Biology subjects

Nowak, J.

Publications and source records attributed to Nowak, J..

16 recordsLinked to original sources

bioRxiv2026

Loss of myosin VI leads to age-dependent changes in the maintenance of heart ventricle mitochondria

Aims We have recently shown that unconventional myosin VI (MVI), a unique actin-based molecular motor, is involved in age-progressive cardiac dysfunction. Here, we aimed at elucidation of mechanisms of MVI involvement in this phenotype. We propose that MVI plays a role as a regulator of mitochondria quality control as well as of their organization and distribution in the heart, an organ with extremely high energy demand. Methods and results We analyzed heart samples from MVI knockout mice (Snell's waltzer) and their heterozygous littermates: newborns (P0), 3-month-old (3m), and 12-month-old (12m) mice. Transmission electron microscopy and immunostaining revealed that loss of MVI affected mitochondria network organization and promoted mitochondria clustering in the perinuclear space. Moreover, accumulation of large functional mitochondria, increase in total mitochondria mass with a concomitant increase in the amount of damaged mitochondria were observed in 12m hearts. The Western blot technique revealed that loss of MVI, at all examined time points, did not affect the activity of AMPK, the key energy balance sensor, and the level of the respiratory chain components. However, in P0 samples it affected the levels of OPA1, involved in mitochondria fusion, and the activity of S6, the downstream effector of mTOR. Also, loss of MVI affected autophagy/mitophagy activity in P0 hearts, leading to accumulation of autophagosomes. A pull-down assay enabled identification of over three hundred potential MVI-binding partners; among them were proteins involved in mitochondria organization and functions, autophagy, ion transport, and cell respiration. Conclusion Taken together, our results indicate that loss of MVI evokes changes disclosed already in newborns that lead to profound alterations in the mitochondria organization and maintenance with age. We believe that accumulation of changes in mitochondria structure, distribution and quality commits to the age-progressing cardiac remodeling and dysfunction.

cell biology↗

bioRxiv2026

Modular Surface Engineering of Feline Parvovirus Virus-Like Particles for Antigen Presentation and Receptor-Mediated Cell Targeting

Virus-like particles (VLPs) are non-infectious, multiprotein nanostructures that mimic the architecture of viruses while lacking genetic material. Their structural versatility and safety profile make them promising platforms for biomedical applications, including vaccine development, targeted drug delivery, and diagnostics. Among various functionalization strategies, click-chemistry has emerged as a precise and efficient method for attaching bioactive molecules to VLP surfaces. In this study, we report the production and functionalization of VLPs derived from the feline panleukopenia virus (FPV), a parvovirus closely related to the well-characterized canine parvovirus (CPV). We demonstrate successful expression, assembly, and purification of FPV VLPs, followed by their modification with fluorophores, peptides, oligonucleotides, and proteins using copper-free click-chemistry. Furthermore, using cryo-EM, we structurally characterize FPV VLP functionalized with antigenic peptides, providing detailed insights for further development of such vaccine prototype. Finally, we demonstrate that our FPV VLP specifically transduce feline and human cells expressing transferrin receptor type 1 (TfR), thus validating their potential as a unique platform for future nanotechnological and therapeutic applications.

bioengineering↗

bioRxiv2025

GLIS3 Marks a Neural-like Progenitor Cell State that Drives Metastasis in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has a high rate of recurrence and metastasis despite intensive therapy. The neural-like progenitor (NRP) transcriptional program is enriched in residual disease after neoadjuvant chemotherapy and radiotherapy; however, the mechanisms for increased NRP expression in the post-treatment setting remain unclear. Here, we find that NRP signatures are strongly enriched in tissue injury and regeneration, and NRP cancer cells co-express transcription factors involved in pancreatic development. We identify both cell autonomous and ligand mediated mechanisms for inducing NRP expression in vitro. We establish and characterize isogenic mouse organoid overexpression models for transcription factors associated with the NRP, classical, and basal-like states. We discovered that Glis3 is a key NRP-associated transcription factor that drives malignant properties including greater clonogenicity, tumor growth, and metastasis compared to isogenic models for the classical and basal-like states. Our work highlights the emergence of clinically relevant developmental regeneration programs in the post-treatment context.

cancer biology↗

bioRxiv2025

Pus7 mutation links tRNA dysregulation to aggressive behavior through activation of the integrated stress response and glycolytic reprogramming

Pseudouridine ({Psi}) is a prevalent RNA modification found in multiple RNA species. It is deposited by {Psi} synthases (Pus) and it can stabilize RNA structures. Patients carrying alterations in the Pus7 gene suffer from developmental delay, intellectual disability, microcephaly, hyperactivity and increased aggression levels. Here we show that the Pus7 mutation in human patient cells and in a Drosophila model is associated with a specific decrease of tRNA:Aspartate (tRNA-Asp) levels, which leads to slow decoding at Aspartate codons. This in turn activates the integrated stress response and induces a metabolic shift towards increased glycolysis and reduced mitochondrial respiration. Elevating tRNA-Asp expression, inhibiting the integrated stress response or dampening the glycolytic pathway is sufficient to rescue the aggressiveness phenotype, demonstrating the involvement of the tRNA-Asp-ISR-glycolysis axis in this behavior. Together our data provide new insights into the molecular defects associated with the loss of Pus7 and suggest potential new avenues for therapeutic treatment.

genetics↗

bioRxiv2024

Glycosaminoglycans act as activators of peptidylarginine deiminase 4

Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that is gathering increasing attention due to its possible involvement in physiological processes as well as in the pathogenesis of diseases like rheumatoid arthritis or thrombosis. PAD4 is activated by calcium ions, but the details of this mechanism are elusive, because in the human body, Ca2+ concentrations are too low for full activity. Given that glycosaminoglycans (GAGs) are also implicated in the development and progression of rheumatoid arthritis, we investigated the activation of PAD4 by GAGs using heparin as a model. We employed activity assays, chromatography techniques, molecular interaction measurements (MST and SPR), FACS, and immunocytochemistry to demonstrate the activation of PAD4 by GAGs. Our data show that PAD4 binds heparin with high affinity and forms high molecular weight complexes with heparin, consistent with heparin-bound tetramer formation. Heparin activates PAD4 by increasing the enzymes Ca2+ affinity threefold. We also show that the effectiveness of activation with heparin depends on the length of GAG used and its negative charge. Direct measurement of heparin binding to PAD4 confirmed tight interaction with nanomolar affinity. Mutagenesis of regions likely responsible for heparin binding showed that dimerization of PAD4 is necessary for efficient activation, but the distinct binding site was not determined as interaction with heparin likely occurs over larger surface of PAD4. Furthermore, we show that other GAG family members, including heparan and chondroitin sulphates, are also able to activate PAD4. We also found that disturbed production of GAGs by CHO cells results in reduced PAD4 binding efficiency. Finally, heparin induces NETosis in hPMNs in concentration-dependent manner, as measured by the release of DNA and citrullination of histone H3. In summary, we identify the first natural coactivator of PAD4, which is present in all individuals, potentially explaining the regulation of PAD4 activity in physiological conditions, and providing new insight into the development of rheumatoid arthritis and other PAD4-related diseases.

biochemistry↗

bioRxiv2024

A model for cortical activity sequences

Networks of neurons in the brain, that act on a timescale of milliseconds, can intrinsically generate reliable sequential activity on slow behavioral timescales of seconds. A possible mechanism for intrinsic sequence generation based on theoretical evidence points to distance-dependent connectivity with correlated spatial asymmetries, establishing an anisotropic network connectivity. We show that networks with such correlated asymmetric connectivity as well as symmetric distance-dependent connectivity match experimental data of connectivity motifs as well as neuronal activity statistics from rat and monkey cortex. At the network level, however, only the correlated asymmetric connectivity pattern generates spatiotemporal activity sequences on behaviorally relevant timescales, while the symmetric connectivity results in transient but stationary spatial bumps of neural activity. Our results strongly support the role of correlated asymmetries in connectivity for the generation of sequential activity in neural networks.

neuroscience↗

bioRxiv2023

The nuclear PIWI-interacting protein Gtsf1 controls the selective degradation of small RNAs in Paramecium

Ciliates undergo developmentally programmed genome elimination, in which small RNAs direct the removal of target DNA segments, including transposable elements. At each sexual generation, the development of the macronucleus (MAC) requires massive and reproducible elimination of a large proportion of the germline micronuclear (MIC) genome, leading to a highly streamlined somatic MAC genome. 25-nt long scnRNAs are produced from the entire germline MIC genome during meiosis, and this initial complex small RNA population is then transported to the maternal MAC, where selection of scnRNAs corresponding to germline (MIC)-specific sequences is thought to take place. Selected scnRNAs, loaded onto the PIWI protein Ptiwi09, guide the deposition of histone H3 post-translational modifications (H3K9me3 and H3K27me3) onto transposable elements in the developing macronucleus, ultimately triggering their specific elimination. How germline-specific MIC scnRNAs are selected remains to be determined. Here, we provide important mechanistic insights into the scnRNA selection pathway by identifying a Paramecium homolog of Gametocyte specific factor 1 (Gtsf1) as essential for the selective degradation of scnRNAs corresponding to retained somatic MAC sequences. Consistently, we also show that Gtsf1 is exclusively localized in the maternal macronucleus and associates with the scnRNA-binding protein Ptiwi09. Furthermore, Gtsf1 is necessary for DNA elimination and correct H3K9me3 and H3K27me3 localization in the new developing macronucleus, demonstrating that the scnRNA selection process is important for genome elimination. We propose that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with nascent RNA transcribed from the maternal MAC genome, similarly to the mechanism suggested for microRNA target-directed degradation in metazoans.

genetics↗

bioRxiv2022

Building an Open Representation for Biological Protocols

Laboratory protocols are critical to biological research and development, yet difficult to communicate and reproduce across projects, investigators, and organizations. While many attempts have been made to address this challenge, there is currently no available protocol representation that is unambiguous enough for precise interpretation and automation, yet simultaneously abstract enough to enable reuse and adaptation. The Protocol Activity Markup Language (PAML) is a free and open protocol representation aiming to address this gap, building on a foundation of UML, Autoprotocol, and SBOL RDF. PAML provides a representation both for protocols and for records of their execution and the resulting data, as well as a framework for exporting from PAML for execution by either humans or laboratory automation. PAML is currently implemented in the form of an RDF knowledge representation, specification document, and Python library, can be exported for execution as either a manual "paper protocol" or Autoprotocol, and is being further developed as an open community effort.

synthetic biology↗

bioRxiv2022

Computational Prediction of Synthetic Circuit Function Across Growth Conditions

A challenge in the design and construction of synthetic genetic circuits is that they will operate within biological systems that have noisy and changing parameter regimes that are largely unmeasurable. The outcome is that these circuits do not operate within design specifications or have a narrow operational envelope in which they can function. This behavior is often observed as a lack of reproducibility in function from day to day or lab to lab. Moreover, this narrow range of operating conditions does not promote reproducible circuit function in deployments where environmental conditions for the chassis are changing, as environmental changes can affect the parameter space in which the circuit is operating. Here we describe a computational method for assessing the robustness of circuit function across broad parameter regions. Previously designed circuits are assessed by this computational method and then circuit performance is measured across multiple growth conditions in budding yeast. The computational predictions are correlated with experimental findings, suggesting that the approach has predictive value for assessing the robustness of a circuit design.

synthetic biology↗

bioRxiv2022

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop

Computational tools addressing various components of design-build-test-learn loops (DBTL) for the construction of synthetic genetic networks exist, but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called DART (Design Assemble Round Trip). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection, and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions.

synthetic biology↗

bioRxiv2022

Highly-Automated, High-Throughput Replication of Yeast-based Logic Circuit Design Assessments

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of S. cerevisiae by Gander, et al. Our experimental campaign used a novel high throughput experimentation framework developed under DARPAs Synergistic Discovery and Design (SD2) program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report, and make some remarks about lessons learned.

synthetic biology↗

bioRxiv2021

A patient-driven clinicogenomic partnership through the Metastatic Prostate Cancer Project

Molecular profiling studies have enabled numerous discoveries for metastatic prostate cancer (MPC), but they have mostly occurred in academic medical institutions focused on select patient populations. We developed the Metastatic Prostate Cancer Project (MPCproject, mpcproject.org), a patient-partnered initiative to empower MPC patients living anywhere in the U.S. and Canada to participate in molecular research and contribute directly to translational discovery. Here we present clinicogenomic results from our partnership with the first 706 MPCproject participants. We found that a patient-centered and remote research strategy enhanced engagement with patients in rural and medically underserved areas. Furthermore, patient-reported data achieved 90% consistency with abstracted health records for therapies and provided a mechanism for patient-partners to share information about their cancer experience not documented in medical records. Among the molecular profiling data from 333 patient-partners (n = 573 samples), whole exome sequencing of 63 tumor samples obtained from hospitals across the U.S. and Canada and 19 plasma cell-free DNA (cfDNA) samples from blood donated remotely recapitulated known findings in MPC and enabled longitudinal study of prostate cancer evolution. Inexpensive ultra-low coverage whole genome sequencing of 318 cfDNA samples from donated blood revealed clinically relevant genomic changes like AR amplification, even in the context of low tumor burden. Collectively, this study illustrates the power of a longitudinal partnership with patients to generate a more representative clinical and molecular understanding of MPC. NoteTo assist our patient-partners and the wider MPC community interpret the results of this study, we have included a glossary of terms in the Supplementary Materials.

cancer biology↗

bioRxiv2020

A device for exploring the full angular excitation space - Can more angular projections improve determination of a molecules 3D-orientation in the presence of noise?

In the past, different methods have been presented to determine the 3D orientation of single molecules in a microscopic set-up by excitation polarization modulation. Using linearly polarized illumination from different directions and thereby measuring different 2D projections enables reconstructing the full 3D orientation. Theoretically, two projections suffice for a full 3D orientation determination if the intensities are properly calibrated. If they are not, a third projection will enable unambiguous orientation measurements. The question arises if three projections already contain the maximum information on the 3D orientation when also considering the limited number of available photons and shot noise in an experiment, or if detecting more projections or even continuously changing the projection direction during a measurement provides more information with an identical number of available photons. To answer this principle question, we constructed a simple device allowing for exploring any projection direction available with a particular microscope objective and tested several different excitation modulation schemes using simulated as well as experimental single molecule data. We found that three different projections in fact already do provide the maximum information also for noisy data. Our results do not indicate a significant improvement in angular precision in comparison to three projections, both when increasing the number of projections and when modulating the projection direction and polarization simultaneously during the measurement. In fluorescence microscopy polarized illumination from different directions enables the determination of the 3D orientation of single molecules by combining the 2D information of different projection directions. Ambiguities that emerge when using only two projections can be eliminated using a third projection. In a systematic study we show that - also considering the limited number of available photons and shot noise in an experiment - three projection directions already contain the maximum information on the 3D orientation. Our results do not indicate a significant improvement in angular precision in comparison to three projections, both when increasing the number of projections and when modulating the projection direction and polarization simultaneously during the measurement.

biophysics↗

bioRxiv2020

Amplitude Analysis of Polarization Modulation Data and 3D-Polarization Demodulation (3D-SPoD)

Super-resolution optical fluctuation imaging (SOFI) is a technique that uses the amplitude of fluorescence correlation data for improved resolution of fluorescence images. Here, we explore if also the amplitude of superresolution by polarisation demodulation (SPoD) data can be used to gain additional information about the underlying structures. Highly organized experimental as well a simulated actin filament data demonstrate a principle information gain from this approach. In addition, we explored theoretically the benefits of analyzing the entire 3D-polarization information instead of only 2D-projections thereof. Due to fundamental principles, the probability of finding parallel orientations is approaching zero in 3D-SPoD in contrast to 2D-approaches. Using the modulation-amplitude based analysis we explored systematically simulated 3D-single molecules data (for which the true structures are known) under different conditions that are typically observed in experiments. We found that this approach can significantly improve the distinction, reconstruction and localization. In addition, these approaches are less sensitive to uncertainties in the knowledge about the true experimental point-spread-function (PSF) used for reconstruction compared to approaches using non-modulated data. Finally, they can effectively remove higher levels of non-modulated back-ground intensity.

biophysics↗

bioRxiv2018

The Therapeutic Antibody Profiler (TAP): Five Computational Developability Guidelines

Therapeutic monoclonal antibodies (mAbs) must not only bind to their target but must also be free from 'developability issues', such as poor stability or high levels of aggregation. While small molecule drug discovery benefits from Lipinski's rule of five to guide the selection of molecules with appropriate biophysical properties, there is currently no in silico analog for antibody design. Here, we model the variable domain structures of a large set of post-Phase I clinical-stage antibody therapeutics (CSTs), and calculate an array of metrics to estimate their typical properties. In each case, we contextualize the CST distribution against a snapshot of the human antibody gene repertoire. We describe guideline values for five metrics thought to be implicated in poor developability: the total length of the Complementarity-Determining Regions (CDRs), the extent and magnitude of surface hydrophobicity, positive charge and negative charge in the CDRs, and asymmetry in the net heavy and light chain surface charges. The guideline cut-offs for each property were derived from the values seen in CSTs, and a flagging system is proposed to identify nonconforming candidates. On two mAb drug discovery sets, we were able to selectively highlight sequences with developability issues. We make available the Therapeutic Antibody Profiler (TAP), an open-source computational tool that builds downloadable homology models of variable domain sequences, tests them against our five developability guidelines, and reports potential sequence liabilities and canonical forms. TAP is freely available at http://opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php.

immunology↗

bioRxiv2018

The SERM/SERD Bazedoxifene Disrupts ESR1 Helix 12 to Overcome Acquired Hormone Resistance in Breast Cancer Cells

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ER) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER+ breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We find BZA possesses improved inhibitory potency against the Y537S and D538G ER mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show that BZAs selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ER mutations.\n\nSignificanceBazedoxifene (BZA) is a potent orally available antiestrogen that is clinically approved for use in hormone replacement therapy (DUAVEE). We explore the efficacy of BZA to inhibit activating somatic mutants of ER that can arise in metastatic breast cancers after prolonged exposure to aromatase inhibitors or tamoxifen therapy. Breast cancer cell line, biophysical, and structural data show that BZA disrupts helix 12 of the ER ligand binding domain to achieve improved potency against Y537S and D538G somatic mutants compared to 4-hydroxytamoxifen.

cancer biology↗