Biology subjects

Mueller, C.

Publications and source records attributed to Mueller, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Comparison of the tolerability of terbium-161 and lutetium-177 in combination with somatostatin analogues in the preclinical setting

Purpose[177Lu]Lu-DOTATATE is an established somatostatin receptor (SSTR) agonist for the treatment of metastasized neuroendocrine neoplasms, while the SSTR antagonist [177Lu]Lu-DOTA-LM3 has only scarcely been employed in clinics. Impressive preclinical data obtained with [161Tb]Tb-DOTA-LM3 in tumor-bearing mice indicated the potential of terbium-161 as an alternative to lutetium-177. The aim of the present study was to compare the tolerability of 161Tb- and 177Lu-based DOTA-LM3 and DOTATATE in immunocompetent mice. MethodsDosimetry calculations were performed based on biodistribution data of the radiopeptides in immunocompetent mice. Treatment-related effects on blood cell counts were assessed on Days 10, 28 and 56 after application of [161Tb]Tb-DOTA-LM3 or [161Tb]Tb-DOTATATE at 20 MBq per mouse. These radiopeptides were also applied at 100 MBq per mouse and the effects compared to those observed after application of the 177Lu-labeled counterparts. Bone marrow smears, blood plasma parameters and organ histology were assessed at the end of the study. ResultsThe absorbed organ dose was commonly higher for the SSTR antagonist than for the SSTR agonist and for terbium-161 over lutetium-177. Application of a therapeutic activity level of 20 MBq [161Tb]Tb-DOTA-LM3 or [161Tb]Tb-DOTATATE was well tolerated without major hematological changes. The injection of 100 MBq of the 161Tb- and 177Lu-based somatostatin analogues affected the blood cell counts, however. The lymphocytes were 40-50% lower in treated mice compared to the untreated controls on Day 10 irrespective of the radionuclide employed. At the same timepoint, thrombocyte and erythrocyte counts were 30-50% and 6-12% lower, respectively, after administration of the SSTR antagonist (p<0.05) while changes were less pronounced in mice injected with the SSTR agonist. All blood cell counts were in the normal range on Day 56. Histological analyses revealed minimal abnormalities in the kidneys, liver and spleen of treated mice. No correlation was observed between the organ dose and frequency of the occurrence of abnormalities. ConclusionHematologic changes were more pronounced in mice treated with the SSTR antagonist than in those treated with the SSTR agonist. Despite the increased absorbed dose delivered by terbium-161 over lutetium-177, [161Tb]Tb-DOTA-LM3 and [161Tb]Tb-DOTATATE should be safe at activity levels that are recommended for their respective 177Lu-based analogues.

cancer biology↗

bioRxiv2024

Development of radiofluorinated MLN-4760 derivatives for PET imaging of the SARS-CoV-2 entry receptor ACE2

PurposeThe angiotensin converting enzyme 2 (ACE2) plays a regulatory role in the cardiovascular system and serves SARS-CoV-2 as an entry receptor. The aim of this study was to synthesize and evaluate radiofluorinated derivatives of the ACE2 inhibitor MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were demonstrated to be suitable for non-invasive imaging of ACE2, potentially enabling a better understanding of its expression dynamics. MethodsBased on computational molecular modeling, the ACE2-binding modes of F-MLN-4760 and F-Aza-MLN-4760 were similar to that of MLN-4760. Co-crystallization of the hACE2/F-MLN-4760 protein complex was performed for confirmation. Displacement experiments using [3H]MLN-4760 enabled the determination of the binding affinities of the synthesized F-MLN-4760 and F-Aza-MLN-4760 to ACE2 expressed in HEK-ACE2 cells. Aryl trimethylstannane-based and pyridine-based radiofluorination precursors were synthesized and used for the preparation of the respective radiotracers. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were evaluated with regard to the uptake in HEK-ACE2 and HEK-ACE cells and in vitro binding to tissue sections of HEK-ACE2 xenografts and normal organs of mice. Biodistribution and PET/CT imaging studies of [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were performed using HEK-ACE2 and HEK-ACE xenografted nude mice. ResultsCrystallography data revealed an equal ACE2-binding mode for F-MLN-4760 as previously found for MLN-4760 and indicated that the same would hold true for F-Aza-MLN-4760. The IC50 values were all in the high nM range, but three-fold lower for F-MLN-4760 and seven-fold lower for F-Aza-MLN-4760 than for MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were obtained in 1.4 {+/-} 0.3 GBq and 0.5 {+/-} 0.1 GBq activity with >99% radiochemical purity in a 5.3% and 1.2% radiochemical yield, respectively. Uptake in HEK-ACE2 cells was higher for [18F]F-MLN-4760 (67 {+/-} 9%) than for [18F]F-Aza-MLN-4760 (37 {+/-} 8%) after 3 h incubation while negligible uptake was seen in HEK-ACE cells (<0.3%). [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 accumulated specifically in HEK-ACE2 xenografts of mice (13 {+/-} 2% IA/g and 15 {+/-} 2% IA/g at 1 h p.i.) with almost no uptake observed in HEK-ACE xenografts (<0.3% IA/g). This was confirmed by PET/CT imaging, which also visualized unspecific accumulation in the gall bladder and intestinal tract. ConclusionBoth radiotracers showed specific and selective binding to ACE2 in vitro and in vivo. [18F]F-MLN-4760 was, however, obtained in higher yields and the ACE2-binding affinity was superior over that of [18F]F-Aza-MLN-4760. [18F]F-MLN-4760 would, thus, be the candidate of choice for further developlment to enable PET imaging of ACE2 in patients.

pharmacology and toxicology↗

bioRxiv2024

Impact of genomic background and developmental state on signaling pathways and response to therapy in Glioblastoma patient-derived cells

Glioblastoma (GBM) tumors represents diverse genomic epigenomic, and transcriptional landscapes, with significant intratumoral heterogeneity that challenges standard of care treatments involving radiation (RT) and the DNA-alkylating agent temozolomide (TMZ). In this study, we employed targeted proteomics to assess the response of a genomically-diverse panel of GBM patient-derived cancer stem cells (CSCs) to astrocytic differentiation, growth factor withdrawal and traditional high fetal bovine serum culture. Our findings revealed a complex crosstalk and co-activation of key oncogenic signaling in CSCs and diverse patterns of response to these external stimuli. Using RNA sequencing and DNA methylation, we observed common adaptations in response to astrocytic differentiation of CSCs across genomically distinct models, including BMP-Smad pathway activation, reduced cholesterol biosynthesis, and upregulation of extracellular matrix components. Notably, we observed that these differentiated CSC progenies retained a subset of stemness genes and the activation of cell survival pathways. We also examined the impact of differentiation state and genomic background on GBM cell sensitivity and transcriptional response to TMZ and RT. Differentiation of CSCs increased resistance to TMZ but not to RT. While transcriptional responses to these treatments were predominantly regulated by p53 in wild-type p53 GBM cells, its transcriptional activity was modulated by the differentiation status and treatment modality. Both mutant and wild-type p53 models exhibited significant activation of a DNA-damage associated interferon response in CSCs and differentiated cells, suggesting this pathway may play a wider role in GBM response to TMZ and RT. Our integrative analysis of the impact of GBM cell developmental states, in the context of genomic and molecular diversity of patient-derived models, provides valuable insights for pre-clinical studies aimed at optimizing treatment strategies.

cancer biology↗

bioRxiv2024

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11), a key enzyme in the ergosterol biosynthesis pathway. They have fungistatic effects against yeasts but fungicidal effects against molds. The molecular basis for this difference remained unknown. The sequence of enzymatic steps required for ergosterol biosynthesis is different in yeasts and molds. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not result in comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3{beta},6-diol, the "toxic diol" responsible for the antifungal effects of azoles in yeasts, is not required for the fungicidal effects in A. fumigatus. In contrast to yeast, where a lack of ERG3 functionality causes azole resistance, A. fumigatus lacking ERG3 becomes more azole susceptible. Mutants lacking mitochondrial complex III functionality, which are less susceptible to the fungicidal effects of azoles, but get strongly inhibited in growth, convert eburicol much more efficiently into the supposedly "toxic diol". Our results support a mechanistic model where the mode of action of azoles against the pathogenic mold A. fumigatus, other than in yeast, relies on the accumulation of eburicol which exerts fungicidal effects by triggering the formation of cell wall carbohydrate patches.

microbiology↗

bioRxiv2024

Focused ultrasound increases gene delivery to deep brain structure following the administration of a recombinant adeno-associated virus in the cerebrospinal fluid

Gene delivery via adeno-associated viral vectors can provide lasting clinical benefits following a one-time treatment. Delivery throughout the brain is needed for the treatment of neurological disorders with widespread pathology, including Alzheimer and Parkinson diseases, and amyotrophic lateral sclerosis. Most gene vectors have poor diffusion in the brain tissue. Furthermore, it is only at high intravenous doses that gene vectors can overcome the blood-brain barrier. In contrast, relatively lower doses of gene vectors injected in the cerebrospinal fluid enable significant transduction of superficial brain regions. The remaining challenge and unmet need of gene therapy is to deliver gene vectors to deep brain structures using a minimally invasive strategy. Here, we demonstrate that non-invasive focused ultrasound blood-brain barrier modulation can increase the delivery of recombinant adeno-associated virus by 5-fold to deep brain structures following injection in the cisterna magna. Delivery of adeno-associated viral vectors to the central nervous system, via administration in the cerebrospinal fluid, is being evaluated in several clinical trials for treating beta-galactosidase-1 deficiency, Batten disease, Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and spinal muscular atrophy. Our findings suggest that the efficacy of gene therapies delivered in the cerebrospinal fluid can be enhanced by targeting brain areas of interest with focused ultrasound. Significance statementAdministration of viral vectors in the cerebrospinal fluid through the cisterna magna is being evaluated in patients with neurological disorders. Focused ultrasound combined with intravenous microbubbles safely increases the permeability of the blood-brain barrier in humans and enables delivery of intravenous adeno-associated virus in non-human primates. Here, we demonstrate that combining these two clinically relevant gene delivery methods, intracisterna magna administration and focused ultrasound with microbubbles, can facilitate gene delivery to superficial and deep brain structures. Our findings have the potential to increase the efficacy of gene therapies, particularly for disorders with brain regions that have remained difficult to reach.

neuroscience↗

bioRxiv2023

Flower visitor groups show differential responses to individual and plot-level chemodiversity with consequences for plant fitness

O_LIChemodiversity, the diversity of specialised metabolites, plays a crucial role in mediating interactions between plants and animals, including insect herbivores and mutualists. Chemodiversity can be observed at both the individual and the population level. However, the impacts of chemodiversity at these two levels on interactions between plants and floral visitors, mainly pollinators and floral herbivores (florivores), are only poorly understood. C_LIO_LIThis study aimed to investigate the effects of chemodiversity at both individual and plot level on pollinators and florivores, examining their mutual interactions. To investigate these questions, we used individuals of the perennial Tanacetum vulgare differing in their terpenoid composition, representing so-called chemotypes. We planted individuals of five distinct chemotypes in a common garden design with homogeneous (five plants of the same chemotype) and heterogeneous (five different chemotypes) plots and observed flower visits in dependence of individual chemotype and plot type. Seeds were collected from a subset of plants and germination rates determined. C_LIO_LIOur findings revealed that chemodiversity at the plot level significantly influenced pollinator visitation, with more visits on plants of heterogeneous plots. We also observed marginally more pollinators on one of the chemotypes grown in homogeneous plots. In contrast, chemotype but not plot type had a significant effect on florivore visits. Pollinator and florivore species richness did not vary with plot type. Furthermore, a negative correlation was observed between pollinator and florivore visits in one year, suggesting competitive interactions between these two groups. Germination rates were positively correlated with pollinator visits and affected by chemotype but not by florivore visits or plot type. C_LIO_LISynthesis. Our study emphasizes the significance of the scale at which different chemical profiles are perceived by flower visitors, potentially influencing the reproductive fitness of plants. Exploring the ecology of these visitors and the varying selection pressures they exert on floral chemistry can help elucidating the evolutionary processes that maintain chemodiversity in natural environments. C_LI

ecology↗

bioRxiv2023

Bioprocess optimization enables enhanced protopanaxadiol production in yeast

Ginsenosides are the primary active metabolites of ginseng and have been used in traditional herbal medicine in Asia for more than 4,000 years. Protopanaxadiol (PPD) is the precursor of dammarane-type ginsenosides, exhibiting different pharmacological activities. However, extraction of PPD from plant material is cumbersome because of its low concentration. Precision fermentation using recombinant yeast is a promising alternative strategy to produce PPD. For increasing PPD production, the medium and fermentation conditions were optimized by a Design of Experiment (DoE) approach. PPD production increased by 7.5-fold in the final cultivation condition compared to the reference condition. The PPD titer reached 1.2 g/L during simple 500 mL shake flask cultivations, the highest PPD production in shake flasks to date. The protocol presented facilitates parallel evaluation of recombinant yeast, thereby contributing to the much-needed sustainable synthesis of the versatile molecule class of triterpenoids.

synthetic biology↗

bioRxiv2023

Chemodiversity in flowers of Tanacetum vulgare has consequences on a florivorous beetle

O_LIThe chemical composition of plant individuals can vary, leading to high intraspecific chemodiversity. Diversity of floral chemistry may impact the responses of flower-feeding insect visitors. C_LIO_LIPlants of Tanacetum vulgare vary significantly in their leaf terpenoid composition, resulting in distinct chemotypes. We investigated the composition of terpenoids and nutritents of flower heads and pollen in plants belonging to three chemotypes, dominated either by {beta}-thujone (BThu), artemisia ketone (Keto) or a mixture of (Z)-myroxide, santolina triene and artemisyl acetate (Myrox). Moreover, we tested the effects of these differences on preferences, weight gain and performance of adults of the shining flower beetle, Olibrus aeneus. C_LIO_LIThe terpenoid composition and diversity of flower heads and pollen significantly differed among individuals belonging to these chemotypes, while total concentrations of terpenoids, sugars, amino acids or lipids of the pollen did not differ. Beetles preferred the BThu over the Myrox chemotype in both olfactory and contact choice assays, while the Keto chemotype was marginally repellent in olfactory assays. The beetles gained the least weight within 48 h and their initial mortality was highest when feeding exclusively on floral tissues of the Myrox chemotype. Short-term weight gain and long-term performance were highest on the BThu chemotype. C_LIO_LIIn conclusion, the beetles showed chemotype-specific responses towards different T. vulgare chemotypes, which may be attributed to the terpenoid composition in flower heads and pollen rather than to differences in nutrient profiles. Both richness and overall diversity are important factors when determining chemodiversity of individual plants and their consequences on interacting insects. C_LI SHORT SUMMARYWe demonstrate that Tanacetum vulgare chemotypes distinguished by their leaf terpenoid profiles also show unique floral and pollen chemotypes based on terpenoid composition and diversity, which affect the preference and performance of a beetle florivore.

ecology↗

bioRxiv2023

Structural insights into human TFIIIC promoter recognition

Transcription factor IIIC (TFIIIC) recruits RNA polymerase (Pol) III to most of its target genes. Recognition of intragenic A- and B-box motifs in tRNA genes by TFIIIC modules {tau}A and {tau}B is the first critical step for tRNA synthesis but is mechanistically poorly understood. Here, we report cryo-EM structures of the human 624 kDa TFIIIC complex unbound and bound to a tRNA gene. The {tau}B module recognizes the B-box via DNA shape and sequence readout through the assembly of multiple winged-helix domains. TFIIIC220 forms an integral part of both {tau}A and {tau}B connecting the two subcomplexes via a [~]550 amino acid residue flexible linker. Our data provide a structural mechanism by which high-affinity B-box recognition anchors TFIIIC to promoter DNA and permits scanning for low-affinity A-boxes and TFIIIB for Pol III activation.

molecular biology↗

bioRxiv2023

Selective effects of estradiol on human corneal endothelial cells

Fuchs endothelial corneal dystrophy (FECD) results from genetic and environmental factors triggering mitochondrial and oxidative stress in corneal endothelial cells (CEnCs) leading to CEnC death and corneal opacification. FECD is more common in women than men, but the basis for this observation is unknown. Because FECD is commonly diagnosed around the time of the menopausal transition in women when estrogen levels decrease precipitously, we studied the effects of the potent estrogen,17-{beta} estradiol (E2) on growth, oxidative stress, and metabolism in primary cultures of human CEnCs (HCEnCs) under conditions of physiologic 2.5% O2 ([O2]2.5) and under hyperoxic stress ([O2]A: room air + 5% CO2). We hypothesized that E2 would counter the stresses of the hyperoxic environment in HCEnCs. HCEnCs were treated {+/-} 10 nM E2 for 7-10 days at [O2]2.5 and [O2]A followed by measurements of cell density, viability, reactive oxygen species (ROS), mitochondrial morphology, oxidative DNA damage, ATP levels, mitochondrial respiration (O2 consumption rate [OCR]), and glycolysis (extracellular acidification rate [ECAR]). There were no significant changes in HCEnC density, viability, ROS levels, oxidative DNA damage, OCR, and ECAR in response to E2 under either O2 condition. We found that E2 disrupted mitochondrial morphology in HCEnCs from female donors but not male donors at the [O2]A condition. ATP levels were significantly higher at [O2]2.5 compared to [O2]A in HCEnCs from female donors only, but were not affected by E2. Our findings demonstrate the overall resilience of primary HCEnCs against hyperoxic stress. The selective detrimental effects of hyperoxia and estradiol on HCEnCs from female but not male donors suggests mechanisms of toxicity based upon cell-sex in addition to hormonal environment.

cell biology↗

bioRxiv2023

Inter-laboratory comparison of plant volatile analyses in the light of intra-specific chemodiversity

IntroductionAssessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses. ObjectivesIn a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps. MethodsMonoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures. ResultsChemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles. ConclusionOur ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.

plant biology↗

bioRxiv2023

The biological activity of bacterial rhamnolipids is linked to their molecular structure

Biosurfactants are amphiphilic compounds of microbial origin with a wide range of industrial applications. Rhamnolipids (RLs) offer a broad range of potential applications as biosurfactants in industry and agriculture. Several studies report a high capacity for controlling different plant pests and pathogens and consider RLs as promising candidates for bio-based plant protection agents. RLs are a class of glycolipids consisting of one (mRLs) or two (dRLs) rhamnose moieties linked to a single or branched {beta}-fatty acid. Due to the diversity of RLs, so far little is known about the relation between molecular structure and biological activity. Engineering the synthesis pathway allowed us to differentiate between the activities of mixtures of pure mRL and of pure dRL congeners and elaborated HPLC techniques further enabled to analyse the activity of single congeners. In a model system with the plant Arabidopsis thaliana and the plant-parasitic nematode Heterodera schachtii we demonstrate that RLs can significantly reduce infection, whereas their impact on the host plant varies depending on their molecular structure. While mRLs reduced plant growth even at low concentration, dRLs showed no or a beneficial impact on plant development. In addition, the effect of RLs on plant H2O2 production was measured as an indicator of plant defense activity. Treatment with mRLs or dRLs at a concentration of 50 ppm increased H2O2 production. Lower concentrations of up to 10 ppm were used to stimulate plants prior to a treatment with water or flagellin (flg22), a bacterial inducer of plant defense responses. At 10 ppm both mRLs and dRLs fostered an increased response to flg22. However, mRLs also led to an increased response to water, the non-inducing negative control, indicating a generally elevated stress level. Neither mRLs nor dRLs induced expression of plant defense marker genes of salicylic acid, jasmonic acid and ethylene pathways within a 1 hour and 48 hours treatment. Due to the negative effect of mRLs on plants further studies were concentrated on dRLs. Treatment of pre-parasitic infective juveniles of H. schachtii revealed that dRLs did not increase mortality even at a very high concentration of 755 ppm. In order to analyse the effect of single dRL congeners nematode infection assays were performed. While dRL congeners with a C10-C8 acyl chain increased nematode infection, dRLs with C10-C12 and C10-C12:1 acyl chains reduced nematode infection even at concentrations below 2 ppm. Plant growth was not reduced by C10-C8 dRLs, but by C10-C12 and C10-C12:1 dRLs at concentrations of 8.3 ppm. H2O2 production was increased compared to the water control upon treatment with C10-C8 dRLs at a concentration of 200 ppm, while C10-C12 and C10-C12:1 dRLs triggered the same effect already at 50 ppm. Our experiments show a clear structure-effect relation. In conclusion, functional assessment and analysis of mode of action of RLs in plants require careful consideration of their molecular structure and composition.

molecular biology↗

bioRxiv2022

Intraspecific chemodiversity provides plant individual- and neighbourhood-mediated associational resistance towards aphids

O_LISome plant species express an extraordinarily high intraspecific diversity in phytochemicals (= chemodiversity). As discussed for biodiversity, higher chemodiversity may provide better protection against environmental stress, including herbivory. However, little is known about whether the resistance of a plant individual towards herbivores is mostly governed by its own chemodiversity or by associational resistance provided by conspecific neighbours. C_LIO_LITo investigate the role of chemodiversity in plant-aphid interactions, we used the Asteraceae Tanacetum vulgare, whose individuals differ pronouncedly in the composition of leaf terpenoids, forming distinct chemotypes. Plants were set-up in a field consisting of 60 plots, each containing five individuals of either the same or different chemotypes. Presence of winged aphids, indicating aphid attraction, and abundance of winged and unwinged aphids, indicating fitness, were scored weekly on each plant, focusing on three commonly occurring aphid species specialised on T. vulgare. During the peak abundance of aphids, leaf samples were taken from all plants for re-analyses of the terpenoid composition and quantification of terpenoid chemodiversity, calculated on an individual plant (Shannon index, Hsind) and plot level (Hsplot). C_LIO_LIAphid attraction was neither influenced by chemotype nor plot-type. The real-time odour environment may be very complex in this setting, impeding clear preferences. In contrast, the abundance was affected by both chemotype and plot-type. On average, more Uroleucon tanaceti aphids were found on plants of two of the chemotypes growing in homogenous compared to heterogenous plots, supporting the associational resistance hypothesis. For Macrosiphoniella tanacetaria the probability of presence on a plant differed between plot-types on one chemotype. Terpenoid chemodiversity expressed as a gradient revealed negative Hsplot effects on U. tanaceti, but a positive correlation of Hsind with the abundance of M. tanacetaria. Aphids of M. fuscoviride were not affected by any level of chemodiversity. C_LIO_LISynthesis. This study shows that not only the chemotype and chemodiversity of individual plants but also that of conspecific neighbours influence plant-herbivore interactions. These effects are highly specific with regard to the plant chemotype, the aphid species as well as its morphs (winged vs. unwinged). Furthermore, our results highlight the importance of analysing chemodiversity at different levels. C_LI

ecology↗

bioRxiv2022

Farnesoid X Receptor suppresses lipid peroxidation and ferroptosis

ObjectiveFerroptosis is a regulated cell death modality that occurs upon iron-dependent lipid peroxidation. The recent decade of research has uncovered many regulators driving ferroptosis as well as cellular gatekeepers preventing ferroptosis. Yet, many processes and networks remain to be elucidated. Methods and resultsIn this study, we performed a chemical screen using small molecules with known mode of action and identified two agonists (Turofexorate and Fexaramine) of the nuclear receptor Farnesoid X Receptor (FXR), also known as NR1H4, to suppress ferroptosis, but not apoptosis or necroptosis. Further, we demonstrate that in liver cells with high FXR protein levels, inhibition of FXR sensitizes cells to undergo ferroptotic cell death, while activation of FXR inhibits ferroptosis. Importantly, FXR also inhibits ferroptosis in ex vivo primary mouse hepatocytes. Activation of FXR by Turofexorate and Fexaramine significantly reduces lipid peroxidation. Mechanistically, overexpression of FXR or activation of FXR by bile acids upregulates the ferroptosis-inhibitory regulators FSP1, PPAR, GPX4, SCD1, and ACSL3 to reduce peroxidized lipids and to counteract ferroptosis. ConclusionIn this study, we demonstrate that activation of FXR inhibits ferroptotic cell death via upregulation of a number of ferroptosis-inhibitory proteins (FSP1, PPAR, GPX4, SCD1, and ACSL3) to reduce lipid peroxidation. Hence, modulating FXR activity may be beneficial to overcome ferroptosis-mediated degenerative diseases.

cell biology↗

bioRxiv2022

CRISPR/Cas9-Mediated Excision of ALS/FTD-Causing Hexanucleotide Repeat Expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro

A hexanucleotide repeat expansion (HRE) consisting of GGGGCC24+ in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are fatal neurodegenerative diseases with no current approved treatments that significantly slow disease progression or extend life expectancy. Several hypotheses have emerged to explain how this HRE causes neuronal death, including C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. In the present study we used a CRISPR/Cas9 gene-editing approach to remove the HRE from the C9ORF72 genomic locus, designing guide RNAs (gRNAs) flanking the HRE, and delivered Cas9 and gRNAs using adeno-associated virus serotype 9 (AAV9) vectors. Here, we demonstrate successful excision of the HRE in C9ORF72 in primary cortical neurons and brains of three mouse models containing the C9ORF72 expanded HRE (ranging from 500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, the major hallmarks of C9-ALS/FTD, making this an extremely attractive therapeutic approach to these diseases.

molecular biology↗

bioRxiv2021

Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency is a life-threatening condition caused by inheritance of the SERPINA1 gene Z variant. This single base pair mutation leads to protein misfolding, ER entrapment and gain of toxic function. Despite the significant unmet medical need presented by this disorder, there remain no approved medicines and the only curative option is liver transplantation. We hypothesized that an unbiased screen of human hepatocytes harbouring the Z mutation (ATZ) using small molecules targeted against protein degradation pathways would uncover novel biological insights of therapeutic relevance. Here we report the results of that screen performed in a patient-derived iPSC model of ATZ. Starting from 1,041 compounds we identified 14 targets capable of reducing polymer burden, including Leucine-rich repeat kinase-2 (LRRK2), a well-studied target in Parkinsons. Genetic deletion of LRRK2 in ATZ mice reduced polymers and associated fibrotic liver disease leading us to test a library of commercially available LRRK2 kinase inhibitors in both patient iPSC and CHO cell models. One of the molecules tested, CZC-25146, reduced polymer load, increased normal AAT secretion and reduced inflammatory cytokines with pharmacokinetic properties supporting its potential use for treating liver diseases. We therefore tested CZC-25146 in the ATZ mouse model and confirmed its efficacy for polymer reduction without signs of toxicity. Mechanistically, in both human and mouse models, our data show CZC-25146 inhibits LRRK2 kinase activity and induces autophagy. Cumulatively, these findings support the use of CZC-25146 and LRRK2 inhibitors in general in hepatic proteopathy disease research and as potential new treatment approaches for patients. One Sentence SummaryA small molecule screen in patient iPSCs with in vivo validation in mice identifies LRRK2 as a new therapeutic target for Alpha-1 Antitrypsin Deficiency.

cell biology↗

bioRxiv2021

bletl - A Python package for integrating microbioreactors in the design-build-test-learn cycle

Microbioreactor (MBR) devices have emerged as powerful cultivation tools for tasks of microbial phenotyping and bioprocess characterization and provide a wealth of online process data in a highly parallelized manner. Such datasets are difficult to interpret in short time by manual workflows. In this study, we present the Python package bletl and show how it enables robust data analyses and the application of machine learning techniques without tedious data parsing and preprocessing. bletl reads raw result files from BioLector I, II and Pro devices to make all the contained information available to Python-based data analysis workflows. Together with standard tooling from the Python scientific computing ecosystem, interactive visualizations and spline-based derivative calculations can be performed. Additionally, we present a new method for unbiased quantification of time-variable specific growth rate [Formula] based on a novel method of unsupervised switchpoint detection with Student-t distributed random walks. With an adequate calibration model, this method enables practitioners to quantify time-variable growth rate with Bayesian uncertainty quantification and automatically detect switch-points that indicate relevant metabolic changes. Finally, we show how time series feature extraction enables the application of machine learning methods to MBR data, resulting in unsupervised phenotype characterization. As an example, t-distributed Stochastic Neighbor Embedding (t-SNE) is performed to visualize datasets comprising a variety of growth/DO/pH phenotypes. Practical ApplicationThe bletl package can be used to analyze microbioreactor datasets in both data analysis and autonomous experimentation workflows. Using the example of BioLector datasets, we show that loading such datasets into commonly used data structures with one line of Python code is a significant improvement over spreadsheet or hand-crafted scripting approaches. On top of established standard data structures, practitioners may continue with their favorite data analysis routines, or make use of the additional analysis functions that we specifically tailored to the analysis of microbioreactor time series. Particularly our function to fit cross-validated smoothing splines can be used for on-line signals from any microbioreactor system and has the potential to improve robustness and objectivity of many data analyses. Likewise, our random walk based [Formula] method for inferring growth rates under uncertainty, but also the time-series feature extraction may be applied to on-line data from other cultivation systems as well.

bioengineering↗

bioRxiv2021

Intergenerational Effects of Early Life Starvation on Life-History, Consumption, and Transcriptomes of a Holometabolous Insect

Intergenerational effects, also known as parental effects in which the offspring phenotype is influenced by the parental phenotype, can occur in response to factors that occur not only in early but also in late parental life. However, little is known about how these parental life stage-specific environments interact with each other and with the offspring environment to influence offspring phenotype, particularly in organisms that realize distinct niches across ontogeny. We examined the effects of parental larval starvation and adult reproductive environment on offspring traits under matching or mismatching offspring larval starvation conditions using the holometabolous, haplo-diploid insect Athalia rosae (turnip sawfly). We show that the parental larval starvation treatment had trait-dependent intergenerational effects on both life-history and consumption traits of offspring larvae, partly in interaction with offspring conditions and sex, while there was no significant effect of parental adult reproductive environment. In addition, while offspring larval starvation led to numerous gene- and pathway-level expression differences, parental larval starvation impacted fewer genes and only the ribosomal pathway. Our findings reveal that parental starvation evokes complex intergenerational effects on offspring life-history traits, consumption patterns as well as gene expression, although the effects are less pronounced than those of offspring starvation.

ecology↗