scholarly journals Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism

2020 ◽  
Vol 86 (11) ◽  
Author(s):  
Salome C. Nies ◽  
Robert Dinger ◽  
Yan Chen ◽  
Gossa G. Wordofa ◽  
Mette Kristensen ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Large Scale ◽  
Nadh Dehydrogenase ◽  
Metabolic Flexibility ◽  
Second Phase ◽  
Content Type ◽  
Depth Analysis ◽  
Nadh Dehydrogenases ◽  
The Impact

ABSTRACT Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. IMPORTANCE While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

scholarly journals The metabolic response ofPseudomonas taiwanensisto NADH dehydrogenase deficiency

2019 ◽  
Author(s):  
Salome C. Nies ◽  
Robert Dinger ◽  
Yan Chen ◽  
Gossa G. Wordofa ◽  
Mette Kristensen ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Large Scale ◽  
Nadh Dehydrogenase ◽  
Metabolic Response ◽  
Metabolic Flexibility ◽  
Second Phase ◽  
Type I ◽  
Depth Analysis ◽  
Nadh Dehydrogenases ◽  
The Impact

AbstractObligate aerobic organisms rely on a functional electron transport chain for energy generation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions.We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobePseudomonas taiwanensisVLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the generated single or double knockout strains displayed none or only a marginal phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants including quantitative physiological experiments, transcript analysis, proteomics and enzyme activity assays revealed distinct responses to type II and type I dehydrogenase deletions. An overall high metabolic flexibility enablesP. taiwanensisto cope with the introduced genetic perturbations and maintain stable phenotypes by rerouting of metabolic fluxes.This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate metabolic engineering efforts.ImportanceWhilePseudomonashas the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and consequently competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants ofPseudomonas taiwanensisVLB120 presented here, provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. The observed great metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse towards a host with controlled and efficient supply of redox cofactors for product synthesis.

scholarly journals Type 2 NADH Dehydrogenase Is the Only Point of Entry for Electrons into theStreptococcus agalactiaeRespiratory Chain and Is a Potential Drug Target

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Andrea M. Lencina ◽  
Thierry Franza ◽  
Matthew J. Sullivan ◽  
Glen C. Ulett ◽  
Deepak S. Ipe ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Drug Target ◽  
Nadh Dehydrogenase ◽  
Potential Drug Target ◽  
Potential Drug ◽  
Intact Cells ◽  
Content Type ◽  
Oxygen Reductase ◽  
Tract Infections

ABSTRACTThe opportunistic pathogenStreptococcus agalactiaeis the major cause of meningitis and sepsis in a newborn’s first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain ofS. agalactiaeconsists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochromebdoxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. AlthoughS. agalactiaegrows well invitroby fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study,S. agalactiaeNDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration inS. agalactiaeand, since it has few side effects, it might be considered a lead compound for therapeutics development.IMPORTANCES. agalactiaeis part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish thatS. agalactiaeutilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochromebdoxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution toS. agalactiaevirulence and propose it as a potential drug target.

scholarly journals Synchronization in 5G networks: a hybrid Bayesian approach toward clock offset/skew estimation and its impact on localization

2021 ◽  
Vol 2021 (1) ◽  
Author(s):  
Meysam Goodarzi ◽  
Darko Cvetkovski ◽  
Nebojsa Maletic ◽  
Jesús Gutiérrez ◽  
Eckhard Grass
Keyword(s):  
Bayesian Approach ◽  
Large Scale ◽  
Hybrid Approach ◽  
Position Estimation ◽  
5G Networks ◽  
Clock Skew ◽  
Depth Analysis ◽  
The Impact ◽  
Mean Square Errors ◽  
Clock Offset

AbstractClock synchronization has always been a major challenge when designing wireless networks. This work focuses on tackling the time synchronization problem in 5G networks by adopting a hybrid Bayesian approach for clock offset and skew estimation. Furthermore, we provide an in-depth analysis of the impact of the proposed approach on a synchronization-sensitive service, i.e., localization. Specifically, we expose the substantial benefit of belief propagation (BP) running on factor graphs (FGs) in achieving precise network-wide synchronization. Moreover, we take advantage of Bayesian recursive filtering (BRF) to mitigate the time-stamping error in pairwise synchronization. Finally, we reveal the merit of hybrid synchronization by dividing a large-scale network into local synchronization domains and applying the most suitable synchronization algorithm (BP- or BRF-based) on each domain. The performance of the hybrid approach is then evaluated in terms of the root mean square errors (RMSEs) of the clock offset, clock skew, and the position estimation. According to the simulations, in spite of the simplifications in the hybrid approach, RMSEs of clock offset, clock skew, and position estimation remain below 10 ns, 1 ppm, and 1.5 m, respectively.

scholarly journals Potentiating Effect of Mandelate and Lactate on Chemically Induced Germination in Members of Bacillus cereus Sensu Lato

2017 ◽  
Vol 83 (24) ◽  
Author(s):  
Alistair H. Bishop
Keyword(s):  
Bacillus Cereus ◽  
Large Scale ◽  
Germination Rate ◽  
Dependent Manner ◽  
Clostridium Sporogenes ◽  
Specific Chemical ◽  
Content Type ◽  
Ph Range ◽  
Significant Discovery ◽  
The Impact

ABSTRACT Endospores of the genus Bacillus can be triggered to germinate by a limited number of chemicals. Mandelate had powerful additive effects on the levels and rates of germination produced in non-heat-shocked spores of Bacillus anthracis strain Sterne, Bacillus cereus, and Bacillus thuringiensis when combined with l-alanine and inosine. Mandelate had no germinant effect on its own but was active with these germinants in a dose-dependent manner at concentrations higher than 0.5 mM. The maximum rate and extent of germination were produced in B. anthracis by 100 mM l-alanine with 10 mM inosine; this was equaled by just 25% of these germinants when supplemented with 10 mM mandelate. Half the maximal germination rate was produced by 40% of the optimum germinant concentrations or 15% of them when supplemented with 0.8 mM mandelate. Germination rates in B. thuringiensis were highest around neutrality, but the potentiating effect of mandelate was maintained over a wider pH range than was germination with l-alanine and inosine alone. For all species, lactate also promoted germination in the presence of l-alanine and inosine; this was further increased by mandelate. Ammonium ions also enhanced l-alanine- and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant. Mandelate appeared to bind to spores while enhancing germination. There was no effect when mandelate was used in conjunction with nonnutrient germinants. No effect was produced with spores of Bacillus subtilis, Clostridium sporogenes, or C. difficile. IMPORTANCE The number of chemicals that can induce germination in the species related to Bacillus cereus has been defined for many years, and they conform to specific chemical types. Although not a germinant itself, mandelate has a structure that is different from these germination-active compounds, and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of B. cereus in foodborne disease and B. anthracis as a threat agent. The destruction of spores of B. anthracis, for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of l-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of “germinate to decontaminate” strategy may thus become more practicable.

Analytical redefinition of DOL and managerial investment decisions

2014 ◽  
Vol 40 (7) ◽  
pp. 734-754 ◽  
Author(s):  
Yoram Kroll ◽  
David Yechiam Aharon
Keyword(s):  
Large Scale ◽  
Investment Decision ◽  
Production Level ◽  
Fixed Cost ◽  
Theoretical Formulation ◽  
Optimal Production ◽  
Demand Shocks ◽  
Content Type ◽  
Operating Leverage ◽  
The Impact

Purpose – The purpose of this paper is to develop alternative analytical measures for the degree of operating leverage (DOL) that reflect the impact of uncertain demand shocks in the product's market on optimal production levels, sales and profits of the firm. Design/methodology/approach – The elasticity measures are constructed according to a theoretical formulation of optimal production level that corresponds to demand shocks for given predetermined levels of fixed cost. Findings – The paper suggests two main findings. First, the analytical marginal DOL is at least twice the traditional DOL depending on the structure of the shock, the production function and demand's elasticity. The traditional DOL is equal to the measure only when large-scale negative demand prompts the firm to abandon production. Second, the paper also provides an analytical measure of DOL in terms of elasticity of profit to sales rather than to production level. Both theoretically and empirically elasticity of profit to sales can be better measured and better reflects risk. Research limitations/implications – This paper should be extended to encompass multiple shocks on demand and supply while investigating the empirical multi variants distribution of the shocks. Practical implications – The model can be used by managers who are well informed about the fixed and variable costs of their firm. The model determines the mean profit- risk trade off which is an important factor in all investment decision problems. Originality/value – Surprisingly and according to the best knowledge, this paper is the first attempt in the literature for alternative analytical DOLs’ formulations that is coherent with basic economic theories of optimal production level under risk.

Buyer-assisted lean intervention in supplier firms: a supplier development approach

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Imranul Hoque
Keyword(s):  
Financial Incentives ◽  
Large Scale ◽  
Qualitative Data ◽  
Research Approach ◽  
Capability Development ◽  
Content Type ◽  
Production Capability ◽  
Lean Tools ◽  
The Right ◽  
The Impact

PurposeThis study aims to investigate how buyer-assisted lean intervention in garment supplier factories affects garment suppliers' productivity and production capability development.Design/methodology/approachEmploying a qualitative research approach and a lean intervention design, a multiple case study method was adopted for this study. Quantitative data on productivity performance and qualitative data on production capability development were collected from a Danish buyer and their four corresponding garment suppliers. Collected data were analysed using standard lean measurement tools and qualitative data analysis techniques.FindingsThis study demonstrates that buyer-assisted lean intervention is a useful strategy for garment suppliers to enhance their productivity and production capability. However, suppliers need to select the right lean tools, ensure seriousness and commitment to lean initiatives, substantial involvement of top management and workers, arrange formal and informal training, provide performance-based financial/non-financial incentives and nurture a learning culture to facilitate suppliers' production capability development.Research limitations/implicationsThis study implemented few lean tools in a single sewing line in four supplier factories for a short intervention duration. Thus, there is a scope for future studies to investigate the impact of the lean intervention on a large scale.Practical implicationsThe findings of this study might bring new insights to the management of buyer and supplier firms concerning how buyers could involve in suppliers' lean intervention initiatives and what suppliers need to ensure to develop production capability.Originality/valueFor the first time, this study engaged a buyer in suppliers' lean intervention initiatives to improve productivity and production capability in the garment industry of a developing country.

Trust, efficacy and ethicacy when testing prisoners for COVID-19

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Steve Lambert ◽  
Dean Wilkinson
Keyword(s):  
Large Scale ◽  
Current System ◽  
Epidemiological Studies ◽  
Careful Consideration ◽  
Power Relationships ◽  
England And Wales ◽  
Content Type ◽  
Large Scale Testing ◽  
The Uk ◽  
The Impact

Purpose The outbreak of the severe acute respiratory syndrome coronavirus 2 virus and subsequent COVID-19 illness has had a major impact on all levels of society internationally. The extent of the impact of COVID-19 on prison staff and prisoners in England and Wales is unknown. Testing for COVID-19 both asymptomatic and symptomatic, as well as for antibodies, to date, has been minimal. The purpose of this paper is to explore the widespread testing of COVID-19 in prisons poses philosophical and ethical questions around trust, efficacy and ethicacy. Design/methodology/approach This paper is both descriptive, providing an overview of the widespread testing of COVID-19 in prisoners in England and Wales, and conceptual in that it discusses and argues the issues associated with large-scale testing. This paper provides a discussion, using comparative studies, of the issues associated with large-scale testing of prisoners across the prison estate in England and Wales (120 prisons). The issues identified in this paper are contextualised through the lens of COVID-19, but they are equally transferrable to epidemiological studies of any pandemic. Given the prevalence of COVID-19 globally and the lack of information about its spread in prisons, at the time of writing this paper, there is a programme of asymptomatic testing of prisoners. However, there remains a paucity of data on the spread of COVID-19 in prisons because of the progress with the ongoing testing programme. Findings The authors argue that the widespread testing of prisoners requires careful consideration of the details regarding who is included in testing, how consent is gained and how tests are administered. This paper outlines and argues the importance of considering the complex nuance of power relationships within the prison system, among prisoner officers, medical staff and prisoners and the detrimental consequences. Practical implications The widespread testing of COVID-19 presents ethical and practical challenges. Careful planning is required when considering the ethics of who should be included in COVID-19 testing, how consent will be gained, who and how tests will be administered and very practical challenges around the recording and assigning of COVID-19 test kits inside the prison. The current system for the general population requires scanning of barcodes and registration using a mobile number; these facilities are not permitted inside a prison. Originality/value This paper looks at the issues associated with mass testing of prisoners for COVID-19. According to the authors’ knowledge, there has not been any research that looks at the issues of testing either in the UK or internationally. The literature available details countries’ responses to the pandemic rather and scientific papers on the development of vaccines. Therefore, this paper is an original review of some of the practicalities that need to be addressed to ensure that testing can be as successful as possible.

scholarly journals Measuring and assessing the impact of using volunteers in UK libraries: issues and methods

2014 ◽  
Vol 15 (3) ◽  
pp. 112-121 ◽  
Author(s):  
Judith Broady-Preston
Keyword(s):  
Systematic Review ◽  
Large Scale ◽  
Third Sector ◽  
Professional Staff ◽  
Research Centre ◽  
Content Type ◽  
Qualitative Approaches ◽  
Starting Point ◽  
Third Sector Organisations ◽  
The Impact

Purpose – The purpose of this paper is to examine a range of issues and methods in relation to measuring the impact of volunteer labour on the design and delivery of all types of library services. With the increasing use of volunteers to deliver library and information services in all sectors, managers need to assess their effectiveness and evaluate the impact of their use in relation to operational service design and delivery, and on the development of the profession and professional practice as a whole. Presented here is an initial scoping study, outlining a range of issues, methods and challenges for more detailed future investigation. Design/methodology/approach – A number of methodological challenges and perspectives are identifiable. Contemporary libraries exhibit increasing similarities with Third Sector organisations, namely a complex stakeholder community, and increasing use of volunteers to supplement or replace services delivered by professional staff. Therefore, a starting point for the research is a systematic review and analysis of the methodologies developed by the Third Sector Research Centre, and those studies in the ESRC contemporary Developing Impact Evaluation strand. As a rich picture is required, both quantitative and qualitative approaches are necessary, with the overall study adopting a mixed methods approach. Findings – This paper reports the findings of the preliminary documentary analysis, literature review and scoping aspects of a large-scale study. Originality/value – Research undertaken to date (June 2014) has failed to identify any published systematic review and examination of these issues.

scholarly journals Physiological relevance, localization and substrate specificity of the alternative (type II) mitochondrial NADH dehydrogenases of Ogataea parapolymorpha

2021 ◽  
Author(s):  
Hannes Juergens ◽  
Álvaro Mielgo-Gómez ◽  
Albert Godoy-Hernández ◽  
Jolanda ter Horst ◽  
Janine M. Nijenhuis ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Respiratory Chain ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Physiological Role ◽  
Proton Pumping ◽  
Type Ii ◽  
Respiratory Chains ◽  
Alternative Type ◽  
Nadh Dehydrogenases

AbstractMitochondria from Ogataea parapolymorpha harbor a branched electron-transport chain containing a proton-pumping Complex I NADH dehydrogenase and three alternative (type II) NADH dehydrogenases (NDH2s). To investigate the physiological role, localization and substrate specificity of these enzymes, growth of various NADH dehydrogenase mutants was quantitatively characterized in shake-flask and chemostat cultures, followed by oxygen-uptake experiments with isolated mitochondria. Furthermore, NAD(P)H:quinone oxidoreduction of the three NDH2s were individually assessed. Our findings show that the O. parapolymorpha respiratory chain contains an internal NADH-accepting NDH2 (Ndh2-1/OpNdi1), at least one external NAD(P)H-accepting enzyme and likely additional mechanisms for respiration-linked oxidation of cytosolic NADH. Metabolic regulation appears to prevent competition between OpNdi1 and Complex I for mitochondrial NADH. With the exception of OpNdi1, the respiratory chain of O. parapolymorpha exhibits metabolic redundancy and tolerates deletion of multiple NADH-dehydrogenase genes without compromising fully respiratory metabolism.ImportanceTo achieve high productivity and yields in microbial bioprocesses, efficient use of the energy substrate is essential. Organisms with branched respiratory chains can respire via the energy-efficient proton-pumping Complex I, or make use of alternative NADH dehydrogenases (NDH2s). The yeast Ogataea parapolymorpha contains three uncharacterized, putative NDH2s which were investigated in this work. We show that O. parapolymorpha contains at least one ‘internal’ NDH2, which provides an alternative to Complex I for mitochondrial NADH oxidation, albeit at a lower efficiency. The use of this NDH2 appeared to be limited to carbon excess conditions and the O. parapolymorpha respiratory chain tolerated multiple deletions without compromising respiratory metabolism, highlighting opportunities for metabolic (redox) engineering. By providing a more comprehensive understanding of the physiological role of NDH2s, including insights into their metabolic capacity, orientation and substrate specificity this study also extends our fundamental understanding of respiration in organisms with branched respiratory chains.

The influence of the structure of employees on sensory quality of restaurants' food

2014 ◽  
Vol 116 (3) ◽  
pp. 527-543 ◽  
Author(s):  
Dragan Tešanovic ◽  
Milovan Krasavcic ◽  
Bojana Miro Kalenjuk ◽  
Milijanko Portic ◽  
Snježana Gagic
Keyword(s):  
Sensory Quality ◽  
Second Phase ◽  
Content Type ◽  
Level Of Education ◽  
Third Phase ◽  
Practical Evaluation ◽  
Quality Of Food ◽  
The Impact ◽  
Practical Implications

Purpose – The aim of this paper is to determine the sensory quality of food in restaurants by professional food evaluators and to research the impact of education, age and number of employees on the quality of food. Design/methodology/approach – In the first phase five trained food tasters evaluated the sensory quality of food. In the second phase, the analysis of the structure of employees was done by establishing their level of education, age and number of employees. In the third phase the regression and correlation analysis was done with the aim to establish the impact of the level of education, age and number of employees on the sensory quality of food. Findings – The sensory evaluation has shown that the evaluated food is of moderate quality. Correlation matrix has shown that the education level of employees has a high impact on the sensory quality of food. There is a correlation between the number of employees, their age and their education. Practical implications – Obtained results are the indicators of the quality of food in restaurants in the region and they can serve for the improvement of quality. They have shown that education and staff training can contribute to a better quality of food. Established methodology can also contribute to the practical evaluation of quality. Originality/value – This paper is reflected on the specific application of methodology of the sensory analysis of food in restaurants. The paper pointed to the impact of employees on the sensory quality of food by statistical methods. Statistical results which point to the great impact of the level of education of employees on the sensory quality of food in restaurants are particularly valuable.

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