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Trieste, Italy

Pena B.,University of Colorado at Denver | Martinelli V.,ICGEB | Jeong M.,University of Colorado at Denver | Bosi S.,University of Trieste | And 6 more authors.
Biomacromolecules | Year: 2016

Heart failure is a morbid disorder characterized by progressive cardiomyocyte (CM) dysfunction and death. Interest in cell-based therapies is growing, but sustainability of injected CMs remains a challenge. To mitigate this, we developed an injectable biomimetic Reverse Thermal Gel (RTG) specifically engineered to support long-term CM survival. This RTG biopolymer provided a solution-based delivery vehicle of CMs, which transitioned to a gel-based matrix shortly after reaching body temperature. In this study we tested the suitability of this biopolymer to sustain CM viability. The RTG was biomolecule-functionalized with poly-l-lysine or laminin. Neonatal rat ventricular myocytes (NRVM) and adult rat ventricular myocytes (ARVM) were cultured in plain-RTG and biomolecule-functionalized-RTG both under 3-dimensional (3D) conditions. Traditional 2D biomolecule-coated dishes were used as controls. We found that the RTG-lysine stimulated NRVM to spread and form heart-like functional syncytia. Regarding cell contraction, in both RTG and RTG-lysine, beating cells were recorded after 21 days. Additionally, more than 50% (p value < 0.05; n = 5) viable ARVMs, characterized by a well-defined cardiac phenotype represented by sarcomeric cross-striations, were found in the RTG-laminin after 8 days. These results exhibit the tremendous potential of a minimally invasive CM transplantation through our designed RTG-cell therapy platform. © 2016 American Chemical Society. Source

Bockor L.,ICGEB | Bockor L.,University of Zagreb | Jelenic S.,University of Zagreb | Malenica N.,University of Zagreb | And 3 more authors.
Food Technology and Biotechnology | Year: 2013

In this work we have constructed an assay plasmid to study intramolecular recombination efficiency in Agrobacterium tumefaciens and compare it to that in Escherichia coli. The assay plasmid used contains direct repeats of incomplete spectinomycin genes that share a 570 bp region of homology and are separated by the ampicillin resistance gene. It enabled us to directly follow generation of deletion and amplification products in A. tumefaciens for the first time. We show here that predominantly recA-independent mechanism contributed to formation of deletion and amplification products between direct repeats in A. tumefaciens. At the same time, deletion and amplification products were detected at similar frequencies, suggesting that dimer formation accompanies deletion reaction probably by a similar intramolecular event. Source

Patra P.K.,Pt JNM Medical College | Chauhan V.S.,ICGEB | Khodiar P.K.,Pt JNM Medical College | Dalla A.R.,Red Cross | Serjeant G.R.,Sickle Cell Trust Jamaica
Journal of Community Genetics | Year: 2011

The aim of this study is to determine the feasibility of large-scale population screening for the sickle cell gene in high risk areas with limited resources. A programme designed to detect the sickle cell trait and sickle cell disease has screened 359,823 subjects among 2,087 (99.7%) of the villages in Raipur District, Chhattisgarh State, India between October 2007 and June 2010. Children aged 3-15 years were initially screened in the villages by solubility tests on fingerprick samples. Venipuncture was performed on subjects with positive solubility tests, and the samples were transferred to Raipur Medical College for alkaline haemoglobin electrophoresis. The sickle cell trait occurred in 33,467 (9.30%) and an SS phenotype in 747 (0.21%). The gene frequencies were not in Hardy- Weinberg equilibrium most likely due to a deficiency of the SS phenotype failing to enter the sampled population from either sickness or early death. Subjects with abnormal haemoglobin genotypes may factor this information into decisions regarding marriage and avoid the risks of having children with sickle cell disease. The techniques described may be a model for other developing societies with limited resources. © Springer-Verlag 2011. Source

Buratti E.,ICGEB | Baralle D.,University of Southampton
Methods in Molecular Biology | Year: 2012

Defects at the level of pre-mRNA splicing represent a common source of disease mutations in almost all known diseases with a genetic aetiology. In general, it is commonly accepted that 15% of all pathogenic mutations are caused by splicing defects. However, this is probably a conservative estimate since clinical practice has only recently begun to routinely assess for this types of abnormalities. Therefore, it is expected that many currently unclassified or apparently harmless genetic variants will really turn out to be splicing-affecting defects. It is also well known that some genes are more susceptible than others to alterations in their splicing processes. Among these genes, one of the most representative is the NF-1 gene. In this gene, almost 50% of all reported disease-causing mutations can be directly attributed to alterations of the pre-mRNA process. In this chapter, we review the splicing process of the NF-1 gene and the most commonly used methods to identify splicing alterations. In particular, we provide practical notes on how to perform this analysis to maximize the chance of correctly identifying aberrant pre-mRNA splicing events in this gene. © 2012 Springer Science+Business Media, LLC. Source

Negro R.,ICGEB | Gobessi S.,ICGEB | Longo P.G.,ICGEB | He Y.,Indiana University | And 3 more authors.
Blood | Year: 2012

A polymorphic variant of the phosphatase PTPN22 has been associated with increased risk for multiple autoimmune diseases. The risk allele is thought to function by diminishing antigen-receptor signals responsible for negative selection of autoreactive lymphocytes. We now show that PTPN22 is markedly overexpressed in chronic lymphocytic leukemia (CLL), a common malignancy of autoreactive B lymphocytes. We also show that overexpression of PTPN22 significantly inhibits antigen-induced apoptosis of primary CLL cells by blocking B-cell receptor (BCR) signaling pathways that negatively regulate lymphocyte survival. More importantly, we show that PTPN22 positively regulates the antiapoptotic AKT kinase, which provides a powerful survival signal to antigen-stimulated CLL cells. This selective uncoupling of AKT from other downstream BCR signaling pathways is a result of inhibition of a negative regulatory circuit involving LYN, CD22, and SHIP. Finally, we show that PTPN22 can be effectively down-regulated by the PKC inhibitors ruboxistaurin and sotrastaurin, resulting in enhanced killing of CLL cells exposed to proapoptotic BCR stimuli. Collectively, these data suggest that PTPN22 overexpression represents a protective mechanism that allows autoantigen-activated CLL cells to escape from negative selection and indicate that this mechanism could be exploited for therapeutic purposes by targeting PTPN22 with PKC inhibitors. © 2012 by The American Society of Hematology. Source

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