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The article describes a diagnostic method, on the basis of which it is possible to assess whether there is a loose connection in the clamping system (detection) and to determine which of them is loose (localization). The diagnostic method is based on comparing the drops in the natural frequencies of a measured system with respect to a reference system, which simulates the desired state. When analysing the drop in natural frequencies in order to locate the loosening, it is necessary to relate this drop to the position of the clamping joints within the considered mode shape.

Musil, M. Comparison of correction methods for mathematical model of generally damped vibrating system. (Porovnanie metod korekcie matematickeho modelu kmitajucej sustavy so vseobecnym tlmenim). Strojnicky Casopis (Slovakia) 56(2): 100-121, 2005 [...]

The evaluation of disk brake squeal is nowadays performed using Finite Element Model. In this standard procedure the thermal effects are omitted. The omission is done because of long computing time of fully coupled brake system thermo-structural transient analysis. This paper is presenting an effective uncoupled thermo-structural FEM procedure. This method is applied on a pin-on-disc system and its time effectiveness is compared to standard full coupled transient analysis.

The paper describes role of non-proportional damping in flutter type instability, demonstrated on simplified disc brake model. The discrete two degrees of freedom system is considered to imply damping induced instability through a system eigenvalues evaluation. The Frequency Response Function (FRF) is further calculated from measurements on the physical disc brake model. From FRF, damping properties are estimated and discussed. Several different loading states of the pad versus disc are considered to show loading impact on FRF and thus damping of the system.

This article discusses the application of so-called ultrasonic quasi-Rayleigh waves to detect surface defects of mechanical constructions, namely plate structures. The application of quasi-Rayleigh waves allows the extension of the scope of detection using conventional ultrasonic methods that are based on bulk waves. This extension means larger distances as well as higher sensitivity of the detection of surface defects such as fatigue or corrosion cracks. An advantage of this method is the transfer of wave energy from one side of a plate to another, which helps to overcome one-sided obstacles (such as cross-pieces, reinforcement elements). The article describes characteristic properties of quasi-Rayleigh waves that are important for the proper (in terms of frequency in particular) design of the excitation of waves towards the structure. FEM simulation results then provide information regarding the sensitivity of the wave response to the presence and sizes of surface defects (perpendicular slots) in an isotropic material with the properties of steel. The theoretical knowledge is set against experimental measurements obtained with the use of a steel plate with cross-pieces welded to it.

This article is devoted to the comparison of the influence of the piezoelectric matrix properties on the magnitude of the resulting charge when a thin piezoelectric membrane of circular cross section, made from aluminium gallium nitride (Al-GaN), is loaded. The size of change of the electric charge was determined by the numerical analysis and the by the change of the properties of the piezoelectric matrix. The matrix constants were obtained from various sources introduced in world databases.

An indirect enzyme immunoassay for rapid detection of Campylobacter jejuni subsp. jejuni O:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 µg/mL, and 8 µg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/µL, limit of quantification being 480 CFU/µL. By testing 5 chromogens. viz. 1,2-benzenediamine, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3′,5,5′-tetramethylbenzidine, bi(4,4′-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3′,5,5′-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain of C. sputorum subsp. sputorum (21.5 %) and with G+ bacterium Micrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according to ČSN ISO 10272 and commercially available Singlepath® Campylobacter GLISA-Rapid Test. [ABSTRACT FROM AUTHOR]

Paper describes the mechanism of vibration of a minimal automotive disc brake model which can lead to unwanted effects known as brake Noise Vibrations and Harshness (NVH). Stick-slip frictional effects are mathematically described by simplification of physical assumptions. Self-excited vibrations due to stick-slip and also stability conditions are defined using a 1 degree of freedom mechanical system. Non-Coulomb nonlinear behavior of the friction force with negative slope is considered. Response is obtained by the numerical solution of ordinary differential equation.

Describes a discrete geologic tomography method based on mixed-integer linear programming. Use of joint inversions to reduce the ambiquity of tomographic studies; Performance of the algorithm on several synthetic data sets; Exploitation of the complementary nature of seismic and georadar data to locate air or water-filled cavities.

Large capacity tanks storing various liquids are important components in distribution and transmission systems. During operation tanks can be subjected to different types of loading. Therefore, maximum attention must be paid to the tank design to capture all possible causes and forms of failures. The article deals with the procedure for seismic resistance of liquid storage tanks which are in accordance with the principles of Eurocode 8 standard. The seismic analysis is performed on flexible (steel) circular vertical ground-supported model of tank containing liquid (water). The main aim is to determine basic seismic characteristics, distributions of hydrodynamic pressure, dynamic properties and response of investigated tank-liquid system subjected to earthquake excitation (El Centro). Seismic analysis and results comparison are carried out on mechanical spring-mass model (Eurocode 8) and finite element model (ANSYS).

Dynamic analysis of a tank containing liquid is a complex problem involving fluid-structure interaction. The tank-liquid system is simplified by an equivalent model in which the total liquid mass is divided into two zones – impulsive and convective. The paper is primary focused on the behavior of the free liquid surface (the convective portion of liquid) subjected to the dynamic loading which may result in liquid spilling or tank wall damage. Therefore, the sufficient freeboard must be required to design. The paper deals with the seismic design of the open cylindrical liquid storage tank with the aim to determine convective dynamic properties (natural frequencies and modes of oscillation), maximum vertical displacements over tank radius and overall response of the liquid to an earthquake. The analysis is performed analytically by applying procedures for the determination of convective effects based on simplified equivalent spring-mass model, numerically response spectrum, and method of motion integrating equations utilizing ANSYS Multiphysics.

During the vibration testing, various types of vibration drivers of different structures are used. Electrodynamic drivers are generally used for smaller excitation forces whereas for bigger excitation forces hydraulic drivers are used. The main disadvantage of drivers can be found in their size. One of the possibilities for size reduction may be a suitable engineering design. The paper deals with the design of a small uniaxial driver which uses an inertial driver as a source of the excitation force. The structure considers the requirements for stiffness changes of the whole system with the aim of available frequency range tuning.

Background: Stroke often impairs upper extremity motor function, with recovery in the sub-acute phase being crucial for regaining independence. This study examines changes in isometric muscle strength, dexterity, and self-care independence during this period, and evaluates the effects of a comprehensive intensive rehabilitation (COMIRESTROKE)., Methods: Individuals in sub-acute stroke recovery and age- and sex-matched controls were assessed for pre- and post-rehabilitation differences in primary outcomes (grip/pinch strength, Nine Hole Peg Test [NHPT], Action Research Arm Test [ARAT]). COMIRESTROKE's effects on primary and secondary outcomes (National Institute of Health Stroke Scale [NIHSS], Modified Rankin Scale [MRS], Functional Independence Measure [FIM]) were evaluated. Outcomes were analyzed for dominant and non-dominant limbs, both regardless of impairment and with a focus on impaired limbs., Results: Fifty-two individuals with stroke (NIHSS 7.51 ± 5.71, age 70.25 ± 12.66 years, 21.36 ± 12.06 days post-stroke) and forty-six controls participated. At baseline, individuals with stroke showed significantly lower strength (dominant grip, key pinch, tip-tip pinch, p adj  < 0.05), higher NHPT scores ( p adj  < 0.05), and lower ARAT scores ( p adj  < 0.001). COMIRESTROKE led to improvements in dominant key pinch, non-dominant tip-tip pinch, NHPT, and both dominant and non-dominant ARAT ( p adj  < 0.05). Notably, non-dominant key pinch improved significantly when considering only impaired hands. Pre- and post-test differences between groups were significant only for ARAT (both limbs), even after adjustment ( p adj  < 0.05). All secondary outcomes (NIHSS, MRS, FIM) showed significant improvement post-COMIRESTROKE ( p adj  < 0.001)., Conclusion: Individuals with stroke exhibit reduced muscle strength and dexterity, impairing independence. However, comprehensive intensive rehabilitation significantly improves these functions. Data are available from the corresponding author upon request and are part of a sub-study of NCT05323916., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 K, P, M, B, J, D, D, L, R, J, J, V and T.)

Summary: Protein design requires information about how mutations affect protein stability. Many web-based predictors are available for this purpose, yet comparing them or using them en masse is difficult. Here, we present BenchStab, a console tool/Python package for easy and quick execution of 19 predictors and result collection on a list of mutants. Moreover, the tool is easily extensible with additional predictors. We created an independent dataset derived from the FireProtDB and evaluated 24 different prediction methods., Availability and Implementation: BenchStab is an open-source Python package available at https://github.com/loschmidt/BenchStab with a detailed README and example usage at https://loschmidt.chemi.muni.cz/benchstab. The BenchStab dataset is available on Zenodo: https://zenodo.org/records/10637728., (© The Author(s) 2024. Published by Oxford University Press.)

Thermostable proteins find their use in numerous biomedical and biotechnological applications. However, the computational design of stable proteins often results in single-point mutations with a limited effect on protein stability. However, the construction of stable multiple-point mutants can prove difficult due to the possibility of antagonistic effects between individual mutations. FireProt protocol enables the automated computational design of highly stable multiple-point mutants. FireProt 2.0 builds on top of the previously published FireProt web, retaining the original functionality and expanding it with several new stabilization strategies. FireProt 2.0 integrates the AlphaFold database and the homology modeling for structure prediction, enabling calculations starting from a sequence. Multiple-point designs are constructed using the Bron-Kerbosch algorithm minimizing the antagonistic effect between the individual mutations. Users can newly limit the FireProt calculation to a set of user-defined mutations, run a saturation mutagenesis of the whole protein or select rigidifying mutations based on B-factors. Evolution-based back-to-consensus strategy is complemented by ancestral sequence reconstruction. FireProt 2.0 is significantly faster and a reworked graphical user interface broadens the tool's availability even to users with older hardware. FireProt 2.0 is freely available at http://loschmidt.chemi.muni.cz/fireprotweb., (© The Author(s) 2023. Published by Oxford University Press.)

In this study, we experimentally evaluated how the feeding behaviour of marbled crayfish Procambarus virginalis is influenced by cues from conspecifics and the round goby Neogobius melanostomus, a fish predator, in tanks that permitted chemical communication but not visual recognition. We used four experimental groups with different combinations in two sub-tanks. The first sub-tank always contained a crayfish and prey (40 individuals of the water louse Asellus aquaticus). The other sub-tanks were set up as follows: (i) empty, serving as a control (C); (ii) with a conspecific crayfish (Cr); (iii) with a round goby (G) to simulate predator-only odour; and (iv) a round goby and three small conspecific crayfish (G + Cr) to simulate the presence of a predator and/or the alarm odour. Two sub-treatments were defined for the fourth group, categorised as 'injured' or 'not injured' depending on whether prey crayfish were visibly injured or not, respectively. We observed a significant decline in the consumption of water lice in the G and G + Cr treatments compared to the C and Cr treatments (up to 47% on average). There were no significant differences in consumption between the G and G + Cr treatments, or C and Cr treatments. No significant differences in food consumption parameters were detected between sub-treatments with 'injured' and 'not injured' conspecific crayfish. Knowledge of modifications in the feeding behaviour of marbled crayfish in the presence of round goby (and fish predators in general) is essential for ecologists attempting to understand the changes and impacts occurring in invaded freshwater ecosystems., (© 2023. The Author(s).)

Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases that contain heme in their active centers allowing both the peroxidatic and catalatic reaction modes. These originally bacterial enzymes are broadly distributed among various fungi allowing them to cope with reactive oxygen species present in the environment or inside the cells. We used various biophysical, biochemical, and bioinformatics methods to investigate differences between catalase-peroxidases originating in thermophilic and mesophilic fungi from different habitats. Our results indicate that the architecture of the active center with a specific post-translational modification is highly similar in mesophilic and thermophilic KatG and also the peroxidatic acitivity with ABTS, guaiacol, and L-DOPA. However, only the thermophilic variant CthedisKatG reveals increased manganese peroxidase activity at elevated temperatures. The catalatic activity releasing molecular oxygen is comparable between CthedisKatG and mesophilic MagKatG1 over a broad temperature range. Two constructed point mutations in the active center were performed selectively blocking the formation of described post-translational modification in the active center. They exhibited a total loss of catalatic activity and changes in the peroxidatic activity. Our results indicate the capacity of bifunctional heme enzymes in the variable reactivity for potential biotech applications.

Protein tunnels are essential in transporting small molecules into the active sites of enzymes. Tunnels' geometrical and physico-chemical properties influence the transport process. The tunnels are attractive hot spots for protein engineering and drug development. However, studying the ligand binding and unbinding using experimental techniques is challenging, while in silico methods come with their limitations, especially in the case of resource-demanding virtual screening pipelines. Caver Web 1.2 is a new version of the web server combining the capabilities for the detection of protein tunnels with the calculation of the ligand trajectories. The new version of the Caver Web server was expanded with the ability to fetch novel ligands from the Integrated Database of Small Molecules and with the fully automated virtual screening pipeline allowing for the fast evaluation of the predefined set of over 4,300 currently approved drugs. The virtual screening pipeline is accompanied by a comprehensive user interface, making it a viable service for the broader spectrum of companies and the academic user community. The web server is freely available for academic use at https://loschmidt.chemi.muni.cz/caverweb., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Protein solubility is an attractive engineering target primarily due to its relation to yields in protein production and manufacturing. Moreover, better knowledge of the mutational effects on protein solubility could connect several serious human diseases with protein aggregation. However, we have limited understanding of the protein structural determinants of solubility, and the available data have mostly been scattered in the literature. Here, we present SoluProtMut DB - the first database containing data on protein solubility changes upon mutations. Our database accommodates 33 000 measurements of 17 000 protein variants in 103 different proteins. The database can serve as an essential source of information for the researchers designing improved protein variants or those developing machine learning tools to predict the effects of mutations on solubility. The database comprises all the previously published solubility datasets and thousands of new data points from recent publications, including deep mutational scanning experiments. Moreover, it features many available experimental conditions known to affect protein solubility. The datasets have been manually curated with substantial corrections, improving suitability for machine learning applications. The database is available at loschmidt.chemi.muni.cz/soluprotmutdb., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

In this study, we focus on a detailed bioinformatics analysis of hyBpox genes, mainly within the genomes of Sclerotiniaceae (Ascomycota, Leotiomycetes), which is a specifically evolved fungal family of necrotrophic host generalists and saprophytic or biotrophic host specialists. Members of the genus Sclerotium produce only sclerotia and no fruiting bodies or spores. Thus, their physiological role for peroxidases remains open. A representative species, S. cepivorum, is a dangerous plant pathogen causing white rot in Allium species, particularly in onions, leeks, and garlic. On a worldwide basis, the white rot caused by this soil-borne fungus is apparently the most serious threat to Allium -crop production. We have also found very similar peroxidase sequences in the related fungus S. sclerotiorum , although with minor yet important modifications in the architecture of its active centre. The presence of ScephyBpox1- specific mRNA was confirmed by transcriptomic analysis. The presence of Hybrid B peroxidase at the protein level as the sole extracellular peroxidase of this fungus was confirmed in the secretome of S. cepivorum through detailed proteomic analyses. This prompted us to systematically search for all available genes coding for Hybrid B heme peroxidases in the whole fungal family of Sclerotiniaceae . We present here a reconstruction of their molecular phylogeny and analyse the unique aspects of their conserved-sequence features and structural folds in corresponding ancestral sequences.

Phenotypic plastic responses to temperature can modulate the kinetic effects of temperature on biological rates and traits and thus play an important role for species adaptation to climate change. However, there is little information on how these plastic responses to temperature can influence trophic interactions. Here, we conducted an experiment using marbled crayfish and their water louse prey to investigate how short-term thermal acclimation at two temperatures (16 and 24°C) modulates the predator functional response. We found that both functional response parameters (search rate and handling time) differed between the two experimental temperatures. However, the sign and magnitudes of these differences strongly depended on acclimation time. Acclimation to 16°C increased handling time and search rate whereas acclimation to 24°C leads to the opposite effects with shorter handling time and lower search rate for acclimated predators. Moreover, the strength of these effects increased with acclimation time so that the differences in search rate and handing time between the two temperatures were reversed between the treatment without acclimation and after 24 h of acclimation. Overall, we found that the magnitude of the acclimation effects can be as strong as the direct kinetic effects of temperature. Our study highlights the importance of taking into account short-term thermal plasticity to improve our understanding of the potential consequences of global warming on species interactions., Competing Interests: The authors declare that they have no conflicts of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

There is a great interest in increasing proteins' stability to widen their usability in numerous biomedical and biotechnological applications. However, native proteins cannot usually withstand the harsh industrial environment, since they are evolved to function under mild conditions. Ancestral sequence reconstruction is a well-established method for deducing the evolutionary history of genes. Besides its applicability to discover the most probable evolutionary ancestors of the modern proteins, ancestral sequence reconstruction has proven to be a useful approach for the design of highly stable proteins. Recently, several computational tools were developed, which make the ancestral reconstruction algorithms accessible to the community, while leaving the most crucial steps of the preparation of the input data on users' side. FireProtASR aims to overcome this obstacle by constructing a fully automated workflow, allowing even the unexperienced users to obtain ancestral sequences based on a sequence query as the only input. FireProtASR is complemented with an interactive, easy-to-use web interface and is freely available at https://loschmidt.chemi.muni.cz/fireprotasr/., (© The Author(s) 2020. Published by Oxford University Press.)

Biological processes tend to dominate the oxygen regime of productive waters. However, in shallow aquatic ecosystems, it is unclear whether the oxygen regime is driven by oxygen production and consumption in the water column or by sediment oxygen demand (SOD). In managed eutrophic ecosystems, this question is especially important in the context of extreme daily oscillations of dissolved oxygen (DO) that could breach physiological limits of heterotrophic aerobic organisms. High-frequency measurement of DO, temperature, global radiation (Gl.Rad.), and pH in a 0.6 m deep, 22 ha eutrophic fishpond Rod (Czech Republic) shows that the oxygen regime depended on the ecosystem state. Over the clearwater period in the early season, the DO level reflected ecosystem heterotrophy with relatively low daily DO oscillations. However, during the summer phytoplankton bloom, the fishpond was primarily autotrophic with extreme DO fluctuation. During late summer, a collapse of the phytoplankton bloom and an associated shift towards heterotrophy and DO deficit frequently occur. In-situ mesocosm experiments in Rod fishpond were conducted throughout 2018 and 2019 growing seasons, to address the importance of SOD to the oxygen regime. We enclosed the water column in transparent and opaque/dark plastic cylinders open or closed to the sediment. The results show that the proportional contribution of SOD to total respiration decreased from 70 to 90% at low phytoplankton biomass (expressed as Chlorophyll-a (Chl-a) concentration) to approximately 10% at phytoplankton bloom. At night, the difference between the oxygen consumption in the cylinders with or without sediment was statistically significant, when the concentration of Chl-a was <100 μg·L -1 . On the contrary, the difference was not significant when the concentration of Chl-a was >100 μg·L -1 . This revealed that the impact of SOD is negligible at high phytoplankton biomass., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personaI relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Enzymes are the natural catalysts that execute biochemical reactions upholding life. Their natural effectiveness has been fine-tuned as a result of millions of years of natural evolution. Such catalytic effectiveness has prompted the use of biocatalysts from multiple sources on different applications, including the industrial production of goods (food and beverages, detergents, textile, and pharmaceutics), environmental protection, and biomedical applications. Natural enzymes often need to be improved by protein engineering to optimize their function in non-native environments. Recent technological advances have greatly facilitated this process by providing the experimental approaches of directed evolution or by enabling computer-assisted applications. Directed evolution mimics the natural selection process in a highly accelerated fashion at the expense of arduous laboratory work and economic resources. Theoretical methods provide predictions and represent an attractive complement to such experiments by waiving their inherent costs. Computational techniques can be used to engineer enzymatic reactivity, substrate specificity and ligand binding, access pathways and ligand transport, and global properties like protein stability, solubility, and flexibility. Theoretical approaches can also identify hotspots on the protein sequence for mutagenesis and predict suitable alternatives for selected positions with expected outcomes. This review covers the latest advances in computational methods for enzyme engineering and presents many successful case studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Protein evolution and protein engineering techniques are of great interest in basic science and industrial applications such as pharmacology, medicine, or biotechnology. Ancestral sequence reconstruction (ASR) is a powerful technique for probing evolutionary relationships and engineering robust proteins with good thermostability and broad substrate specificity. The following protocol describes the setting up and execution of an automated FireProt ASR workflow using a dedicated web site. The service allows for inference of ancestral proteins automatically, from a single protein sequence. Once a protein sequence is submitted, the server will build a dataset of homology sequences, perform a multiple sequence alignment (MSA), build a phylogenetic tree, and reconstruct ancestral nodes. The protocol is also highly flexible and allows for multiple forms of input, advanced settings, and the ability to start jobs from: (i) a single sequence, (ii) a set of homologous sequences, (iii) an MSA, and (iv) a phylogenetic tree. This approach automates all necessary steps and offers a way for novices with limited exposure to ASR techniques to improve the properties of a protein of interest. The technique can even be used to introduce catalytic promiscuity into an enzyme. A web server for accessing the fully automated workflow is freely accessible at https://loschmidt.chemi.muni.cz/fireprotasr/. © 2021 Wiley Periodicals LLC. Basic Protocol: ASR using the Web Server FireProt ASR ., (© 2021 Wiley Periodicals LLC.)

The majority of naturally occurring proteins have evolved to function under mild conditions inside the living organisms. One of the critical obstacles for the use of proteins in biotechnological applications is their insufficient stability at elevated temperatures or in the presence of salts. Since experimental screening for stabilizing mutations is typically laborious and expensive, in silico predictors are often used for narrowing down the mutational landscape. The recent advances in machine learning and artificial intelligence further facilitate the development of such computational tools. However, the accuracy of these predictors strongly depends on the quality and amount of data used for training and testing, which have often been reported as the current bottleneck of the approach. To address this problem, we present a novel database of experimental thermostability data for single-point mutants FireProtDB. The database combines the published datasets, data extracted manually from the recent literature, and the data collected in our laboratory. Its user interface is designed to facilitate both types of the expected use: (i) the interactive explorations of individual entries on the level of a protein or mutation and (ii) the construction of highly customized and machine learning-friendly datasets using advanced searching and filtering. The database is freely available at https://loschmidt.chemi.muni.cz/fireprotdb., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Biodiesel is a mixture of esters of fatty acids (most often palmitic, stearic and oleic) and lower alcohols (in our work butanol) produced by transesterification. It is a renewable source of energy, prepared from triacylglycerides, which are contained in vegetable oils and animal fats. This work focuses on alkaline catalyzed transesterification of rapeseed oil with butanol and comparison of two catalysts (potassium hydroxide and potassium tert-butoxide). In industry is usually transesterification of rapeseed oil carried out like reaction catalyzed by potassium hydroxide. Potassium hydroxide have high content of K 2 CO 3 , KHCO 3 and water. Moreover water is formed by neutralization of potassium hydroxide with free fatty acids contained in oil. In cause of tert-butoxide catalyzed reaction, it is not possible because tert-butoxide have not a OH - aniont, which is needed for water forming. The influence of various conditions (addition of water, temperature of separation, intensity of stirring and type of catalyst) on butanolysis process was studied for both catalysts. For both catalysts dependence of conversions on time were plotted. When tert-butoxide was used, satisfactory phase separation was not achieved. The only way was separation of hot crude reaction mixture without adding water. Ester formed by this method had high content of free glycerol and soaps, but reached higher conversion. The best results were obtained with KOH and subsequent separation of cold crude reaction mixture with the addition of water and slow stirring. The difference between reactions catalyzed by potassium hydroxide and potassium tert-butoxide was described., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

An important message taken from human genome sequencing projects is that the human population exhibits approximately 99.9% genetic similarity. Variations in the remaining parts of the genome determine our identity, trace our history and reveal our heritage. The precise delineation of phenotypically causal variants plays a key role in providing accurate personalized diagnosis, prognosis, and treatment of inherited diseases. Several computational methods for achieving such delineation have been reported recently. However, their ability to pinpoint potentially deleterious variants is limited by the fact that their mechanisms of prediction do not account for the existence of different categories of variants. Consequently, their output is biased towards the variant categories that are most strongly represented in the variant databases. Moreover, most such methods provide numeric scores but not binary predictions of the deleteriousness of variants or confidence scores that would be more easily understood by users. We have constructed three datasets covering different types of disease-related variants, which were divided across five categories: (i) regulatory, (ii) splicing, (iii) missense, (iv) synonymous, and (v) nonsense variants. These datasets were used to develop category-optimal decision thresholds and to evaluate six tools for variant prioritization: CADD, DANN, FATHMM, FitCons, FunSeq2 and GWAVA. This evaluation revealed some important advantages of the category-based approach. The results obtained with the five best-performing tools were then combined into a consensus score. Additional comparative analyses showed that in the case of missense variations, protein-based predictors perform better than DNA sequence-based predictors. A user-friendly web interface was developed that provides easy access to the five tools' predictions, and their consensus scores, in a user-understandable format tailored to the specific features of different categories of variations. To enable comprehensive evaluation of variants, the predictions are complemented with annotations from eight databases. The web server is freely available to the community at http://loschmidt.chemi.muni.cz/predictsnp2.

The aim of the present prospective, single centre observational study was to describe the profile and prognosis of patients hospitalised with chronic heart failure and to determine the value of discharge blood pressure and cholesterol for long-term survival. From among 2,346 hospitalised patients, 320 (13.6%) suffered from chronic heart failure and 28 (8.8%) died during hospitalisation. The in-patient mortality rate was similar to that in patients not suffering from chronic heart failure (P = 0, 3). Of 292 patients who were discharged, 162 (55%) died during the subsequent 5 years. The predetermined parameters of pure prognosis were associated with lower diastolic blood pressure (P = 0.008) and lower cholesterol (P = 0.012). A poor prognosis was associated with lower systolic blood pressure plus lower cholesterol and lower diastolic blood pressure and lower cholesterol. Other independent prognostic parameters were older age (P < 0.001), higher heart rate (P = 0.02), higher creatinine (P < 0.001), higher urea (P < 0.001), higher uric acid (P < 0.001), lower hemoglobin (P = 0.02), lower ejection fraction (P = 0.080), and a history of ischemic heart disease (P < 0.01). Patients suffering from chronic heart failure and discharged home have a worse prognosis if their systolic and/or diastolic blood pressures and/or cholesterol levels are too low. The optimal values seem to be levels that are around the recommended targets, that is a systolic BP of 140 mmHg, diastolic BP of 90 mmHg, and a cholesterol level of 5 mmol/L.

Enzyme-linked immunosorbent assay (ELISA) enabled a more precise serological typing of isolates of red clover necrotic mosaic virus (RCNMV) originating from Slovak and Czech Republics, Poland, Sweden and Great Britain. We found 5 isolates of serotype A, 14 isolates of serotype B, and 6 isolates of serotype C. Some isolates represented mixtures of serotypes, namely A+B (1 isolate), A+C (1), B+C (7), and A+B+C (5). ELISA was found to be a more suitable method of serotype identification of RCNMV isolates than the double diffusion agar gel test for its higher sensitivity and greater selectivity.

Enzyme-linked immunosorbent assay (ELISA) was used to determine the degree of serological specificity of two serotypes of broad bean stain virus (BBSV) and their relationship to red clover mottle virus (RCMV). Optimal conditions for the differentiation by ELISA of the two viruses and the two BBSV serotypes were established. BBSV isolates from Vicia sativa belonged to serotype I, those from pea plants (F1, Kow 60) to serotype II, and isolates from Lens culinaris differed from these two serotypes. ELISA revealed no antigenic differences between 22 RCMV isolates which showed the same degree of serological relationship to both BBSV serotypes.

Detection of three isolates of red clover necrotic mosaic virus (RCNMV) representing A, B, and C serotypes was experimentally proved in 18 host plant species by enzyme-linked immunosorbent assay (ELISA). In all plant species tested, the homologous serotype reactions showed high selectivity. Individual virus serotypes could be reliably detected in the extracts of infected plants only with the homologous IgG fraction. Group specific detection of RCNMV without serotype determination was possible using the mixture of IgG directed to all virus serotypes occurring in the region of investigation. Intensity of positive reaction of optimally diluted IgG with the extracts from infected plants differed markedly from that of negative reaction and from the reaction background. The latter depended on the quality of serum used for the IgG preparation. For detection of small amounts of RCNMV, virus infectivity test on indicator plants was more sensitive than ELISA.