Biology subjects

Lechner, M.

Publications and source records attributed to Lechner, M..

11 recordsLinked to original sources

bioRxiv2026

Pulsed-SILAC in single mouse embryos reveals early embryonic protein synthesis dynamics and phosphosite regulation

Early embryogenesis relies extensively on maternally deposited products until zygotic genome activation, yet the dynamics for the synthesis of new proteins in mammalian embryos remains poorly characterized. To address this, we applied pulsed stable isotope labelling by amino acids in cell culture (pSILAC) combined with narrow-window data-independent acquisition mass spectrometry to single mouse oocytes and embryos to resolve de novo protein synthesis during early embryogenesis. This revealed that the maternal proteome is not a static reservoir, with components of the subcortical maternal complex and amino acid transporters SLC7A1/2 being actively synthesized during the earliest developmental stages. Furthermore, phosphoproteomic analysis identified hundreds of previously unreported phosphosites and extensive regulation during the oocyte-to-embryo transition. Notably, phosphorylation of the PRC2-interacting KLP motif of EZHIP emerged as a potential regulatory mechanism, with modification of this region reducing EZHIP-PRC2 interaction and coinciding with H3K27me3 remodelling. Together, single embryo pSILAC revealed a maternal proteome that is continuously synthesized, recycled, and post-translationally regulated during early embryogenesis.

cell biology↗

bioRxiv2026

scRNA-seq and genomics analyses reveal key mechanisms of inverted papilloma-associated sinonasal squamous cell carcinoma malignant transformation

Sinonasal inverted papilloma (IP) carries a 10% risk of malignant transformation to IP-associated sinonasal squamous cell carcinoma (IP-SNSCC), yet the molecular and immune drivers of this progression remain poorly defined. This study integrates single-cell RNA sequencing, multiplexed spatial proteomics, whole-exome sequencing, and functional assays across IP and IP-SNSCC cohorts to define mechanisms of malignant transformation. Respiratory epithelial basal cells are identified as the putative cell of origin, with progression marked by recurrent CDKN2A loss and TP53 mutations. Spatial profiling reveals immune reorganization at the lesional interface in IP-SNSCC, characterized by enrichment of alternatively activated M2 macrophages. CXCL14 is shown to directly induce an immunosuppressive myeloid phenotype that suppresses T-cell IFN{gamma} production through an IDO-pathway-dependent mechanism. Integration of these multimodal datasets defines a previously unrecognized CXCL14-IDO mechanism that constrains anti-tumor immunity at the tumor-stroma interface. These findings establish IDO-targeted immunomodulation as a rational adjuvant strategy and provide a comprehensive molecular framework for understanding IP-SNSCC pathogenesis.

genomics↗

bioRxiv2025

Single-cell proteomics of pancreatic islet cells reveals type 1 diabetes and donor-specific features

Type 1 diabetes mellitus (T1DM) is the most common severe chronic disease in children and adolescents and requires life-long exogenous insulin treatment. Investigating key differences between the pancreas of healthy and T1DM patients is key to help uncover new treatments. Here we performed a single-cell proteomic (SCP) analysis of purified pancreatic islets from several healthy and T1DM donors at a depth of > 4000 protein quantified. We employed tools designed for single-cell mRNA sequencing (scRNAseq) for cell type annotation highlighting discrepancies between mRNA and protein markers. Subsequently, we defined a subset of markers useful for future proteomics analyses of pancreatic islets. This marker list was employed to refine the annotations of our combined dataset, which we exploited with a machine learning approach to successfully transfer annotations into a new islet dataset. Lastly, we showcase the applicability and value of SCP in a clinical context by providing novel insights into cell type-specific differences between healthy and T1DM islets as well as between healthy islets of different donors. Our study is the first SCP analysis of pancreatic islets from multiple donors and T1DM islets, performed at this level of analytical depth. This provides methodological and biological insight as well as a reference dataset for future SCP studies and for any researcher interested in T1DM and in pancreatic islet physiology.

biochemistry↗

bioRxiv2025

In vivo CRISPR screening identifies NF1/RASA1/TP53 co-mutations and downstream MEK signaling as a common key mechanism of sinonasal tumorigenesis

Genomic alterations driving tumorigenesis in sinonasal malignancies remain largely unexplored. Here, we perform an in vivo loss-of-function screen using a pooled custom single-guide library delivered to the sinonasal cavity by adeno-associated virus vector to identify cancer driver genes across diverse sinonasal malignancies. This approach yielded sinonasal malignancies with diverse histologies, including sinonasal squamous cell carcinoma, adenocarcinoma, poorly differentiated sinonasal carcinoma, and sinonasal neuroendocrine tumors characteristic of olfactory neuroblastoma. Surprisingly, rather than observing distinct sgRNA profiles across sinonasal tumor subtypes, common recurrent mutations were identified in Nf1 (79%), Rasa1 (74%), and Trp53 (68%) across malignancies with distinct histologies. Utilizing an orthogonal approach, we confirmed that Nf1/Trp53 were required for sinonasal tumorigenesis. Given that loss-of-function in NF1 and RASA1 may lead to increased Ras activity and downstream MEK signaling, we tested small molecule targeting of the RAS-MAPK pathway in sinonasal malignancies. Indeed, both tumor cell lines derived from our loss-of-function approach as well as from human sinonasal malignancies displayed significant sensitivity to MEK inhibition in standard in vitro culture and organoid models. These findings demonstrate that loss of NF1 and RASA1-mediated Ras-GAP activity leads to Ras activation and downstream MEK signaling which is a potential common target throughout major sinonasal tumor subtypes.

cancer biology↗

bioRxiv2024

Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows

This study presents a comparative analysis of three LysC homologues from Achromobacter lyticus, Pseudomonas aeruginosa, and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing a protein aggregation capture (PAC) workflow with HeLa cell lysate, we assessed the enzymes cleavage specificity, digestion efficiency, and performance across various experimental conditions. Results showed that while all three homologues exhbihited high cleavage specificity at lysine residues, A. lyticus LysC outperformed the two others with its superior peptide identification, digestion efficiency, and protein coverage, especially at short digestion times. Combination of A. lyticus LysC and trypsin demonstrated that importance of LysC for signifancanlty minimizing missed cleavage rates in tryptic digests. This study underscores A. lyticus LysCs potential as an optimal choice for enhancing mass spectrometry-based proteomics.

biochemistry↗

bioRxiv2024

Global analysis of protein turnover dynamics in single cells

Even with recent improvements in sample preparation and instrumentation, single-cell proteomics (SCP) analyses mostly measure protein abundances, making the field unidimensional. In this study, we employ a pulsed stable isotope labeling by amino acids in cell culture (SILAC) approach to simultaneously evaluate protein abundance and turnover in single cells (SC-pSILAC). Using state-of-the-art SCP workflow, we demonstrated that two SILAC labels are detectable from [~]4000 proteins in single HeLa cells recapitulating known biology. We investigated drug effects on global and specific protein turnover in single cells and performed a large-scale time-series SC-pSILAC analysis of undirected differentiation of human induced pluripotent stem cells (iPSC) encompassing six sampling times over two months and analyzed >1000 cells. Abundance measurements highlighted cell-specific markers of stem cells and various organ-specific cell types. Protein turnover dynamics highlighted differentiation-specific co-regulation of core members of protein complexes with core histone turnover discriminating dividing and non-dividing cells with potential in stem cell and cancer research. Our study represents the most comprehensive SCP analysis to date, offering new insights into cellular diversity and pioneering functional measurements beyond protein abundance. This method distinguishes SCP from other single-cell omics approaches and enhances its scientific relevance in biological research in a multidimensional manner.

systems biology↗

bioRxiv2023

Using Unstructured Crowd-sourced Data to Evaluate Urban Tolerance of Terrestrial Native Species within a California Mega-City

In response to biodiversity loss and biotic community homogenization in urbanized landscapes, City managers around the world are increasingly working to conserve and increase urban biodiversity. Accordingly, around the world, previously extirpated species are (re)colonizing and otherwise infiltrating urban landscapes, while once abundant species are in various states of decline. Tracking the occurrence of traditionally urban intolerant species and loss of traditionally urban tolerant species should be a management goal of urban areas, but we generally lack tools to study this phenomenon. To address this, we first used species occurrences from iNaturalist, a large collaborative dataset of species observations, to measure an urban association index (UAI) for 967 native animal species that occur in the city of Los Angeles. On average, the occurrence of native species was negatively associated with our composite measure of urban intensity, with the exception of snails and slugs, which instead occur more frequently in areas of increased urban intensity. Next, we assessed 8,348 0.25 x 0.25 mile grids across the City of Los Angeles to determine the average grid-level UAI scores (i.e., a summary of the UAIs present in a grid cell, which we term Community Urban Tolerance Index or CUTI). We found that areas of higher urban intensity host more urban tolerant species, but also that taxonomic groups differ in their aggregate tolerance of urban areas, and that spatial patterns of tolerance vary between groups (e.g., mammals are not the same as birds). The framework established here was designed to be iteratively reevaluated by city managers of Los Angeles in order to track the progress of initiatives to preserve and encourage urban biodiversity, but can be rescaled to sample different regions within the city or different cities altogether to provide a valuable tool for city managers globally.

ecology↗

bioRxiv2023

Immune-Epithelial Dynamics and Tissue Remodeling in Chronically Inflamed Nasal Epithelium via Multi-scaled Transcriptomics

Chronic rhinosinusitis (CRS) is a common inflammatory disease of the sinonasal cavity that affects millions of individuals worldwide. The complex pathophysiology of CRS remains poorly understood, with emerging evidence implicating the orchestration between diverse immune and epithelial cell types in disease progression. We applied single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to both dissociated and intact, freshly isolated sinonasal human tissues to investigate the cellular and molecular heterogeneity of CRS with and without nasal polyp formation compared to non-CRS control samples. Our findings reveal a mechanism for macrophage-eosinophil recruitment into the nasal mucosa, systematic dysregulation of CD4+ and CD8+ T cells, and enrichment of mast cell populations to the upper airway tissues with intricate interactions between mast cells and CD4 T cells. Additionally, we identify immune-epithelial interactions and dysregulation, particularly involving understudied basal progenitor cells and Tuft chemosensory cells. We further describe a distinct basal cell differential trajectory in CRS patients with nasal polyps (NP), and link it to NP formation through immune-epithelial remodeling. By harnessing stringent patient tissue selection and advanced technologies, our study unveils novel aspects of CRS pathophysiology, and sheds light onto both intricate immune and epithelial cell interactions within the disrupted CRS tissue microenvironment and promising targets for therapeutic intervention. These findings expand upon existing knowledge of nasal inflammation and provide a comprehensive resource towards understanding the cellular and molecular mechanisms underlying this uniquely complex disease entity, and beyond.

immunology↗

bioRxiv2022

Notch2 signaling guides B cells away from germinal centers towards marginal zone B cell and plasma cell differentiation

Notch2 signaling has a profound role in driving the development of Marginal Zone B (MZB) cells. We recently demonstrated that Follicular B (FoB) cells act as precursors for MZB cells in mice, but the mechanistic aspects of this differentiation pathway are still elusive. By studying Notch signaling in CBF:H2B-Venus Notch-reporter mice, we show that most B cells receive a Notch signal, which is highest in MZB cells. However, surprisingly, around one-third of MZB cells seem to lose their Notch signal with time. Conditional deletion or constitutive activation of Notch2 in mice upon T-cell-dependent (TD) immunization unraveled an interplay between antigen-induced activation and Notch2 signaling, in which FoB cells that turn off the Notch pathway enter germinal centers, whereas FoB cells with high Notch signals undergo MZB cell or plasmablast differentiation. Input of experimental data into a mathematical modeling framework reveals that MZB cells regularly emerge from antigen-activated FoB cells in a Notch2-dependent manner upon TD immunization.

immunology↗

bioRxiv2020

Tol-Pal system and Rgs proteins interact to promote unipolar growth and cell division in Sinorhizobium meliloti

Sinorhizobium meliloti is an -proteobacterium belonging to the Rhizobiales. Bacteria from this order elongate their cell wall at the new cell pole, generated by cell division. Screening for protein interaction partners of the previously characterized polar growth factors RgsP and RgsM, we identified the inner membrane components of the Tol-Pal system (TolQ and TolR) and novel Rgs (rhizobial growth and septation) proteins with unknown functions. TolQ, Pal and all Rgs proteins, except for RgsE, were indispensable for S. meliloti cell growth. Six of the Rgs proteins, TolQ and Pal localized to the growing cell pole in the cell elongation phase and to the septum in pre-divisional cells, and three Rgs proteins localized to growing cell pole only. The FtsN-like protein RgsS contains a conserved SPOR domain and is indispensable at the early stages of cell division. The components of the Tol-Pal system were required at the late stages of cell division. RgsE, a homolog of the Agrobacterium tumefaciens growth pole ring protein GPR, has an important role in maintaining the normal growth rate and rod cell shape. RgsD is a novel periplasmic protein with the ability to bind peptidoglycan. Analysis of the phylogenetic distribution of novel Rgs proteins showed that they are conserved in Rhizobiales and mostly absent from other -proteobacterial orders, suggesting a conserved role of these proteins in polar growth. IMPORTANCEBacterial cell proliferation involves cell growth and septum formation followed by cell division. For cell growth, bacteria have evolved different complex mechanisms. The most prevalent growth mode of rod shaped bacteria is cell elongation by incorporating new peptidoglycan in a dispersed manner along the sidewall. A small share of rod-shaped bacteria, including the -proteobacterial Rhizobiales, grow unipolarly. Here, we identified and initially characterized a set of Rgs (rhizobial growth and septation) proteins, which are involved in cell division and unipolar growth of Sinorhizobium meliloti and highly conserved in Rhizobiales. Our data expand the knowledge of components of the polarly localized machinery driving cell wall growth and suggest a complex of Rgs proteins with components of the divisome, differing in composition between the polar cell elongation zone and the septum.

microbiology↗

bioRxiv2018

Pan-cancer deconvolution of cellular composition identifies molecular correlates of antitumour immunity and checkpoint blockade response

The nature and extent of immune cell infiltration into solid tumours are key determinants of therapeutic response. Here, using a novel DNA methylation-based approach to tumour cell fraction deconvolution, we report the integrated analysis of tumour composition and genomics across a wide spectrum of solid cancers. Initially studying head and neck squamous cell carcinoma, we identify two distinct tumour subgroups: immune hot and immune cold, which display differing prognosis, mutation burden, cytokine signalling, cytolytic activity, and oncogenic driver events. We demonstrate the existence of such tumour subgroups pan-cancer, link clonal-neoantigen burden to hot tumours, and show that transcriptional signatures of hot tumours are selectively engaged in immunotherapy responders. We also find that treatment-naive hot tumours are markedly enriched for known immune-resistance genomic alterations and define a catalogue of novel and known mediators of active antitumour immunity, deriving biomarkers and potential targets for precision immunotherapy.

cancer biology↗