scholarly journals Evaluation of DNA Primase DnaG as a Potential Target for Antibiotics

2013 ◽  
Vol 58 (3) ◽  
pp. 1699-1706 ◽  
Author(s):  
Aneta Kuron ◽  
Malgorzata Korycka-Machala ◽  
Anna Brzostek ◽  
Marcin Nowosielski ◽  
Aidan Doherty ◽  
...  
Keyword(s):  
Standard Laboratory ◽  
Content Type ◽  
Growth And Survival ◽  
Inducible Promoters ◽  
Growth Defects ◽  
Laboratory Conditions ◽  
Recombination System ◽  
Conditional Mutant ◽  
Single Deletion ◽  
Low Levels

ABSTRACTMycobacteria contain genes for several DNA-dependent RNA primases, includingdnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. Anin silicoanalysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-typednaGcannot be deleted from the chromosome ofMycobacterium smegmatiswithout disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identifiedM. smegmatisAEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of nativednaGinM. smegmatiswas tolerated only after the integration of an extra intact copy of theM. smegmatisorMycobacterium tuberculosisdnaGgene, under the control of chemically inducible promoters, into theattBsite of the chromosome.M. tuberculosisandM. smegmatisDnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably,M. smegmatisbacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target.

scholarly journals Evaluation of NAD+-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

2007 ◽  
Vol 51 (8) ◽  
pp. 2888-2897 ◽  
Author(s):  
Malgorzata Korycka-Machala ◽  
Ewelina Rychta ◽  
Anna Brzostek ◽  
Heather R. Sayer ◽  
Anna Rumijowska-Galewicz ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Bacteriophage T4 ◽  
Dna Ligase ◽  
Extra Copy ◽  
Dna Ligases ◽  
Inducible Promoters ◽  
Recombination System ◽  
Conditional Mutant ◽  
Mutant Strains ◽  
Low Levels

ABSTRACT Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD+-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD+-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD+-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

scholarly journals Gene Essentiality Analyzed byIn VivoTransposon Mutagenesis and Machine Learning in a Stable Haploid Isolate ofCandida albicans

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Ella Shtifman Segal ◽  
Vladimir Gritsenko ◽  
Anton Levitan ◽  
Bhawna Yadav ◽  
Naama Dror ◽  
...  
Keyword(s):  
Machine Learning ◽  
Candida Albicans ◽  
Fungal Pathogens ◽  
Essential Genes ◽  
Antifungal Drugs ◽  
Model Organisms ◽  
Standard Laboratory ◽  
Genome Database ◽  
Content Type ◽  
Laboratory Conditions

ABSTRACTKnowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungusCandida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicansessential genes, including 1,195 with high “essentiality confidence” scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential inC. albicanswere 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogensC. albicans,Aspergillus fumigatus,Cryptococcus neoformans, andHistoplasma capsulatumand highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies.IMPORTANCEComprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power ofin vivotransposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogenCandida albicansand to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicansgenes that are essential for growth under standard laboratory conditions. Among theseC. albicansessential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

When do job insecure employees adapt to change?

2019 ◽  
Vol 25 (3) ◽  
pp. 271-286 ◽  
Author(s):  
Kristi N. Lavigne ◽  
Victoria L. Whitaker ◽  
Dustin K. Jundt ◽  
Mindy K. Shoss
Keyword(s):  
Job Insecurity ◽  
Small Sample ◽  
Adaptive Performance ◽  
Work Behaviors ◽  
Content Type ◽  
Work Tasks ◽  
Main Effect ◽  
Low Levels ◽  
The Impact ◽  
The Relationship

Purpose The purpose of this paper is to examine the relationship between job insecurity and adaptive performance (AP), contingent on changes to core work tasks, which we position as a situational cue to employees regarding important work behaviors. Design/methodology/approach Employees and their supervisors were invited to participate in the study. Supervisors were asked to provide ratings of employees’ AP and changes to core tasks; employees reported on job insecurity. Findings As predicted, changes to core tasks moderated the relationship between job insecurity and AP. Job insecurity was negatively related to AP for those experiencing low levels of change, but was not related to AP for those experiencing high levels of change. Counter to expectations, no main effect of job insecurity was found. Research limitations/implications This study employed a fairly small sample of workers from two organizations, which could limit generalizability. Practical implications The study identifies changes to core tasks as a boundary condition for the job insecurity–AP relationship. Findings suggest that organizations may not observe deleterious consequences of job insecurity on AP when changes to core tasks are high. Originality/value Few researchers have examined boundary conditions of the impact of job insecurity on AP. Furthermore, inconsistent findings regarding the link between job insecurity and AP have emerged. This study fills the gap and expands upon previous research by examining changes to core tasks as a condition under which job insecurity does not pose an issue for AP.

Employee emotional displays in the extended service encounter

2019 ◽  
Vol 31 (1) ◽  
pp. 115-136 ◽  
Author(s):  
Magnus Soderlund ◽  
Hanna Berg
Keyword(s):  
Marketing Communications ◽  
Emotional Expressions ◽  
Service Firms ◽  
Still Images ◽  
Content Type ◽  
Emotional Displays ◽  
Service Setting ◽  
Low Levels ◽  
The Impact ◽  
Practical Implications

Purpose The purpose of this paper is to examine the impact of happiness expressed by service firm employees when they are depicted in marketing communications materials, such as printed ads and videos. Design/methodology/approach Two experiments were conducted in a fitness service setting, in which employee display of happiness was manipulated (low vs high). Findings Both experiments showed that expressions of high levels of happiness produced a more positive attitude toward the service employee than expressions of low levels of happiness. Moreover, the impact of the expression of happiness on the evaluation of the employee was mediated by several variables, which suggests that the influence of depicted employees’ emotional expressions can take several routes. Practical implications The results imply that service firms should not only be mindful about which specific employee they select for appearing in marketing communications materials, they should also pay attention to the emotional displays of selected employees. Originality/value The present study contributes to previous research by assessing a set of potential mediators to explain why displays of happiness influence consumers, and by examining these effects in a marketing communications setting in which the customer is exposed to still images or video-based representations of the employee. The present study also focuses explicitly on happiness rather than on smiles.

scholarly journals Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis

2010 ◽  
Vol 10 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Francisco J. Piña ◽  
Allyson F. O'Donnell ◽  
Silvere Pagant ◽  
Hai Lan Piao ◽  
John P. Miller ◽  
...  
Keyword(s):  
Environmental Stress ◽  
Complex Function ◽  
Content Type ◽  
Growth Defects ◽  
Proton Atpase ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Specific Proteins ◽  
A Subunit ◽  
Split Ubiquitin

ABSTRACT Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1 Δ hph2 Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71 Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1 Δ hph2 Δ and hph1 Δ hph2 Δ sec71 Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1 Δ hph2 Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

scholarly journals Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance

2016 ◽  
Vol 198 (7) ◽  
pp. 1087-1100 ◽  
Author(s):  
Gursonika Binepal ◽  
Kamal Gill ◽  
Paula Crowley ◽  
Martha Cordova ◽  
L. Jeannine Brady ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Streptococcus Mutans ◽  
Transport System ◽  
Biofilm Formation ◽  
Cellular Response ◽  
Pathogenic Bacteria ◽  
Transport Systems ◽  
Content Type ◽  
Growth And Survival ◽  
Dental Biofilm

ABSTRACTPotassium (K+) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K+and a variety of K+transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K+acquisition inStreptococcus mutansand the importance of K+homeostasis for its virulence attributes. TheS. mutansgenome harbors four putative K+transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K+cotransporter (GlnQHMP), and a channel-like K+transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K+] less than 5 mM eliminated biofilm formation inS. mutans. The functionality of the Trk2 system was confirmed by complementing anEscherichia coliTK2420 mutant strain, which resulted in significant K+accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K+-dependent cellular response ofS. mutansto environment stresses.IMPORTANCEBiofilm formation and stress tolerance are important virulence properties of caries-causingStreptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment ofS. mutans. K+is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K+transporters inS. mutans. We identified the most important system for K+homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K+for the activity of biofilm-forming enzymes, which explains why such high levels of K+would favor biofilm formation.

scholarly journals Coordination of K+Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

2013 ◽  
Vol 12 (5) ◽  
pp. 684-696 ◽  
Author(s):  
Alberto Rivetta ◽  
Kenneth E. Allen ◽  
Carolyn W. Slayman ◽  
Clifford L. Slayman
Keyword(s):  
Atp Hydrolysis ◽  
Model Organism ◽  
Carbon Starvation ◽  
Transmembrane Helices ◽  
Content Type ◽  
Biochemical Genetic ◽  
Low Levels ◽  
Electrophysiological Characterization ◽  
Low K ◽  
Potassium Limitation

ABSTRACTFungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organismNeurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the geneHAK1to be much more highly regulated than isTRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K+] and/or the coexpression ofHAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K+and by coexpression ofTRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ∼500:1,viz., 10- to 50-fold larger than that in K+- and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K+stress by means of progressively increasing transporter affinity for extracellular K+. The underlying observations are (iv) that K+influx via Trk1p remains nearly constant at ∼9 mM/h when extracellular K+is progressively depleted below 0.05 mM and (v) that K+influx via Hak1p remains at ∼3 mM/h when extracellular K+is depleted below 0.1 mM.

Resolution of brain edema in severe brain injury at controlled high and low intracranial pressures

1984 ◽  
Vol 61 (4) ◽  
pp. 707-712 ◽  
Author(s):  
Meihong Cao ◽  
He Lisheng ◽  
Sun Shouzheng
Keyword(s):  
Cerebrospinal Fluid ◽  
Brain Injury ◽  
Brain Edema ◽  
External Ventricular Drainage ◽  
Blue Dye ◽  
Severe Brain Injury ◽  
Content Type ◽  
Intracranial Pressures ◽  
Low Levels ◽  
Cerebrospinal Fluid Protein

✓ A series of 87 patients with severe brain injury were studied. Intracranial pressure (ICP) monitoring and external ventricular drainage were used to control ICP at high and low levels. Clearance of ytterbium-169-labeled diethylenetriaminepentaacetic acid (169Yb-DTPA), Evans blue dye, and ventricular cerebrospinal fluid protein was measured at the two ICP levels over consecutive periods of 4 hours to confirm clearance of brain edema. The results support the hypothesis that brain edema is in part absorbed in the cerebrospinal fluid via transventricular flow.

Directional predictability between returns and volume in cryptocurrencies markets

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Panos Fousekis ◽  
Vasilis Grigoriadis
Keyword(s):  
High Volume ◽  
Stationary Time Series ◽  
Temporal Association ◽  
Informational Efficiency ◽  
Content Type ◽  
Low Levels ◽  
The Cross ◽  
Trading Volumes ◽  
Directional Predictability ◽  
The Empirical Analysis

Purpose This paper aims to identify and quantify directional predictability between returns and volume in major cryptocurrencies markets. Design/methodology/approach The empirical analysis relies on the cross-quantilogram approach that allows one to assess the temporal (lag-lead) association between two stationary time series at different parts of their joint distribution. The data are daily prices and trading volumes from four markets (Bitcoin, Ethereum, Ripple and Litecoin). Findings Extreme returns either positive or negative tend to lead high volume levels. Low levels of trading activity have in general no information content about future returns; high levels, however, tend to precede extreme positive returns. Originality/value This is the first work that uses the cross-quantilogram approach to assess the temporal association between returns and volume in cryptocurrencies markets. The findings provide new insights about the informational efficiency of these markets and the traders’ strategies.

Comparing retail formats in an emerging market

2014 ◽  
Vol 6 (1) ◽  
pp. 48-69 ◽  
Author(s):  
Gordhan K. Saini ◽  
Arvind Sahay
Keyword(s):  
Purchase Intention ◽  
Emerging Market ◽  
Retail Store ◽  
Policy Implications ◽  
Content Type ◽  
Consumer Responses ◽  
Consumer Purchase Intention ◽  
Low Levels ◽  
Low Price Guarantee ◽  
Retail Formats

Purpose – This study aims to examine the importance of credit and low price guarantee (LPG) on consumer purchase intention across types of retail store formats in an emerging market context. Design/methodology/approach – A 2 (kirana/modern retail)×2 (high/low LPG)×2 (credit/no credit) experimental design was used for this study. A sample of 200 respondents was asked about their purchase intention for a newly introduced hypothetical toothpaste brand and six hypotheses were tested. Findings – Findings show that credit and level of LPG determine consumer's purchase intention across store formats. The presence of credit and high LPG increases the purchase intention; however, relatively importance of these two varies by type of store. The absence of credit at kirana store definitely reduces the buying intention, while same is not true for modern retail store, where level of LPG is more important than the credit. Interestingly, buyer is likely to discount high LPG for a month's credit offered by a kirana store. Practical implications – The study can help practitioners and scholars to understand consumer responses to credit and LPG in buying decisions, and subsequently in designing a better product offer at a particular store format in emerging markets. Originality/value – Important insights are provided about the consumer behavior resulting from the presence or absence of credit and high or low levels of LPG in an emerging market context. The study also has public policy implications in a country where FDI in retail is a hotly debated topic.

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