Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies

2001 ◽  
Vol 354 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Ashlee PERRY ◽  
Lu-Yun LIAN ◽  
Nigel S. SCRUTTON
Keyword(s):  
Simple Procedure ◽  
Factor Xa ◽  
Pseudomonas Oleovorans ◽  
Terminal Domain ◽  
Binding Domains ◽  
Redox Active ◽  
Charge Transfer Excitation ◽  
The Individual ◽  
High Level ◽  
Difference Absorption

A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756–1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276nm; from J⊘rgensen's electronegativity theory, the 240nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin.

scholarly journals Recombinant two-iron rubredoxin of Pseudomonas oleovorans: overexpression, purification and characterization by optical, CD and 113Cd NMR spectroscopies

1997 ◽  
Vol 328 (1) ◽  
pp. 131-136 ◽  
Author(s):  
Ho Joon LEE ◽  
Lu-Yun LIAN ◽  
S. Nigel SCRUTTON
Keyword(s):  
Chemical Exchange ◽  
Chemical Shifts ◽  
Pseudomonas Oleovorans ◽  
Redox Active ◽  
Step Procedure ◽  
High Frequency Shift ◽  
The Difference ◽  
Ferrous Ammonium ◽  
High Level ◽  
Difference Absorption

The gene (alk G) encoding the two-iron rubredoxin of Pseudomonas oleovorans was amplified from genomic DNA by PCR and subcloned into the expression vector pKK223-3. The vector directed the high-level production of rubredoxin in Escherichia coli. A simple three-step procedure was used to purify recombinant rubredoxin in the 1Fe form. 1Fe-rubredoxin was readily converted to the 2Fe, apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of ferrous ammonium sulphate or CdCl2 respectively. Recombinant 1Fe and 2Fe rubredoxins are redox-active and able to transfer electrons from reduced spinach ferredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the cadmium-substituted protein are similar to those reported for cadmium-substituted Desulfovibrio gigas rubredoxin [Henehan, Poutney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of cadmium-substituted rubredoxin revealed the presence of four Gaussian-resolved maxima at 207, 228, 241 and 280 nm; the 241 nm band is attributable, from J⊘rgensen's electronegativity theory, to a CysS-CdII charge-transfer excitation. The 113Cd NMR spectrum of the 113Cd-substituted rubredoxin contains two 113Cd resonances with chemical shifts located at 732.3 and 730 p.p.m. The broader linewidth and high frequency shift of the resonance at 730 p.p.m. indicates that the Cd2+ ion is undergoing chemical exchange and, consistent with the difference absorption spectra, is bound less tightly than the Cd2+ ion, giving rise to the chemical shift at 732.3 p.p.m.

Territorial Distribution of Alcohol and Drug Addictions Mortality Concerning Regional Disparities in the Slovak Republic from Year 1996 to Year 2015

2020 ◽  
Keyword(s):  
Mortality Rate ◽  
Slovak Republic ◽  
Regional Disparities ◽  
Point Of View ◽  
Extreme Position ◽  
Data Set ◽  
Female Sex ◽  
Male Sex ◽  
The Individual ◽  
High Level

BACKGROUND: This paper deals with territorial distribution of the alcohol and drug addictions mortality at a level of the districts of the Slovak Republic. AIM: The aim of the paper is to explore the relations within the administrative territorial division of the Slovak Republic, that is, between the individual districts and hence, to reveal possibly hidden relation in alcohol and drug mortality. METHODS: The analysis is divided and executed into the two fragments – one belongs to the female sex, the other one belongs to the male sex. The standardised mortality rate is computed according to a sequence of the mathematical relations. The Euclidean distance is employed to compute the similarity within each pair of a whole data set. The cluster analysis examines is performed. The clusters are created by means of the mutual distances of the districts. The data is collected from the database of the Statistical Office of the Slovak Republic for all the districts of the Slovak Republic. The covered time span begins in the year 1996 and ends in the year 2015. RESULTS: The most substantial point is that the Slovak Republic possesses the regional disparities in a field of mortality expressed by the standardised mortality rate computed particularly for the diagnoses assigned to the alcohol and drug addictions at a considerably high level. However, the female sex and the male sex have the different outcome. The Bratislava III District keeps absolutely the most extreme position. It forms an own cluster for the both sexes too. The Topoľčany District bears a similar extreme position from a point of view of the male sex. All the Bratislava districts keep their mutual notable dissimilarity. Contrariwise, evaluation of a development of the regional disparities among the districts looks like notably heterogeneously. CONCLUSIONS: There are considerable regional discrepancies throughout the districts of the Slovak Republic. Hence, it is necessary to create a common platform how to proceed with the solution of this issue.

Quantitative indicators of air traffic controllers’ attitude to the danger of errors

2020 ◽  
pp. 68-78
Author(s):  
O. M. Reva ◽  
V. V. Kamyshin ◽  
S. P. Borsuk ◽  
V. A. Shulhin ◽  
A. V. Nevynitsyn
Keyword(s):  
Human Factor ◽  
Differential Method ◽  
Air Traffic ◽  
Professional Activity ◽  
Aviation Accidents ◽  
Air Traffic Controllers ◽  
The Difference ◽  
Level Of Significance ◽  
The Individual ◽  
High Level

The negative and persistent impact of the human factor on the statistics of aviation accidents and serious incidents makes proactive studies of the attitude of “front line” aviation operators (air traffic controllers, flight crewmembers) to dangerous actions or professional conditions as a key component of the current paradigm of ICAO safety concept. This “attitude” is determined through the indicators of the influence of the human factor on decision-making, which also include the systems of preferences of air traffic controllers on the indicators and characteristics of professional activity, illustrating both the individual perception of potential risks and dangers, and the peculiarities of generalized group thinking that have developed in a particular society. Preference systems are an ordered (ranked) series of n = 21 errors: from the most dangerous to the least dangerous and characterize only the danger preference of one error over another. The degree of this preference is determined only by the difference in the ranks of the errors and does not answer the question of how much time one error is more dangerous in relation to another. The differential method for identifying the comparative danger of errors, as well as the multistep technology for identifying and filtering out marginal opinions were applied. From the initial sample of m = 37 professional air traffic controllers, two subgroups mB=20 and mG=7 people were identified with statisti-cally significant at a high level of significance within the group consistency of opinions a = 1%. Nonpara-metric optimization of the corresponding group preference systems resulted in Kemeny’s medians, in which the related (middle) ranks were missing. Based on these medians, weighted coefficients of error hazards were determined by the mathematical prioritization method. It is substantiated that with the ac-cepted accuracy of calculations, the results obtained at the second iteration of this method are more ac-ceptable. The values of the error hazard coefficients, together with their ranks established in the preference systems, allow a more complete quantitative and qualitative analysis of the attitude of both individual air traffic controllers and their professional groups to hazardous actions or conditions.

scholarly journals Automatic Labeled Dialogue Generation for Nursing Record Systems

2020 ◽  
Vol 10 (3) ◽  
pp. 62
Author(s):  
Tittaya Mairittha ◽  
Nattaya Mairittha ◽  
Sozo Inoue
Keyword(s):  
Data Augmentation ◽  
Short Term Memory ◽  
Generative Models ◽  
Abstract Knowledge ◽  
Augmentation Techniques ◽  
Nursing Record ◽  
Long Short Term Memory ◽  
The Individual ◽  
High Level ◽  
Embedding Methods

The integration of digital voice assistants in nursing residences is becoming increasingly important to facilitate nursing productivity with documentation. A key idea behind this system is training natural language understanding (NLU) modules that enable the machine to classify the purpose of the user utterance (intent) and extract pieces of valuable information present in the utterance (entity). One of the main obstacles when creating robust NLU is the lack of sufficient labeled data, which generally relies on human labeling. This process is cost-intensive and time-consuming, particularly in the high-level nursing care domain, which requires abstract knowledge. In this paper, we propose an automatic dialogue labeling framework of NLU tasks, specifically for nursing record systems. First, we apply data augmentation techniques to create a collection of variant sample utterances. The individual evaluation result strongly shows a stratification rate, with regard to both fluency and accuracy in utterances. We also investigate the possibility of applying deep generative models for our augmented dataset. The preliminary character-based model based on long short-term memory (LSTM) obtains an accuracy of 90% and generates various reasonable texts with BLEU scores of 0.76. Secondly, we introduce an idea for intent and entity labeling by using feature embeddings and semantic similarity-based clustering. We also empirically evaluate different embedding methods for learning good representations that are most suitable to use with our data and clustering tasks. Experimental results show that fastText embeddings produce strong performances both for intent labeling and on entity labeling, which achieves an accuracy level of 0.79 and 0.78 f1-scores and 0.67 and 0.61 silhouette scores, respectively.

scholarly journals Opportunities and Challenges of Green-Lean: An Integrated System for Sustainable Construction

2020 ◽  
Vol 12 (11) ◽  
pp. 4460 ◽  
Author(s):  
Mohammadsoroush Tafazzoli ◽  
Ehsan Mousavi ◽  
Sharareh Kermanshachi
Keyword(s):  
Cost Savings ◽  
Integrated Approach ◽  
Integrated System ◽  
Waste Minimization ◽  
Sustainable Construction ◽  
Future Research ◽  
Safety Improvement ◽  
The Individual ◽  
High Level ◽  
Industry Professionals

Although the two concepts of lean and sustainable construction have been developed due to different incentives, and they do not pursue the same exact goals, there exists considerable commonality between them. This paper discusses the potentials for integrating the two approaches and their practices and how the resulting synergy from combining the two methods can potentially lead to higher levels of fulfilling the individual goals of each of them. Some limitations and challenges to implementing the integrated approach are also discussed. Based on a comprehensive review of existing papers related to sustainable and lean construction topics, the commonality between the two approaches is discussed and grouped in five categories of (1) cost savings, (2) waste minimization, (3) Jobsite safety improvement, (4) reduced energy consumption, and (5) customers’ satisfaction improvement. The challenges of this integration are similarly identified and discussed in the four main categories of (1) additional initial costs to the project, (2) difficulty of providing specialized expertise, (3) contractors’ unwillingness to adopt the additional requirements, and (4) challenges to establish a high level of teamwork. Industry professionals were then interviewed to rank the elements in each of the two categories of opportunities and challenges. The results of the study highlight how future research can pursue the development of a new Green-Lean approach by investing in the communalities and meeting the challenges of this integration.

scholarly journals Children’s Astronomy. Development of the Shape of the Earth Concept in Polish Children between 5 and 10 Years of Age

2021 ◽  
Vol 11 (2) ◽  
pp. 75
Author(s):  
Jan Amos Jelinek
Keyword(s):  
Mental Model ◽  
Cultural Influences ◽  
Test Results ◽  
The Earth ◽  
Spherical Earth ◽  
The Individual ◽  
High Level ◽  
Cognitive Problems ◽  
Teaching Situations ◽  
Model Approach

The Earth’s shape concept develops as consecutive cognitive problems (e.g., the location of people and trees on the spherical Earth) are gradually resolved. Establishing the order of problem solving may be important for the organisation of teaching situations. This study attempted to determine the sequence of problems to be resolved based on tasks included in the EARTH2 test. The study covered a group of 444 children between 5 and 10 years of age. It captured the order in which children solve cognitive problems on the way to constructing a science-like concept. The test results were compared with previous studies. The importance of cultural influences connected to significant differences (24%) in test results was emphasised. Attention was drawn to the problem of the consistency of the mental model approach highlighted in the literature. The analysis of the individual sets of answers provided a high level of consistency of indications referring to the same model (36%), emphasising the importance of the concept of mental models.

Bioinformatics analysis of bacteriophage and prophage endolysin domains

Biologia ◽  
2014 ◽  
Vol 69 (5) ◽  
Author(s):  
Barbora Vidová ◽  
Zuzana Šramková ◽  
Lenka Tišáková ◽  
Michaela Oravkinová ◽  
Andrej Godány
Keyword(s):  
Antimicrobial Properties ◽  
In Silico Analysis ◽  
Multiple Sequence ◽  
Catalytic Domains ◽  
Binding Domains ◽  
Multiresistant Bacteria ◽  
Or Gene ◽  
The Individual ◽  
Conserved Amino Acids ◽  
Sequence Types

AbstractEndolysins as a class of antibacterial enzymes are expected to become a very useful tool for many purposes to control spreading of, e.g., multiresistant bacteria in different environments. Their antimicrobial properties could be broadened or altered by mutagenesis, domain swapping or gene shuffling. Therefore, the specific designing of endolysins to achieve their desired properties is challenging. This work is focused on the in silico analysis of protein domains presence in sequences of phage and prophage endolysins, followed by the study of variety of domain combinations in the individual endolysin types. The multiple sequence alignment of endolysin sequences revealed the recognition of sequence types with typical domain arrangement and conserved amino acids, divided according to the target substrate in bacterial cell walls. The five protein families of catalytic domains are specifically occurring in dependence of bacterial Gram-type. The presence, types and numbers of binding domains within endolysin sequences were also studied. The obtained results enable a more targeted design of endolysins with required antimicrobial properties.

scholarly journals Structure-Function Analysis of the Endoplasmic Reticulum Oxidoreductase TMX3 Reveals Interdomain Stabilization of the N-terminal Redox-active Domain

2007 ◽  
Vol 282 (46) ◽  
pp. 33859-33867 ◽  
Author(s):  
Johannes Haugstetter ◽  
Michael Andreas Maurer ◽  
Thomas Blicher ◽  
Martin Pagac ◽  
Gerhard Wider ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Disulfide Isomerase ◽  
Function Analysis ◽  
Transmembrane Protein ◽  
Reduced Form ◽  
Disulfide Isomerase ◽  
Binding Domains ◽  
Redox Active ◽  
Protein Disulfide ◽  
Protein Disulfide Isomerase Family

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b′). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb′, we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb′, the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.

scholarly journals Healthy ageing, resilience and wellbeing

2017 ◽  
Vol 26 (6) ◽  
pp. 579-583 ◽  
Author(s):  
T. D. Cosco ◽  
K. Howse ◽  
C. Brayne
Keyword(s):  
Population Level ◽  
Healthy Ageing ◽  
Ageing Population ◽  
Environmental Resources ◽  
Great Achievement ◽  
Health Concept ◽  
Ageing Model ◽  
The Face ◽  
The Individual ◽  
High Level

The extension of life does not appear to be slowing, representing a great achievement for mankind as well as a challenge for ageing populations. As we move towards an increasingly older population we will need to find novel ways for individuals to make the best of the challenges they face, as the likelihood of encountering some form of adversity increases with age. Resilience theories share a common idea that individuals who manage to navigate adversity and maintain high levels of functioning demonstrate resilience. Traditional models of healthy ageing suggest that having a high level of functioning across a number of domains is a requirement. The addition of adversity to the healthy ageing model via resilience makes this concept much more accessible and more amenable to the ageing population. Through asset-based approaches, such as the invoking of individual, social and environmental resources, it is hoped that greater resilience can be fostered at a population level. Interventions aimed at fostering greater resilience may take many forms; however, there is great potential to increase social and environmental resources through public policy interventions. The wellbeing of the individual must be the focus of these efforts; quality of life is an integral component to the enjoyment of additional years and should not be overlooked. Therefore, it will become increasingly important to use resilience as a public health concept and to intervene through policy to foster greater resilience by increasing resources available to older people. Fostering wellbeing in the face of increasing adversity has significant implications for ageing individuals and society as a whole.

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