Publications by authors named "Lasse Hyldgaard Klausen"

12 Publications

  • Page 1 of 1

Long-distance electron transfer in a filamentous Gram-positive bacterium.

Nat Commun 2021 03 17;12(1):1709. Epub 2021 Mar 17.

Interdisciplinary Nanoscience Center (iNANO), Sino-Danish Center for Education and Research (SDC), Aarhus University, Aarhus, Denmark.

Long-distance extracellular electron transfer has been observed in Gram-negative bacteria and plays roles in both natural and engineering processes. The electron transfer can be mediated by conductive protein appendages (in short unicellular bacteria such as Geobacter species) or by conductive cell envelopes (in filamentous multicellular cable bacteria). Here we show that Lysinibacillus varians GY32, a filamentous unicellular Gram-positive bacterium, is capable of bidirectional extracellular electron transfer. In microbial fuel cells, L. varians can form centimetre-range conductive cellular networks and, when grown on graphite electrodes, the cells can reach a remarkable length of 1.08 mm. Atomic force microscopy and microelectrode analyses suggest that the conductivity is linked to pili-like protein appendages. Our results show that long-distance electron transfer is not limited to Gram-negative bacteria.
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http://dx.doi.org/10.1038/s41467-021-21709-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969598PMC
March 2021

Growing vertical aligned mesoporous silica thin film on nanoporous substrate for enhanced degradation, drug delivery and bioactivity.

Bioact Mater 2021 May 10;6(5):1452-1463. Epub 2020 Nov 10.

State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University, Xi'an, 710032, China.

Mesoporous silica thin film has been widely used in various fields, particularly the medical implant coating for drug delivery. However, some drawbacks remain with the films produced by traditional method (evaporation-induced self-assembly, EISA), such as the poor permeability caused by their horizontal aligned mesochannels. In this study, the vertical aligned mesoporous silica thin film (VMSTF) is uniformly grown alongside the walls of titania nanotubes array via a biphase stratification growth method, resulting in a hierarchical two-layered nanotubular structure. Due to the exposure of opened mesopores, VMSTF exhibits more appealing performances, including rapid degradation, efficient small-molecular drug (dexamethasone) loading and release, enhanced early adhesion and osteogenic differentiation of MC3T3-E1 cells. This is the first time successfully depositing VMSTF on nanoporous substrate and our findings suggest that the VMSTF may be a promising candidate for bone implant surface coating to obtain bioactive performances.
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http://dx.doi.org/10.1016/j.bioactmat.2020.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670213PMC
May 2021

In situ Surface Charge Density Visualization of Self-assembled DNA Nanostructures after Ion Exchange.

Chemphyschem 2020 07 8;21(13):1474-1482. Epub 2020 Jun 8.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark.

The charge density of DNA is a key parameter in strand hybridization and for the interactions occurring between DNA and molecules in biological systems. Due to the intricate structure of DNA, visualization of the surface charge density of DNA nanostructures under physiological conditions was not previously possible. Here, we perform a simultaneous analysis of the topography and surface charge density of DNA nanostructures using atomic force microscopy and scanning ion conductance microscopy. The effect of in situ ion exchange using various alkali metal ions is tested with respect to the adsorption of DNA origami onto mica, and a quantitative study of surface charge density reveals ion exchange phenomena in mica as a key parameter in DNA adsorption. This is important for structure-function studies of DNA nanostructures. The research provides an efficient approach to study surface charge density of DNA origami nanostructures and other biological molecules at a single molecule level.
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http://dx.doi.org/10.1002/cphc.201901168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891384PMC
July 2020

Electroactive Scaffolds for Neurogenesis and Myogenesis: Graphene-Based Nanomaterials.

Small 2018 11 27;14(48):e1801983. Epub 2018 Sep 27.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000, Aarhus C, Denmark.

One of the major issues in tissue engineering is constructing a functional scaffold to support cell growth and also provide proper synergistic guidance cues. Graphene-based nanomaterials have emerged as biocompatible and electroactive scaffolds for neurogenesis and myogenesis, due to their excellent tunable chemical, physical, and mechanical properties. This review first assesses the recent investigations focusing on the fabrication and applications of graphene-based nanomaterials for neurogenesis and myogenesis, in the form of either 2D films, 3D scaffolds, or composite architectures. Besides, because of their outstanding electrical properties, graphene family materials are particularly suitable for designing electroactive scaffolds that could provide proper electrical stimulation (i.e., electrical or photo stimuli) to promote the regeneration of excitable neurons and muscle cells. Therefore, the effects and mechanism of electrical and/or photo stimulations on neurogenesis and myogenesis are followed. Furthermore, studies on their biocompatibilities and toxicities especially to neural and muscle cells are evaluated. Finally, the future challenges and perspectives in facilitating the development of clinical translation of graphene-family nanomaterials in treating neurodegenerative and muscle diseases are discussed.
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http://dx.doi.org/10.1002/smll.201801983DOI Listing
November 2018

In vitro single-cell dissection revealing the interior structure of cable bacteria.

Proc Natl Acad Sci U S A 2018 08 6;115(34):8517-8522. Epub 2018 Aug 6.

Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark;

Filamentous Desulfobulbaceae bacteria were recently discovered as long-range transporters of electrons from sulfide to oxygen in marine sediments. The long-range electron transfer through these cable bacteria has created considerable interests, but it has also raised many questions, such as what structural basis will be required to enable micrometer-sized cells to build into centimeter-long continuous filaments? Here we dissected cable bacteria cells in vitro by atomic force microscopy and further explored the interior, which is normally hidden behind the outer membrane. Using nanoscale topographical and mechanical maps, different types of bacterial cell-cell junctions and strings along the cable length were identified. More important, these strings were found to be continuous along the bacterial cells passing through the cell-cell junctions. This indicates that the strings serve an important function in maintaining integrity of individual cable bacteria cells as a united filament. Furthermore, ridges in the outer membrane are found to envelop the individual strings at cell-cell junctions, and they are proposed to strengthen the junctions. Finally, we propose a model for the division and growth of the cable bacteria, which illustrate the possible structural requirements for the formation of centimeter-length filaments in the recently discovered cable bacteria.
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http://dx.doi.org/10.1073/pnas.1807562115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112711PMC
August 2018

Rapid Growth of Acetylated Aβ(16-20) into Macroscopic Crystals.

ACS Nano 2018 06 22;12(6):5408-5416. Epub 2018 May 22.

Interdisciplinary Nanoscience Center , Aarhus University , Aarhus 8000 , Denmark.

Aberrant assembly of the amyloid-β (Aβ) is responsible for the development of Alzheimer's disease, but can also be exploited to obtain highly functional biomaterials. The short Aβ fragment, KLVFF (Aβ), is crucial for Aβ assembly and considered to be an Aβ aggregation inhibitor. Here, we show that acetylation of KLVFF turns it into an extremely fast self-assembling molecule, reaching macroscopic ( i.e., mm) size in seconds. We show that KLVFF is metastable and that the self-assembly can be directed toward a crystalline or fibrillar phase simply through chemical modification, via acetylation or amidation of the peptide. Amidated KLVFF can form amyloid fibrils; we observed folding events of such fibrils occurring in as little as 60 ms. The ability of single KLVFF molecules to rapidly assemble as highly ordered macroscopic structures makes it a promising candidate for applications as a rapid-forming templating material.
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http://dx.doi.org/10.1021/acsnano.8b00448DOI Listing
June 2018

Direct measurement of surface charge distribution in phase separating supported lipid bilayers.

Nanoscale 2018 Mar;10(9):4538-4544

State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, 92 West Da-Zhi Street, Harbin, 150001, China.

The local surface charge density of the cell membrane influences regulation and localization of membrane proteins. The local surface charge density could, until recently, not be measured directly under physiological conditions, and it was largely a hypothetical yet very important parameter. Here we use unsaturated lipids of a distinct charge (DOTAP, DOPC, and DOPG) and a neutral fully saturated lipid (DPPC) to create model membranes with phase separating domains of a defined charge. We then apply quantitative surface charge microscopy (QSCM) to investigate the local surface charge density; this is a technique based on a scanning ion conductance microscope (SICM) capable of measuring surface charge density with nanoscale lateral resolution. We are able to clearly distinguish lipid domains from charge and topography in all three model membranes. The measured surface charge densities furthermore reveal that disordered domains formed by charged lipids are in fact not only impure, but also incorporate uncharged saturated lipids. We estimate that at least 30% of disordered domains in DOPG : DPPC and DOTAP : DPPC will be DPPC. These ratios could present a limit for the formation of charged domains in lipid membranes.
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http://dx.doi.org/10.1039/c7nr09522hDOI Listing
March 2018

High-vacuum optical platform for cryo-CLEM (HOPE): A new solution for non-integrated multiscale correlative light and electron microscopy.

J Struct Biol 2018 01 4;201(1):63-75. Epub 2017 Nov 4.

Center for Biological Imaging, Core Facilities for Protein Science, Institute of Biophysics, CAS, Beijing, China; University of Chinese Academy of Sciences, Beijing, China; National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address:

Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The 'touch-free' specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.
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http://dx.doi.org/10.1016/j.jsb.2017.11.002DOI Listing
January 2018

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy.

Nat Commun 2016 08 26;7:12447. Epub 2016 Aug 26.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.

Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.
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http://dx.doi.org/10.1038/ncomms12447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007656PMC
August 2016

Strong interactions with polyethylenimine-coated human serum albumin nanoparticles (PEI-HSA NPs) alter α-synuclein conformation and aggregation kinetics.

Nanoscale 2015 Dec;7(46):19627-40

Interdisciplinary Nanoscience Centre (iNANO) and Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 14, DK - 8000 Aarhus C, Denmark.

The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different levels of interactions had different effects on αSN aggregation. While the weakly interacting HSA NPs did not alter the aggregation kinetic parameters of αSN, the rate of primary nucleation increased in the presence of PEI-HSA NPs. The aggregation rate changed in a PEI-HSA NP-concentration dependent and size independent manner and led to fibrils which were covered with small aggregates. Furthermore, PEI-HSA NPs reduced the level of membrane-perturbing oligomers and reduced oligomer toxicity in cell assays, highlighting a potential role for NPs in reducing αSN pathogenicity in vivo. Collectively, our results highlight the fact that a simple modification of NPs can strongly modulate interactions with target proteins, which may have important and positive implications in NP safety.
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http://dx.doi.org/10.1039/c5nr05663bDOI Listing
December 2015

Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion.

Int J Nanomedicine 2015 24;10:2335-46. Epub 2015 Mar 24.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.

Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA) for sustained gene silencing in nearby cells, the layer-by-layer (LbL) approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA) nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP). The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more importantly, a gradual release of the film allowed the siRNA to be maintained on the titanium surface over approximately 1 week. In vitro transfection revealed that the LbL film-associated siRNA could consistently suppress GFP expression in H1299 without showing significant cytotoxicity. The LbL film loading with osteogenic siRNA could dramatically increase the osteogenic differentiation in MG63. In conclusion, LbL technology can potentially modify titanium surfaces with specific gene-regulatory siRNAs to enhance biofunction.
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http://dx.doi.org/10.2147/IJN.S76513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378287PMC
June 2016

The importance of being capped: Terminal capping of an amyloidogenic peptide affects fibrillation propensity and fibril morphology.

Biochemistry 2014 Nov 31;53(44):6968-80. Epub 2014 Oct 31.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Gustav Wieds Vej 14, DK-8000 Aarhus, Denmark.

The formation of aggregated fibrillar β-sheet structures has been proposed to be a generic feature of proteins. Aggregation propensity is highly sequence dependent, and often only part of the protein is incorporated into the fibril core. Therefore, shorter peptide fragments corresponding to the fibril core are attractive fibrillation models. The use of peptide models introduces new termini into the fibrils, yet little attention has been paid to the role these termini may play in fibrillation. Here, we report that terminal modifications of a 10-residue peptide fragment of human islet amyloid polypeptide strongly affect fibrillation kinetics and the resulting fibril morphology. Capping of the N-terminus abolishes fibrillation, while C-terminal capping results in fibrils with a twisted morphology. Peptides with either both termini free or both termini capped form flat fibrils. Molecular dynamics simulations reveal that the N-terminal acetyl cap folds up and interacts with the peptide's hydrophobic side chains, while the uncapped N-terminus in the C-terminally capped version results in twisting of the fibrils due to charge repulsion from the free N-termini. Our results highlight the role of terminal interactions in fibrillation of small peptides and provide molecular insight into the consequences of C-terminal modifications frequently found in peptide hormones in vivo.
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http://dx.doi.org/10.1021/bi500674uDOI Listing
November 2014