Biology subjects

Koch, M.

Publications and source records attributed to Koch, M..

69 records · Page 4Linked to original sources

bioRxiv2019

Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries

Protein engineering through directed evolution facilitates the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries with minimal hands-on time and error-free, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

bioengineering↗

bioRxiv2019

Similarity-guided Monte Carlo Tree Search for bio-retrosynthesis

Metabolic engineering aims to produce chemicals of interest from living organisms, to advance towards greener chemistry. Despite efforts, the research and development process is still long and costly and efficient computational design tools are required to explore the chemical biosynthetic space. Here, we propose to explore the bio-retrosynthesis space using an Artificial Intelligence based approach relying on the Monte Carlo Tree Search reinforcement learning method, guided by chemical similarity. We implement this method in RetroPath RL, an open-source and modular command line tool. We validate it on a golden dataset of 20 manually curated experimental pathways as well as on a larger dataset of 152 successful metabolic engineering projects. Moreover, we provide a novel feature, that suggests potential media supplements to complement the enzymatic synthesis plan.

bioinformatics↗

bioRxiv2019

Large scale active-learning-guided exploration to maximize cell-free production

Lysate-based cell-free systems have become a major platform to study gene expression but batch-to-batch variation makes protein production difficult to predict. Here we describe an active learning approach to explore a combinatorial space of ~4,000,000 cell-free compositions, maximizing protein production and identifying critical parameters involved in cell-free productivity. We also provide a one-step-method to achieve high quality predictions for protein production using minimal experimental effort regardless of the lysate quality.

synthetic biology↗

bioRxiv2019

The effects of dyslipidaemia and cholesterol modulation on erythrocyte susceptibility to malaria parasite infection

Malaria disease commences when blood-stage parasites, called merozoites, invade human red blood cells (RBCs). Whilst the process of invasion is traditionally seen as being entirely merozoite-driven, emerging data suggests RBC biophysical properties markedly influence invasion. Cholesterol is a major determinant of cell membrane biophysical properties. We set out to assess whether cholesterol content in the RBC membrane affects susceptibility to merozoite invasion. Here we demonstrate that RBC bending modulus (a measure of deformability) is markedly affected by artificial modulation of cholesterol content and negatively correlated with merozoite invasion efficiency. Contextualising this observation, we tested a mouse model of hypercholesterolemia and human clinical samples from patients with a range of serum cholesterol concentrations for parasite susceptibility. Hypercholesterolaemia in both human and murine subjects had little effect merozoite invasion efficiency. Furthermore, on testing, RBC cholesterol content in both murine and human hypercholesterolaemia settings was found to be unchanged from normal controls. Serum cholesterol is, therefore, unlikely to impact on RBC susceptibility to merozoite entry. Our work, however, suggests that native polymorphisms that affect RBC membrane lipid composition would be expected to affect parasite entry. This supports investigation of RBC biophysical properties in endemic settings, which may yet identify naturally protective lipid-related polymorphisms.

microbiology↗

bioRxiv2019

Metabolic Perceptrons for Neural Computing in Biological Systems

AbstractSynthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene expression regulation for information processing using digital logic. Here, we present a new approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model trained on experimental data, we finally implement two four-input \"perceptrons\" for desired binary classification of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.

synthetic biology↗

bioRxiv2019

Substrate-rigidity dependent migration of an idealized twitching bacterium

Mechanical properties of the extracellular matrix are important determinants of cellular migration in diverse processes, such as immune response, wound healing, and cancer metastasis. Moreover, recent studies indicate that even bacterial surface colonization can depend on the mechanics of the substrate. Here, we focus on physical mechanisms that can give rise to substrate-rigidity dependent migration. We study a \"twitcher\", a cell driven by extension-retraction cycles, to idealize bacteria and perhaps eukaryotic cells that employ a slip-stick mode of motion. The twitcher is asymmetric and always pulls itself forward at its front. Analytical calculations show that the migration speed of a twitcher depends non-linearly on substrate rigidity. For soft substrates, deformations do not lead to build-up of significant force and the migration speed is therefore determined by stochastic adhesion unbinding. For rigid substrates, forced adhesion rupture determines the migration speed. Depending on the force-sensitivity of front and rear adhesions, forced bond rupture implies an increase or a decrease of the migration speed. A requirement for the occurrence of rigidity-dependent stick-slip migration is a \"sticky\" substrate, with binding rates being an order of magnitude larger than unbinding rates in absence of force. Computer simulations show that small stall forces of the driving machinery lead to a reduced movement on high rigidities, regardless of force-sensitivities of bonds. The simulations also confirm the occurrence of rigidity-dependent migration speed in a generic model for slip-stick migration of cells.

biophysics↗

bioRxiv2019

Restored alveolar epithelial differentiation and reversed human lung fibrosis upon Notch inhibition

Alveolar epithelial cell type II (AEC2) injury underlies idiopathic pulmonary fibrosis (IPF). Here we show increased Notch1 signaling in AEC2s in human IPF and IPF models, causing enhanced proliferation and de-differentiation of AEC2s. As a result, we observed defective surfactant protein (SP)-B/C processing, elevated alveolar surface tension, repetitive alveolar collapse and development of lung fibrosis. Similar changes were encountered upon pharmacological inhibition of SP-B/C processing in vivo by pepstatin A. Inhibition of Notch signaling in cultured human IPF precision cut lung slices improved surfactant processing capacity of AEC2s and reversed fibrosis. Notch1 therefore offers as novel therapeutic target.\n\nOne sentence summaryNotch1 inhibition restores alveolar epithelial differentiation and surface tension and reverses matrix deposition in lung fibrosis

cell biology↗

bioRxiv2019

Ultra-thin fluorocarbon foils optimize multiscale imaging of three-dimensional native and optically cleared specimens

In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast, and achieve an optimal resolution is minimizing the impact of changes of the refractive index along an optical path. Many light sheet fluorescence microscopes operate with a large chamber that contains an aqueous immersion medium and an inner specimen holder that contains the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimize the impact of the specimen holder on the optical quality, we use multi-facetted cuvettes fabricated with vacuum-formed ultra-thin fluorocarbon (FEP) foils The ultra-thin FEP-foil cuvettes have a wall thickness of about 12 {micro}m. They are resilient to fluidic exchanges, durable, mechanically stable and yet flexible. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs, thick tissue sections and dense organoid clusters. The cuvettes outperform many other sample-mounting techniques in terms of full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding, as well as their applicability for multiple-view imaging and automated segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The ultra-thin cuvettes allow the fixation and staining of the specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.

cell biology↗

bioRxiv2018

Macrophages protect against loss of adipose tissue during cancer cachexia

Cancer cachexia represents a central obstacle in medical oncology as it is associated with poor therapy response and reduced overall survival. Systemic inflammation is considered to be a key driver of cancer cachexia, however, clinical studies with anti-inflammatory drugs failed to show a robust cachexia-inhibiting effect. To address this contradiction, we investigated the functional importance of innate immune cells for hepatocellular carcinoma (HCC)-associated cachexia. To this end, we used a transgenic HCC mouse model intercrossed with mice harboring a defect in myeloid cell-mediated inflammation. We identified robust cachexia in the HCC mouse model as evidenced by a marked loss of visceral fat and lean mass. Computed tomography-based analyses demonstrated that a subgroup of human HCC patients displays reduced visceral fat mass, complementing the murine data. While the myeloid cell-mediated inflammation defect resulted in reduced expression of pro-inflammatory cytokines in the serum of HCC-bearing mice, this unexpectedly did not translate into diminished, but rather enhanced cachexia-associated fat loss. Defective myeloid cell-mediated inflammation was associated with decreased macrophage abundance in visceral adipose tissue, suggesting a role for local macrophages in the regulation of cancer-induced fat loss. Taken together, myeloid cell-mediated inflammation displays a rather unexpected beneficial function in a murine HCC model. These results demonstrate that immune cells are capable of protecting the host against cancer-induced tissue wasting, adding a further layer of complexity to the pathogenesis of cachexia and providing a potential explanation for the contradictory results of clinical studies with anti-inflammatory drugs.

cancer biology↗

bioRxiv2018

Plug-and-Play Metabolic Transducers Expand the Chemical Detection Space of Cell-Free Biosensors

Cell-free transcription-translation systems have great potential for biosensing, yet the range of detectable chemicals is limited. Here we provide a framework to expand the range of molecules detectable by cell-free biosensors by combining synthetic metabolic cascades with transcription factor-based networks. These hybrid cell-free biosensors are highly-sensitive and have a fast response and high-dynamic range. This work provides a foundation to engineer modular cell-free biosensors tailored for many applications.

synthetic biology↗

bioRxiv2018

Capturing variation impact on molecular interactions: the IMEx Consortium mutations data set

The current wealth of genomic variation data identified at the nucleotide level has provided us with the challenge of understanding by which mechanisms amino acid variation affects cellular processes. These effects may manifest as distinct phenotypic differences between individuals or result in the development of disease. Physical interactions between molecules are the linking steps underlying most, if not all, cellular processes. Understanding the effects that amino acid variation of a molecules sequence has on its molecular interactions is a key step towards connecting a full mechanistic characterization of nonsynonymous variation to cellular phenotype. Here we present an open access resource created by IMEx database curators over 14 years, featuring 28,000 annotations fully describing the effect of individual point sequence changes on physical protein interactions. We describe how this resource was built, the formats in which the data content is provided and offer a descriptive analysis of the data set. The data set is publicly available through the IntAct website at www.ebi.ac.uk/intact/resources/datasets#mutationDs and is being enhanced with every monthly release.

bioinformatics↗

bioRxiv2017

Molecular structures enumeration and virtual screening in the chemical space with RetroPath2.0

BackgroundNetwork generation tools coupled with chemical reaction rules have been mainly developed for synthesis planning and more recently for metabolic engineering. Using the same core algorithm, these tools apply a set of rules to a source set of compounds, stopping when a sink set of compounds has been produced. When using the appropriate sink, source and rules, this core algorithm can be used for a variety of applications beyond those it has been developed for.\n\nResultsHere, we showcase the use of the open source workflow RetroPath2.0. First, we mathematically prove that we can generate all structural isomers of a molecule using a reduced set of reaction rules. We then use this enumeration strategy to screen the chemical space around a set of monomers and predict their glass transition temperatures, as well as around aminoglycosides to search structures maximizing antibacterial activity. We also perform a screening around aminoglycosides with enzymatic reaction rules to ensure biosynthetic accessibility. We finally use our workflow on an E. coli model to complete E. coli metabolome, with novel molecules generated using promiscuous enzymatic reaction rules. These novel molecules are searched on the MS spectra of an E. coli cell lysate interfacing our workflow with OpenMS through the KNIME analytics platform.\n\nConclusionWe provide an easy to use and modify, modular, and open-source workflow. We demonstrate its versatility through a variety of use cases including, molecular structure enumeration, virtual screening in the chemical space, and metabolome completion. Because it is open source and freely available on MyExperiment.org, workflow community contributions should likely expand further the features of the tool, even beyond the use cases presented in the paper.

systems biology↗

bioRxiv2017

Post-transcriptional modulation of Dscam1 enhances axonal growth in development and after injury

Injury to the adult central nervous systems (CNS) results in severe long-term disability because damaged CNS connections rarely regenerate. Although several axon regeneration regulators have been proposed, intrinsic regenerative mechanisms remain largely unexplored. Here, we use a Drosophila CNS injury model to identify a novel pro-regeneration signaling pathway. We conducted a genetic screen of approximately three hundred candidate genes and identified three strong inducers of axonal growth and regeneration: the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism.

neuroscience↗

bioRxiv2017

ALPK1 And TIFA Dependent Innate Immune Response Triggered By The Helicobacter Pylori Type IV Secretion System

Activation of transcription factor NF-{kappa}B is a hallmark of infection with the gastric pathogen Helicobacter pylori and associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous hits involved in H. pylori-, but not IL-1{beta}- and TNF-- dependent NF-{kappa}B regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry and mutant H. pylori strains, identified the H. pylori metabolite D-glycero-{beta}-D-manno-heptose 1,7-bisphosphate ({beta}HBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-{kappa}B activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-{kappa}B activation, TIFA phosphorylation as well as TIFAsome formation depended on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as core of a novel innate immune pathway. ALPK1-TIFA-mediated NF-{kappa}B activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogens T4SS.

molecular biology↗

bioRxiv2017

Post-transcriptional regulation of adult CNS axonal regeneration by Cpeb1

Adult mammalian CNS neurons are unable to regenerate following axonal injury, leading to permanent functional impairments. Yet, the reasons underlying this regeneration failure are not fully understood. Here, we study the transcriptome and proteome shortly after spinal cord injury. Profiling of the total and ribosome-bound RNA in injured and naive spinal cords identify a substantial post-transcriptional regulation of gene expression. In particular, transcripts associated with nervous system development were downregulated in the total RNA-fraction while remaining stably loaded onto ribosomes. Interestingly, motif association analysis of post-transcriptionally regulated transcripts identified the cytoplasmic polyadenylation element (CPE) as enriched in a subset of these transcripts that was more resistant to injury-induced reduction at transcriptome level. Modulation of these transcripts by overexpression of the CPE binding protein, Cpeb1, in mouse and Drosophila CNS neurons promoted axonal regeneration following injury. Our study uncovers a global conserved post-transcriptional mechanism enhancing regeneration of injured CNS axons.

neuroscience↗