Presence of microplastics (MPs) in wastewater has posed a huge ecosystem risk. Constructed wetlands (CWs) can effectively intercept MPs, while with MPs accumulation the response of CWs' performance is still unclear. In order to evaluate those effects, we conducted a 370-day experiment using CW microcosms fed with different levels (0, 10, 100, and 1000 μg/L) of polystyrene (PS) MPs (diameter: 50-100 μm). Results showed that nitrogen removal efficiency was increased (by 3.9%-24.7%) during the first 60 days and then decreased (by 7.1%-41.3%) with MPs accumulating, but no obvious change in COD and TP removal was observed. From further analysis, MPs accumulation changed the biofilm composition (TOC content increased from 41.4% to 52.7%), substrate porosity (electrical resistivity increased by 1.2-2.4 folds), and oxygen mass transfer (|KLa,O2| increased from 3.5% to 18.6%). Moreover, the microbial dynamics presented a higher abundance of nitrifiers (Nitrospira and Nitrosomonas) during the 60-day experiment and a lower abundance in the last days, while denitrifiers (Thauera, Thiobacillus, and Anaerolinea) had a high relative abundance throughout the experiment, being consistent with the variation of nitrification and denitrification rates. Finally, structural equation model analysis proved that due to the changes of substrate characteristics and microbial compositions and activities, the obvious decrease in nitrification efficiency was a direct reason for the decline of nitrogen removal during 370-day MPs accumulation. Overall, our study first prove that MPs accumulation can cause a series of changes in physicochemical and microbial characteristics of substrate, and ultimately affect the nitrogen-transforming process in CWs. Although our conclusions were based on the lab-scale CWs being different from the real wetlands, we hope that the conclusions can provide the effective regulatory strategies to guide the control of MPs in the actual wetlands.

• Klíčová slova
• Constructed wetlands, Microbial composition & activity, Microplastics, Nitrogen removal, Substrate characteristic,
• MeSH
• denitrifikace MeSH
• dusík analýza   MeSH
• ekosystém MeSH
• mikroplasty * MeSH
• mokřady * MeSH
• odpad tekutý - odstraňování MeSH
• odpadní voda analýza   MeSH
• plastické hmoty MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dusík MeSH
• mikroplasty * MeSH
• odpadní voda MeSH
• plastické hmoty MeSH

Chondroitin sulfate proteoglycans inhibit regeneration, neuroprotection, and plasticity following spinal cord injury. The development of a second-generation chondroitinase ABC enzyme, capable of being secreted from mammalian cells (mChABC), has facilitated the functional recovery of animals following severe spinal trauma. The genetically modified enzyme has been shown to efficiently break down the inhibitory extracellular matrix surrounding cells at the site of injury, while facilitating cellular integration and axonal growth. However, the activity profile of the enzyme in relation to the original bacterial chondroitinase (bChABC) has not been determined. Here, we characterize the activity profile of mChABC and compare it to bChABC, both enzymes having been maintained under physiologically relevant conditions for the duration of the experiment. We show that this genetically modified enzyme can be secreted reliably and robustly in high yields from a mammalian cell line. The modifications made to the cDNA of the enzyme have not altered the functional activity of mChABC compared to bChABC, ensuring that it has optimal activity on chondroitin sulfate-A, with an optimal pH at 8.0 and temperature at 37 °C. However, mChABC shows superior thermostability compared to bChABC, ensuring that the recombinant enzyme operates with enhanced activity over a variety of physiologically relevant substrates and temperatures compared to the widely used bacterial alternative without substantially altering its kinetic output. The determination that mChABC can function with greater robustness under physiological conditions than bChABC is an important step in the further development of this auspicious treatment strategy toward a clinical application.

• Publikační typ
• časopisecké články MeSH

An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.

• MeSH
• Agrobacterium tumefaciens enzymologie   genetika   metabolismus   MeSH
• aktivace enzymů MeSH
• biologické modely MeSH
• Bradyrhizobium enzymologie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fylogeneze MeSH
• hydrolasy klasifikace   genetika   metabolismus   fyziologie   MeSH
• mutantní proteiny klasifikace   genetika   metabolismus   MeSH
• Mycobacterium bovis enzymologie   genetika   metabolismus   MeSH
• Mycobacterium smegmatis genetika   metabolismus   MeSH
• Rhodococcus enzymologie   genetika   metabolismus   MeSH
• Sphingobacterium enzymologie   genetika   metabolismus   MeSH
• substrátová specifita MeSH
• Xanthobacter enzymologie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• mutantní proteiny MeSH

Chronic volume overload leads to cardiac hypertrophy and later to heart failure (HF), which are both associated with increased risk of cardiac arrhythmias. The goal of this study was to describe changes in myocardial morphology and to characterize arrhythmogenic substrate in rat model of developing HF due to volume overload. An arteriovenous fistula (AVF) was created in male Wistar rats between the inferior vena cava and abdominal aorta using needle technique. Myocardial morphology, tissue fibrosis, and connexin43 distribution, localization and phosphorylation were examined using confocal microscopy and Western blotting in the stage of compensated hypertrophy (11 weeks), and decompensated HF (21 weeks). Heart to body weight (BW) ratio was 89% and 133% higher in AVF rats at 11 and 21 weeks, respectively. At 21 weeks but not 11 weeks, AVF rats had pulmonary congestion (increased lung to BW ratio) indicating presence of decompensated HF. The myocytes in left ventricular midmyocardium were significantly thicker (+8% and +45%) and longer (+88% and +97%). Despite extensive hypertrophy, there was no excessive fibrosis in the AVF ventricles. Distribution and localization of connexin43 were similar between groups, but its phosphorylation was significantly lower in AVF hearts at 21st week, but not 11th week, suggesting that HF, rather than hypertrophy contributes to the connexin43 hypophosphorylation. In conclusion, volume overload leads to extensive eccentric hypertrophy, but not to myocardial fibrosis. Increased vulnerability to arrhythmia in this HF model is possibly related to gap junction remodeling with hypophosphorylation of connexin43.

• MeSH
• arteriovenózní píštěl komplikace   metabolismus   patologie   MeSH
• chronická nemoc MeSH
• fosforylace MeSH
• kardiomegalie komplikace   metabolismus   patologie   MeSH
• konexin 43 biosyntéza   MeSH
• krysa rodu Rattus MeSH
• modely nemocí na zvířatech * MeSH
• myokard metabolismus   patologie   MeSH
• potkani Wistar MeSH
• srdeční arytmie etiologie   metabolismus   patologie   MeSH
• srdeční selhání metabolismus   patologie   MeSH
• tepový objem fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• konexin 43 MeSH

A fast method for preparing of silver particle layers on glass substrates with high application potential for using in surface enhanced Raman spectroscopy (SERS) is introduced. Silver particle layers deposited on glass cover slips were generated in one-step process by reduction of silver nitrate using several reducing agents (ethylene glycol, glycerol, maltose, lactose and glucose) under ultrasonic irradiation. This technique allows the formation of homogeneous layers of silver particles with sizes from 80nm up to several hundred nanometers depending on the nature of the used reducing agent. Additionally, the presented method is not susceptible to impurities on the substrate surface and it does not need any additives to capture or stabilize the silver particles on the glass surface. The characteristics of prepared silver layers on glass substrate by the above mentioned sonochemical approach was compared with chemically prepared ones. The prepared layers were tested as substrates for SERS using adenine as a model analyte. The factor of Raman signal enhancement reached up to 5·10(5). On the contrary, the chemically prepared silver layers does not exhibit almost any pronounced Raman signal enhancement. Presented sonochemical approach for preparation of silver particle layers is fast, simple, robust, and is better suited for reproducible fabrication functional SERS substrates than chemical one.

• Klíčová slova
• Glass substrate, Silver particles layer, Sonochemical, Surface enhanced Raman spectroscopy,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

• MeSH
• beta-N-acetylhexosaminidasy chemie   metabolismus   MeSH
• fylogeneze MeSH
• glykosylace MeSH
• katalytická doména MeSH
• kinetika MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• reprodukovatelnost výsledků MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• Talaromyces enzymologie   MeSH
• výpočetní biologie * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH

Alumina substrate can be found in electronic components used in portable electronic devices. The material is radiation sensitive and can be applied in dosimetry using thermally or optically stimulated luminescence. Electronic portable devices such as mobile phones, USB flash discs, mp3 players, etc., which are worn close to the body, can represent personal dosemeters for members of the general public in situations of large-scale radiation accidents or malevolent acts with radioactive materials. This study investigated dosimetric properties of alumina substrates and aspects of using mobile phones as personal dosemeters. The alumina substrates exhibited favourable dosimetry characteristics. However, anomalous fading had to be properly corrected in order to achieve sufficient precision in dose estimate. Trial dose reconstruction performed by means of two mobile phones proved that mobile phones can be used for reconstruction of personal doses.

• MeSH
• elektronika přístrojové vybavení   MeSH
• luminiscence MeSH
• mobilní telefon přístrojové vybavení   MeSH
• oxid hlinitý chemie   MeSH
• radiační účinky MeSH
• radiometrie přístrojové vybavení   MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oxid hlinitý MeSH

The haloacid dehalogenase (HAD) superfamily is one of the largest known groups of enzymes and the majority of its members catalyze the hydrolysis of phosphoric acid monoesters into a phosphate ion and an alcohol. Despite the fact that sequence similarity between HAD phosphatases is generally very low, the members of the family possess some characteristic features, such as a Rossmann-like fold, HAD signature motifs or the requirement for Mg2+ ion as an obligatory cofactor. This study focuses on a new hypothetical HAD phosphatase from Thermococcus thioreducens. The protein crystallized in space group P21212, with unit-cell parameters a = 66.3, b = 117.0, c = 33.8 Å, and the crystals contained one molecule in the asymmetric unit. The protein structure was determined by X-ray crystallography and was refined to 1.75 Å resolution. The structure revealed a putative active site common to all HAD members. Computational docking into the crystal structure was used to propose substrates of the enzyme. The activity of this thermophilic enzyme towards several of the selected substrates was confirmed at temperatures of 37°C as well as 60°C.

• Klíčová slova
• HAD superfamily, crystal structure, docking, hypothetical phosphatase, phosphatase assay,
• MeSH
• fosfatasy chemie   MeSH
• hydrolasy chemie   MeSH
• katalytická doména MeSH
• kinetika MeSH
• krystalografie rentgenová metody   MeSH
• molekulární modely MeSH
• substrátová specifita MeSH
• Thermococcus enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2-haloacid dehalogenase MeSH Prohlížeč
• fosfatasy MeSH
• hydrolasy MeSH

The perovskite ytterbium ferrite is a new ferroelectric semiconductor material. We presented the temperature induced current-voltage (I-V) characteristics of the Al/YbFeO3-δ/p-Si/Al hetero-junction. The orthoferrite YbFeO3-δthin films were deposited on a single crystal p-type Si substrate by a radio frequency magnetron sputtering system. The potential barrier height (BH)and ideality factornof the heterojunction were obtained by thermionic emission current method based on the recommendations in the literature. The fact that the calculated slopes ofI-Vcurves become temperature independent implying that the field emission current mechanism takes place across the device, which has been explained by the presence of the spatial inhomogeneity of BHs or potential fluctuations. Moreover, a tunneling transmission coefficient value of 26.67 was obtained for the ferroelectric YbFeO3-δlayer at the Al/p-Si interface.

• Klíčová slova
• YbFeO3, current–voltage (I–V) characteristics, field emission, hetero-junction, perovskite oxide,
• Publikační typ
• časopisecké články MeSH

We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.

• MeSH
• Agrobacterium tumefaciens enzymologie   genetika   metabolismus   MeSH
• alkany metabolismus   MeSH
• DNA bakterií chemie   genetika   MeSH
• estery metabolismus   MeSH
• hydrolasy genetika   izolace a purifikace   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• rostliny mikrobiologie   MeSH
• sekvenční analýza DNA MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkany MeSH
• DNA bakterií MeSH
• estery MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH