Biology subjects

Ehlers, A.

Publications and source records attributed to Ehlers, A..

4 recordsLinked to original sources

bioRxiv2025

Asymmetrical glycoengineering of monoclonal antibodies: new insights in a-gal immunogenicity

Glycosylation heterogeneity in therapeutic monoclonal antibodies (mAbs) necessitates precise glycoengineering to optimize effector functions, such as those mediated by Fc{gamma}-receptors (Fc{gamma}Rs). Current enzymatic remodeling methods are limited in scope, producing only a narrow spectrum of glycoforms and struggling to achieve complete control over the synthesis of defined asymmetrical structures, which are often critical for enhanced mAb efficacy. We report a multi-step, fully enzymatic platform for the solution-phase synthesis of a comprehensive library of biantennary mAb glycoforms with complete control over the 1,3- and 1,6-mannose arm architecture. The strategy relies on two key catalytic steps: the use of the single domain of the {beta}-N-acetylglucosaminidase StrH (GH20-2) to achieve the regioselective hydrolysis of the GlcNAc residue on the 1,3-mannose arm of the intact antibody, thereby introducing asymmetry; and exploiting the intrinsic, broad acceptor flexibility of GnT-I to reinstall the 1,3-mannose arm GlcNAc even after the 1,6-mannose arm has been selectively extended and terminally capped with Neu5Ac or the -gal epitope. This robust, two-stage enzymatic remodeling methodology, demonstrated on infliximab and cetuximab, unlocks the complete chemical space of biantennary Fc-glycans for comprehensive, functional studies. We employed these well-defined glycovariants to determine the minimum structural requirements for anti--gal IgE binding and systematically evaluate binding affinities toward Fc{gamma}Rs, providing essential insights for the rational design of next-generation immunotherapeutics.

biochemistry↗

bioRxiv2022

Chromosome 8 gain drives poor patient outcome via expression of 4E-BP1 in Ewing sarcoma

Chromosome 8 (chr8) gains are common in cancer. However, their potential contribution to tumor heterogeneity is largely unexplored. Ewing sarcoma (EwS) is characterized by pathognomonic FET::ETS fusions but a general paucity of other recurrent somatic mutations that could explain the observed clinical diversity. In EwS, chr8 gains are the second most common genetic alteration rendering EwS an ideal model to investigate the relevance of chr8 gains in an otherwise silent genomic context. Here, we report that chr8 gain-driven gene expression patterns correlate with poor overall survival of EwS patients. This effect is predominantly mediated by increased expression of the translation initiation factor binding protein 4E-BP1 encoded by EIF4EBP1 on chr8. High EIF4EBP1 expression showed the strongest association with poor patient survival among all chr8-encoded genes and correlated with chr8 gains in EwS tumors. Similar findings were made in numerous entities of The Cancer Genome Atlas (TCGA). Integrated multi-omics profiling uncovered that 4E-BP1 orchestrates a pro-proliferative proteomic network. Consistently, silencing of 4E-BP1 in the EwS model reduced cell proliferation, clonogenicity, spheroidal growth in vitro, and tumorigenesis in vivo. Drug screens and functional assays revealed that high 4E-BP1 expression sensitizes for pharmacological CDK4/6 inhibition in preclinical models. Collectively, we establish chr8 gains and high 4E-BP1 expression as prognostic biomarkers in EwS and demonstrate that their association with patient outcome is primarily mediated by 4E-BP1 orchestrating a pro-proliferative proteomic network sensitizing EwS for CDK4/6 inhibitors. Our data suggest that testing for chr8 gains may improve risk-stratification and therapeutic management in EwS and other cancers.

cancer biology↗

bioRxiv2022

Single-nuclei Transcriptome of Human Adipose Tissue Reveals Metabolically Distinct Depot-Specific Adipose Progenitor Subpopulations

Single-cell and single-nuclei RNA sequencing data (scRNAseq and snRNAseq, respectively) have revealed substantial heterogeneity in the AT (AT) cellular landscape in rodents and humans depending on depot and disease status. We used snRNAseq to characterize the cellular landscape of human visceral (VAT) and subcutaneous AT (SAT) samples from lean subjects and subjects with obesity. We identified multiple cell types in the AT cellular repertoire, including three major AT stromal cell (ASC) subpopulations, multiple types of adipocyte (ADIPO) populations that retain properties similar to ASC, endothelial cell (EC), T-cell, and macrophage (MAC) populations that are in concordance and expand upon other published datasets. ADIP and EC are more prominent in SAT compared to VAT which has a higher proportion of ASC. Of two dominant ASC subpopulations, one (inflammatory-mesothelial, IM- ASC) is present in VAT, but absent in SAT, while the other (fibroadipogenic, FA-ASC) is present in both VAT and SAT. Both populations retain their properties in in vitro culture and have adipogenic capacity with different metabolic features. Informatic and in situ studies support ADIP derived from IM- and FA- ASC are found in human VAT. We also identified a wide range of EC subtypes in human AT with features of lymphatic, venous, and arterial EC, with identification of a PRDM16 expression EC population with features of an EC progenitor. Immune cell populations match recent experimental validation of lipid activated macrophage (LAM) phenotypes, TIM4 macrophages, and a prominent population of MRC1/CD206+ resident AT macrophages with gene expression signatures related to glucocorticoid activation. Overall, our study demonstrates depot-specific cellular diversity in human VAT and SAT in which distinct ASC subpopulations may differently contribute to AT dysfunction in obesity. Also, our results highlight an unprecedented EC heterogeneity suggesting AT EC as highly specialized cells and potentially important regulators of depot-specific functions. METHODS STATEMENTHuman subjects provided informed consent and were enrolled with approval from Institutional Review Boards at the University of Michigan and Veterans Affairs Ann Arbor Healthcare System. Enrollment, consent, and all aspects of human subject research were carried out in accordance with the Belmont Report from the National Research Act of 1974, and the Declaration of Helsinki set forth by the World Medical Association. This manuscript contains no human participants names or other HIPAA identifiers.

molecular biology↗

bioRxiv2020

Effective psychological treatment for PTSD changes the dynamics of specific large-scale brain networks

AO_SCPLOWBSTRACTC_SCPLOWVery little is known about the role of effective cognitive therapy in reversing imbalances in brain activity after trauma. We hypothesised that exaggerated threat perception characteristic of post-traumatic stress disorder (PTSD), and subsequent recovery from this disorder, are underpinned by changes in the dynamics of large-scale brain networks. Here, we use a novel data-driven approach with high temporal precision to find recurring brain networks from fMRI data and estimate when these networks become active during exposure to either trauma reminders or neutral pictures. We found that PTSD patients spend less time in two default mode sub-networks in contrast to trauma-exposed healthy controls, and that PTSD symptom severity correlates positively with time spent in the salience network during exposure to trauma reminders. The former are important for different aspects of self-referential processing and the latter for detection of threat. Importantly, the decreased time in the default mode sub-networks is rebalanced after successful cognitive therapy for PTSD. Our results show that remittance of PTSD through trauma-focused cognitive therapy is associated with the successful reinstatement of a healthy balance in self-referential and threat detection brain networks.

neuroscience↗