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Müller, M.

Publications and source records attributed to Müller, M..

At least 19 recordsLinked to original sources
bioRxiv2026

Super-Resolution Optical Sectioning Microscopy Visualizes Nanopores in the Plasma Membrane of Endothelial Cells in situ

The ultrastructure of endothelial cells (ECs) "in situ" is of great interest due to their involvement in many physiological processes. In some organs, these cells form transcellular pores or fenestrae, allowing for the rapid exchange of molecules between blood and interstitium. Despite their importance, no optical images of these dynamic morphological structures have yet been acquired in situ. Major obstacles to their in-situ imaging are the lack of specifical labels for fenestrae and their size well below the optical diffraction limit. Here, we report how we have overcome these challenges and managed to visualize the EC ultrastructure in situ in 25 {micro}m thick liver sections. To enable this, a lipophilic, fluorescent membrane dye was infused into the portal vein of murine livers to stain the sinusoidal ECs before the organ was harvested. Tissue sections were subsequently imaged using a novel, super-resolution optical-sectioning structured illumination microscope (OS-SIM), providing approx. 170 nm spatial resolution with significantly faster image acquisition compared to confocal microscopy.

biophysics↗
bioRxiv2026

Identification of a large class of cancer-germline microproteins as a source of immunotherapeutic targets

Classical cancer germline-antigens (CGAs) are proteins that are expressed in the male germinal line but not in somatic tissues, and that can also become expressed in tumors. However, the vast majority of testis-specific transcripts are long non-coding RNAs (lncRNAs) rather than protein-coding genes. Since recent studies have shown that many lncRNAs contain non-canonical open reading frames (ncORFs) that are translated into small proteins, or microproteins, there could be a large class of non-canonical cancer-germline antigens (ncCGAs) that remains to be discovered. Here, we integrate ribosome profiling from human testis and cancer cell lines with paired tumor/normal transcriptomes from 917 patients across eight common cancer types to define a comprehensive catalog of ncCGAs. This set comprises 235 ncCGAs encoded by lncRNAs or mRNA untranslated regions (5UTRs and 3UTRs), compared to 192 canonical CGAs (cCGAs) with similar expression patterns. We show that ncCGAs are evolutionary young, consistent with recent de novo emergence in the rapidly evolving male germline. Moreover, a large fraction is expressed across multiple patients and cancer types, indicating recurrent reactivation mechanisms in tumors. We further find that ncCGAs are frequently located in cancer-amplified regions or associated with MYC or E2F-regulated pathways, which may explain their expression in cancer. Finally, we provide strong evidence that a subset of ncCGAs give rise to potentially immunogenic HLA class I bound peptides. Together, our results describe a previously unexplored class of tumor-restricted antigens with potential applications in cancer immunotherapy.

cancer biology↗
bioRxiv2026

Dosage compensation defects due to roX RNA deletion are rescued by recalibration of X/autosome stoichiometry

Metazoa evolved regulatory networks to balance the expression of their sex chromosomes. In Drosophila, males have a single gene-rich X chromosome, whereas females have two. Balanced X/autosome expression is essential for viability, and in male flies is achieved by activation of genes on the X through the male-specific-lethal (MSL) dosage compensation complex (DCC). This ribonucleoprotein assembly contains long, non-coding roX RNAs. To dissect the functional requirements of roX in a cell-based system, we deleted the roX2 gene in male S2 cells and selected two independent lines lacking detectable roX RNA. In the absence of roX, the remaining MSL protein complex was unable to associate with known or newly identified binding sites and thus failed to activate transcription. Surprisingly, the X/autosome expression ratio appeared nevertheless compensated. Cytogenetic and genomic analyses revealed that both roX-deficient cell populations had acquired additional X chromosomes. Apparently, chromosome gains due to mis-segregation made up for the loss of DCC-mediated dosage compensation. Interestingly, ectopic expression of full-length roX2, but not of shortened derivatives, fully restored DCC binding and normalized the karyotype. These findings illustrate that X chromosome dosage compensation is critical for viability even in cultured cells and provide a striking example of rapid evolution under stringent selection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/707606v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1bbf907org.highwire.dtl.DTLVardef@1da43a9org.highwire.dtl.DTLVardef@903326org.highwire.dtl.DTLVardef@10e3865_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗
bioRxiv2026

Human T Cells From Resistant Hypertension Patients Promote Hypertension via TNF in humanized mice

ObjectivePreclinical studies suggest a pivotal role of adaptive immunity, particularly T cells, in hypertension. However, due to the multifactorial pathogenesis, there is still no definitive evidence for a causal role of T cells in the development of hypertension in humans. We sought to determine whether T cells from patients with treatment-resistant hypertension (TRH) directly modulate blood pressure and vascular function in vivo. MethodsPeripheral blood mononuclear cells (PBMCs) from TRH patients and healthy controls (HC) were adoptively transferred into immunodeficient NSG-(KbDb)^null mice. Hypertension was induced by angiotensin II infusion for 14 days and monitored continuously by radiotelemetry. ResultFollowing T cell engraftment, blood pressure was assessed at baseline and during AngII infusion in both groups of recipient mice. At baseline, systolic blood pressure did not differ between both groups. However, mice receiving TRH-PBMCs developed a significantly higher systolic blood pressure following AngII compared with HC-PBMC recipients. Endothelial dysfunction in isolated perfused kidneys was more pronounced in AngII-challenged TRH-PBMC recipients compared to HC-PBMC recipients. TRH-PBMC recipients displayed elevated effector memory CD4 T cells and Th17 frequencies in spleen and kidney, along with markedly increased renal expression of human T cell-derived TNF. Overnight incubation of mouse aortic rings with human TNF induced endothelial dysfunction, indicating a causal role of T cell-derived TNF. As a proof of concept, TNF inhibition attenuated AngII-induced hypertension in TRH-PBMC-engrafted mice. ConclusionT cells from patients with treatment-resistant hypertension promote an exaggerated hypertensive response and endothelial dysfunction in PBMC-engrafted humanized mice, promoted by TNF-mediated mechanisms. These findings provide evidence that T cell-derived TNF may contribute to the pathogenesis of human hypertension.

immunology↗
bioRxiv2026

L-pentahomoserine correlates with therapy outcome in esophageal cancer and promotes metabolic adaptations that support cell survival under nutrient-deprived conditions

Esophageal adenocarcinoma (EAC) is the sixth-leading cause of cancer-related death. Although pyrimidine analogue-based neoadjuvant and adjuvant therapies are widely used, patient responses remain variable. Emerging evidence indicates that bacteria-derived metabolites influence tumor biology and therapy outcomes. To identify non-canonical plasma metabolites linked to cancer biology, we performed correlation analyses between untargeted metabolomics profiles and overall survival. This approach revealed a bacterial metabolite called L-pentahomoserine, or L-2-amino-5-hydroxypentanoic acid (L-2A5HPA), to be positively associated with overall survival. Notably, L-2A5HPA promoted cell survival under nutrient limitation by redirecting glucose metabolism towards aspartate and pyrimidine biosynthesis. In vitro, L-2A5HPA uptake varied among cell lines and was controlled by stereospecific transporters. Furthermore, metabolic profiling in mouse models of liver cancer showed different levels of L-2A5HPA and a strong correlation with pyrimidine intermediates, dihydroorotate and orotate. The link between L-2A5HPA, pyrimidine nucleotide metabolism, and cell survival provides mechanistic insight into its association with patient outcome. Our findings position L-2A5HPA as a metabolite with potential to become a prognostic biomarker for EAC and underscores its role in metabolic adaptation under nutrient-deprived conditions.

cancer biology↗
bioRxiv2026

Regulatory Mechanisms of Maternal Imprinting at the Dlk1-Dio3 Domain

Genomic imprinting is an epigenetic process causing parent-of-origin specific gene expression. The Dlk1-Dio3 domain is one of the largest imprinted clusters. While DNA methylation at an intergenic CpG-island (IG-CGI) within the imprinting control region (ICR) controls expression from the paternal chromosome, mechanisms regulating the unmethylated maternal chromosome remain unknown. Within the transcriptional regulatory element (IG-TRE) of the ICR, deletions identified a minimal region in vitro exhibiting both silencing and enhancing activity, with SOX2 and ZFP281 contributing to enhancer function on the maternal chromosome. In vivo, however, this deletion did not affect maternal expression in embryos; instead it activated Dlk1 on both parental chromosomes. Combining deletion of this IG-TRE with the lethal IG-CGI deletion rescued lethality by balancing Dlk1 expression, despite persistent maternal gene upregulation. These results demonstrate that loss of expression at this domain is more detrimental than gain highlighting the importance of in vivo analysis. Identification of active regulatory factors on the unmethylated maternal chromosome challenges the prevailing view that imprinting is primarily a methylation-driven phenomenon, further revealing the sophisticated hierarchical mechanisms governing imprinting control.

genetics↗
bioRxiv2025

Genetic characterization of the apterous Life Span Enhancer in Drosophila melanogaster

The selector gene apterous (ap) of Drosophila melanogaster is best known for its prominent role in wing formation. apnull flies are viable but have no wings. However, apnull flies are associated with another phenotype whose analysis was largely neglected in the past 40 years: adults die precociously 2-3 days after eclosion. We have recently published a comprehensive analysis on the physiology of this striking phenotype. We showed that Ap protein is essential early in metamorphosis during hindgut remodeling where it is required for rectal papillae formation in the rectal ampulla. Wild-type flies have four rectal papillae that are involved in selective reabsorption of essential ions and metabolites from the primary urine produced by the Malpighian tubules. In apnull flies, rectal papillae formation is aborted prematurely. The corresponding papillar cells remain stuck in the rectal lumen. This leads to complete intestinal blockage and a cascade of pathologies resulting in premature death. In this study, we genetically identify and functionally dissect a novel tissue specific cis- regulatory element that directs Ap expression in the hindgut. This Life Span Enhancer (apLSE) maps to a [~]400 bp DNA fragment that is activated via only two small regulatory modules. Genetic analysis of apLSE alleles proves that they are essential for papilla formation and thus for adult survival. Our studies solve the 110-year-old enigma about the ap syndrome. They establish the apLSE as a key player linking hindgut morphogenesis and survival. We propose that several aspects of the ap syndrome are a consequence of drastic changes in endocrine homeostasis.

developmental biology↗
bioRxiv2025

Intrinsic specificity of a 'core' Tip60 acetyltransferase complex in Drosophila

The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histone N-terminal tail domains. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A, or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzymes mode of action.

biochemistry↗
bioRxiv2025

Intestinal control of feeding initiation in Drosophila melanogaster

The interplay between feeding and excretion is essential for organismal nutrition and survival, yet their mechanistic coupling remains poorly understood. At the onset of life, feeding must be initiated while developmental waste products - the meconium - need to be eliminated. Using Drosophila as a model system, we explored the in vivo mechanisms coordinating these processes. We developed novel behavioral assays for newly eclosed flies and discovered that, similar to neonatal mammals, Drosophila excrete their meconium shortly after eclosion. Remarkably, feeding initiation occurs only after partial meconium elimination. We identified a cis-regulatory element associated with the apterous gene, which, when disrupted, prevents both meconium excretion and adult feeding initiation. These flies develop hindgut obstruction (ileus), avoid food, and exhibit increased proboscis extension sleep - a behaviour we found plays a functional role in waste clearance under normal conditions. Through experimental inhibition of meconium excretion, we established that this process is prerequisite for feeding initiation, suggesting a gut-to-brain signaling circuit that couples these fundamental physiological processes. The progression of phenotypes we observed parallels the hallmarks of mechanical ileus in humans. Our findings reveal previously unrecognized links between intestinal clearance, feeding behavior, and survival, with potential implications for understanding similar processes across species.

developmental biology↗
bioRxiv2025

Induced pluripotent stem cell-derived human macrophages as an infection model for Trypanosoma cruzi

Chagas disease, caused by the parasite Trypanosoma cruzi, affects millions of people globally. Unfortunately, the available treatment options, especially for the chronic stage of the disease, are suboptimal. Given the chronic nature of the disease and the elusive nature of the parasite, there is a high need for new and safer drugs that deliver sterile cure. Posaconazole was a promising lead in the drug discovery pipeline but ultimately failed in clinical trials due to patient relapses. This failure illustrates the need for a drug screening assay that can predict sterile cure by assessing recrudescence after treatment. Here, we used human induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) as host cells for T. cruzi. The iMACs were highly susceptible to infection by the parasites. By combining red fluorescent protein (RFP)-expressing iMACs with mNeonGreen-expressing T. cruzi, we were able to monitor the dynamics of the infection through live cell imaging. The activity of the compounds benznidazole and posaconazole was consistent with the results of an established infection system using mouse primary macrophages. The post-mitotic nature of iMACs makes them suitable host cells for long-term assays needed to assess recrudescence of parasites. Moreover, their human origin, stable genetic background, and capacity for genetic modification make the iMACs excellent host cells for studying host-pathogen interaction. Author summaryThe parasite Trypanosoma cruzi, the causative agent of Chagas disease, is a global health concern affecting millions each year. Infection with T. cruzi can cause chronic disease, often remaining asymptomatic for decades before resulting in severe cardiac or gastro-intestinal pathologies. To date, only benznidazole and nifurtimox are used for treatment of the infection, but both drugs are suboptimal for curing the chronic stage. Posaconazole showed great promise in preclinical studies but failed to achieve sterile cure in clinical trials, causing patient relapses. These disappointing results underline the need for drug screening assays able to predict sterile cure by evaluating recrudescence post-treatment. We used human induced pluripotent stem cell derived macrophages as host cells for T. cruzi and testing of trypanocidal compounds. This model can be used for long-term in vitro screening assays to find new drug candidates against Chagas disease. The human origin of these cells combined with the possibility of upscaling their production make them great host cells for drug screening campaigns.

microbiology↗
bioRxiv2025

Historic Genomes Uncover Demographic Shifts and Kinship Structures in Post-Roman Central Europe

Many European towns and villages trace their origins to Early Medieval foundations. In former Roman territories, their emergence has traditionally been linked to mass migrations from outside the Roman Empire. However, recent studies have emphasised local continuity with some individual-level mobility. We generated and analysed 248 historic genomes from Late Roman (3rd and 4th century CE) and Early Medieval (5th-8th century CE) burial sites in southern Germany, comparing them to over 2,500 contemporary and Iron Age genomes in addition to 1,344 modern-day genomes from Germany, Italy and Great-Britain. Despite small inferred Early Medieval period community sizes, genetic diversity exceeded that of modern German cities. In the Altheim graveyard, established in the 5th century by a group of Northern European descent, we inferred a demographic shift in the 6th century with the integration of newcomers with ancestry typical of a nearby Roman military camp, likely as a result of the collapse of Roman state structures. We reconstructed multigenerational pedigrees and, using a novel approach to infer ancestry of unsampled relatives, inferred immediate intermarriage between incoming and local groups, with a distinct tendency for men from former Roman background marrying women of northern descent. Burial proximity correlates strongly with kinship, in some cases spanning six generations. These communities were organized around small family units, exhibited loosely patrilineal or bilateral descent patterns, practiced reproductive monogamy, and avoided close-kin marriages. Such practices reflect broader transformations in family structures that began during the Late Roman period, were transferred to small agrarian societies in the Early Medieval period, and continued to shape European societies. By the 7th century, ongoing admixture had shaped genetic diversity patterns into those resembling Central Europe today.

genetics↗
bioRxiv2025

The Pseudomonas aeruginosa Tse4 toxin assembles ion-selective and voltage-sensitive ion channels to couple membrane depolarisation with K+ efflux

Pseudomonas aeruginosa employs the Type VI secretion system (T6SS) to outcompete other bacteria in its environment. Among the effectors secreted by the T6SS of P. aeruginosa PAO1, Tse4 is known for its potent antibacterial activity. This study elucidates the molecular function of Tse4, which promotes cell depolarisation in competing bacteria. Our results show that Tse4 spontaneously incorporates into lipid monolayers and forms multiionic channels in planar bilayers, with either ohmic conduction or diode-like rectifying currents and a preference for cations over anions. These observations allow us to propose a model of action whereby Tse4 channels couple cell depolarisation with K+ efflux. These insights into Tse4s pore-forming activity enhance our understanding of bacterial competition and exemplify a finely tuned antibacterial strategy, coupling its ability to cause membrane depolarisation with potassium efflux that synergises with other T6SS effectors. These results highlight the sophistication of Pseudomonas aeruginosas competitive arsenal.

microbiology↗
bioRxiv2025

Mapping the complete influenza A virus infection cycle through single vRNP imaging

Cell-to-cell heterogeneity is a common feature of viral infection that can generate enormous complexity, complicating understanding of infection progression and interpretation of differences between viral variants. To overcome these challenges, we developed a technology to visualize the infection cycle of human influenza A virus (IAV) from start-to-finish in individual living cells with unprecedented spatial and temporal resolution, which identified numerous distinct pathways through which infection can progress. We show that heterogeneous viral gene expression drives infection cycle heterogeneity, and identified genome packaging, vRNP transcriptional activation and host cell division as major determinants of gene expression variability. Our work maps out the complete IAV infection cycle, identifies the origin of infection heterogeneity and provides a broadly-applicable technology for studying IAV and other viruses.

cell biology↗
bioRxiv2024

Ideal efficacy photoswitches for TRPC4/5 channels harness high potency for spatiotemporally-resolved control of TRPC function in live tissues

Directly probing the endogenous biological roles of target proteins with high spatial and temporal resolution, as non-invasively and reproducibly as possible, is a shared conceptual goal for research across many fields, as well as for targeted therapies. Here we describe the rational conceptual design and test-case practical implementation of a photopharmacological paradigm to empower high-performance photomodulation studies in vivo. TRPC4/5 ion channels are involved in many spatiotemporally resolved circuits, from pain and anxiety, to reproductive signaling, digestion, and obesity. To unpick their biology requires spatiotemporally precise tools, which were lacking. We developed "ideal efficacy photoswitch" ligands to control their diverse functions in situ. These E{leftrightarrows}Z-photoswitchable ligands bias TRPC[4]/5 channel activity with exquisite photocontrol, from strong agonism under 360 nm, to low agonism at 385 nm, to strong antagonism at 410-460 nm. Cryo-EM structures of both TRPC4 and TRPC5 with both Z-agonists and E-antagonists support the rationale for efficacy switching through competitive E/Z isomer binding. Crucially, since the E/Z ratio is exclusively determined by the light wavelength applied, their channel photocontrol is exclusively wavelength-dependent, yet drug-concentration-independent: so is reproducible from cell culture to >millimetre-depth tissues. Indeed, we were able to photocontrol both direct and downstream TRPC4/5 biology in cell lines or primary cells in culture, from calcium flux, to primary neuron excitability and adrenaline release; and even in tissues, photoswitching small intestine motility and peristalsis. The TRPC4/5 ligands we develop will thus unlock a range of high-precision investigations in TRP biology. More broadly, we propose that the success of this efficacy photoswitch program, from concept to tissue level translation, is mainly a consequence of how biology has evolved proteins for efficacy control. We therefore foresee that a variety of functionally responsive protein targets, not only sensory and signaling ion channels and receptors, will be amenable to similarly high-performance photocontrol even in vivo, if a new generation of reagent development adopts this paradigm of ideal efficacy photoswitching. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/602451v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1f8e527org.highwire.dtl.DTLVardef@18c72d0org.highwire.dtl.DTLVardef@1c5d648org.highwire.dtl.DTLVardef@177203d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗
bioRxiv2023

Association with TFIIIC limits MYCN localization in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA Polymerase II

MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II. While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYCs effects on transcription. Here we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organization at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localization to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localize to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA.

molecular biology↗
bioRxiv2023

Transcriptional control of compartmental boundary positioning during Drosophila wing development

The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene engrailed (en) and dorsal fate by the selector gene apterous (ap). While the en expression domain is set up during embryogenesis, ap expression begins only during early wing development. Thus, the correct establishment of the ap expression pattern relative to en must be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the early ap enhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, and the HOX gene Antennapedia (Antp) contributes to early wing development. Together, our results provide a comprehensive molecular basis of early ap activation and the developmental consequences of its misregulation, shedding light on how compartmental boundaries are set up during development.

developmental biology↗
bioRxiv2023

Induced pluripotent stem cell-derived human macrophages as an infection model for Leishmania donovani

The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimise the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening. Author SummaryYearly, up two million people in poverty-stricken areas contract leishmaniasis, a disease caused by parasites of the genus Leishmania. When an infected sandfly takes a blood meal, Leishmania parasites enter the host where they are taken up by macrophages. Inside the macrophage, Leishmania parasites establish a niche where they can proliferate. Although this infection often leads to disability or death, the drugs currently available are lacking due to toxic side effects, high expenses or difficulties in usage. Drug screening assays that are currently used for compound screening often rely on mouse peritoneal macrophages. We have generated human induced pluripotent stem cell derived macrophages and used these as new host cells for Leishmania donovani in the testing of anti-leishmanial compounds. This model has many advantages. For one, it allows us to work with human cells, mimicking the natural infection more closely than possible with murine cells. Secondly, it allows to obtain bigger batches of uniform cells for screening campaigns. Finally, this approach aligns with the principle of 3R, replacing the use of animals for cultivation of Leishmania and drug screening purposes.

cell biology↗
bioRxiv2023

Functional comparisons of the virus sensor RIG-I from humans, the microbat Myotis daubentonii, and the megabat Rousettus aegyptiacus, and their response to SARS-CoV-2 infection

Bats (order Chiroptera) are a major reservoir for emerging and re-emerging zoonotic viruses. Their tolerance towards highly pathogenic human viruses led to the hypothesis that bats may possess an especially active antiviral interferon (IFN) system. Here, we cloned and functionally characterized the virus RNA sensor, Retinoic Acid-Inducible Gene-I (RIG-I), from the "microbat" Myotis daubentonii (suborder Yangochiroptera) and the "megabat" Rousettus aegyptiacus (suborder Yinpterochiroptera), and compared them to the human ortholog. Our data show that the overall sequence and domain organization is highly conserved and that all three RIG-I orthologs can mediate a similar IFN induction in response to viral RNA at 37{degrees} and 39{degrees}C, but not at 30{degrees}C. Like human RIG-I, bat RIG-Is were optimally activated by double stranded RNA containing a 5-triphosphate end and required Mitochondrial Antiviral-Signalling Protein (MAVS) for antiviral signalling. Moreover, the RIG-I orthologs of humans and of R. aegyptiacus, but not of M. daubentonii, enable innate immune sensing of SARS-CoV-2 infection. Our results thus show that microbats and megabats express a RIG-I that is not substantially different from the human counterpart with respect to function, temperature dependency, antiviral signaling, and RNA ligand properties, and that human and megabat RIG-I are able to sense SARS-CoV-2 infection. IMPORTANCEA common hypothesis holds that bats (order Chiroptera) are outstanding reservoirs for zoonotic viruses because of a special antiviral interferon (IFN) system. However, functional studies about key components of the bat IFN system are rare. RIG-I is a cellular sensor for viral RNA signatures that activates the antiviral signalling chain to induce IFN. We cloned and functionally characterized RIG-I genes from representatives of the suborders Yangochiroptera and Yinpterochiroptera. The bat RIG-Is were conserved in their sequence and domain organization, and similar to human RIG-I in (i) mediating virus- and IFN-activated gene expression, (ii) antiviral signalling, (iii) temperature dependence, and (iv) recognition of RNA ligands. Moreover, RIG-I of Rousettus aegyptiacus (suborder Yinpterochiroptera) and of humans were found to recognize SARS-CoV-2 infection. Thus, members of both bat suborders encode RIG-Is that are comparable to their human counterpart. The ability of bats to harbour zoonotic viruses therefore seems due to other features.

immunology↗