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Imaging the cell entry and activity of anthrax toxins
No full textAnthrax is a severe zoonosis, which may also affect humans, caused by pathogenic strains of the
Gram-positive, spore forming Bacillus anthracis. The bacterium secretes a three-components toxic
complex consisting of the protective antigen (PA), the lethal factor (LF) and the edema factor (EF).
These elements combine to form lethal toxin (LeTx: PA+LF) and edema toxin (EdTx: PA+EF). LF is a
zinc metalloprotease that cleaves mitogen-activated protein kinase kinases (MEKs), thereby
interfering with the MAPK cascade; EF is a calmodulin-activated adenylate cyclase, which catalyses
the formation of cAMP, thus altering cell signalling and tissue ion fluxes.
PA binds its specific cellular receptors, then it is proteolytically activated by cellular proteases and
self-associates into homo-oligomers capable of binding LF and EF. These complexes enter surface
rafts, are endocytosed and reach late endosomal compartments, whose acid luminal pH causes a
conformational change, which results in LF and EF release into the cell cytosol.
Here, the activity of anthrax toxins, in particular of EF, is monitored with fluorescent imaging
techniques and particular attention is focused on the cell entry and trafficking of anthrax toxins
using LF and EF chimerae C-terminally fused to the EGFP or mCherry fluorescent proteins.L’antrace è una zoonosi causata da Bacillus anthracis, un batterio Gram-positivo in grado di
formare spore. Il batterio è in grado di secernere tre proteine, chiamate antigene protettivo (PA),
fattore letale (LF) e fattore edematoso (EF), che si assemblano in maniera binaria per formare due
complessi tossici chiamati tossina letale (LeTx: PA+LF) e tossina edematosa (EdTx: PA+EF) . LF è
una zinco-metalloproteasi che taglia la maggior parte delle isoforme delle mitogen-activated
protein kinase kinases (MEKs), interferendo con questa importante via di segnale intracellulare; EF,
invece, è una adenilato ciclasi calmodulina dipendente che catalizza un forte aumento di cAMP,
importante secondo messaggero, portando ad una alterazione dell’equilibrio osmotico della
cellula.
PA, dopo essersi legato a specifici recettori cellulari, viene tagliato da proteasi cellulari, ciò ne
promuove l’attivazione e porta all’assemblamento di omo-oligomeri in grado di legare LF ed EF. Il
complesso viene quindi internalizzato attraverso il processo di endocitosi e raggiunge i
compartimenti tardivi, il cui pH acido favorisce un riarrangiamento conformazionale che porta alla
traslocazione delle subunità catalitiche nel citoplasma cellulare.
In questa tesi, l’attività delle tossine, in particolare di EF, è monitorata con diverse tecniche di
microscopia e particolare attenzione è rivolta al processo di entrata e trafficking nella cellula
facendo uso di chimere di LF ed EF fuse al C-terminale con proteine fluorescenti
Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras
No full textTo investigate the cell entry and intracellular trafficking of anthrax oedema
factor (EF) and lethal factor (LF), they were C-terminally fused to the enhanced
green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent
proteins. Both chimeras bound to the surface of BHK cells treated with protective
antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and
the two anthrax factors were found to traffic along the same endocytic route and
with identical kinetics, indicating that their intracellular path is essentially
dictated by PA. Colocalization studies indicated that anthrax toxins enter
caveolin-1 containing compartments and then endosomes marked by
phoshatidylinositol 3-phoshate and Rab5, but not by early endosome antigen 1 and
transferrin. After 40 min, both EF and LF chimeras were observed to localize
within late compartments. Eventually, LF and EF appeared in the cytosol with a
time-course consistent with translocation from late endosomes. Only the EGFP
derivatives reached the cytosol because they are translocated by the PA channel,
while the mCherry derivatives are not. This difference is attributed to a higher
resistance of mCherry to unfolding. After translocation, LF disperses in the
cytosol, while EF localizes on the cytosolic face of late endosomes
The first non Clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain
Full text linkThe genome of Weissella oryzae SG25T was recently sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. The typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains could be identified. The BoNT family of neurotoxins is rapidly growing, but this was the first indication of the possible expression of a BoNT toxin outside the Clostridium genus. We performed molecular modeling and dynamics simulations showing that the 50 kDa N-terminal domain folds very similarly to the metalloprotease domain of BoNT/B, whilst the binding part is different. However, neither the recombinant metalloprotease nor the binding domains showed cross-reactivity with the standard antisera that define the seven serotypes of BoNTs. We found that the purified Weissella metalloprotease cleaves VAMP at a single site untouched by the other VAMP-specific BoNTs. This site is a unique Trp-Trp peptide bond located within the juxtamembrane segment of VAMP which is essential for neurotransmitter release. Therefore, the present study identifies the first non-Clostridial BoNT-like metalloprotease that cleaves VAMP at a novel and relevant site and we propose to label it BoNT/Wo
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
Espressione, purificazione e caratterizzazioner del fattore letale di Bacillus anthracis fuso con Green Fluorescent Protein
Full text linkBacillus anthracis, è in grado di produrre diversi fattori di virulenza, fondamentali per la patogenesi e l'instaurarsi dell'infezione, i principali sono rappresentati da due tossine: il fattore edematoso (EF) ed il fattore letale (LF), insieme all'antigene protettivo (PA), vanno a combinarsi per formare la tossina edematosa e quella letale, rispettivamente.
In questo lavoro, in particolare, ci siamo soffermati su quest'ultimo; ne abbiamo indotto l'espressione da parte di cellule batteriche come proteina di fusione con una variante fluorescente della green fluorescent protein, EGFP, abbiamo purificato la chimera e intossicato cellule in coltura con lo scopo ultimo di seguirne il processo di internalizzazione mediante microscopia a fluorescenza.ope
Envenomations by bothrops and crotalus snakes induce the release of mitochondrial alarmins
Full text linkSkeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as 'danger' signals. These are known as 'alarmins', and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different time-course of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial 'alarmins' might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations
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
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