Biology subjects

Schubert, K.

Publications and source records attributed to Schubert, K..

9 recordsLinked to original sources

bioRxiv2026

From Lab Notes to Linked Data: MeSyTo for Ontology-Driven Metadata in Toxicological Omics

Toxicological omics studies require comprehensive metadata to support reproducibility, interoperability, and regulatory reuse. However, metadata requirements differ across public repositories, reporting frameworks, and laboratory workflows, resulting in inconsistent annotation and limited data integration. To address this challenge, we developed MeSyTo (Metadata for Systems Toxicology), an ontology-driven framework for harmonizing metadata across toxicological omics. Metadata concepts from public repositories, the OECD Omics Reporting Framework (OORF), community standards, and institutional workflows were semantically aligned and implemented as the MeSyTo Metadata Model (MMM). The MMM serves as the basis for the automatic generation of SHACL validation shapes and framework-specific metadata profiles, while curated value sets are represented as SKOS controlled vocabularies to support metadata collection and validation. The current implementation comprises 105 ontology classes and 527 data properties and supports transcriptomics, proteomics, and metabolomics. A prototype web application demonstrates ontology-driven metadata collection with integrated semantic validation and ontology-based term resolution. The ontology, validation shapes, controlled vocabularies, generation scripts, and software are publicly available as open-source resources. MeSyTo provides a reusable semantic foundation for harmonized, machine-actionable metadata and facilitates repository submission, regulatory reporting, and interoperable data exchange across toxicological omics studies.

bioinformatics↗

bioRxiv2024

Tumor-associated macrophages enhance tumor innervation and spinal cord repair

AO_SCPLOWBSTRACTC_SCPLOWTumor-associated macrophages (TAM) enhance cancer progression by promoting angiogenesis, extracellular matrix (ECM) remodeling, and immune suppression. Nerve infiltration is a hallmark of various cancers and is known to directly contribute to tumor growth. However, the role of TAM in promoting intratumoral nerve growth remains poorly understood. In this study, we demonstrate that TAM expressed a distinct "neural growth" gene signature. TAM actively enhance neural growth within tumors and directly promote neurites outgrowth. We identify secreted phosphoprotein 1 (Spp1) as a key mediator of TAM-driven neural growth activity, which triggers neuronal mTORC2 signaling. Leveraging this new neural growth function, which added to the TAM wound healing properties, we explored TAM potential to repair central nervous system. Adoptive transfer of in vitro-generated TAM in a severe complete-compressive-contusive spinal cord injury (scSCI) model, not only repaired the damaged neural parenchyma by improving tissue oxygenation, ECM remodeling, and dampening chronic inflammation, but also resulted in neural regrowth and partial functional motor recovery. Proteomic analysis and subsequent functional validation confirmed that TAM-induced spinal cord regeneration is mediated through the activation of neural mTORC2 signaling pathways. Collectively, our data unveil a previously unrecognized role of TAM in tumor innervation, neural growth, and neural tissue repair.

neuroscience↗

bioRxiv2024

The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37{degrees}C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence. Author SummaryMany pathogens carry extrachromosomal DNA elements known as plasmids, which encode genes that confer bacterial virulence or antimicrobial resistance (AMR). Acquisition of these plasmids by bacteria can lead to the emergence of new pathogenic traits and the spread of AMR, yet the mechanisms by which plasmids are retained in bacterial populations particularly in the absence of selective pressure remain incompletely understood. Here we show that the major bacterial polyadenylase enzyme PAP I, encoded by the pcnB gene, is critical for the human pathogen Yersinia pseudotuberculosis to maintain its native virulence plasmid as well as AMR plasmids. Very little is known about the process of polyadenylation in bacteria, or the post-transcriptional addition of adenosine residues to the 3 end of transcripts. This study represents the first demonstration that PAP I-mediated polyadenylation contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2023

Effects of benzothiazinone and ethambutol on the integrity of the corynebacterial cell envelope

The mycomembrane (MM) is a hydrophobic layer formed by mycolic acids covering the surface of Mycobacteria and related species. This group includes important pathogens such as Mycobacterium tuberculosis, Corynebacterium diphtheriae, but also the biotechnologically important strain Corynebacterium glutamicum. The MM contributes to the impermeability of the cell envelope and thereby also protects bacteria from antibiotics. This makes biosynthesis of the MM an attractive target for antibiotic intervention. The first line anti-tuberculosis drug ethambutol (EMB) interferes with the synthesis of the arabinogalactan (AG), which is a structural scaffold for covalently attached mycolic acids that form the inner leaflet of the MM. Similarly, the new drug candidate, benzothiazinone 043 (BTZ) affects the synthesis of the AG component of the cell wall. We previously showed that C. glutamicum cells treated with a sublethal concentration of EMB lose the integrity of the MM. In this study we examined the effects of a sublethal concentration of BTZ. Our work shows that 1 {micro}g ml-1 BTZ efficiently blocks the apical growth machinery and reduces cell proliferation, however the integrity of the MM is largely preserved and the effects of {beta}-lactam antibiotics are only additive, not synergistic. Transmission electron microscopy (TEM) analysis revealed a distinct middle layer in the septum of control cells considered to be the inner leaflet of the MM covalently attached to the AG. It functions as a greasy slide for the lateral flow of mycolic acids in the outer leaflet. This layer was not detectable in the septa of BTZ or EMB treated cells, which suggests that the greasy slide is impaired and the confluency of the MM is thereby reduced. In addition, we observed that EMB treated cells have a thicker and less electron dense peptidoglycan (PG) layer consistent with the report that EMB also inhibits the glutamate racemase MurI. We conclude that EMB and BTZ have distinct differences in their modes of action. While EMB and BTZ both effectively block elongation growth, BTZ also strongly reduces septal cell wall synthesis. This renders BTZ treated cells likely more tolerant to antibiotics that act on growing bacteria.

microbiology↗

bioRxiv2023

Universal features of Nsp1-mediated translational shutdown by coronaviruses

Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses - SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV - using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.

biochemistry↗

bioRxiv2021

Release, rescue and recycling: termination of translation in mammalian mitochondria

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single particle cryo-EM as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondria release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present near-atomic models detailing the process of mitochondrial ribosome recycling, to explain how a dedicated elongation factor, mtEFG2, has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process. (134 words)

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2018

A conserved Lkb1 signaling axis modulates TORC2 signaling in budding yeast

Nutrient availability, growth rate and cell size are closely linked. For example, in budding yeast, the rate of cell growth is proportional to nutrient availability, cell size is proportional to growth rate, and growth rate is proportional to cell size. Thus, cells grow slowly in poor nutrients and are nearly half the size of cells growing in rich nutrients. Moreover, large cells grow faster than small cells. A signaling network that surrounds Tor kinase complex 2 (TORC2) plays an important role in enforcing these proportional relationships. Cells that lack components of the TORC2 network fail to modulate their growth rate or size in response to changes in nutrient availability. Here, we show that budding yeast homologs of the Lkb1 tumor suppressor kinase are required for normal modulation of TORC2 signaling and in response to changes in carbon source. Lkb1 kinases activate Snf1/AMPK to initiate transcription of genes required for utilization of poor carbon sources. However, Lkb1 influences TORC2 signaling via a novel pathway that is independent of Snf1/AMPK. Of the three Lkb1 homologs in budding yeast, Elm1 plays the most important role in modulating TORC2. Elm1 activates a pair of related kinases called Gin4 and Hsl1. Previous work found that loss of Gin4 and Hsl1 causes cells to undergo unrestrained growth during a prolonged mitotic arrest, which suggests that play a role in linking cell cycle progression to cell growth. We found that Gin4 and Hsl1 also control the TORC2 network. In addition, Gin4 and Hsl1 are themselves influenced by signals from the TORC2 network, consistent with previous work showing that the TORC2 network constitutes a feedback loop. Together, the data suggest a model in which the TORC2 network sets growth rate in response to carbon source, while also relaying signals via Gin4 and Hsl1 that set the critical amount of growth required for cell cycle progression. This kind of close linkage between control of cell growth and size would suggest a simple mechanistic explanation for the proportional relationship between cell size and growth rate.

cell biology↗

bioRxiv2017

Cell size and growth rate are modulated by TORC2-dependent signals

The size of all cells, from bacteria to vertebrates, is proportional to the growth rate set by nutrient availability, but the underlying mechanisms are unknown. Here, we show that nutrients modulate TORC2 signaling, and that cell size is proportional to TORC2 signaling in budding yeast. The TORC2 network controls production of ceramide lipids, which play roles in signaling. We discovered that ceramide-dependent signals control both growth rate and cell size. Thus, cells that can not make ceramides fail to modulate their growth rate or size in response to changes in nutrients. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is embedded in a feedback loop that controls TORC2 signaling and plays an important role in mechanisms that modulate TORC2 signaling in response to nutrients. Together, the data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals arising from the TORC2 network.

cell biology↗