1,354,139 research outputs found
High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins
No full textElectrophoresis. 1995 Jan;16(1):101-4.
High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins.
Rossini K, Rizzi C, Sandri M, Bruson A, Carraro U.
Source
Department of Biomedical Sciences, University of Padova, Italy.
Abstract
In mammals myosin heavy chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemistry. Electrophoretic analysis is the only method for quantitating MHC profiles in single myofibers and/or cryostat sections of biopsied muscle. We present a method for SDS-PAGE of adult rat skeletal muscle which resolves MHC into four bands: 1, 2B, 2X, and 2A from the faster to the slower migrating band. Furthermore, embryonic MHC can be also resolved in a complex mixture of isomyosins, e.g. developing or regenerating muscles. The method does not involve preparation of gradient gels or electrophoresis at low temperature. Improved reproducibility is obtained by: (i) modification of the sample buffer; (ii) use of 7% polyacrylamide in the separating gel; (iii) control of pH of running buffer by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, silver and immunoblot staining. Resolution is sufficient to permit transblotting of separated MHC after SDS-PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides an improved method to separate MHC and quantitate MHC2X and MHCemb in complex mixtures of MHC from a few cryostat sections of normal and diseased muscle.
PMID:
7737081
[PubMed - indexed for MEDLINE
Macrophages regulate proliferation and differentiation of satellite cells
No full textBiochem Biophys Res Commun. 1994 Aug 15;202(3):1688-96.
Macrophages regulate proliferation and differentiation of satellite cells.
Cantini M, Massimino ML, Bruson A, Catani C, Dalla Libera L, Carraro U.
Source
Department of Biomedical Sciences, University of Padova, Italy.
Abstract
We used an in vitro model to investigate whether macrophages stimulate satellite cells proliferation. Satellite cells were obtained by tryptic digestion of adult muscle. Macrophages were obtained from peritoneal cavity by wash after injection of thioglycolate broth. Macrophages and satellite cells cocultures showed an increased number of differentiated myotubes as compared to control cultures. Moreover, in conditions of myoblast colony growth, the addition of macrophage-conditioned medium resulted in a greater number of muscle cell colonies, which are richer in large and differentiated myotubes. The experiments with macrophage-conditioned media suggest that the increased muscle cell proliferation and differentiation is mediated by soluble factor(s) released by macrophages. These results demonstrate that besides their scavenger role macrophages play a pivotal role in myoblast proliferation during muscle regeneration.
PMID:
8060358
[PubMed - indexed for MEDLINE
Sviluppo di metodi per l'identificazione e l'analisi di sequenze geniche fetali nella circolazione materna per una diagnosi prenatale non invasiva
No full textDuring last decade, molecular biology evolution has lead to a deep knowledge of human genome allowing the in-utero diagnosis of a large number of genetic diseases. Now adays performing a prenatal diagnosis of chromosomal abnormalities and of genetic diseases, requires genetic material from the fetus and this is possible only by invasive procedures such as amniocentesis and chorionic villus sampling. The risk of miscarriage of these technics is between 0.5% and 1% and considering these limits, new alternative and non-invasive screening methods, based on ultrasound and/or biochemical parameters of maternal blood, were developed. The disavantages of these methods are the probabilistic aspect of the results.
There was an increasing interest in a new approach that could keep to zero the risk of miscarriage, with an extension of fetal genetic materials screening to a large number of women at the beginning of pregnancy, leading a decreased physic and psychological impact.
In 1997 it was demonstrated the presence of cell-free fetal DNA (cffDNA) in the maternal blood and this has opened up new possibilities for a non-invasive prenatal diagnosis (NIPD).
The cell-free fetal DNA originates from apoptotic trophoblasts on placenta’s surface and comprises around 3-6% of total cell-free DNA in maternal circulation. The cffDNA consists of short DNA fragments (<200bp). Fetal DNA can be detected from 32 days of gestation (though only reliably from 7 weeks) and it’s concentration increases with gestational age (21% per week during the first three months) with a sharp peak during last 8 weeks of pregnancy. The cffDNA is completely removed after delivery.
The main problem related to the application of this NIPD is the high maternal background, so researchers have initially focused their attention on the detection of sequences that are absent in the maternal genome, such as DNA sequences from chromosome Y of male fetuses, the determination of fetal RhD genotype in women RhD-negative, and the detection of paternal mutations in maternal plasma.
Although, the Real Time qPCR amplification of fetal sequences is the widespread method for cffDNA study, there are still a lot of studies focused on the development different protocols for detection of minimal differences between maternal and fetal DNA.
The aim of this PhD thesis is the study and setup of different methods to identify fetal genomic sequences in maternal circulation.
The research project was divided in three steps: the first one was focused on the validation of Real-Time qPCR protocol, applied to the detection and quantification of total free DNA and of cffDNA in pregnancies with a male fetus; the second was focused on the develop of a reliable protocol for the identification of fetal genomic sequences useful to recognize cffDNA in pregnancies with a female fetus, by the mini-sequencing technics; finally, the last step was focused on the validation of an analytical protocol to verify the presence/absence of paternal mutations in maternal plasma using nested PCR allele-specific.
The results demonstrated here for each technic, show a high sensibility and resolution capacity, together with an easy and rapid handling. Furthermore, the combination of the two technics (Real-Time qPCR and mini-sequencing reaction) was essential in order to exclude the presence of a paternal mutation in the fetal genome in every case where a mutation wasn’t identified. The ability to exclude the presence of the paternal mutation in the fetus by analysis of maternal plasma avoid the utilization of invasive prenatal tests and, at the same time, reassures the couple early in the course of the pregnancy about the recurrence of the disease, thus avoiding significant anxiety.Negli ultimi anni l'evoluzione della biologia molecolare ha permesso una conoscenza sempre maggiore del genoma umano e consente ormai la diagnosi in utero di un numero crescente di patologie genetiche. Al momento attuale la diagnostica prenatale di anomalie cromosomiche o di malattie genetiche richiede il prelievo di cellule fetali ottenibili con metodiche invasive, l'amniocentesi e la villocentesi. Queste tecniche comportano un rischio di provocare un aborto compreso fra 0.5% e 1%. Di fronte a questi limiti delle metodiche invasive sono stati messi a punto alcuni metodi di screening alternativi non invasivi, basati su parametri ecografici fetali e/o biochimici su sangue materno, che però hanno il limite di fornire risposte solo di tipo probabilistico.
L'introduzione di un test realmente alternativo di diagnosi prenatale non invasiva che permetta di eliminare il rischio di aborto legato all’amniocentesi e alla villocentesi è fortemente attesa. Infatti un simile test permetterebbe una estensione dell'analisi del materiale genetico fetale a molte più donne all'inizio della gravidanza e diminuirebbe l'impatto fisico e psicologico di questo tipo di indagine.
Nel 1997 uno studio ha dimostrato la presenza di DNA libero fetale (cell-free fetal DNA, cffDNA) nel plasma materno e ha evidenziato la sua potenzialità per una diagnosi prenatale non invasiva (NIPD). Durante i 15 anni successivi, si è sviluppata la ricerca di base sulle caratteristiche del DNA libero fetale e molti ricercatori hanno studiato la biologia e il significato clinico del trasporto di cffDNA nel circolo materno.
Il DNA libero fetale origina dall’apoptosi dei trofoblasti costantemente rimpiazzati sulla superficie della placenta e comprende circa il 3-6% del DNA libero totale nella circolazione materna. E’ stato inoltre osservato che è costituito da corti frammenti di DNA (<200bp) piuttosto che interi cromosomi e che la sua concentrazione aumenta gradualmente durante la gravidanza: è rilevabile nella circolazione materna già a 32 giorni circa di gestazione (in quantità affidabile solo dopo le 7 settimane) e aumenta del 21% ogni settimana nel primo trimestre, con un forte picco durante le ultime 8 settimane di gravidanza per poi venire rapidamente rimosso dalla circolazione materna subito dopo il parto.
L’utilizzo del DNA fetale libero come mezzo per la diagnosi prenatale non invasiva di malattie genetiche fino ad ora è fortemente limitato dall’elevato background di DNA materno pertanto le prime strategie messe a punto per l’identificazione del DNA libero fetale si sono basate sul rilevamento di sequenze assenti nel genoma materno, come le sequenze Y-specifiche, la determinazione del genotipo RhD fetale in donne RhD-negative e l’identificazione di sequenze fetali con mutazioni ereditate dal padre.
L’amplificazione tramite Real Time qPCR di sequenze unicamente fetali nel plasma di donne gravide rappresenta ad oggi il metodo di elezione per lo studio del DNA libero fetale, ma numerosi sforzi sono stati realizzati per la messa a punto di sistemi sempre più sensibili ed universali per rilevare le più piccole differenze tra il DNA fetale libero e quello materno, al fine di consentire un utilizzo su larga scala del cffDNA per la diagnosi prenatale non invasiva.
Lo scopo di questa ricerca è stato quindi, la messa a punto di protocolli validi per l’identificazione e l’analisi di sequenze geniche fetali nella circolazione materna.
Il progetto è stato quindi suddiviso in tre fasi: la prima fase ha comportato la messa a punto di opportuni protocolli di Real-Time qPCR per il rilevamento e la quantificazione sia del DNA libero totale estratto sia del cffDNA estratto in gravidanze con feto di sesso maschile; la seconda fase del progetto si è occupata di testare e sviluppare un protocollo affidabile per l’identificazione di sequenze geniche fetali utili al riconoscimento di cffDNA nelle gravidanze con feto di sesso femminile, mediante l’utilizzo della tecnica di mini-sequencing; l’ultima fase del progetto si è concentrata infine sulla messa a punto di un protocollo di verifica della presenza/assenza di mutazioni paterne in plasma materno mediante nested PCR allele-specifica.
L’analisi dei risultati ha dimostrato, per ogni tecnica sviluppata, elevate sensibilità e capacità di risoluzione accoppiate a velocità e facilità di esecuzione, confermandone quindi l’applicabilità per una diagnosi prenatale non invasiva. Inoltre l’utilizzo in combinazione delle due tecniche (Real-Time qPCR e mini-sequencing) per la conferma della presenza del cffDNA nel campione in analisi, si è rilevata indispensabile per poter escludere che il feto abbia ereditato la mutazione paterna, in tutti i casi in cui non viene identificata la mutazione. L’esclusione della mutazione paterna nel feto attraverso l’analisi su plasma materno permetterebbe quindi di evitare l’utilizzo di procedure invasive per il prelievo di cellule fetali e avere questa informazione alla 9-10 settimana di gravidanza consentirebbe di rassicurare la coppia prima di qualsiasi altro test diagnostico di routine
Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.
No full textCell Biochem Funct. 1995 Jun;13(2):99-104.
Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.
Carraro U, Bruson A, Catani C, Dalla Libera L, Massimino ML, Rizzi C, Rossini K, Sandri M, Cantini M.
Source
University of Padova, CNR Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, Italy.
Abstract
Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.
PMID:
7538914
[PubMed - indexed for MEDLINE
Polymorphisms of the SCN1A gene in children and adolescents with primary headache and idiopathic or cryptogenic epilepsy: is there a linkage?
No full textThe purpose of this study was to evaluate the distribution of the polymorphisms of the SCN1A gene in a series of children and adolescents with primary headache and idiopathic or cryptogenic epilepsy compared to controls. Five non-synonymous exonic polymorphisms (1748A > T, 2656T > C, 3199A > G, 5771G > A, 5864T > C) of the SCN1A gene were selected and their genotyping was performed, by high resolution melting (HRM), in 49 cases and 100 controls. We found that among the five polymorphisms, only 3199A > G was a true polymorphism. We did not find a statistically significant difference between distribution of 3199A > G genotypes between cases and controls. We excluded the role of the SCN1A gene in the pathogenesis of comorbidity between headache (especially migraine) and epilepsy. The SCN1A gene is a major gene in different epilepsies and epilepsy syndromes; theHRM could be the new methodology, more rapid and efficacious, for molecular analysis of the SCN1A gene. © The Author(s) 2011
High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins
No full textExperimental and numerical mechanical characterisation of additively manufactured polymeric lattice structures under uniaxial tensile load
No full textAdditive manufacturing enables the production of lighter, more robust components with intricate features like lattice structures. However, since the mechanical behaviour of lattice structures is not fully characterized, the application of such potential is limited today. The challenge with lattice structures tensile tests is defining a suitable design that fits the standard requirements and process characteristics. In the polymeric powder bed fusion process, the problem is to produce powder-free geometries and to avoid stress concentrations zones, adapting the specimen accordingly. In this regard, numerical simulation may provide insightful information and support the analysis of the deformation mechanisms. This paper analyses a new tensile sample for lattice structures using finite element analysis. The sample is designed following the EN ISO 527 standard prescriptions. An area with a controlled gradation of the lattice relative density is designed to ensure both powder-free voids and fracture localization within the lattice specimen gauge length. Experimental tests are performed to validate the numerical results using a modified body cubic centred topology with two different strut diameters. The specimens are produced in polyamide by powder bed AM process. Due to the complexity of the lattice design, a digital image correlation is used to compute the full range of strains at the macroscopic level. Experimental and numerical strain maps results showed a good agreement. The recorded deviation was attributed to the process-induced defect, such as the geometrical accuracy that, if compensated, boosted the capability of the numerical model to predict the mechanical behaviour of the lattice structure
Interview with Maestro Renato Bruson, Conducted During the Masterclass in Parma (October 25-28, 2024)
No full textThis interview with Maestro Renato Bruson was conducted during a masterclass at his house-museum in Parma in October 2024. Bruson, one of the greatest baritones of the 20th century, is celebrated for his expressive singing and deep respect for the interpretative power of the word. Despite humble beginnings and no early access to music education, he rose to international fame with his debut in Verdi’s Il trovatore and later at the Metropolitan Opera. He credits his success to his dedication, seriousness, and his chamber music training under Professor Elena Fava Ceriati, which refined his interpretative sensibility. Bruson emphasized humility, consistency, and the avoidance of shortcuts in a singer’s journey. He warned against the negative influence of the media on young artists and stressed the importance of respecting one’s own vocal limits. For him, vocal technique, emotional depth, and the ability to move the audience are central to true artistry. He does not believe in predefined national schools of singing but sees taste and style as cultural variations. His message to future generations is simple yet powerful: “Study, study, study.
Effect of 3D grading thickness on mechanical and deformation behaviour of gyroid structures produced via powder bed fusion with electron beam
No full textThe production of complex geometries with geometrical tuned features is made possible by Additive manufacturing (AM) processes. Powder Bed Fusion using Electron Beam (PBF-EB) is one of the AM techniques for metallic components, which stands out for its ability to fabricate intricate structures with high-performance materials. One example is surface-based architectures known as Triply Periodic Minimal Surfaces (TPMS), where structural walls are defined by a specific thickness. The mechanical behaviour and deformation mechanisms of TPMS are governed by both the geometry and the wall thickness of the structure. Conventional TPMS designs typically employ a uniform or one-dimensional thickness gradient, which constrains their performance under varied loading conditions. This study explores a novel approach involving three-dimensional thickness gradation, aiming to enhance structural integrity, improve load-bearing capacity, and enable functional optimisation. The gyroid surface, a widely studied TPMS for applications ranging from lightweight aerospace components to biomedical implants, is used as a reference geometry. Three types of initiator surface (diagonal plane, cross-shape, and sphere) are employed to create spatial variations in wall thickness between predefined minimum and maximum values. Samples are fabricated using the PBF-EB process, and the resulting structures are characterised via X-ray computed tomography to assess morphometric parameters. These parameters are then correlated with mechanical properties and deformation mechanisms and compared against gyroid TPMS with uniform thickness. The results reveal a significant influence of 3D thickness variation on performance, offering new insights for the design and additive manufacturing of next-generation TPMS structures with tailored mechanical responses
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