Effects of the Sex Steroid Hormone Estradiol on Biofilm Growth of Cystic Fibrosis Pseudomonas aeruginosa Isolates.

Women with cystic fibrosis (CF) have a significantly lower life expectancy compared to men, which is indicated by an earlier impairment of lung function due to chronic colonization with biofilm formed by Pseudomonas aeruginosa. There is growing evidence that blood serum concentrations of the steroid sex hormone estradiol (E2) correlate with the occurrence of pulmonary exacerbations in CF but also play a role in the mucoid switch of P. aeruginosa. This study aims to shed light on possible microbiological reasons for sexual dimorphism in CF by investigating the influence of E2 on biofilm formation of P. aeruginosa CF isolates. For this purpose, 10 CF isolates of the respiratory tract derived from different CF patients have been treated with E2 in a microtiter plate biofilm model. Biofilms have been examined by crystal violet assays, field emission scanning electron microscopy (FE-SEM), 3D laser scanning microscopy (LSM), and quorum sensing (QS) reporter assays of the supernatants taken from biofilms. This allowed us to simultaneously investigate the effects of E2 on attached biofilm mass, biofilm ultrastructure, and QS activity. Upon E2 treatment, six out of 10 investigated CF isolates showed an increase of attached biofilm mass, whereas biofilms from two tested non-CF laboratory strains (PAO1 and ATCC19660) did not. Moreover, FE-SEM and 3D LSM analyses of the E2 responsive CF biofilms revealed ultrastructural remodeling of biofilm structure at different scales with increased formation of prominent biofilm spots, enhanced coverage with extracellular polymeric substance (EPS), and extended average surface roughness. QS activity measurements performed in biofilm supernatants via luminescence acyl homoserine lactone (AHL) reporter assays further showed that E2 treatment may also modulate QS signaling, as shown in an E2 sensitive CF isolate. Together, our results suggest the biofilm modulating effects of E2 on various clinical CF isolates that are documented by both biomass and ultrastructural changes of biofilms. The gained new insight into the influence of steroid hormones on P. aeruginosa biofilm phenotypes might pave the way for novel future approaches in personalized medicine based on the patients' sex and hormonal status.

Phase Transitions and Formation of a Monolayer-Type Structure in Thin Oligothiophene Films: Exploration with a Combined In Situ X-ray Diffraction and Electrical Measurements.

A combination of in situ electrical and grazing-incidence X-ray diffraction (GIXD) is a powerful tool for studies of correlations between the microstructure and charge transport in thin organic films. The information provided by such experimental approach can help optimizing the performance of the films as active layers of organic electronic devices. In this work, such combination of techniques was used to investigate the phase transitions in vacuum-deposited thin films of a common organic semiconductor dihexyl-quarterthiophene (DH4T). A transition from the initial highly crystalline phase to a mesophase was detected upon heating, while only a partial backward transition was observed upon cooling to room temperature. In situ electrical conductivity measurements revealed the impact of both transitions on charge transport. This is partly accounted for by the fact that the initial crystalline phase is characterized by inclination of molecules in the plane perpendicular to the π-π stacking direction, whereas the mesophase is built of molecules tilted in the direction of π-π stacking. Importantly, in addition to the two phases of DH4T characteristic of the bulk, a third interfacial substrate-stabilized monolayer-type phase was observed. The existence of such interfacial structure can have important implications for the charge mobility, being especially favorable for lateral two-dimensional charge transport in the organic field-effect transistors geometry.

Multimodal microscopy-based identification of surface nanobubbles.

HYPOTHESIS: Surface nanobubbles, which were controversially discussed in the literature, promise a number of outstanding applications, and their presence may hamper nanoscale processes at solid-aqueous interfaces. A most crucial and yet unsolved question is the rapid and conclusive identification of gas-filled (surface) nanobubbles. We hypothesize that surface nanobubbles and oil nanodroplets can be conclusively differentiated in co-localization experiments with atomic force microscopy (AFM) and time-resolved fluorescence microscopy by localizing tracer fluorophores and analyzing their fluorescence lifetimes. EXPERIMENTS: Combined AFM and fluorescence lifetime imaging microscopy (FLIM) were conducted to localize the various interfaces labelled by the reporter dye rhodamine 6G (Rh6G). The dependence of the fluorescence lifetime of Rh6G on its local environment was determined for air/water, water/glass and polysiloxane/water interfaces under different conditions. FINDINGS: In in situ co-localization experiments, surface nanobubbles labeled with Rh6G were probed by AFM with high spatial resolution and were differentiated from polysiloxane droplets as well as contamination originating from lubricant-coated syringe needles owing to the characteristic short fluorescence lifetime of Rh6G at the gas/water interface observed in FLIM. In particular, this approach lends itself to conclusively identify and rapidly differentiate these gas-filled entities from adsorbed contamination, such as siloxane-based oil nanodroplets.

Thickness-Encoded Micropatterns in One-Component Thermoresponsive Polymer Brushes for Culture and Triggered Release of Pancreatic Tumor Cell Monolayers and Spheroids.

Fabrication, characterization, and application of micropatterned one-component poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes for monolayer cell and spheroid culture and temperature-triggered release are reported. Micropatterns of various shapes and sizes were designed to possess a unique functionality imparted by thermoresponsive thin PDEGMA patches, which are cell adhesive at 37 °C, embedded in a much thicker cell-resistant PDEGMA matrix that does not exhibit measurable thermoresponsive properties. Depending on the cell seeding density, PaTu 8988t human pancreatic tumor cells or spheroids were cultured area-selectively, confined by the 40 ± 4 nm thick passivating PDEGMA matrix, and could be released on demand by a mild thermally triggered brush swelling in the 5 ± 1 nm thin regions. As shown by surface plasmon resonance (SPR) measurements, in contrast to the thinner brushes, the thicker brushes exhibited virtually no fibronectin adhesive properties at 37 °C, whereas at 25 °C, both areas showed similar protein resistant behavior. The quasi-2D thickness-encoded micropatterns were shown to be useful templates for the growth of 3D multicellular aggregates. Thermally induced release after 5 days of incubation afforded 3D cell spheroids comprising up to 99% viable cells demonstrating that the system can be used as a 3D spheroid in vitro model for basic tumor research and anticancer drug screenings.

Fluorimetric Detection of G-Quadruplex DNA in Solution and Adsorbed on Surfaces with a Selective Trinuclear Cyanine Dye.

Quadruplex DNA, which is a relevant target for anticancer therapies, may alter its conformation because of interactions with interfaces. In pursuit of a versatile methodology to probe adsorption-induced conformational changes, the interaction between a fluorescent [2.2.2]heptamethinecyanine dye and quadruplex DNA (G4-DNA) was studied in solution and on surfaces. In solution, the cyanine dye exhibits a strong light-up effect upon the association with G4-DNA without interference from double-stranded DNA. In addition, a terminal π-stacking as a binding mode between the cyanine dye and G4-DNA is concluded using NMR spectroscopy. To unravel the effects of adsorption on the conformation of quadruplex-DNA, G4-DNA, and double-stranded and single-stranded DNA were adsorbed to positively charged poly(allylamine) hydrochloride (PAH) surfaces, both in planar and in constrained 55 nm diameter aluminum oxide nanopore formats. All DNA forms showed a very strong affinity to the PAH surfaces as shown by surface plasmon resonance and reflectometric interference spectroscopy. The significant increase of the fluorescence emission intensity of the cyanine light-up probe observed exclusively for surface immobilized G4-DNA affords evidence for the adsorption of G4-DNA on PAH with retained quadruplex conformation.

Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche.

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyer's patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyer's patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyer's patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research.

Detailed Study of BSA Adsorption on Micro- and Nanocrystalline Diamond/ß-SiC Composite Gradient Films by Time-Resolved Fluorescence Microscopy.

The adsorption of bovine serum albumin (BSA) on micro- and nanocrystalline diamond/ß-SiC composite films synthesized using the hot filament chemical vapor deposition (HFCVD) technique has been investigated by confocal fluorescence lifetime imaging microscopy. BSA labeled with fluorescein isothiocyanate (FITC) was employed as a probe. The BSAFITC conjugate was found to preferentially adsorb on both O-/OH-terminated microcrystalline and nanocrystalline diamond compared to the OH-terminated ß-SiC, resulting in an increasing amount of BSA adsorbed to the gradient surfaces with an increasing diamond/ß-SiC ratio. The different strength of adsorption (>30 times for diamond with a grain size of 570 nm) coincides with different surface energy parameters and differing conformational changes upon adsorption. Fluorescence data of the adsorbed BSAFITC on the gradient film with different diamond coverage show a four-exponential decay with decay times of 3.71, 2.54, 0.66, and 0.13 ns for a grain size of 570 nm. The different decay times are attributed to the fluorescence of thiourea fluorescein residuals of linked FITC distributed in BSA with different dye-dye and dye-surface distances. The longest decay time was found to correlate linearly with the diamond grain size. The fluorescence of BSAFITC undergoes external dynamic fluorescence quenching on the diamond surface by H- and/or sp2-defects and/or by amorphous carbon or graphite phases. An acceleration of the internal fluorescence concentration quenching in BSAFITC because of structural changes of albumin due to adsorption, is concluded to be a secondary contributor. These results suggest that the micro- and nanocrystalline diamond/ß-SiC composite gradient films can be utilized to spatially control protein adsorption and diamond crystallite size, which facilitates systematic studies at these interesting (bio)interfaces.

The Effect of Size and Geometry of Poly(acrylamide) Brush-Based Micropatterns on the Behavior of Cells.

In this study, the fabrication, detailed characterization, and application of long-term stable micropatterned bio-interfaces of passivating poly(acrylamide) (PAAm) brushes on transparent gold for application in the study of cell-surface interactions is reported. The micropatterns were fabricated by microcontact printing of an initiator for surface-initiated atom transfer radical polymerization (SI-ATRP), SI-ATRP of acrylamide, and subsequently backfilling of the unfunctionalized areas of 400-2500 µm(2) size and systematically altered number of corners with octadecanethiol. As verified by surface plasmon resonance spectroscopy, the physisorption of fibronectin (FN) was restricted to the adhesive areas. Exploiting this platform, the effect of micropattern geometry and size of cell-adhesive FN areas surrounded by passivating PAAm brushes on transparent gold substrates on the attachment of cells and cytoskeleton alignment was investigated at the single-cell level. Exceptional long-term stability of the patterned PAAm brushes and arrays of adhesive areas, in which human pancreatic tumor cells (Patu 8988T) and fibroblast cells (NIH 3T3) were confined for more than one week, was observed. Adhesive areas of 1600 µm(2) or less constrained the cell shape and caused focal adhesions to accumulate in the corners of the pattern. These changes were most obvious for the PatuT cells in adhesive areas of ∼900 µm(2), in which the actin filaments were aligned, following the boundary of the pattern, and merged in the focal adhesions concentrated in the corners of the pattern. NIH 3T3 cells possessed a larger cell area, which led to an optimal cytoskeleton alignment in adhesive patterns of ∼1600 µm(2). The alignment of the cytoskeleton was found to be less pronounced in cells on larger adhesive areas, where the PatuT cells spread similarly to cells on unpatterned substrates. By contrast, the NIH 3T3 cells were found to stretch even on larger adhesive areas, spanning from one corner to the other. The long-term stability under cell culture conditions of the patterns introduced here will also be useful for long-term studies of single and multiple cells, cell motility in toxicity assays, and stem cell differentiation.

Surface Nanobubbles Studied by Time-Resolved Fluorescence Microscopy Methods Combined with AFM: The Impact of Surface Treatment on Nanobubble Nucleation.

The impact of surface treatment and modification on surface nanobubble nucleation in water has been addressed by a new combination of fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM). In this study, rhodamine 6G (Rh6G)-labeled surface nanobubbles nucleated by the ethanol-water exchange were studied on differently cleaned borosilicate glass, silanized glass as well as self-assembled monolayers on transparent gold by combined AFM-FLIM. While the AFM data confirmed earlier reports on surface nanobubble nucleation, size, and apparent contact angles in dependence of the underlying substrate, the colocalization of these elevated features with highly fluorescent features observed in confocal intensity images added new information. By analyzing the characteristic contributions to the excited state lifetime of Rh6G in decay curves obtained from time-correlated single photon counting (TCSPC) experiments, the characteristic short-lived (<600 ps) component of could be associated with an emission at the gas-water interface. Its colocalization with nanobubble-like features in the AFM height images provides evidence for the observation of gas-filled surface nanobubbles. While piranha-cleaned glass supported nanobubbles, milder UV-ozone or oxygen plasma treatment afforded glass-water interfaces, where no nanobubbles were observed by combined AFM-FLIM. Finally, the number density of nanobubbles scaled inversely with increasing surface hydrophobicity.

Photoinduced formation of stable Ag-nanoparticles from a ternary ligand-DNA-Ag(+) complex.

The combination of (i) the light-harvesting nature and excited-state redox reactivity of a cationic DNA intercalator, (ii) a conjugated Ag(+)-binding crown ether, and (iii) the stabilizing effect of DNA on AgNPs in one integral ternary complex enables the mild photoinduced formation of Ag nanoparticles.

Molecular beacon modified sensor chips for oligonucleotide detection with optical readout.

Three different surface bound molecular beacons (MBs) were investigated using surface plasmon fluorescence spectroscopy (SPFS) as an optical readout technique. While MB1 and MB2, both consisting of 36 bases, differed only in the length of the linker for surface attachment, the significantly longer MB3, consisting of 56 bases, comprised an entirely different sequence. For sensor chip preparation, the MBs were chemisorbed on gold via thiol anchors together with different thiol spacers. The influence of important parameters, such as the length of the MBs, the length of the linker between the MBs and the gold surface, the length and nature of the thiol spacers, and the ratio between the MBs and the thiol spacers was studied. After hybridization with the target, the fluorophore of the longer MB3 was oriented close to the surface, and the shorter MBs were standing more or less upright, leading to a larger increase in fluorescence intensity. Fluorescence microscopy revealed a homogeneous distribution of the MBs on the surface. The sensor chips could be used for simple and fast detection of target molecules with a limit of detection in the larger picomolar range. The response time was between 5 and 20 min. Furthermore, it was possible to distinguish between fully complementary and singly mismatched targets. While rinsing with buffer solution after hybridization with target did not result in any signal decrease, complete dehybridization could be carried out by intense rinsing with pure water. The MB modified sensor chips could be prepared in a repeatable manner and reused many times without significant decrease in performance.

Construction of three-dimensional DNA hydrogels from linear building blocks.

A three-dimensional DNA hydrogel was generated by self-assembly of short linear double-stranded DNA (dsDNA) building blocks equipped with sticky ends. The resulting DNA hydrogel is thermoresponsive and the length of the supramolecular dsDNA structures varies with temperature. The average diffusion coefficients of the supramolecular dsDNA structures formed by self-assembly were determined by diffusion-ordered NMR spectroscopy (DOSY NMR) for temperatures higher than 60 °C. Temperature-dependent rheological measurements revealed a gel point of 42±1 °C. Below this temperature, the resulting material behaved as a true gel of high viscosity with values for the storage modulus G' being significantly larger than that for the loss modulus G''. Frequency-dependent rheological measurements at 20 °C revealed a mesh size (ξ) of 15 nm. AFM analysis of the diluted hydrogel in the dry state showed densely packed structures of entangled chains, which are also expected to contain multiple interlocked rings and catenanes.

Controlled surface chemistry of diamond/ß-SiC composite films for preferential protein adsorption.

Diamond and SiC both process extraordinary biocompatible, electronic, and chemical properties. A combination of diamond and SiC may lead to highly stable materials, e.g., for implants or biosensors with excellent sensing properties. Here we report on the controllable surface chemistry of diamond/ß-SiC composite films and its effect on protein adsorption. For systematic and high-throughput investigations, novel diamond/ß-SiC composite films with gradient composition have been synthesized using the hot filament chemical vapor deposition (HFCVD) technique. As revealed by scanning electron microscopy (SEM), the diamond/ß-SiC ratio of the composite films shows a continuous change from pure diamond to ß-SiC over a length of ∼ 10 mm on the surface. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed to unveil the surface termination of chemically oxidized and hydrogen treated surfaces. The surface chemistry of the composite films was found to depend on diamond/ß-SiC ratio and the surface treatment. As observed by confocal fluorescence microscopy, albumin and fibrinogen were preferentially adsorbed from buffer: after surface oxidation, the proteins preferred to adsorb on diamond rather than on ß-SiC, resulting in an increasing amount of proteins adsorbed to the gradient surfaces with increasing diamond/ß-SiC ratio. By contrast, for hydrogen-treated surfaces, the proteins preferentially adsorbed on ß-SiC, leading to a decreasing amount of albumin adsorbed on the gradient surfaces with increasing diamond/ß-SiC ratio. The mechanism of preferential protein adsorption is discussed by considering the hydrogen bonding of the water self-association network to OH-terminated surfaces and the change of the polar surface energy component, which was determined according to the van Oss method. These results suggest that the diamond/ß-SiC gradient film can be a promising material for biomedical applications which require good biocompatibility and selective adsorption of proteins and cells to direct cell migration.

Poly(ester-ether)s: II. Properties of electrospun nanofibres from polydioxanone and poly(methyl dioxanone) blends and human fibroblast cellular proliferation.

This article deals with an in-depth study of the thermal, mechanical and degradation behaviours of nanofibres from polydioxanone (PDX) and polydl-3-methyl-1,4-dioxan-2-one (PMeDX) and a comparison with their blend films. Varying ratios of both polymers were blended and electrospun from solution. Electrospun fibres exhibited a melting transition at 109 °C independently of the PMeDX content, which corresponds to the melting of PDX nanofibres. As a result of the drawing process, PMeDX had a reduced plasticizing effect on PDX. In general, it was observed that overall crystallinity of the fibres decreased from 53% to 36% with increasing PMeDX content and this impacted on their mechanical properties. The Young's moduli decreased as the PMeDX content of the fibres increased. However, an increase in strain at break and peak stress was noted as a result of a decrease in the fibre diameter. AFM images of the electrospun fibres showed an increasing degree of morphological heterogeneity with increasing PMeDX content. Thermal degradation studies showed that electrospun mats were thermally more stable than blend films, as confirmed by a two-fold increase in activation energy. The hydrolytic degradation of the electrospun mats conducted in phosphate buffer solution at 37 °C showed that the degradation followed a surface erosion mechanism as opposed to bulk degradation observed for blend films. Degradation of fibres was found to be mainly dependent on their diameter. On the other hand, the degradation of blend films depended on the overall crystallinity of the blends. Electrospun PDX/PMeDX nanofibrous scaffolds were also subjected to cell viability studies with human dermal fibroblasts, in which they did not show illicit response and demonstrated excellent cell attachment and proliferation.

High resolution imaging of surface patterns of single bacterial cells.

We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.