- by wang, y., Zhao, Q., Lai, W. K., Yu, H.Single-cell proteomics has emerged as a powerful approach for characterizing cellular heterogeneity. Here, we present an optimized scProteomic workflow that enhances proteome coverage and quantification by refining liquid chromatography (LC) conditions across platforms (both Evosep and nanoElute2). Importantly, our results show that using our optimized LC conditions, even with Bruker timsTOF HT, a machine not designed for scProteomics applications, we achieved solid performance with single cell samples that allows meaningful biological discoveries. First, we compared power to detect differentially-expressed genes/proteins […]
- by Messa, G. M., Liu, P., Napolitano, F., tegner, j., Gao, X., Orlando, V.Circadian metabolism arises from complex, time-dependent interactions across organs, yet experimental characterization in humans remains limited. Current whole-body metabolic models are either too large for dynamic simulation or insufficiently detailed to capture temporal physiology. We developed a multi-tissue human metabolic reconstruction, HGEM1.19+, an extensively curated model for dynamic simulation. We introduced Thermo-Dynamic Flux Balance Analysis (tdFBA), a novel formulation that integrates thermodynamic constraints, organ-specific enzyme capacities, solubility limits, osmotic balance and pH buffering. Murine circadian transcriptomics and metabolomics were integrated […]
- by Stroek, R., Gabriels, M., Winkelhorst, M., van den Broek, M., Pabst, M., Jourdin, L., Daran, J.-M., Bajic, D.Biofilm-based microbial electrosynthesis (MES) is a promising technology that converts CO2 into industrially relevant organic compounds using renewable energy sources. The highest performing MES systems reported to date consist of biofilm-driven microbial communities. However, for a successful deployment of this technology, we still need to overcome key challenges, including the long colonization time of the biocathode and the difficulty of controlling the product profile. To address these challenges, it is crucial to better understand the key microbial components responsible for […]
- by Chen, L., Murillo-de-Ozores, A. R., Park, E., Ou, S.-M., Knepper, M.Vasopressin regulates transcription of the aquaporin-2 gene (Aqp2) in collecting duct principal cells. To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP, a vasopressin analog, these cells exhibited low or undetectable GFP and Aqp2 expression in all cells. dDAVP stimulation (1nM dDAVP for 48hrs) markedly increased both GFP and Aqp2 […]
- by El Kassem, G., Sieber, A., Klinger, B., Uhlitz, F., Steinbrecht, D., van Bentum, M., Hillmer, J., von Schlichting, J., Schaefer, R., Bluethgen, N., Boettcher, M.The MAPK pathway is a central signaling cascade whose dysregulation contributes to numerous diseases. While its upstream regulation is well studied, the mechanisms by which MAPK activation leads to diverse transcriptional outcomes remain incompletely understood. To address this shortcoming, we mapped the target gene sets controlled by 22 RAF-inducible transcription factors using targeted Perturb-seq and integrated these data with time-resolved transcriptional profiling. Network reconstruction revealed a topology dominated by two central hubs, EGR1 and FOS, which co-regulate partially overlapping target […]
- by Eya'ane Meva, F., Qahash, T., Arigana, J., Llinas, M.BackgroundThe development of new drugs against afflictions that disproportionately impact poorly resourced areas around the globe is an expensive endeavor. As cost-effective alternatives, strategic combinations of approved drugs can be used to enhance the efficacy against Plasmodium falciparum. Understanding the metabolic consequences of such combinations is essential for optimizing treatment strategies and delaying drug resistance. MethodsAn integrated metabolomic and pharmacological analysis was performed on P. falciparum exposed to chloroquine (CQ), pyrimethamine (PY), sulfadoxine (SD), and their combinations (SDPY and SDCQ). […]
- by Sahu, K., Davis, H. M., Lu, J., Villalobos, C. A., Heyman, A., Simsek, E., You, L.Pattern formation underlies organization and function across biological systems. A major challenge in understanding and controlling these systems lies in making accurate, photorealistic predictions of emergent patterns that reflect both the governing biophysical rules and the fine-scaled visual features observed experimentally. Such predictions are essential for interpreting experimental observations and for guiding rational design in contexts where patterning governs system behavior, such as microbial consortia, tissue morphogenesis, and spatially structured biomanufacturing. Mechanistic models can reproduce global morphological patterns but lack […]
- by Marquez-Zavala, E., Di Bartolomeu, F., Machado, D.Microbial biotechnology has the potential to address several societal issues through the sustainable production of industrially relevant compounds. Despite decades of successful cases, rational engineering of microbial metabolism is still a complex process due to the fine balance between nutrient supply, allocation of cellular resources, energy demand and redox balancing. In this work, we implemented a text-mining workflow for metabolic engineering and compiled a database of experimentally validated strain design strategies from over 15.000 research articles, which includes information on […]
- by Heydari, T., Bashth, O., Fernandes, J., Sabbineni, B., Aguilar-Hidalgo, D., Chen, J., Shakiba, N., Edelstein-Keshet, L., Zandstra, P. W.Collective behavior is a defining property of multicellular systems, where coordinated outcomes emerge from local cell-cell interactions. Yet the quantitative rules linking single-cell decision-making to tissue-scale organization remain poorly resolved. Here, we develop a quantitative framework that defines an order parameter predicting when initially disordered colonies undergo a transition to ordered fate alignment and when minimal, localized inputs can redirect their collective state. This analysis reveals a distinct control regime in which multicellular assemblies become susceptible to a single engineered […]
- by Masser, S., Lotteritsch, T., Ludwig, A. E., Halwachs, B., Annerer, E., Mussbacher, M., Stelzl, U.Both, protein arginine methyltransferases (PRMTs) and protein kinases are critical regulators of cellular processes and frequently dysregulated in malignancy. To systematically study the crosstalk of the two regulatory enzyme classes, we used parallel yeast two-hybrid matrix screening and defined 45 interactions connecting 4 PRMTs and 20 human kinases. The PRMT-kinase network revealed a strong association between PRMTs and cell cycle/mitogen-activated kinases. Notably, the PRMT5-CDK4 emerged as the most prominent functional cell cycle link through integrative data analyses of different high-throughput […]
- by Do, T. H. L., Lohoff, C., Jung, F., Kummer, S., Pohly, M. F., Scheinost, S., Huellein, J., Benes, V., Huber, W., Lu, J., Zenz, T.Pathway inhibitors are a backbone of cancer treatment. The configuration of pathway dependencies varies from tumour to tumour. Better treatment for individual patients could be designed if the pathway wiring were readily measurable. Here, we characterise the transcriptional responses of 116 lymphoma patient samples exposed to ten drug perturbations. We used factor analysis to decompose individual and shared drug effects, thereby generating a pathway connectivity map of chronic lymphocytic leukemia (CLL). The expression profiles of the major disease subgroups, defined […]
- by Handakumbura, P. P., Rivas Ubach, A., Battu, A., Anthony, W. E., Schultz, K., McClure, R., Varga, T., Jansson, C., Egbert, R.The rhizosphere microbiome directly influences plant health and acclimation to extreme environments, yet plant-microbe interactions in the rhizosphere have proven complex and difficult to study. We present RhizoGrid, a new methodological framework that integrates a 3D-printed pot structure with spatial measurements of root structure, metabollite and taxonomy to detect links between metabolites and microbes along a soil-grown root system. The RhizoGrid identifies microhabitats hidden belowground. Using the food, forage, and bioenergy crop sorghum, we showcase how the RhizoGrid opens new […]
- by Bhatia, H. S., Simons, L. H. A., Kuemmerle, L. B., McCabe, C., Jansen, S., He, Z., Ali, M., Jeridi, D., Todorov, M. I., Hoeher, L., Padmarasu, S., Park, H. E., Thevis, J. F., Bartos, L. M., Singh, I., Ussar, S., Gorgulu, K., Alguel, H., Roberts, K., Bayraktar, O. A., Brendel, M., Theis, F. J., Treutlein, B., Regev, A., Erturk, A.Single-cell transcriptomics has transformed tissue analysis, yet current methods struggle to integrate whole-tissue 3D architecture. Conventional techniques restrict molecular profiling to pre-selected 2D sections, losing systemic context and introducing anatomical bias by sampling less than 0.001% of a whole organism. To overcome these challenges, we developed DISCO-seq, a tissue-clearing chemistry that enables superior RNA accessibility compared to fresh or fixed tissues. DISCO-seq integrates whole-organ or organism 3D imaging with both untargeted and targeted transcriptomics, yielding high-quality RNA from cleared tissues […]
- by Jeanrenaud, A. C. S. N., Arnau Soler, A., Ghauri, A., Marenholz, I., Mertins, P., Beyer, K., Worm, M., Lee, Y.-A.BackgroundHazelnut allergy is a major cause of food-induced anaphylaxis yet remains poorly defined at the molecular level. ObjectiveWe aimed to identify molecular differences between individuals with primary hazelnut allergy and nonallergic controls by investigating a comprehensive spectrum of omics profiles in immune cells. MethodsWe analysed DNA methylation, transcriptomic and proteomic profiles in hazelnut-stimulated and unstimulated immune cells. ResultsAcross analyses, we identified 80 differentially methylated signatures, 125 differentially expressed genes, and 11 differentially secreted proteins associated with hazelnut allergy. DNA methylation […]
- by Mezzanotte, V., Cimatti, M., Burrascano, S., Di Marco, M.Climate change is a major driver of global biodiversity loss, and Europe is no exception with several areas exposed to high climate velocity and/or magnitude. Within the rapidly warming Europe, Italy is facing particularly high risk as part of the Mediterranean region with potentially dramatic consequences for its diverse habitat types. While species-level effects of climate risk are widely investigated, habitat-level exposure to climate change has rarely been assessed. This reduces the comprehensiveness of habitat state assessment under the Habitats […]
- by Thayer, E. A., Shipman, G., Rivera-Cardona, J., Mahajan, T., Teo, Q. W., Paez, J. S., Lederer, J., Chen, J., Wu, N. C., Maslov, S., Brooke, C. B.Type I and III interferons (IFNs) are among the first lines of defense against viral infections, yet they are generally only produced by a tiny fraction of infected cells. Here, we show that variability in tonic cell signaling significantly influences cells ability to produce IFN upon stimulation with the synthetic double-stranded RNA, polyinosinic:polycytidylic acid (pIC). Using single-cell approaches, we found that members of the activator protein (AP)-1 transcription factor were implicated in IFNL1 expression predisposition. This guided us to investigate […]
- by Levin, N., Mohammed, S.To date, collision-induced dissociation and methods based on electron transfer dissociation are considered the standard approaches for the mass spectrometry analysis of N- and O-glycopeptides, respectively, allowing for identification of both peptide and glycan compositions. In recent years, alternative fragmentation methods such as ultraviolet photodissociation (UVPD) and more energetic versions of electron-based techniques (such as electron ionisation dissociation, EID) have been shown to be useful for the analysis of glycopeptides, producing rich information on the glycopeptide structure, including types of […]
- by Andrews, C. J., Costa, E. J., Janer Carattini, G. L., DelRosso, N. V., Fujimori, T., Shimasawa, M., Bintu, L.Dynamic gene expression pulses enable adaptive response to stimuli and can be generated in natural and synthetic systems. Controlling these dynamics typically involves circuits consisting of multiple genes and transcription factors (TFs). Here, we discover a new class of bifunctional transcriptional effector domains that can first activate and subsequently repress the same gene, producing dynamic gene expression pulses from a single input. These pulse dynamics arise from distinct, temporally separated chromatin states defined by active and repressive chromatin modifications. The […]
- by Novoa, J., Pazos, F., Chagoyen, M.Despite their low individual prevalence, rare diseases collectively pose a significant health burden, affecting millions of people worldwide. These conditions often result from single-gene mutations, yet the cellular contexts in which these alterations act remain largely unknown–information crucial for improving diagnosis and treatment. As patient-derived samples are scarce, we use single-cell RNA sequencing (scRNA-seq) data from non-diseased tissues to identify relevant cell populations. We introduce Cell4Rare, a computational framework that integrates these healthy scRNA-seq datasets with known phenotype-associated genes to […]
- by Canalejo, F. J., Nogales, J.Beyond the heterologous expression of genes to generate microbes with novel properties, evolutionary engineering offers a complementary approach by exploiting adaptive processes to refine and expand cellular functionalities in biotechnology. A promising source for the generation of novel metabolic functions is the so-called underground metabolism, i.e., the subnetwork conformed by catalytically inefficient promiscuous enzymatic activities without an apparent physiological role. In this work, the potential of this underground metabolism as a source of novel phenotypes has been assessed in the […]
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