Biology subjects

Frey, K.

Publications and source records attributed to Frey, K..

8 recordsLinked to original sources

bioRxiv2025

The developing tendon and enthesis are hypoxic and rely on hypoxia-inducible factor 1a (Hif1a) during postnatal development

The tendon-bone enthesis is a specialized fibrocartilaginous tissue crucial for muscle-to-bone force transmission, yet its postnatal development is not fully understood. Emerging evidence suggests hypoxia plays a pivotal role in enthesis maturation akin to its function in skeletal growth plates, with Hypoxia Inducible Factor 1 alpha (HIF-1) acting as a key regulator of cellular adaptation (e.g., cell survival, extracellular matrix (ECM) deposition). Here, we investigated the spatial and temporal dynamics of hypoxia in the murine Achilles tendon enthesis and elucidated the role of Hif1a in enthesis cell survival and ECM formation using Scleraxis-lineage conditional knockout (ScxCre; Hif1a cKO) mice. We found that while neonatal tendons rapidly resolve hypoxia after birth, the enthesis maintains a hypoxic niche through postnatal day 5, mirroring a gradient observed in growth plates. Disruption of HIF-1 in enthesis-resident cells resulted in pronounced deficits in grip strength, abnormal tendon-bone attachment morphology, disrupted calcaneal architecture, impaired mineralization, and significant ECM disorganization. Histological analyses revealed persistent cell death and loss of the characteristic fibrocartilaginous gradient in cKO entheses, including dysregulated collagen alignment. In vitro, HIF-1-deficient tendon fibroblasts exhibited blunted transcriptional responses to hypoxia, altered metabolic gene expression, and changes in ECM deposition. Collectively, our findings illuminate hypoxia as a sustained niche in the postnatal enthesis, with HIF-1 critically required for cell survival, ECM organization, and enthesis structural integrity. This work advances our understanding of enthesis biology and provides insights relevant to tendon-bone attachment disorders and regenerative strategies.

developmental biology↗

bioRxiv2024

L-Asparaginase treatment induces reversible immunoregulatory and immunosuppressive effects in non-malignant B cells in a model of T-cell dependent B cell activation

Metabolic reprogramming is critical for immune cell adaptation upon activation to exert full functionality with amino acids being a key metabolic factor. While L-asparagine is a non- essential amino acid it turns out to be conditionally essential in malignant B cells due to defective asparagine synthetase making L-asparaginase a commonly used chemotherapeutic agent. Off-target enzymatic activity including glutaminolysis impacting crucial immune function. However, its effects on healthy B cells remain unclear, therefore in this study, we explored how L-asparaginase modulates the biology and function of CD40-activated B cells, using an in-vitro model. B cells from healthy donors were treated with increasing L- asparaginase concentrations and analyzed for proliferation, immune phenotype, and metabolic changes. Results showed L-asparaginase reduced B cell proliferation and homotypic clustering without inducing apoptosis, instead impairing metabolic pathways, lowering glycolysis and oxidative phosphorylation, and reducing surface markers associated with antigen-presenting cell (APC) function. Functional assays confirmed that L-asparaginase- treated B cells had diminished ability to activate T cells. Supplementing with asparagine or glutamine restored B cell proliferation and function, with glutamine slightly more effective than asparagine. Interestingly, L-asparaginase induced a regulatory B cell phenotype, marked by CD24+CD38+CD27+ expression and increased interleukin-10 and TGF-beta, suggesting a potential immunosuppressive mechanism. These findings indicate that L-asparaginase not only affects malignant cells but also impacts the function of non-malignant B cells, proposing potential therapeutic applications in B cell-driven autoimmune disorders. Further studies are needed to explore its effects at lower, clinically relevant concentrations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/625617v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1ae8d10org.highwire.dtl.DTLVardef@af405borg.highwire.dtl.DTLVardef@1aa4c0eorg.highwire.dtl.DTLVardef@16750ea_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Mapping the dynamic cell surface interactome of high-density lipoprotein reveals Aminopeptidase N as modulator of its endothelial uptake

Heterogeneous high-density lipoprotein (HDL) particles, which can contain hundreds of proteins, affect human health and disease through dynamic molecular interactions with cell surface proteins. How HDL mediates its long-range signaling functions and interactions with various cell types is largely unknown. Due to the complexity of HDL, we hypothesize that multiple receptors engage with HDL particles resulting in condition-dependent receptor-HDL interaction clusters at the cell surface. Here we used the mass spectrometry-based and light-controlled proximity labeling strategy LUX-MS in a discovery-driven manner to decode HDL-receptor interactions. Surfaceome nanoscale organization analysis of hepatocytes and endothelial cells using LUX-MS revealed that the previously known HDL-binding protein scavenger receptor SCRB1 is embedded in a cell surface protein community, which we term HDL synapse. Modulating the endothelial HDL synapse, composed of 60 proteins, by silencing individual members showed that the HDL synapse can be assembled in the absence of SCRB1 and that the members are interlinked. The aminopeptidase AMPN (also known as CD13) was identified as an HDL synapse member that directly influences HDL uptake into the primary human aortic endothelial cells (HAECs). Our data indicate that preformed cell surface residing protein complexes modulate HDL function and suggest new theragnostic opportunities.

systems biology↗

bioRxiv2022

Decoding functional high-density lipoprotein particle surfaceome interactions

High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport in the human body and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDLs journey throughout the body, its function is mediated through interactions with cell surface receptors on different cell types. Using four different cellular model systems HDL is interacting with, we comparatively analyzed their surfaceomes to define the HDL receptome. Surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAEC) revealed the main currently known HDL-receptor scavenger receptor B1 (SCRB1), as well as 154 shared cell surface receptors representing potential HDL interaction receptor candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF modulated surfaceome of HAEC using auto-CSC technology. VEGF-A treatment led to a remodeling of the surfaceome of HAEC cells including the previously reported higher surfaceome abundance of SCRB1 upon treatment. 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using HATRIC-based ligand receptor capturing (HATRIC-LRC) technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK), which we show is directly contributing to endothelial HDL binding and uptake. Subsequent proximity ligation assays (PLA) demonstrate furthermore the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence functional HDL binding and uptake. List of human gene and protein names discussed in the paperABCA1: Phospholipid-transporting ATPase ABCA1 (ABCA1) ABCA7: Phospholipid-transporting ATPase ABCA7 (ABCA7) ABCG1: ATB-binding cassette G1 (ABCG1) ACVR2A: Activin receptor type-2A (AVR2A) APOA1: Apolipoprotein A1 (APOA1) APOB: Apolipoprotein B (APOB) APOC3: Apolipoprotein C-III (APOC3) APOH: Beta-2-glycoprotein 1 (APOH) APOM: Apolipoprotein M (APOM) ATP5MF: Ecto-F1-ATPase (ATPK) CD4: T-cell surface glycoprotein CD4 (CD4) CD14: Monocyte differentiation antigen CD14 (CD14) CD36: platelet glycoprotein 4 (CD36) EGFR: Epidermal growth factor receptor (EGFR) ITGAM: Integrin alpha-M (ITAM) ITGAV: Integrin alpha-V (ITAV) KDR: Vascular endothelial growth factor receptor 2 (VGFR2) KIT: Mast/stem cell growth factor receptor Kit (KIT) MEGF8: Multiple epidermal growth factor-like domains protein 8 (MEGF8) MEGF10: Multiple epidermal growth factor-like domains protein 10 (MEGF10) MERTK: tyrosine-protein kinase Mer (MERTK) MRC2: C-type mannose receptor 2 (MRC2) MSR1: Macrophage scavenger receptor types I and II (MSRE) PCDHAC1: Protocadherin alpha-C1 (PCDC1) PLTP: Phospholipid transfer protein (PLTP) S1PR1: sphingosine 1-phosphate receptor 1 (S1PR1) S1PR2: sphingosine 1-phosphate receptor 2 (S1PR2) S1PR3: sphingosine 1-phosphate receptor 3 (S1PR3) SCARA3: Scavenger receptor class A member 3 (SCAR3) SCARB1: Scavenger receptor B1 (SCRB1) SLC8A3: Sodium/calcium exchanger 2 (NAC2) TF: Transferrin (TRFE) TFRC: Transferrin receptor protein 1 (TFR1) VEGFA: vascular endothelial growth factor A (VEGF-A) Significance statementA molecular understanding of the high-density lipoprotein (HDL) receptome would provide the rational basis for modulating reverse cholesterol transport. Here we used cellular model systems mimicking tissues involved in HDL-trafficking and reverse cholesterol transport to define the HDL receptome. The receptor tyrosine-protein kinase Mer (MERTK) was identified as a novel mediator of HDL binding and uptake in human aortic endothelial cells. Endothelial MERTK resides within the surfaceome proximal to SCRB1 representing a potential co-receptor. The molecular nanoscale organization of the cell-specific surfaceome may influence cellular HDL interactions and functionality.

molecular biology↗

bioRxiv2020

Reference-based QUantification Of gene Dispensability (QUOD)

BackgroundDispensability of genes in a phylogenetic lineage, e.g. a species, genus, or higher-level clade, is gaining relevance as most genome sequencing projects move to a pangenome level. Most analyses classify genes as core genes, which are present in all investigated individual genomes, and dispensable genes, which only occur in a single or a few investigated genomes. The binary classification as core or dispensable is often based on arbitrary cutoffs of presence/absence in the analysed genomes. Even when extended to conditionally dispensable, this concept still requires the assignment of genes to distinct groups. ResultsHere, we present a new method which overcomes this distinct classification by quantifying gene dispensability and present a dedicated tool for reference-based QUantification Of gene Dispensability (QUOD). As a proof of concept, sequence data of 966 Arabidopsis thaliana accessions (Ath-966) were processed to calculate a gene-specific dispensability score for each gene based on normalised coverage in read mappings. We validated this score by comparison of highly conserved Benchmarking Universal Single Copy Orthologs (BUSCOs) to all other genes. The average scores of BUSCOs were significantly lower than the scores of non-BUSCOs. Analysis of variation demonstrated lower variation values between replicates of a single accession than between iteratively, randomly selected accessions from the whole dataset Ath-966. Functional investigations revealed defense and antimicrobial response genes among the genes with high-dispensability scores. ConclusionsInstead of classifying a gene as core or dispensable, QUOD assigns a dispensability score to each gene. Hence, QUOD facilitates the identification of candidate dispensable genes, associated with high dispensability scores, which often underlie lineage-specific adaptation to varying environmental conditions.

bioinformatics↗

bioRxiv2019

Animal, fungi, and plant genome sequences harbour different non-canonical splice sites

Most protein encoding genes in eukaryotes contain introns which are interwoven with exons. After transcription, introns need to be removed in order to generate the final mRNA which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5 end and AG at the 3 end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations which have been known for years. During the last years, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here we expand systematic investigations of non-canonical splice site combinations in plants to all eukaryotes by analysing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences such as a substantially increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts could be one explanation for this observation. In addition, high numbers of GA-AG splice site combinations were observed in Eurytemora affinis and Oikopleura dioica. A variant in one U1 snRNA isoform might allow the recognition of GA as 5 splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3 splice site compared to the 5 splice site across animals, fungi, and plants.

genomics↗

bioRxiv2018

A Chromosome-level Sequence Assembly Reveals the Structure of the Arabidopsis thaliana Nd-1 Genome and its Gene Set

BackgroundIn addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species.\n\nResultsHere we provide a chromosome-arm level assembly of the A. thaliana accession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate a de novo assembly. In addition, we assembled the chondrome and plastome sequences.\n\nConclusionsDetailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. This de novo assembly extends the known proportion of the A. thaliana pan-genome.

genomics↗

bioRxiv2018

Pharmacokinetics, Side Effects, and Anti-Hyperalgesic Efficacy of The Mglu5 Antagonist Fenobam.

Metabotropic glutamate receptor 5 (mGlu5) has been shown to modulate nociception in animals, but no mGlu5 antagonists have been developed commercially as analgesics. The mGlu5 antagonist fenobam [N-(3-chlorophenyl)-N-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] was originally evaluated for development as a non-benzodiazepine anxiolytic. Fenobam is analgesic in numerous mouse pain models, acting exclusively via mGlu5 blockade. Furthermore, fenobam showed no signs of analgesic tolerance with up to two weeks of daily dosing in mice. Analgesic effects of fenobam in humans have not been reported. The purpose of this investigation was to evaluate fenobam pharmacokinetics and analgesic effects in humans. We first evaluated single-dose oral fenobam disposition in a parallel-group dose-escalation study in healthy volunteers. A second investigation tested the analgesic effects of fenobam in an established experimental human pain model of cutaneous sensitization utilizing capsaicin cream and heat, in a double-blind placebo-controlled study. The primary outcome measure was the area of hyperalgesia and allodynia around the area applied with heat/capsaicin. Secondary outcome measures included nociception, measured as pain rating on a visual analog scale, heat-pain detection threshold, and effects on cognition and mood. Fenobam plasma exposures showed considerable inter-individual variability, and were not linear with dose. Fenobam reduced sensitization vs placebo at a single time-point (peak plasma concentration); we found no other difference between fenobam and placebo. Our results suggest highly variable fenobam disposition, and minimal analgesic effects at the dose tested. We suggest that future studies testing analgesic effects of mGlu5 blockade are warranted, but such studies should employ molecules with improved pharmacokinetic profiles.

neuroscience↗