3D Bioprinting of Prevascularized Full-Thickness Gelatin-Alginate Structures with Embedded Co-Cultures.

The use of bioprinting allows the creation of complex three-dimensional cell laden grafts with spatial placements of different cell lines. However, a major challenge is insufficient nutrient transfer, especially with the increased size of the graft causing necrosis and reduced proliferation. A possibility to improve nutrient support is the integration of tubular structures for reducing diffusion paths. In this study the influence of prevascularization in full-thickness grafts on cell growth with a variation of cultivation style and cellular composition was investigated. To perform this, the rheological properties of the used gelatin-alginate hydrogel as well as possibilities to improve growth conditions in the hydrogel were assessed. Prevascularized grafts were manufactured using a pneumatic extrusion-based bioprinter with a coaxial extrusion tool. The prevascularized grafts were statically and dynamically cultured with a monoculture of HepG2 cells. Additionally, a co-culture of HepG2 cells, fibroblasts and HUVEC-TERT2 was created while HUVEC-TERT2s were concentrically placed around the hollow channels. A static culture of prevascularized grafts showed short-term improvements in cell proliferation compared to avascular grafts, while a perfusion-based culture showed improvements in mid-term cultivation times. The cultivation of the co-culture indicated the formation of vascular structures from the hollow channels toward avascular areas. According to these results, the integration of prevascular structures show beneficial effects for the in vitro cultivation of bioprinted grafts for which its impact can be increased in larger grafts.

Assessment of Phenotype Relevant Amino Acid Residues in TEM-ß-Lactamases by Mathematical Modelling and Experimental Approval.

Single substitutions or combinations of them alter the hydrolytic activity towards specific ß-lactam-antibiotics and ß-lactamase inhibitors of TEM-ß-lactamases. The sequences and phenotypic classification of allelic TEM variants, as provided by the NCBI National Database of Antibiotic Resistant Organisms, does not attribute phenotypes to all variants. Some entries are doubtful as the data assessment differs strongly between the studies or no data on the methodology are provided at all. This complicates mathematical and bioinformatic predictions of phenotypes that rely on the database. The present work aimed to prove the role of specific substitutions on the resistance phenotype of TEM variants in, to our knowledge, the most extensive mutagenesis study. In parallel, the predictive power of extrapolation algorithms was assessed. Most well-known substitutions with direct impact on the phenotype could be reproduced, both mathematically and experimentally. Most discrepancies were found for supportive substitutions, where some resulted in antagonistic effects in contrast to previously described synergism. The mathematical modelling proved to predict the strongest phenotype-relevant substitutions accurately but showed difficulties in identifying less prevalent but still phenotype transforming ones. In general, mutations increasing cephalosporin resistance resulted in increased sensitivity to ß-lactamase inhibitors and vice versa. Combining substitutions related to cephalosporin and ß-lactamase inhibitor resistance in almost all cases increased BLI susceptibility, indicating the rarity of the combined phenotype.

A Genotype-Phenotype Correlation Study of SHV ß-Lactamases Offers New Insight into SHV Resistance Profiles.

The SHV ß-lactamases (BLs) have undergone strong allele diversification that has changed their substrate specificities. Based on 147 NCBI entries for SHV alleles, in silico mathematical models predicted 5 positions as relevant for the ß-lactamase inhibitor (BLI)-resistant (2br) phenotype, 12 positions as relevant for the extended-spectrum BL (ESBL) (2be) phenotype, and 2 positions as related solely to the narrow-spectrum (2b) phenotype. These positions and six additional positions described in other studies (including one promoter mutation) were systematically substituted and investigated for their substrate specificities in a BL-free Escherichia coli background, representing, to our knowledge, the most comprehensive substrate and substitution analysis for SHV alleles to date. An in vitro analysis confirmed the essentiality of positions 238 and 179 for the 2be phenotype and of position 69 for the 2br phenotype. The E240K and E240R substitutions, which do not occur alone in known 2br SHV variants, led to a 2br phenotype, indicating a latent BLI resistance potential of these substitutions. The M129V, A234G, S271I, and R292Q substitutions conferred latent resistance to cefotaxime. In addition, seven positions that were found not always to be associated with the ESBL phenotype resulted in increased resistance to ceftaroline. We also observed that coupling of a strong promoter (IS26) to an A146V mutant with the 2b phenotype resulted in highly increased resistance to BLIs, cefepime, and ceftaroline but not to third-generation cephalosporins, indicating that SHV enzymes represent an underestimated risk for empirical therapies that use piperacillin-tazobactam or cefepime to treat different infectious diseases caused by Gram-negative bacteria.

On the Control of Chromophore Orientation, Supramolecular Structure, and Thermodynamic Stability of an Amphiphilic Pyridyl-Thiazol upon Lateral Compression and Spacer Length Variation.

The supramolecular structure essentially determines the properties of organic thin films. Therefore, it is of utmost importance to understand the influence of molecular structure modifications on supramolecular structure formation. In this article, we demonstrate how to tune molecular orientations of amphiphilic 4-hydroxy thiazole derivatives by means of the Langmuir-Blodgett (LB) technique and how this depends on the length of an alkylic spacer between the thiazole chromophore and the polar anchor group. Therefore, we characterize their corresponding supramolecular structures, thermodynamic, absorption, and fluorescence properties. Particularly, the polarization-dependence of the fluorescence is analyzed to deduce molecular orientations and their possible changes after annealing, i.e., to characterize the thermodynamic stability of the individual solid state phases. Because the investigated thiazoles are amphiphilic, the different solid state phases can be formed and be controlled by means of the Langmuir-Blodgett (LB) technique. This technique also allows to deduce atomistic supramolecular structure motives of the individual solid phases and to characterize their thermodynamic stabilities. Utilizing the LB technique, we demonstrate that subtle molecular changes, like the variation in spacer length, can yield entirely different solid state phases with distinct supramolecular structures and properties.

Development of a QCM-D biosensor for Ochratoxin A detection in red wine.

Ochratoxin A (OTA), a highly toxic compound, is one of the most widely spread mycotoxins that contaminates a large variety of agricultural commodities. Due to its presence in the food chain, it imposes a hazard on both human and animal health. Therefore, there is a need for precise, fast and simple methods for toxin quantification. Herein, a novel sensor based on a quartz crystal microbalance with dissipation monitoring (QCM-D) and antibodies for specific analyte recognition was developed for rapid and sensitive detection of OTA in red wine. The combination of indirect competitive assay with QCM-D gives a straightforward device, which can simultaneously measure frequency (Δf) and dissipation (ΔD) changes resulting in detailed information about the mass attached to the sensor surface as well as conformational changes, viscoelastic properties and the hydration state of the film. Small molecules (such as OTA) suffer from poor LOD due to the high concentration of primary antibody needed to generate adequate signal. In the present study, amplification of the QCM-D signal was obtained by applying secondary antibodies conjugated with gold nanoparticles (AuNPs). Thanks to this, a linear detection range of 0.2-40ngmL-1 has been achieved with an excellent LOD of 0.16ngmL-1, which is one order of magnitude lower than LOD specified by European Union legislation concerning the limit of OTA in food. Moreover, a matrix effect (caused by the occurrence of polyphenols in wine) and associated non-specific interactions with the sensor surface was completely eliminated by a simple pre-treatment of the wine with the addition of 3% poly(vinylpyrrolidone) (PVP).

A cell-based biosensor for nanomaterials cytotoxicity assessment in three dimensional cell culture.

Nanoparticles (NPs) are widely used in consumer and medicinal products. The high prevalence of nanoparticles in the environment raises concerns regarding their effects on human health, but there is limited knowledge about how NPs interact with cells or tissues. Because the European Union has called for a substantial reduction of animal experiments for scientific purposes (Directive 2010/63), increased efforts are required to develop in vitro models to evaluate potentially hazardous agents. Here, we describe a new cell-based biosensor for the evaluation of NPs cytotoxicity. The new biosensor is based on transgenic human hepatoblastoma cells (HepG2) that express a secreted form of alkaline phosphatase (SEAP) as a reporter protein whose expression is induced upon activation of a stress response pathway controlled by the transcription regulator nuclear factor-κB (NF-κB). The NF-κB_HepG2 sensor cells were cultured in a Matrigel-based three dimensional environment to simulate the in vivo situation. The new biosensor cells offer the advantage of generating fast and reproducible readout at lower concentrations and shorter incubation time than conventional viability assays, avoid possible interaction between nanomaterials and assay compounds, therefore, minimize generation of false positive or negative results and indicate mechanism of toxicity through NF-κB signaling.

Fast and sensitive detection of ochratoxin A in red wine by nanoparticle-enhanced SPR.

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL(-1) and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL(-1) by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied.

Sensitive and rapid detection of aflatoxin M1 in milk utilizing enhanced SPR and p(HEMA) brushes.

The rapid and sensitive detection of aflatoxin M1 (AFM1) in milk by using surface plasmon resonance (SPR) biosensor is reported. This low molecular weight mycotoxin is analyzed using an indirect competitive immunoassay that is amplified by secondary antibodies conjugated with Au nanoparticles. In order to prevent fouling on the sensor surface by the constituents present in analyzed milk samples, an interface with poly(2-hydroxyethyl methacrylate) p(HEMA) brush was employed. The study presents a comparison of performance characteristics of p(HEMA)-based sensor with a regularly used polyethylene glycol-based architecture relying on mixed thiol self-assembled monolayer. Both sensors are characterized in terms of surface mass density of immobilized AFM1 conjugate as well as affinity bound primary and secondary antibodies. The efficiency of the amplification strategy based on Au nanoparticle is discussed. The biosensor allowed for highly sensitive detection of AFM1 in milk with a limit of detection (LOD) as low as 18pgmL(-1) with the analysis time of 55min.

Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro.

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, we have developed and tested three dimensional environments for cells using different types of hydrogels including transglutaminase-cross-linked gelatin, collagen type I, and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, analysis of urea and albumin biosynthesis revealed that the created system allowed the immortalized liver cell line HepG2 to re-establish normal hepatocyte-like properties which were not observed under the conditions of conventional cell cultures. This study presents a scalable technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates a higher cytotoxic effect when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that the cell environment has significant influence on cell sensitivity and that liver spheroid is a useful and novel tool to examine nanoparticle dosing effect even at the level of in vitro studies. Therefore, this system can be applied to a wide variety of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions.

A sensitive sensor cell line for the detection of oxidative stress responses in cultured human keratinocytes.

In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

Multi-objective optimization and design of experiments as tools to tailor molecularly imprinted polymers specific for glucuronic acid.

We present a multi-objective optimization of the binding properties of a molecularly imprinted polymer (MIP) which specifically binds glucuronic acid (GA). A design of experiments approach is used to improve four different parameters that describe the binding properties of the polymer. Eleven different methacrylamide-co-ethyleneglycol dimethacrylate polymers imprinted with GA were synthesized according to a full factorial experimental design plan with 3 influencing factors (degree of cross-linking, molar equivalent of monomer to template and initiator concentration). These polymers were characterized by adsorption of the radiolabeled target analyte in methanol:water 9:1. The binding parameters were computed to optimize the polymer composition, taking into account four objective variables: the maximum binding capacity at high (Bmax) and low (B2) analyte concentrations, the equilibrium constant K50, and the imprinting factor (IF, binding to MIP/binding to NIP). With the multi-objective optimization method based on a desirability approach the composition of a twelfth "ideal" polymer could be predicted. This predicted polymer with highest "desirability" was synthesized with a composition of 0.65 mol% of initiator and a 1:4:20 ratio of template:functional monomers:cross-linker (T:M:X) (80% of cross-linking), and found to be the overall best MIP. Improvements over the original starting polymer were a 6 times lower K50, which corresponds to higher affinity, 20% higher capacity at low analyte concentration (B2), 40% higher capacity (Bmax) and 1.3 times increased imprinting factor (IF). Binding assays were also performed in aqueous solvents. Good binding properties were obtained in pure water with an imprinting factor of 3.2. Thus, this polymer is potentially applicable to biological samples like urine where glucuronides occur.

A whole-cell biosensor as in vitro alternative to skin irritation tests.

This study presents the time-resolved detection of chemically induced stress upon intracellular signaling cascades by using genetically modified sensor cells based on the human keratinocyte cell line HaCaT. The cells were stably transfected with a HSP72-GFP reporter gene construct to create an optical sensor cell line expressing a stress-inducible reporter protein. The time- and dose-dependent performance of the sensor cells is demonstrated and discussed in comparison to a label-free impedimetric monitoring approach (electric cell-substrate impedance sensing, ECIS). Moreover, a microfluidic platform was established based on µSlidesI(0,4)Luer to allow for a convenient, sterile and incubator-independent time-lapse microscopic observation of the sensor cells. Cell growth was successfully achieved in this microfluidic setup and the cellular response to a cytotoxic substance could be followed in real-time and in a non-invasive, sensitive manner. This study paves the way for the development of micro-total analysis systems that combine optical and impedimetric readouts to enable an overall quantitative characterization of changes in cell metabolism and morphology as a response to toxin exposure. By recording multiple parameters, a detailed discrimination between competing stress- or growth-related mechanisms is possible, thereby presenting an entirely new in vitro alternative to skin irritation tests.

Development of fluorescent array based on sol-gel/chitosan encapsulated acetylcholinesterase and pH sensitive oxazol-5-one derivative.

A highly sensitive fluorescent enzyme array for quantitative acetylcholine detection is developed. The enzyme array has been constructed by spotting of pH sensitive fluorophore 2-phenyl-4-[4-(1,4,7,10-tetraoxa-13-azacycloopentadecyl)benzylidene]oxazol-5-one and acetylcholinesterase doped in tetraethoxysilane/chitosan matrix via a microarrayer. The constructed tetraethoxysilane/chitosan network provided a microenvironment in which the enzyme molecule was active biologically. The optimal operational conditions for the array developed were investigated. The response of the developed biosensor array to acetylcholine was highly reproducible (RSD = 3.27%, n = 6). A good linearity was observed for acetylcholine in the concentrations up to 1 × 10(-8) M, with a detection limit of 0.27 × 10(-8) M.