The First Observation of the Filamentous Fungus Neurospora crassa Growing in the Roots of the Grass Brachypodium distachyon.

The model organism Neurospora crassa has been cultivated in laboratories since the 1920s and its saprotrophic lifestyle has been established for decades. However, beyond their role as saprotrophs, fungi engage in intricate relationships with plants, showcasing diverse connections ranging from mutualistic to pathogenic. Although N. crassa has been extensively investigated under laboratory conditions, its ecological characteristics remain largely unknown. In contrast, Brachypodium distachyon, a sweet grass closely related to significant crops, demonstrates remarkable ecological flexibility and participates in a variety of fungal interactions, encompassing both mutualistic and harmful associations. Through a comprehensive microscopic analysis using electron, fluorescence, and confocal laser scanning microscopy, we discovered a novel endophytic interaction between N. crassa and B. distachyon roots, where fungal hyphae not only thrive in the apoplastic space and vascular bundle but also may colonize plant root cells. This new and so far hidden trait of one of the most important fungal model organisms greatly enhances our view of N. crassa, opening new perspectives concerning the fungus' ecological role. In addition, we present a new tool for studying plant-fungus interspecies communication, combining two well-established model systems, which improves our possibilities of experimental design on the molecular level.

Satiety controls behavior in Hydra through an interplay of pre-enteric and central nervous system-like neuron populations.

Hunger and satiety can have an influence on decision-making, sensory processing, and motor behavior by altering the internal state of the brain. This process necessitates the integration of peripheral sensory stimuli into the central nervous system. Here, we show how animals without a central nervous system such as the cnidarian Hydra measure and integrate satiety into neuronal circuits and which specific neuronal populations are involved. We demonstrate that this simple nervous system, previously referred to as diffuse, has an endodermal subpopulation (N4) similar to the enteric nervous system (feeding-associated behavior) and an ectodermal population (N3) that performs central nervous system-like functions (physiology/motor). This view of a supposedly simple nervous system could open an important window into the origin of more complex nervous systems.

Non-professional efferocytosis of Salmonella-infected intestinal epithelial cells in the neonatal host.

The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal intestine. Instead, here, we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighboring epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid cocultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis, and identified the involvement of the "eat-me" signals and adaptors phosphatidylserine and C1q as well as the "eat-me" receptors integrin-αv (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighboring epithelial cells may represent a previously unrecognized mechanism of neonatal antimicrobial host defense to maintain barrier integrity.

Effects of benzothiazinone and ethambutol on the integrity of the corynebacterial cell envelope.

The mycomembrane (MM) is a mycolic acid layer covering the surface of Mycobacteria and related species. This group includes important pathogens such as Mycobacterium tuberculosis, Corynebacterium diphtheriae, but also the biotechnologically important strain Corynebacterium glutamicum. Biosynthesis of the MM is an attractive target for antibiotic intervention. The first line anti-tuberculosis drug ethambutol (EMB) and the new drug candidate, benzothiazinone 043 (BTZ) interfere with the synthesis of the arabinogalactan (AG), which is a structural scaffold for covalently attached mycolic acids that form the inner leaflet of the MM. We previously showed that C. glutamicum cells treated with a sublethal concentration of EMB lose the integrity of the MM. In this study we examined the effects of BTZ on the cell envelope. Our work shows that BTZ efficiently blocks the apical growth machinery, however effects in combinatorial treatment with ß-lactam antibiotics are only additive, not synergistic. Transmission electron microscopy (TEM) analysis revealed a distinct middle layer in the septum of control cells considered to be the inner leaflet of the MM covalently attached to the AG. This layer was not detectable in the septa of BTZ or EMB treated cells. In addition, we observed that EMB treated cells have a thicker and less electron dense peptidoglycan (PG). While EMB and BTZ both effectively block elongation growth, BTZ also strongly reduces septal cell wall synthesis, slowing down growth effectively. This renders BTZ treated cells likely more tolerant to antibiotics that act on growing bacteria.

How do barley plants with impaired photosynthetic light acclimation survive under high-light stress?

MAIN CONCLUSION: WHIRLY1 deficient barley plants surviving growth at high irradiance displayed increased non-radiative energy dissipation, enhanced contents of zeaxanthin and the flavonoid lutonarin, but no changes in α-tocopherol nor glutathione. Plants are able to acclimate to environmental conditions to optimize their functions. With the exception of obligate shade plants, they can adjust their photosynthetic apparatus and the morphology and anatomy of their leaves to irradiance. Barley (Hordeum vulgare L., cv. Golden Promise) plants with reduced abundance of the protein WHIRLY1 were recently shown to be unable to acclimatise important components of the photosynthetic apparatus to high light. Nevertheless, these plants did not show symptoms of photoinhibition. High-light (HL) grown WHIRLY1 knockdown plants showed clear signs of exposure to excessive irradiance such as a low epoxidation state of the violaxanthin cycle pigments and an early light saturation of electron transport. These responses were underlined by a very large xanthophyll cycle pool size and by an increased number of plastoglobules. Whereas zeaxanthin increased with HL stress, α-tocopherol, which is another lipophilic antioxidant, showed no response to excessive light. Also the content of the hydrophilic antioxidant glutathione showed no increase in W1 plants as compared to the wild type, whereas the flavone lutonarin was induced in W1 plants. HPLC analysis of removed epidermal tissue indicated that the largest part of lutonarin was presumably located in the mesophyll. Since lutonarin is a better antioxidant than saponarin, the major flavone present in barley leaves, it is concluded that lutonarin accumulated as a response to oxidative stress. It is also concluded that zeaxanthin and lutonarin may have served as antioxidants in the WHIRLY1 knockdown plants, contributing to their survival in HL despite their restricted HL acclimation.

Newly Established Genetic System for Functional Analysis of MetSV.

The linear chromosome of the Methanosarcina spherical virus with 10,567 bp exhibits 22 ORFs with mostly unknown functions. Annotation using common tools and databases predicted functions for a few genes like the type B DNA polymerase (MetSVORF07) or the small (MetSVORF15) and major (MetSVORF16) capsid proteins. For verification of assigned functions of additional ORFs, biochemical or genetic approaches were found to be essential. Consequently, we established a genetic system for MetSV by cloning its genome into the E. coli plasmid pCR-XL-2. Comparisons of candidate plasmids with the MetSV reference based on Nanopore sequencing revealed several mutations of yet unknown provenance with an impact on protein-coding sequences. Linear MetSV inserts were generated by BamHI restriction, purified and transformed in Methanosarcina mazei by an optimized liposome-mediated transformation protocol. Analysis of resulting MetSV virions by TEM imaging and infection experiments demonstrated no significant differences between plasmid-born viruses and native MetSV particles regarding their morphology or lytic behavior. The functionality of the genetic system was tested by the generation of a ΔMetSVORF09 mutant that was still infectious. Our genetic system of MetSV, the first functional system for a virus of methanoarchaea, now allows us to obtain deeper insights into MetSV protein functions and virus-host interactions.

Apical bulkheads accumulate as adaptive response to impaired bile flow in liver disease.

Hepatocytes form bile canaliculi that dynamically respond to the signalling activity of bile acids and bile flow. Little is known about their responses to intraluminal pressure. During embryonic development, hepatocytes assemble apical bulkheads that increase the canalicular resistance to intraluminal pressure. Here, we investigate whether they also protect bile canaliculi against elevated pressure upon impaired bile flow in adult liver. Apical bulkheads accumulate upon bile flow obstruction in mouse models and patients with primary sclerosing cholangitis (PSC). Their loss under these conditions leads to abnormally dilated canaliculi, resembling liver cell rosettes described in other hepatic diseases. 3D reconstruction reveals that these structures are sections of cysts and tubes formed by hepatocytes. Mathematical modelling establishes that they positively correlate with canalicular pressure and occur in early PSC stages. Using primary hepatocytes and 3D organoids, we demonstrate that excessive canalicular pressure causes the loss of apical bulkheads and formation of rosettes. Our results suggest that apical bulkheads are a protective mechanism of hepatocytes against impaired bile flow, highlighting the role of canalicular pressure in liver diseases.

Four Novel Caudoviricetes Bacteriophages Isolated from Baltic Sea Water Infect Colonizers of Aurelia aurita.

The moon jellyfish Aurelia aurita is associated with a highly diverse microbiota changing with provenance, tissue, and life stage. While the crucial relevance of bacteria to host fitness is well known, bacteriophages have often been neglected. Here, we aimed to isolate virulent phages targeting bacteria that are part of the A. aurita-associated microbiota. Four phages (Pseudomonas phage BSwM KMM1, Citrobacter phages BSwM KMM2-BSwM KMM4) were isolated from the Baltic Sea water column and characterized. Phages KMM2/3/4 infected representatives of Citrobacter, Shigella, and Escherichia (Enterobacteriaceae), whereas KMM1 showed a remarkably broad host range, infecting Gram-negative Pseudomonas as well as Gram-positive Staphylococcus. All phages showed an up to 99% adsorption to host cells within 5 min, short latent periods (around 30 min), large burst sizes (mean of 128 pfu/cell), and high efficiency of plating (EOP > 0.5), demonstrating decent virulence, efficiency, and infectivity. Transmission electron microscopy and viral genome analysis revealed that all phages are novel species and belong to the class of Caudoviricetes harboring a tail and linear double-stranded DNA (formerly known as Siphovirus-like (KMM3) and Myovirus-like (KMM1/2/4) bacteriophages) with genome sizes between 50 and 138 kbp. In the future, these isolates will allow manipulation of the A. aurita-associated microbiota and provide new insights into phage impact on the multicellular host.

Dual-RNAseq Analysis Unravels Virus-Host Interactions of MetSV and Methanosarcina mazei.

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6, MetSVORF5, MetSVORF2, and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study's results led to the first preliminary infection model of MetSV infecting M. mazei, summarizing the key infection steps as follows: replication, assembly, and host cell lysis.

Bacteroides muris sp. nov. isolated from the cecum of wild-derived house mice.

Two bacterial strains, KH365_2T and KH569_7, were isolated from the cecum contents of wild-derived house mice. The strains were characterized as Gram-negative, rod-shaped, strictly anaerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains were most closely related to Bacteroides uniformis ATCC 8492T. Whole genome sequences of KH365_2T and KH569_7 strains have a DNA G + C content of 46.02% and 46.03% mol, respectively. Most morphological and biochemical characteristics did not differ between the newly isolated strains and classified Bacteroides strains. However, the average nucleotide identity (ANI) and dDNA-DNA hybridization (dDDH) values clearly distinguished the two strains from described members of the genus Bacteroides. Here, we present the phylogeny, morphology, and physiology of a novel species of the genus Bacteroides and propose the name Bacteroides muris sp. nov., with KH365_2T (DSM 114231T = CCUG 76277T) as type strain.

[Mediastinal pseudotumor-necrotizing and sclerosing mediastinitis with pseudomembranes]. / Mediastinaler Pseudotumor ­ nekrotisierende und sklerosierende Mediastinitis mit Pseudomembranen.

Mediastinal tumors present a rather rare phenomenon with a variety of underlying causes. Important differential diagnoses include benign and malignant tumors as well as idiopathic sclerosing mediastinitis and parasitic infection.Here, we present the case of a mediastinal pseudotumor with compression of the superior vena cava in a young adult male who spent several years abroad in Brazil. Clinicians suspected either a mediastinal lymphoma, malignant mesenchymal tumor, teratoma, sarcoidosis, or tuberculosis. Biopsy tissue failed to provide a definite diagnosis. The subsequently resected mediastinal mass showed a necrotizing, granulomatous inflammation with prominent eosinophilia and sclerosis. Membranous structures were detected in the necrotic areas, which presented as collagen bundles ultrastructurally. Therefore, these membranes were proven to be constituents of lipid pseudomembranes. Consequently the lesion shows characteristics of sclerosing mediastinitis next to characteristics of adipose tissue necrosis with lipid pseudomembranes. Parasitic infection could not be proven.

The plastid-nucleus localized DNA-binding protein WHIRLY1 is required for acclimation of barley leaves to high light.

MAIN CONCLUSION: In accordance with a key role of WHIRLY1 in light-acclimation mechanisms, typical features of acclimation to high light, including photosynthesis and leaf morphology, are compromised in WHIRLY1 deficient plants. Acclimation to the environment requires efficient communication between chloroplasts and the nucleus. Previous studies indicated that the plastid-nucleus located WHIRLY1 protein is required for the communication between plastids and the nucleus in situations of high light exposure. To investigate the consequences of WHIRLY1 deficiency on the light acclimation of photosynthesis and leaf anatomy, transgenic barley plants with an RNAi-mediated knockdown of HvWHIRLY1 were compared to wild-type plants when growing at low and high irradiance. While wild-type plants showed the typical light acclimation responses, i.e. higher photosynthetic capacity and thicker leaves, the WHIRLY1 deficient plants were not able to respond to differences in irradiance. The results revealed a systemic role of WHIRLY1 in light acclimation by coordinating responses at the level of the chloroplast and the level of leaf morphology.

SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo.

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads.

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.

Three-dimensional spatially resolved geometrical and functional models of human liver tissue reveal new aspects of NAFLD progression.

Early disease diagnosis is key to the effective treatment of diseases. Histopathological analysis of human biopsies is the gold standard to diagnose tissue alterations. However, this approach has low resolution and overlooks 3D (three-dimensional) structural changes resulting from functional alterations. Here, we applied multiphoton imaging, 3D digital reconstructions and computational simulations to generate spatially resolved geometrical and functional models of human liver tissue at different stages of non-alcoholic fatty liver disease (NAFLD). We identified a set of morphometric cellular and tissue parameters correlated with disease progression, and discover profound topological defects in the 3D bile canalicular (BC) network. Personalized biliary fluid dynamic simulations predicted an increased pericentral biliary pressure and micro-cholestasis, consistent with elevated cholestatic biomarkers in patients' sera. Our spatially resolved models of human liver tissue can contribute to high-definition medicine by identifying quantitative multiparametric cellular and tissue signatures to define disease progression and provide new insights into NAFLD pathophysiology.

Correlative single-molecule localization microscopy and electron tomography reveals endosome nanoscale domains.

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-color SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor.

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.

Enhanced Permeability and Retention-like Extravasation of Nanoparticles from the Vasculature into Tuberculosis Granulomas in Zebrafish and Mouse Models.

The enhanced permeability and retention (EPR) effect is the only described mechanism enabling nanoparticles (NPs) flowing in blood to reach tumors by a passive targeting mechanism. Here, using the transparent zebrafish model infected with Mycobacterium marinum we show that an EPR-like process also occurs allowing different types of NPs to extravasate from the vasculature to reach granulomas that assemble during tuberculosis (TB) infection. PEGylated liposomes and other NP types cross endothelial barriers near infection sites within minutes after injection and accumulate close to granulomas. Although ∼100 and 190 nm NPs concentrated most in granulomas, even ∼700 nm liposomes reached these infection sites in significant numbers. We show by confocal microscopy that NPs can concentrate in small aggregates in foci on the luminal side of the endothelium adjacent to the granulomas. These spots are connected to larger foci of NPs on the ablumenal side of these blood vessels. EM analysis suggests that NPs cross the endothelium via the paracellular route. PEGylated NPs also accumulated efficiently in granulomas in a mouse model of TB infection with Mycobacterium tuberculosis, arguing that the zebrafish embryo model can be used to predict NP behavior in mammalian hosts. In earlier studies we and others showed that uptake of NPs by macrophages that are attracted to infection foci is one pathway for NPs to reach TB granulomas. This study reveals that when NPs are designed to avoid macrophage uptake, they can also efficiently target granulomas via an alternative mechanism that resembles EPR.

A seven-helix protein constitutes stress granules crucial for regulating translation during human-to-mosquito transmission of Plasmodium falciparum.

The complex life-cycle of the human malaria parasite Plasmodium falciparum requires a high degree of tight coordination allowing the parasite to adapt to changing environments. One of the major challenges for the parasite is the human-to-mosquito transmission, which starts with the differentiation of blood stage parasites into the transmissible gametocytes, followed by the rapid conversion of the gametocytes into gametes, once they are taken up by the blood-feeding Anopheles vector. In order to pre-adapt to this change of host, the gametocytes store transcripts in stress granules that encode proteins needed for parasite development in the mosquito. Here we report on a novel stress granule component, the seven-helix protein 7-Helix-1. The protein, a homolog of the human stress response regulator LanC-like 2, accumulates in stress granules of female gametocytes and interacts with ribonucleoproteins, such as CITH, DOZI, and PABP1. Malaria parasites lacking 7-Helix-1 are significantly impaired in female gametogenesis and thus transmission to the mosquito. Lack of 7-Helix-1 further leads to a deregulation of components required for protein synthesis. Consistently, inhibitors of translation could mimic the 7-Helix-1 loss-of-function phenotype. 7-Helix-1 forms a complex with the RNA-binding protein Puf2, a translational regulator of the female-specific antigen Pfs25, as well as with pfs25-coding mRNA. In accord, gametocytes deficient of 7-Helix-1 exhibit impaired Pfs25 synthesis. Our data demonstrate that 7-Helix-1 constitutes stress granules crucial for regulating the synthesis of proteins needed for life-cycle progression of Plasmodium in the mosquito vector.

Murine alveolar epithelial cells and their lentivirus-mediated immortalisation.

In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air-blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800Ω ·cm². Transport across the air-blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω·cm²) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies.