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EcsAB ATP-binding kassett (ABC) transporter sin funksjon i stressrespons hos Staphylococcus haemolyticus
Get PDFStaphylococcus haemolyticus er et økende problem innen humanmedisin hvor den ofte forårsaker hudinfeksjoner i mennesker med svekket immunforsvar. Samtidig gjør dens virulensegenskaper, som biofilmdannelse og antibiotikaresistens, behandling utfordrende. Derfor har alternative behandlingsmetoder som bakteriociner, små antimikrobielle peptider, blitt undersøkt om de kan bidra i å behandle infeksjoner av S. haemolyticus. Et nytt konstruert bakteriocin hybrid 1 (H1), er vist å være aktiv mot denne arten. Likevel har det blitt sett at resistente celler er utbredt når bakteriocinet testes in vitro, på grunn av celler med spontane mutasjoner i regulator av sigma-E protease (rseP) genet og/eller et gen kodende for en ABC-transporter kalt effekt på eksoproteiner, defekt i kompetanse og sporulering AB (ecsAB). Bakteriocinet H1 er vist å drepe målceller via en interaksjon med RseP, og EcsAB har en viktig men ukjent kobling til RseP. Genene for disse er vist å være viktige for stressrespons, virulens og biofilmdannelse i flere andre arter, men funksjonen i S. haemolyticus er ukjent.
I denne oppgaven var målet å få en økt forståelse for hvilken rolle EcsAB har i biofilmdannelse og stressrespons til S. haemolyticus, og som følge undersøke om H1- resistente mutanter har svekket levedyktighet. Dette ble gjort ved å undersøke og sammenligne fenotypiske forskjeller mellom villtype og mutanter med ikke funksjonelt EcsB. Virulensegenskaper, slik som motstandsevne mot ulike antimikrobielle midler og evne til å danne biofilm ble undersøkt. For å få en dypere innsikt i genregulering ble forskjeller i mRNA nivåer av ulike gener hos mutant og villtype undersøkt med og uten stress. Dette ble undersøkt ved å utsette cellene for bakteriocinet garvicin KS (GarKS) og undersøke genuttrykk av et utvalg sentrale gener med revers transkriptase-kvantitativ polymerasekjedereaksjon (RT-qPCR).
Resultatene viste at mutanter av S. haemolyticus med ikke fungerende EcsAB hadde en svekket evne til å danne biofilm sammenlignet med villtype og svakere motstandsevne mot GarKS. I tillegg viste RT-qPCR analysen signifikante forskjeller i uttrykk av DUF418 (protein med ukjent funksjon) og stafylokokk respiratorisk respons A (srrA), der villtype hadde en høyere oppregulering av disse genene under stress enn det sett for ecsB-mutanten. Dette kan tyde på at reguleringen av disse genene er sterkt knyttet til EcsB og at disse genene er viktig for normal stressregulering hos S. haemolyticus.Staphylococcus haemolyticus is an increasing problem in human medicine where it often causes skin infections in people with weakened immune systems. At the same time, its virulence factors such as biofilm formation and antibiotic resistance make treatment challenging. Therefore, alternative treatment options such as bacteriocins, small antimicrobial peptides, have been investigated to see if they can treat S. haemolyticus infections. A newly engineered bacteriocin hybrid 1 (H1) has been shown to be active against this species. Nevertheless, it has been observed that resistant cells are prevalent when the bacteriocin is tested in vitro due to cells with spontaneous mutations in the regulator of sigma-E protease (rseP) and/or an ABC-transporter named effect on exoproteins, defect in competence and sporulation AB (ecsAB). These genes have been shown to be important for stress response, virulence, and biofilm formation in several other species, but the functions in S. haemolyticus are still unknown.
In this thesis, the aim was to gain an increased understanding of the role EcsAB has in biofilm formation and stress response in S. haemolyticus, and as a result investigate whether H1- resistant mutants have impaired fitness. This was done by examining and comparing phenotypic differences between wild type and mutants with non-functional EcsB. Virulence properties, such as resistance to various antimicrobial agents and ability to form biofilm were investigated. To gain a deeper insight into gene regulation, differences in the mRNA level of various genes in mutant and wild type strains were investigated with and without stress. This was investigated by exposing the cells to the bacteriocin garvicin KS (GarKS) and examining gene expression levels of a selection of genes by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
The results showed that mutants of S. haemolyticus with non-functioning EcsAB had a weakened ability to form biofilm compared to wild type and weaker resistance to GarKS. In addition, RT-qPCR analysis showed significant differences in the expression of DUF418 and srrA, where wild type had a higher up-regulation of these genes under stressful conditions than seen for the ecsB mutant. This may indicate that the regulation of these genes is strongly linked to EcsB and that these genes are important for normal stress regulation in S. haemolyticus
Rollen til RseP i biofilmdannelse og stresstoleranse i Staphylococcus haemolyticus
Get PDFStaphylococcus haemolyticus er en bakterie som er en del av den vanlige hudmikrobiotaen til mennesker. Samtidig er den ofte en årsak til infeksjoner hos mennesker med nedsatt immunforsvar eller personer med medisinske implantater. Blant koagulasenegative stafylokokker er denne arten en av de som oftest isoleres fra kliniske blodprøver. I tillegg til sin evne til å danne biofilm er antibiotikaresistens stadig mer utbredt hos S. haemolyticus, noe som gjør behandling utfordrende. Derfor har bakteriociner blitt undersøkt som en mulig tilleggsbehandling og/eller alternativ til antibiotika i behandling av infeksjoner forårsaket av S. haemolyticus. Spesielt lovende er medlemmer av en familie bakteriociner kalt LsbB familien. Disse bakteriocinene har en enkel og liten struktur, og er derfor relativt enkle å modifisere og syntetisere. Ved hjelp av peptiddesign ble bakteriocinet hybrid 1 (H1) konstruert ved å kombinere sekvenser fra to forskjellige bakteriociner fra LsbB familien. Dette nye konstruerte bakteriocinet er vist å ha spesielt god antimikrobiell aktivitet mot S. haemolyticus.
Bakteriocinet H1 er tenkt å interagere med membranproteinet regulator av sigma-E protease (RseP) for å drepe målcellen, og en mutasjon i dette genet som endrer struktur og/eller interagerende aminosyrer gjør at bakteriocinet ikke lenger fungerer. Funksjonen til RseP i S. haemolyticus er ikke kjent, og det er sett at spontane mutasjoner i rseP genet lett kan oppstå. RseP er kjent å være involvert i blant annet stressrespons, virulens, og biofilmdannelse hos en rekke bakterier, og det antas derfor at resistensutvikling kommer på bekostning av levedyktighet og virulens.
Formålet med dette studiet var å undersøke om S. haemolyticus uten RseP, som er resistente mot H1, hadde redusert motstandsdyktighet, i form av sensitivitet mot ulike kjemiske stoffer og antibiotika. Det ble også undersøkt forskjeller i evnen til å danne biofilm. For å få bedre innsikt i de molekylære mekanismene bak forskjellene ble genuttrykk mellom mutant og villtype av utvalgte gener målt med revers transkripsjon (RT) kvantitativ PCR (qPCR).
I arbeidet ble det isolert muterte stammer av S. haemolyticus resistente mot H1, og sekvensering av disse viste at alle hadde mutasjoner knyttet til rseP. Fenotypiske tester viste at rseP mutantene hadde lavere motstandsdyktighet mot stress og betydelig svekket evne til å danne biofilm. Analyse av genuttrykk viste at villtypen kan respondere på stressende forhold ved å oppregulere srrA og nedregulerer ecsB, noe som ikke ble sett hos rseP mutanten. Det ble også sett at genuttrykk var høyere for mutanten enn det sett for villtypen, noe som tyder på at normalt utrykk av disse genene avhenger av funksjonelt RseP. Disse funnene gir verdifull innsikt i bruken av bakteriocinet H1 til behandling av infeksjoner forårsaket av S. haemolyticus, samt konsekvensene av resistente celler. Videre kan studien bidra til en dypere forståelse av de mekanismene som reguleres av RseP, og hvordan proteinet kan brukes som et antimikrobielt mål. Dette vil kunne ha betydning for utviklingen av nye og mer effektive behandlingsmetoder for infeksjoner forårsaket av S. haemolyticus.Staphylococcus haemolyticus is a commensal bacterium that is part of the normal skin microbiota of humans. At the same time, it is often a cause of infections in people with weakened immune systems or people with medical implants. Among coagulase-negative staphylococci, this species is one of those most frequently from clinical blood samples. In addition to its ability to form biofilms, antibiotic resistance is becoming more widespread in S. haemolyticus, which makes treatment challenging. Therefore, bacteriocins have been investigated as a possible adjunctive treatment and/or alternative to antibiotics for the treatment of infections caused by S. haemolyticus. Particularly promising are members of a family of bacteriocins called the LsbB family. These bacteriocins are small, have a simple structure and are therefore relatively easy to modify and synthesize. Using peptide design, the bacteriocin hybrid 1 (H1) was constructed by combining sequences from two different bacteriocins from the LsbB family. This new engineered bacteriocin has been shown to have particularly good antimicrobial activity against S. haemolyticus.
The bacteriocin H1 has been thought to interact with the membrane protein regulator of sigma-E protease (RseP) to kill the target cell, and mutations in this gene that change the structure and/or interacting amino acids render cells resistant to the bacteriocin. The function of RseP in S. haemolyticus is not well understood, and it has been observed that spontaneous mutants of rseP can arise easily. RseP is known to be involved in, among other functions, stress response, virulence, and biofilm formation in several species, and it is therefore likely that resistance carry a fitness cost, such as reduced fitness and virulence.
The aim of this study was to investigate whether S. haemolyticus with mutated RseP, which is resistant to H1, had increased susceptibility to various chemical substances and antibiotics. Differences in the ability to form biofilms were also investigated. To gain better insight into the molecular mechanisms behind the differences, gene expression between mutant and wild type of selected genes was measured by reverse transcroption quantitative PCR.
In the work, S. haemolyticus strains resistant to H1 were isolated, and sequencing of these showed that all had a mutation associated with rseP. Phenotypic tests showed that rseP mutants had lower resistance to stress and a significantly reduced ability to form biofilm. Analysis of gene expression showed that the wild type can respond to stressful conditions by upregulating srrA and downregulating ecsB, which was not observed for the rseP mutant. It was also noted that gene expression was higher for the mutant than that seen for the wild type, which suggests that normal expression of these genes depends on functional RseP. These findings provide valuable insight into the use of the bacteriocin H1 in the treatment of infections caused by S. haemolyticus, as well as the consequences of resistant cells. Further, this study contributes to a deeper understanding of the mechanisms regulated by RseP, and how the protein can be used as an antimicrobial target. This could have an impact on the development of new and more effective treatment methods for infections caused by S. haemolyticus
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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