Biology subjects

Roth, C.

Publications and source records attributed to Roth, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Less is more: Hormetic and selective antimicrobial activity of a thymoquinone-standardized black seed oil ThymoQuin in gut microbiota models

The gut microbiome plays a central role in host metabolism, immune function, and overall health, with disruptions in microbial composition (dysbiosis) being associated with a range of metabolic, inflammatory, and infectious conditions [1,2]. Consequently, strategies aiming to modulate the microbiome require selective activity that preserves beneficial commensals while limiting pathogenic organisms [3]. In this context, ThymoQuin(R)--a cold-pressed, standardized black cumin (Nigella sativa) seed oil developed by TriNutra Ltd. and defined by [≥]3% thymoquinone (TQ), controlled p-cymene levels, and low free fatty acids ([≤]1.25%)--was evaluated for its microbiome-relevant activity. In vitro minimum bactericidal concentration (MBC) assays across three independent batches demonstrated a biphasic, dose-dependent response. At intermediate concentrations (0.25-0.5%), Streptococcus thermophilus was strongly stimulated (up to 53-fold) and Lactiplantibacillus plantarum fully preserved, while Klebsiella pneumoniae was effectively reduced (>94%). Akkermansia muciniphila exhibited stable viability at concentrations below 1%, with reductions only observed at 1%. This is notable given its role as a mucin-degrading commensal that has been linked to metabolic health, but whose abundance may vary across physiological and disease contexts [4,5]. At concentrations [≥]1%, selective effects diminished, resulting in broader antimicrobial activity and reduced specificity. These findings indicate a defined concentration range in which selective microbiome modulation is maintained, whereas higher thymoquinone levels may increase the risk of non-selective detrimental effect on microbes.

microbiology↗

bioRxiv2026

Conformational dynamics of plasmepsin X during inhibitor binding

The aspartic protease plasmepsin X (PMX) of the parasite Plasmodium is a promising drug target for novel malaria therapies. Two potent inhibitors of PMX are WM382 and WM4, which both include a guanidinium group that is in contact with the two catalytic aspartates of PMX in the bound complexes. In structural representations of the inhibitors, the guanidinium group is typically depicted as uncharged. However, pKa predictions with standard tools presented in this article indicate that the guanidinium groups of WM382 and WM4 are protonated and, thus, positively charged in the bound complexes. This positive charge is counterbalanced by a negatively charged catalytic aspartate D266 in PMX of Plasmodium falciparum, while the second catalytic aspartate D457 is uncharged. To investigate the interplay of the conformational dynamics of PMX and inhibitor (un)binding, we performed Hamiltonian replica exchange molecular dynamics (H-REMD) simulations starting from the predicted protonation state of the PMX-WM382 complex. On eight unbinding pathways enabled by weakened interactions of PMX and WM382 in the H-REMD simulations, we observed a dominant route of exit of the inhibitor from the binding pocket with a coupling to conformational changes in the "flap" of PMX, a {beta}-hairpin located above the binding pocket. In the bound complex, the flap adopts a closed conformation in which it tightly interacts with and covers the inhibitor. On the dominant route observed in our simulations, unbinding involves an open conformation of the flap that allows the inhibitor to exit the binding pocket. After unbinding, the flap adopts an occluded conformation in which the binding site is blocked by a bulky aromatic sidechain.

biophysics↗

bioRxiv2025

Various Optimization Strategies for the Isolation of Mitochondria from Sprague-Dawley Rat Liver Tissue

Mitochondria are key organelles that establish a very large free energy drop for the ATP hydrolysis reaction in the cytoplasm of cells, which provides energy required for the cell maintenance of homeostasis. They are commonly isolated from living tissue as it makes them easier to interrogate at the biochemical level. Thus, isolated mitochondria of sufficient quality are desired to improve translational impact. To gain a greater insight into the isolation process and optimize our current isolation protocol for quality, we implemented various fine-tuning modifications to our standard homogenization, centrifugation, and purification steps with isolated rat hepatocyte mitochondria. The following modifications we tested were: i) different homogenization speeds (10,000, 14,000, and 18,000 rpm) with varying time intervals (10, 20, and 30 sec), ii) addition of an additional purification spin, and iii) use of density gradients to further purify the isolated mitochondria from non-mitochondrial contaminants. Mitochondrial quality was approximated using the well-established respiratory control ratio (RCR). The data reveal that our original protocol yields isolated mitochondria with acceptable quality and the optimization attempts produced similar or worse mitochondrial isolates. In addition, an extra purification spin decreased RCR values and therefore is not recommended. We note that the use of density gradients did not improve the RCR, but it did remove presumed peroxisomal contamination. While this protocol can be further enhanced using additional metrics, the data indicate that our current isolation protocol is sufficiently effective since additional modifications did not yield major improvements.

biochemistry↗

bioRxiv2025

An Integrative Approach to Develop and Characterise Antibodies Against the Cancer Associated Antigen Sialyl Lewis A (CA 19-9)

BackgroundSialyl Lewis A (sLeA), or the CA 19-9 marker, is a tetrasaccharide and a tumour-associated carbohydrate antigen (TACA) overexpressed and abnormally secreted as a serum-borne marker in gastrointestinal malignancies. CA 19-9 is the best validated and only FDA-approved serologic marker clinically used to monitor recurrence, progression, and therapy efficiency in pancreatic ductal adenocarcinoma (PDAC) patients. Due to its altered expression on cancer cells, sLeA is also an attractive target for antibody development. Although recent clinical trials have demonstrated insufficient efficacy of the fully human anti-sLeA 5B1 (MVT-5873) format as a stand-alone drug or an adjuvant therapy in PDAC [1], its safety profile and unique expression in additional malignancies keep CA 19-9 an attractive TACA. Hence, we set out to explore the use of synthetic sLeA to develop novel monoclonal antibodies (mAbs) with improved sLeA recognition and better efficacy. MethodsTwo mAbs targeting sLeA were generated through mice immunisation with synthetic sLeA glycoconjugates, synthetic glycan arrays, and hybridoma technology. We then compared the antigen-binding properties of the newly developed mAbs with the widely used mAb 1116-NS-19- 9 via synthetic glycan arrays, immunohistochemistry (IHC), X-ray crystallography, molecular dynamics (MD) simulation, and Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) spectroscopy. ResultsThe newly generated mAbs demonstrated improved affinity and specificity for both synthetic and native sLeA, surpassing the performance of the established mAb 1116-NS-19-9. First, synthetic glycan arrays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) assays confirmed superior antigen-binding properties to synthetic sLeA. In particular, the mAb designated GB11 demonstrated markedly enhanced binding to native sLeA ectopically expressed in B16 melanoma cells. To elucidate the structural origin of GB11s improved antigen binding, we conducted high-resolution mapping of the molecular recognition patterns between sLeA and the different antibodies using X-ray crystallography and STD NMR. These analyses revealed subtle yet critical differences in the glycan engagement and identified key structural features underlying GB11s enhanced recognition of sLeA. MD simulations further supported these observations, indicating distinct orientations of sLeA within the binding pockets of each mAb. ConclusionOur results suggest better recognition of the sLeA antigen by the newly generated GB11 antibody and provide a detailed high-resolution elucidation of the molecular interactions behind it. Our study may provide a novel tool with improved theranostic properties against sLeA-overexpressing malignancies.

cancer biology↗

bioRxiv2025

Two genetically linked Arabidopsis TIR-type NLRs are required for immunity and interact with NLRs encoded in a segmentally duplicated genomic region

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors detect pathogen-secreted effectors inside host cells and induce a robust immune response, typically involving hypersensitive cell death. NLR genes are often genetically linked and can function as pairs or within larger NLR networks. We previously showed that the truncated Toll/Interleukin-1 Receptor (TIR)-type NLR TIR-NB13 (TN13) is required for resistance of Arabidopsis to Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. TN13 is genetically linked to a full length TIR-NB-LRR (TNL) gene on chromosome 3. Here, we show that TN13, and its genetically linked TNL both localize to the ER membrane via N-terminal transmembrane domains, are required for resistance to Pst DC3000 ({Delta}AvrPto/AvrPtoB) and interact with each other in transient expression assays in Nicotiana benthamiana. In contrast to TN13, the full length TNL, which we named TN13-INTERACTING TNL1 (TNT1), induces an autoactive cell death response when expressed in N. benthamiana that depends on an atypical MHV motif in its NB-ARC domain, as well as the EDS1/SAG101/NRG1 module. TN13 and TNT1 furthermore interact with phylogenetically related NLRs encoded by a segmentally duplicated region on chromosome 5. Our data suggest that both TN13 and the genetically linked TNT1 could be part of a larger TIR-type NLR immune regulatory network, in which TNT1 contributes to basal immunity and might function as an autoactive death switch to induce cell death upon pathogen detection. SIGNIFICANCE STATEMENTThe ER membrane localized truncated TIR-NLR TN13 and the genomically linked full length TNL TNT1 are required for plant disease resistance and form heteromeric associations with phylogenetically related NLRs, encoded by a segmentally duplicated chromosomal region.

plant biology↗

bioRxiv2025

Human Lung Alveolar Model with an Autologous Innate and Adaptive Immune Compartment

Lung-resident immune cells, spanning both innate and adaptive compartments, preserve the integrity of the respiratory barrier, but become pathogenic if dysregulated1. Current in vitro organoid models aim to replicate interactions between the alveolar epithelium and immune cells but have not yet incorporated lung-specific immune cells critical for tissue residency2. Here we address this shortcoming by describing human lung alveolar immuno-organoids (LIO) that contain an autologous tissue-resident lymphoid compartment, primarily composed of tissue-resident memory T cells (TRMs). Additionally, we introduce lung alveolar immuno-organoids with myeloid cells (LIOM), which include both TRMs and a macrophage-rich alveolar myeloid compartment. The resident immune cells formed a stable immune-epithelial system, frequently interacting with the epithelium and promoting a regenerative alveolar transcriptomic profile. To understand how dysregulated inflammation perturbed the respiratory barrier, we simulated T-cell-mediated inflammation in LIOs and LIOMs and used single-cell transcriptomic analyses to uncover the molecular mechanisms driving immune responses. The presence of innate cells induced a shift in T cell identity from cytotoxic to immunosuppressive, reducing epithelial cell killing and inflammation. Based on insights obtained with bulk RNA-seq data from the phase 3 IMpower150 trial, we tested whether LIOM cultures could model clinically-relevant but poorly understood pulmonary side effects caused by immunotherapies such as the checkpoint inhibitor atezolizumab3. We observed a decrease in immunosuppressive T cells and identified gene signatures that matched the transcriptomic profile of patients with drug-induced pneumonitis. Given its effectiveness in capturing outcomes and mechanisms associated with a prevalent pulmonary disease, this system unlocks opportunities for studying a wide range of immune-related pathologies in the lung.

cell biology↗

bioRxiv2024

A humanized neuronal model system reveals key roles for manganese in neuronal endocytosis, calcium flux and mitochondrial bioenergetics

Manganese (Mn) is an essential trace metal that is necessary for life. Its duality as both a crucial micronutrient and potential neurotoxicant necessitates tight control of intracellular and extracellular Mn levels. Dysregulation of Mn is implicated in a broad range of human diseases, from neurodevelopmental sequelae related to Mn levels in drinking water, to acquired forms of manganism, rare inherited Mn transportopathies and more common disorders such as Parkinsons and Alzheimers disease. Despite the clear association between Mn dysregulation and neurodevelopmental or neurodegenerative diseases, the underlying cellular mechanisms that govern neuropathology remain poorly understood. We established an induced pluripotent stem cells-derived midbrain neuronal system from SLC39A14, SLC39A8, and SLC30A10 patients to better understand the neuronal sequelae of Mn dysregulation. By integrating transcriptomic and functional approaches, we show that Mn dyshomeostasis leads to dysregulation of key cellular pathways that are crucial to normal neuronal function, including defects in mitochondrial bioenergetics, calcium signalling, endocytosis, and glycosylation, as well as cellular stress and early neurodegeneration. Our humanized model has enhanced understanding of the role of Mn in the human brain, and the consequences of both acquired and genetic disorders associated with Mn dysregulation. Better understanding of these underlying pathophysiological processes will identify potential targets for future therapeutic intervention.

neuroscience↗

bioRxiv2024

An in vivo and in vitro spatiotemporal atlas of human midbrain development

The dopaminergic system has key roles in human physiology and is implicated in a broad range of neurological and neuropsychiatric conditions that are increasingly investigated using induced pluripotent stem cell-derived midbrain models. To determine the similarity of such models to human systems, we undertook single cell and spatial profiling of first and second trimester fetal midbrain and compared it to in vitro midbrain models. Our initial histological analysis of second trimester fetal midbrain revealed structural complexity already similar to that of adult tissue, although this similarity did not fully extend to transcriptional activity. Moreover, we show that in vitro models recapitulate the transcriptional activity of late first trimester fetal midbrain, while 3D models replicate the spatial organization and cellular microenvironments of first and second trimester fetal midbrain. Understanding the extent of human tissue recapitulation in midbrain laboratory models is essential to justify their use as biological proxies.

neuroscience↗

bioRxiv2024

Epigenomic manipulation reveals the relationship between locus specific chromatin dynamics and gene expression

Dysregulation of epigenetic processes leads to a plethora of abnormalities including disease states such as cancer. Therapies focused on epigenetic modulation alter gene expression to correct dysfunction, though the mechanisms and perpetuation of these states is unknown. Here, we use integrated epigenomics and three-dimensional chromatin structure-function analyses after acute histone deacetylase inhibitor cancer drug treatment (suberoylanilide hydroxamic acid in lung cancer cells). Treatment induced substantial (13%) genomic rearrangement that rebounds despite persistent gene expression changes and spreading of acetylation. The chromatin functional landscape (accessibility, active transcription modification, and gene expression) is controlled and locus-specific, while chromatin contacts are globally altered resulting in a moderate weakening of topologically associating domains. Chromatin states are more dynamic at transcriptionally active loci while genes with reduced expression are epigenetically stable suggesting chromatin architectural turnover and nucleosome remodeling is locus-specific and underlies the bidirectional expression changes. Thus, local 3D chromatin and genome structural dynamics is integral for loci regulation in response to epigenomic perturbation. The partial persistence of these altered features may have larger implications for efficacy of epigenetic drugs in amelioration of disease states.

cancer biology↗

bioRxiv2024

Role of van der Waals, electrostatic, and hydrogen-bond interactions for the relative stability of cellulose Iβ and II crystals

Naturally occuring cellulose I{beta} with its characteristic parallel orientation of cellulose chains is less stable than cellulose II, in which neighbouring pairs of chains are oriented antiparallel to each other. While the distinct hydrogen-bond patterns of these two cellulose crystal forms are well established, the energetic role of the hydrogen bonds for crystal stability, in comparison to the van der Waals and overall electrostatic interactions in the crystals, is a matter of current debate. In this article, we investigate the relative stability of cellulose I{beta} and II in energy minimizations with classical force fields. We find that the larger stability of cellulose II results from clearly stronger electrostatic interchain energies that are only partially compensated by stronger van der Waals interchain energies in cellulose I{beta}. In addition, we show that a multipole description of hydrogen bonds that includes the whole COH groups of donor and acceptor oxygen atoms leads to consistent interchain hydrogen-bond energies that account for roughly 70% and 75% of the interchain electrostatics in cellulose I{beta} and II, respectively.

biophysics↗

bioRxiv2023

Vaccinia virus infection induces concurrent alterations in host chromatin architecture, accessibility, and gene expression

Genomic DNA folds into complex configurations that produce particular local and global structures thought to profoundly impact genome function. To understand the dynamic nature of this relationship, we investigated the extent of host chromatin structural and functional changes in response to a viral agent. We performed comprehensive assessments of host architecture (Hi-C), accessibility (ATAC-seq), and gene expression (RNA-seq) in a paired manner in response to attenuated vaccinia (smallpox) virus. Over time, infection significantly increased long-range intra-chromosomal interactions and decreased chromatin accessibility. Fine-scale accessibility changes were independent of broad-scale chromatin compartment exchange, which increased (up to 12% of the genome) over time, underscoring potential independent mechanisms for global and local chromatin reorganization. The majority of differentially expressed genes, including those downregulated in immune responses, had concurrent alterations in local accessibility and loop domain restructuring. Increased B compartmentalization, intra-chromosomal interactions, and decreased inter-chromosomal interactions and chromatin accessibility together indicate that infection converts the host genome into a more condensed state with nearly equal bidirectional differential gene expression. These changes in host chromatin features may have implications for developing efficacious anti-viral countermeasures. Overall, our empirical data provides evidence of orchestrated concurrent alterations in chromatin architecture, accessibility, and gene expression in response to infection, further reinforcing the notion of coordinated structure-function dynamics of the genome.

genomics↗

bioRxiv2023

Simulating Interclonal Interactions in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of cancers, accounting for 37% of B-cell tumors globally. DLBCL is known to be a heterogeneous disease, resulting in variable clinical presentations and the development of drug resistance. One underexplored aspect of drug resistance is the evolving dynamics between parental and drug-resistant clones with the same microenvironment. In this work, the effects of interclonal interactions between two cell populations - one sensitive to treatment and another resistant to treatment - on tumor growth behaviors were explored through a mathematical model. In vitro cultures of mixed DLBCL populations demonstrated cooperative interactions and revealed the need for modifying the model to account for complex interactions. Multiple best-fit models derived from in vitro data indicated a difference in steady-state behaviors based on therapy administrations in simulations. The model and methods may serve as a tool in understanding the behaviors of heterogeneous tumors and in identifying the optimal therapeutic regimen to eliminate cancer cell populations using computer-guided simulations. ImportanceThe cellular makeup of tumors can play a vital role in its growth and cancer development. In this work, two different types of cell populations of diffuse large B-cell lymphoma (DLBCL) were studied together to understand how they interact with each other in cultures. In mixed cultures, both types of cells cooperated with each other and increased their growth in complex manners. A mathematical model was created to simulate the growth behavior of mixed cultures. The model can potentially be used to predict future cell behavior and help in identifying more effective therapy regimens to maximize tumor cell reduction.

cancer biology↗

bioRxiv2023

Diffusion Tensor Imaging: Influence of segmentation on fiber tracking in the supraspinatus muscle - an inter-operator reliability analysis

The ability of muscle to generate force depends on its architecture and quality. MR-based diffusion tensor imaging of muscle (mDTI) is an innovative approach for showing the fiber arrangement for the whole muscle volume. For accurate calculations of fiber metrics, muscle segmentation prior to tractography seems mandatory. Since segmentation is known to be operator dependent, it is important to understand how segmentation affects tractography. The aim of this study was to compare the results of deterministic fiber tracking based on muscle models generated by two independent operators. In addition, this study compares the results with a segmentation-free approach. Fifteen subjects underwent mDTI of the right shoulder. The results showed that mDTI can be successfully applied to complex joints such as the human shoulder. Furthermore, operators segmentation did not influence the results of fiber tracking and showed excellent intraclass correlation estimates ([≥] 0.975). As an exploratory approach, the segmentation-free fiber tracking showed significant differences in terms of mean fascicle length. Based on these findings, we assume that tractography does not require a detailed muscle segmentation. Furthermore, it implies that mDTI and automatic segmentation approaches or even a segmentation-free analysis can be considered for evaluation of muscle changes.

physiology↗

bioRxiv2023

Novel taxonomic and functional diversity of bacteria from the upper digestive tract of chicken

Strategies to increase the production rate of chicken for human consumption alter the natural process of microbial colonisation and the nutritional performance of the animal. The lack of sufficient reference genomes limits the interpretation of sequencing data and restrain the study of complex functions. In this study, 43 strains obtained from crop, jejunum and ileum of chicken were isolated, characterised and genome analysed to observe their metabolic profiles, adaptive strategies and to serve as future references. Eight isolates represent new species that colonise the upper gut intestinal tract and present consistent adaptations that enable us to predict their ecological role, expanding our knowledge on the adaptive functions. Molecular characterisation confirmed the classification of Clostridium anaerovisceris sp. nov. (DSM 113860= LMG 32675), Clostridium butanolproducens (DSM 115076= LMG 32878), Ligilactobacillus hohenheimius sp. nov. (DSM 113870= LMG 32876), Limosilactobacillus gallus sp. nov. (DSM 113833 =LMG 32623), Limosilactobacillus avium sp. nov. (DSM 113849= LMG 32671), Limosilactobacillus pullum sp. nov. (DSM 115077 =LMG 32877), Limosilactobacillus digestus sp. nov. (DSM113835= LMG 32625) and Limosilactobacillus difficile sp. nov. (DSM 114195= LMG 32875). Strains of Limosilactobacillus were found to be more abundant in the crop, while Ligilactobacillus dominated the ileal digesta. Isolates from crop encode a high number of glycosidases specialized in complex polysaccharides compared to strains isolated from jejunum and ileum. These results represent the first approach for the isolation and detection of bacteria from the chickens upper digestive tract and describe the functional diversity of bacteria inhabiting these regions, improving the potential handling of chicken microbiota with biotechnological applications.

microbiology↗

bioRxiv2023

Improved Quality Metrics for Association and Reproducibility in Chromatin Accessibility Data Using Mutual Information

BackgroundCorrelation metrics are widely utilized in genomics analysis and often implemented with little regard to assumptions of normality, homoscedasticity, and independence of values. This is especially true when comparing values between replicated sequencing experiments that probe chromatin accessibility, such as assays for transposase-accessible chromatin via sequencing (ATAC-seq). Such data can possess several regions across the human genome with little to no sequencing depth and are thus non-normal with a large portion of zero values. Despite distributed use in the epigenomics field, few studies have evaluated and benchmarked how correlation and association statistics behave across ATAC-seq experiments with known differences or the effects of removing specific outliers from the data. Here, we developed a computational simulation of ATAC-seq data to elucidate the behavior of correlation statistics and to compare their accuracy under set conditions of reproducibility. ResultsUsing these simulations, we monitored the behavior of several correlation statistics, including the Pearsons R and Spearmans{rho} coefficients as well as Kendalls{tau} and Top-Down correlation. We also test the behavior of association measures, including the coefficient of determination R2, Kendalls W, and normalized mutual information. Our experiments reveal an insensitivity of most statistics, including Spearmans{rho} , Kendalls{tau} , and Kendalls W, to increasing differences between simulated ATAC-seq replicates. The removal of co-zeros (regions lacking mapped sequenced reads) between simulated experiments greatly improves the estimates of correlation and association. After removing co-zeros, the R2 coefficient and normalized mutual information display the best performance, having a closer one-to-one relationship with the known portion of shared, enhanced loci between simulated replicates. When comparing values between experimental ATAC-seq data using a random forest model, mutual information best predicts ATAC-seq replicate relationships. ConclusionsCollectively, this study demonstrates how measures of correlation and association can behave in epigenomics experiments. We provide improved strategies for quantifying relationships in these increasingly prevalent and important chromatin accessibility assays.

bioinformatics↗

bioRxiv2022

Amoeba Predation of Cryptococcus: A Quantitative and Population Genomic Evaluation of the Accidental Pathogen Hypothesis

The "Amoeboid Predator-Fungal Animal Virulence Hypothesis" posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal pathogens. In this hypothesis, selection to avoid predation by amoeba inadvertently selects for traits that contribute to fungal escape from phagocytic immune cells. Here, we investigate this hypothesis in the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans. Applying quantitative trait locus (QTL) mapping and comparative genomics, we discovered a cross-species QTL region that is responsible for variation in resistance to amoeba predation. In C. neoformans, this same QTL was found to have pleiotropic effects on melanization, an established virulence factor. Through fine mapping and population genomic comparisons, we identified the gene encoding the transcription factor Bzp4 that underlies this pleiotropic QTL and we show that decreased expression of this gene reduces melanization and increases susceptibility to amoeba predation. Despite the joint effects of BZP4 on amoeba resistance and melanin production, we find no relationship between BZP4 genotype and escape from macrophages or virulence in murine models of disease. Our findings provide new perspectives on how microbial ecology shapes the genetic architecture of fungal virulence, and suggests the need for more nuanced models for the evolution of pathogenesis that account for the complexities of both microbe-microbe and microbe-host interactions. Author summaryA prominent hypothesis for the evolution of many environmental pathogens proposes that opportunistic pathogenesis is an "accidental" by-product of selection to survive encounters with microbial predators. Chief among the predators that have been suggested as relevant to the evolution of virulence are phagocytic amoebae. Amoebae share many characteristics with macrophages and other primary immune cells that microbial pathogens encounter during infection of animal hosts. This has led to the suggestion that amoebae may act as "training grounds" for both bacterial and fungal pathogens. In this study we test key tenets of the accidental pathogen hypothesis by examining two related questions: "Do alleles important for survival in the face of amoeba predation correspond to known virulence genes? And does genetic variation that increases resistance to amoeba predation increase virulence potential?" We carried out quantitative trait locus (QTL) mapping in two species of the human fungal pathogen Cryptococcus and identified an orthologous QTL, shared by the two species, where allelic variation is a key predictor of resistance to amoeba predation. In C. neoformans we show that this QTL corresponds to a deletion upstream of a transcription factor gene, BZP4. Variation at BZP4 also predicts melanin synthesis, another trait implicated in Cryptococcus virulence. Although BZP4 genotype is a strong predictor of resistance to amoeba predation, we find no correlation between genetic variation at this locus and the ability to proliferate in macrophages or to kill animal hosts. Our findings suggest that the evolutionary landscape of fungal virulence is complex, and highlights the importance of accounting for natural genetic variation when evaluating evolutionary hypotheses.

genomics↗

bioRxiv2022

Differentiation of human induced pluripotent stem cells into cortical neural stem cells

Efficient and effective methods for converting human induced pluripotent stem cells (iPSC) into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells (NSCs). We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/{beta}-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of ICC Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

developmental biology↗

bioRxiv2022

Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogen Cryptococcus deneoformans

We recently reported transposon mutagenesis as a significant driver of spontaneous mutations in the human fungal pathogen Cryptococcus deneoformans during murine infection. Mutations caused by transposable element (TE) insertion into reporter genes were dramatically elevated at high temperature (37{degrees} versus 30{degrees}) in vitro, suggesting that heat stress stimulates TE mobility in the Cryptococcus genome. To explore the genome-wide impact of TE mobilization, we generated transposon accumulation lines by in vitro passage of C. deneoformans strain XL280 for multiple generations at both 30{degrees} and at the host-relevant temperature of 37{degrees}. Utilizing whole-genome sequencing, we identified native TE copies and mapped multiple de novo TE insertions in these lines. Movements of the T1 DNA transposon occurred at both temperatures with a strong bias for insertion between gene-coding regions. By contrast, the Tcn12 retrotransposon integrated primarily within genes and movement occurred exclusively at 37{degrees}. In addition, we observed a dramatic amplification in copy number of the Cnl1 (C. neoformans LINE-1) retrotransposon in sub-telomeric regions under heat-stress conditions. Comparing TE mutations to other sequence variations detected in passaged lines, the increase in genomic changes at elevated temperature was primarily due to mobilization of the retroelements Tcn12 and Cnl1. Finally, we found multiple TE movements (T1, Tcn12 and Cnl1) in the genomes of single C. deneoformans isolates recovered from infected mice, providing evidence that mobile elements are likely to facilitate microevolution and rapid adaptation during infection. Significance StatementRising global temperatures and climate change are predicted to increase fungal diseases in plants and mammals. However, the impact of heat stress on genetic changes in environmental fungi is largely unexplored. Environmental stressors can stimulate the movement of mobile DNA elements (transposons) within the genome to alter the genetic landscape. This report provides a genome-wide assessment of heat stress-induced transposon mobilization in the human fungal pathogen Cryptococcus. Transposon copies accumulated in genomes more rapidly following growth at the higher, host-relevant temperature. Additionally, movements of multiple elements were detected in the genomes of cryptococci recovered from infected mice. These findings suggest that heat stress-stimulated transposon mobility contributes to rapid adaptive changes in fungi both in the environment and during infection.

microbiology↗