%0 Journal Article %J Bioconjug Chem %D 2017 %T Synthesis of Lipophilic Core-Shell FeO@SiO@Au Nanoparticles and Polymeric Entrapment into Nanomicelles: A Novel Nanosystem for in Vivo Active Targeting and Magnetic Resonance-Photoacoustic Dual Imaging. %A Monaco, Ilaria %A Arena, Francesca %A Biffi, Stefania %A Locatelli, Erica %A Bortot, Barbara %A La Cava, Francesca %A Marini, Giada Maria %A Severini, Giovanni Maria %A Terreno, Enzo %A Comes Franchini, Mauro %K Animals %K Cell Proliferation %K Female %K Ferric Compounds %K Folic Acid %K Gold %K Humans %K Image Processing, Computer-Assisted %K Magnetic Resonance Imaging %K Magnetite Nanoparticles %K Mice %K Mice, Inbred BALB C %K Mice, Nude %K Micelles %K Multimodal Imaging %K Ovarian Neoplasms %K Photoacoustic Techniques %K Polymers %K Silicon Dioxide %K Tumor Cells, Cultured %K Xenograft Model Antitumor Assays %X

In this work, iron/silica/gold core-shell nanoparticles (FeO@SiO@Au NPs) characterized by magnetic and optical properties have been synthesized to obtain a promising theranostic platform. To improve their biocompatibility, the obtained multilayer nanoparticles have been entrapped in polymeric micelles, decorated with folic acid moieties, and tested in vivo for photoacoustic and magnetic resonance imaging detection of ovarian cancer.

%B Bioconjug Chem %V 28 %P 1382-1390 %8 2017 05 17 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/28453929?dopt=Abstract %R 10.1021/acs.bioconjchem.7b00076 %0 Journal Article %J Pediatr Rheumatol Online J %D 2015 %T Genetic profiling of autoinflammatory disorders in patients with periodic fever: a prospective study. %A De Pieri, Carlo %A Vuch, Josef %A De Martino, Eleonora %A Bianco, Anna M %A Ronfani, Luca %A Athanasakis, Emmanouil %A Bortot, Barbara %A Crovella, Sergio %A Taddio, Andrea %A Severini, Giovanni M %A Tommasini, Alberto %K Adolescent %K Carrier Proteins %K Child %K Cryopyrin-Associated Periodic Syndromes %K Cytoskeletal Proteins %K Familial Mediterranean Fever %K Female %K Fever %K Gene Expression Profiling %K Genotype %K Hereditary Autoinflammatory Diseases %K Humans %K Intracellular Signaling Peptides and Proteins %K Logistic Models %K Male %K Mutation %K Phosphotransferases (Alcohol Group Acceptor) %K Prospective Studies %K Receptors, Tumor Necrosis Factor, Type I %K Syndrome %X

BACKGROUND: Periodic fever syndromes (PFS) are an emerging group of autoinflammatory disorders. Clinical overlap exists and multiple genetic analyses may be needed to assist diagnosis. We evaluated the diagnostic value of a 5-gene sequencing panel (5GP) in patients with undiagnosed PFS.

METHODS: Simultaneous double strand Sanger sequencing of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12 genes was performed in 42 patients with unexplained PFS. Clinical features were correlated with genetic results.

RESULTS: None of 42 patients analyzed displayed a causative genotype. However, single or multiple genetic variants of uncertain significance were detected in 24 subjects. Only in 5 subjects a definite diagnosis was made by taking into account both genetic and clinical data (2 TRAPS syndrome; 2 FMF; 1 FCAS). Statistical analysis showed that patients carrying genetic variants in one or more of the five selected genes displayed a significantly lower response to glucocorticoids compared with subjects who had completely negative genetic results.

CONCLUSIONS: The sequencing of multiple genes is of little help in the diagnostics of PFS and can often lead to results of uncertain interpretation, thus the clinically driven sequencing of single genes should remain the recommended approach. However, the presence of single or multiple genetic variants of uncertain significance, even if not allowing any specific diagnosis, correlated with a poorer response to glucocorticoids, possibly indicating a multifactorial subgroup of PFS with differential response to pharmacological treatment.

%B Pediatr Rheumatol Online J %V 13 %P 11 %8 2015 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/25866490?dopt=Abstract %R 10.1186/s12969-015-0006-z %0 Journal Article %J Mol Cell Biochem %D 2012 %T Β-hexosaminidase over-expression affects lysosomal glycohydrolases expression and glycosphingolipid metabolism in mammalian cells. %A Tancini, Brunella %A Magini, Alessandro %A Bortot, Barbara %A Polchi, Alice %A Urbanelli, Lorena %A Sonnino, Sandro %A Severini, Giovanni Maria %A Emiliani, Carla %K Animals %K beta-Hexosaminidase alpha Chain %K Cell Membrane %K Exocytosis %K Fibroblasts %K Glycoside Hydrolases %K Glycosphingolipids %K Humans %K Lysosomes %K Mice %K NIH 3T3 Cells %K Transfection %X

Lysosomes are not only degrading organelles but also involved in other critical cellular processes. In addition, active lysosomal glycohydrolases have been detected in an extra-lysosomal compartment: the presence of glycohydrolases on the plasma membrane (PM) has been widely demonstrated, and a possible role on the modification of the cell surface glycosphingolipids (GSL) participating in the modulation of cell functions such as cell-to-cell interactions and signal transduction pathways has been proposed. On this basis, the coordinated expression of lysosomal glycohydrolases and their translocation to the PM appear to be crucial for many cellular events. In this paper, we report evidence for the existence of a coordinated mechanism regulating the expression/activity of both lysosomal and PM-associated glycohydrolases. We show that the over-expression of the acidic glycohydrolase β-hexosaminidase α-subunit in mouse NIH/3T3 fibroblasts induces the increased expression of the Hex β-subunit necessary to form the active isoenzyme dimers as well as of other glycohydrolases participating in the GSL catabolism, such as β-galactosidase and β-glucocerebrosidase. More interestingly, this regulatory effect was also extended to the PM-associated hydrolases. In addition, transfected cells displayed a rearrangement of the GSL expression pattern that cannot be simply explained by the increased activity of a single enzyme. These observations clearly indicate that the expression level of metabolically related glycohydrolases is regulated in a coordinated manner and this regulation mechanism also involves the PM-associated isoforms.

%B Mol Cell Biochem %V 363 %P 109-18 %8 2012 Apr %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/22147196?dopt=Abstract %R 10.1007/s11010-011-1163-0 %0 Journal Article %J Diagn Mol Pathol %D 2011 %T High-throughput genotyping robot-assisted method for mutation detection in patients with hypertrophic cardiomyopathy. %A Bortot, Barbara %A Athanasakis, Emmanouil %A Brun, Francesca %A Rizzotti, Diego %A Mestroni, Luisa %A Sinagra, Gianfranco %A Severini, Giovanni Maria %K Cardiomyopathy, Hypertrophic %K DNA Mutational Analysis %K Genetic Predisposition to Disease %K Genetic Testing %K Genotyping Techniques %K High-Throughput Nucleotide Sequencing %K Humans %K Muscle Proteins %K Mutation %K Robotics %X

Hypertrophic cardiomyopathy (HCM) is the most frequent autosomal dominant genetic heart muscle disease and the most common cause of sudden cardiac death in young people (under 30 y of age), who are often unaware of their underlying condition. Genetic screening is now considered a fundamental tool for clinical management of HCM families. However, the high genetic heterogeneity of HCM makes genetic screening very expensive. Here, we propose a new high-throughput genotyping method based on a HCM 96-well sequencing plate for the analysis of 8 of the most frequent HCM-causing sarcomeric genes by automating several processes required for direct sequencing, using a commercially available robotic systems and routinely used instruments. To assess the efficiency of the robot-assisted method, we have analyzed the entire coding sequence and flanking intronic sequences of the 8 sarcomeric genes in samples from 18 patients affected by HCM and their relatives, which revealed 9 different mutations, 3 of which were novel. The automated, robot-assisted assembling of polymerase chain reaction, purification of polymerase chain reaction products, and assembly of sequencing reactions resulted in a substantial saving of time, reagent costs, and reduction of human errors, and can therefore be proposed as a primary strategy for mutation identification in HCM genetic screening in many medical genetic laboratories.

%B Diagn Mol Pathol %V 20 %P 175-9 %8 2011 Sep %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/21817903?dopt=Abstract %R 10.1097/PDM.0b013e31820b34fb %0 Journal Article %J Diagn Mol Pathol %D 2011 %T Quantification of heteroplasmic mitochondrial DNA mutations for DNA samples in the low picogram range by nested real-time ARMS-qPCR. %A Biffi, Stefania %A Bortot, Barbara %A Carrozzi, Marco %A Severini, Giovanni Maria %K Child %K DNA, Mitochondrial %K Humans %K Mitochondrial Diseases %K Mutation %K Polymerase Chain Reaction %K Sensitivity and Specificity %X

In many mitochondrial diseases, different clinical manifestations are related to tissue-specific distribution of mutated mitochondrial DNA (mtDNA). In this study, we describe an assay for the determination of mutated mtDNA copy number in small clinical samples, using standard polymerase chain reaction (PCR) followed by SYBR Green real-time allelic-specific PCR [amplification refractory mutation system-quantitative PCR (ARMS-qPCR)]. To assess the degree of heteroplasmy in a patient harboring 2 cosegregating mtDNA mutations (4415A>G and 9922A>C) starting from picogram amounts of DNA, we first amplified the mutated target sequence by standard PCR, and then analyzed it by real-time ARMS-qPCR. To validate this method, we analyzed by real-time ARMS-qPCR the PCR amplification products derived from different mixtures containing known proportions of mutant and wild-type cloned mtDNA fragments. The correlation coefficient of 0.994 between expected and observed values for the percentage of mutant A4415G confirms that the relative proportion of mutated and wild-type mtDNA was maintained after the first PCR amplification. This method allows the precise quantification of heteroplasmic mutations in DNA samples extracted from hairs, urine, small stomach biopsies, and, more importantly, single-muscle fiber, with a limit of detection close to 0.5%. This nested real-time ARMS-PCR represents a rapid, efficient, and less expensive method for the detection and quantification of heteroplasmic mutant mtDNA, even in very small clinical samples.

%B Diagn Mol Pathol %V 20 %P 117-22 %8 2011 Jun %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/21532488?dopt=Abstract %R 10.1097/PDM.0b013e3181efe2c6