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Biomedical subjects

Paolo Fortina

Publications and source records attributed to Paolo Fortina.

At least 19 recordsLinked to original sources
PubMed2007

Multiplex pyrosequencing for DNA variation analysis.

Pyrosequencing technique has been widely used to perform both single-nucleotide polymorphism detection and quantitative DNA methylation analysis. Simplex Pyrosequencing is sufficient to interrogate more than one polymorphic site if these gene variants are within the reach of the sequencing reaction. For polymorphisms far apart from each other or located on different genes, multiple simplex analyses are required. To reduce the number of simplex reactions, multiplex Pyrosequencing becomes a useful alternative method. The multiplex reaction is performed in the presence of single or multiple templates with several sequencing primers. Factors such as primer selection for the PCR and Pyrosequencing reaction, generation of optimal nucleotide dispensation order, use of internal and external controls, preparation of instrumentation, and Pyrogram interpretation are essential to the success of the multiplexing. In this chapter, the mouse 45S rRNA gene is used to present two general multiplex Pyrosequencing protocols for determining DNA methylation and allele frequency in the spacer promoter region of this gene.

Animals↗
PubMed2006

Combinatorial sequencing-by-hybridization: analysis of the NF1 gene.

Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant disorders, is caused by mutations in the NF1 gene. A variety of methods are currently used in clinical settings to define disease-causing mutations. We describe microarray-based combinatorial sequencing-by-hybridization (cSBH), which overcomes some disadvantages associated with other techniques. Sequence readout of 2 kb was achieved on a single slide, with detection of base substitutions, insertions and small deletions. In addition, cSBH analysis of the entire NF1 gene demonstrates reproducibility, efficiency and reduced time; therefore, representing an alternative to extensive DNA sequence characterization.

Chromosome Disorders↗
PubMed2006

Opportunities for near-infrared thermal ablation of colorectal metastases by guanylyl cyclase C-targeted gold nanoshells.

Colorectal cancer is the third most common malignancy and the second most common cause of cancer-related mortality worldwide. While surgery remains the mainstay of therapy, approximately 50% of patients who undergo resection develop parenchymal metastatic disease. Unfortunately, current therapeutic regimens offer little improvement to the survival of patients with parenchymal metastases in the liver and lung. In that context, there is a significant unrealized opportunity at the intersection of engineering and biology for the development of novel targeted therapeutic approaches to colorectal cancer metastases. This opportunity exploits the discovery that an intestinal receptor, guanylyl cyclase C, which mediates diarrhea induced by bacterial heat-stable enterotoxins (STs), is over-expressed by metastatic colorectal tumors only. Moreover, it leverages recent advances in the fabrication of metal nanoshells with defined thicknesses absorb near-infrared (NIR) light, resulting in resonance and transfer of thermal energies of more than 40 degrees C. Thus, the conjugation of ST to gold nanoshells, which can undergo resonance excitation by NIR light and emit heat, represents a previously unrecognized approach for the targeted therapy of parenchymal colorectal cancer metastases, specifically to the liver and lung. This article discusses the potential of ST-targeted nanoshells for NIR thermal ablation of metastatic colorectal tumors and highlights the significant challenges and solutions linked to the translation of this emerging technology to patient care.

Biomarkers, Tumor↗
PubMed2006

L-selenomethionine modulates high LET radiation-induced alterations of gene expression in cultured human thyroid cells.

L-selenomethionine (SeM) is emerging as a highly effective protective agent against radiation-induced biological effects. We have shown its protective effect on space radiation-induced death of MCF-10 cells as well as on space radiation-induced transformation of HTori-3 cells. The present study was aimed at elucidation of molecular mechanisms and cellular pathways involved in SeM-mediated radioprotection. Human thyroid epithelial cells (HTori-3 cells), in the presence or absence of SeM, were exposed to a non-toxic or a slightly toxic radiation dose from 1 GeV/n iron ions (10 cGy and 20 cGy, respectively). Total RNA was prepared and changes in gene expression were analyzed using microarray technology. Our analysis has revealed a dramatic effect of SeM on alterations of gene expression caused by space radiation. This study provides a basis for furthering our knowledge about radiation-induced molecular and cellular changes that lead to cellular transformation and death.

Aerospace Medicine↗
PubMed2005

Ethanol potentiates HIV-1 gp120-induced apoptosis in human neurons via both the death receptor and NMDA receptor pathways.

Neuronal loss is a hallmark of AIDS dementia syndromes. Human immunodeficiency virus type I (HIV-1)-specific proteins may induce neuronal apoptosis, but the signal transduction of HIV-1 gp120-induced, direct neuronal apoptosis remains unclear. Ethanol (EtOH) is considered to be an environmental co-factor in AIDS development. However, whether EtOH abuse in patients with AIDS increases neuronal dysfunction is still uncertain. Using pure, differentiated, and post-mitotic NT2.N-derived human neurons, we investigated the mechanisms of HIV-1 and/or EtOH-related direct neuronal injury and the molecular interactions between HIV-1-specific proteins and EtOH. It was demonstrated that NT2.N neurons were susceptible to HIV-1 Bal (R5-tropic strain) gp120-induced direct cell death. Of importance, EtOH induced cell death in human neurons in a clinically-relevant dose range and EtOH strongly potentiated HIV-1 gp120-induced neuronal injury at low and moderate concentrations. Furthermore, this potentiation of neurotoxicity could be blocked by N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B) antagonists. We analyzed human genomic profiles in these human neurons, using Affymetrix genomics technology, to elucidate the apoptotic pathways involved in HIV-1- and EtOH-related neurodegeneration. Our findings indicated significant over-expression of selected apoptosis functional genes. Significant up-regulation of TRAF5 gene expression may play an essential role in triggering potentiation by EtOH of HIV-1 gp120-induced neuronal apoptosis at early stages of interaction. These studies suggested that two primary apoptotic pathways, death receptor (extrinsic) and NMDA receptor (intrinsic)-related programmed cell-death pathways, are both involved in the potentiation by EtOH of HIV-1 gp120-induced direct human neuronal death. Thus, these data suggest rationally-designed, molecular targets for potential anti-HIV-1 neuroprotection.

AIDS Dementia Complex↗
PubMed2005

Micropillar array chip for integrated white blood cell isolation and PCR.

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 microm in cross-section, etched to a depth of 80 microm with adjacent pillars being separated by 3.5 microm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip.

Cell Separation↗
PubMed2005

Nanobiotechnology: the promise and reality of new approaches to molecular recognition.

Nanobiotechnology is the convergence of engineering and molecular biology that is leading to a new class of multifunctional devices and systems for biological and chemical analysis with better sensitivity and specificity and a higher rate of recognition. Nano-objects with important analytical applications include nanotubes, nanochannels, nanoparticles, nanopores and nanocapacitors. Here, we take a critical look at the subset of recent developments in this area relevant to molecular recognition. Potential benefits of using nano-objects (nanotubes, quantum dots, nanorods and nanoprisms) and nanodevices (nanocapacitors, nanopores and nanocantilevers) leading to an expanded range of label multiplexing are described along with potential applications in future diagnostics. We also speculate on further pathways in nanotechnology development and the emergence of order in this somewhat chaotic, yet promising, new field.

Biotechnology↗
PubMed2005

Region-specific detection of neuroblastoma loss of heterozygosity at multiple loci simultaneously using a SNP-based tag-array platform.

Many cancers are characterized by chromosomal aberrations that may be predictive of disease outcome. Human neuroblastomas are characterized by somatically acquired copy number changes, including loss of heterozygosity (LOH) at multiple chromosomal loci, and these aberrations are strongly associated with clinical phenotype including patient outcome. We developed a method to assess region-specific LOH by genotyping multiple SNPs simultaneously in DNA from tumor tissues. We identified informative SNPs at an average 293-kb density across nine regions of recurrent LOH in human neuroblastomas. We also identified SNPs in two copy number neutral regions, as well as two regions of copy number gain. SNPs were PCR-amplified in 12-plex reactions and used in solution-phase single-nucleotide extension incorporating tagged dideoxynucleotides. Each extension primer had 5' complementarity to one of 2000 oligonucleotides on a commercially available tag-array platform allowing for solid-phase sorting and identification of individual SNPs. This approach allowed for simultaneous detection of multiple regions of LOH in six human neuroblastoma-derived cell lines, and, more importantly, 14 human neuroblastoma primary tumors. Concordance with conventional genotyping was nearly absolute. Detection of LOH in this assay may not require comparison to matched normal DNAs because of the redundancy of informative SNPs in each region. The customized tag-array system for LOH detection described here is rapid, results in parallel assessment of multiple genomic alterations, and may speed identification of and/or assaying prognostically relevant DNA copy number alterations in many human cancers.

Humans↗
PubMed2005

Miniaturized detection technology in molecular diagnostics.

Miniaturization of genetic tests represents the convergence of molecular biology and engineering and is leading to a new class of small analyzers and test systems for genetic testing with improved analytical characteristics. Miniaturization initially focused on devices that contained micrometer-sized features designed for a particular analytical purpose (e.g., filters for cell isolation and chips for capillary electrophoresis). Now, the focus is shifting to analytical applications based on nano-sized objects such as nanotubes, nanochannels, nanoparticles, nanopores and nanocapacitors. These nanofabricated objects provide new tools for sequencing of nucleic acids and rapid, multiplexed, nucleic acid detection.

Animals↗
PubMed2004

Identification of APC gene mutations in colorectal cancer using universal microarray-based combinatorial sequencing-by-hybridization.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited form of colorectal cancer, caused mostly by mutations in the APC gene. Due to the wide variety of mutations found and the large size of the APC gene, several methods of mutation detection are used, which can be time consuming and costly. Here we demonstrate a new method of mutation detection in the APC gene using an array-based approach termed combinatorial sequencing-by-hybridization (cSBH). In cSBH, a universal probe set is attached to a support and a second one is in solution. Two-probe ligation occurs when a DNA strand from the target PCR product consecutively anneals to both unlabeled array-bound and solution-phase dye-labeled probe, creating all target-complementary long-labeled probes attached to the surface. A standard array reader scores fluorescent signals at each array position. Cell lines and patient DNA with known APC gene mutations were analyzed using a cSBH-based HyChip trade mark product. Results show that this universal hexamer (6-mer) chip can successfully detect a range of mutations. Results are very robust for a continuous readout of 3.6 kb from a PCR target, with 99.97% accuracy on a single HyChip trade mark slide. cSBH is a fast, cost-efficient method for first stage mutation screening in the APC or any other gene.

Adenomatous Polyposis Coli↗
PubMed2004

Gene expression profiling during the transition to failure in TNF-alpha over-expressing mice demonstrates the development of autoimmune myocarditis.

Transgenic mice with cardiac-specific over-expression of tumor necrosis factor-alpha (TNF1.6) progress to dilated heart failure. A significant inflammatory response precedes functional deterioration, and may contribute to cardiac damage in this model. To evaluate the underlying molecular mechanisms, we assessed the gene expression in six groups of mouse hearts defined by age, gender, and phenotype (n = 3/group) using Affymetrix microarray analysis. Phenotype was defined as compensated (in young TNF1.6) or decompensated (in older TNF1.6) via echocardiogram. Of the >1000 transcripts altered in the compensated hearts (fold change > 2, P < 0.05 vs. wild-type (WT)), 102 were identified as immune response genes, 20 of which function in antigen presentation and processing. When comparing the compensated and decompensated hearts, >50 genes were differentially regulated, including seven immunoglobulin genes. Real-time reverse transcriptase-polymerase chain reaction and cDNA microarray confirmed the Affymetrix data. Mac3+ macrophages, CD4+ T and CD45/B220+ B-cells were identified in both compensated and decompensated hearts. However, a large amount of IgG was found deposited in areas devoid of B-lymphocytes in the myocardium of decompensated TNF1.6 mice; no such accumulation was seen in the compensated or age-matched controls. Furthermore, nuclei density analyses showed a two-fold increase in the myocardium of both compensated and decompensated TNF1.6 mice (vs. WT). This study suggests that TNF-alpha over-expression activates not only the inflammatory response, but also humoral immune responses within the transgenic hearts. The autoimmune response occurs concomitantly with cardiac decompensation and may participate in triggering the transition to failure in TNF1.6 mice.

Animals↗
PubMed2004

Surface effects on PCR reactions in multichip microfluidic platforms.

We evaluated the compatibility of several common plastics, commercially available plastic tubing and disposable syringes which might be useful in the construction of microfluidic platforms with respect to the polymerase chain reaction (PCR). A simple and inexpensive plastic test module was constructed in order to evaluate some of the construction plastics. We also investigated the effect of addition of PEG 8000 to PCR reaction mixtures on the compatibility of materials. These studies identified several common plastics, plastic tubing, and disposable syringes which were compatible with the PCR reaction.

Equipment Design↗
PubMed2004

Beta-thalassemia microelectronic chip: a fast and accurate method for mutation detection.

BACKGROUND: beta-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the beta-thalassemia alleles in the Mediterranean area. METHODS: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes. RESULTS: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip. CONCLUSIONS: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing beta-thalassemia in other regions of the world.

Fluorometry↗
PubMed2004

Increased amplification efficiency of microchip-based PCR by dynamic surface passivation.

Surface passivation is critical for effective PCR using silicon-glass chips. We tested a dynamic polymer-based surface passivation method. Polyethylene glycol 8000 (PEG 8000) or polyvinylpyrrolidone 40 (PVP-40) applied at 0.75% (w/v) in the reaction mixture produced significant surface passivation effects using either native or SiO2-precoated silicon-glass chips. PCR amplification was achieved from human genomic DNA as a template as well as from human lymphocytes. The dynamic surface passivation effect of PEG 8000 remained similar under both conditions. Dynamic surface passivation offers a simple and cost-effective method to make microfabricated silicon-glass chips PCR friendly. It can also be used in combination with static passivation (silicon oxide surface layer) to further improve PCR performance using silicon-glass PCR chips.

Cell Separation↗
PubMed2004

Mutation detection using ligase chain reaction in passivated silicon-glass microchips and microchip capillary electrophoresis.

The ligase chain reaction (LCR) following PCR is one of the most sensitive and specific methods for detecting mutations, especially single nucleotide polymorphisms (SNPs). Performing LCR in microchips remains a challenge because of the inhibitory effect of the internal surfaces of silicon-glass microchips. We have tested a dynamic polymer-based surface passivation method for LCR conducted in oxide-coated silicon-glass microchips. The combination of polyvinylpyrrolidone 40 (PVP-40) at 0.75% (w/v) with an excess of the ligase produced successful LCR in the silicon-glass microchips, with yields of ligated primers comparable to reactions performed in conventional reaction tubes. Ligated primers were detected and quantified simply and conveniently using microchip capillary electrophoresis.

DNA Mutational Analysis↗
PubMed2003

Genotyping on a thermal gradient DNA chip.

Silicon-based chips with discrete, independently temperature-controlled islands have been developed for use in DNA microarray hybridization studies. Each island, containing a heater made of a diffusion layer and a temperature sensor based on a p-n junction, is created on a silicon dioxide/nitride surface by anisotropic etching. Different reactive groups are subsequently added to the surface of the islands, and allele-specific oligonucleotide probes are attached to discrete spots on the chip. Hybridization is performed with Cy5-tagged single-stranded targets derived by PCR from genomic DNA. Results are assessed by measuring fluorescence of bound dye-tagged targets after hybridization and washing. Temperatures at each island can be set at different values to obtain optimal distinction between perfect matches and mismatches. This approach facilitates definition of optimal temperatures for probe/target annealing and for distinction between perfectly matched versus mismatched solution-phase targets. The thermal gradient DNA chips were then tested for genotyping, and the results for four different loci in two genes are presented. Unambiguous typing was achieved for clinically relevant loci within the factor VII and hemochromatosis genes.

Factor VII↗