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- 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.
- ItemImpact of mucus modulation by N-acetylcysteine on nanoparticle toxicity(Amsterdam : Elsevier, 2023) Meziu, Enkeleda; Shehu, Kristela; Koch, Marcus; Schneider, Marc; Kraegeloh, AnnetteHuman respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.
- ItemHuman glabrous skin contains crystallized urea dendriform structures in the stratum corneum which affect the hydration levels(Oxford : Wiley-Blackwell, 2023) Infante, Victor Hugo Pacagnelli; Bennewitz, Roland; Kröger, Marius; Meinke, Martina C.; Darvin, Maxim E.Glabrous skin is hair-free skin with a high density of sweat glands, which is found on the palms, and soles of mammalians, covered with a thick stratum corneum. Dry hands are often an occupational problem which deserves attention from dermatologists. Urea is found in the skin as a component of the natural moisturizing factor and of sweat. We report the discovery of dendrimer structures of crystalized urea in the stratum corneum of palmar glabrous skin using laser scanning microscopy. The chemical and structural nature of the urea crystallites was investigated in vivo by non-invasive techniques. The relation of crystallization to skin hydration was explored. We analysed the index finger, small finger and tenar palmar area of 18 study participants using non-invasive optical methods, such as laser scanning microscopy, Raman microspectroscopy and two-photon tomography. Skin hydration was measured using corneometry. Crystalline urea structures were found in the stratum corneum of about two-thirds of the participants. Participants with a higher density of crystallized urea structures exhibited a lower skin hydration. The chemical nature and the crystalline structure of the urea were confirmed by Raman microspectroscopy and by second harmonic generated signals in two-photon tomography. The presence of urea dendrimer crystals in the glabrous skin seems to reduce the water binding capacity leading to dry hands. These findings highlight a new direction in understanding the mechanisms leading to dry hands and open opportunities for the development of better moisturizers and hand disinfection products and for diagnostic of dry skin.
- ItemA high-throughput 3D kinetic killing assay(Weinheim : Wiley-VCH, 2023) Zhao, Renping; Yanamandra, Archana K.; Qu, Bin[No abstract available]
- ItemIn vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1(San Francisco, California, US : PLOS, 2023) Blanch-Asensio, Marc; Dey, Sourik; Sankaran, ShrikrishnanLactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.
- ItemElectroactive nanoinjection platform for intracellular delivery and gene silencing(London : Biomed Central, 2023) Shokouhi, Ali-Reza; Chen, Yaping; Yoh, Hao Zhe; Murayama, Takahide; Suu, Koukou; Morikawa, Yasuhiro; Brenker, Jason; Alan, Tuncay; Voelcker, Nicolas H.; Elnathan, RoeyBackground: Nanoinjection—the process of intracellular delivery using vertically configured nanostructures—is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell’s intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation. Results: Through both theoretical simulations and experiments, we demonstrate the capability of the ENI platform to locally perforate GPE-86 mouse fibroblast cells and efficiently inject a diverse range of membrane-impermeable biomolecules with efficacy of 62.5% (antibody), 55.5% (mRNA), and 51.8% (plasmid DNA), with minimal impact on cells’ viability post nanoscale-EP (> 90%). We also show gene silencing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of 41.3%. Conclusions: We anticipate that our non-viral and low-voltage ENI platform is set to offer a new safe path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene silencing. Graphical abstract: [Figure not available: see fulltext.]
- ItemBreast Cancer Stem Cell–Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands(Philadelphia, Pa. : AACR, 2023) Raute, Katrin; Strietz, Juliane; Parigiani, Maria Alejandra; Andrieux, Geoffroy; Thomas, Oliver S.; Kistner, Klaus M.; Zintchenko, Marina; Aichele, Peter; Hofmann, Maike; Zhou, Houjiang; Weber, Wilfried; Boerries, Melanie; Swamy, Mahima; Maurer, Jochen; Minguet, SusanaThere are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γ δ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γ δ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γ δ T cells. Indeed, neither promigratory engineered γ δ T cells, nor anti–PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γ δ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.
- ItemDynamic fine-tuning of CAR-T cell therapy(London : Nature Publishing Group, 2023) Trehin, Pierre V.M.; Muñoz-Guamuro, Geisler; Weber, Wilfried[No abstract available]