Biology subjects

Mueller, R.

Publications and source records attributed to Mueller, R..

47 records · Page 3Linked to original sources

bioRxiv2020

Biosynthesis of cittilins, unusual ribosomally synthesized and post-translationally modified peptides from Myxococcus xanthus

Cittilins are secondary metabolites from myxobacteria comprised of three L-tyrosines and one L-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We confirm the roles of the biosynthetic genes responsible for the formation of cittilins using targeted gene inactivation and heterologous expression in Streptomyces. We also report first steps towards the biochemical characterization of the proposed biosynthetic pathway in vitro. An investigation of the cellular uptake properties of cittilin A connected it to a potential biological function as an inhibitor of the prokaryotic carbon storage regulator A (CsrA). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/114512v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@11da6b5org.highwire.dtl.DTLVardef@1abc508org.highwire.dtl.DTLVardef@14bb22org.highwire.dtl.DTLVardef@109cedd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

The Digital Mouse: why computational modelling of mouse models of disease can improve translation

Computational models can be used to study the mechanistic phenomena of disease. Current mechanistic computer simulation models mainly focus on (patho)physiology in humans. However, often data and experimental findings from preclinical studies are used as input to develop such models. Biological processes underlying age-related chronic diseases are studied in animal models. The translation of these observations to clinical applications is not trivial. As part of a group of international scientists working in the COST Action network MouseAGE, we argue that in order to boost the translation of pre-clinical research we need to develop accurate in silico counterparts of the in vivo animal models. The Digital Mouse is proposed as framework to support the development of evidence-based medicine, for example to develop geroprotectors, which are drugs that target fundamental mechanisms of ageing. HighlightsO_LIComputational modelling of human (patho)physiology is advancing rapidly, often using and extrapolating experimental findings from preclinical disease models. C_LIO_LIThe lack of in silico models to support in vivo modelling in mice is a missing link in current approaches to study complex, chronic diseases. C_LIO_LIThe development of mechanistic computational models to simulate disease in mice can boost the discovery of novel therapeutic interventions. C_LIO_LIThe Digital Mouse is proposed as a framework to implement this ambition. The development of a Digital Mouse Frailty Index (DM:FI) to study aging and age-related diseases is provided as an example. C_LI

bioengineering↗

bioRxiv2020

Scaffold pore geometry influences bone-like tissue formation in dynamic cell culture conditions

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. Here, monodisperse templated scaffolds (MTSC) were fabricated and used as a well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress (WSS) using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cells differentiation, bone-like tissue formation, structure and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and micro-computed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared to static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, while MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared to MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs. Impact statementAging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction towards the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization.

bioengineering↗

bioRxiv2019

Tau deposition is associated with imaging patterns of tissue calcification in the P301L mouse model of human tauopathy

Brain calcification is associated with several neurodegenerative proteinopathies. Here, we report a new phenotype of intracranial calcification in transgenic P301L mice overexpressing 4 repeat tau. P301L mice (Thy1.2) of 3, 5, 9 and 18-25 months-of-age and age-matched non-transgenic littermates were assessed using in vivo/ex vivo magnetic resonance imaging (MRI) with a gradient recalled echo sequence and micro computed tomography (CT). Susceptibility weighted images computed from the gradient recalled echo data revealed regional hypointensities in the hippocampus, cortex, caudate nucleus and thalamus of P301L mice, which in corresponding phase images indicated diamagnetic lesions. Concomitantly, {micro}CT detected hyperdense lesions. Occurrence of diamagnetic susceptibility lesions in the hippocampus, increased with age. Immunochemical staining of brain sections revealed bone protein-positive deposits. Furthermore, intra-neuronal and vessel-associated protein-containing nodules co-localized with phosphorylated-tau (AT8 and AT100) in the hippocampus. Protein-containing nodules were detected also in the thalamus in the absence of phosphorylated-tau deposition. In contrast, osteocalcin-containing nodules were vessel-associated, indicating ossified vessels, in the thalamus in absence of phosphorylated-tau. In summary, MRI and {micro}CT demonstrated imaging pattern of intracranial calcification, concomitant with immunohistochemical evidence of formation of protein deposits containing bone proteins along with phosphorylated-tau in the P301L mouse model of human tauopathy. The P301L mouse model may thus serve as a future model to study the pathogenesis of brain calcifications in tauopathies.

neuroscience↗

bioRxiv2019

Ribosome-targeting antibiotics impair T cell effector function and ameliorate autoimmunity by blocking mitochondrial protein synthesis

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive, for reasons that remain unclear. Here we show that linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of Th17 effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromises the integrity of the electron transport chain (ETC). Ultimately, this leads to loss of mitochondrial metabolism and cytokine production in differentiating Th cells. In accordance, mice lacking Gfm1 in T cells are protected from EAE, demonstrating that this pathway plays a key role in maintaining T cell function and pathogenicity.

immunology↗

bioRxiv2019

Discovery of a polybrominated aromatic secondary metabolite from a planctomycete points at an ambivalent interaction with its macroalgae host

The roles of the majority of bacterial secondary metabolites, especially those from uncommon sources are yet elusive even though many of these compounds show striking biological activities. To further investigate the secondary metabolite repertoire of underexploited bacterial families, we chose to analyze a novel representative of the yet untapped bacterial phylum Planctomycetes for the production of secondary metabolites under laboratory culture conditions. Development of a planctomycetal high density cultivation technique in combination with high resolution mass spectrometric analysis revealed Planctomycetales strain 10988 to produce the plant toxin 3,5 dibromo p-anisic acid. This molecule represents the first secondary metabolite reported from any planctomycete. Genome mining revealed the biosynthetic origin of this doubly brominated secondary metabolite and a biosynthesis model for the com-pound was devised. Comparison of the biosynthetic route to biosynthetic gene clusters responsible for formation of polybrominated small aromatic compounds reveals evidence for an evolutionary link, while the compounds herbicidal activity points towards an ambivalent role of the metabolite in the planctomycetal ecosystem.

microbiology↗

bioRxiv2019

Evaluation of longitudinal time-lapsed in vivo micro-CT for monitoring fracture healing in mouse femur defect models

Longitudinal in vivo micro-computed tomography (micro-CT) is of interest to non-invasively capture the healing process of individual animals in preclinical fracture healing studies. However, it is not known whether longitudinal imaging itself has an impact on callus formation and remodeling. In this study, a scan group received weekly micro-CT measurements (week 0-6), whereas controls were only scanned post-operatively and at week 5 and 6. Registration of consecutive scans using a branching scheme (bridged vs. unbridged defect) combined with a two-threshold approach enabled assessment of localized bone turnover and mineralization kinetics relevant for monitoring callus remodeling. Weekly micro-CT application did not significantly change any of the assessed callus parameters in the defect and periosteal volumes. This was supported by histomorphometry showing only small amounts of cartilage residuals in both groups, indicating progression towards the end of the healing period. Also, immunohistochemical staining of Sclerostin, previously associated with mediating adverse radiation effects on bone, did not reveal differences between groups.\n\nThe established longitudinal in vivo micro-CT-based approach allows monitoring of healing phases in mouse femur defect models without significant effects of anesthesia, handling and radiation on callus properties. Therefore, this study supports application of longitudinal in vivo micro-CT for healing-phase-specific monitoring of fracture repair in mice.

bioengineering↗

bioRxiv2019

Microbial community-level features linked to divergent carbon flows during early litter decomposition in a constant environment

During plant litter decomposition in soils, carbon has two general fates: return to the atmosphere via microbial respiration or transport into soil where long-term storage may occur. Discovering microbial community features that drive carbon fate from litter decomposition may improve modeling and management of soil carbon. This concept assumes there are features (or underlying processes) that are widespread among disparate communities, and therefore amenable to modeling. We tested this assumption using an epidemiological approach in which two contrasting patterns of carbon flow in laboratory microcosms were delineated as functional states and diverse microbial communities representing each state were compared to discover shared features linked to carbon fate. Microbial communities from 206 soil samples from the southwestern United States were inoculated on plant litter in microcosms, and carbon flow was measured as cumulative carbon dioxide (CO2) and dissolved organic carbon (DOC) after 44 days. Carbon flow varied widely among the microcosms, with a 2-fold range in cumulative CO2 efflux and a 5-fold range in DOC quantity. Bacteria, not fungi, were the strongest drivers of DOC variation. The most significant community-level feature linked to DOC abundance was bacterial richness--the same feature linked to carbon fate in human-gut microbiome studies. This proof-of-principle study under controlled conditions suggests common features driving carbon flow in disparate microbial communities can be identified, motivating further exploration of underlying mechanisms that may influence carbon fate in natural ecosystems.

microbiology↗

bioRxiv2018

Sustained oscillations of epithelial cell sheets

Morphological changes during development, tissue repair, and disease largely rely on coordinated cell movements and are controlled by the tissue environment. Epithelial cell sheets are often subjected to large scale deformation during tissue formation. The active mechanical environment in which epithelial cells operate have the ability to promote collective oscillations, but how these cellular movements are generated and relate to collective migration remains unclear. Here, combining in vitro experiments and computational modelling we describe a novel mode of collective oscillations in confined epithelial tissues where the oscillatory motion is the dominant contribution to the cellular movements. We show that epithelial cells exhibit large-scale coherent oscillations when constrained within micro-patterns of varying shapes and sizes, and that their period and amplitude are set by the smallest confinement dimension. Using molecular perturbations, we then demonstrate that force transmission at cell-cell junctions and its coupling to cell polarity are pivotal for the generation of these collective movements. We find that the resulting tissue deformations are sufficient to trigger mechanotransduction within cells, potentially affecting a wide range of cellular processes.

biophysics↗

bioRxiv2018

Watching antibiotics in action: Exploiting time-lapse microfluidic microscopy as a tool for target-drug interaction studies in Mycobacterium

Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria. The genus Mycobacterium encompasses both human and animal pathogens that cause severe diseases and have profound impacts on global health and the world economy. Here, we used a novel system of microfluidics, fluorescence microscopy and target-tagged fluorescent reporter strains of M. smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid, nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, and griselimycin caused replisome instability with subsequent over-initiation of chromosome replication and over-relaxation of the nucleoid. This work is an example of using a microscopy-based approach to evaluate the activity of potential replication inhibitors and provides mechanistic insights into their modes of action. Our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.). Because proteins involved in the DNA replication are well conserved among bacteria (including mycobacterial species), the properties of various replication inhibitors observed here in fast-growing M. smegmatis may be easily extrapolated to slow-growing pathogenic tubercle bacilli, such as M. tuberculosis.\n\nSignificanceThe growing problem of bacterial resistance to antibiotics and the emergence of new strains that are resistant to multiple drugs raise the need to explore new antibiotics and re-evaluate the existing options. Here, we present a system that allows the action of antibiotics to be monitored at the single-cell level. Such studies are important in the light of bacterial heterogeneity, which may be enhanced in unfavorable conditions, such as under antibiotic treatment. Moreover, our studies provide mechanistic insights into the action modes of the tested compounds. As combined therapies have recently gained increased interest, it is also notable that our described system may help researchers identify the best combination of antimicrobials for use against infections caused by a variety of bacteria.

microbiology↗

bioRxiv2018

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly sequenced whole genomes) and culture-based resistance profiles (10,991 of 11,087 isolates were comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns could be observed including increasing resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species such as conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene-drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into a resource, GEAR-base, available for academic research use free of charge at https://gear-base.com.

genomics↗