1,721,036 research outputs found
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Full text link“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
Full text linkWe 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
Full text linkWe 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
Localization matters : new insights into spatio-temporal regulation of type VI secretion systems
Full text linkSubcellular organization is important for bacterial cell physiology. Especially, bacterial secretion systems are tightly regulated in a temporal and spatial manner to efficiently fulfill their function. Among them, the contact-dependent Type VI secretion (T6SS) has an important role in inter-bacterial competitions and pathogenicity of Gram-negative bacteria.
T6SS translocates effector proteins into target cells using the contraction of a long cytosolic sheath, which pushes an inner tube together with a sharp tip and associated effectors across target cell membranes. This mode of action allows bacteria to use T6SS against a broad range of prokaryotic and eukaryotic organisms. However, the contact-dependency limits the target range and the efficiency of effector translocation is low because only a small number of effectors can be delivered per one round of T6SS assembly.
Recent advances in live-cell fluorescence and super resolution microscopy led to the revelation that T6SS activation patterns and dynamics are surprisingly diverse in different bacteria. These differences in T6SS assembly dynamics likely reflect different strategies to overcome the disadvantages of T6SS mode of action. However, the spatio-temporal regulation behind these different T6SS firing patterns are not well understood.
My PhD thesis provides new insights into how different subcellular localizations of T6SS assembly are achieved.
The Threonine phosphorylation pathway (TPP) is a unique posttranslational regulation mechanism, which allows Pseudomonas aeruginosa to activate its T6SS apparatus in response to membrane damage inflicted by an attack from neighboring bacteria and to localize it to the site of attack. While the involved components are identified, it is not clear how the periplasmic sensor module integrates spatial and temporal information for precise and fast T6SS assembly initiation. To test if relocation of TPP components from outer to inner membrane (IM) is important for T6SS activation, I changed their subcellular localization by mutating their N-terminal signal sequences. Relocation of one TPP component to IM indeed hyper-activated T6SS assembly, however, the exact mechanism of T6SS localization remains to be elucidated.
In collaboration with Prof. Kevin Foster, University of Oxford, we tested the benefit and cost of TPP-dependent localization of T6SS during bacterial competitions. Our results from in silico and imaging experiments suggested that P. aeruginosa uses TPP to kill competing bacteria by localized and repeated T6SS assemblies and thus inflicting more damage than it encounters from attacking competitors.
In collaboration with Prof. Petr Broz, University of Lausanne, we characterized the unique Francisella pathogenicity island (FPI), which encodes a non-canonical T6SS essential for phagosomal escape. The FPI lacks a specialized unfoldase required for recycling of contracted sheaths and for dynamics of canonical T6SS. Furthermore, the FPI encodes genes with unknown function. By live-cell fluorescence microscopy, we showed that F. novicida T6SS dynamics is comparable to canonical T6SS dynamics. Moreover, we found that general-purpose unfoldase ClpB recycles contracted sheaths and is essential for phagosomal escape in vivo. By analyzing T6SS dynamics and virulence of single deletion mutants in vitro and in vivo, we could group FPI components with unknown function into structural components, which are required for T6SS function, and putative effectors, which are critical for virulence but not for T6SS assembly.
Moreover, we showed that F. novicida T6SS assembles exclusively at bacterial poles. This unique polar localization raised the question of how Francisella T6SS is localized to the poles and whether it is important for T6SS function. I analyzed the dynamics of membrane complex formation, which is the first step of T6SS assembly, by live-cell fluorescence microscopy and structured illumination microscopy. I showed that the membrane complex is stably formed on the poles even in the absence of other FPI components. In addition, the membrane complex formation was insufficient to initiate sheath assembly indicating that additional signals are required to activate T6SS in F. novicida.
To investigate the contribution of FPI components and localization of T6SS to Francisella virulence in more detail, I established Galleria mellonella larvae as infection model. Besides, I constructed two expression plasmids for F. novicida, which are mobilized by conjugation and have tetracyline inducible promoters for tunable gene expression. These new tools will be invaluable in the future research of mechanism required for F. novicida pathogenesis
Oligomeric structures of metabolic protein assemblies - Structure-function analysis of acetyl-CoA carboxylase and urease
Full text linkOligomeric protein assemblies play a pivotal role in metabolism. The advantages of such complexes are increased stability, protection from degradation and additional options for regulation through allostery. The two enzymes discussed in this thesis utilize oligomerization as a central mechanism, to shield from damaging conditions and control their activity.
Urease is a nickel-metalloenzyme with varying assembly structures expressed in all branches of life except animals and is an essential part of the nitrogen cycle. It catalyzes the breakdown of urea into ammonia, which is used as a nitrogen source. Oligomerization of urease leads to the local increase of ammonia in a concentrated area and contributes to urease stability in extreme environments. Ureases have a huge impact on agricultural practices, but their function as virulence factors in pathogens also makes them important drug targets.
Acetyl-CoA Carboxylase (ACC) catalyzes the first and rate limiting step in the production of fatty acids. Acetyl-CoA is carboxylated by ACC in two consecutive and distinct reactions, forming malonyl-CoA. Fatty acid synthase (FAS) uses malonyl-CoA as a precursor for the formation of fatty acids. Eukaryotic ACCs are multienzymes, which contain all the catalytic domains in a single polypeptide chain. Human ACC is a dimer and oligomerizes into a filament in its most active state. Upregulation of ACC activity has been linked to cancer, metabolic syndrome and viral infections.
The aim of this thesis is the investigation of the mechanisms leading to oligomerization of these essential enzymes by using cryo-electron microscopy (cryo-EM).
In chapter two, the nickel –metalloenzyme urease of Yersinia enterocolitica is revealed to form a tetramer-of-trimers. A similar assembly has only been observed for the pathogen Helicobacter pylori, where it is described as a crucial factor in survival of acidic environments. Including higher order aberration correction in data processing of cryoEM movies, a reconstruction of Y. enterocolitica urease at 1.98 Å resolution was obtained. The structural data revealed an oligomerization loop and a specific alpha-helix as central elements involved in the oligomerization. At better than 2.0 Å resolution, so far only reached by single-particle analysis for four others proteins, salt bridges, alternate conformations and protonation are visualized for the entire protein, and in particular the active site. The two nickel ions in the active site are at a distance of only 3.2 Å, much closer than in other urease active sites described so far by X-ray crystallography. Frame-wise analysis of cryoEM movies indicates the onset of radiation-damage in the environment of the Ni2+ ions during electron-beam exposure as the cause of a distorted overall bimetal center structure.
The mechanism of allosteric regulation of ACC is discussed in chapter three, uncovering the mechanism behind citrate-induced filament formation. Prior and newly collected cryoEM data of ACC filaments were processed using advanced algorithms. Corrections for higher order aberrations resulted in a 3.84 Å resolution reconstruction, which supported identification of a potential citrate interaction region. Site specific point mutants of residues in the suspected citrate binding pocket showed decreased activity in in vitro activity assays. Citrate acts on a pocket of positively charged residues in the central domain of the multienzyme. This central domain region has no catalytic properties, but was observed to function as a hinge where large conformational changes occur. Binding of citrate influences the conformational ensemble of the central domain region, creating an interface for other dimers of ACC to dock and form a filament. This filament locks ACC in an active conformation and is the most active state of the protein.
The findings of this thesis show how different proteins employ oligomerization to preserve or augment their function. Ureases grant protection from acidic environments through their assembly while citrate-induced filament formation increases ACC activity
Cohesive properties of the caulobacter crescentus holdfast adhesin are regulated by a novel c-di-GMP effector protein
Full text linkMany bacteria grow in aquatic environments such as oceans, lakes and rivers. These environments pose special challenges, as nutrient availability is poor and hard to exploit due to constant water flow. Therefore, most bacteria grow on surfaces and have developed mechanisms that anchor them on an adequate nutrient source in the moment of encounter. Yet, they need to effectively disperse when conditions become unfavorable. The switch between motile and sessile lifestyles is controlled by the global second messenger c-di-GMP, which suppresses motility and promotes sessility. Here, we use the aquatic bacterium Caulobacter crescentus as a model to dissect c-di-GMP mediated lifestyle changes and surface attachment. C. crescentus has a biphasic life cycle that comprises both a motile and a sessile phase. A motile swarmer cell can develop into a sessile surface attached stalked cell by expressing a polar exopolysaccharide-based structure called the holdfast that shows remarkably strong adhesive properties. Recent studies have shown that holdfast biogenesis requires c-di-GMP and is initiated either upon surface contact or as part of the developmental program. However, the underlying molecular mechanisms are not understood.
In an unbiased screen for c-di-GMP binding proteins in C. crescentus we identified a novel c-di-GMP effector of unknown function and confirmed specific binding to c-di-GMP in vitro. Orthologous genes of this effector were found in different phyla and showed a strong association with exopolysaccharide synthesis genes. Its importance in holdfast biogenesis could be experimentally confirmed and the protein was named HfsK following the terminology used for holdfast synthesis proteins. Cells deficient in HfsK produced holdfast but could not retain the adhesin on the cell envelope, resulting in a strong surface adhesion defect and failure in surface colonization. Furthermore, a strong deformation of the mutant holdfast was observed when it was subject to shear stress, indicating a decrease in holdfast cohesion forces. HfsK is a member of an uncharacterized subclass of Gcn5-related-N-acetyltransferases, a diverse enzyme family that could potentially acylate the holdfast exopolysaccharide. Mutations in genes encoding holdfast anchor proteins that are predicted to form membrane-anchored filaments exhibited a similar phenotype. Based on our findings we propose a model in which the anchor filaments and the holdfast form a strong interaction network that relies on HfsK-mediated chemical modification of the holdfast exopolysaccharide.
In accordance with its proposed function in holdfast biogenesis, HfsK predominately localized to the cell membrane. The protein delocalized into the cytosol for a short period during the cell cycle, coinciding with holdfast biogenesis and with an upshift of cellular c-di-GMP levels. HfsK mutants impaired in c-di-GMP binding remained membrane associated throughout the cell cycle indicating that c-di-GMP binding controls the localization of this protein. A role in HfsK control could be attributed to a short stretch of amino acids at the C-terminus. This part of the protein is involved in c-di-GMP binding and is required for HfsK localization and activity. Functional analysis revealed a clear correlation between HfsK activity and subcellular localization. HfsK mutants that remained membrane-associated were mostly found to be active, while variants exclusively localizing to the cytosol failed to support proper holdfast formation. We propose that c-di-GMP binding to the C-terminus of HfsK leads to its delocalization and concomitant inactivation.
Finally, we show that overexpression of the glycosyltransferase HfsJ restored holdfast biogenesis in a strain lacking c-di-GMP. This exposed HfsJ as catalyst of the rate-limiting step of holdfast biogenesis when c-di-GMP levels are low. Together with recent findings that HfsJ directly binds c-di-GMP this suggested furthermore that HfsJ is the main effector through which holdfast synthesis is activated upon a cellular upshift of c-di-GMP.
This work establishes two pathways through which c-di-GMP can activate and possibly modulate the holdfast adhesin of C. crescentus and provides novel insight into the basis of the holdfast strong adhesive properties
A calcium-responsive kinase induces the acute-to-chronic lifestyle switch in "Pseudomonas aeruginosa"
Full text linkVirulence of pathogenic bacteria is a highly controlled process to facilitate invasion and survival in host tissues. While many of the regulatory pathways controlling virulence behavior have been defined in detail, information about host signals modulating these processes is still scarce. The opportunistic pathogen Pseudomonas aeruginosa causes acute and chronic infections in humans and is the leading cause of morbidity in cystic fibrosis (CF) patients. Disease progression is typically associated with the loss of acute virulence and the emergence of biofilms and chronic behavior. This acute-to-chronic switch is governed by the global Gac/Rsm pathway, which assembles around the central Gac two-component system and several associated sensor histidine kinases. The signal(s) and mechanisms involved in Gac/Rsm activation and induction of chronic behavior are not known. Using a newly developed Gac/Rsm-responsive dual reporter system we show that calcium stimulates the signaling cascade via the action of the Gac-associated hybrid histidine kinase LadS. Specific activation of LadS by calcium ions depends on its histidine kinase activity and on the periplasmic DISMED2 sensor domain. Our data suggest that calcium sensing by LadS evolved as an adaptation to the opportunistic behavior of P. aeruginosa. Consistent with a role for LadS in chronic infections, most clinical isolates from CF airways remain calcium-responsive during decades of evolution in the human host. We propose that elevated calcium levels in host tissue acts as trigger to induce the acute-to-chronic virulence switch during persistent P. aeruginosa infections. By establishing calcium signaling in host-pathogen interaction these results add to a growing body of evidence indicating that calcium signaling plays an important role not only in eukaryotic but also in prokaryotic cells
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