Biology subjects

Werner, T.

Publications and source records attributed to Werner, T..

15 recordsLinked to original sources

bioRxiv2026

Clade III HIPP genes encode plasmodesmata-targeted proteins with pleiotropic functions in regulating plant development.

Heavy metal-associated isoprenylated plant proteins (HIPPs) are encoded by large gene families, which have diversified specifically in vascular plants. Their physiological functions and molecular mode of activity are currently largely unknown. In this study, we characterize a group of phylogenetically closely related genes HIPP32, HIPP33, and HIPP34 in Arabidopsis thaliana, revealing their essential roles in controlling diverse developmental pathways. Through comprehensive genetic analyses, we demonstrate that these genes exhibit partially overlapping pleiotropic functions, influencing multiple aspects of plant growth such as embryogenesis, maintenance of apical meristems, root architecture, shoot branching, leaf morphogenesis and floral organ formation. Transcriptomic profiling of hipp mutants identified significant deregulation in several regulatory pathways involved in plant hormone responses, with a specific impact on auxin signaling processes. Interestingly, we show that the analyzed HIPP proteins localize very specifically to plasmodesmata, suggesting their potential function in regulating intercellular communication in shaping plant development.

plant biology↗

bioRxiv2024

Proteomic profiling of IDH-wildtype Glioblastoma Tissue and Serum uncovers prognostic Subtypes and Marker Candidates

BackgroundIDH-wildtype glioblastoma (GBM) is the most prevalent primary brain cancer with a 5-year survival rate below 10%. Despite combined treatment through extensive resection and radiochemotherapy, nine out of ten patients develop recurrences. The lack of targeted treatment options and reliable diagnostic markers for recurrent tumors remain major challenges. Methods & AimsIn this study, we present the proteomic characterization of tissue and serum from 55 initial GBM tumors and five matching recurrences, which we investigated for proteomic tumor subtypes and proteomic signatures associated with recurrence. ResultsPrimary tumors revealed four distinct subgroups through hierarchical clustering: a neuronal cluster with elevated mature neuron markers, an innate immunity cluster with increased protease expression, a mixed cluster, and a stem-cell cluster. Neurodevelopmental and inflammatory processes were identified as key factors influencing clustering, with proteolytic activity increasing relative to the degree of inflammation. An analysis comprising proteins with lower coverage confirmed and expanded this pattern. Patients in the neuronal cluster exhibited significantly longer survival compared to those in the stem-cell cluster. In a patient-matched differential expression analysis, five recurrent tumors displayed significantly altered protein expression compared to their primary counterparts, emphasizing the proteomic plasticity of recurrent tumors. Investigation of serum proteomes before and after surgery, using a depletion-based protocol, revealed highly patient-specific and stable proteome compositions, despite a notable increase in inflammation markers post-surgery. However, the levels of circulating proteolytic products matched to the proteolytic activity within the tissue and one fragment of proteolysis activated receptor 2 (PAR2) consistently dropped in abundance after removal of inflamed tumors. ConclusionOverall, we describe a large proteomic GBM cohort. We identified distinct tumor subgroups, molecular patterns of recurrence, and matching proteomic patterns in the bloodstream, which may improve risk prediction for recurrent GBM.

cancer biology↗

bioRxiv2024

Proteomic Characterization of Intrahepatic Cholangiocarcinoma Identifies Distinct Subgroups and Proteins Associated with Time-To-Recurrence

Background & AimsIntrahepatic cholangiocarcinoma (ICC) is a poorly understood cancer with dismal survival and high recurrence rates. ICCs are often detected in advanced stages. Surgical resection is the most important first-line treatment but limited to non-advanced cases, whereas chemotherapy provides only a moderate benefit. The proteome biology of ICC has only been scarcely studied and the prognostic value of initial ICCs proteomic features for the time-to-recurrence (TTR) remains unclear. MethodsWe dissected formalin-fixed, paraffin-embedded samples from 80 tumor- and 77 matching adjacent non-malignant (TANM) tissues. All samples were measured via liquid-chromatography mass-spectrometry (LC-MS/MS) in data independent acquisition mode (DIA). ResultsTumor- and TANM tissue showed strongly different biologies and DNA-repair, translation, and matrisomal processes were upregulated in ICC. In a hierarchical clustering analysis, we determined two proteomic subgroups of ICC, which showed significantly diverging TTRs. Cluster 1, which is associated with a beneficial prognosis, was enriched for matrisomal processes and proteolytic processing, while cluster 2 showed increased RNA and protein turnover. In a second, independent Cox proportional hazards model analysis, we identified individual proteins whose expression correlates with TTR distribution. Proteins with a positive hazard ratio were mainly involved in carbon/glucose metabolism and protein turnover. Conversely, proteins associated with a low hazard ratio were mostly linked to the extracellular matrix. Additional proteome profiling of patient-derived xenograft tumor models of ICC successfully distinguished tumor and stromal proteins and provided insights into cell-matrix interactions. ConclusionsWe successfully determine the proteome biology of ICC and present two proteome clusters in ICC patients with significantly different TTR rates and distinct biological motifs. A xenograft model confirmed the importance of tumor-stroma interactions for this cancer.

cancer biology↗

bioRxiv2023

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

evolutionary biology↗

bioRxiv2023

Integration of Thermal Proteome Profiling with phosphoproteomic and transcriptomic data via mechanistic network models decodes the molecular response to PARP inhibition

The deregulation of complex diseases often spans multiple molecular processes. A multimodal functional characterization of these processes can shed light on the disease mechanisms and the effect of drugs. Thermal Proteome Profiling (TPP) is a mass-spectrometry based technique assessing changes in thermal protein stability that can serve as proxies of functional changes of the proteome. These unique insights of TPP can complement those obtained by other omics technologies. Here, we show how TPP can be integrated with phosphoproteomics and transcriptomics in a network-based approach using COSMOS, a framework for causal integration of multi-omics, to provide an integrated view of transcription factors, kinases and proteins with altered thermal stability. This allowed us to recover known mechanistic consequences of PARP inhibition in ovarian cancer cells on cell cycle and DNA damage response in detail and to uncover new insights into drug response mechanisms related to interferon and hippo signaling. We found that TPP complements the other omics data and allowed us to obtain a network model with higher coverage of the main underlying mechanisms. These results illustrate the added value of TPP, and more generally the power of network models to integrate the information provided by different omics technologies. We anticipate that this strategy can be used to broadly integrate functional proteomics with other omics to study complex molecular processes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/553354v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@af952eorg.highwire.dtl.DTLVardef@16b1d1borg.highwire.dtl.DTLVardef@1440787org.highwire.dtl.DTLVardef@14a4227_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2022

An integrated cell barcoding and computational analysis pipeline for scalable analysis of differentiation at single-cell resolution

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping of in vitro cell types to in vivo developmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas of in vitro differentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor protein TMEM88 as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show that TMEM88 is required for differentiation of diverse endoderm and mesoderm cell lineages in vitro and that TMEM88 knockout in vivo results in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology.

genomics↗

bioRxiv2022

The use of non-model Drosophila species to study natural variation in TOR pathway signaling

Nutrition and growth are strongly linked, but not much is known about how nutrition leads to growth. To understand the connection between nutrition through the diet, growth, and proliferation, we need to study the phenotypes resulting from the activation and inhibition of central metabolic pathways. One of the most highly conserved metabolic pathways across eukaryotes is the Target of Rapamycin (TOR) pathway, whose primary role is to detect the availability of nutrients and to either induce or halt cellular growth. Here we used the model organism Drosophila melanogaster (D. mel.) and three non-model Drosophila species with different dietary needs, Drosophila guttifera (D. gut.), Drosophila deflecta (D. def.), and Drosophila tripunctata (D. tri.), to study the effects of dietary amino acid availability on fecundity and longevity. In addition, we inhibited the Target of Rapamycin (TOR) pathway, using rapamycin, to test how the inhibition interplays with the nutritional stimuli in these four fruit fly species. We hypothesized that the inhibition of the TOR pathway would reverse the phenotypes observed under conditions of overfeeding. Our results show that female fecundity increased with higher yeast availability in all four species but decreased in response to TOR inhibition. The longevity data were more varied: most species experienced an increase in median lifespan in both genders with an increase in yeast availability, while the lifespan of D. mel. females decreased. When exposed to the TOR inhibitor rapamycin, the life spans of most species decreased, except for D. tri, while we observed a major reduction in fecundity across all species. The obtained data can benefit future studies on the evolution of metabolism by showing the potential of using non-model species to track changes in metabolism. Particularly, our data show the possibility to use relatively closely related Drosophila species to gain insight on the evolution of TOR signaling.

physiology↗

bioRxiv2021

DrosoPhyla: genomic resources for drosophilid phylogeny and systematics

The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4000 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the groups and genera in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred well-supported group and species trees for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

evolutionary biology↗

bioRxiv2021

Butterfly eyespots evolved via co-option of the antennal gene-regulatory network

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via co-option of the antennal gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster with antennae relative to multiple other tissues. Furthermore, three genes essential for eyespot development (Distal-less (Dll), spalt (sal), and Antennapedia (Antp)) share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots and antennae, and also legs and wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely re-used the ancient antennal GRN, a network previously implicated also in the development of legs and wings.

evolutionary biology↗

bioRxiv2020

Impaired cytotoxic CD8+ T cell response in elderly COVID-19 patients

SARS-CoV-2 infection induces a T cell response that most likely contributes to virus control in COVID-19 patients, but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B, as well as perforin within different effector CD8+ T cell subsets. PD-1 expressing CD8+ T cells also produced cytotoxic molecules during acute infection indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provides valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development. ImportanceCytotoxic T cells are responsible for the elimination of infected cells and are key players for the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group in comparison to younger patients.

immunology↗

bioRxiv2020

The regulation of a pigmentation gene in the formation of complex color patterns in Drosophila abdomens

Changes in cis-regulatory modules (CRMs) that control developmental gene expression patterns have been implicated in the evolution of animal morphology1-6. However, the genetic mechanisms underlying complex morphological traits remain largely unknown. Here we investigated the molecular mechanisms that induce the pigmentation gene yellow (y) in a complex spot and shade pattern on the abdomen of the quinaria group species Drosophila guttifera. We show that the y expression pattern is controlled by only one CRM, which contains a stripe-inducing CRM at its core. We identified several developmental genes that may collectively interact with the CRM to orchestrate the patterning in the pupal abdomen of D. guttifera. We further show that the core CRM is conserved among D. guttifera and the closely related quinaria group species Drosophila deflecta, which displays a similarly spotted abdominal pigment pattern. Our data suggest that besides direct activation of patterns in distinct spots, abdominal spot patterns in Drosophila species may have evolved through partial repression of an ancestral stripe pattern, leaving isolated spots behind. Abdominal pigment patterns of extant quinaria group species support the partial repression hypothesis and further emphasize the modularity of the D. guttifera pattern.

developmental biology↗

bioRxiv2020

From simplicity to complexity: The gain or loss of spot rows underlies the morphological diversity of three Drosophila species

To understand how novel animal patterning emerged, one needs to ask how the development of color patterns has changed among diverging species. Here we examine three species of fruit flies - Drosophila guttifera (D. guttifera), Drosophila palustris (D. palustris), and Drosophila subpalustris (D. subpalustris) - displaying a varying number of abdominal spot rows that were either gained or lost throughout evolutionary time. Through in situ hybridization, we examine the mRNA expression patterns for the pigmentation genes Dopa decarboxylase (Ddc), tan (t), and yellow (y) during pupal development. Our results show that Ddc, t, and y are co-expressed in identical patterns, each foreshadowing the adult abdominal spots in D. guttifera, D. palustris, and D. subpalustris.

developmental biology↗

bioRxiv2018

Proteome thermal stability reflects organ physiology and identifies drug-target engagement in vivo

Studying biological processes at a molecular level and monitoring drug-target interactions is established for simple cell systems but challenging in vivo. We introduce and apply a methodology for proteome-wide thermal stability measurements to characterize organ physiology and activity of many fundamental biological processes across tissues, such as energy metabolism and protein homeostasis. This method, termed tissue thermal proteome profiling (tissue-TPP), also enabled target and off-target identification and occupancy measurements in tissues derived from animals dosed with the non-covalent histone deacetylase inhibitor, panobinostat. Finally, we devised blood-CETSA, a thermal stability-based method to monitor target engagement in whole blood. Our study generates the first proteome-wide map of protein thermal stability in tissue and provides tools that will be of great impact for translational research.

cell biology↗

bioRxiv2018

Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

Nucleotide triphosphates (NTPs) regulate numerous biochemical processes in cells as (co-)substrates, allosteric modulators, biosynthetic precursors, and signaling molecules1-3. Apart from its roles as energy source fueling cellular biochemistry, adenosine triphosphate (ATP), the most abundant NTP in cells, has been reported to affect macromolecular assemblies, such as protein complexes4,5 and membrane-less organelles6-8. Moreover, both ATP and guanosine triphosphate (GTP) have recently been shown to dissolve protein aggregates9. However, system-wide studies to characterize NTP-interactions under conditions approximating the native cellular environment are lacking, which limits our perspective of the diverse physiological roles of NTPs. Here, we have mapped and quantified proteome-wide NTP-interactions by assessing thermal stability and solubility of proteins using mechanically disrupted cells. Our results reveal diverse biological roles of ATP depending on its concentration. We found that ATP specifically interacts with proteins that utilize it as substrate or allosteric modulator at doses lower than 500 M, while it affects protein-protein interactions of protein complexes at mildly higher concentrations (between 1-2 mM). At high concentrations (> 2 mM), ATP modulates the solubility state of a quarter of the insoluble proteome, consisting of positively charged, intrinsically disordered, nucleic acid binding proteins, which are part of membrane-less organelles. The extent of solubilization depends on the localization of proteins to different membrane-less organelles. Furthermore, we uncover that ATP regulates protein-DNA interactions of the Barrier to autointegration factor (BANF1). Our data provides the first quantitative proteome-wide map of ATP affecting protein structure and protein complex stability and solubility, providing unique clues on its role in protein phase transitions.

systems biology↗

bioRxiv2017

Universal Panel Testing of Pancreatic Cancer Cases for Cancer Predisposition

Background and AimsGenes associated with hereditary breast and ovarian cancer (HBOC) and colorectal cancer (CRC) susceptibility have been shown to play a role in pancreatic cancer susceptibility. Germline genetic testing of pancreatic cancer cases could be beneficial for at-risk relatives with pathogenic variants in established HBOC and CRC genes, but it is unclear what proportion of pancreatic cancer cases harbor pathogenic variants in these genes.\n\nMethods66 pancreatic cancer cases, unselected for family history and diagnosed at the Huntsman Cancer Hospital (HCH), were sequenced on a custom 34-gene panel including known HBOC and CRC genes. A second set of 156 unselected HCH pancreatic cancer cases were sequenced on an expanded 59-gene panel (n=95) or with a custom 14-gene clinical panel (n=61). Sequencing data from both sets of pancreatic cancer cases, the pancreatic cancer cases of the Cancer Genome Atlas (TCGA), and an unselected pancreatic cancer screen from the Mayo Clinic were combined in a meta-analysis to estimate the proportion of carriers with pathogenic and variants of uncertain significance.\n\nResultsApproximately 8.9% of unselected pancreatic cancer cases at the HCH carried a variant with potential HBOC or CRC screening recommendations. A meta-analysis of unselected pancreatic cancer cases revealed that approximately 10.5% carry a pathogenic variant or HiP-VUS.\n\nConclusionWith the inclusion of both HBOC and CRC susceptibility genes in a panel test, unselected pancreatic cancer cases have a high enough percentage of carriers to rationalize genetic testing for identification of variants that could be further used in cascade testing of healthy relatives to increase HBOC and CRC surveillance measures.

genetics↗