Biology subjects

Becker, C.

Publications and source records attributed to Becker, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Transdermal Delivery of Ultradeformable Cationic Liposomes Complexed with miR211-5p (UCL-211) Stabilizes BRAFV600E+ Melanocytic Nevi

Small non-coding RNAs (e.g. siRNA, miRNA) are involved in a variety of melanocyte-associated skin conditions and act as drivers for alterations in gene expression within melanocytes. These molecular changes can potentially affect the cellular stability of melanocytes and promote their oncogenic transformation. Thus, small RNAs can be considered as therapeutic targets for these conditions, however, their topical delivery to the melanocytes through the epidermal barrier is challenging. We synthesized and extensively evaluated ultradeformable cationic liposome (UCLs) carriers complexed with synthetic microRNAs (miR211-5p; UCL-211) for transdermal delivery to melanocytes. UCL-211 complexes were characterized for their physicochemical properties, encapsulation efficiency, and deformability, which revealed a significant advantage over conventional liposomal carriers. Increased expression of miR211-5p stabilizes melanocytic nevi and keeps them in growth-arrested state. We did a comprehensive assessment of cellular delivery, and biological activity of the miR211-5p carried by UCL-211 in vitro and their permeation through the epidermis of intact skin using ex vivo human skin tissue explants. We also demonstrated, in vivo, that topical delivery of miR211-5p by UCL-211 stabilized BRAFV600E+ nevi melanocytes in a benign nevi state.

pharmacology and toxicology↗

bioRxiv2024

Altered Hormone and Bioactive Lipid Plasma Profile in Rodent Models of Polycystic Ovarian Syndrome Revealed by Targeted Mass Spectrometry

Polycystic ovarian syndrome (PCOS) symptoms include excessive body or facial hair, irregular periods, reduced fertility, and reoccurring pregnancy loss. Hyperandrogenism and chronic inflammation are hallmarks of PCOS, which is diagnosed by analysing steroid hormones in the blood. Studies suggest that bioactive lipids are contributing to chronic inflammation. To research PCOS, animal models, such as letrozole- and dihydrotestosterone-treated rats, are used. They display similar ovarian and metabolic characteristics, although plasma lipid profiles have not been determined. Therefore, in order to validate the use of these models for PCOS, we have optimised a mass spectrometry based targeted lipidomics workflow which increases the sensitivity of measuring these lipids in rat plasma. Our analysis shows that letrozole caused a significant elevation of 5-androstene-3,17-dione and testosterone. Dihydrotestosterone treatment resulted in increased dehydroepiandrosterone-sulphate and allopregnanolone but a reduction in testosterone, progesterone, pregnenolone, and D-sphingosine. In both models 25-hydroxycholesterol and leukotriene C4 were significantly diminished and 4-cholesten-3-one was significantly increased, and these particular metabolites are not known to be changed in human PCOS. These results suggest that the plasma lipids of these rodent models have some changes akin to human PCOS and other changes that are not.

biochemistry↗

bioRxiv2024

Sub-functionalization and epigenetic regulation of a biosynthetic gene cluster in Solanaceae

Biosynthetic gene clusters (BGCs) are sets of often heterologous genes that are genetically and functionally linked. Among eukaryotes, BGCs are most common in plants and fungi, where they ensure the co-expression of the different enzymes coordinating the biosynthesis of specialised metabolites. Here, we report on a newly identified withanolide BGC in Physalis grisea (ground-cherry), a member of the nightshade family (Solanaceae). A combination of transcriptomic, epigenomic and metabolic analyses, revealed that, following a duplication event, this BGC evolved two tissue-specifically expressed sub-clusters, containing eight pairs of paralogs that contribute to related but distinct biochemical processes; this sub- functionalisation is tightly associated with epigenetic features and the local chromatin environment. The two sub-clusters appear strictly isolated from each other at the chromatin structural level, each forming a highly self-interacting chromatin domain with tissue-dependent levels of condensation. This correlates with gene expression exclusively in either above- or below-ground tissue, thus spatially separating the production of different withanolide compounds. By comparative phylogenomic analysis, we show that the withanolide BGC most likely evolved before the diversification of the Solanaceae family and underwent lineage- specific diversifications and losses. The tissue-specific sub-functionalisation is common to species of the Physalideae tribe but distinct from other, independent duplication events outside of this clade. In sum, our study reports on the first instance of an epigenetically modulated sub-functionalisation within a BGC and sheds light on the biosynthesis of withanolides, a highly diverse group of steroidal triterpenoids important in plant defence and amenable to pharmaceutical applications due to their anti-inflammatory, antibiotic and anti- cancer properties.

plant biology↗

bioRxiv2024

ApoE4 disrupts intracellular trafficking and iron homeostasis in an improved iPSC-based model of human brain endothelial cells

Transferrin receptor in brain endothelial cells can deliver therapeutic antibodies to the brain via transcytosis across the blood-brain barrier. Whether receptor transport remains intact in Alzheimers disease is still a major open question. Here, we investigated whether apolipoprotein E4 (ApoE4), the major genetic risk factor for Alzheimers disease, altered intracellular transport in human brain endothelial cells. To achieve this, we first developed an optimized protocol for induced pluripotent stem cells based on a defined chemical cocktail and extracellular-matrix support to differentiate brain endothelial cells (iCE-BECs). Multi-omic profiling and functional transport assays showed that iCE-BECs have a brain endothelial gene signature and recapitulate receptor-mediated transcytosis of a clinically validated BrainshuttleTM antibody against transferrin receptor. Engineered iCE-BECs homozygous for ApoE4 had altered spatiotemporal organization of early endosomes, increased transferrin receptor expression and reduced cytoplasmic iron. Our data revealed that ApoE4 can impact intracellular transport and iron homeostasis at the BBB in a cell-autonomous manner. This finding could be relevant for the brain delivery of therapeutic antibodies for Alzheimers disease.

cell biology↗

bioRxiv2024

Integrated multi-model analysis of intestinal inflammation exposes key molecular features of preclinical and clinical IBD

BackgroundInflammatory bowel disease (IBD) is a chronic inflammatory condition of the intestine with a complex and multifaceted pathogenesis. While various animal models exist to study specific disease mechanisms relevant to human IBD, a comprehensive comparative framework linking these to IBD pathophysiology is lacking. ObjectiveIn our study, we aimed at providing a framework that delineates common and unique features encountered in 13 widely used mouse models comparing them with human IBD to identify translatable pathways in model-cohort pairs. Another aim of our study was to provide an explorable resource for looking up gene and pathway level changes in mouse models assisting in hypothesis testing and minimizing animal burden abiding by the 3R principals. DesignWe employed comparative transcriptomic analyses with curated and a priori statistical correlative methods between mouse models versus established as well as own patient datasets at both bulk and single cell levels. ResultsWe identify IBD-related pathways, ontologies, and cellular processes that are translatable between mouse models and patient cohorts. Moreover, we identify, known and novel IBD-associated subcellular mechanisms and how they are recapitulated in specific mouse models. ConclusionOur findings provide a valuable resource for selecting the most appropriate experimental paradigm to model unique features of IBD pathomechanisms, allowing analysis at the tissue, cellular, and subcellular levels. What is already known on this topicPreclinical modelling of IBD is key to the discovery of pathomechanisms and the evaluation of therapeutic approaches. However, individual models do not recapitulate the complexity of the disease and comprehensive studies comparing modelling paradigms with human IBD are lacking. What this study addsOur study provides a comparative analysis of thirteen commonly used intestinal inflammation models, identifying core-conserved pathways between mouse models and IBD patient cohorts. In addition, our study shows how specific pathways involved in IBD are recapitulated in specific mouse models and introduces a web tool to analyse the models. How this study might affect research, practice or policyBy identifying conserved and discrepant pathways between specific mouse models and IBD patient cohorts, our analysis platform provides an invaluable resource for translational IBD research.

systems biology↗

bioRxiv2024

Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

Tumor-associated macrophages (TAMs) shape the tumor microenvironment (TME) and exert a decisive impact on anti-tumor immunity. Understanding TAM function is therefore critical to understand anti-tumor immune responses and to design immunotherapies. Here, we describe the transcription factor ZEB1, a well-known driver of epithelial-to-mesenchymal transition, as an intrinsic regulator of TAM function in adaptive anti-tumor immunity. By combining cell type-specific deletion of Zeb1 with syngeneic models of colorectal and pancreatic cancer, we discovered an unexpected function of ZEB1 in the TAM-mediated control of T cell trafficking. ZEB1 supports secretion of a subset of chemokines including CCL2 and CCL22 by promoting their transcription and translation as well as by safeguarding protein processing. ZEB1 thereby elevates cytotoxic T cell (CTL) recruitment in vitro and in vivo and fosters immunosurveillance during tumor as well as lung metastatic outgrowth. Our study spotlights ZEB1 as a crucial facilitator of adaptive anti-tumor immunity and uncovers a potential therapeutic window of opportunity for cytokine-guided enhancement of CTL infiltration into tumors and metastases.

cancer biology↗

bioRxiv2024

Evolution and diversification of the momilactone biosynthetic gene cluster in the genus Oryza

Plants are master chemists and collectively are able to produce hundreds of thousands of different organic compounds. The genes underlying the biosynthesis of many specialized metabolites are organized in biosynthetic gene clusters (BGCs), which is hypothesized to ensure their faithful co-inheritance and to facilitate their coordinated expression. In rice, momilactones are diterpenoids that act in plant defence and various organismic interactions. Many of the genes essential for momilactone biosynthesis are grouped in a BGC. Here, we apply comparative genomics of diploid and allotetraploid Oryza species to reconstruct the species-specific architecture, evolutionary trajectory, and sub-functionalisation of the momilactone biosynthetic gene cluster (MBGC) in the Oryza genus. Our data show that the evolution of the MBGC is marked by lineage-specific rearrangements and gene copy number variation, as well as by occasional cluster loss. We identified a distinct cluster architecture in O. coarctata, which represents the first instance of an alternative architecture of the MBGC in Oryza and strengthens the idea of a common origin of the cluster in Oryza and the distantly related genus Echinochloa. Our research illustrates the evolutionary and functional dynamics of a biosynthetic gene cluster within a plant genus.

plant biology↗

bioRxiv2023

The lactonase BxdA mediates metabolic adaptation of maize root bacteria to benzoxazinoids

Root exudates contain secondary metabolites that affect the plants root microbiome. How microbes cope with these bioactive compounds, and how this ability shapes root microbiomes remain largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound (6-methoxy-benzoxazolin-2-one, MBOA) in the maize rhizosphere to 2-amino-7-methoxyphenoxazin-3-one (AMPO). By contrast, bacteria isolated from Arabidopsis, which does not produce benzoxazinoids, were unable to metabolise MBOA. Among Microbacteria strains, this differential metabolisation allowed to identify a conserved gene cluster containing the lactonase bxdA. BxdA converts MBOA to AMPO in vitro and we show that this capacity provided bacteria a growth benefit under carbon-limiting conditions. Together these results reveal that maize root bacteria - through BxdA - are metabolically adapted to the benzoxazinoids of their host. We propose that metabolic adaptation to plant-specialised compounds shapes root bacterial communities across the plant kingdom.

microbiology↗

bioRxiv2023

Haplotype independence contributes to evolvability in the long-term absence of sex in a mite

Some unique asexual species persist over time and contradict the consensus that sex is a prerequisite for long-term evolutionary survival. How they escape the dead-end fate remains enigmatic. Here, we generated a haplotype-resolved genome assembly based on a single individual and collected genomic data from worldwide populations of the parthenogenetic diploid oribatid mite Platynothrus peltifer to identify signatures of persistence without sex. We found that haplotypes diverge independently since the transition to asexuality at least 20 my ago. Multiple lines of evidence indicate disparate evolutionary trajectories between haplotypic blocks. Our findings imply that such haplotypic independence can lead to non-canonical routes of evolvability, helping some species to adapt, diversify and persist for millions of years in the absence of sex. One-Sentence SummaryFunctionally different chromosome sets in an asexual mite species showcase a survival strategy spanning millions of years.

evolutionary biology↗

bioRxiv2023

Monitoring hPSC genomic stability in the chromosome 20q region by ddPCR

Copy number increases involving chromosome 20q with gain of the gene BCL2L1 are a prevalent form of genomic instability in hPSC. In addition to large aneuploidies, findings in this region often include microamplifications that are too small to detect by G-banded karyotyping. Gene editing procedures warrant especially close monitoring of 20q genomic stability because they involve p53-activating stressors that select for the survival of BCL2L1-aneuploid cells. Here we describe an optimized strategy for detecting BCL2L1 copy number increases in hPSC cultures using duplexed droplet digital PCR (ddPCR) with genomic DNA or cell lysate as the starting material. The procedure consists of droplet generation, thermocycling, droplet reading and data analysis. The expected result is a copy number estimate derived by comparing the number of droplets positive for BCL2L1 to the number positive for a reference template, PVRL2. This procedure generates same-day screening results for 1 to 96 samples, providing a convenient option for screening hPSC cultures that is easily integrated into a gene editing workflow.

cell biology↗

bioRxiv2023

Transposon dynamics in the emerging oilseed crop Thlaspi arvense

Genome evolution is partly driven by the mobility of transposable elements (TEs) which often leads to deleterious effects, but their activity can also facilitate genetic novelty and catalyze local adaptation. We explored how the intraspecific diversity of TE polymorphisms is shaping the broad geographic success and adaptation capacity of the emerging oil crop Thlaspi arvense. We achieved this by classifying the TE inventory of this species based on a high-quality genome assembly, age estimation of retrotransposon TE families and a comprehensive assessment of their mobilization potential. Our survey of TE insertion polymorphisms (TIPs) captured 280 accessions from 12 regions across the Northern hemisphere. We quantified over 90,000 TIPs, with their distribution mirroring genetic differentiation as measured by single nucleotide polymorphisms (SNPs). The number and types of mobile TE families vary substantially across populations, but there are also shared patterns common to all accessions. We found that Ty3/Athila elements are the main drivers of TE diversity in T. arvense populations, while a single Ty1/Alesia lineage might be particularly important for molding transcriptome divergence. We further observed that the number of retrotransposon TIPs is associated with variation at genes related to epigenetic regulation while DNA transposons are associated with variation at a Heat Shock Protein (HSP19). We propose that the high rate of mobilization activity can be harnessed for targeted gene expression diversification, which may ultimately present a toolbox for the potential use of transposition in breeding and domestication of T. arvense.

genomics↗

bioRxiv2022

The common oral parasitic protozoan Entamoeba gingivalis forms cysts in response to antibiotic treatment

The eukaryotic protozoan Entamoeba gingivalis (E.g.) is strongly associated with inflamed periodontal pockets. Unlike other obligate anaerobic Entamoeba species, it is considered to not have a life cycle of actively dividing trophozoites and dormant cysts. Accordingly, it has been regarded as non-infectious. To investigate if E.g is capable of encystation in response to adverse environmental conditions, we cultivated clinical isolates of E.g. collected from inflamed periodontal pockets in antibiotics for 8 days. The cytomorphological and ultrastructure forms of the amoeba were investigated by transmission and scanning electron microscopy to reveal cyst formation. We observed exocysts and the encapsulated trophozoids separated by an intra-cystic space, a dense poorly vesiculated cytoplasma and polygonal surface areas of cysts. The cysts walls were composed of chitin. Cysts were conspicuously smaller compared to trophozoids and lacked pseudo- and filipodia. We did not observe multi-nucleated trophozoids after antibiotic induces encystation. Cyst formation in E.g may explain why established treatment approaches often do not stop periodontal tissue destruction during periodontitis and periimplantitis.

microbiology↗

bioRxiv2022

Global Endometrial DNA Multi-omics Analysis Reveals Insights into mQTL Regulation and Associated Endometriosis Disease Risk

Endometriosis is a leading cause of pain and infertility affecting millions of women globally. Identifying biologic and genetic effects on DNA methylation (DNAm) in endometrium increases understanding of mechanisms that influence gene regulation predisposing to endometriosis and offers an opportunity for novel therapeutic target discovery. Herein, we characterize variation in endometrial DNAm and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genome-wide genotype data and methylation at 759,345 DNAm sites in endometrial samples from 984 deeply-phenotyped participants. We identify significant differences in DNAm profiles between menstrual cycle phases and at four DNAm sites between stage III/IV endometriosis and controls. We estimate that 15.4% of the variation in endometriosis is captured by DNAm, and identify DNAm networks associated with endometriosis. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independent cis-mQTL including some tissue-specific effects. We find significant differences in DNAm profiles between endometriosis sub- phenotypes and a significant association between genetic regulation of methylation in endometrium and disease risk, providing functional evidence for genomic targets contributing to endometriosis risk and pathogenesis.

systems biology↗

bioRxiv2022

Epigenetic variation in the Lombardy poplar along climatic gradients is independent of genetic structure and persists across clonal reproduction

- Environmental changes can trigger phenotypic variation in plants through epigenetic mechanisms, but strong genetic influences make it difficult to isolate and study epigenetic effects. Clonal trees with low genetic variation, such as the Lombardy poplar (Populus nigra cv. Italica Duroi), offer a unique system to study epigenetic variation associated with the environment. - We collected cuttings (ramets) of Lombardy poplar along a wide geographical range in Europe. We performed whole-genome-bisulfite sequencing of 164 ramets grown in a common garden and of a subset of 35 of the original parental individuals. Using historical bioclimatic data, we tested the relationship between DNA methylation and climatic gradients. - We found that average methylation levels in TEs and promoter regions correlate with biologically relevant climatic variables. Furthermore, we observed that DNA methylation was transmitted to the next clonal generation, but a fraction of the methylome changed relatively fast when comparing the parental individuals with the clonal offspring. - Our results suggest that the poplar methylome is a dynamic layer of information that can be transmitted to the clonal offspring and potentially affect how poplars acclimate to new environmental conditions.

ecology↗

bioRxiv2022

High-resolution methylome analysis in the clonal Populus nigra cv. 'Italica' reveals environmentally sensitive hotspots and drought-responsive TE superfamilies

ABSTRACT/SUMMARYO_LIDNA methylation is environment-sensitive and can mediate plant stress responses. In long-lived trees, changing environments might cumulatively shape the methylome landscape over their lifetime. However, because high-resolution methylome studies usually focus on single environments, it remains unclear to what extent the methylation responses are generic or stress-specific, and how this relates to their long-term stability. C_LIO_LIHere, we studied the methylome plasticity of a single poplar genotype, Populus nigra cv. Italica. Adult poplar trees with diverse environmental histories were clonally propagated, and the ramets exposed to experimental cold, heat, drought, herbivory, rust infection and salicylic acid treatments. Then, we identified and compared stress-induced vs. naturally occurring DNA methylation changes using whole genome bisulfite sequencing data. C_LIO_LIMethylation changes mainly targeted transposable elements and when occurring in CG/CHG contexts, the same regions were often affected by multiple stresses, indicating a generic response. Drought triggered a unique CHH hypermethylation response in transposable elements, affecting entire superfamilies and often occurring near drought-responsive genes. Stress-induced methylation variation in CG/CHG contexts showed striking overlap with methylation differences observed between trees from distinct geographical locations. C_LIO_LIAltogether, our results indicate that generic methylome stress responses can persist as epialleles in nature while some environments trigger more transient but large and specific responses, with possible functional consequences. C_LI

genomics↗

bioRxiv2022

Functional precision profiling reveals non-mutational rewiring of kinase signaling networks in colorectal cancer

BackgroundDespite major advances in the development of targeted therapies, precision (immuno)oncology approaches for patients with colorectal cancer continue to lag behind other solid cancers. Functional precision oncology - a strategy that is based on perturbing primary tumor cells from cancer patients with drugs - could provide an alternate road forward to personalize treatment. MethodsWe extend here the functional precision oncology paradigm to measuring phosphoproteome landscapes using patient-derived organoids (PDOs). We first employed steady-state multi-omics (exome sequencing, RNA sequencing, and proteomics) and single-cell characterization of the PDOs. The PDOs were then perturbed with kinase inhibitors (MEKi, PI3Ki, mTORi, TBKi, BRAFi, and TAKi), and large-scale phosphoproteomics profiling using data-independent acquisition was carried out. Further, we used imaging mass-cytometry-based single-cell proteomic profiling of the primary tumors to characterize cellular composition of the tumor-microenvironment (TME) and to quantify heterocellular signaling crosstalk. ResultsWe show that kinase inhibitors induce profound off-target effects resulting in a crosstalk with oncogenic and immune-related pathways. Reconstruction of the topologies of the kinase networks revealed that the patient-specific rewiring of the central EGFR-RAS-MAPK network is unaffected by mutations. Moreover, we show non-genetic heterogeneity of the PDOs and patient- and inhibitor-specific upregulation of stemness and differentiation genes by kinase inhibitors. We complemented our functional profiling by spatial proteomics profiling of the primary tumors using imaging mass cytometry. We quantify spatial heterocellular crosstalk and tumor-immune cell interactions, showing an avoidance of PD1+ immune cells and PD-L1+ tumor cells. ConclusionsCollectively, we provide a multi-modal framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno)-oncology in colorectal cancer.

cancer biology↗

bioRxiv2022

An intragenic FAT1 regulatory element deleted in muscular dystrophy patients drives muscle and mesenchyme expression during development

Fat1 is an atypical cadherin playing multiple roles that influence tissue morphogenesis. During mouse development Fat1 is required to modulate muscle morphogenesis through complementary activities in myogenic cells, muscle-associated connective tissue, and motor neurons, ablation of which leads to regionalized muscle phenotypes. We previously identified copy number variants (CNV) deleting an intragenic conserved non-coding element (CNE) in the human FAT1 locus, that were enriched among muscular dystrophy patients with symptoms resembling those of Facioscapulohumeral Dystrophy (FSHD), compared to healthy individuals. Since such deletions of a putative cis-regulatory element had the potential to cause tissue-specific depletion of FAT1, they were postulated to act as symptom modifiers. However, activity of this CNE has not been functionally explored so far. To investigate the possible regulatory activity of this Fat1-CNE, we engineered transgenic mice in which it drives expression of a bi-cistronic reporter comprising the CRE-recombinase (Cre) and a myristilated-tdTomato fluorescent protein. The tissue-specific pattern of cre and tomato expression indicates that this enhancer has bipotential character, and drives expression in skeletal muscle and in muscle-associated mesenchymal cells. We extended our analysis of one of the transgenic lines, which exhibits enhanced expression in mesenchymal cells at extremities of subsets of muscles matching the map of Fat1-dependent muscles. This transgenic line exhibits highly selective CRE-mediated excision in scattered cells within the Tomato-positive territory hotspots. This represents a novel tool to genetically explore the diversity of muscle-associated mesenchymal lineages.

developmental biology↗

bioRxiv2022

A receptor-independent signaling pathway for BDNF

In addition to its well-known receptor-mediated function in cell survival, differentiation and growth, we report that the extracellular brain-derived neurotrophic factor (BDNF) also controls the intracellular KEAP1-NRF2 cytoprotective system by a receptor-independent pathway. Extracellular BDNF can cross the cell membrane as it possesses a protein-translocation domain, also known as cell-penetrating peptide. This membrane crossing process is energy-independent, ruling out endocytosis and receptor-dependent mechanisms. Once in the cytosol, BDNF binds to KEAP1 with a nanomolar affinity, enabling nuclear translocation of NRF2 and transcription of NRF2-target genes. BDNF is thus a major regulator of NRF2 activation. A dysfunction of this BDNF-KEAP1-NRF2 pathway may be involved in most diseases where antioxidant and cytoprotective functions are altered. This novel form of communication, whereby a receptor ligand protein exerts a biological activity by crossing the cell membrane, opens new avenues for cell signaling.

cell biology↗