Angelo Minucci | Università Cattolica del Sacro Cuore (Catholic University of the Sacred Heart) (original) (raw)

Papers by Angelo Minucci

Research paper thumbnail of DNA from buccal swab is suitable for rapid genotyping of angiotensin-converting enzyme insertion/deletion (I/D) polymorphism

Clinica chimica acta; international journal of clinical chemistry

Plasma angiotensin-converting enzyme (ACE) variability between individuals is the results of an i... more Plasma angiotensin-converting enzyme (ACE) variability between individuals is the results of an insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene. The I and D alleles differ for the presence or absence of a 288bp Alu sequence DNA fragment. The present paper regards the development of a single-tube High Resolution Melting Analysis (HRMA), applied to DNA extracted by buccal swabs, for determining three ACE I/I, I/D, D/D genotypes, in order to obtain a rapid and high throughput method. This method takes advantage by the presence of the 288bp DNA fragment. Primers design was performed taking into account the possible different efficiency of allele I amplification compared to allele D, avoiding the misclassification of I/D with D/D genotypes. 50 samples previously genotyped by "conventional" PCR protocol already published in literature were 100% concordant with the HRMA results, showing high reproducibility, sensitivity and specificity. ACE genotypes were disti...

Research paper thumbnail of Evaluation of plasma thiolic groups and reactive oxygen metabolites in critically ill patients

Critical Care, 2008

all 10 cities including the rural areas of the province of Kerman. All data were finally analyzed... more all 10 cities including the rural areas of the province of Kerman. All data were finally analyzed by SPSS software (version 11.5). Results On the basis of recorded statistical analysis, the mortality cases of human rabies in the province of Kerman during one decade was 10 persons (eight males and two females). One-half of them (50%) were bitten by dogs and the others (50%) by foxes. Among the reported deaths, 40% were from Kahnooj county (Jiroft region). The reported data indicated that 21,546 persons were bitten by animals during 10 years in the province of Kerman. The mean of age of the people who were bitten by dogs was 24.80 years (SD = ±14.6), while the mean age of the people who were bitten by foxes was 57.25 years (SD = ±1.50). There was a significant difference between the mean age of these two groups of the people (P < 0.05). The most frequent rate of injured people was reported in the age group 10-19 years old and the frequency rate of males (76.00%) was more than females (24.00%). Therefore, there was a statistically significant difference between males and females in this study (P < 0.01). About 60% of all persons that were bitten by animals were from rural areas and 40% of them were from urban areas (P < 0.05). Among the people who were bitten and injured by animals during one decade in the province of Kerman, 85.70% of them were not treated by the rabies prophylaxis treatment regimen. Among all of them who were bitten by animals, 50% were injured through hands and feet, 40%

Research paper thumbnail of Incidental Finding of a Homozygous p.M348K Asymptomatic Italian Patient Confirms the Many Faces of Cystic Fibrosis

Case Reports in Genetics, 2015

Cystic fibrosis (CF; OMIM number 219700) is an autosomal recessive disease caused by mutations in... more Cystic fibrosis (CF; OMIM number 219700) is an autosomal recessive disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which results in abnormal viscous mucoid secretions in multiple organs and whose main clinical features are pancreatic insufficiency, chronic endobronchial infection, and male infertility. We report the case of a 47-year-old apparently normal male resulting in homozygosity for the mutation p.M348K from nonconsanguineous parents. The proband was screened using a standard panel of 70 different tested on NanoChip 400 platform. The massive parallel pyrosequencing on 454 JS machine allowed the second level analysis. The patient was firstly screened with two different platforms available in our laboratory, obtaining an ambiguous signal for the p.R347P mutation. For this reason we decided to clarify the discordant result of CFTR status by Next Generation Sequencing (NGS) using 454 Junior instrument. The patient is resulted no carrier of the p.R347P mutation, but NGS highlighted a homozygous substitution from T&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;A at position 1043 in the coding region, causing an amino acid substitution from methionine to lysine (p.M348K). Casual finding of p.M348K homozygote mutation in an individual, without any feature of classical or nonclassical CF form, allowed us to confirm that p.M348K is a benign rare polymorphism.

Research paper thumbnail of Description of an Automated Method for Urea Nitrogen Determination in Bronchoalveolar Lavage Fluid (BALF) of Neonates and Infants

Journal of laboratory automation, Jan 13, 2015

Bronchoalveolar lavage (BAL) partially recovers both the instilled saline and the alveolar fluid,... more Bronchoalveolar lavage (BAL) partially recovers both the instilled saline and the alveolar fluid, so-called epithelial lining fluid (ELF), but a correction for the dilution due to the BAL technique itself is needed to know the amount of recovered ELF. In this regard, urea nitrogen may be useful and has been proposed to calculate ELF. The aim of the present study was to develop and validate a new method to measure urea nitrogen in BAL fluid (BALF). We used 19 BALF samples obtained from neonates and infants with different respiratory conditions. The urea nitrogen assay was carried out on Cobas c311 analyzer (Roche Diagnostics). A validation study shows that the method is perfectly linear (R(2) = 0.999), sensitive (limit of detection = 0.055 mg/dL; limit of quantification = 0.16 mg/dL), repeatable (low = 0.15 ± 0.02, 13.3%; high = 1.80 ± 0.02, 1.1%), reproducible (low = 0.14 ± 0.02, 14.2 %; high = 1.76 ± 0.04, 2.2 %) with accuracy ranging between 93-96%. Our results support the robustn...

Research paper thumbnail of Effect of whole body hypothermia on inflammation and surfactant function in asphyxiated neonates

The European respiratory journal, 2014

Hypothermia has become an evidence-based treatment for neonates with hypoxic-ischaemic encephalop... more Hypothermia has become an evidence-based treatment for neonates with hypoxic-ischaemic encephalopathy (HIE) . The usefulness of hypothermia is due to several mechanisms and among these the reduction in inflammation seems to play a relevant role . When applied as whole body hypothermia (WBH), cooling may affect other organs as well. Recent data showed better respiratory outcomes and trends towards lower inflammation in WBH-treated preterm lambs [4], suggesting its possible usefulness to reduce lung injury through the modulation of the inflammatory pathway. By contrast, experiments with hibernating animals have shown that temperature induces significant adaptive changes to the surfactant composition and structure . Nevertheless, no data are currently available in humans. Two case reports have recently described an infant [6] and an adult [7] with severe lung injury, whose ventilation had been facilitated by concurrent hypothermia. Since hypothermia is an accepted therapy only for HIE, we designed a preliminary translational study to investigate the effect of WBH on inflammation and surfactant status in neonates with HIE unaffected by any pulmonary injury. LETTER IN PRESS | CORRECTED PROOF 1 .

Research paper thumbnail of Co-inheritance of G6PD and PK deficiencies in a neonate carrying a Novel UGT1A1 genotype associated to Crigler-Najjar type II syndrome

Pediatric Blood & Cancer, 2015

Abbreviations: CHA, Chronic hemolytic anemia; G6PD, Glucose-6-phosphate dehydrogenase; PK, Pyruva... more Abbreviations: CHA, Chronic hemolytic anemia; G6PD, Glucose-6-phosphate dehydrogenase; PK, Pyruvate kinase; PK-LR, Pyruvate kinase, liver and RBC; TSB, Total serum bilirubin; UGT1A1, Uridine-5 0 -diphosphate glucuronosyltransferase isoform 1A1

Research paper thumbnail of A preliminary Quality Control (QC) for next generation sequencing (NGS) library evaluation turns out to be a very useful tool for a rapid detection of BRCA1/2 deleterious mutations

Clinica Chimica Acta, 2014

Background: Recent advances in next generation sequencing (NGS) technology have enabled comprehen... more Background: Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the entire genomic region of BRCA1/2 genes and, to date, many studies report the effectiveness of these technologies. Here we show that Gene Scan (GS) labeling Quality Control (QC), performed before massive parallel pyrosequencing, coupled with Multiple Amplicon Quantification software (MAQ-S) analysis is a rapid and powerful tool in the detection of deleterious BRCA mutations carried by different patients. Methods: GS labeling QC assay was performed according to the manufacturers' instructions and MAQ-S software was employed for analysis results. Results: GS labeling QC was able to detect 14 different BRCA frameshift mutations in our patients. In addition, two novel BRCA mutations (c.1893_1894insTTAAGCCCACAAAT in BRCA1 gene and c.9413_9414insT in BRCA2 gene) were identified. Conclusion: We prove that a simple QC step may represent a valid and useful tool for a rapid detection of frameshift mutations in BRCA genes. For this reason, we recommend using this approach before massive parallel sequencing.

Research paper thumbnail of A prolonged neonatal jaundice associated with a rare G6PD mutation

Pediatric Blood & Cancer, 2009

Research paper thumbnail of Novel human pathological mutations. Gene symbol: MYBPC3. Disease: cardiomyopathy, hypertrophic

Human genetics, 2010

... Tiago Matos, H. Caria, H. Teixeira, G. Fialho Centre of Genetics and Molecular Biology, ICAT,... more ... Tiago Matos, H. Caria, H. Teixeira, G. Fialho Centre of Genetics and Molecular Biology, ICAT, Campus da FCUL, Campo Grande 1749-016, Lisbon, Portugal, e-mail: tiagomorimatos@gmail.com, Tel.: +351-217-500006, Fax: +351-217-500172 ...

Research paper thumbnail of Secretory phospholipase A2 pathway during pediatric acute respiratory distress syndrome: A preliminary study

Pediatric Critical Care Medicine, 2011

To verify if secretory phospholipase A2 (sPLA2) is increased in pediatric acute respiratory distr... more To verify if secretory phospholipase A2 (sPLA2) is increased in pediatric acute respiratory distress syndrome (ARDS) triggered or not by respiratory syncytial virus infection and to clarify how the enzyme may influence the disease severity and the degree of ventilatory support. Prospective pilot study. Two academic pediatric intensive care units. All infants &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 6 months old hospitalized for severe respiratory syncytial virus bronchiolitis, who developed ARDS (respiratory syncytial virus-ARDS group); all infants &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 6 months old diagnosed with ARDS secondary to other causes (ARDS group); and infants &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 6 months old who needed ventilation for reasons other than any lung disease (control group). None. We enrolled six respiratory syncytial virus -ARDS babies, five ARDS babies, and six control infants. The sPLA2 activity and tumor necrosis factor (TNF)-α were significantly higher in the bronchoalveolar lavage of ARDS infants. Worst oxygenation, ventilation, and longer pediatric intensive care unit stay and ventilation time were present in ARDS babies. No differences were found in Clara cell secretory protein and in serum cytokines levels. Because there is no correlation between bronchoalveolar lavage protein content (a marker of permeability) and sPLA2, the enzyme seems mainly produced in the alveoli. TNF-α, the main inductor of sPLA2 expression, significantly correlates with the enzyme level in the bronchoalveolar lavage. Significant positive correlations exist between sPLA2, TNF-α and oxygen need, mean airway pressure, ventilatory index, and the Murray&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s lung injury score. Negative correlations were also found between sPLA2, TNF-α, and Pao2/Fio2 ratio. The sPLA2 and TNF-α are increased in ARDS and seem correlated with clinical severity, higher oxygen requirement, and more aggressive ventilation. This correlation confirms findings from adult experience and should guide further investigations on pediatric ARDS pathophysiology.

Research paper thumbnail of Feasibility of extracellular competitive inhibition of phospholipase A2 in neonatal and pediatric lung injury

Pediatric Anesthesia, 2011

Research paper thumbnail of Homocysteine Lowering by Folate-Rich Diet or Pharmacological Supplementations in Subjects with Moderate Hyperhomocysteinemia

Nutrients, 2013

Background/Objectives: To compare the efficacy of a diet rich in natural folate and of two differ... more Background/Objectives: To compare the efficacy of a diet rich in natural folate and of two different folic acid supplementation protocols in subjects with "moderate" hyperhomocysteinemia, also taking into account C677T polymorphism of

Research paper thumbnail of Effect of aqueous cigarette smoke extract on the chemiluminescence kinetics of polymorphonuclear leukocytes and on their glycolytic and phagocytic activity

Luminescence, 2001

Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as sa... more Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as saliva or fluid lining alveolar spaces, and can act on both cellular and extracellular compartments. In this paper we have analysed the effect of aqueous smoke extract on some metabolic and functional aspects of polymorphonuclear leukocytes. In particular, the following cellular aspects were studied: chemiluminescence, glycolysis, membrane fluidity and microscopic interaction with zymosan particles. While chemiluminescence and glycolytic activity are highly inhibited, no effect of smoke extract on membrane fluidity was observed. Moreover, the response of luminol-dependent chemiluminescence was significantly delayed, while that of lucigenin-dependent chemiluminescence was anticipated. Furthermore, the phagocytic ability of neutrophils pretreated with aqueous smoke extract was also significantly hindered. All these results might indicate that the finely tuned activity of polymorphonuclear leukocytes is somehow hampered by the aqueous extract of cigarette smoke in a way which makes these cells less effective against bacteria and more noxious towards surrounding tissues.

Research paper thumbnail of Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione

Luminescence, 2005

Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many ... more Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

Research paper thumbnail of PCR experion automated electrophoresis system to detect Listeria monocytogenes in foods

Journal of Separation Science, 2009

Listeria monocytogenes is frequently found as a contaminant in raw and ready-to-eat foods. The ab... more Listeria monocytogenes is frequently found as a contaminant in raw and ready-to-eat foods. The ability of L. monocytogenes to multiply at refrigeration temperatures and to grow in a wide range of pH values is of particular concern for food safety. According to the European Union regulation on microbiological criteria for foodstuffs, L. monocytogenes must be absent in some categories of ready-to-eat foods. The standard microbiological method for L. monocytogenes detection in foods (ISO 11290-1: 1996 (ISO, International Organization for Standardization)) is cost and time consuming. Developments of rapid, cost-effective and automated diagnostic methods to detect food-borne pathogens in foods continue to be a major concern for the industry and public health. The aim of this study was the development of a rapid, sensitive and specific molecular detection method for L. monocytogenes. To this purpose, we have applied a capillary electrophoresis method to a PCR protocol (PCR-EES (EES, experion automated electrophoresis system)) for detecting L. monocytogenes in food. In particular, a microfluidic chip-based automated electrophoresis system (experion automated electrophoresis system, Bio-Rad Laboratories, USA) was used for the rapid and automatic analysis of the amplicons. Fifty naturally contaminated samples were analysed with this method and the results were compared with those obtained with ISO method. Moreover, the microfluidic chip-based automated electrophoresis system was compared with classical gel electrophoresis (PCR-CGE). The results showed that after 24 h of culture enrichment, the PCR-EES showed a relative accuracy of 100% with ISO, while using PCR-CGE decreased it down to 96%. After 48 h of enrichment, both PCR-EES and PCR-CGE showed an accuracy of 100% with ISO.

Research paper thumbnail of Identification of RFLP G6PD mutations by using microcapillary electrophoretic chips (Experion TM )

Journal of Separation Science, 2008

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most wellknown human genetic de... more Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most wellknown human genetic defects, identified in more than 400 million individuals in the world. To date, no commercial kits are available for the mutation screening of this disease. Seventy G6PD-deficient Italian individuals admitted to the Laboratory of Clinical Molecular Biology of Hospital "Agostino Gemelli" of Rome were screened for the most frequent Italian mutations, by means of allele-specific PCR, followed by restriction fragment length electrophoresis. The present study compares two techniques for the identification of restriction patterns: agarose gel electrophoresis versus Experion system. When the first screening was negative, the entire G6PD gene was sequenced using the ABI 3100 Avant Instrumentation. The G6PD variants identified and their frequencies were the following: G6PD Mediterranean (75.7%), G6PD Seattle (7.1%), G6PD A -202 + 376 (7.1%), and G6PD Cassano (2.8%). In addition, we identified by direct sequencing two new mutations, namely Buenos Aires and Rignano. With the Experion method, the size band determination was more accurate than that obtained by gel electrophoresis. The Experion system resulted as a valid, easy, and reproducible diagnostic method for the screening of G6PD mutation as compared with the agarose electrophoretic analysis.

Research paper thumbnail of Is capillary electrophoresis on microchip devices able to genotype uridine diphosphate glucuronosyltransferase 1A1 TATA-box polymorphisms?

Journal of Separation Science, 2014

In this commentary, we focused our attention on capillary electrophoresis. It achieves the effici... more In this commentary, we focused our attention on capillary electrophoresis. It achieves the efficient separation of molecular species by the application of high voltages to samples in solution. Actually, capillary electrophoresis can be performed on microchip devices, based on an automated and miniaturized electrophoresis system, based on lab-on-a-chip technology. By this technology it is possible to separate nucleic acid fragments (DNA or RNA) with respect to sizing accuracy and sizing resolution. Currently, two automated capillary electrophoresis on microchips devices are available: the Agilent 2100 Bioanalyzer and the Experion™ Automated Electrophoresis System. In this study, we evaluated if the CE is able to distinguish the three uridine diphosphate glucuronosyltransferase 1A1 TATA-box genotypes.

Research paper thumbnail of Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins

Journal of Dentistry, 2007

Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacryla... more Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.

Research paper thumbnail of Two novel CYP21A2 missense mutations in Italian patients with 21-hydroxylase deficiency: Identification and functional characterisation

IUBMB Life, 2009

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal... more Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia (CAH), an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. In this report, we report the characterisation of two novel CYP21A2 missense mutations (p.H119R and p.I194N) found in two unrelated Italian patients with nonclassic (NC) CAH clinical diagnosis. Functional in vitro assays for mutagenized CYP21 enzymes were performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained for p.H119R and p.I194N mutants allowed to classify them as NC-CAH associated mutations. These results correlate with the rate of severity of the patients' disease. Finally, the new p.H119R and p.I194N mutations should be included in the panel of those already listed for association with the NC form of 21-hydroxylase deficiency. 2009 IUBMB IUBMB Life, 61(3): 229-235, 2009

Research paper thumbnail of Glucose-6-phosphate dehydrogenase laboratory assay: How, when, and why?

IUBMB Life, 2009

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells.... more Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells. Although some different laboratory techniques or methods are employed for the biochemical screening, a strict relationship between biochemists, clinicians, and molecular biologists is necessary for a definitive diagnosis. This article represents an overview on the current laboratory tests finalized to the screening or to the definitive diagnosis of G6PD-deficiency, underlying the problems regarding the biochemical and molecular identification of heterozygote females other than those regarding the standardization of the clinical and laboratory diagnostic procedures. Finally, this review is aimed to give a flow-chart for the complete diagnostic approach of G6PD-deficiency.

Research paper thumbnail of DNA from buccal swab is suitable for rapid genotyping of angiotensin-converting enzyme insertion/deletion (I/D) polymorphism

Clinica chimica acta; international journal of clinical chemistry

Plasma angiotensin-converting enzyme (ACE) variability between individuals is the results of an i... more Plasma angiotensin-converting enzyme (ACE) variability between individuals is the results of an insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene. The I and D alleles differ for the presence or absence of a 288bp Alu sequence DNA fragment. The present paper regards the development of a single-tube High Resolution Melting Analysis (HRMA), applied to DNA extracted by buccal swabs, for determining three ACE I/I, I/D, D/D genotypes, in order to obtain a rapid and high throughput method. This method takes advantage by the presence of the 288bp DNA fragment. Primers design was performed taking into account the possible different efficiency of allele I amplification compared to allele D, avoiding the misclassification of I/D with D/D genotypes. 50 samples previously genotyped by "conventional" PCR protocol already published in literature were 100% concordant with the HRMA results, showing high reproducibility, sensitivity and specificity. ACE genotypes were disti...

Research paper thumbnail of Evaluation of plasma thiolic groups and reactive oxygen metabolites in critically ill patients

Critical Care, 2008

all 10 cities including the rural areas of the province of Kerman. All data were finally analyzed... more all 10 cities including the rural areas of the province of Kerman. All data were finally analyzed by SPSS software (version 11.5). Results On the basis of recorded statistical analysis, the mortality cases of human rabies in the province of Kerman during one decade was 10 persons (eight males and two females). One-half of them (50%) were bitten by dogs and the others (50%) by foxes. Among the reported deaths, 40% were from Kahnooj county (Jiroft region). The reported data indicated that 21,546 persons were bitten by animals during 10 years in the province of Kerman. The mean of age of the people who were bitten by dogs was 24.80 years (SD = ±14.6), while the mean age of the people who were bitten by foxes was 57.25 years (SD = ±1.50). There was a significant difference between the mean age of these two groups of the people (P < 0.05). The most frequent rate of injured people was reported in the age group 10-19 years old and the frequency rate of males (76.00%) was more than females (24.00%). Therefore, there was a statistically significant difference between males and females in this study (P < 0.01). About 60% of all persons that were bitten by animals were from rural areas and 40% of them were from urban areas (P < 0.05). Among the people who were bitten and injured by animals during one decade in the province of Kerman, 85.70% of them were not treated by the rabies prophylaxis treatment regimen. Among all of them who were bitten by animals, 50% were injured through hands and feet, 40%

Research paper thumbnail of Incidental Finding of a Homozygous p.M348K Asymptomatic Italian Patient Confirms the Many Faces of Cystic Fibrosis

Case Reports in Genetics, 2015

Cystic fibrosis (CF; OMIM number 219700) is an autosomal recessive disease caused by mutations in... more Cystic fibrosis (CF; OMIM number 219700) is an autosomal recessive disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which results in abnormal viscous mucoid secretions in multiple organs and whose main clinical features are pancreatic insufficiency, chronic endobronchial infection, and male infertility. We report the case of a 47-year-old apparently normal male resulting in homozygosity for the mutation p.M348K from nonconsanguineous parents. The proband was screened using a standard panel of 70 different tested on NanoChip 400 platform. The massive parallel pyrosequencing on 454 JS machine allowed the second level analysis. The patient was firstly screened with two different platforms available in our laboratory, obtaining an ambiguous signal for the p.R347P mutation. For this reason we decided to clarify the discordant result of CFTR status by Next Generation Sequencing (NGS) using 454 Junior instrument. The patient is resulted no carrier of the p.R347P mutation, but NGS highlighted a homozygous substitution from T&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;A at position 1043 in the coding region, causing an amino acid substitution from methionine to lysine (p.M348K). Casual finding of p.M348K homozygote mutation in an individual, without any feature of classical or nonclassical CF form, allowed us to confirm that p.M348K is a benign rare polymorphism.

Research paper thumbnail of Description of an Automated Method for Urea Nitrogen Determination in Bronchoalveolar Lavage Fluid (BALF) of Neonates and Infants

Journal of laboratory automation, Jan 13, 2015

Bronchoalveolar lavage (BAL) partially recovers both the instilled saline and the alveolar fluid,... more Bronchoalveolar lavage (BAL) partially recovers both the instilled saline and the alveolar fluid, so-called epithelial lining fluid (ELF), but a correction for the dilution due to the BAL technique itself is needed to know the amount of recovered ELF. In this regard, urea nitrogen may be useful and has been proposed to calculate ELF. The aim of the present study was to develop and validate a new method to measure urea nitrogen in BAL fluid (BALF). We used 19 BALF samples obtained from neonates and infants with different respiratory conditions. The urea nitrogen assay was carried out on Cobas c311 analyzer (Roche Diagnostics). A validation study shows that the method is perfectly linear (R(2) = 0.999), sensitive (limit of detection = 0.055 mg/dL; limit of quantification = 0.16 mg/dL), repeatable (low = 0.15 ± 0.02, 13.3%; high = 1.80 ± 0.02, 1.1%), reproducible (low = 0.14 ± 0.02, 14.2 %; high = 1.76 ± 0.04, 2.2 %) with accuracy ranging between 93-96%. Our results support the robustn...

Research paper thumbnail of Effect of whole body hypothermia on inflammation and surfactant function in asphyxiated neonates

The European respiratory journal, 2014

Hypothermia has become an evidence-based treatment for neonates with hypoxic-ischaemic encephalop... more Hypothermia has become an evidence-based treatment for neonates with hypoxic-ischaemic encephalopathy (HIE) . The usefulness of hypothermia is due to several mechanisms and among these the reduction in inflammation seems to play a relevant role . When applied as whole body hypothermia (WBH), cooling may affect other organs as well. Recent data showed better respiratory outcomes and trends towards lower inflammation in WBH-treated preterm lambs [4], suggesting its possible usefulness to reduce lung injury through the modulation of the inflammatory pathway. By contrast, experiments with hibernating animals have shown that temperature induces significant adaptive changes to the surfactant composition and structure . Nevertheless, no data are currently available in humans. Two case reports have recently described an infant [6] and an adult [7] with severe lung injury, whose ventilation had been facilitated by concurrent hypothermia. Since hypothermia is an accepted therapy only for HIE, we designed a preliminary translational study to investigate the effect of WBH on inflammation and surfactant status in neonates with HIE unaffected by any pulmonary injury. LETTER IN PRESS | CORRECTED PROOF 1 .

Research paper thumbnail of Co-inheritance of G6PD and PK deficiencies in a neonate carrying a Novel UGT1A1 genotype associated to Crigler-Najjar type II syndrome

Pediatric Blood & Cancer, 2015

Abbreviations: CHA, Chronic hemolytic anemia; G6PD, Glucose-6-phosphate dehydrogenase; PK, Pyruva... more Abbreviations: CHA, Chronic hemolytic anemia; G6PD, Glucose-6-phosphate dehydrogenase; PK, Pyruvate kinase; PK-LR, Pyruvate kinase, liver and RBC; TSB, Total serum bilirubin; UGT1A1, Uridine-5 0 -diphosphate glucuronosyltransferase isoform 1A1

Research paper thumbnail of A preliminary Quality Control (QC) for next generation sequencing (NGS) library evaluation turns out to be a very useful tool for a rapid detection of BRCA1/2 deleterious mutations

Clinica Chimica Acta, 2014

Background: Recent advances in next generation sequencing (NGS) technology have enabled comprehen... more Background: Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the entire genomic region of BRCA1/2 genes and, to date, many studies report the effectiveness of these technologies. Here we show that Gene Scan (GS) labeling Quality Control (QC), performed before massive parallel pyrosequencing, coupled with Multiple Amplicon Quantification software (MAQ-S) analysis is a rapid and powerful tool in the detection of deleterious BRCA mutations carried by different patients. Methods: GS labeling QC assay was performed according to the manufacturers' instructions and MAQ-S software was employed for analysis results. Results: GS labeling QC was able to detect 14 different BRCA frameshift mutations in our patients. In addition, two novel BRCA mutations (c.1893_1894insTTAAGCCCACAAAT in BRCA1 gene and c.9413_9414insT in BRCA2 gene) were identified. Conclusion: We prove that a simple QC step may represent a valid and useful tool for a rapid detection of frameshift mutations in BRCA genes. For this reason, we recommend using this approach before massive parallel sequencing.

Research paper thumbnail of A prolonged neonatal jaundice associated with a rare G6PD mutation

Pediatric Blood & Cancer, 2009

Research paper thumbnail of Novel human pathological mutations. Gene symbol: MYBPC3. Disease: cardiomyopathy, hypertrophic

Human genetics, 2010

... Tiago Matos, H. Caria, H. Teixeira, G. Fialho Centre of Genetics and Molecular Biology, ICAT,... more ... Tiago Matos, H. Caria, H. Teixeira, G. Fialho Centre of Genetics and Molecular Biology, ICAT, Campus da FCUL, Campo Grande 1749-016, Lisbon, Portugal, e-mail: tiagomorimatos@gmail.com, Tel.: +351-217-500006, Fax: +351-217-500172 ...

Research paper thumbnail of Secretory phospholipase A2 pathway during pediatric acute respiratory distress syndrome: A preliminary study

Pediatric Critical Care Medicine, 2011

To verify if secretory phospholipase A2 (sPLA2) is increased in pediatric acute respiratory distr... more To verify if secretory phospholipase A2 (sPLA2) is increased in pediatric acute respiratory distress syndrome (ARDS) triggered or not by respiratory syncytial virus infection and to clarify how the enzyme may influence the disease severity and the degree of ventilatory support. Prospective pilot study. Two academic pediatric intensive care units. All infants &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 6 months old hospitalized for severe respiratory syncytial virus bronchiolitis, who developed ARDS (respiratory syncytial virus-ARDS group); all infants &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 6 months old diagnosed with ARDS secondary to other causes (ARDS group); and infants &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 6 months old who needed ventilation for reasons other than any lung disease (control group). None. We enrolled six respiratory syncytial virus -ARDS babies, five ARDS babies, and six control infants. The sPLA2 activity and tumor necrosis factor (TNF)-α were significantly higher in the bronchoalveolar lavage of ARDS infants. Worst oxygenation, ventilation, and longer pediatric intensive care unit stay and ventilation time were present in ARDS babies. No differences were found in Clara cell secretory protein and in serum cytokines levels. Because there is no correlation between bronchoalveolar lavage protein content (a marker of permeability) and sPLA2, the enzyme seems mainly produced in the alveoli. TNF-α, the main inductor of sPLA2 expression, significantly correlates with the enzyme level in the bronchoalveolar lavage. Significant positive correlations exist between sPLA2, TNF-α and oxygen need, mean airway pressure, ventilatory index, and the Murray&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s lung injury score. Negative correlations were also found between sPLA2, TNF-α, and Pao2/Fio2 ratio. The sPLA2 and TNF-α are increased in ARDS and seem correlated with clinical severity, higher oxygen requirement, and more aggressive ventilation. This correlation confirms findings from adult experience and should guide further investigations on pediatric ARDS pathophysiology.

Research paper thumbnail of Feasibility of extracellular competitive inhibition of phospholipase A2 in neonatal and pediatric lung injury

Pediatric Anesthesia, 2011

Research paper thumbnail of Homocysteine Lowering by Folate-Rich Diet or Pharmacological Supplementations in Subjects with Moderate Hyperhomocysteinemia

Nutrients, 2013

Background/Objectives: To compare the efficacy of a diet rich in natural folate and of two differ... more Background/Objectives: To compare the efficacy of a diet rich in natural folate and of two different folic acid supplementation protocols in subjects with "moderate" hyperhomocysteinemia, also taking into account C677T polymorphism of

Research paper thumbnail of Effect of aqueous cigarette smoke extract on the chemiluminescence kinetics of polymorphonuclear leukocytes and on their glycolytic and phagocytic activity

Luminescence, 2001

Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as sa... more Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as saliva or fluid lining alveolar spaces, and can act on both cellular and extracellular compartments. In this paper we have analysed the effect of aqueous smoke extract on some metabolic and functional aspects of polymorphonuclear leukocytes. In particular, the following cellular aspects were studied: chemiluminescence, glycolysis, membrane fluidity and microscopic interaction with zymosan particles. While chemiluminescence and glycolytic activity are highly inhibited, no effect of smoke extract on membrane fluidity was observed. Moreover, the response of luminol-dependent chemiluminescence was significantly delayed, while that of lucigenin-dependent chemiluminescence was anticipated. Furthermore, the phagocytic ability of neutrophils pretreated with aqueous smoke extract was also significantly hindered. All these results might indicate that the finely tuned activity of polymorphonuclear leukocytes is somehow hampered by the aqueous extract of cigarette smoke in a way which makes these cells less effective against bacteria and more noxious towards surrounding tissues.

Research paper thumbnail of Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione

Luminescence, 2005

Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many ... more Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

Research paper thumbnail of PCR experion automated electrophoresis system to detect Listeria monocytogenes in foods

Journal of Separation Science, 2009

Listeria monocytogenes is frequently found as a contaminant in raw and ready-to-eat foods. The ab... more Listeria monocytogenes is frequently found as a contaminant in raw and ready-to-eat foods. The ability of L. monocytogenes to multiply at refrigeration temperatures and to grow in a wide range of pH values is of particular concern for food safety. According to the European Union regulation on microbiological criteria for foodstuffs, L. monocytogenes must be absent in some categories of ready-to-eat foods. The standard microbiological method for L. monocytogenes detection in foods (ISO 11290-1: 1996 (ISO, International Organization for Standardization)) is cost and time consuming. Developments of rapid, cost-effective and automated diagnostic methods to detect food-borne pathogens in foods continue to be a major concern for the industry and public health. The aim of this study was the development of a rapid, sensitive and specific molecular detection method for L. monocytogenes. To this purpose, we have applied a capillary electrophoresis method to a PCR protocol (PCR-EES (EES, experion automated electrophoresis system)) for detecting L. monocytogenes in food. In particular, a microfluidic chip-based automated electrophoresis system (experion automated electrophoresis system, Bio-Rad Laboratories, USA) was used for the rapid and automatic analysis of the amplicons. Fifty naturally contaminated samples were analysed with this method and the results were compared with those obtained with ISO method. Moreover, the microfluidic chip-based automated electrophoresis system was compared with classical gel electrophoresis (PCR-CGE). The results showed that after 24 h of culture enrichment, the PCR-EES showed a relative accuracy of 100% with ISO, while using PCR-CGE decreased it down to 96%. After 48 h of enrichment, both PCR-EES and PCR-CGE showed an accuracy of 100% with ISO.

Research paper thumbnail of Identification of RFLP G6PD mutations by using microcapillary electrophoretic chips (Experion TM )

Journal of Separation Science, 2008

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most wellknown human genetic de... more Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most wellknown human genetic defects, identified in more than 400 million individuals in the world. To date, no commercial kits are available for the mutation screening of this disease. Seventy G6PD-deficient Italian individuals admitted to the Laboratory of Clinical Molecular Biology of Hospital "Agostino Gemelli" of Rome were screened for the most frequent Italian mutations, by means of allele-specific PCR, followed by restriction fragment length electrophoresis. The present study compares two techniques for the identification of restriction patterns: agarose gel electrophoresis versus Experion system. When the first screening was negative, the entire G6PD gene was sequenced using the ABI 3100 Avant Instrumentation. The G6PD variants identified and their frequencies were the following: G6PD Mediterranean (75.7%), G6PD Seattle (7.1%), G6PD A -202 + 376 (7.1%), and G6PD Cassano (2.8%). In addition, we identified by direct sequencing two new mutations, namely Buenos Aires and Rignano. With the Experion method, the size band determination was more accurate than that obtained by gel electrophoresis. The Experion system resulted as a valid, easy, and reproducible diagnostic method for the screening of G6PD mutation as compared with the agarose electrophoretic analysis.

Research paper thumbnail of Is capillary electrophoresis on microchip devices able to genotype uridine diphosphate glucuronosyltransferase 1A1 TATA-box polymorphisms?

Journal of Separation Science, 2014

In this commentary, we focused our attention on capillary electrophoresis. It achieves the effici... more In this commentary, we focused our attention on capillary electrophoresis. It achieves the efficient separation of molecular species by the application of high voltages to samples in solution. Actually, capillary electrophoresis can be performed on microchip devices, based on an automated and miniaturized electrophoresis system, based on lab-on-a-chip technology. By this technology it is possible to separate nucleic acid fragments (DNA or RNA) with respect to sizing accuracy and sizing resolution. Currently, two automated capillary electrophoresis on microchips devices are available: the Agilent 2100 Bioanalyzer and the Experion™ Automated Electrophoresis System. In this study, we evaluated if the CE is able to distinguish the three uridine diphosphate glucuronosyltransferase 1A1 TATA-box genotypes.

Research paper thumbnail of Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins

Journal of Dentistry, 2007

Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacryla... more Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.

Research paper thumbnail of Two novel CYP21A2 missense mutations in Italian patients with 21-hydroxylase deficiency: Identification and functional characterisation

IUBMB Life, 2009

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal... more Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia (CAH), an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. In this report, we report the characterisation of two novel CYP21A2 missense mutations (p.H119R and p.I194N) found in two unrelated Italian patients with nonclassic (NC) CAH clinical diagnosis. Functional in vitro assays for mutagenized CYP21 enzymes were performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained for p.H119R and p.I194N mutants allowed to classify them as NC-CAH associated mutations. These results correlate with the rate of severity of the patients' disease. Finally, the new p.H119R and p.I194N mutations should be included in the panel of those already listed for association with the NC form of 21-hydroxylase deficiency. 2009 IUBMB IUBMB Life, 61(3): 229-235, 2009

Research paper thumbnail of Glucose-6-phosphate dehydrogenase laboratory assay: How, when, and why?

IUBMB Life, 2009

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells.... more Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells. Although some different laboratory techniques or methods are employed for the biochemical screening, a strict relationship between biochemists, clinicians, and molecular biologists is necessary for a definitive diagnosis. This article represents an overview on the current laboratory tests finalized to the screening or to the definitive diagnosis of G6PD-deficiency, underlying the problems regarding the biochemical and molecular identification of heterozygote females other than those regarding the standardization of the clinical and laboratory diagnostic procedures. Finally, this review is aimed to give a flow-chart for the complete diagnostic approach of G6PD-deficiency.