Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection.
Cell reports methods
2022; 2 (10): 100298
Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.
View details for DOI 10.1016/j.crmeth.2022.100298
View details for PubMedID 36313809
Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells.
Proceedings of the National Academy of Sciences of the United States of America
Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.
View details for PubMedID 30232265
Interfacial mechanisms for stability of surfactant-laden films
2017; 12 (5)
Thin liquid films are central to everyday life. They are ubiquitous in modern technology (pharmaceuticals, coatings), consumer products (foams, emulsions) and also serve vital biological functions (tear film of the eye, pulmonary surfactants in the lung). A common feature in all these examples is the presence of surface-active molecules at the air-liquid interface. Though they form only molecular-thin layers, these surfactants produce complex surface stresses on the free surface, which have important consequences for the dynamics and stability of the underlying thin liquid film. Here we conduct simple thinning experiments to explore the fundamental mechanisms that allow the surfactant molecules to slow the gravity-driven drainage of the underlying film. We present a simple model that works for both soluble and insoluble surfactant systems in the limit of negligible adsorption-desorption dynamics. We show that surfactants with finite surface rheology influence bulk flow through viscoelastic interfacial stresses, while surfactants with inviscid surfaces achieve stability through opposing surface-tension induced Marangoni flows.
View details for DOI 10.1371/journal.pone.0175753
View details for Web of Science ID 000401487700007
View details for PubMedID 28520734
- Hand-powered ultralow-cost paper centrifuge NATURE BIOMEDICAL ENGINEERING 2017; 1 (1)
Instability and Breakup of Model Tear Films
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2016; 57 (3): 949-958
An experimental platform to replicate the human tear film on a contact lens is presented. The influence of interfacial viscoelasticity in stabilizing in vitro model tear films against breakup and dewetting is investigated using this instrument.Model tear films consisting of bovine meibomian lipids (meibum) spread on either PBS or artificial tear solution (ATS) are created. The interfacial shear rheology of these films is measured as a function of temperature. The dewetting dynamics of these films is then investigated using the Interfacial Dewetting and Drainage Optical Platform (i-DDrOP) on top of silicone hydrogel (SiHy) contact lenses at 23 and 35°C. The film breakup times are evaluated using two parameters: onset of film breakup, Tonset for thick films (∼100 μm), and tear breakup times, TBU for thin films (∼1 μm). Thin film thinning rates as a result of evaporation are also calculated.The ATS/meibum films have the largest surface rheology and correspondingly show the largest Tonset times at both 23 and 35°C. The parameter TBU is also significantly larger for ATS/meibum (TBU ∼ 40 seconds) compared with that of ATS and PBS/meibum films (TBU ∼ 30 seconds) at room temperature. However, at 35°C, all three model tear films exhibit similar TBU ∼ 17 seconds and average rate of thinning of -4 μm/minute.Tear film stability is influenced by both surface rheology and evaporation. The in vitro tear breakup times and thinning rates of model tear films at 35°C are in good agreement with in vivo measurements previously reported, highlighting the utility of the i-DDrOP for in vitro tear film breakup research.
View details for DOI 10.1167/iovs.15-18064
View details for Web of Science ID 000374860600026
View details for PubMedID 26943158