1,720,973 research outputs found
Molecular mechanisms impairing biosynthesis and function of hemostatic proteins
No full textThe gene mutations leading to hemorrhagic disorders provide peculiar models to elucidate molecular mechanisms underlying protein biosynthesis, and the relationship between the structure and function.
The research activity has been focused on the molecular defects leading to severe deficiency of Factor IX (FIX), a serine protease with a key role in the intrinsic pathway of blood coagulation, and of von Willebrand factor (VWF), a large multimeric protein essential for the primary hemostasis.
In particular, I investigated mechanisms due to three different molecular defects such as nonsense and missense mutations in F9 gene, associated with type I hemophilia B, and an in-frame deletion in the VWF gene, displaying a dominant-negative effect.
Results from investigations in patients’ plasma and the expression of nonsense FIX variants in eukaryotic cells led to the demonstration of trace levels of full-length FIX molecules even in the presence of nonsense mutations through a mechanism of ribosome read-through. The efficiency was dependent of the specific nonsense mutation and on it sequence context. Moreover, I investigated the susceptibility of a panel of nonsense FIX mutations to the induction of readthrough by aminoglycosides. The data suggested a direct relationship between the spontaneous and the drug-induced readthrough. Overall data indicated that not all nonsense mutations can be considered truly “null-mutations”, a finding that have pathophysiological implications.
The severe p.Tyr450Cys mutation in the carboxyl-terminal region of coagulation FIX was chosen as model to study the interplay between impaired protein biosynthesis and/or function caused by missense mutations in relation to specific protein regions, which has been poorly investigated. Results from the expression of a panel of recombinant variants demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl-terminus mutations produce life-threatening or mild phenotypes in the presence of similarly reduced protein amounts.
Finally I contributed to the characterization of the dominant inheritance in VWD, due to two essential process in VWF dimerization and multimerization VWD can express dominant-negative features. Previous study characterized and demonstrate d the modulation of this dominant effect. In our study we reproduced in vivo the effect of dominance and through a creation of an artificial mutation, we demonstrated in vitro an in vivo the key role of interaction between wild type and mutant protein monomers during dimerization and or multimerization, We believe that our finding have general implication for the dominant forms of VWD, the most frequent inherited bleeding disorders in humans
SiMPLOD, a structure-integrated database of collagen lysyl hydroxylase (LH/PLOD) enzyme variants
No full textPLOD genes encode for procollagen lysyl hydroxylase enzymes (LH/PLOD), a family of proteins essential for collagen biosynthesis. Several mutations affect these genes causing severe disorders, such as Ehlers-Danlos and Bruck syndrome, as well a connective tissue disease with phenotype resembling osteogenesis imperfecta caused by lack of LH3 functions. The recently determined three-dimensional structures of the full-length human LH3/PLOD3 isoform, together with the structure of a fragment of a viral LH/PLOD homolog, are now allowing molecular mapping of the numerous disease-causing mutations, providing insights often suitable for the interpretation of the resulting disease phenotypes. However, the added value of molecular structure interpretation is affected by the limited accessibility of complex molecular data to scientific communities lacking direct expertise in structural biology. In this work, we present SiMPLOD (Structurally-integrated database for Mutations of PLOD genes), a publicly-available manually-curated online database with an embedded molecular viewer interface for the visualization and interpretation of LH/PLOD mutations on available molecular models. Each SiMPLOD entry is accompanied by manual annotations extrapolated from literature references and comments about the localization of the amino acid variants on the molecular structure. Additional links to the appropriate online resources for clinically-relevant as well as biochemical data are also provided in a standardized format. The web application is available at http://fornerislab.unipv.it/SiMPLOD. This article is protected by copyright. All rights reserved
Cationic lipid nanosystems as carriers for nucleic acids
No full textSolid lipid nanoparticles (SLN) consisting of tristearin or tribehenin, and monoolein aqueous dispersions (MAD) consisting of glyceril-monoolein have been studied as potential nanocarriers for nucleic acids. The cationic character of nanocarriers was obtained by adding cationic surfactants, such as diisobutylphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA) or PEG-15 Cocopolyamine (PCPA), to the lipid composition. The products were characterized in terms of size and morphology by Cryo-TEM and PCS. The charge properties were determined by measuring the zeta potential. Our experimental protocol enabled us to obtain homogeneous and stable cationic nanosystems within 3-6 months from production.
Assessment of cytotoxicity on HepG2 cells by MTT assays indicated that MAD preparations were less toxic than SLN, and in general PCPA-containing formulations are less cytotoxic than DEBDA-containing ones.
The formation of electrostatic complex with Salmon Sperm or plasmid DNA, used as model nucleic acids, was evaluated by electrophoresis on agarose gel. The results confirmed that all the formulations studied are able to form the complex.
Finally, we investigated the ability of SLN and MAD to deliver DNA into HepG2 cells, and to this purpose we exploited expression plasmids for the green fluorescent protein or the firefly luciferase. Although with reduced efficiency, the results showed that the produced nanocarriers are able to convey plasmids within cells.
The data obtained encourage further study aimed at improving these new formulations and proposing them as novel in vitro transfection reagents with potential application to in vivo delivery of nucleic acids
Responsiveness of hemophilia B- causing non sense mutations to ribosome readthrough-inducing drugs strictly depends on the nucleotide and prrotein context
No full textBACKGRUOUND
Nonsense mutations, caused by premature termination codons, are relatively frequent in Haemophilia (>10%) and are considered as “null mutations”. However, they have been found also in moderate/mild Haemophiliacs and, as compared with large gene deletions, are associated with lower risk for developing inhibitors. This observation point to the presence of residual expression levels arising from “ribosome readthrough” over nonsense triplets, whose sequence context has an impact on readthrough efficiency, as we have shown in a very small cohort of Haemophilia B (HB) patients. Drugs inducing readthrough such as aminoglycosides are proposed as potential therapy.
AIM
To investigate secreted and functional levels of factor IX (FIX) produced by an extended panel of HB nonsense mutations.
METHODS
Recombinant FIX (rFIX) nonsense variants were expressed in HEK293 cells, and secreted and intracellular protein levels, as well as protein isoforms, were evaluated by ELISA and Western Blotting analysis. Residual activity was assessed by coagulant assays.
RESULTS
We investigated a panel of F9 mutations (R75X, L103X, R162X, W240X, R294X, R298X, Y330X, Q370X, R379X, R384X), associated to severe/moderate HB, representing the vast majority of patients with nonsense mutations in HB (324 patients out of 467, corresponding to ~70%). Appreciable levels of secreted FIX were detected for the R162X, W240X, R294X, R298X, Y330X mutants, with truncated isoforms being the large predominance. Truncated forms for Q370X, R379X and R384X were observed in cell lysates only, which suggests misfolding and intracellular retention. Noticeably, the full-length FIX form was appreciable for the R162X variant, indicating the occurrence of spontaneous readthrough, which was significantly increased by the use of aminoglycosides. Moreover, aminoglycosides promoted the synthesis of full-length FIX in the presence of the R75X, Y330X, Q370X, R379X, R384X mutations, not undergoing appreciable spontaneous readthrough. Intriguingly, we investigated the peculiar W240X nonsense mutation, caused by two different termination codons, which showed a differential impact of readthrough in restoring the full-length protein depending on the sequence context. Preliminary coagulant and functional assays revealed that only a few variants were prone to undergo efficient readthrough in terms of secretion and coagulant function.
CONCLUSIONS
These data demonstrate that nonsense mutations can be associated to residual FIX levels through a mechanism of “productive” readthrough. The identification of “leaky” nonsense mutations, and thus of patients with trace FIX levels and potentially “high responders” to readthrough-inducing drugs, might help diagnosis and treatment
Nanosistemi lipidici per la veicolazione di acidi nucleici
No full textNanoparticelle solide lipidiche (SLN), costituite da tristearina oppure da tribehenina, e cubosomi costituiti da glicerilmonoleina sono stati studiati come possibili nanosistemi cationici destinati a veicolare acidi nucleici. Il carattere cationico è stato ottenuto aggiungendo DEBDA e PCPA, quali tensioattivi cationici. Le formulazioni prodotte sono state caratterizzate dal punto di vista morfologico e dimensionale mediante Cryo-TEM e PCS. Le proprietà di carica sono state determinate misurando il potenziale zeta. Nelle condizioni sperimentali utilizzate, è stato possibile realizzare SLN e cubosomi cationici omogenei e stabili entro 3-6 mesi dalla produzione.
SLN e cubosomi sono stati sottoposti a saggio MTT di citotossicità in vitro su cellule HepG2 dal quale si evince che le formulazioni con PCPA sono meno citotossiche rispetto a quelle con DEBDA e che i cubosomi presentano limitata citotossicità.
La formazione del complesso elettrostatico con DNA Salmon Sperm, utilizzato come acido nucleico modello, è stata valutata mediante elettroforesi su gel d'agarosio. I risultati ottenuti confermano che tutte le formulazioni studiate sono in grado di formare il complesso.
Infine, sono stati realizzati studi di transfezione in vitro su cellule HepG2 utilizzando un plasmide codificante per GFP ed uno codificante per la proteina luciferasi.
I risultati ottenuti dimostrano che i nanosistemi considerati possono veicolare plasmidi a livello endocellulare.
Gli incoraggianti dati ottenuti invitano a proseguire gli studi dai punti di vista conoscitivo e applicativo, allo scopo di realizzare reagenti di transfezione in vitro alternativi a quelli attualmente disponibili e di individuare possibili applicazioni in vivo
Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension
No full textRecombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In this regard, human embryonic kidney 293 (HEK293) cells constitute one of the main standard lab-scale mammalian hosts for recombinant protein production since these cells are relatively easy to handle, scale-up, and transfect. Here, we present a detailed protocol for the cost-effective, reproducible, and scalable implementation of HEK293 cell cultures in suspension (suitable for commercially available HEK293 cells, HEK293-F) for high-quantity recombinant production of secreted soluble multi-domain proteins. In addition, the protocol is optimized for a Monday-to-Friday maintenance schedule, thus simplifying and streamlining the work of operators responsible for cell culture maintenance
Nuove strategie per la veicolazione di acidi nucleici: nanosistemi cationici a matrice lipidica
No full textNegli ultimi anni, la terapia genica si è dimostrata molto efficace nella cura di numerose patologie di origine genetica, virale o neoplastica, nonostante la somministrazione per via sistemica di oligonucleotidi terapeutici risulti ancora complessa.
La nostra ricerca ha previsto la produzione di nanoparticelle solide lipidiche (SLN), costituite da tristearina oppure da tribehenina, e di cubosomi costituiti da glicerilmonoleina, come possibili nanosistemi cationici per la veicolazione di acidi nucleici. Il carattere cationico è stato ottenuto aggiungendo DEBDA e PCPA quali tensioattivi cationici.
Le formulazioni ottenute sono state successivamente caratterizzate in dimensioni e morfologia mediante spettroscopia di fotocorrelazione (PCS) e microscopia elettronica di crio-trasmissione (Cryo-TEM). Le proprietà di carica sono state determinate misurando il potenziale zeta. Nelle condizioni sperimentali utilizzate, è stato possibile realizzare SLN e cubosomi cationici omogenei e stabili entro 3-6 mesi dalla produzione.
SLN e cubosomi sono stati sottoposti a saggio MTT di citotossicità in vitro su cellule HepG2. Le formulazioni costituite da PCPA sono risultate meno citotossiche rispetto a quelle costituite da DEBDA e i cubosomi hanno evidenziato una limitata citotossicità.
È stata inoltre valutata la formazione del complesso elettrostatico con DNA Salmon Sperm e un plasmide codificante per il fattore IX della coagulazione mediante elettroforesi su gel d'agarosio.
I risultati ottenuti confermano che tutte le formulazioni sono in grado di formare il complesso.
Sono stati infine realizzati studi di transfezione in vitro su cellule HepG2 utilizzando un plasmide codificante per GFP e un plasmide codificante per la proteina luciferasi. I risultati ottenuti dimostrano che i nanosistemi considerati sono in grado di veicolare plasmidi a livello endocellulare. Le SLN Tristearina-PCPA 1.5% presentano un'efficienza di transfezione del 11.3% rispetto a LipofectaminaTM inducendo minore citotossicità, mentre i complessi realizzati utilizzando le SLN Tribehenina-PCPA 1.5% aumentano la proliferazione delle cellule HepG2.
Gli incoraggianti dati ottenuti invitano a proseguire gli studi migliorando gli elementi conoscitivi ed applicativi, allo scopo di realizzare reagenti di transfezione in vitro alternativi a quelli attualmente disponibili e individuare possibili applicazioni in vivo
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
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