Inflammation plays a crucial role in AML progression, but excessive activation of cell-intrinsic inflammatory pathways can also trigger cell death. IRF2BP2 is a chromatin regulator implicated in AML pathogenesis, although its precise role in this disease is not fully understood. In this study, we demonstrate that IRF2BP2 interacts with the AP-1 heterodimer ATF7/JDP2, which is involved in activating inflammatory pathways in AML cells. We show that IRF2BP2 is recruited by the ATF7/JDP2 dimer to chromatin and counteracts its gene-activating function. Loss of IRF2BP2 leads to overactivation of inflammatory pathways, resulting in strongly reduced proliferation. Our research indicates that a precise equilibrium between activating and repressive transcriptional mechanisms creates a pro-oncogenic inflammatory environment in AML cells. The ATF7/JDP2-IRF2BP2 regulatory axis is likely a key regulator of this process and may therefore represent a promising therapeutic vulnerability for AML. Thus, our study provides new insights into the molecular mechanisms underlying AML pathogenesis and identifies a potential therapeutic target for AML treatment.

molecular biology↗

bioRxiv2023

Latitudinal scaling of aggregation with abundance and its consequences for coexistence in species rich forests

The search for simple principles underlying the complex spatial structure and dynamics of plant communities is a long-standing challenge in ecology1-6. In particular, the relationship between the spatial distribution of plants and species coexistence is challenging to resolve in species-rich communities7-9. Analysing the spatial patterns of tree species in 21 large forest plots, we find that rare species tend to be more spatially aggregated than common species, and a latitudinal gradient in the strength of this negative correlations that increases from tropical to temperate forests. Our analysis suggests that latitudinal gradients in animal seed dispersal10 and mycorrhizal associations11,12,13 may jointly generate this intriguing pattern. To assess the consequences of negative aggregation-abundance correlations for species coexistence, we present here a framework to incorporate the observed spatial patterns into population models8 along with an analytical solution for the local extinction risk14 of species invading from low abundances in dependence of spatial structure, demographic parameters, and immigration. For example, the stabilizing effect of the observed spatial patterns reduced the local extinction risk of species when rare almost by a factor of two. Our approach opens up new avenues for integrating observed spatial patterns into mathematical theory, and our findings demonstrate that spatial patterns, such as species aggregation and segregation, can contribute substantially to coexistence in species-rich communities. This underscores the need to understand the interactions between multiple ecological processes and spatial patterns in greater detail.

ecology↗

bioRxiv2023

The salt-and-pepper pattern in mouse blastocysts is compatible with signalling beyond the nearest neighbours.

Embryos develop in a concerted sequence of spatio-temporal arrangements of cells. In the preimplantation mouse embryo, the distribution of the cells in the inner cell mass evolves from a salt-and-pepper pattern to spatial segregation of two distinct cell types. The exact properties of the salt-and-pepper pattern have not been analysed so far. We investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. A combination of spatial statistics and agent-based modelling reveals that the cell fate distribution follows a local clustering pattern. Using ordinary differential equations modelling, we show that this pattern can be established by a distance-based signalling mechanism enabling cells to integrate information from the whole inner cell mass into their cell fate decision. Our work highlights the importance of longer-range signalling to ensure coordinated decisions in groups of cells to successfully build embryos.

developmental biology↗

bioRxiv2023

Self-organized BMP signaling dynamics underlie the development and evolution of tetrapod digit patterns

Repeating patterns of synovial joints are a highly conserved feature of articulated digits, with variations in joint number and location giving rise to a diverse range of digit morphologies and limb functions across the tetrapod clade. During development, joints form iteratively within the growing digit ray, as a population of distal progenitors alternately specifies joint and phalanx cell fates to segment the digit into distinct elements. Whilst numerous molecular pathways have been implicated in this fate choice, it remains unclear how they give rise to a repeating pattern. Here, using single cell RNA-sequencing and spatial gene expression profiling, we investigate the transcriptional dynamics of interphalangeal joint specification in vivo. Combined with mathematical modelling, we predict that interactions within the BMP signaling pathway - between the ligand GDF5, the inhibitor NOG, and the intracellular effector pSMAD - result in a self-organizing Turing system that forms periodic joint patterns. Our model is able to recapitulate the spatiotemporal gene expression dynamics observed in vivo, as well as phenocopy digit malformations caused by BMP pathway perturbations. By contrasting in silico simulations with in vivo morphometrics of two morphologically distinct digits, we show how changes in signaling parameters and growth dynamics can result in variations in the size and number of phalanges. Together, our results reveal a self-organizing mechanism that underpins tetrapod digit patterning and its evolvability, and, more broadly, illustrate how Turing systems based on a single molecular pathway may generate complex repetitive patterns in a wide variety of organisms.

developmental biology↗

bioRxiv2023

Cell targeting and immunostimulatory properties of a novel Fcγ-receptor independent agonistic anti-CD40 antibody in rhesus macaques

Targeting CD40 by agonistic antibodies used as vaccine adjuvants or for cancer immunotherapy is a strategy to stimulate immune responses. The majority of studied agonistic anti-human CD40 antibodies require crosslinking of their Fc region to inhibitory Fc{gamma}RIIb to induce immune stimulation although this has been associated with toxicity in previous studies. Here we introduce an agonistic anti-human CD40 monoclonal IgG1 antibody (MAB273) unique in its specificity to the CD40L binding site of CD40 but devoid of Fc{gamma}-receptor binding, we demonstrate rapid binding of MAB273 to B cells and dendritic cells resulting in strong activation in vitro on human cells and in vivo in rhesus macaques. Dissemination of fluorescently labeled MAB273 after subcutaneous administration was found predominantly at the site of injection and specific draining lymph nodes. Phenotypic cell differentiation and upregulation of genes associated with immune activation were found in the targeted tissues. Antigen-specific T cell responses were enhanced by MAB273 when given in a prime-boost regimen and for boosting low preexisting responses. MAB273 may therefore be a promising immunostimulatory adjuvant that warrants future testing for therapeutic and prophylactic vaccination strategies.

immunology↗

bioRxiv2023

Fcgamma-receptor-independent controlled activation of CD40 canonical signaling by novel therpeutic antibodies for cancer therapy

Activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Agonistic anti-CD40 antibodies currently in development require Fc{gamma}-receptor-mediated crosslinking of CD40 molecules for meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely Fc-independent manner. These novel Fc-silenced anti-CD40 antibodies induce upregulation of costimulatory receptors CD80 and CD86 and cytokine release by dendritic cells with an efficacy exceeding that of existing antibodies. Binding to the CD40L interaction region on CD40 appears to be a prerequisite to achieving such strong activities. Finally, the most active identified anti-CD40 antibody shows evidence of activity in terms of the expected markers of canonical CD40 signaling when injected in humanized mice. There are no signs of obvious toxicities whereas the clinical-stage anti-CD40 antibody CP-870,893 induced severe signs of toxicity in these animals despite a lower dose compared with the novel Fc-silenced canonical agonist. These studies thus demonstrate potent activation of antigen-presenting cells with anti-CD40 antibodies lacking Fc{gamma}-receptor-binding activity and open the possibility of an efficacious and safe combination therapy for cancer patients. One Sentence SummaryTreatment of antigen-presenting cells and humanized mice with novel Fc-silenced CD40 antibodies demonstrates an Fc{gamma}-receptor-independent canonical agonistic mode of action for therapeutic use.

immunology↗

bioRxiv2022

Modular cell type organization of cortical areas revealed by in situ sequencing

The cortex is composed of neuronal types with diverse gene expression that are organized into specialized cortical areas. These areas, each with characteristic cytoarchitecture (Brodmann 1909; Vogt and Vogt 1919; Von Bonin 1947), connectivity (Zingg et al. 2014; Harris et al. 2019), and neuronal activity (Schwarz et al. 2008; Ferrarini et al. 2009; He et al. 2009; Meunier et al. 2010; Bertolero et al. 2015), are wired into modular networks (Zingg et al. 2014; Harris et al. 2019; Huang et al. 2020). However, it remains unclear whether cortical areas and their modular organization can be similarly defined by their transcriptomic signatures and how such signatures are established in development. Here we used BARseq, a high-throughput in situ sequencing technique, to interrogate the expression of 104 cell type marker genes in 10.3 million cells, including 4,194,658 cortical neurons over nine mouse forebrain hemispheres at cellular resolution. De novo clustering of gene expression in single neurons revealed transcriptomic types that were consistent with previous single-cell RNAseq studies(Yao et al. 2021a; Yao et al. 2021b). Gene expression and the distribution of fine-grained cell types vary along the contours of cortical areas, and the composition of transcriptomic types are highly predictive of cortical area identity. Moreover, areas with similar compositions of transcriptomic types, which we defined as cortical modules, overlap with areas that are highly connected, suggesting that the same modular organization is reflected in both transcriptomic signatures and connectivity. To explore how the transcriptomic profiles of cortical neurons depend on development, we compared the cell type distributions after neonatal binocular enucleation. Strikingly, binocular enucleation caused the cell type compositional profiles of visual areas to shift towards neighboring areas within the same cortical module, suggesting that peripheral inputs sharpen the distinct transcriptomic identities of areas within cortical modules. Enabled by the high-throughput, low-cost, and reproducibility of BARseq, our study provides a proof-of-principle for using large-scale in situ sequencing to reveal brain-wide molecular architecture and to understand its development.

neuroscience↗

bioRxiv2022

Peptide-mediated inhibition of the transcriptional regulator Elongin BC induces apoptosis in cancer cells

Inhibition of protein-protein interactions (PPIs) via designed peptides is an effective strategy to interfere with their biological functions. The Elongin BC heterodimer (ELOB/C) is involved in transcription elongation and protein turnover by PPIs that involve the so-called BC-box. ELOB and ELOC are commonly upregulated in cancer and essential for cancer cell growth, making them attractive drug targets. However, no strategy has been established to inhibit their functions in cells, so far. Here, we report a peptide that mimics a high-affinity BC-box and tightly binds to the ELOB/C dimer (kD = 0.45 {+/-} 0.03 nM). Our peptide blocks the association of ELOB/C with its interaction partners, both in vitro and in the cellular environment. Cancer cells treated with this peptide inhibitor show decreased cell viability, altered cell cycle and increased apoptosis. Therefore, our work proposes that blocking the BC-box binding pocket of ELOB/C is a feasible strategy to impair the function of the ELOB/C heterodimer and inhibit cancer cell growth. Our peptide inhibitor promises novel mechanistic insights into the biological function of the ELOB/C dimer and offers a starting point for therapeutics linked to ELOB/C dysfunction.

cancer biology↗

bioRxiv2022

The Influences of Stomatal Size and Density on Rice Drought, Salinity and VPD Resilience

O_LIA warming climate coupled with reductions in water availability and rising salinity are increasingly affecting rice yields (Oryza sativa L.). Elevated temperatures are causing vapour pressure deficit (VPD) rises, leading to stomata closure, further reducing plant productivity and cooling. It is unclear which conformation of stomatal size (SS) and stomatal density (SD) will best suit these future environmental extremes. C_LIO_LITo understand the influence of stomatal characteristics on rice abiotic stress tolerance, we screened the stomatal characteristics of 72 traditionally-bred varieties. We found significant variation in SS, SD and maximal stomatal conductance (gsmax) but did not identify any varieties with SD and gsmax as low as the genetically manipulated stomatal development mutant OsEPF1oe. C_LIO_LITraditionally-bred varieties with high SD and small SS (resulting in high gsmax) typically had lower biomasses, and these plants were more resilient to drought than low SD and large SS plants, which were physically larger. None of the varieties tested were as resilient to drought or salinity as low SD OsEPF1oe mutants. High SD and small SS rice displayed faster stomatal closure during rising VPD, but photosynthesis and plant cooling were reduced. C_LIO_LICompromises will be required when choosing rice SS and SD to tackle multiple future environmental stresses. C_LI

plant biology↗

bioRxiv2022

The kinase activity of the cancer stem cell marker DCLK1 drives gastric cancer progression by reprogramming the stromal tumor landscape

Gastric cancer (GC) is the 3rd leading cause of cancer mortality worldwide, therefore providing novel diagnostic and treatment options is crucial for at risk groups. The serine/threonine kinase doublecortin-like kinase 1 (DCLK1) is a proposed driver of GC with frequent amplification and somatic missense mutations yet the molecular mechanism how DCLK1 mediates tumorigenesis is poorly understood. We report how DCLK1 expression orchestrates complementary cancer cell intrinsic and extrinsic processes leading to a comprehensive pro-invasive and pro-metastatic reprogramming of cancer cells and tumor stroma in a DCLK1 kinase-dependent manner. Mechanistically, we identify the chemokine CXCL12 as a key promoter of the pro-tumorigenic properties downstream of DCLK1. Importantly, inhibition of the DCLK1 kinase domain reverses the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a promising, targetable master regulator of GC. TeaserDCLK1 is a druggable cancer driver of GC

cancer biology↗

bioRxiv2021

Transcriptional networks underpinning ploidy related increased leaf potassium in neo-tetraploids

Neo-tetraploid Arabidopsis thaliana have elevated leaf potassium (K) driven by processes within the root. The root transcriptome of neo-tetraploids is distinct from diploids, with evidence of altered K homeostasis. Mutational analysis revealed that the canonical K-uptake transporters AKT1 and HAK5 are not required for this elevated leaf K in neo-tetraploids, while the endodermis, root hairs, and SOS signaling are. Contrasting the root transcriptomes of neo-tetraploids and diploids of mutants that block the neo-tetraploid K phenotype, allowed us to identify 91 differentially expressed genes associated with elevated leaf K in neo-tetraploids. This set of genes connects WGD to elevated leaf K, and is enriched in functions such as cell wall and Casparian strip development, and ion-transport, in the endodermis, root hairs, and procambium. This gene set provides tools to test the intriguing idea of recreating the physiological effects of WGD within a diploid genome.

plant biology↗

bioRxiv2021

Two engineered AAV capsid variants for efficient transduction of human cortical neurons directly converted from iPSC

Recombinant adeno-associated virus (AAV) is the most widely used vector for gene therapy in clinical trials. To increase transduction efficiency and specificity, novel engineered AAV variants with modified capsid sequences are evaluated in human cell cultures and non-human primates. In the present study, we tested two novel AAV capsid variants, AAV2-NNPTPSR and AAV9-NVVRSSS, in human cortical neurons, which were directly converted from human induced pluripotent stem cells and cocultured with rat primary astrocytes. AAV2-NNPTPSR variant efficiently transduced both induced human cortical glutamatergic neurons and induced human cortical GABAergic interneurons. By contrast, AAV9-NVVRSSS variant transduced both induced human cortical neurons and cocultured rat primary astrocytes. High viral titers (1x10E5 viral genomes per cell) caused a significant decrease in viability of induced human cortical neurons. Low viral titers (1x10E4 viral genomes per cell) lead to a significant increase in the neuronal activity marker c-Fos in transduced human neurons following treatment with a potassium channel blocker, which may indicate functional alterations induced by viral transduction and/or transgene expression.

neuroscience↗