1,720,964 research outputs found
Ingegnerizzazione della proteina di matrice M1 del virus dell'influenza per la produzione di virus-like particles (VLPs) a scopo vaccinale
Full text linkInfluenza viruses represent a significant public health problem, since they are the etiological agents of acute respiratory illness that can occur in annual epidemics or in occasionally pandemic events. Influenza viruses constantly evolve by the accumulation of point mutations at the level of their antigenic determinants hemagglutinin and neuraminidase (antigenic drift), or as a result of a genetic reassortment (antigenic shift), usually happening when avian and human viruses co-infect an intermediate host. The high rate of mutations, the possibility of reassortment of gene segments and the wide variety of influenza viruses explain their ability to overcome the host immunity, acquired by previously vaccination (Wong et al., 2005). Moreover, the difficulty of developing vaccines capable of
inducing a long-lasting immune response leads to the need of up-dating the vaccine formulation every year (Wong et al., 2005). On the other hand, molecular evolution processes determine an increasing emergence of drug-resistant viruses that restrict the efficacy of the antiviral drugs. Indeed, vaccination remains the most effective preventative strategy to reduce the chance of infection from influenza virus and its complications. In this context, vaccines based on virus-like particles (VLPs) are extremely promising. In fact VLPs, while being antigenically identical to the infectious virus, lack pathogenicity. VLPs are obtained by expressing specific viral structural proteins, that display the ability to bud from cells, in the absence of any other viral component (such as retroviral Gag and\ud
papillomavirus L1) (Zeltins, 2013). VLPs can be used per se or as “platforms” for the presentation of immunodominant epitopes. In the case of influenza viruses, the matrix protein M1 represents the major driving force of viral budding process (Gomez-Puertas et al., 2000). However, the expression of M1 alone does not allow the production of VLPs. Among other reasons, this feature is due to the lack in M1 of a membrane targeting signal (Wang et al., 2010a). In this context, during
the viral infection, it is the second matrix protein, M2, which is charachterized by a targeting signal and can physically interact with M1, that brings the latest in contact with the plasma membrane (Chen et al., 2008). Moreover, M2 is also able to facilitate the curvature of the membrane and the scission of the envelope of the budding viral particle from the cell surface (Rossman et al., 2010), a function that is linked to the cellular complex ESCRT (endosomal sorting complex required for
transport)-III, in the case of most enveloped viruses (Calistri et al., 2009b). The overall aim of our work was to optimize the production of influenza VLPs based on M1, by identifying the minimal sequences requested to render M1 able to bud independently from M2 co-expression. To this end, we created different recombinant M1 proteins characterized by the ability to interact with different ESCRT components and to directly contact the plasma membrane, in the absence of M2. Specifically, we generated constructs expressing M1 fused to the myristilation signal of HIV-1 Gag and containing short proline rich domains (Ldomains) capable of interacting with either Tsg101 (PTAP), AIP1/Alix (YPDL), both cellular proteins able to interact with ESCRT-III and involved in the budding
of several RNA enveloped viruses (Calistri et al., 2009b), or to a motif
(PTAPPEY) known to be extremely efficient in mediating the production of VLPs (Strack et al., 2000). Moreover, we directly fused at the C-terminus of M1 specific ESCRT-III proteins, namely CHMP3 and CHMP4B. In this way, we were able to demonstrated that: i) the HIV-1 Gag myristilation signal, added to the N-terminus of M1 (Myr-M1), confers it the ability to contact the plasma membrane; ii) the insertion of the small proline rich domain PTAP allows Myr-M1 to form VLPs and iii) the fusion between M1 and the cellular protein CHMP4B allows M1 to bud from the cells, independently from the presence of the myristilation signal. Furthermore, we were able to demonstrated that M2 alone and the M2-M1 fusion
protein can self-assemble and bud into virus-like particles. In this context, by engineering specific mutants of the Gag proteins of the feline immunodeficiency virus (FIV), previously described in our laboratory (Calistri et al., 2009a), we were able to demonstrated that indeed M2, and in particular its cytoplasmic region, can act as a substitute of ESCRT-III components for the budding of the influenza virus. Finally, VLPs based on M2, M2-M1 and M1-Myr-CHMP4B, were used in a pilot experiment performed in mice, in order to analyze their immunogenicity.
Interestingly, we were able to detected specific antibodies directed against the protein M1, even though in a small number of tested animals. However, the obtained data are encouraging, since the purification of the employed VLPs, which are still under characterization in terms of density and morphology, was not optimal, thus affecting the obtained results. Finally, we also generated different hemagglutinin (HA) mutants characterized by the absence of the globular head (headless-HA). Indeed, it is well known that the conserved stalk domain of influenza HA is highly immunogenic and confers a broadly immune response (Steel et al., 2010). We demonstrated that headless HA can be incorporated into
engineered VLPs, based on FIV Gag protein. Currently, we are analyzing the possibility of incorporating headless-HA protein also in M1-based VLPs. Next, after setting up the appropriate purification protocol, we would test these M1- based VLPs, together with the FIV-based one, in the mouse model. In conclusion, overall our data provide on one hand important biological insights into the function/characteristics of the influenza virus M1 and M2 proteins and, on the other hand useful information for the development and improvement of influenza-based VLPs to be employed in vaccination strategies
Dissecting the Role of K61/K59 Residue in VPS4 Functions
No full textESCRTs (Endosomal Sorting Complexes Required for Transport) are required for the formation of the intraluminal vesicles in the multivesicular bodies and are involved in other topologically similar processes such as cytokinesis, nuclear envelope sealing and viral egress. The final complex ESCRT-III is disassembled by the recruitment and activation of the AAA-ATPase VPS4 to the endosomal membranes. This recruitment is due to the binding of VPS4 N-terminal MIT with MIM1 and MIM2 domains present in the CHMPs proteins. By analyzing different cellular membrane remodeling events in which VPS4 is involved, here we provide evidence that the K61 residue, mapping within the MIT domain of VPS4B (K59 in VPS4A), is involved in VPS4 functioning. Posttranslational modifications of this residue might modulate MIT-MIM2 binding affinity and, as a consequence, VPS4 functions
Supercritical carbon dioxide combined with high power ultrasound as innovate drying process for chicken breast
No full textThis work explores the feasibility to apply supercritical CO2 (SC - CO2) drying alone or in combination with High-Power Ultrasounds (SC - CO2 + HPU) to improve the shelf life and safety of raw chicken meat. SC - CO2 + HPU drying process revealed the fastest water removal and higher rehydration capacity. A complete inactivation (6 log CFU/g) of mesophilic bacteria and yeasts and moulds was achieved with both the SC - CO2 processes, while oven drying at 75 degrees C, used as control, showed only a limited inactivation (4 log CFU/g). The SC - CO2 processes were efficient also for the inactivation of inoculated Salmonella. The retention of Vitamins B1, B2, B3 and B12 after the SC - CO2 drying demonstrated the preservation of fresh like properties in terms of nutrients. Colour analysis showed a change in colour comparable to traditional cooking techniques. Results demonstrate that SC - CO2-based processes may be used as innovative technologies to dry chicken and make it microbiologically safe, while maintaining nutritional properties
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
- …
