1,721,012 research outputs found
Study of the mechanism of action of botulinum neurotoxins to develop inhibitors and to improve their pharmacological application
Full text linkSeven antigenically different botulinum neurotoxin types (BoNT/A through /G) and many subtypes (BoNT/A1, BoNT/A2, etc.) constitute a growing family of bacterial exotoxins that specifically paralyze the cholinergic peripheral nerve terminals of vertebrates. Most notably, BoNTs intoxicate the neuromuscular junction, thereby causing a severe neuro-muscular paralysis known as botulism.
Despite the heterogeneity of their primary sequence, BoNTs are structurally and functionally conserved and composed of a 50 kDa light chain (L) and a 100 kDa heavy chain (H), linked via a unique, and fundamental, disulphide bridge. The C-terminal (HC, 50 kDa) and the N-terminal halves (HN, 50 kDa) of H constitute a sophisticated nanomolecular machine that mediates both the neurospecific binding of the molecule to peripheral nerve endings and the delivery of L into the neuronal cytosol. L is a zinc-dependent protease that specifically cleaves SNARE proteins (SNAP-25 (synaptosomal-associated protein of 25 kDa), VAMP (vesicle-associated membrane protein) and Stx (syntaxin)), the three proteins that form the SNARE complex which is the core of the nanomachine that mediates the fusion of synaptic vesicles (SV) with the presynaptic membrane, thus allowing neurotransmitter release. Cleavage causes impairment of SNARE complex assembly/function and thus a blockade of neuroexocytosis, which results in the flaccid paralysis typical of botulism. Patients can die for respiratory failure but, if vital functions are maintained by intensive care, they fully recover as the botulism neuroparalysis is completely reversible.
The mechanism of action of BoNTs can be conveniently divided into five fundamental steps: 1) binding to nerve terminals, 2) internalization by SV recycling, 3) pH-dependent translocation of L into the cytoplasm, 4) reduction of the interchain disulphide bond and 5) hydrolysis of SNARE proteins. BoNT/A and /E cleave SNAP-25, BoNT/B, /D, /F and /G cleave VAMP. BoNT/C is unique because it cleaves two substrates, SNAP-25 and syntaxins.
BoNTs are the most poisonous substances known to vertebrates, and are classified as potential biological weapons. Currently, the only treatment available consists in passive immunisation with antisera raised against the seven main toxin types. Unfortunately, antisera are variably reactive against subtypes and, moreover, no licensed vaccine are available for human use. This situation has promoted an intense research to develop new antitoxins.
At the same time, neurospecificity and reversibility of action make BoNTs the therapeutic of choice for the treatment of a heterogeneous number of human diseases characterized by the hyperactivity of peripheral nerve terminals.
Given this dichotomy of BoNTs, the aim of my PhD has been double: i) to develop pan-inhibitors that would prevent/treat botulism and ii) to better understand the toxin functioning in vivo to improve its use in human therapy.
i) BoNT’s intoxication strictly depends on the reduction of the interchain disulphide bond. Without it, L remains attached to H and cannot exert its catalytic activity. By using a pharmacological approach, I found that the Thioredoxin-Thioredoxin Reductase (Trx-TrxR) system is responsible for the reduction of all BoNT serotypes and that inhibitors of Trx-TrxR strongly reduce their neurotoxicity in vitro and in vivo in a model that recapitulates clinical botulism. These results are remarkable because they show for the first time that the different BoNTs can be inhibited by a single drug (a pan-inhibitor) by impacting on their common mechanism of action.
Following the same concept, the trafficking of BoNTs within the synaptic terminal represents another rational target to develop pan-inhibitors. Recently, it was reported that the chemical compound EGA inhibits pathogens or toxins that enter cells via acidic endosomes. Since also BoNTs have a similar requirement to trigger the translocation of L into the cytosol, I tested the activity of EGA and I found that it significantly inhibits the neurotoxic activity of BoNT/A, BoNT/B and BoNT/D in vitro and in vivo, tested because are serotypes frequently involved in human and animal botulism, respectively. Interestingly, none of the main steps underlying toxin’s cellular mechanism is directly affected by the drug. Rather, I provided indirect evidence that EGA interferes with the sorting of BoNTs inside nerve terminals, hampering their trafficking toward acidic compartments essential for L translocation.
Together, these studies show that BoNT’s activity can be significantly mitigated independently from their intertypic differences by using drugs targeting common steps of their mechanism of action. These inhibitors represent lead compounds for the development of new drugs against botulism.
ii) BoNTs are successful human therapeutic agents. Despite their use is almost invariably restricted to BoNT/A and BoNT/B, recent data on human volunteers suggest that BoNT/C can be used to treat non-responder individuals with similarly effective pharmacological outcomes. However, little is known about the mechanism by which BoNT/C paralyzes peripheral nerve terminals in vivo. In fact, at variance from all the other BoNTs, BoNT/C cleaves two substrates, SNAP-25 and syntaxin-1A/1B. Therefore, I undertook a study to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to BoNT/C activity in vivo. I took advantage from a recent publication where two triple-mutated BoNT/C L, L200W/M221W/I226W (BoNT/C α-3W) and S51T/R52N/N53P (BoNT/C α-51), were reported to cleave selectively syntaxins.
Thanks to a collaboration with Dr. T. Binz, I received the full-length BoNT/C mutants produced by recombinant methods and I tested their biochemical and toxicological properties. I found that both mutants cleave syntaxin with similar efficiency with respect to wild type BoNT/C (BoNT/C-wt), but unexpectedly, they maintain a residual activity on SNAP-25 which is higher for BoNT/C α-3W than for BoNT/C α-51. Interestingly, this different activity on SNAP-25 strictly correlates with the lethality of mutant toxins in vivo. At the same time, the proteolysis of syntaxin provides a substantial and prolonged neuromuscular impairment without the complete blockage of neurotransmission. These results suggest that SNAP-25 cleavage is the main determinant of BoNT/C neuroparalyzing activity and that BoNT/C derivatives with selective activity for syntaxins represent an appealing strategy to develop BoNTs endowed with long lasting activity and a wide safety margin
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
Electrophysiological Recordings of Evoked End-Plate Potential on Murine Neuro-muscular Synapse Preparations
Full text linkNeuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure and functionality. Electrophysiology is the most accurate experimental procedure to study NMJ physiology and it largely contributed to the elucidation of synaptic transmission basic principles. Many electrophysiological techniques have been developed to study NMJ physiology and physiopathology. In this paper, we describe an ex vivo tissue preparation for electrophysiology that can be applied to investigate nerve-muscle transmission functionality in mice. It is routinely used in our laboratory to study presynaptic neurotoxins, antitoxins, and to monitor NMJ degeneration and regeneration. This is a broadly applicable technique which can also be adopted to investigate alterations of NMJ activity in mouse models of neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes
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
Mouse Phrenic Nerve Hemidiaphragm Assay (MPN)
Full text linkThe neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their biochemical nature, the site they target (either the nerve or the muscle) and their mechanism of action, substances affecting NMJ produce very specific alterations of neuromuscular functionality. Here we provide a detailed protocol to isolate the diaphragmatic muscle from mice and to set up two autonomously innervated hemidiaphragms. This preparation can be used to study bioactive substances like toxins, venoms and neuroactive molecules of various origin, or to measure the force of skeletal muscle contraction. The 'mouse phrenic nerve hemidiaphragm assay' (MPN) is an established model of ex vivo NMJ and recapitulates the complexity of neuromuscular transmission in a system easy to control and to manipulate, thus representing a valuable tool to study both NMJ physiology and the mechanism of action of toxins and other molecules acting at this synapse
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