Engineering the Cellular Microenvironment: Integrating Three-Dimensional Nontopographical and Two-Dimensional Biochemical Cues for Precise Control of Cellular Behavior.

The development of biomaterials capable of regulating cellular processes and guiding cell fate decisions has broad implications in tissue engineering, regenerative medicine, and cell-based assays for drug development and disease modeling. Recent studies have shown that three-dimensional (3D) nanoscale physical cues such as nanotopography can modulate various cellular processes like adhesion and endocytosis by inducing nanoscale curvature on the plasma and nuclear membranes. Two-dimensional (2D) biochemical cues such as protein micropatterns can also regulate cell function and fate by controlling cellular geometries. Development of biomaterials with precise control over nanoscale physical and biochemical cues can significantly influence programming cell function and fate. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. Our findings demonstrate the precise engineering of single cell architectures through 2D micropatterning on nanopillar platforms. We show that the coupling between the nuclear and cell shape is disrupted on nanopillar surfaces compared to flat surfaces. Furthermore, our results suggest that cell elongation on nanopillars enhances nanopillar-induced endocytosis. We believe our platform serves as a versatile tool for further explorations into programming cell function and fate through combined physical cues that create nanoscale curvature on cell membranes and biochemical cues that control the geometry of the cell.

Cable bacteria: widespread filamentous electroactive microorganisms protecting environments.

Cable bacteria have been identified and detected worldwide since their discovery in marine sediments in Aarhus Bay, Denmark. Their activity can account for the majority of oxygen consumption and sulfide depletion in sediments, and they induce sulfate accumulation, pH excursions, and the generation of electric fields. In addition, they can affect the fluxes of other elements such as calcium, iron, manganese, nitrogen, and phosphorous. Recent developments in our understanding of the impact of cable bacteria on element cycling have revealed their positive contributions to mitigating environmental problems, such as recovering self-purification capacity, enhancing petroleum hydrocarbon degradation, alleviating phosphorus eutrophication, delaying euxinia, and reducing methane emission. We highlight recent research outcomes on their distribution, state-of-the-art findings on their physiological characteristics, and ecological contributions.

A NanoCurvS platform for quantitative and multiplex analysis of curvature-sensing proteins.

The cell membrane is characterized by a rich variety of topographical features such as local protrusions or invaginations. Curvature-sensing proteins, including the Bin/Amphiphysin/Rvs (BAR) or epsin N-terminal homology (ENTH) family proteins, sense the bending sharpness and the positive/negative sign of these topographical features to induce subsequent intracellular signaling. A number of assays have been developed to study curvature-sensing properties of proteins in vitro, but it is still challenging to probe low curvature regime with the diameter of curvature from hundreds of nanometers to micrometers. It is particularly difficult to generate negative membrane curvatures with well-defined curvature values in the low curvature regime. In this work, we develop a nanostructure-based curvature sensing (NanoCurvS) platform that enables quantitative and multiplex analysis of curvature-sensitive proteins in the low curvature regime, in both negative and positive directions. We use NanoCurvS to quantitatively measure the sensing range of a negative curvature-sensing protein IRSp53 (an I-BAR protein) and a positive curvature-sensing protein FBP17 (an F-BAR protein). We find that, in cell lysates, the I-BAR domain of IRSp53 is able to sense shallow negative curvatures with the diameter-of-curvature up to 1500 nm, a range much wider than previously expected. NanoCurvS is also used to probe the autoinhibition effect of IRSp53 and the phosphorylation effect of FBP17. Therefore, the NanoCurvS platform provides a robust, multiplex, and easy-to-use tool for quantitative analysis of both positive and negative curvature-sensing proteins.

Folding Double-Stranded DNA into Designed Shapes with Triplex-Forming Oligonucleotides.

The compaction and organization of genomic DNA is a central mechanism in eukaryotic cells, but engineered architectural control over double-stranded DNA (dsDNA) is notably challenging. Here, long dsDNA templates are folded into designed shapes via triplex-mediated self-assembly. Triplex-forming oligonucleotides (TFOs) bind purines in dsDNA via normal or reverse Hoogsteen interactions. In the triplex origami methodology, these non-canonical interactions are programmed to compact dsDNA (linear or plasmid) into well-defined objects, which demonstrate a variety of structural features: hollow and raster-filled, single- and multi-layered, with custom curvatures and geometries, and featuring lattice-free, square-, or honeycomb-pleated internal arrangements. Surprisingly, the length of integrated and free-standing dsDNA loops can be modulated with near-perfect efficiency; from hundreds down to only six bp (2 nm). The inherent rigidity of dsDNA promotes structural robustness and non-periodic structures of almost 25.000 nt are therefore formed with fewer unique starting materials, compared to other DNA-based self-assembly methods. Densely triplexed structures also resist degradation by DNase I. Triplex-mediated dsDNA folding is methodologically straightforward and orthogonal to Watson-Crick-based methods. Moreover, it enables unprecedented spatial control over dsDNA templates.

Liquid-Phase Friction of Two-Dimensional Molybdenum Disulfide at the Atomic Scale.

Tribological properties depend strongly on environmental conditions such as temperature, humidity, and operation liquid. However, the origin of the liquid effect on friction remains largely unexplored. Herein, taking molybdenum disulfide (MoS2) as a model system, we explored the nanoscale friction of MoS2 in polar (water) and nonpolar (dodecane) liquids through friction force microscopy. The friction force exhibits a similar layer-dependent behavior in liquids as in air; i.e., thinner samples have a larger friction force. Interestingly, friction is significantly influenced by the polarity of the liquid, and it is larger in polar water than in nonpolar dodecane. Atomically resolved friction images together with atomistic simulations reveal that the polarity of the liquid has a substantial effect on friction behavior, where liquid molecule arrangement and hydrogen-bond formation lead to a higher resistance in polar water in comparison to that in nonpolar dodecane. This work provides insights into the friction on two-dimensional layered materials in liquids and holds great promise for future low-friction technologies.

Emerging high-ammonia­nitrogen wastewater remediation by biological treatment and photocatalysis techniques.

The bacterial and photocatalysis techniques have been widely applied into the remediation of ammonia nitrogen wastewater. Although traditional microbial methods had been verified useful; more efficient, energy-saving and controllable candidate treatment methods are still urgently needed to cover the increasingly diverse ammonia nitrogen pollution cases. The bacterial treatment technique for ammonia nitrogen mainly depends on the ammonia nitrogen oxidation-reduction (e.g. nitrification, denitrification) by nitrifying bacteria and denitrifying bacteria, but these reactions suffer from slow denitrifying kinetic process and uncontrolled disproportionation reaction. In comparison, the photocatalysis technique based on photoelectrons is more efficient and has some advantages, such as low temperature reaction and long life, while the photocatalysis technique can not perform multiple complex biochemical reactions. Despite much scientific knowledge obtained about this issue recently, such research has yet not been widely adopted in the industry because of many concerns about subsequent catalyst stability and economic feasibility. This review summarized and discussed the very recent achievements and key problems on remediation of high-ammonia­nitrogen wastewater and oxidation driven by bacterial treatment and photocatalysis techniques, as well as the most promising future directions for these two techniques, especially the potential of jointly bacterial-photocatalysis techniques.

Nanoscale Surface Topography Reduces Focal Adhesions and Cell Stiffness by Enhancing Integrin Endocytosis.

Both substrate stiffness and surface topography regulate cell behavior through mechanotransduction signaling pathways. Such intertwined effects suggest that engineered surface topographies might substitute or cancel the effects of substrate stiffness in biomedical applications. However, the mechanisms by which cells recognize topographical features are not fully understood. Here we demonstrate that the presence of nanotopography drastically alters cell behavior such that neurons and stem cells cultured on rigid glass substrates behave as if they were on soft hydrogels. With atomic force microscopy, we show that rigid nanotopography resembles the effects of soft hydrogels in reducing cell stiffness and membrane tension. Further, we reveal that nanotopography reduces focal adhesions and cell stiffness by enhancing the endocytosis and the subsequent removal of integrin receptors. This mechanistic understanding will support the rational design of nanotopography that directs cells on rigid materials to behave as if they were on soft ones.

Characterization of DNA-protein complexes by nanoparticle tracking analysis and their association with systemic lupus erythematosus.

Nanotechnology enables investigations of single biomacromolecules, but technical challenges have limited the application in liquid biopsies, for example, blood plasma. Nonetheless, tools to characterize single molecular species in such samples represent a significant unmet need with the increasing appreciation of the physiological importance of protein structural changes at nanometer scale. Mannose-binding lectin (MBL) is an oligomeric plasma protein and part of the innate immune system through its ability to activate complement. MBL also serves a role as a scavenger for cellular debris, especially DNA. This may link functions of MBL with several inflammatory diseases in which cell-free DNA now appears to play a role, but mechanistic insight has been lacking. By making nanoparticle tracking analysis possible in human plasma, we now show that superoligomeric structures of MBL form nanoparticles with DNA. These oligomers correlate with disease activity in systemic lupus erythematosus patients. With the direct quantification of the hydrodynamic radius, calculations following the principles of Taylor dispersion in the blood stream connect the size of these complexes to endothelial inflammation, which is among the most important morbidities in lupus. Mechanistic insight from an animal model of lupus supported that DNA-stabilized superoligomers stimulate the formation of germinal center B cells and drive loss of immunological tolerance. The formation involves an inverse relationship between the concentration of MBL superoligomers and antibodies to double-stranded DNA. Our approach implicates the structure of DNA-protein nanoparticulates in the pathobiology of autoimmune diseases.

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

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.

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

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.

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

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.

Dynamic Manipulation of Cell Membrane Curvature by Light-Driven Reshaping of Azopolymer.

Local curvatures on the cell membrane serve as signaling hubs that promote curvature-dependent protein interactions and modulate a variety of cellular processes including endocytosis, exocytosis, and the actin cytoskeleton. However, precisely controlling the location and the degree of membrane curvature in live cells has not been possible until recently, where studies show that nanofabricated vertical structures on a substrate can imprint their shapes on the cell membrane to induce well-defined curvatures in adherent cells. Nevertheless, the intrinsic static nature of these engineered nanostructures prevents dynamic modulation of membrane curvatures. In this work, we engineer light-responsive polymer structures whose shape can be dynamically modulated by light and thus change the induced-membrane curvatures on-demand. Specifically, we fabricate three-dimensional azobenzene-based polymer structures that change from a vertical pillar to an elongated vertical bar shape upon green light illumination. We observe that U2OS cells cultured on azopolymer nanostructures rapidly respond to the topographical change of the substrate underneath. The dynamically induced high membrane curvatures at bar ends promote local accumulation of actin fibers and actin nucleator Arp2/3 complex. The ability to dynamically manipulate the membrane curvature and analyze protein response in real-time provides a new way to study curvature-dependent processes in live cells.

Lipidoid-polymer hybrid nanoparticles loaded with TNF siRNA suppress inflammation after intra-articular administration in a murine experimental arthritis model.

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.

A nanostructure platform for live-cell manipulation of membrane curvature.

Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy (FIB-SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.

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

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.

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

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.

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

Aberrant assembly of the amyloid-ß (Aß) is responsible for the development of Alzheimer's disease, but can also be exploited to obtain highly functional biomaterials. The short Aß fragment, KLVFF (Aß16-20), is crucial for Aß assembly and considered to be an Aß 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.

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

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.

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

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.

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

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.