Impact of Plastic-Related Compounds on P-Glycoprotein and Breast Cancer Resistance Protein In Vitro.

Plastic in oceans degrades to microplastics and nanoplastics, causing various problems for marine fauna and flora. Recently, microplastic has been detected in blood, breast milk and placenta, underlining their ability to enter the human body with still unknown effects. In addition, plastic contains other compounds such as plasticizers, antioxidants or lubricants, whose impact on human health is also elusive. On the cellular level, two transporters involved in cell protection and detoxification of xenobiotic compounds are the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). Despite the great importance of these proteins to maintain the correct cellular balance, their interaction with plastic and related products is evasive. In this study, the possible interaction between different plastic-related compounds and these two transporters was investigated. Applying virtual compound screening and molecular docking of more than 1000 commercially available plastic compounds, we identified candidates most probably interacting with these two transporters. Cytotoxicity and uptake assays confirmed their toxic interaction on P-glycoprotein-overexpressing CEM/ADR5000 and BCRP-overexpressing MDA-MD-231-BCRP cell lines. To specifically visualize the results obtained on the P-glycoprotein inhibitor 2,2'-methylenebis(6-tert-butyl-4-methylphenol), we performed live cell time-lapse microscopy. Confocal fluorescence microscopy was used to understand the behavior of the molecule and the consequences that it has on the uptake of the well-known substrate doxorubicin and, in comparison, with the known inhibitor verapamil. Based on the results, we provide evidence that the compound in question is an inhibitor of the P-glycoprotein. Moreover, it is also possible that 2,2'-methylenebis(6-tert-butyl-4-methylphenol), together with three other compounds, may also inhibit the breast cancer resistance protein. This discovery implies that plastic-related compounds can not only harm the human body but can also inhibit detoxifying efflux pumps, which increases their toxic potential as these transporters lose their physiological functions.

Snake venom-defined fibrin architecture dictates fibroblast survival and differentiation.

Fibrin is the provisional matrix formed after injury, setting the trajectory for the subsequent stages of wound healing. It is commonly used as a wound sealant and a natural hydrogel for three-dimensional (3D) biophysical studies. However, the traditional thrombin-driven fibrin systems are poorly controlled. Therefore, the precise roles of fibrin's biophysical properties on fibroblast functions, which underlie healing outcomes, are unknown. Here, we establish a snake venom-controlled fibrin system with precisely and independently tuned architectural and mechanical properties. Employing this defined system, we show that fibrin architecture influences fibroblast survival, spreading phenotype, and differentiation. A fine fibrin architecture is a key prerequisite for fibroblast differentiation, while a coarse architecture induces cell loss and disengages fibroblast's sensitivity towards TGF-ß1. Our results demonstrate that snake venom-controlled fibrin can precisely control fibroblast differentiation. Applying these biophysical principles to fibrin sealants has translational significance in regenerative medicine and tissue engineering.

Effect of low aspect ratio one-dimensional nanoparticles on properties of photocrosslinked alginate nanocomposite hydrogels.

One-dimensional nanoparticles such as fibers, wires, represent an exciting type of filler for obtaining nanocomposite hydrogels due to their high aspect ratio. Unlike continuous fibers, in composites with short fibers, stress transfer occurs not only at the cylindrical surface of the fiber but also at the fiber ends. This work aimed to study the influence of the length of low aspect ratio one-dimensional nanoparticles on the rheological, swelling and thermal properties of nanocomposite hydrogels. We synthesized nanocomposite hydrogels via photopolymerization of methacrylated alginate macromonomers and aluminum oxide nanoparticles, nanowires and nanofibers as fillers. The main difference between the nanoparticles lies in their length. Longer nanoparticles (nanofibers) provided a glassier structure and a more significant reinforcement to the hydrogel, for example, at the concentration of 0.5 wt% the complex modulus increased approximately 2-fold and 8-fold for nanowires and nanofibers, respectively. Similarly, the incorporation of nanofibers produced hydrogels with a lower swelling capacity (2.5-fold decrease), which was attributed to a more connected network structure due to the higher aspect ratio of the nanofibers. Finally, both nanoparticle types produced an increase in the activation energy of thermal degradation.

Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons drives overconsumption of palatable food and obesity.

Palatable food can promote overfeeding beyond homeostatic requirements, thereby constituting a major risk to obesity. Here, the lack of cannabinoid type 1 receptor (CB1) in dorsal telencephalic glutamatergic neurons (Glu-CB1-KO) abrogated the overconsumption of palatable food and the development of obesity. On low-fat diet, no genotype differences were observed. However, under palatable food conditions, Glu-CB1-KO mice showed decreased body weight and food intake. Notably, Glu-CB1-KO mice were protected from alterations in the reward system after high-fat diet feeding. Interestingly, obese wild-type mice showed a superior olfactory detection as compared to mutant mice, suggesting a link between overconsumption of palatable food and olfactory function. Reconstitution of CB1 expression in olfactory cortex in high-fat diet-fed Glu-CB1-KO mice using viral gene delivery partially reversed the lean phenotype concomitantly with improved odor perception. These findings indicate that CB1 in cortical glutamatergic neurons regulates hedonic feeding, whereby a critical role of the olfactory cortex was uncovered as an underlying mechanism.

Deciphering Design Principles of Förster Resonance Energy Transfer-Based Protease Substrates: Thermolysin-Like Protease from Geobacillus stearothermophilus as a Test Case.

Protease activity is frequently assayed using short peptides that are equipped with a Förster resonance energy transfer (FRET) reporter system. Many frequently used donor-acceptor pairs are excited in the ultraviolet range and suffer from low extinction coefficients and quantum yields, limiting their usefulness in applications where a high sensitivity is required. A large number of alternative chromophores are available that are excited in the visible range, for example, based on xanthene or cyanine core structures. These alternatives are not only larger in size but also more hydrophobic. Here, we show that the hydrophobicity of these chromophores not only affects the solubility of the resulting FRET-labeled peptides but also their kinetic parameters in a model enzymatic reaction. In detail, we have compared two series of 4-8 amino acid long peptides, designed to serve as substrates for the thermolysin-like protease (TLP-ste) from Geobacillus stearothermophilus. These peptides were equipped with a carboxyfluorescein donor and either Cy5 or its sulfonated derivative Alexa Fluor 647 as the acceptor. We show that the turnover rate k cat is largely unaffected by the choice of the acceptor fluorophore, whereas the K M value is significantly lower for the Cy5- than for the Alexa Fluor 647-labeled substrates. TLP-ste is a rather nonspecific protease with a large number of hydrophobic amino acids surrounding the catalytic site, so that the fluorophore itself may form additional interactions with the enzyme. This hypothesis is supported by the result that the difference between Cy5- and Alexa Fluor 647-labeled substrates becomes less pronounced with increasing peptide length, that is, when the fluorophore is positioned at a larger distance from the catalytic site. These results suggest that fluorophores may become an integral part of FRET-labeled peptide substrates and that K M and k cat values are generally only valid for a specific combination of the peptide sequence and FRET pair.

Interfacial Activation of Candida antarctica Lipase B: Combined Evidence from Experiment and Simulation.

Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Candida antarctica lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small "lid" (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents.

Single-enzyme kinetics with fluorogenic substrates: lessons learnt and future directions.

Single-molecule fluorescence techniques have developed into powerful tools for studying the kinetics of biological reactions at the single-molecule level. Using fluorogenic substrates, enzymatic reactions can be observed in real-time with single-turnover resolution. The turnover sequence contains all kinetic information, giving access to reaction substeps and dynamic processes such as fluctuations in the reaction rate. Despite their clearly proven potential, the accuracy of current measurements is limited by the availability of substrates with 1:1 stoichiometry and the signal-to-noise ratio of the measurement. In this review we summarize the state-of-the-art and discuss these limitations using experiments performed with α-chymotrypsin as an example. We are further providing an overview of recent efforts aimed at the improvement of fluorogenic substrates and the development of new detection schemes. These detection schemes utilize nanophotonic structures such as zero mode waveguides or nanoantennas. Nanophotonic approaches reduce the size of the effective detection volume and are a powerful strategy to increase the signal-to-noise ratio. We believe that a combination of improved substrates and novel detection schemes will pave the way for performing accurate single-enzyme experiments in biologically relevant conditions.

Robust sensor for turbidity measurement from light scattering and absorbing liquids.

Internationally standardized turbidity measurements for probing solid particles in liquid are problematic in the case of simultaneous light scattering and absorption. A method and a sensor to determine the turbidity in the presence of light absorption are presented. The developed sensor makes use of the total internal reflection of a laser beam at the liquid-prism interface, and the turbidity is assessed using the concept of laser speckle pattern. Using average filtering in speckle data analyzing the observed dynamic speckle pattern, which is due to light scattering from particles and the static speckle due to stray light of the sensor, can be separated from each other. Good correlation between the standard deviation of dynamic speckle and turbidity value for nonabsorbing and for absorbing liquids was observed. The sensor is suggested, for instance, for the measurement of ill-behaved as well as small-volume turbid liquids in both medicine and process industry.