Publications by authors named "Jan Mueller"

41 Publications

Functional Analysis of Non-Genetic Resistance to Platinum in Epithelial Ovarian Cancer Reveals a Role for the MBD3-NuRD Complex in Resistance Development.

Cancers (Basel) 2021 Jul 28;13(15). Epub 2021 Jul 28.

Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany.

Epithelial ovarian cancer (EOC) is the most lethal disease of the female reproductive tract, and although most patients respond to the initial treatment with platinum (cPt)-based compounds, relapse is very common. We investigated the role of epigenetic changes in cPt-sensitive and -resistant EOC cell lines and found distinct differences in their enhancer landscape. Clinical data revealed that two genes (JAK1 and FGF10), which gained large enhancer clusters in resistant EOC cell lines, could provide novel biomarkers for early patient stratification with statistical independence for JAK1. To modulate the enhancer remodeling process and prevent the acquisition of cPt resistance in EOC cells, we performed a chromatin-focused RNAi screen in the presence of cPt. We identified subunits of the Nucleosome Remodeling and Deacetylase (NuRD) complex as critical factors sensitizing the EOC cell line A2780 to platinum treatment. Suppression of the Methyl-CpG Binding Domain Protein 3 (MBD3) sensitized cells and prevented the establishment of resistance under prolonged cPt exposure through alterations of H3K27ac at enhancer regions, which are differentially regulated in cPt-resistant cells, leading to a less aggressive phenotype. Our work establishes JAK1 as an independent prognostic marker and the NuRD complex as a potential target for combinational therapy.
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http://dx.doi.org/10.3390/cancers13153801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345099PMC
July 2021

Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion.

Elife 2020 05 11;9. Epub 2020 May 11.

Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.

Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.
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http://dx.doi.org/10.7554/eLife.55351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239657PMC
May 2020

The value of chest magnetic resonance imaging compared to chest radiographs with and without additional lung ultrasound in children with complicated pneumonia.

PLoS One 2020 19;15(3):e0230252. Epub 2020 Mar 19.

Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany.

Introduction: In children with pneumonia, chest x-ray (CXR) is typically the first imaging modality used for diagnostic work-up. Repeated CXR or computed tomography (CT) are often necessary if complications such as abscesses or empyema arise, thus increasing radiation exposure. The aim of this retrospective study was to evaluate the potential of radiation-free chest magnetic resonance imaging (MRI) to detect complications at baseline and follow-up, compared to CXR with and without additional lung ultrasound (LUS).

Methods: Paired MRI and CXR scans were retrospectively reviewed by two blinded readers for presence and severity of pulmonary abscess, consolidation, bronchial wall thickening, mucus plugging and pleural effusion/empyema using a chest MRI scoring system. The scores for MRI and CXR were compared at baseline and follow-up. Furthermore, the MRI scores at baseline with and without contrast media were evaluated.

Results: 33 pediatric patients (6.3±4.6 years), who had 33 paired MRI and CXR scans at baseline and 12 at follow-up were included. MRI detected significantly more lung abscess formations with a prevalence of 72.7% compared to 27.3% by CXR at baseline (p = 0.001), whereas CXR+LUS was nearly as good as MRI. MRI also showed a higher sensitivity in detecting empyema (p = 0.003). At follow-up, MRI also showed a slightly better sensitivity regarding residual abscesses. The overall severity of disease was rated higher on MRI. Contrast material did not improve detection of abscesses or empyema by MRI.

Conclusion: CXR and LUS seem to be sufficient in most cases. In cases where LUS cannot be realized or the combination of CXR+LUS might be not sufficient, MRI, as a radiation free modality, should be preferred to CT. Furthermore, the admission of contrast media is not mandatory in this context.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230252PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082029PMC
June 2020

Transient localization of the Arp2/3 complex initiates neuronal dendrite branching .

Development 2019 04 4;146(7). Epub 2019 Apr 4.

Deutsches Zentrum für Neurodegenerative Erkrankungen e.V./German Center for Neurodegenerative Diseases (DZNE), 53127 Bonn, Germany

The formation of neuronal dendrite branches is fundamental for the wiring and function of the nervous system. Indeed, dendrite branching enhances the coverage of the neuron's receptive field and modulates the initial processing of incoming stimuli. Complex dendrite patterns are achieved through a dynamic process of branch formation, branch extension and retraction. The first step towards branch formation is the generation of a dynamic filopodium-like branchlet. The mechanisms underlying the initiation of dendrite branchlets are therefore crucial to the shaping of dendrites. Through time-lapse imaging of the subcellular localization of actin during the process of branching of larva sensory neurons, combined with genetic analysis and electron tomography, we have identified the Actin-related protein (Arp) 2/3 complex as the major actin nucleator involved in the initiation of dendrite branchlet formation, under the control of the activator WAVE and of the small GTPase Rac1. Transient recruitment of an Arp2/3 component marks the site of branchlet initiation These data position the activation of Arp2/3 as an early hub for the initiation of branchlet formation.
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http://dx.doi.org/10.1242/dev.171397DOI Listing
April 2019

On the relation between filament density, force generation, and protrusion rate in mesenchymal cell motility.

Mol Biol Cell 2018 11 29;29(22):2674-2686. Epub 2018 Aug 29.

Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.

Lamellipodia are flat membrane protrusions formed during mesenchymal motion. Polymerization at the leading edge assembles the actin filament network and generates protrusion force. How this force is supported by the network and how the assembly rate is shared between protrusion and network retrograde flow determines the protrusion rate. We use mathematical modeling to understand experiments changing the F-actin density in lamellipodia of B16-F1 melanoma cells by modulation of Arp2/3 complex activity or knockout of the formins FMNL2 and FMNL3. Cells respond to a reduction of density with a decrease of protrusion velocity, an increase in the ratio of force to filament number, but constant network assembly rate. The relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network explain the experimental observations. The formins act as filament nucleators and elongators with differential rates. Modulation of their activity suggests an effect on network assembly rate. Contrary to these expectations, the effect of changes in elongator composition is much weaker than the consequences of the density change. We conclude that the force acting on the leading edge membrane is the force required to drive F-actin network retrograde flow.
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http://dx.doi.org/10.1091/mbc.E18-02-0082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249830PMC
November 2018

Automated MR-based lung volume segmentation in population-based whole-body MR imaging: correlation with clinical characteristics, pulmonary function testing and obstructive lung disease.

Eur Radiol 2019 Mar 27;29(3):1595-1606. Epub 2018 Aug 27.

Department of Diagnostic and Interventional Radiology, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany.

Objectives: Whole-body MR imaging is increasingly utilised; although for lung dedicated sequences are often not included, the chest is typically imaged. Our objective was to determine the clinical utility of lung volumes derived from non-dedicated MRI sequences in the population-based KORA-FF4 cohort study.

Methods: 400 subjects (56.4 ± 9.2 years, 57.6% males) underwent whole-body MRI including a coronal T1-DIXON-VIBE sequence in inspiration breath-hold, originally acquired for fat quantification. Based on MRI, lung volumes were derived using an automated framework and related to common predictors, pulmonary function tests (PFT; spirometry and pulmonary gas exchange, n = 214) and obstructive lung disease.

Results: MRI-based lung volume was 4.0 ± 1.1 L, which was 64.8 ± 14.9% of predicted total lung capacity (TLC) and 124.4 ± 27.9% of functional residual capacity. In multivariate analysis, it was positively associated with age, male, current smoking and height. Among PFT indices, MRI-based lung volume correlated best with TLC, alveolar volume and residual volume (RV; r = 0.57 each), while it was negatively correlated to FEV/FVC (r = 0.36) and transfer factor for carbon monoxide (r = 0.16). Combining the strongest PFT parameters, RV and FEV/FVC remained independently and incrementally associated with MRI-based lung volume (β = 0.50, p = 0.04 and β = - 0.02, p = 0.02, respectively) explaining 32% of the variability. For the identification of subjects with obstructive lung disease, height-indexed MRI-based lung volume yielded an AUC of 0.673-0.654.

Conclusion: Lung volume derived from non-dedicated whole-body MRI is independently associated with RV and FEV/FVC. Furthermore, its moderate accuracy for obstructive lung disease indicates that it may be a promising tool to assess pulmonary health in whole-body imaging when PFT is not available.

Key Points: • Although whole-body MRI often does not include dedicated lung sequences, lung volume can be automatically derived using dedicated segmentation algorithms • Lung volume derived from whole-body MRI correlates with typical predictors and risk factors of respiratory function including smoking and represents about 65% of total lung capacity and 125% of the functional residual capacity • Lung volume derived from whole-body MRI is independently associated with residual volume and the ratio of forced expiratory volume in 1 s to forced vital capacity and may allow detection of obstructive lung disease.
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http://dx.doi.org/10.1007/s00330-018-5659-9DOI Listing
March 2019

Performance of Magnetic Resonance Susceptibility-Weighted Imaging for Detection of Calcifications in Patients With Hepatic Echinococcosis.

J Comput Assist Tomogr 2018 Mar/Apr;42(2):211-215

Objective: We evaluated the performance of susceptibility-weighted imaging (SWI) for identification of hepatic calcifications in alveolar echinococcosis and cystic echinococcosis.

Methods: The SWI images of 58 lesions in 40 patients (age, 49 ± 14 y) with alveolar echinococcosis (n = 22) or cystic echinococcosis (n = 18) were reviewed for calcifications. First, calcifications were suggested by visual assessment. Second, ratios of minimum intralesional intensity and mean lumbar muscle intensity were recorded. Computed tomography (CT) served as the criterion standard.

Results: Thirty-seven lesions showed calcifications on CT. Susceptibility-weighted imaging provided a sensitivity of 89.2% (95% confidence interval [CI], 50.1-75.7) and a specificity of 57.1% (95% CI, 34.4-77.4) for calcifications detected by visual assessment. Receiver operating characteristic curves demonstrated a sensitivity of 67.6% and a specificity of 85.0% for an intensity ratio of 0.61. A specificity of 100% (95% CI, 80.8-100) and a sensitivity of 84.5% (95% CI, 67.3-93.2) were achieved by SWI for calcifications with a density greater than 184 HU in CT.

Conclusions: Identification of hepatic calcifications is possible with SWI. Susceptibility-weighted imaging offers the potential to reduce the need for of CT imaging for evaluation of echinococcosis.
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http://dx.doi.org/10.1097/RCT.0000000000000687DOI Listing
April 2018

Load Adaptation of Lamellipodial Actin Networks.

Cell 2017 Sep 31;171(1):188-200.e16. Epub 2017 Aug 31.

Institute of Science and Technology Austria (IST Austria), am Campus 1, 3400 Klosterneuburg, Austria. Electronic address:

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.
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http://dx.doi.org/10.1016/j.cell.2017.07.051DOI Listing
September 2017

FMNL formins boost lamellipodial force generation.

Nat Commun 2017 03 22;8:14832. Epub 2017 Mar 22.

Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.
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http://dx.doi.org/10.1038/ncomms14832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364437PMC
March 2017

Cement leakage in pedicle screw augmentation: a prospective analysis of 98 patients and 474 augmented pedicle screws.

J Neurosurg Spine 2016 Jul 4;25(1):103-9. Epub 2016 Mar 4.

Department of Neurosurgery, University Medicine Greifswald.

OBJECTIVE Loosening and pullout of pedicle screws are well-known problems in pedicle screw fixation surgery. Augmentation of pedicle screws with bone cement, first described as early as 1975, increases the pedicle-screw interface and pullout force in osteoporotic vertebrae. The aim of the present study was to identify cement leakage and pulmonary embolism rates in a large prospective single-center series of pedicle screw augmentations. METHODS All patients who underwent cement-augmented pedicle screw placement between May 2006 and October 2010 at the authors' institution were included in this prospective cohort study. Perivertebral cement leakage and pulmonary cement embolism were evaluated with a CT scan of the area of operation and with a radiograph of the chest, respectively. RESULTS A total of 98 patients underwent placement of cement-augmented pedicle screws; 474 augmented screws were inserted in 237 vertebrae. No symptomatic perivertebral cement leakage or symptomatic pulmonary cement embolism was observed, but asymptomatic perivertebral cement leakage was seen in 88 patients (93.6%) and in 165 augmented vertebrae (73.3%). Cement leakage most often occurred in the perivertebral venous system. Clinically asymptomatic pulmonary cement embolism was found in 4 patients (4.1%). CONCLUSIONS Perivertebral cement leakage often occurs in pedicle screw augmentation, but in most cases, it is clinically asymptomatic. Cement augmentation should be performed under continuous fluoroscopy to avoid high-volume leakage. Alternative strategies, such as use of expandable screws, should be examined in more detail for patients at high risk of screw loosening.
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http://dx.doi.org/10.3171/2015.10.SPINE15511DOI Listing
July 2016

Efficient Photochemical Approaches for Spatially Resolved Surface Functionalization.

Angew Chem Int Ed Engl 2015 Sep 1;54(39):11388-403. Epub 2015 Sep 1.

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstrasse 18, 76131 Karlsruhe (Germany).

Materials interfaces--with a gas, a liquid, or another solid--are highly important for advanced applications. Besides their topological design, controlling interactions at these interfaces is typically realized by tuning the chemical composition of the materials surface. In areas such as nanoscience or biology, it is, however, highly desirable to impart heterogeneously distributed properties. Photopatterning, more than micro- and nanoprinting methods, is often the method of choice for precise functionalization, especially in terms of versatility. Recently, a range of new or rediscovered photochemistry approaches have been applied to precision surface functionalization, with the common aim of increasing efficiency and resolution while concomitantly lowering the amount of required energy. A survey of such methods is presented in this Review, with a focus on those we have explored.
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http://dx.doi.org/10.1002/anie.201504920DOI Listing
September 2015

Visible-Light-Induced Click Chemistry.

Angew Chem Int Ed Engl 2015 Aug 15;54(35):10284-8. Epub 2015 Jul 15.

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstrasse 18, 76128 Karlsruhe (Germany).

A rapid and catalyst-free cycloaddition system for visible-light-induced click chemistry is reported. A readily accessible photoreactive 2H-azirine moiety was designed to absorb light at wavelengths above 400 nm. Irradiation with low-energy light sources thus enables efficient small-molecule synthesis with a diverse range of multiple-bond-containing compounds. Moreover, in order to demonstrate the efficiency of the current approach, quantitative ligation of the photoactivatable chromophore with functional polymeric substrates was performed and full conversion with irradiation times of only 1 min at ambient conditions was achieved. The current report thus presents a highly efficient method for applications involving selective cycloaddition to electron-deficient multiple-bond-containing materials.
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http://dx.doi.org/10.1002/anie.201504716DOI Listing
August 2015

Photo-induced sequence defined macromolecules via hetero bifunctional synthons.

Chem Commun (Camb) 2015 Feb;51(10):1799-802

Soft Matter Synthesis Laboratory, Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.

We report the first photochemical protocol for the generation of sequence defined macromolecules employing two hetero bifunctional photoreactive synthons, exploiting the orthogonal nature of photochemical - via the use of caged dienes - and thermally driven ligation protocols. We demonstrate that the iterative alternating synthon addition to an initial bifunctional core under irradiation at ambient temperature enables the generation of a macromolecule with up to 10 units (M = 3231.58 g mol(-1), Đ = 1.00). The resulting macromolecules are monodisperse and feature absolute chain end fidelity. The unit-by-unit construction of the macromolecule is evidenced by Nuclear Magnetic Resonance Spectroscopy, Electrospray Ionization Mass Spectrometry and Size Exclusion Chromatography. The fundamental principle demonstrated herein paves the way for employing photochemical strategies for the design of sequence defined polymers.
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http://dx.doi.org/10.1039/c4cc08756aDOI Listing
February 2015

Tridimensional surface roughness analysis after resin infiltration of (deproteinized) natural subsurface carious lesions.

Clin Oral Investig 2015 Jul 9;19(6):1473-83. Epub 2014 Dec 9.

Centre for Operative Dentistry, Periodontology, and Endodontology, University of Dental Medicine and Oral Health, Danube Private University (DPU), Steiner Landstraße 124, 3500, Krems, Austria.

Objectives: The objectives of this study were to evaluate ex vivo the effects of resin infiltration on the areal surface roughness of natural non-cavitated proximal subsurface lesions with or without previous deproteinization and to determine differences between E2 and D1 lesions or between premolars and molars.

Materials And Methods: Forty premolars and 40 molars with proximal carious lesions and macroscopically intact surfaces (International Caries Detection and Assessment System (ICDAS) II; code 2) were radiologically assessed and randomly allocated to four groups (with 20 E2 and 20 D1 lesions, respectively). In each group, 10 lesions were deproteinized (NaOCl; 1%) before etching (HCl; 15%) and resin infiltration (Icon). Areal surface roughness (Sa) at the most demineralized lesion part (DIAGNOdent) was evaluated topometrically before and after deproteinization, after etching, and after infiltration using focus variation 3D scanning microscopy.

Results: Pretreatment with NaOCl (n = 40) had no significant effects on Sa (p = 0.208), but resulted in significantly differing Sa values between premolars and molars after etching (p = 0.011). Regarding the effects between etching and baseline, significantly differing Sa values (p = 0.0498) were found for premolars and molars (n = 40/40); Sa after resin infiltration (compared to etching) differed significantly between premolars and molars (p = 0.009). No treatment regimen lead to differences among the radiological grades (E2 vs. D1; p > 0.106).

Conclusions: Resin infiltration showed only minor effects on Sa values of etched subsurface lesions (p < 0.170) and did neither equal nor improve baseline surface roughness (p > 0.401) of the different tooth types.

Clinical Relevance: Deproteinization should be recommended before etching and infiltration, even if surface roughness of infiltrated advanced (pre-)molar lesions will not be improved.
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http://dx.doi.org/10.1007/s00784-014-1372-5DOI Listing
July 2015

A disulfide intercalator toolbox for the site-directed modification of polypeptides.

Chemistry 2015 Jan 30;21(1):228-38. Epub 2014 Oct 30.

Institute of Organic Chemistry III, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm (Germany), Fax: (+49) 731-5022883.

A disulfide intercalator toolbox was developed for site-specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole-SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live-cell imaging.
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http://dx.doi.org/10.1002/chem.201403965DOI Listing
January 2015

Fluorescent polymers from non-fluorescent photoreactive monomers.

Chem Commun (Camb) 2014 Dec 30;50(99):15681-4. Epub 2014 Oct 30.

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany.

A facile, fast and ambient-temperature avenue towards highly fluorescent polymers is introduced via polymerizing non-fluorescent photoreactive monomers based on light-induced NITEC chemistry, providing a platform technology for fluorescent polymers. The resulting polypyrazolines were analyzed in depth and the photo-triggered step-growth process was monitored in a detailed kinetic study.
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http://dx.doi.org/10.1039/c4cc07792jDOI Listing
December 2014

Ursodeoxycholyl lysophosphatidylethanolamide attenuates hepatofibrogenesis by impairment of TGF-β1/Smad2/3 signalling.

Br J Pharmacol 2014 Nov 5;171(22):5113-26. Epub 2014 Sep 5.

Department of Internal Medicine IV, Gastroenterology and Hepatology, University of Heidelberg, Heidelberg, Germany.

Background And Purpose: Chronic hepatic inflammation results in liver fibrosis. As effective anti-fibrogenic agents are lacking, we investigated ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), a synthetic bile acid-phospholipid conjugate with anti-inflammatory and anti-apoptotic properties for tis effects on hepatic fibrogenesis.

Experimental Approach: To stimulate fibrogenesis, LX2 hepatic stellate cells were cultured with conditioned medium from CL48 liver cells after exposure to stress-inducing conditions - methionine-choline-deficient (MCD) medium or TNFα/cycloheximide (CHX) - with or without UDCA-LPE preincubation. Anti-fibrogenic effects of UDCA-LPE were further studied in CL48 and LX2 cells and in primary human hepatic stellate cells (HHStec) directly exposed to TGF-β1. To test UDCA-LPE in vivo, C57BL/6 mice were fed a MCD diet for 11 weeks followed by 30 mg·kg(-1) UDCA-LPE 3× per week for 2.5 weeks.

Key Results: Expression of α-smooth muscle actin (α-SMA), α1-collagen, vimentin and TGF-β1 was down-regulated by up to 93% by UDCA-LPE in LX-2 cells cultured with conditioned medium. Also, UDCA-LPE inhibited Smad3 phosphorylation in CL48 cells incubated with MCD medium or TNFα/CHX and in LX2 cells exposed to conditioned medium. UDCA-LPE also decreased phosphorylated Smad3 and Smad2 directly induced by TGF-β1. Inhibition of TGF-β1/Smad2/3 signalling with down-regulation of target genes was confirmed in HHStec. In vivo, UDCA-LPE decreased hepatic α-SMA, α1-collagen and TGF-β1 expression and markedly lowered α-SMA protein and collagen deposition in MCD mice.

Conclusions And Implications: By blocking TGF-β1/Smad2/3 signalling, UDCA-LPE suppressed key mediators of hepatic fibrogenesis. Thus, UDCA-LPE could be suitable for prevention of fibrotic progression of chronic liver disease.
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http://dx.doi.org/10.1111/bph.12837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253459PMC
November 2014

Photo-patterning of non-fouling polymers and biomolecules on paper.

Adv Mater 2014 Jun 9;26(24):4087-92. Epub 2014 Apr 9.

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128, Karlsruhe, Germany; Institut für Biologische Grenzflächen (IBG), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.

Functional cellulose substrates with tetrazole moieties are generated to serve as universal platforms for the spatio-temporal immobilization of synthetic ultra-low fouling polymer brushes and protein species via a nitrile imine-mediated tetrazole-ene cycloaddition (NITEC)-based protocol. Poly(carboxybetaine acrylamide) brushes are grafted from initiators photo-patterned by NITEC utilizing single electron transfer living radical polymerization. Streptavidin is photo-immobilized with remarkable efficiency, opening the possibility to generate new materials for biomedical and biosensing applications.
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http://dx.doi.org/10.1002/adma.201401006DOI Listing
June 2014

Adaptable hetero Diels-Alder networks for fast self-healing under mild conditions.

Adv Mater 2014 Jun 21;26(21):3561-6. Epub 2014 Mar 21.

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76131 Karlsruhe, Germany, und Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.

A novel adaptable network based on the reversible hetero Diels-Alder reaction of a cyanodithioester and cyclopentadiene is presented. Reversible between 50-120 °C, the adjustable and self-healing features of the network are evidenced via temperature dependent rheology experiments and repetitive tensile tests whereas the network's chemical structure is explored by temperature dependent (1) H MAS-NMR spectroscopy.
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http://dx.doi.org/10.1002/adma.201306258DOI Listing
June 2014

Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

PLoS Biol 2014 Jan 14;12(1):e1001765. Epub 2014 Jan 14.

Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria.

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.
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http://dx.doi.org/10.1371/journal.pbio.1001765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891563PMC
January 2014

Controlled cell adhesion on poly(dopamine) interfaces photopatterned with non-fouling brushes.

Adv Mater 2013 Nov 3;25(42):6123-7. Epub 2013 Sep 3.

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstrasse 18, 76131, Karlsruhe, Germany; Zell- und Neurobiologie, Zoologisches Institut, Karlsruhe Institute of Technology (KIT), Haid-und-Neu-Str. 9, Karlsruhe and Institut für Funktionelle Grenzflächen (IFG), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.

Bioinspired poly(dopamine) (PDA) films are merged with antifouling poly(MeOEGMA) brushes utilizing a nitrile imine-mediated tetrazole-ene cycloaddition (NITEC)-based phototriggered surface encoding protocol. The antifouling brushes were photopatterned on PDA surfaces, leading cells to form confluent layers in the non-irradiated sections, while no adhesion occurred on the brushes resulting in a remarkably precise cell pattern. The presented strategy paves the way for the design of tailor-made patterned cell interfaces.
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http://dx.doi.org/10.1002/adma.201302492DOI Listing
November 2013

Frequency and phenotype of human circulating and intrahepatic natural killer cell subsets is differentially regulated according to stage of chronic liver disease.

Digestion 2013 14;88(1):1-16. Epub 2013 Jun 14.

Department of Medicine III, RWTH University Hospital Aachen, Aachen, Germany.

Background/aims: Experimental liver injury models have indicated that natural killer (NK) cells are critical regulators of inflammation and fibrosis. However, data on NK cells and subsets in patients with liver diseases are limited. We thus comprehensively characterized peripheral and hepatic NK cell subsets in patients with chronic liver diseases (CLDs) of different etiologies and fibrosis stages.

Methods: NK cells and other lymphocyte populations were characterized by FACS in 189 CLD patients (71 non-cirrhosis, 118 cirrhosis) and 153 healthy controls in blood and liver biopsies (n = 40).

Results: In contrast to other lymphocyte subsets, circulating NK cells were generally reduced in CLD patients. Patients with fibrosis displayed a distinct increase of CD16- NK cells in blood and of the CD16+ NK cell subset in liver. Patients with cirrhosis had overall lymphopenia, including reduced peripheral NK cells. Most pronounced shifts in NK cell subsets in blood and liver were found in cholestatic and autoimmune CLDs. Blood NK cells and subsets correlated with liver function, and inversely with fibrosis markers and inflammatory cytokines.

Conclusions: The close association of human NK cells with disease severity and the intrahepatic accumulation of CD16+ NK cells in early fibrosis favor the concept of beneficial NK cell functions in hepatofibrogenesis.
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http://dx.doi.org/10.1159/000350821DOI Listing
March 2014

Light-induced modular ligation of conventional RAFT polymers.

Angew Chem Int Ed Engl 2013 Jan 22;52(2):762-6. Epub 2012 Oct 22.

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology, Deutschland.

Making light work of RAFT conjugation: a non-activated RAFT agent at the end of RAFT polymers can readily be coupled with ortho-quinodimethanes (photoenols) in a photo-triggered Diels-Alder reaction under mild conditions without catalyst. The method is universal and opens the door for the conjugation of a large number of RAFT-prepared polymers with photoenol-functionalized (macro)molecules. (RAFT=reversible addition-fragmentation chain transfer.).
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http://dx.doi.org/10.1002/anie.201206905DOI Listing
January 2013

Direct determination of actin polarity in the cell.

J Mol Biol 2012 Jun 26;419(5):359-68. Epub 2012 Mar 26.

Structural Biology Research Center and Division of Biological Science, Graduate School of Sciences, Nagoya University, Nagoya 464-8601, Japan.

Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subcellular compartments is therefore required to establish their local function. Myosin binding has previously been the sole method of polarity determination. Here, we report the first direct determination of actin filament polarity in the cell without myosin binding. Negatively stained cytoskeletons of lamellipodia were analyzed by adapting electron tomography and a single particle analysis for filamentous complexes. The results of the stained cytoskeletons confirmed that all actin filament ends facing the cell membrane were the barbed ends. In general, this approach should be applicable to the analysis of actin polarity in tomograms of the actin cytoskeleton.
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http://dx.doi.org/10.1016/j.jmb.2012.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370650PMC
June 2012

Actin branching in the initiation and maintenance of lamellipodia.

J Cell Sci 2012 Jun 19;125(Pt 11):2775-85. Epub 2012 Mar 19.

Institute of Molecular Biotechnology, Vienna, Austria.

Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 μm. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.9 nm resolution, which was comparable with that derived for the in vitro actin-Arp2/3 complex. Lamellipodium initiation was monitored in an intracellular wound-healing model and was found to involve branching from the sides of actin filaments oriented parallel to the plasmalemma. Many filament plus ends, presumably capped, terminated behind the lamellipodium tip and localized on the dorsal and ventral surfaces of the actin network. These findings reveal how branching events initiate and maintain a network of actin filaments of variable length, and provide the first structural model of the branch junction in vivo. A possible role of filament capping in generating the lamellipodium leaflet is discussed and a mathematical model of protrusion is also presented.
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http://dx.doi.org/10.1242/jcs.107623DOI Listing
June 2012

Ursodeoxycholyl lysophosphatidylethanolamide improves steatosis and inflammation in murine models of nonalcoholic fatty liver disease.

Hepatology 2012 May 18;55(5):1369-78. Epub 2012 Mar 18.

Department of Internal Medicine IV, Gastroenterology and Hepatology, University of Heidelberg, Heidelberg, Germany.

Unlabelled: Hepatic fat accumulation and changes in lipid composition are hallmarks of nonalcoholic fatty liver disease (NAFLD). As an experimental approach for treatment of NAFLD, we synthesized the bile acid-phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE). Previous work demonstrated profound hepatoprotective properties of the conjugate in vitro and in vivo. Here we investigated the effects of UDCA-LPE in two nutritional mouse models of NAFLD. C57BL/6 mice were fed a high-fat diet (HFD) for 28 weeks, resulting in steatosis with hyperlipidemia. In a second model, mice received a methionin-choline-deficient (MCD) diet for up to 11 weeks, which induced advanced nonalcoholic steatohepatitis (NASH). Establishment of liver injury was followed by intraperitoneal injections of 30 mg/kg UDCA-LPE three times a week for different time periods. UDCA-LPE ameliorated both HFD- and MCD-induced increases in alanine aminotransferase (ALT) values near to normalization. As for metabolic parameters, UDCA-LPE reduced elevated serum triglyceride and cholesterol values in HFD mice. Liver histology showed improvement of steatosis in HFD and MCD mice concomitant with reductions in hepatic triglyceride and cholesterol levels. Additionally, the conjugate lowered serum caspase-8 activity in both models and decreased lipid hydroperoxides in MCD mice. Abundance of proinflammatory lysophosphatidylcholine (LPC), which was detectable in both HFD and MCD mice, was reduced by UDCA-LPE. Quantitative reverse transcriptase-polymerase chain reaction qRT-PCR of liver specimens revealed that UDCA-LPE strongly down-regulated inflammatory genes and modified the expression of genes involved in lipid metabolism.

Conclusion: The current study demonstrates that UDCA-LPE improves hepatic injury at different stages of NAFLD. By concurrently lowering hepatic lipid overloading as well as susceptibility of hepatocytes toward inflammatory stimuli, the conjugate may be able to ameliorate disease progression. Thus, UDCA-LPE represents a promising compound suitable for the treatment of NAFLD.
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http://dx.doi.org/10.1002/hep.25531DOI Listing
May 2012

Cofilin cooperates with fascin to disassemble filopodial actin filaments.

J Cell Sci 2011 Oct;124(Pt 19):3305-18

Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.

Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.
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http://dx.doi.org/10.1242/jcs.086934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074248PMC
October 2011

Surface substance loss of subsurface bovine enamel lesions after different steps of the resinous infiltration technique: a 3D topography analysis.

Odontology 2012 Jul 16;100(2):172-80. Epub 2011 Jun 16.

Department of Operative Dentistry and Periodontology, University School of Dental Medicine, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Surface substance loss of subsurface enamel lesions before (baseline/demineralization) and after each step of the infiltration technique was evaluated by means of a three-dimensional focus variation. Eighty enamel specimens were prepared and partially varnished (control). Non-varnished areas were demineralized (pH 4.95; 28 days), and etched with phosphoric acid gel (20%; 5 s). Specimens were randomly assigned to eight groups (n = 10), and were infiltrated using four resinous materials. In subgroups 1, polymerization and finishing with abrasive polishing strips followed. In subgroups 2, excess material was removed before polymerization (E1/E2-Excite, Vivadent; F1/F2-Fortify, Bisco; G1/G2-Glaze & Bond, DMG; I1/I2-Icon, DMG). Topometrical evaluation revealed a negligible substance loss of demineralized enamel. After etching, mean (±SD) differences of height decreased uniformly (-6.6 ± 2.0 μm; p = 0.089; ANOVA). For infiltrated lesions, DH of subgroups 1 was comparable to the etched lesions, with a significant increase (compared to etched lesions) in subgroups 2 (1.1 ± 0.1 μm; p < 0.001; t test). Within the limitations of this study, it is concluded that etching of initial subsurface lesions will result in significant surface substance loss; removal of excess material before light-curing should simplify the infiltration procedure, and this will avoid any abrasion resulting from polishing procedures.
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http://dx.doi.org/10.1007/s10266-011-0031-4DOI Listing
July 2012
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