Biology subjects

Schmidt, M.

Publications and source records attributed to Schmidt, M..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Deep Proteome Profiling Reveals Signatures of Age and Sex Differences in Paw Skin and Sciatic Nerve of Naïve Mice

The age and sex of studied animals profoundly impact experimental outcomes in animal-based preclinical biomedical research. However, most preclinical studies in mice use a wide-spanning age range from 4 to 14 weeks and do not assess study parameters in male and female mice in parallel. This raises concerns regarding reproducibility and neglects potentially relevant age and sex differences. Furthermore, the molecular setup of tissues in dependence of age and sex is unknown in naive mice. Here, we employed an optimized quantitative proteomics workflow in order to deeply profile mouse paw skin and sciatic nerve (SCN) - two tissues, which are crucially implicated in nociception and pain as well as diverse diseases induced by inflammation, trauma, and demyelination. Remarkably, we uncovered significant differences when comparing (i) male and female mice, and, in parallel, (ii) adolescent mice (4 weeks) with adult mice (14 weeks). Age was identified as a major discriminator of analyzed samples irrespective of tissue type. Moreover, our analysis enabled us to decipher protein subsets and networks that exhibit differential abundance in dependence on the age and/or sex of mice. Notably, among these were proteins and signaling pathways with known relevance for (patho)physiology, such as homeostasis and epidermal signaling in skin and, in SCN, multiple myelin proteins and regulators of neuronal development. In addition, extensive comparisons with available databases revealed that we quantified approx. 50% of gene products that were implicated in distinct skin diseases and pain, many of which exhibited significant abundance changes in dependence on age and/or sex. Taken together, our study emphasizes the need for accurate age matching and uncovers hitherto unknown sex and age differences at the level of proteins and protein networks. Overall, we provide a unique systems biology proteome resource, which facilitates mechanistic insights into somatosensory and skin biology in dependence on age and sex - a prerequisite for successful preclinical studies in mouse disease models. Graphic workflow O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/498721v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@fc5aaforg.highwire.dtl.DTLVardef@1a5b87dorg.highwire.dtl.DTLVardef@f353e9org.highwire.dtl.DTLVardef@109f6e5_HPS_FORMAT_FIGEXP M_FIG C_FIG The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

systems biology↗

bioRxiv2022

Genomic, transcriptomic, and metabolomic analysis of Traditional Chinese Medicine plant Oldenlandia corymbosa reveals the biosynthesis and mode of action of anti-cancer metabolites

Natural products from traditional medicinal plants are valuable candidates for clinical cancer therapy. Plants from the Oldenlandia-Hedyotis complex are popular ingredients of Traditional Chinese Medicine (TCM), however a major hurdle in the plant bioprospecting process of TCM plants is that the active metabolites, their biosynthetic pathways, and mode of action are often unknown. We show that Oldenlandia corymbosa extracts are active against breast cancer cell lines. To study the genes involved in the biosynthesis of active compounds in this medicinal plant, we assembled a high-quality genome. We show that the main active compound is ursolic acid and that abiotic stresses cause changes in anti-cancer activity, metabolite composition, and gene expression of plants. To reveal the mode of action of ursolic acid, we show that cancer cells undergo mitotic catastrophe, and we identify three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking.

plant biology↗

bioRxiv2022

Species-specific cutaneous protein signatures upon incision injury and correlation with distinct pain-related phenotypes in humans

Pain after surgery is common, and its management remains a clinical challenge. Severe acute and prolonged post-surgical pain impairs immediate recovery and leads to long-term consequences like chronic pain, opioid dependency, and reduced quality of life. Althought rodent pain incision models exist, translation to patients is still hampered. To bridge this gap, we combined sensory phenotyping with quantitative proteomics and protein networks in humans and mice after skin incision representing an established model for surgical pain. Initially, we revealed, for the first time, similarities and differences of protein-protein interaction (PPI) networks across both species. Next, we comprehensively phenotyped humans for pain-related symptoms and observed phenotypes with incision-induced proteome changes. Remarkably, post-incision PPI-networks differed between volunteers with small incision-related hyperalgesic areas ("Low responder") versus those with large areas ("High responder"). The latter exhibited a pronounced proteolytic environment associated with persistent inflammation, while an anti-inflammatory protein signature was observed in Low responders. Taken together, we provide unprecedented insights into peripheral processes relevant for developing hyperalgesia and pain after incision. This knowledge will immensely facilitate bidirectional translational pain studies and guide future research on the pathophysiology of pain after surgery and the discovery of novel targets for its treatment and prevention.

neuroscience↗

bioRxiv2022

Escherichia coli YigI is a conserved γ-proteobacterial acyl-CoA thioesterase permitting metabolism of unusual fatty acid substrates

Thioesterases play a critical role in metabolism, membrane biosynthesis, and overall homeostasis for all domains of life. In this present study, we characterize a putative thioesterase from Escherichia coli MG1655 and define its role as a cytosolic enzyme. Building on structure-guided functional predictions, we show that YigI is a medium- to -long chain acyl-CoA thioesterase that is involved in the degradation of conjugated linoleic acid (CLA) in vivo, showing overlapping specificity with two previously defined E. coli thioesterases TesB and FadM. We then bioinformatically identify the regulatory relationships that induce YigI expression, which include: an acidic environment, high oxygen availability, and exposure to aminoglycosides. Our findings define a role for YigI and shed light on why the E. coli genome harbors numerous thioesterases with closely related functions. ImportancePrevious research has shown that long chain acyl-CoA thioesterases are needed for E. coli to grow in the presence of carbon sources such as conjugated linoleic acid, but that E. coli must possess at least one such enzyme that had not previously been characterized. Building off of structure-guided function predictions, we showed that the poorly annotated protein YigI is indeed the previously unidentified third acyl CoA thioesterase. We found that the three potentially overlapping acyl-CoA thioesterases appear to be induced by non-overlapping conditions, and use that information as a starting point for identifying the precise functions catalyzed by each such thioesterase, which is an important prerequisite for their industrial application and for more accurate metabolic modeling of E. coli.

microbiology↗

bioRxiv2021

Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop- out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCEUnderstanding the genetic basis of P. putidas diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators--which are of particular interest for metabolic engineering.

microbiology↗

bioRxiv2021

Long-term culture system for deep-sea mussels Gigantidas childressi

The simulation of deep-sea conditions in laboratories is technically challenging but necessary for experiments that aim at a deeper understanding of physiological mechanisms or host-symbiont interactions of deep-sea organisms. In a proof-of-concept study, we designed a recirculating system for long-term culture (>2 years) of deep-sea mussels Gigantidas childressi (previously Bathymodiolus childressi). Mussels were automatically (and safely) supplied with a maximum stable level of ~60 {micro}M methane in seawater using a novel methane-air mixing system. Experimental animals also received daily doses of live microalgae. Condition indices of cultured G. childressi remained high over years, and low shell thickness growth could be detected, which is indicative of positive energy budgets. Using stable isotope data, we demonstrate that G. childressi in our culture system gained energy, both, from digestion of methane oxidizing endosymbionts and from digesting particulate food (microalgae). Limitations of the system, as well as opportunities for future experimental approaches involving deep-sea mussels are discussed.

ecology↗

bioRxiv2021

Reconstitution of ATP-dependent 3'-cleavage of mammalian pre-mRNA reveals a central role of RBBP6

The 3 ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction, endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of fourteen polypeptides essential and two stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, Cleavage Factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

biochemistry↗

bioRxiv2021

Potassium-chelating drug sodium polystyrene sulfonate enhances lysosomal function and suppresses proteotoxicity

Lysosomes are crucial for degradation and recycling of damaged proteins and cellular components. Therapeutic strategies enhancing lysosomal function are a promising approach for aging and age-related neurodegenerative diseases. Here, we show that an FDA approved drug sodium polystyrene sulfonate (SPS), used to reduce high blood potassium in humans, enhances lysosomal function both in C.elegans and in human neuronal cells. Enhanced lysosomal function following SPS treatment is accompanied by the suppression of proteotoxicity caused by expression of the neurotoxic peptides A{beta} and TAU. Additionaly, treatment with SPS imparts health benefits as it significantly increases lifespan in C.elegans. Overall our work supports the potential use of SPS as a prospective geroprotective intervention. HIGHLIGHTSO_LISodium polystyrene sulfonate improves pH-dependent processing of lysosomal cargo, enhances proteotoxic stress resistance and extends lifespan in C. elegans C_LIO_LISodium polystyrene sulfonate boosts lysosomal function in human neuronal cells and reduces level of aggregation-associated phosphorylated-TAU C_LI

cell biology↗

bioRxiv2021

Parkinsonian gait effects with DBS are associated with pallido-peduncular axis activation

BackgroundDeep brain stimulation (DBS) targeting the subthalamic nucleus (STN) often shows variable outcomes on treating gait dysfunction in Parkinsons disease (PD). Such variability may stem from which specific neuronal pathways are modulated by DBS and the extent to which those pathways are modulated relative to one another. ObjectiveLeveraging ultra-high-field (7T) imaging data and subject-specific computational models, this study investigated how activation of seven distinct pathways in and around STN, including the pallidopeduncular and pedunculopallidal pathways, affect step length at clinically-optimized STN-DBS settings. MethodsPersonalized computational models were developed for 10 subjects with a clinical diagnosis of PD and with bilateral STN-DBS implants. ResultsThe subject-specific pathway activation models showed a significant positive association between activation of the pedunculopallidal pathway and increased step length, and negative association on step length with pallidopeduncular pathway and hyperdirect pathway activation. ConclusionsThe STN region includes multiple pathways, including fibers of passage to and from the mesencephalic locomotor area. Future clinical optimization of STN-DBS should consider these fibers of passage in the context of treating parkinsonian gait.

neuroscience↗

bioRxiv2021

Before trilobite legs: Pygmaclypeatus daziensis reconsidered and the ancestral appendicular organization of Cambrian artiopods

The Cambrian Stage 3 Chengjiang biota in South China is one of the most influential Konservat-Lagerstatten worldwide thanks to the fossilization of diverse non-biomineralizing organisms through pyritization. Despite their contributions to understanding the evolution of early animals, several Chengjiang species remain poorly known due to their scarcity and/or incomplete preservation. Here, we use micro-computed tomography to reveal in detail the ventral appendage organization of the enigmatic non-trilobite artiopod Pygmaclypeatus daziensis - one of the rarest euarthropods in Chengjiang - and explore its functional ecology and broader evolutionary significance. Pygmaclypeatus daziensis possesses a set of uniramous antennae and 14 pairs of post-antennal biramous appendages, the latter of which show an unexpectedly high degree of heteronomy based on the localized differentiation of the protopodite, endopodite and exopodite along the antero-posterior body axis. The small body size (less than 2 cm), presence of delicate spinose endites, and well-developed exopodites with multiple paddle-shaped lamellae on the appendages of P. daziensis indicate a nekto-benthic mode of life, and a scavenging/detritus feeding strategy. Pygmaclypeatus daziensis shows that appendage heteronomy is phylogenetically widespread within Artiopoda - the megadiverse clade that includes trilobites and their relatives with non-biomineralizing exoskeletons - and suggests that a single exopodite lobe with paddle-like lamellae is ancestral for this clade. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/456779v1_ufig1.gif" ALT="Figure 1"> View larger version (111K): org.highwire.dtl.DTLVardef@b4ddaaorg.highwire.dtl.DTLVardef@44f686org.highwire.dtl.DTLVardef@1950095org.highwire.dtl.DTLVardef@1596739_HPS_FORMAT_FIGEXP M_FIG C_FIG Cover image: Morphological reconstruction of the non-trilobite artiopod Pygmaclypeatus daziensis from the early Cambrian (Stage 3) Chengjiang biota in south China. Artwork by Holly Sullivan (https://www.sulscientific.com/).

paleontology↗

bioRxiv2021

Phage co-transport with hyphal-riding bacteria fuels bacterial invasion in water-unsaturated microbial ecosystems

Non-motile microbes enter new habitats often by co-transport with motile microorganisms. Here, we report on the ability of hyphal-riding bacteria to co-transport lytic phages and utilize them as weapons during colonization of new water-unsaturated habitats. This is comparable to the concept of biological invasions in macroecology. In analogy to invasion frameworks in plant and animal ecology, we tailored spatially organized, water-unsaturated model microcosms using hyphae of Pythium ultimum as invasion paths and flagellated soil-bacterium Pseudomonas putida KT2440 as carrier for co-transport of Escherichia virus T4. P. putida KT2440 efficiently dispersed along P. ultimum to new habitats and dispatched T4 phages across air gaps transporting {approx} 0.6 phages bacteria-1. No T4 displacement along hyphae was observed in the absence of carrier bacteria. If E. coli occupied the new habitat, T4 co-transport fueled the fitness of invading P. putida KT2440, while the absence of phage co-transport led to poor colonization followed by extinction. Our data emphasize the importance of hyphal transport of bacteria and associated phages in regulating fitness and composition of microbial populations in water-unsaturated systems. As such co-transport mirrors macroecological invasion processes, we recommend hyphosphere systems with motile bacteria and co-transported phages as models for testing hypotheses in invasion ecology.

microbiology↗

bioRxiv2021

Phenotypic plasticity and genetic control in colorectal cancer evolution

Cancer evolution is driven by natural selection acting upon phenotypic trait variation. However, the extent to which phenotypic variation within a tumour is a consequence of intra-tumour genetic heterogeneity remains undetermined. Here we show that colorectal cancer cells frequently have highly plastic phenotypic traits in vivo in patient tumours. We measured the degree to which trait variation reflects genetic ancestry by quantifying the phylogenetic signal of gene expression across 297 samples with multi-region paired whole genome and transcriptome sequencing collected from 27 primary colorectal cancers. Within-tumour phylogenetic signal for genes and pathways was detected only infrequently, suggesting that the majority of intra-tumour variation in gene expression programmes was not strongly heritable. Expression quantitative trait loci analyses (eQTL) identified a small number of putative mechanisms of genetic control of gene expression due to the cis-acting coding, non-coding and structural genetic alteration, but most gene expression variation was not explained by our genetic analysis. Leveraging matched chromatin-accessibility sequencing data, enhancer mutations with cis regulatory effects on gene expression were associated with a change in chromatin accessibility, indicating that non-coding variation can have phenotypic consequence through modulation of the 3D architecture of the genome. This study maps the evolution of transcriptional variation during cancer evolution, highlighting that intra-tumour phenotypic plasticity is pervasive in colorectal malignancies, and may play key roles in further tumour evolution, from metastasis to therapy resistance.

cancer biology↗

bioRxiv2021

Assessment of the evolutionary consequence of putative driver mutations in colorectal cancer with spatial multiomic data

Cancer genomic medicine relies on targeting driver genes. However, current catalogues of cancer drivers are mostly based on indirect measurements of mutation frequencies, positions or types, rather than their effect on clonal expansions in vivo. Moreover, non-genetic drivers are largely unknown, as are the epigenetic and transcriptomic effects of genetic drivers. Here we perform spatial computational inference on multiomic data with matched whole-genome sequencing, ATAC-seq and RNA-seq. Using 436 samples, we directly quantify the contribution, or lack thereof, of putative driver genes to subclonal expansions in vivo in 30 colorectal carcinomas (4-33 samples per patient, median=15). Although subclonal neutral evolution was widespread (13/26 cases with sufficient data), there were cases with clear evidence of subclonal selection (6/26) in which we measured epigenetic and transcriptomic differences between subclones in vivo. In 7/26 cases we could not distinguish between neutral or selective evolution with the available data. We identified expanding subclones that were not driven by known genetic alterations, and propose candidate epigenetic drivers. We identified the distinguishing patterns of genomic heterogeneity produced in fast, exponentially growing tumours (7/26) versus neoplasms growing only at the periphery (19/26), as well as identifying clonally intermixed (16/28 cases with sufficient data) versus segregated malignancies (10/28). Our model-based approach measures genetic and non-genetic subclonal selection, or lack thereof, in space and time and allows in vivo comparisons of the emergent phenotypic properties of subclones within human tumours.

cancer biology↗

bioRxiv2021

The co-evolution of the genome and epigenome in colorectal cancer

Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts, DNA mutations do not fully explain malignant evolution. Here we study the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,373 samples from 30 primary cancers and 9 concomitant adenomas and generated 1,212 chromatin accessibility profiles, 527 whole-genomes and 297 whole-transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent chromatin changes in regulatory regions of cancer drivers with otherwise no mutation. Genome-wide alterations in transcription factor binding accessibility involved CTCF, downregulation of interferon, and increased accessibility for SOX and HOX, indicating developmental genes reactivation. Epigenetic aberrations were heritable, distinguishing adenomas from cancers. Mutational signature analysis showed the epigenome influencing DNA mutation accumulation. This study provides a map of (epi)genetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.

cancer biology↗

bioRxiv2021

Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

Human pluripotent stem cell derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for derivation of skeletal muscle cells and their utility in three-dimensional skeletal muscle organoid formation as well as skeletal muscle tissue engineering. Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors of hypaxial origin followed by primary and secondary fetal myogenesis into hypaxial muscle with development of a satellite cell pool and evidence for innervation in vitro. Skeletal muscle organoids faithfully recapitulate all steps of embryonic myogenesis in 3D Tissue engineered muscle exhibits organotypic maturation and function, advanced by thyroid hormone. Regenerative competence was demonstrated in a cardiotoxin injury model with evidence of satellite cell activation as underlying mechanism. Collectively, we introduce a hypaxial muscle model with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease, and repair.

cell biology↗

bioRxiv2021

Correction of a pathogenic mutation in iPSCs derived from a patient with Christianson syndrome using CRISPR/Cas9 genome editing

SLC9A6 (also termed NHE6) encodes the endosomal Na+/H+ exchanger 6 (NHE6). Pathogenic, loss-of-function mutations in NHE6 cause the X-linked neurogenetic disorder Christianson syndrome (CS). We developed induced pluripotent stem cell (iPSC) lines derived from a patient with CS and from a biologically related control. The patient with CS contained the nonsense mutation c.1569G>A (p.(W523X)), which caused a significant reduction in NHE6 mRNA and a lack of detectable NHE6 protein in CS iPSCs in comparison to control iPSCs. To establish a cell model for study of CS with an isogenic control, we corrected the c.1569G>A mutation to the NHE6 reference genome sequence using CRISPR/Cas9-mediated homology directed repair knock-in methodology. Multiple subclonal lines were generated, and notably, NHE6 protein was expressed in all analyzed c.1569G>A (p.(W523X)) genome-corrected iPSC lines. This CS iPSC model together with the associated biologically related and isogenic control cell lines will serve as a valuable resource for both basic and translational studies in CS.

genetics↗

bioRxiv2021

Vitamin C inhibits SARS coronavirus-2 main protease essential for viral replication

There is an urgent need for anti-viral agents that treat and/or prevent Covid-19 caused by SARS-Coronavirus (CoV-2) infections. The replication of the SARS CoV-2 is dependent on the activity of two cysteine proteases, a papain-like protease, PL-pro, and the 3C-like protease known as main protease Mpro or 3CLpro. The shortest and the safest path to clinical use is the repurposing of drugs with binding affinity to PLpro or 3CLpro that have an established safety profile in humans. Several studies have reported crystal structures of SARS-CoV-2 main protease in complex with FDA approved drugs such as those used in treatment of hepatitis C. Here, we report the crystal structure of 3CLpro in complex Vitamin C (L-ascorbate) bound to the proteins active site at 2.5 [A]ngstrom resolution. The crystal structure of the Vitamin C 3CLpro complex may aid future studies on the effect of Vitamin C not only on the coronavirus main protease but on related proteases of other infectious viruses.

biophysics↗

bioRxiv2021

Generation of Pathogenic TPP1 Mutations in Human Stem Cells as a Model for CLN2 Disease

Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is an autosomal recessive neurodegenerative disorder generally with onset at 2 to 4 years of age and characterized by seizures, loss of vision, progressive motor and mental decline, and premature death. CLN2 disease is caused by loss-of-function mutations in the tripeptidyl peptidase 1 (TPP1) gene leading to deficiency in TPP1 enzyme activity. Approximately 60% of patients have one of two pathogenic variants (c.509-1G>C or c.622C>T [p.(Arg208*)]). In order to generate a human stem cell model of CLN2 disease, we used CRISPR/Cas9-mediated knock-in technology to introduce these mutations in a homozygous state into H9 human embryonic stem cells. Heterozygous lines of the c.622C>T (p.(Arg208*)) mutation were also generated, which included a heterozygous mutant with a wild-type allele and different compound heterozygous coding mutants resulting from indels on one allele. We describe the methodology that led to the generation of the lines and provide data on the initial validation and characterization of these CLN2 disease models. Notably, both mutant lines (c.509-1G>C and c.622C>T [p.(Arg208*)]) in the homozygous state were shown to have reduced or absent protein, respectively, and deficiency of TPP1 enzyme activity. These models, which we are making available for wide-spread sharing, will be useful for future studies of molecular and cellular mechanisms underlying CLN2 disease and for therapeutic development.

cell biology↗