Biology subjects

Wolf, R.

Publications and source records attributed to Wolf, R..

11 recordsLinked to original sources

bioRxiv2026

Development and characterisation of an optimised in vitro differentiation protocol for deriving hepatocyte-like cells from mouse embryonic stem cells

IntroductionReliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. MethodsHepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. ResultsMorphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. DiscussionThese findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

cell biology↗

bioRxiv2022

Defining the in vivo mechanism of air pollutant toxicity using murine stress response biomarkers.

BackgroundAir pollution can cause a wide range of serious human diseases. For the informed instigation of interventions which prevent these outcomes there is an urgent need to develop robust in vivo biomarkers which provide insights into mechanisms of toxicity and relate pollutants to specific adverse outcomes. ObjectiveTo exemplify the application of in vivo stress response reporters in establishing mechanisms of air pollution toxicity and the application of this knowledge in epidemiological studies and potentially in disease prevention. MethodsMurine stress-reporter models (oxidative stress/inflammation, DNA damage and Ah receptor -AhR-activity) and primary mouse and primary human nasal cells were exposed to chemicals present in diesel exhaust emissions, particulate matter (PM) standards (PM2.5-SRM2975, PM10-SRM1648b) or fresh roadside PM10. Stress reporter activity was analysed by luminescence assays and histochemical approaches in a panel of murine tissues. Biochemical, genetic and pharmacological approaches were used to establish the mechanism of the stress responses observed. Pneumococcal adhesion was assessed in exposed primary human nasal epithelial cells (HPNEpC). ResultsNitro-PAHs induced Hmox1 and CYP1a1 reporters in a time- and dose-dependent, cell- and tissue-specific manner. NRF2 pathway mediated this Hmox1-reporter induction. SRM1658b, but not SRM2975, was a potent inducer of NRF2-dependent Hmox1 reporter activity in lung macrophages. Combined use of HPNEpC and in vivo reporters demonstrated that London roadside PM10 particles induced pneumococcal infection in HPNEpC mediated by oxidative stress responses. DiscussionThe combined use of in vivo reporter models with HPNEpC provides a robust approach to define the relationship between air pollutant exposure and health risks. These models can be used to hazard ranking environmental pollutants by considering the complexity of mechanisms of toxicity. These data will facilitate the relationship between toxic potential and the level of pollutant exposure in populations to be established and potentially extremely valuable tools for intervention studies.

pharmacology and toxicology↗

bioRxiv2022

Role of 5-HT2A, 5-HT2C, 5-HT1A and TAAR1 receptors in the head twitch response induced by 5-hydroxytryptophan and psilocybin: Translational implications.

There is increasing interest in the therapeutic potential of psilocybin in psychiatric disorders. In common with other serotonergic psychedelics, psilocybin is thought to act via the 5-HT2A receptor (5-HT2AR). Serotonin is the endogenous ligand of 5-HTR. In rodents, the serotonin precursor, 5-hydroxytryptophan (5-HTP), and psilocybin, induce a characteristic head twitch response (HTR), which is correlated with the human psychedelic trip in intensity and duration. We examined the role of other serotonergic receptors and the trace amine associated receptor 1 (TAAR1) in modulating HTR induced by 5-HTP and psilocybin. Male C57BL/6J mice (11 weeks old, ~30g) were administered 5-HTP, 50-250 mg/kg intraperitoneally (i.p.) or 200 mg/kg i.p. after pretreatment with 5-HT/TAAR1 receptor modulators. Psilocybin was administered at 0.1-51.2 mg/kg i.p. or at 4.4 mg/kg i.p. preceded by 5-HT/TAAR1 receptor modulators. HTR was assessed in a custom-built magnetometer. 5-HTP and psilocybin induced a dose dependent increase in the frequency of HTR over 20 minutes with attenuation by the 5-HT2AR antagonist, M100907 (volanserin), and the 5-HT1AR agonist, 8-OH-DPAT. The 5-HT2CR antagonist, RS102221, enhanced HTR at lower doses but reduced it at higher doses for 5-HTP and psilocybin. The TAAR1 antagonist, EPPTB, reduced 5-HTP-but not psilocybin-induced HTR. We have confirmed the key role of 5-HT2AR in HTR and have demonstrated an effect of 5-HT1AR and a bimodal contribution of 5-HT2CR as well as a role of TAAR1 in modulating HTR induced by 5-HTP. Compounds that modulate HTR induced by psychedelics have a potentially important role in the emerging therapeutic use of these compounds. Significance StatementWe have confirmed the key role of 5-HT2AR in in the induction of HTR by 5-HTP and psilocybin, have demonstrated the effect of a 5-HT1AR agonist to attenuate HTR and a bimodal contribution of 5-HT2CR as well as a role of TAAR1 in modulating HTR induced by 5-HTP. Compounds that modulate HTR induced by psychedelics have a potentially important role in the emerging therapeutic use of these compounds. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/501026v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1812b88org.highwire.dtl.DTLVardef@1511eb4org.highwire.dtl.DTLVardef@b6c868org.highwire.dtl.DTLVardef@1f3292_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities

A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of -diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere -diversity loss, but can be driven by {beta}-diversity decay across space and time.

ecology↗

bioRxiv2021

Human ventromedial prefrontal cortex lesions enhance expectation-related pain modulation

Pain is strongly modulated by expectations and beliefs. Research across species indicates that subregions of the ventromedial prefrontal cortex (VMPFC) play a fundamental role in learning and generating predictions about valued outcomes. Consistent with this overarching framework, neuroimaging studies of experimental pain indicate that VMPFC activation tracks expectations of pain relief and statistically mediates expectation-related reductions in responses to painful stimuli across a distributed pain processing network. However, lesion studies in preclinical models and in humans with refractory chronic pain suggest that VMPFC may play a more general role in representing the affective and motivational qualities of pain that contribute to its strong aversive drive. To test whether VMPFC is necessary for pain processing in general, or instead plays a more specific role in the modulation of pain by expectations, we studied responses to experimental heat pain in five adults with bilateral surgical lesions of VMPFC and twenty healthy adults without brain damage. All participants underwent quantitative sensory testing (QST) to characterize pain sensitivity, followed by a pain expectancy task. Participants were instructed that auditory cues would be followed by heat calibrated to elicit low or high pain. Following a conditioning phase, each cue was intermittently paired with a single temperature calibrated to elicit moderate pain. We compared ratings of moderate heat stimuli and subjective expectancy ratings as a function of cue to evaluate whether VMPFC lesions impact cue-based expectancy and expectancy effects on pain intensity and unpleasantness. We also analyzed QST measures to evaluate whether VMPFC lesions were associated with overall shifts in pain sensitivity. Compared to adults without brain damage, individuals with VMPFC lesions reported larger differences in expectations as a function of pain-predictive cues, and stronger cue-based modulation of pain ratings, particularly for ratings of pain unpleasantness. There were no group differences in pain sensitivity, nor in the relationship between pain and autonomic arousal, indicating that the impact of VMPFC lesions is specific to expectancy-based modulation of pain unpleasantness. Our findings suggest that the VMPFC is not essential for basic subjective and physiological responses to painful stimuli. Rather, our findings of significantly enhanced cue- related expectancy effects may suggest that VMPFC plays an important role in updating expectations or integrating sensory information with expectations to guide subjective judgements about pain. Taken together, these results expand our understanding VMPFCs contribution to pain and highlight the role of VMPFC in integrating cognitive representations with sensory information to yield affective responses.

neuroscience↗

bioRxiv2021

A parasite's paradise: Biotrophic species prevail oomycete community composition in tree canopies

Oomycetes (Stramenopiles, Protista) are among the most severe plant pathogens, comprising species with a high economic and ecologic impact on forest ecosystems. Their diversity and community structures are well studied in terrestrial habitats, but tree canopies as huge and diverse habitats have been widely neglected. A recent study highlighted distinct oomycete communities in the canopy region compared to forest soils when taking oomycete abundances into account, in contrast to the homogeneity at the incidence level. It remains however unknown if this homogeneity also leads to a functional homogenisation among microhabitats. In this study, we supplemented functional traits to oomycete canopy and ground communities, which were determined over a time period of two years with a metabarcoding approach. Our results showed that even though most oomycetes occurred in all habitats, a strong discrepancy between the strata and correspondingly the distribution of oomycete lifestyles could be observed, which was constant over time. Obligate biotrophic species, exclusively feeding on living host tissue, dominated the canopy region, implying tree canopies to be a hitherto neglected reservoir for parasitic protists. Parasites highly specialised on hosts that were not sampled could be determined in high abundances in the canopy and the surrounding air, challenging the strict host dependencies ruled for some oomycetes. Our findings further contribute to the understanding of oomycete ecosystem functioning in forest ecosystems.

microbiology↗

bioRxiv2021

On the phenology of protists: Recurrent patterns reveal seasonal variation of protistan (Rhizaria: Cercozoa, Endomyxa) communities in tree canopies

Tree canopies are colonized by billions of highly specialized microorganisms that are well adapted to the extreme microclimatic conditions, caused by diurnal fluctuations and seasonal changes. In this study we investigated seasonality patterns of protists in tree canopies of a temperate floodplain forest via high-throughput sequencing with group-specific primers for the phyla Cercozoa and Endomyxa. We observed consistent seasonality and identified divergent spring and autumn taxa. Tree crowns were characterized by a dominance of bacterivores and omnivores, while eukaryvores gained a distinctly larger share in litter and soil communities on the ground. Seasonality was largest among communities detected on the foliar surface. Higher variance within alpha diversity of foliar communities in spring indicated greater heterogeneity during community assembly. However, communities underwent distinct changes during the aging of leaves in autumn, reflecting recurring phenological changes during microbial colonization of leaves. Surprisingly, endomyxan root pathogens appeared to be exceptionally abundant across tree canopies during autumn season, demonstrating a potential role of the canopy surface as an important reservoir for wind-dispersed propagules. Overall, about 80% of detected OTUs could not be assigned to known species - representing only a fraction of dozens of microeukaryotic taxa whose canopy inhabitants are waiting to be discovered.

ecology↗

bioRxiv2020

To the canopy and beyond: Air samples reveal wind dispersal as a driver of ubiquitous protistan pathogen assembly in tree canopies

We analyzed air dispersal of the protistan phyla Cercozoa and Oomycota with an air sampler near the ground (~2 m) and in tree crowns (~25 m) of three tree species (oak, linden and ash) in a temperate floodplain forest in March (before leafing) and May (after leaf unfolding) with a cultivation-independent high throughput metabarcoding approach. Both, Cercozoa and Oomycota, contain important pathogens of forest trees and other vegetation. We found a high diversity of Cercozoa and Oomycota in air samples with 122 and 81 OTUs, respectively. Especially oomycetes showed a high temporal variation in beta diversity between both sampling dates. Differences in community composition between air samples in tree canopies and close to the ground were however negligible, and also tree species identity did not affect communities in air samples, indicating that the distribution of protistan propagules through the air was not spatially restricted in the forest ecosystem. OTUs of plant pathogens, whose host species that did not occur in the forest, demonstrate wind dispersal of propagules from outside the forest biome. Overall, our results lead to a better understanding of the stochastic processes of wind dispersal of protists and protistan pathogens, a prerequisite to understand the mechanisms of their community assembly in forest ecosystems. ImportanceWind dispersal has been shown to play a crucial role in protistan community assembly. The protistan taxa Cercozoa and Oomycota contain important plant parasites with a major ecologic and economic impact. However, comprehensive assessments of cercozoan and oomycete diversity in forest air samples were lacking. Using a cultivation-independent high throughput metabarcoding approach, we analyzed cercozoan and oomycete air dispersal in forest floors and the canopy region - a potential filter for microbial propagules. Our study provides insights into the diversity and community assembly of protists within the air, contributing to a better understanding which factors drive the distribution of plant pathogens within forest ecosystems.

ecology↗

bioRxiv2019

Freewater EstimatoR using iNtErpolated iniTialization (FERNET): Toward Accurate Estimation of Free Water in Peritumoral Region Using Single-Shell Diffusion MRI Data

Characterization of healthy versus pathological tissue is a key concern when modeling tissue microstructure in the peritumoral area, confounded by the presence of free water (e.g., edema). Most methods that model tissue microstructure are either based on advanced acquisition schemes not readily available in the clinic, or are not designed to address the challenge of edema. This underscores the need for a robust free water elimination (FWE) method that estimates free water in pathological tissue but can be used with clinically prevalent single-shell diffusion tensor imaging data. FWE in single-shell data requires the fitting of a bi-compartment model, which is an ill-posed problem. Its solution requires optimization, which relies on an initialization step. We propose a novel initialization approach for FWE, FERNET, which improves the estimation of free water in edematous and infiltrated peritumoral regions, using single-shell diffusion MRI data. The method has been extensively investigated on simulated data and healthy and brain tumor datasets, demonstrating its applicability on clinically acquired data. Additionally, it has been applied to data from brain tumor patients to demonstrate the improvement in tractography in the peritumoral region.

bioengineering↗

bioRxiv2019

Development of a hybrid Bayesian network model for predicting acute fish toxicity using multiple lines of evidence

A Bayesian network was developed for predicting the acute toxicity intervals of chemical substances to fish, based on information on fish embryo toxicity (FET) in combination with other information. This model can support the use of FET data in a Weight-of-Evidence (WOE) approach for replacing the use of juvenile fish. The BN predicted correct toxicity intervals for 69%-80% of the tested substances. The model was most sensitive to components quantified by toxicity data, and least sensitive to components quantified by expert knowledge. The model is publicly available through a web interface. Further development of this model should include additional lines of evidence, refinement of the discretisation, and training with a larger dataset for weighting of the lines of evidence. A refined version of this model can be a useful tool for predicting acute fish toxicity, and a contribution to more quantitative WOE approaches for ecotoxicology and environmental assessment more generally.\n\nHighlightsO_LIA Bayesian network (BN) was developed to predict the toxicity of chemicals to fish\nC_LIO_LIThe BN uses fish embryo toxicity data in a quantitative weight-of-evidence approach\nC_LIO_LIThe BN integrates physical, chemical and toxicological properties of chemicals\nC_LIO_LICorrect toxicity intervals were predicted for 69-80% of test cases\nC_LIO_LIThe BN is publicly available for demonstration and testing through a web interface\nC_LI

pharmacology and toxicology↗

bioRxiv2018

Altering the temporal regulation of one transcription factor drives sensory trade-offs

Size trade-offs of visual versus olfactory organs is a pervasive feature of animal evolution. Comparing Drosophila species, we find that larger eyes correlate with smaller antennae, where olfactory organs reside, and narrower faces. We demonstrate that this tradeoff arises through differential subdivision of the head primordium into visual versus non-visual fields. Specification of the visual field requires a highly-conserved eye development gene called eyeless in flies and Pax6 in humans. We discover that changes in the temporal regulation of eyeless expression during development is a conserved mechanism for sensory trade-offs within and between Drosophila species. We identify a natural single nucleotide polymorphism in the cis-regulatory region of eyeless that is sufficient to alter its temporal regulation and eye size. Because Pax6 is a conserved regulator of sensory placode subdivision, we propose that alterations in the mutual repression between sensory territories is a conserved mechanism for sensory trade-offs in animals.

developmental biology↗