The use and detection of quantum dots as nanotracers in environmental fate studies of engineered nanoparticles.

Investigations of the behavior and effects of engineered nanoparticles (ENPs) on human health and the environment need detailed knowledge of their fate and transport in environmental compartments. Such studies are highly challenging due to low environmental concentrations, varying size distribution of the particles and the interference with the natural background. A strategy to overcome these limits is to use mimics of ENPs with unique detectable properties that match the properties of the ENPs as nanotracers. A special class of ENPs that can be tracked are quantum dots (QDs). QDs are composed of different metals, metalloids, or more recently also carbon (e.g., graphene), that result in unique optical properties. This allows the tracking of such particles by fluorescence microscopic and photometric techniques. Many types of QDs consist of heavy elements, allowing to track and visualize these particles also by electron microscopy and to quantitate the particles indirectly based on these elements. QDs can also be surface modified in various ways which enable them to be used as a label or as traceable mimics for ENPs. This review reflects a broad range of methods to synthesize and modify QDs based on metals, metalloids, and graphene for studying the environmental fate of nanoparticles and discusses and compares analytical methods that can be used for tracking and quantifying QDs. In addition, we review applications of QDs as ENP mimics in environmental studies of surface waters, soils, microorganisms, and plants with respect to the applied analytical techniques.

Isolation and Characterization of Cell Envelope Fragments Comprising Archaeal S-Layer Proteins.

The outermost component of cell envelopes of most bacteria and almost all archaea comprise a protein lattice, which is termed Surface (S-)layer. The S-layer lattice constitutes a highly porous structure with regularly arranged pores in the nm-range. Some archaea thrive in extreme milieus, thus producing highly stable S-layer protein lattices that aid in protecting the organisms. In the present study, fragments of the cell envelope from the hyperthermophilic acidophilic archaeon Saccharolobus solfataricus P2 (SSO) have been isolated by two different methods and characterized. The organization of the fragments and the molecular sieving properties have been elucidated by transmission electron microscopy and by determining the retention efficiency of proteins varying in size, respectively. The porosity of the archaeal S-layer fragments was determined to be 45%. S-layer fragments of SSO showed a retention efficiency of up to 100% for proteins having a molecular mass of ≥ 66 kDa. Moreover, the extraction costs for SSO fragments have been reduced by more than 80% compared to conventional methods, which makes the use of these archaeal S-layer material economically attractive.

Screening for Best Neuronal-Glial Differentiation Protocols of Neuralizing Agents Using a Multi-Sized Microfluidic Embryoid Body Array.

Stem cell technology and embryonic stem cell models are of great interest in biomedical research since they provide deeper insights into, e.g., neurogenesis and early mammalian brain development. Despite their great scientific potential, the reliable establishment of three-dimensional embryoid bodies (EBs) remains a major challenge, and the current lack of standardization and comparability is still limiting a broader application and translation of stem cell technology. Among others, a vital aspect for the reliable formation of EBs is optimizing differentiation protocols since organized differentiation is influenced by soluble inducers and EB size. A microfluidic biochip array was employed to automate cell loading and optimize directed neuronal and astrocytic differentiation protocols using murine P19 embryoid bodies to facilitate reliable embryonic stem cell differentiation. Our gravity-driven microfluidic size-controlled embryoid body-on-a-chip system allows (a) the robust operation and cultivation of up to 90 EBs in parallel and (b) the reproducible generation of five increasing sizes ranging from 300 µm to 1000 µm diameters. A comparative study adds two differentiation-inducers such as retinoic acid and EC23 to size-controlled embryoid bodies to identify the optimal differentiation protocol. Our study revealed a 1.4 to 1.9-fold higher neuron and astrocyte expression in larger embryoid bodies (above 750 µm) over smaller-sized EBs (below 450 µm), thus highlighting the importance of EB size in the establishment of robust neurodevelopmental in vitro models.

The current state of the art in internal additive materials and quantum dots for improving efficiency and stability against humidity in perovskite solar cells.

The remarkable optoelectronic capabilities of perovskite structures enable the achievement of astonishingly high-power conversion efficiencies on the laboratory scale. However, a critical bottleneck of perovskite solar cells is their sensitivity to the surrounding humid environment affecting drastically their long-term stability. Internal additive materials together with surface passivation, polymer-mixed perovskite, and quantum dots, have been investigated as possible strategies to enhance device stability even in unfavorable conditions. Quantum dots (QDs) in perovskite solar cells enable power conversion efficiencies to approach 20%, making such solar cells competitive to silicon-based ones. This mini-review summarized the role of such QDs in the perovskite layer, hole-transporting layer (HTL), and electron-transporting layer (ETL), demonstrating the continuous improvement of device efficiencies.

Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence-An Experimental Study of the Human Voltage-Dependent Anion Channel.

Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage-dependent anion channel (VDAC), which is a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function that quantifies the feasibility of the formation of the translation initiation complex through the ribosome-mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome is proposed. The scoring function enables one to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at University of Vienna, Austria.

A critical review of the environmental impacts of manufactured nano-objects on earthworm species.

The presence of manufactured nano-objects (MNOs) in various consumer or their (future large-scale) use as nanoagrochemical have increased with the rapid development of nanotechnology and therefore, concerns associated with its possible ecotoxicological effects are also arising. MNOs are releasing along the product life cycle, consequently accumulating in soils and other environmental matrices, and potentially leading to adverse effects on soil biota and their associated processes. Earthworms, of the group of Oligochaetes, are an ecologically significant group of organisms and play an important role in soil remediation, as well as acting as a potential vector for trophic transfer of MNOs through the food chain. This review presents a comprehensive and critical overview of toxic effects of MNOs on earthworms in soil system. We reviewed pathways of MNOs in agriculture soil environment with its expected production, release, and bioaccumulation. Furthermore, we thoroughly examined scientific literature from last ten years and critically evaluated the potential ecotoxicity of 16 different metal oxide or carbon-based MNO types. Various adverse effects on the different earthworm life stages have been reported, including reduction in growth rate, changes in biochemical and molecular markers, reproduction and survival rate. Importantly, this literature review reveals the scarcity of long-term toxicological data needed to actually characterize MNOs risks, as well as an understanding of mechanisms causing toxicity to earthworm species. This review sheds light on this knowledge gap as investigating bio-nano interplay in soil environment improves our major understanding for safer applications of MNOs in the agriculture environment.

Investigations on inhibitory effects of nickel and cobalt salts on the decolorization of textile dyes by the white rot fungus Phanerochaete velutina.

Organic aromatic compounds used for dyeing and coloring in the textile industry are persistent and hazardous pollutants that must be treated before they are discharged into rivers and surface waters. Therefore, we investigated the potential of the white rot fungus Phanerochaete velutina to decolorize commonly used reactive dyes. The fungus decolorized in average 55% of Reactive Orange 16 (RO-16) after 14 days at a maximum rate of 0.09 d-1 and a half-life of 8 days. Furthermore, we determined the inhibitory effects of co-present inorganic contaminants Nickel (Ni) and Cobalt (Co) salts on the decolorization potential and determined IC50 values of 5.55 mg l-1 for Co and a weaker inhibition by Ni starting from a concentration of 20 mg l-1. In the decolorization assay for Remazol Brilliant Blue R (RBBR) we observed the interference of a metabolite of P. velutina, which did not allow us to investigate the kinetics of the reaction. The formation of the metabolite, however, could be used to obtain IC50 values of 3.37 mg l-1 for Co and 7.58 mg l-1 for Ni. Our results show that living white rot fungi, such as P. velutina, can be used for remediation of dye polluted wastewater, alternatively to enzyme mixtures, even in the co-presence of heavy metals.

Functional proteoliposome-like structure derived from simultaneous evisceration and enucleation of T-lymphoblastoid A3R5.7 cells: A top-down story.

Structurally-reduced cells and cell-derived structures are powerful tools for membrane studies. Using this approach, we probed whether a cell, without its nucleus and cytoplasm, is still capable of undergoing CD4-mediated membrane fusion. For this, we needed a cell-derived structure, akin to a giant liposome functionalised with CD4 and chemokine receptors. We present a method for the simultaneous removal of cytoplasmic and nuclear material from cells presenting CD4, CCR5, and CXCR4, using Colcemid treatment followed by hypotonic cytolysis, and then enriched using preparative flow cytometry. We show that the resultant cell membrane remains intact, retains presentation of CD4, CCR5, and CXCR4, and is still capable of CD4-mediated membrane fusion with a target cell. Finally, we detail how this protocol was developed, as well as how such samples should be handled for storage and assays. We envision the use of such systems for host-pathogen interaction studies, and the development of targeted delivery vehicles.

Supported polymer/lipid hybrid bilayers formation resembles a lipid-like dynamic by reducing the molecular weight of the polymer.

Amphiphilic block copolymers form self-assembled bilayers even in combination with phospholipids. They represent an attractive alternative to native lipid-based membrane systems for supported bilayer formation with applications in biomedical research, sensoring and drug delivery. Their enhanced stability and excellent mechanical properties are linked to their higher molecular weight which generates thicker bilayers. Hypothesis: It is hypothesized that reducing the molecular weight of the polymer facilitates the formation of a thinner, more homogeneous polymer/lipid hybrid bilayer which would benefit the formation of supported bilayers on silicon oxide. Experiment: We investigated hybrid bilayers composed of mixtures of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and increasing amounts of a low molecular weight polybutadiene-b-polyethylene oxide copolymer (1050 g/mol). By assessing the bilayer thickness and the molecular packing behavior we sought to demonstrate how reducing the polymer molecular weight increases the tendency to form supported hybrid bilayers in a lipid-like manner. Findings: The formation of a supported hybrid bilayers occurs at polymer contents <70 mol% in a lipid-like fashion and is proportional to the cohesive forces between the bilayer components and inversely related to the bilayer hydrophobic core thickness and the extended brush regime of the PEGylated polymeric headgroup.

Constitutive activation of integrin αvß3 contributes to anoikis resistance of ovarian cancer cells.

Epithelial ovarian cancer involves the shedding of single tumor cells or spheroids from the primary tumor into ascites, followed by their survival, and transit to the sites of metastatic colonization within the peritoneal cavity. During their flotation, anchorage-dependent epithelial-type tumor cells gain anoikis resistance, implicating integrins, including αvß3. In this study, we explored anoikis escape, cisplatin resistance, and prosurvival signaling as a function of the αvß3 transmembrane conformational activation state in cells suspended in ascites. A high-affinity and constitutively signaling-competent αvß3 variant, which harbored unclasped transmembrane domains, was found to confer delayed anoikis onset, enhanced cisplatin resistance, and reduced cell proliferation in ascites or 3D-hydrogels, involving p27kip upregulation. Moreover, it promoted EGF-R expression and activation, prosurvival signaling, implicating FAK, src, and PKB/Akt. This led to the induction of the anti-apoptotic factors Bcl-2 and survivin suppressing caspase activation, compared to a signaling-incapable αvß3 variant displaying firmly associated transmembrane domains. Dissecting the mechanistic players for αvß3-dependent survival and peritoneal metastasis of ascitic ovarian cancer spheroids is of paramount importance to target their anchorage independence by reversing anoikis resistance and blocking αvß3-triggered prosurvival signaling.

Mobility and fate of ligand stabilized semiconductor nanoparticles in landfill leachates.

Semiconductor quantum dots (QDs) are nanocrystals used in diverse optoelectronics. At the end of their useful life they are likely to end up in landfills, where they could be mobilzed by infiltrating rain water. In this work, spectroscopic and light scattering techniques were employed to investigate the environmental fate of QDs exposed to leachates from Austrian landfill sites containing municipal solid and bulky wastes. Brij-58-coated CdSe QDs, a model for surfactant stabilized hydrophobic nanoparticles, primarily sedimented before being degraded on a slower timescale in the course of 6 months. In contrast, N-acetyl-l-cystein-coated CdTe QDs, which represent electrostatically stabilized nanoparticles with a small covalently linked stabilizing molecule, mainly underwent a degradation mechanism that was accelerated by temperature. 71-95 % of this QD type was still dispersed in all leachates after 6 months at low temperature. Leachate temperature and composition, such as the DOC, as well as the used particle coating determined the mechanistic route of clearance of sedimentation versus degradation. Our study shows, that mechanistic investigations are necessary to determine the persistence of nanoparticles depending on their coatings in waste matrices which can be further used to assess hazardous risks of such nanowastes.

Controllable cell manipulation in a microfluidic pipette-tip design using capacitive coupling of electric fields.

Systems designed toward cell manipulation by electric fields are inherently challenged by energy dissipation along the electrode-electrolyte interface. A promising remedy is the introduction of high-k electrode passivation, enabling efficient capacitive coupling of electric fields into biological samples. We present the implementation of this strategy in a reusable pipette tip design featuring a 10 µl chamber volume for life science applications. Prototype validation and comparison to conductive gold-coated electrodes reveal a consistent and controllable biological effect that significantly increases the reproducibility of lysis events. The system provides precise descriptions of HEK-293 lysis dependency to variables such as field strength, frequency, and conductivity. Over 80% of cells were reversibly electroporated with minimal electrical lysis over a broad range of field settings. Successful transfection requires exponential decay pulses and showcases how modulating capacitive coupling can advance our understanding of fundamental mechanics in the field of electroporation.

The Usual Suspects 2019: of Chips, Droplets, Synthesis, and Artificial Cells.

Synthetic biology aims to understand fundamental biological processes in more detail than possible for actual living cells. Synthetic biology can combat decomposition and build-up of artificial experimental models under precisely controlled and defined environmental and biochemical conditions. Microfluidic systems can provide the tools to improve and refine existing synthetic systems because they allow control and manipulation of liquids on a micro- and nanoscale. In addition, chip-based approaches are predisposed for synthetic biology applications since they present an opportune technological toolkit capable of fully automated high throughput and content screening under low reagent consumption. This review critically highlights the latest updates in microfluidic cell-free and cell-based protein synthesis as well as the progress on chip-based artificial cells. Even though progress is slow for microfluidic synthetic biology, microfluidic systems are valuable tools for synthetic biology and may one day help to give answers to long asked questions of fundamental cell biology and life itself.

Effect of Spheroidal Age on Sorafenib Diffusivity and Toxicity in a 3D HepG2 Spheroid Model.

The enhanced predictive power of 3D multi-cellular spheroids in comparison to conventional monolayer cultures makes them a promising drug screening tool. However, clinical translation for pharmacology and toxicology is lagging its technological progression. Even though spheroids show a biological complexity resembling native tissue, standardization and validation of drug screening protocols are influenced by continuously changing physiological parameters during spheroid formation. Such cellular heterogeneities impede the comparability of drug efficacy studies and toxicological screenings. In this paper, we demonstrated that aside from already well-established physiological parameters, spheroidal age is an additional critical parameter that impacts drug diffusivity and toxicity in 3D cell culture models. HepG2 spheroids were generated and maintained on a self-assembled ultra-low attachment nanobiointerface and characterized regarding time-dependent changes in morphology, functionality as well as anti-cancer drug resistance. We demonstrated that spheroidal aging directly influences drug response due to the evolution of spheroid micro-structure and organo-typic functions, that alter inward diffusion, thus drug uptake.

Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids.

The assessment of drug-dose responses is vital for the prediction of unwanted toxicological effects in modern medicine. Three-dimensional (3D) cell cultures techniques can provide in vivo-like spheroids and microtissues that resemble natural tumor function. However, formation of necrotic core and diffusion limitation of chemical compounds within these models can reduce the reproducibility and precision of standard bioassay protocols used to test two-dimensional (2D) cell cultures. Nonetheless, the accurate prediction of detrimental effects of test compounds based on functional bioassays is essential for the development of new efficient therapeutic strategies. For instance, alamarBlue® is a widely-used commercially available redox indicator dye that can evaluate metabolic activity and cellular health status in a single-step procedure however, suitability and optimization of this bioassay must be determined for each individual application scenario. Here, we optimized the standard alamarBlue® proliferation/viability protocol for tumor spheroid cultures to enhance assay precision during toxicological drug screening. We optimized the original protocol of alamarBlue® assay that usually suggests an incubation time of 2-4 hours. The key modifications of the protocol for spheroid cultures are as follows: •Aspiration of cell culture medium before drug exposure.•Replacement of drug-supplemented medium with 10% (v/v) alamarBlue® reagent mixed with culture medium.•Increase of incubation period to 24 h at 37 °C protected from light.

Cell-Free Approaches in Synthetic Biology Utilizing Microfluidics.

Synthetic biology is a rapidly growing multidisciplinary branch of science which aims to mimic complex biological systems by creating similar forms. Constructing an artificial system requires optimization at the gene and protein levels to allow the formation of entire biological pathways. Advances in cell-free synthetic biology have helped in discovering new genes, proteins, and pathways bypassing the complexity of the complex pathway interactions in living cells. Furthermore, this method is cost- and time-effective with access to the cellular protein factory without the membrane boundaries. The freedom of design, full automation, and mimicking of in vivo systems reveal advantages of synthetic biology that can improve the molecular understanding of processes, relevant for life science applications. In parallel, in vitro approaches have enhanced our understanding of the living system. This review highlights the recent evolution of cell-free gene design, proteins, and cells integrated with microfluidic platforms as a promising technology, which has allowed for the transformation of the concept of bioprocesses. Although several challenges remain, the manipulation of biological synthetic machinery in microfluidic devices as suitable 'homes' for in vitro protein synthesis has been proposed as a pioneering approach for the development of new platforms, relevant in biomedical and diagnostic contexts towards even the sensing and monitoring of environmental issues.