Jens Wohlmann

• Stein, Johannes; Ericsson, Maria; Nofal, Michel; Magni, Lorenzo; Aufmkolk, Sarah & McMillan, Ryan B. [Vis alle 16 forfattere av denne artikkelen] (2025). Cryosectioning-enhanced super-resolution microscopy for single-protein imaging across cells and tissues. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 122(32). doi: 10.1073/pnas.2504578122. Fulltekst i vitenarkiv Vis sammendrag

  DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) enables nanoscale imaging with virtually unlimited multiplexing and molecular counting. Here, we address challenges, such as variable imaging performance and target accessibility, that can limit its broader applicability. Specifically, we enhance its capacity for robust single-protein imaging and molecular counting by optimizing the integration of total internal reflection fluorescence microscopy with physical sectioning, in particular, Tokuyasu cryosectioning. Our method, tomographic and kinetically enhanced DNA-PAINT (tkPAINT), achieves 3 nm localization precision across diverse samples, enhanced imager binding, and improved cellular integrity. tkPAINT can facilitate molecular counting with DNA-PAINT inside the nucleus, as demonstrated through its quantification of the in situ abundance of RNA Polymerase II in both HeLa cells as well as mouse tissues. Anticipating that tkPAINT could become a versatile tool for the exploration of biomolecular organization and interactions across cells and tissues, we also demonstrate its capacity to support multiplexing, multimodal targeting of proteins and nucleic acids, and three-dimensional (3D) imaging.

• Wohlmann, Jens (2024). Expanding the field of view – a simple approach for interactive visualisation of electron microscopy data. Journal of Cell Science. ISSN 0021-9533. 137(20). doi: 10.1242/jcs.262198. Fulltekst i vitenarkiv Vis sammendrag

  The unparalleled resolving power of electron microscopy is both a blessing and a curse. At 30,000× magnification, 1 µm corresponds to 3 cm in the image and the field of view is only a few micrometres or less, resulting in an inevitable reduction in the spatial data available in an image. Consequently, the gain in resolution is at the cost of loss of the contextual ‘reference space’, which is crucial for understanding the embedded structures of interest. This problem is particularly pronounced in immunoelectron microscopy, where the detection of a gold particle is crucial for the localisation of specific molecules. The common solution of presenting high-magnification and overview images side by side often insufficiently represents the cellular environment. To address these limitations, we propose here an interactive visualization strategy inspired by digital maps and GPS modules which enables seamless transitions between different magnifications by dynamically linking virtual low magnification overview images with primary high-resolution data. By enabling dynamic browsing, it offers the potential for a deeper understanding of cellular landscapes leading to more comprehensive analysis of the primary ultrastructural data.

• Resseguier, Julien Alain Andre; Nguyen-Chi, Mai; Wohlmann, Jens; Rigaudeau, Dimitri; Salinas, Irene & Oehlers, Stefan H. [Vis alle 13 forfattere av denne artikkelen] (2023). Identification of a pharyngeal mucosal lymphoid organ in zebrafish and other teleosts: Tonsils in fish? Science Advances. 9(44), s. 1–18. doi: 10.1126/sciadv.adj0101. Fulltekst i vitenarkiv Vis sammendrag

  The constant exposure of the fish branchial cavity to aquatic pathogens causes local mucosal immune responses to be extremely important for their survival. Here, we used a marker for T lymphocytes/natural killer (NK) cells (ZAP70) and advanced imaging techniques to investigate the lymphoid architecture of the zebrafish branchial cavity. We identified a sub-pharyngeal lymphoid organ, which we tentatively named “Nemausean lymphoid organ” (NELO). NELO is enriched in T/NK cells, plasma/B cells, and antigen-presenting cells embedded in a network of reticulated epithelial cells. The presence of activated T cells and lymphocyte proliferation, but not V(D)J recombination or hematopoiesis, suggests that NELO is a secondary lymphoid organ. In response to infection, NELO displays structural changes including the formation of T/NK cell clusters. NELO and gill lymphoid tissues form a cohesive unit within a large mucosal lymphoid network. Collectively, we reveal an unreported mucosal lymphoid organ reminiscent of mammalian tonsils that evolved in multiple teleost fish families.

• Bhandari, Madhavi; Soria-Carrera, Héctor; Wohlmann, Jens; Dal, Nils-Jørgen; Fuente, Jesús M. de la & Martín-Rapún, Rafael [Vis alle 8 forfattere av denne artikkelen] (2023). Subcellular localization and therapeutic efficacy of polymeric micellar nanoparticles encapsulating bedaquiline for tuberculosis treatment in zebrafish. Biomaterials Science. ISSN 2047-4830. 11(6), s. 2103–2114. doi: 10.1039/d2bm01835g. Fulltekst i vitenarkiv Vis sammendrag

  The combination drug regimens that have long been used to treat tuberculosis (TB), caused by Mycobacterium tuberculosis, are fraught with problems such as frequent administration, long duration of treatment, and harsh adverse effects, leading to the emergence of multidrug resistance. Moreover, there is no effective preventive vaccine against TB infection. In this context, nanoparticles (NPs) have emerged as a potential alternative method for drug delivery. Encapsulating antibiotics in biodegradable NPs has been shown to provide effective therapy and reduced toxicity against M. tuberculosis in different mammalian models, when compared to conventional free drug administration. Here, we evaluate the localization, therapeutic efficacy and toxic effects of polymeric micellar NPs encapsulating a promising but highly hydrophobic and toxic antitubercular drug bedaquiline (BQ) in zebrafish embryos infected with Mycobacterium marinum. Our study shows that the NP formulation of BQ improves survival and reduces bacterial burden in the infected embryos after treatment when compared to its free form. The intravenously injected BQ NPs have short circulation times due to their rapid and efficient uptake into the endothelial cells, as observed by correlative light and electron microscopy (CLEM).

• Dal, Nils-Jørgen; Schäfer, Gabriela; Thompson, Andrew; Schmitt, Sascha; Redinger, Natalja & Rodriguez, Noelia Alonso [Vis alle 16 forfattere av denne artikkelen] (2023). Π-Π interactions stabilize PeptoMicelle-based formulations of Pretomanid derivatives leading to promising therapy against tuberculosis in zebrafish and mouse models. Journal of Controlled Release. ISSN 0168-3659. 354, s. 851–868. doi: 10.1016/j.jconrel.2023.01.037. Fulltekst i vitenarkiv Vis sammendrag

  Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic and had limited bioavailability, we expected that their micellar formulations would overcome these limitations, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by π-π interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these π-π-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model almost completely eradicated the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug-loaded PeptoMicelles significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.

• Griffiths, Gareth Wyn; Gruenberg, Jean; Marsh, Mark; Wohlmann, Jens; Jones, Arwyn T. & Parton, Robert G. (2022). Nanoparticle entry into cells; the cell biology weak link. Advanced Drug Delivery Reviews. ISSN 0169-409X. 188. doi: 10.1016/j.addr.2022.114403. Fulltekst i vitenarkiv
• Dal, Nils-Jørgen Knudsen; Speth, Martin Tobias; Johann, Kerstin; Barz, Matthias; Beauvineau, Claire & Wohlmann, Jens [Vis alle 10 forfattere av denne artikkelen] (2022). The zebrafish embryo as an in vivo model for screening nanoparticle-formulated lipophilic anti-tuberculosis compounds. Disease Models and Mechanisms. ISSN 1754-8403. 15(1). doi: 10.1242/dmm.049147. Fulltekst i vitenarkiv Vis sammendrag

  ABSTRACT With the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models, such as mice, is time consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model for evaluating the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified as possessing anti-TB activity in vitro. To aid solubilization, compounds were formulated in biocompatible polymeric micelles (PMs). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating the in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited for pinpointing promising compounds for further development.

• Barcytė, Dovilė; Eikrem, Wenche; Engesmo, Anette; Seoane, Sergio; Wohlmann, Jens & Horák, Aleš [Vis alle 8 forfattere av denne artikkelen] (2021). Olisthodiscus represents a new class of Ochrophyta. Journal of Phycology. ISSN 0022-3646. 57(4), s. 1094–1118. doi: 10.1111/jpy.13155. Fulltekst i vitenarkiv Vis sammendrag

  The phylogenetic diversity of Ochrophyta, a diverse and ecologically important radiation of algae, is still incompletely understood even at the level of the principal lineages. One taxon that has eluded simple classification is the marine flagellate genus Olisthodiscus. We investigated Olisthodiscus luteus K-0444 and documented its morphological and genetic differences from the NIES-15 strain, which we described as Olisthodiscus tomasii sp. nov. Phylogenetic analyses of combined 18S and 28S rRNA sequences confirmed that Olisthodiscus constitutes a separate, deep, ochrophyte lineage, but its position could not be resolved. To overcome this problem, we sequenced the plastid genome of O. luteus K-0444 and used the new data in multigene phylogenetic analyses, which suggested that Olisthodiscus is a sister lineage of the class Pinguiophyceae within a broader clade additionally including Chrysophyceae, Synchromophyceae, and Eustigmatophyceae. Surprisingly, the Olisthodiscus plastid genome contained three genes, ycf80, cysT, and cysW, inherited from the rhodophyte ancestor of the ochrophyte plastid yet lost from all other ochrophyte groups studied so far. Combined with nuclear genes for CysA and Sbp proteins, Olisthodiscus is the only known ochrophyte possessing a plastidial sulfate transporter SulT. In addition, the finding of a cemA gene in the Olisthodiscus plastid genome and an updated phylogenetic analysis ruled out the previously proposed hypothesis invoking horizontal cemA transfer from a green algal plastid into Synurales. Altogether, Olisthodiscus clearly represents a novel phylogenetically distinct ochrophyte lineage, which we have proposed as a new class, Olisthodiscophyceae.

• Resseguier, Julien; Levraud, Jean-Pierre; Dal, Nils-Jørgen Knudsen; Fenaroli, Federico; Primard, Charlotte & Wohlmann, Jens [Vis alle 10 forfattere av denne artikkelen] (2021). Biodistribution of surfactant-free poly(lactic-acid) nanoparticles and uptake by endothelial cells and phagocytes in zebrafish: Evidence for endothelium to macrophage transfer. Journal of Controlled Release. ISSN 0168-3659. 331, s. 228–245. doi: 10.1016/j.jconrel.2021.01.006. Fulltekst i vitenarkiv Vis sammendrag

  In the development of therapeutic nanoparticles (NP), there is a large gap between in vitro testing and in vivo experimentation. Despite its prominence as a model, the mouse shows severe limitations for imaging NP and the cells with which they interact. Recently, the transparent zebrafish larva, which is well suited for high-resolution live-imaging, has emerged as a powerful alternative model to investigate the in vivo behavior of NP. Poly(D,L lactic acid) (PLA) is widely accepted as a safe polymer to prepare therapeutic NP. However, to prevent aggregation, many NP require surfactants, which may have undesirable biological effects. Here, we evaluate ‘safe-by-design’, surfactant-free PLA-NP that were injected intravenously into zebrafish larvae. Interaction of fluorescent NPs with different cell types labelled in reporter animals could be followed in real-time at high resolution; furthermore, by encapsulating colloidal gold into the matrix of PLA-NP we could follow their fate in more detail by electron microscopy, from uptake to degradation. The rapid clearance of fluorescent PLA-NP from the circulation coincided with internalization by endothelial cells lining the whole vasculature and macrophages. After 30 min, when no NP remained in circulation, we observed that macrophages continued to internalize significant amounts of NP. More detailed video-imaging revealed a new mechanism of NP transfer where NP are transmitted along with parts of the cytoplasm from endothelial cells to macrophages.

• Kocere, Agnese; Resseguier, Julien; Wohlmann, Jens; Skjeldal, Frode Miltzow; Khan, Shanawaz & Speth, Martin [Vis alle 14 forfattere av denne artikkelen] (2020). Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis. EBioMedicine. 58, s. 1–12. doi: 10.1016/j.ebiom.2020.102902. Fulltekst i vitenarkiv Vis sammendrag

  Background The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. Methods Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. Findings Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. Interpretation The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells.

• Dal, Nils-Jørgen Knudsen; Kocere, Agnese; Wohlmann, Jens; Herck, Simon Van; Bauer, Tobias A & Resseguier, Julien [Vis alle 11 forfattere av denne artikkelen] (2020). Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle–Cell Interactions. Small. ISSN 1613-6810. 16(5), s. 1–12. doi: 10.1002/smll.201906719. Fulltekst i vitenarkiv Vis sammendrag

  The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real‐time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.

• Bargen, Kristine Von; Scraba, Mirella; Kramer, I.; Ketterer, M.; Nehls, Christian & Krokowski, Sina [Vis alle 22 forfattere av denne artikkelen] (2019). Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor. Cellular Microbiology. ISSN 1462-5814. 21(1), s. 1–18. doi: 10.1111/cmi.12958. Fulltekst i vitenarkiv Vis sammendrag

  Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R . equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R . equi to multiply. This observation is mirrored in the fact that virulent and avirulent R . equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences.

• Geerds, Christina; Wohlmann, Jens; Haas, A & Niemann, H (2014). Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs. Acta Crystallographica. Section F : Structural Biology and Crystallization Communications. doi: 10.1107/S2053230X14009911. Fulltekst i vitenarkiv
• Sydor, Tobias; Bargen, Kristine Von; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto & Wohlmann, Jens [Vis alle 14 forfattere av denne artikkelen] (2013). Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length. Cellular Microbiology. ISSN 1462-5814. doi: 10.1111/cmi.12050. Fulltekst i vitenarkiv
• Bargen, Kristine Von; Wohlmann, Jens; Taylor, GA; Utermohlen, Olaf & Haas, A (2011). Nitric oxide-mediated intracellular growth restriction of pathogenic Rhodococcus equi can be prevented by iron. Infection and Immunity. ISSN 0019-9567. doi: 10.1128/IAI.00983-10. Fulltekst i vitenarkiv
• Hsu, Fong-Fu; Wohlmann, Jens; Turk, J. & Haas, A (2011). Structural definition of trehalose 6-monomycolates and trehalose 6,6'-dimycolates from the pathogen Rhodococcus equi by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. Journal of the American Society for Mass Spectrometry. ISSN 1044-0305. doi: 10.1007/s13361-011-0240-7. Fulltekst i vitenarkiv

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• Khan, Ayyad Ahmad Zartasht; Fineide, Fredrik; Wohlmann, Jens; Gundersen, Kjell Gunnar; Gundersen, Morten & Kolko, Miriam [Vis alle 7 forfattere av denne artikkelen] (2025). Demodex folliculorum . doi: 10.3390/diagnostics15121520. Fulltekst i vitenarkiv Vis sammendrag

  Herein, we present scanning electron microscopy imagery of Demodex folliculorum on the eyelashes of a patient with a two-year history of dry, burning, and watery eyes. Demodex mites are part of the normal human skin flora, inhabiting hair follicles and sebaceous glands. However, in some individuals, they may contribute to ocular surface diseases, including blepharitis and dry eye disease. Symptoms often include itching, photophobia, and a foreign body sensation. The pathogenic role of Demodex is not fully understood but may involve microabrasions, gland obstruction, hypersensitivity reactions, and bacterial dysbiosis. The presence of collarettes at the base of eyelashes is a diagnostic hallmark. Although optimal treatment remains debated, options include topical tea tree oil, ivermectin, and a recently FDA-approved drug lotilaner. Our patient responded favorably to a two-month regimen of tea tree oil-based eyelid wipes. This case underscores the clinical relevance of Demodex infestation in chronic ocular discomfort and highlights the importance of diagnostics.

• Mascherin, Elisa; Karlsen, Markus; Wohlmann, Jens; Reseland, Janne Elin & Linke, Dirk (2025). In vitro toxicology and antimicrobial efficacy of Q-FIELD™, an innovative photocatalytic additive for paints. Fulltekst i vitenarkiv

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