Biology subjects

Zimmermann, N.

Publications and source records attributed to Zimmermann, N..

9 recordsLinked to original sources

bioRxiv2025

Phylogenetic tree inference from single-cell RNA sequencing data

Single-cell RNA sequencing technologies enable the large-scale measurement of gene expression profiles at the individual cell level to assess cellular diversity and function. In oncology, leveraging these single-cell transcriptomic data to reconstruct the phylogenetic relationships among cancer cells can provide insights into tumor evolution, metastasis formation, and the development of treatment resistance. However, phylogenetic inference from single-cell RNA sequencing is challenging due to sparse and noisy data and large dataset sizes. We present a novel tree inference method designed for such data that takes reference and alternative read counts of single-nucleotide variants and reconstructs a phylogenetic tree of the sequenced cells via maximum likelihood using a random-scan greedy search. To overcome local optima in the search, our algorithm alternates between two different tree representations: cell lineage trees, where cells are represented by nodes and mutations are attached to edges, and mutation trees, where mutation nodes encode the mutational events and cells are attached to them. Because a local optimum in one tree space generally does not correspond to a local optimum in the other space, we maximize the likelihood by switching between the two tree spaces until convergence is achieved in both. We demonstrate superior performance of our approach on simulated data with complex clonal architectures compared to existing methods. Furthermore, we show its applicability to cancer single-cell RNA sequencing data, which allows us to link evolutionary trajectories of cells to their gene expression profiles.

genomics↗

bioRxiv2025

Screening of FDA-approved small molecules to discover inhibitors of the Pseudomonas aeruginosa quorum-sensing enzyme, PqsE

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections. Due to its heightened resistance to a broad range of antibiotics, there is great need for new antimicrobial agents that are effective in treating P. aeruginosa infections. One attractive option for exploring new antibiotic targets is the quorum sensing (QS) pathway, as it governs many of the pathogenic behaviors that allow P. aeruginosa to stage and maintain infections. Within the QS pathway, there is a key protein-protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa, also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. These findings encourage drug discovery efforts to identify small molecules that bind in the active site of PqsE and induce a conformational change similar to the ones induced by particular amino acid substitutions in the active site. Here, we performed a fluorescence polarization screen to identify molecules that bind in the active site of PqsE. We screened a library of FDA-approved drugs, with the intention of potentially repurposing a known molecule for the treatment of P. aeruginosa infections. Three molecules were identified, two of which showed inhibitory activity that was consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile, and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in an in vivo enzyme activity assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein-protein interaction via binding in the PqsE active site.

biochemistry↗

bioRxiv2024

PTPN11 Mutation Clonal Hierarchy in Acute Myeloid Leukemia

Mutations in protein tyrosine phosphatase non-receptor type 11 (PTPN11) have been considered late acquired mutations in acute myeloid leukemia (AML) development. To interrogate the ontogeny of PTPN11 mutations, we utilized single-cell DNA sequencing and identified that PTPN11 mutations can occur as initiating events in some AML patients when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The co-driver role of PTPN11 mutations was confirmed in a novel murine model that exhibits an AML phenotype with early expansion of a diverse set of variably differentiated myeloid cells that engrafted into immunodeficient and immunocompetent mice. This immune diversity was reconstituted from early precursor cells when engrafted into immunodeficient mice. Moreover, immune diversity was also observed in the blast component of patient samples with NPM1 and PTPN11 mutations, providing novel antigen targets for immune based approaches in this subset of AML that is resistant to multiple targeted therapies.

cancer biology↗

bioRxiv2024

Pathophysiology of hypereosinophilia-associated heart disease

BackgroundCardiac complications in patients with hypereosinophilia cause significant morbidity and mortality. However, mechanisms of how eosinophilic inflammation causes heart damage are poorly understood. MethodsWe developed a model of hypereosinophilia-associated heart disease by challenging hypereosinophilic mice with peptide from the cardiac myosin heavy chain. Disease outcomes were measured by histology, immunohistochemistry, flow cytometry, and measurement of cells and biomarkers in peripheral blood. Eosinophil dependence was determined by using eosinophil-deficient mice ({Delta}dblGATA). Single cells from heart were subjected to single cell RNA sequencing to assess cell composition, subtypes and expression profiles. ResultsMice challenged with myocarditic and control peptide had peripheral blood leukocytosis, but only those challenged with myocarditic peptide had heart inflammation. Heart tissue was infiltrated by eosinophil-rich inflammatory infiltrates associated with cardiomyocyte damage. Disease penetrance and severity were dependent on the presence of eosinophils. Single cell RNA sequencing showed enrichment of myeloid cells, T-cells and granulocytes (neutrophils and eosinophils) in the myocarditic mice. Macrophages were M2 skewed, and eosinophils had an activated phenotype. Gene enrichment analysis identified several pathways potentially involved in pathophysiology of disease. ConclusionEosinophils are required for heart damage in hypereosinophilia-associated heart disease. Additionally, myeloid cells, granulocytes and T-cell cooperatively or independently participate in the pathogenesis of hypereosinophilia-associated heart disease.

immunology↗

bioRxiv2024

Interaction between ciliary component proteins from Chlamydomonas revealed by CRISPR/CAS9, cryo-electron tomography and mass spectrometry

To understand molecular mechanism of ciliary beating motion, knowledge of location, interaction and dynamics of >400 component proteins are indispensable. While recent progress of structural biology revealed conformation and localization of >100 proteins, we still need to investigate their networking, art of their interaction and assembly mechanism. We applied CRISPR/CAS9 genome editing technique to the green algae Chlamydomonas to engineer a deletion mutant of a ciliary component, FAP263, located at the distal protrusion, and examined it structurally by cryo-electron tomography (cryo-ET) and mass spectrometry (MS). Cryo-ET and atomic model fitting demonstrated that the FAP263 deletion mutant lacks additional components, FAP78, and FAP184. Unassigned density near FAP263 in the cryo-ET map of WT cilia is likely FAP151, as suggested by cross-linking mass spectrometry. Based on the structure, we modeled how these four proteins might form a complex. Furthermore, it was shown that dynein f phosphorylation is inhibited in the FAP263 mutant, indicating an important role of this protein complex for dynein f phosphorylation. Our study demonstrates a novel approach to investigate protein networking inside cilia.

cell biology↗

bioRxiv2022

ATP induced conformational change of axonemal outer dynein arms studied by cryo-electron tomography

Axonemal dyneins in the outer dynein arm (ODA) generate force for ciliary beating. We analyzed three states of ODA during the power stroke cycle using in situ cryo-electron tomography, subtomogram averaging and classification. These states of force generation depict the pre-power stroke, post-power stroke conformations and an intermediate state. Comparison of these conformations to published in vitro atomic structures of cytoplasmic dynein, ODA and Shulin-ODA complex showed that the orientation and position of the dynein head and linker differs. Our analysis shows that all dynein linkers in the in the absence of ATP interact with AAA3/AAA4, indicating interaction to the adjacent B-tubule direct dynein orientation. For the pre-power stroke conformation, we found changes of the tail anchored on the A-tubule. We built pseudo-atomic models from high-resolution structures to generate a best fitting atomic model to the in-situ pre- and post-power stroke ODA, thereby showing that the Shulin-ODA display similar conformation as the active pre-power stroke ODA conformation in the axoneme.

molecular biology↗

bioRxiv2021

The interaction of climate history and evolution impacts alpine biodiversity assembly differently in freshwater and on land

Quaternary climate fluctuations can affect biodiversity assembly through speciation in two non-mutually-exclusive ways: a glacial species pump, where isolation in glacial refugia accelerates allopatric speciation, and adaptive radiation during ice-free periods. Here we detected biogeographic and genetic signatures associated with both mechanisms in the generation of the European Alps biodiversity. Age distributions of endemic and widespread species within aquatic and terrestrial taxa (amphipods, fishes, amphibians, butterflies and flowering plants) revealed that endemic fish evolved only in lakes, are highly sympatric and mainly of Holocene age, consistent with adaptive radiation. Endemic amphipods are ancient, suggesting preglacial radiation with limited range expansion and local Pleistocene survival, perhaps facilitated by a groundwater-dwelling lifestyle. Terrestrial endemics are mostly of Pleistocene age, and are thus more consistent with the glacial species pump. The lack of evidence for Holocene adaptive radiation in the terrestrial biome may be attributable to a faster range expansion of these taxa after glacial retreats, though fewer stable environments may also have contributed to differences between terrestrial areas and lakes. The high proportion of young, endemic species make the Alps vulnerable to climate change, but the mechanisms and consequences of species loss will likely differ between biomes because of their distinct histories.

evolutionary biology↗

bioRxiv2019

Heart Disease in a Mutant Mouse Model of Spontaneous Eosinophilic Myocarditis Maps to Three Highly Significant Loci

BackgroundHeart disease (HD) is the major cause of morbidity and mortality in patients with hypereosinophilic diseases. Due to a lack of adequate animal models, our understanding of the pathophysiology of eosinophil-mediated diseases with heart complications is limited. We have discovered a mouse mutant, now maintained on an A/J inbred background, that spontaneously develops hypereosinophilia in multiple organs. Cellular infiltration into the heart causes an eosinophilic myocarditis, with affected mice of the mutant line (i.e., A/JHD) demonstrating extensive myocardial damage and remodeling that leads to HD and premature death, usually by 15-weeks old. Maintaining the A/JHD line for many generations established that the HD trait was heritable and implied the mode of inheritance was not too complex. Backcross and intercross populations generated from mating A/JHD males with females from four different inbred strains produced recombinant populations with highly variable rates of affected offspring, ranging from none in C57BL/6J intercrosses, to a few mice with HD using 129S1/SvImJ intercrosses and C57BL/6J backcrosses, but nearly 8% of intercrosses and >17% of backcrosses from SJL/J related populations developed HD. Linkage analyses of these SJL/J derived recombinants identified three highly significant loci: a recessive locus mapping to distal chromosome 5 (LOD=4.88; named Emhd1 for eosinophilic myocarditis to heart disease-1); and two dominant variants mapping to chromosome 17, one (Emhd2; LOD=7.51) proximal to the major histocompatibility complex, and a second (Emhd3; LOD=6.89) that includes the major histocompatibility region. Haplotype analysis identified the specific crossovers that defined the Emhd1 (2.65Mb), Emhd2 (8.46Mb) and Emhd3 (14.59Mb) intervals. These results indicate the HD trait in this mutant mouse model of eosinophilic myocarditis is oligogenic with reduced penetrance, due to multiple segregating variants and possibly additional genetic or nongenetic factors. The A/JHD mouse model represents a unique and valuable tool to understand the interplay of causal factors that underlie the pathology of this newly discovered eosinophil-associated disease with cardiac complications.

genetics↗

bioRxiv2019

Highly efficient genome editing in primary bronchial epithelial cells establishes FOXJ1 as essential for ciliation in human airways

The structure and composition of the bronchial epithelium is altered in respiratory diseases such as COPD and asthma, in which goblet cell hyperplasia and reduced numbers of ciliated cells impair mucociliary clearance. We describe a robust genome editing pipeline to interrogate modulators of primary human bronchial epithelial cell (HBEC) differentiation and function. By employing plasmid- and virus-free delivery of CRISPR/Cas9 to human airway basal cells we achieve highly efficient gene inactivation without the need for positive selection. Genome edited cells are differentiated at air liquid interface (ALI) into a pseudo-stratified epithelium. We focus on profiling ciliation using imaging cytometry coupled to confocal microscopy and immunohistochemistry. To our knowledge, this is the first study to describe highly efficient genome editing of ALI cultured primary HBECs. As proof of concept, we establish that inactivation of the gene encoding the transcription factor FOXJ1 in primary human airway basal cells precludes ciliation in ALI differentiated bronchial epithelia.

molecular biology↗