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- ItemTime‐Dependent Cation Selectivity of Titanium Carbide MXene in Aqueous Solution(Weinheim : Wiley-VCH, 2022) Wang, Lei; Torkamanzadeh, Mohammad; Majed, Ahmad; Zhang, Yuan; Wang, Qingsong; Breitung, Ben; Feng, Guang; Naguib, Michael; Presser, VolkerElectrochemical ion separation is a promising technology to recover valuable ionic species from water. Pseudocapacitive materials, especially 2D materials, are up-and-coming electrodes for electrochemical ion separation. For implementation, it is essential to understand the interplay of the intrinsic preference of a specific ion (by charge/size), kinetic ion preference (by mobility), and crystal structure changes. Ti3C2Tz MXene is chosen here to investigate its selective behavior toward alkali and alkaline earth cations. Utilizing an online inductively coupled plasma system, it is found that Ti3C2Tz shows a time-dependent selectivity feature. In the early stage of charging (up to about 50 min), K+ is preferred, while ultimately Ca2+ and Mg2+ uptake dominate; this unique phenomenon is related to dehydration energy barriers and the ion exchange effect between divalent and monovalent cations. Given the wide variety of MXenes, this work opens the door to a new avenue where selective ion-separation with MXene can be further engineered and optimized.
- ItemMechanically Stable, Binder‐Free, and Free‐Standing Vanadium Trioxide/Carbon Hybrid Fiber Electrodes for Lithium‐Ion Batteries(Weinheim : Wiley-VCH, 2023) Bornamehr, Behnoosh; Gallei, Markus; Husmann, Samantha; Presser, VolkerBinder is a crucial component in present-day battery electrodes but commonly contains fluorine and requires coating processing using organic (often toxic) solvents. Preparing binder-free electrodes is an attractive strategy to make battery electrode production and its end-of-use waste greener and safer. Herein, electrospinning is employed to prepare binder-free and self-standing electrodes. Such electrodes often suffer from low flexibility, and the correlation between performance and flexibility is usually overlooked. Processing parameters affect the mechanical properties of the electrodes, and for the first time it is reported that mechanical flexibility directly influences the electrochemical performance of the electrode. The importance is highlighted when processing parameters advantageous to powder materials, such as a higher heat treatment temperature, harm self-standing electrodes due to deterioration of fiber flexibility. Other strategies, such as conductive carbon addition, can be employed to improve the cell performance, but their effect on the mechanical properties of the electrodes must be considered. Rapid heat treatment achieves self-standing V2O3 with a capacity of 250 mAh g−1 at 250 mA g−1 and 390 mAh g−1 at 10 mA g−1
- ItemCharacterization of immune cell migration using microfabrication(Berlin ; Heidelberg : Springer, 2021) Vesperini, Doriane; Montalvo, Galia; Qu, Bin; Lautenschläger, FranziskaThe immune system provides our defense against pathogens and aberrant cells, including tumorigenic and infected cells. Motility is one of the fundamental characteristics that enable immune cells to find invading pathogens, control tissue damage, and eliminate primary developing tumors, even in the absence of external treatments. These processes are termed “immune surveillance.” Migration disorders of immune cells are related to autoimmune diseases, chronic inflammation, and tumor evasion. It is therefore essential to characterize immune cell motility in different physiologically and pathologically relevant scenarios to understand the regulatory mechanisms of functionality of immune responses. This review is focused on immune cell migration, to define the underlying mechanisms and the corresponding investigative approaches. We highlight the challenges that immune cells encounter in vivo, and the microfabrication methods to mimic particular aspects of their microenvironment. We discuss the advantages and disadvantages of the proposed tools, and provide information on how to access them. Furthermore, we summarize the directional cues that regulate individual immune cell migration, and discuss the behavior of immune cells in a complex environment composed of multiple directional cues.
- ItemEncapsulation of bacteria in bilayer Pluronic thin film hydrogels: A safe format for engineered living materials(Amsterdam : Elsevier, 2023) Bhusari, Shardul; Kim, Juhyun; Polizzi, Karen; Sankaran, Shrikrishnan; del Campo, AránzazuIn engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.
- ItemDiscovery of a high-performance phage-derived promoter/repressor system for probiotic lactobacillus engineering(Cold Spring Harbor : Cold Spring Harbor Laboratory, NY, 2023) Blanch-Asensio, Marc; Tadimarri, Varun Sai; Wilk, Alina; Sankaran, ShrikrishnanBackground: The Lactobacillus family comprises many species of great importance for the food and healthcare industries, with numerous strains identified as beneficial for humans and used as probiotics. Hence, there is a growing interest in engineering these probiotic bacteria as live biotherapeutics for animals and humans. However, the genetic parts needed to regulate gene expression in these bacteria remain limited compared to model bacteria like E. coli or B. subtilis. To address this deficit, in this study, we selected and tested several bacteriophage-derived genetic parts with the potential to regulate transcription in lactobacilli. Results: We screened genetic parts from 6 different lactobacilli-infecting phages and identified one promoter/repressor system with unprecedented functionality in L. plantarum WCFS1. The phage-derived promoter was found to achieve expression levels nearly 9-fold higher than the previously reported strongest promoter in this strain and the repressor was able to almost completely repress this expression by reducing it nearly 500-fold. Conclusions: The new parts and insights gained from their engineering will enhance the genetic programmability of lactobacilli for healthcare and industrial applications. Competing Interest Statement: A patent application has been filed based on the results of this work (Application no. is DE 102022 119024.2).
- ItemSelf-Adhesive Silicone Microstructures for the Treatment of Tympanic Membrane Perforations(Weinheim : Wiley-VCH, 2021) Lana, Gabriela Moreira; Sorg, Katharina; Wenzel, Gentiana Ioana; Hecker, Dietmar; Hensel, René; Schick, Bernhard; Kruttwig, Klaus; Arzt, EduardInspired by the gecko foot, polymeric microstructures have demonstrated reliable dry adhesion to both stiff objects and sensitive surfaces such as skin. Microstructured silicone patches are proposed, herein, for the treatment of tympanic membrane perforations with the aim of serving as an alternative for current surgical procedures that require anesthesia and ear canal packing. Sylgard 184 PDMS micropillars of 20 μm in diameter and 60 μm in length are topped by a Soft Skin Adhesive (SSA) MG7-1010 terminal layer, of about 25 μm thickness. The adhesion is evaluated by specially designed tack tests against explanted murine eardrums and, for comparison, against a rigid substrate. Functional effects are evaluated using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAE). The adhesion strength of the microstructure and unstructured controls to explanted murine tympanic membranes is comparable (typically 12 kPa), but the microstructured patches are easier to handle by the surgeon. For the first time, partial recovery of hearing performance is measured immediately after patch application. The novel patches adhere without the need for further fixation, removing the need for ear canal packing. The proposed material design holds great promise for improving clinical treatments of tympanic membrane perforations.
- ItemMyxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections(Basel : MDPI, 2020) Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K.; Müller, Rolf; Fuhrmann, GregorIn 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
- ItemQuantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy(Basel : MDPI, 2021) Peckys, Diana B.; Gaa, Daniel; de Jonge, NielsCurrently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.
- ItemGelation Kinetics and Mechanical Properties of Thiol-Tetrazole Methylsulfone Hydrogels Designed for Cell Encapsulation(Weinheim : Wiley-VCH, 2022) de Miguel‐Jiménez, Adrián; Ebeling, Bastian; Paez, Julieta I.; Fink‐Straube, Claudia; Pearson, Samuel; del Campo, AránzazuHydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.
- ItemNovel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli(Oxford : Wiley-Blackwell, 2023) Dey, Sourik; Blanch‐Asensio, Marc; Balaji Kuttae, Sanjana; Sankaran, ShrikrishnanLactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA, from a phylogenetically distant bacterium, Salmonella typhimurium, which drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tuneable transient genetically modified organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.
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