Biology subjects

Zimmermann, S.

Publications and source records attributed to Zimmermann, S..

12 recordsLinked to original sources

bioRxiv2026

ERECTA signaling controls the timing of Arabidopsis Guard Cell maturation at the embryonic leaf tip

While cell identities are established early during embryogenesis, these cells remain immature until germination, and the mechanisms enforcing this developmental pause are poorly understood. Embryonic stomatal cells provide a model to study this pause as the stomatal transcription factor FAMA, normally sufficient for Guard Cell maturation in seedlings, can not drive maturation in the Arabidopsis embryo. Here we show that FAMAs ability to drive maturation depends on leaf polarity and adaxial stomatal cells can progress further in their lineage. We next find that ERECTA-family receptor signaling, which controls stomatal patterning, also suppresses embryonic stomatal maturation. In er erl1 erl2 mutants, cell pairs at the cotyledon tip acquire characteristics of maturing guard cells: cell wall reinforcement, pore-associated thickening, and expression of late lineage markers as identified by whole embryo transcriptomics. This precocious maturation however remains incomplete: many GC markers remain absent, and cells lack an open pore and mature vacuoles. Genetic analysis shows that partial maturation requires but is not limited by low levels of FAMA. Restriction of maturation to the cotyledon tip correlates with locally elevated ERECTA-family receptor abundance, while high auxin appears dispensable for this. Finally, we show that EPFL-ER signaling mediates leaf tip Guard Cell size postembryonically as well. Altogether, we identify ERECTA signaling as a local brake on embryonic stomatal cell maturation, discovering another way to push precocious stomatal cell maturation that results in a complex, partially mature cell state that provide insights into the limitations on cell embryonic cell maturation.

plant biology↗

bioRxiv2025

Impact of Mutations Affecting 4'-Fluorouridine Susceptibility on Fitness and Treatment Outcomes for Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic alphavirus. Members of the Alphavirus genus are found across the globe, transmitted by arthropod vectors, and cause significant disease burdens in humans and animals. There are currently no FDA-approved antivirals against any member of the Alphavirus genus. While a vaccine exists against chikungunya virus (CHIKV), a member of the arthitogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). 4-Fluorouridine (4-FlU, EIDD-2749) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. 4-FlU can potently inhibit VEEV-TC83 replication, with submicromolar potency in cell culture. However, the emergence of antiviral resistance represents a hurdle for antiviral drug development and the implementation of effective treatment strategies. Here, we have identified novel mutations in the VEEV nsP4 RNA-dependent RNA polymerase that reduce susceptibility to 4-FlU, including P187A, Q191L, L289F, and T296I. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of these mutations on fitness and pathogenicity. In addition, we assessed the impact of mutations reducing sensitivity to 4-FlU in a mouse model. Although mutations against 4-FlU arise quickly in vitro, treatment can still alleviate severe disease and lethal encephalitis. Together, these data highlight the promising therapeutic potential of 4-FlU for the treatment of alphavirus encephalitis. ImportanceVEEV is one of several mosquito-spread viruses that can cause serious brain infections in people and animals. Unlike CHIKV, against which an approved vaccine exists, there are no countermeasures to prevent or treat VEEV infections or block its close relatives, EEEV and WEEV. 4-FlU inhibits many different RNA viruses, including VEEV, and is currently being developed to treat multiple viral infections. A major problem with small-molecule antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we identified mutations in VEEVs RNA-dependent RNA polymerase (nsP4) that confer reduced susceptibility to 4-FlU. We then engineered these mutations into full-length infectious clones and assessed if viruses encoding for these mutations were still pathogenic. In addition, we treated infected mice with 4-FlU and measured how well the compound inhibited virus replication and prevented severe disease, even when mice were infected with viruses harboring mutations that reduced susceptibility to 4-FlU. Although VEEV can develop moderate resistance to 4-FlU in vitro, administration of 4-FlU still reduced severe disease and prevented lethality in the animals infected with viruses that possess mutations that decrease susceptibility to 4-FlU. These results suggest that 4-FlU has strong potential as a future treatment for alphavirus infections like VEEV encephalitis.

microbiology↗

bioRxiv2025

Timing of independence is explained by movement ability, but depends on how independence is defined for a long-lived raptor

Many young animals must gain independence from parental care by acquiring the necessary skills for survival. Thus, the rate of skill acquisition can predict how long juveniles remain with their parents before emigrating from the natal territory. Yet in some systems, young animals acquire adult-like skills and the capacity to be independent long before they emigrate. This raises the question of why the timing of emigration decouples from the rate of skill acquisition, resulting in extended parental care. We addressed this by leveraging GPS tracking data from juvenile golden eagles (Aquila chrysaetos), which learn to fly and to hunt while remaining with their parents for 61-283 days after fledging. Young golden eagles often go on excursions without their parents outside of their natal territories before emigration. We expected flight performance to predict the timing of excursions better than the timing of emigration, which should be more influenced by external factors. In a set of time-to-event models, we predicted the rate of excursions and of emigration using metrics of soaring flight performance or of daily movement. Contrary to our prediction, we found that soaring performance failed to predict the rate of either excursions or emigration, whereas daily movement predicted both rates, but inconsistently. This was likely because those coarser metrics captured motivation and weather in addition to flight skills. Thus external factors seem more likely to explain the timing of independence and internal development--in this case of flight skills--may be completed quickly. Juveniles may delay emigration not due to lack of skill, but because the costs of leaving parental care outweigh the benefits. The difference we discovered between when individuals were capable of independence and when they committed to it may obscure the more general link between skill acquisition and the end of parental care in other systems as well.

animal behavior and cognition↗

bioRxiv2024

When monkeys meet an ANYmal robot in the wild

Animal-robot interaction studies have been of increasing interest in research, but most of these studies have involved robots interacting with insects, birds, and frogs in laboratory settings. To date, only two studies used non-human primates and no behavioral study has tested the social integration of a robot in a group of wild primates. To fill this gap, we studied the interactions between the quadruped ANYmal robot and a group of 37 wild vervet monkeys in South Africa. The ANYmal robot is a remote-controlled sheep-sized robot with an open box of food on its back. We gradually introduced the robot to the monkeys following five different steps over 6 days for a total exposition time of about 10h. The monkeys habituated to the robot very quickly with six individuals eating the food in the robots box from the second day. A few individuals, mostly juveniles, emitted alarm calls towards the robot. In total, seven individuals, high rankers, spent time near the robot and 21 monkeys approached the robot from a greater distance. High rankers displayed significantly more vigilant and self-centered behaviors and they, with females and juveniles, ate more food in the robots box compared to low rankers, males, and adults conversely. This study offers exciting perspectives on the phenomena of social acceptance of machines in mammalian societies and the automation of field data collection.

animal behavior and cognition↗

bioRxiv2023

Mass lysis of bacterial predators drives the enrichment of antibiotic resistance in soil microbial communities

While studies on anthropogenic activities and antibiotic resistance are numerous, the impact of microbial interactions on resistance in complex communities remains uncertain. Here we demonstrate a correlation between the presence of Myxococcus xanthus in natural soil communities and the abundance of antibiotic-resistant bacteria. Further, introducing M. xanthus isolates also enriches antibiotic resistance. This is due to the mass lysis of M. xanthus cells, which results in a toxic environment that fosters the proliferation of pre-existing resistant bacteria rather than de novo resistance evolution. Metagenomic analysis revealed that this enrichment is not limited to the tested antibiotics in culture-based methods, indicating its broader relevance. Crucially, these findings go beyond laboratory settings, showing M. xanthus introduction enriches resistant isolates in natural soil communities. Finally, we demonstrate that the mass lysis of M. xanthus cells during starvation-induced development--key aspect of the lifecycle of M. xanthus--also results in the enrichment of antibiotic resistance in soil communities. Together, we demonstrate how life-history traits in bacterial predators, like M. xanthus, significantly impact antibiotic resistomes in nature. This study also highlights the complex dynamics at play in the evolution and maintenance of antibiotic resistance, emphasizing the role of interspecies interactions in shaping antibiotic resistance profiles.

evolutionary biology↗

bioRxiv2023

Tumor-reactive clonotype dynamics underlying clinical response to TIL therapy in melanoma

The profiles, specificity and dynamics of tumor-specific clonotypes that are associated with clinical response to adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) remain unclear. Using single-cell RNA/TCR-sequencing, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients treated with TIL-ACT. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, which were more effectively mobilized upon in vitro expansion, yielding products with higher numbers of tumor-specific CD8+ cells, which also preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes, and with cell products mostly composed of blood-borne clonotypes mainly persisting in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost the specific signatures of states originally exhibited in tumors, including exhaustion, and in responders acquired an intermediate exhausted effector state after tumor engraftment, revealing important functional cell reinvigoration.

cancer biology↗

bioRxiv2022

Tumor Microenvironment Cellular Crosstalk Predicts Response to Adoptive TIL Therapy in Melanoma

Adoptive cell therapy (ACT) using ex vivo expanded tumor-infiltrating T lymphocytes (TILs) can mediate responses in metastatic melanoma, but long-term efficacy remains limited to a fraction of patients. Here we interrogated tumor-microenvironment (TME) cellular states and interactions of longitudinal samples from 13 metastatic melanoma patients treated with TIL-ACT in our clinical study (NCT03475134). We performed single-cell RNA-seq and spatial proteomic analyses in pre- and post-ACT tumor tissues and showed that responders exhibited higher tumor cell-intrinsic immunogenicity. Also, endogenous CD8+ TILs and myeloid cells of responders were characterized by increased cytotoxicity, exhaustion and costimulation and type-I IFN signaling, respectively. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders have rich baseline intratumoral and stromal tumor-reactive T-cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study reveals CD8+ T-cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials. One-Sentence SummaryResponse to adoptive TIL therapy in melanoma is determined by CD8+ TIL-myeloid cell networks

immunology↗

bioRxiv2022

A drop-on-demand bioprinting approach to spatially arrange multiple cell types and monitor their cell-cell interactions towards vascularization based on endothelial cells and mesenchymal stem cells

Spheroids, organoids, or highly-dense cell-laden droplets are often used as building blocks for bioprinting, but so far little is known about the spatio-temporal cellular interactions post printing. We present a drop-on-demand approach to study the biological interactions of such building blocks in micrometer dimensions. Droplets (containing approximately 700 cells in 10 nl) of multiple cell types are patterned in a 3D hydrogel matrix with a precision of less than 70 m. It is applied to investigate interactions of cell types relevant for vascularization approaches. We show that a gap of 200 m between droplets containing endothelial cells (HUVECs) and adipose-derived mesenchymal stem cells (ASCs) leads to decreased sprouting of HUVECs towards ASCs and increased growth from ASCs towards HUVECs. For mixed aggregates containing both cell types, cellular interconnections of ASCs with up to approximately 0.8 millimeter length and inhibition of HUVEC sprouting are observed. When ASCs are differentiated into smooth muscle cells (SMCs), HUVECs display decreased sprouting towards SMCs in separate aggregates, whereas no cellular interconnections or inhibition of HUVEC sprouting are detected for mixed aggregates. These findings demonstrate that this approach acts as a new tool to investigate cell-cell interactions of different cell types in 3D bioprinted constructs.

bioengineering↗

bioRxiv2022

Integrated workflow of down-scaled Smart-seq2 and absolute quantitative, single-cell digital PCR validates lack of representability of low-abundant transcripts

Single-cell RNA sequencing (scRNA-seq) can unmask transcriptional heterogeneity facilitating the detection of rare subpopulations at unprecedented resolution. In response to challenges related to coverage and quantity of transcriptome analysis, the lack of unbiased and absolutely quantitative validation methods hampers further improvements. Digital PCR (dPCR) represents such a method as we could show that the inherent partitioning enhances molecular detections by increasing effective mRNA concentrations. We developed a scRT-ddPCR method and validated it using two breast cancer cell lines, MCF7 and BT-474, and bulk methods. ErbB2, a low-abundant transcript in MCF7 cells, suffers from dropouts in scRNA-seq and thus calculated fold changes are biased. Using our scRT-ddPCR, we could improve the detection of ErbB2 and based on the absolute counts obtained we could validate the scRNA-seq fold change. We think this workflow is a valuable addition to the single-cell transcriptomic research toolbox and could even become a new standard in fold change validation because of its reliability, ease of use and increased sensitivity.

molecular biology↗

bioRxiv2021

Reversal of the renal hyperglycemic memory by targeting sustained tubular p21 expression

A major therapeutic obstacle in diabetes mellitus is the metabolic or hyperglycemic memory: the persistence of impaired organ function despite improvement of blood glucose. Therapies reversing the hyperglycemic memory and thus improving already established organ-dysfunction are lacking, but urgently needed considering the increasing prevalence of diabetes mellitus worldwide. Here we show that glucose-mediated changes in gene expression largely persist in diabetic kidney disease (DKD) despite reversing hyperglycemia. The senescence-associated cyclin-dependent kinase inhibitor p21 (Cdkn1a) was the top hit among genes persistently induced by hyperglycemia and was associated with sustained induction of the p53-p21 pathway. Persistent p21 induction was confirmed in various animal models, in several independent human samples and in in vitro models. Tubular p21 expression and urinary p21-levels were associated with DKD severity and remained elevated despite improved blood glucose levels in humans, suggesting that p21 may be a biomarker indicating persistent ("memorized") kidney damage. Glucose-mediated p21 induction and tubular senescence were enhanced in mice with reduced levels of the disease resolving protease activated protein C (aPC). Mechanistically, glucose-induced and sustained tubular p21 expression is linked with demethylation of its promoter and reduced DNMT1 expression. aPC reverses already established p21 expression independent of its anticoagulant function through receptor signaling. Accordingly, new pharmacological approaches specifically mimicking aPC signaling (3K3A-aPC, parmodulin-2) enabled the reversal of glucose-mediated sustained tubular p21 expression, tubular senescence, and DKD. Thus, p21-dependent tubular senescence contributes to the hyperglycemic memory but can be therapeutically targeted. Single sentence summaryaPC signaling targets persistent p21 expression and tubular senescence and reverses the hyperglycemic memory in diabetic kidney disease.

molecular biology↗

bioRxiv2020

Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion, creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (surveyor assay), mRNA rtPCR assessing altered gene expression and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing altered bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process. Author summaryI completed my undergraduate degree in biochemistry at the University of Ulm and finished my master's degree in pharmaceutical biotechnology at the University of Ulm and University of applied science of Biberach with a focus on biotechnology, toxicology and molecular biology. For my master thesis, I went to the University of Freiburg to the department of microsystems engineering, where I developed a novel workflow for cell line development. I stayed at the institute for my doctorate, but changed my scientific focus to the development of the emulsion coupling technology, which is a powerful tool for the quantitative and highly parallel measurement of protein and protein interactions. I am generally interested in being involved in the development of innovative molecular biological methods that can be used to gain new insights about biological issues. I am particularly curious to unravel the complex and often poorly understood protein interaction pathways that are the cornerstone of understanding cellular functionality and are a fundamental necessity to describe life mechanistically.

cell biology↗

bioRxiv2017

ALPK1 And TIFA Dependent Innate Immune Response Triggered By The Helicobacter Pylori Type IV Secretion System

Activation of transcription factor NF-{kappa}B is a hallmark of infection with the gastric pathogen Helicobacter pylori and associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous hits involved in H. pylori-, but not IL-1{beta}- and TNF-- dependent NF-{kappa}B regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry and mutant H. pylori strains, identified the H. pylori metabolite D-glycero-{beta}-D-manno-heptose 1,7-bisphosphate ({beta}HBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-{kappa}B activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-{kappa}B activation, TIFA phosphorylation as well as TIFAsome formation depended on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as core of a novel innate immune pathway. ALPK1-TIFA-mediated NF-{kappa}B activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogens T4SS.

molecular biology↗