Extreme thermal stability of the antiGFP nanobody - GFP complex.

OBJECTIVE: The green fluorescent protein (GFP) and its derivatives are widely used in biomedical research. The manipulation of GFP-tagged proteins by GFP-specific binders, e.g. single-domain antibodies (nanobodies), is of increasing significance. It is therefore important to better understand the properties of antiGFP-GFP interaction in order to establish methodological applications. In this work the interaction of superfolder GFP (sfGFP) and its enhancer nanobody (aGFPenh) was characterized further. RESULTS: Previous calorimetric experiments demonstrated that the aGFPenh nanobody binds strongly to sfGFP with a nanomolar affinity. Here we show that this interaction results in a substantial structural stabilization of aGFPenh reflected in a significant increase of its melting temperature by almost 30 °C. The thermal stability of the sfGFP-aGFPenh complex is close to 85 °C in the pH range 7.0-8.5. For therapeutic applications thermoresistance is often an essential factor. Our results suggest that methodologies based on GFP-aGFP interaction can be applied under a wide range of physicochemical conditions. The aGFPenh nanobody seems to be suitable for manipulating sfGFP-labeled targets even in extreme thermophilic organisms.

Ecotoxic emissions generated by illegal burning of household waste.

The practice of burning household waste including different types of plastic is illegal in Hungary, still an existing problem. As environmental consequences are hardly known, this study attempts to give an initial estimation of the ecotoxicity generated during controlled combustion of different waste types. These samples included polystyrene (PS), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyurethane (PU), oriented strand board (OSB) and rag (RAG). Ecotoxicological profiling was completed using the following test battery: Vibrio fischeri bioluminescence inhibition assay, Daphnia magna immobility test and the seedling emergence assay. Also, genotoxicity of plastic waste samples was assessed using the SOS Chromotest. Concerning main pollutants in the samples, the samples could be distinguished as 'PAH-type' and 'heavy metal-type' samples. PVC, PU and PS samples showed the highest toxicity in the Vibrio and Daphnia assays. The PVC sample was characterized by an extremely high cadmium concentration (22.4 µg/L), PS, PP and PU samples on the contrary had high total PAH content. While Vibrio and Daphnia showed comparable sensitivity, the phytotoxicity assay had no response for any of the samples tested. Samples originating from the controlled burning of different plastic types such as PU, PVC, PS and PP were classified as genotoxic, PS sample showed extremely high genotoxicity. Genotoxicity expressed as SOSIF showed strong correlation with most of the PAHs detected.

Flagellin-based electrochemical sensing layer for arsenic detection in water.

Regular monitoring of arsenic concentrations in water sources is essential due to the severe health effects. Our goal was to develop a rapidly responding, sensitive and stable sensing layer for the detection of arsenic. We have designed flagellin-based arsenic binding proteins capable of forming stable filament structures with high surface binding site densities. The D3 domain of Salmonella typhimurium flagellin was replaced with an arsenic-binding peptide motif of different bacterial ArsR transcriptional repressor factors. We have shown that the fusion proteins developed retain their polymerization ability and have thermal stability similar to that of wild-type filament. The strong arsenic binding capacity of the monomeric proteins was confirmed by isothermal titration calorimetry (ITC), and dissociation constants (Kd) of a few hundred nM were obtained for all three variants. As-binding fibers were immobilized on the surface of a gold electrode and used as a working electrode in cyclic voltammetry (CV) experiments to detect inorganic arsenic near the maximum allowable concentration (MAC) level. Based on these results, it can be concluded that the stable arsenic-binding flagellin variant can be used as a rapidly responding, sensitive, but simple sensing layer in a field device for the MAC-level detection of arsenic in natural waters.

Sensing Layer for Ni Detection in Water Created by Immobilization of Bioengineered Flagellar Nanotubes on Gold Surfaces.

The environmental monitoring of Ni is targeted at a threshold limit value of 0.34 µM, as set by the World Health Organization. This sensitivity target can usually only be met by time-consuming and expensive laboratory measurements. There is a need for inexpensive, field-applicable methods, even if they are only used for signaling the necessity of a more accurate laboratory investigation. In this work, bioengineered, protein-based sensing layers were developed for Ni detection in water. Two bacterial Ni-binding flagellin variants were fabricated using genetic engineering, and their applicability as Ni-sensitive biochip coatings was tested. Nanotubes of mutant flagellins were built by in vitro polymerization. A large surface density of the nanotubes on the sensor surface was achieved by covalent immobilization chemistry based on a dithiobis(succimidyl propionate) cross-linking method. The formation and density of the sensing layer was monitored and verified by spectroscopic ellipsometry and atomic force microscopy. Cyclic voltammetry (CV) measurements revealed a Ni sensitivity below 1 µM. It was also shown that, even after two months of storage, the used sensors can be regenerated and reused by rinsing in a 10 mM solution of ethylenediaminetetraacetic acid at room temperature.

Eco- and genotoxicity profiling of a rapeseed biodiesel using a battery of bioassays.

Biodiesel is considered an important renewable energy source but still there is some controversy about its environmental toxicity, especially to aquatic life. In our study, the toxicity of water soluble fraction of biodiesel was evaluated in relatively low concentrations using a battery of bioassays: Vibrio fischeri bioluminescence inhibition, Sinapis alba root growth inhibition, Daphnia magna immobilization, boar semen live/dead ratio and DNA fragmentation and Unio pictorum micronucleus test. While the S. alba test indicated nutritive (stimulating) effect of the sample, the biodiesel exerted toxic effect in the aquatic tests. D. magna was the most sensitive with EC50 value of 0.0226%. For genotoxicity assessment, the mussel micronucleus test (MNT) was applied, detecting considerable genotoxic potential of the biodiesel sample: it elucidated micronuclei formation already at low concentration of 3.3%. Although this test has never been employed in biodiesel eco/genotoxicity assessments, it seems a promising tool, based on its appropriate sensitivity, and representativity.

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity - A flow cytometric study.

Local extreme climatic conditions occurring as a result of global climate change may interfere with the reproduction of animals. In the present study fish spermatozoa were incubated at different temperatures (20, 25, 30 and 40 °C) for 10 and 30 minutes, respectively and plasma membrane integrity and mitochondrial membrane potential changes were evaluated with flow cytometry using SYBR-14/PI and Mitotracker Deep Red FM fluorescent dyes. No significant differences were found in plasma membrane integrity at either incubation temperatures or time points. Mitotracker Deep Red FM histogram profiles indicating mitochondrial activity showed significant (p < 0.001) alterations in all cases of higher (25, 30 and 40 °C) temperature treatments as compared to the samples incubated at 20 °C. Our results indicate that fish spermatozoa exposed to high temperatures suffer sublethal damage that cannot be detected with conventional, vital staining techniques.

Flow cytometric detection of oxidative DNA damage in fish spermatozoa exposed to cadmium - Short communication.

The aim of the present pilot study was to apply a flow cytometric assay, the so-called OxyDNA test, to determine the level of oxidative DNA damage in fish spermatozoa exposed to different concentrations (0.01-10,000 mg/L) of cadmium. Milt was collected from three randomly selected Prussian carp (Carassius auratus gibelio) males. Oxidative DNA damage was assessed with the OxyDNA kit and using flow cytometry. The ratio of OxyDNA-positive events increased significantly at higher cadmium concentrations. The results indicate that direct contact of fish spermatozoa with cadmium-polluted water initiates genotoxic damage.

A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus 'Nicoletti assay'.

The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value.

Mitochondrial toxicity of triclosan on mammalian cells.

Effects of triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol) on mammalian cells were investigated using human peripheral blood mono nuclear cells (PBMC), keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine pancreatic islets (MIN-6) and neuroblastoma cells (MNA) as targets. We show that triclosan (1-10 µg ml-1) depolarised the mitochondria, upshifted the rate of glucose consumption in PMBC, HaCaT, PK-15 and MNA, and subsequently induced metabolic acidosis. Triclosan induced a regression of insulin producing pancreatic islets into tiny pycnotic cells and necrotic death. Short exposure to low concentrations of triclosan (30 min, ≤1 µg/ml) paralyzed the high amplitude tail beating and progressive motility of spermatozoa, within 30 min exposure, depolarized the spermatozoan mitochondria and hyperpolarised the acrosome region of the sperm head and the flagellar fibrous sheath (distal part of the flagellum). Experiments with isolated rat liver mitochondria showed that triclosan impaired oxidative phosphorylation, downshifted ATP synthesis, uncoupled respiration and provoked excessive oxygen uptake. These exposure concentrations are 100-1000 fold lower that those permitted in consumer goods. The mitochondriotoxic mechanism of triclosan differs from that of valinomycin, cereulide and the enniatins by not involving potassium ionophoric activity.

Ecotoxicity and genotoxicity assessment of exhaust particulates from diesel-powered buses.

Diesel exhaust is one of the major sources of fine and ultra-fine particulate matter in urban air. Toxicity of diesel-powered engine emissions has been quite widely assessed; however, much less information is available on their ecotoxicity. In our study, the kinetic version of the Vibrio fischeri bioluminescence inhibition bioassay based on the ISO 21338:2010 standard was used to characterise the ecotoxicity of diesel-powered buses. It is a direct contact test in which solid samples are tested in suspension and test organisms are in direct contact with toxic particles. The age of the selected buses fell into a wide range; the oldest one was produced in 1987. Diesel engines of different emission standards (Euro0-Euro4) were included. Measured EC50 values of Euro0-Euro1 engine emissions fell into the same range, 1.24-0.96 µg ml(-1), respectively. On the contrary, emission of Euro4 vehicle proved to be non-toxic. Genotoxic potential of the samples was also estimated, using the colorimetric SOS-chromotest™. Genotoxicity was detected also for Euro0 and Euro1 buses, showing correlation with the ecotoxic potential. The fact that the particulates from Euro4 vehicles did not show ecotoxic/genotoxic effect implies that replacing old Euro1 and Euro2 buses can be a highly effective solution for reducing environmental hazard of automotive emissions. The whole-aerosol testing method is a cheap alternative that can be used in engine developments and emission control.