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Biomedical subjects

M Falconi

Publications and source records attributed to M Falconi.

At least 19 recordsLinked to original sources

Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies.

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six-transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd-numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge-pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt-bridge networks with a significant shift from inter- to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even-numbered helices can undergo a face rotation, whereas odd-numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor.

Animals↗

TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system.

The need for standards in the management of patients with endocrine tumors of the digestive system prompted the European Neuroendocrine Tumor Society (ENETS) to organize a first Consensus Conference, which was held in Frascati (Rome) and was based on the recently published ENETS guidelines on the diagnosis and treatment of digestive neuroendocrine tumors (NET). Here, we report the tumor-node-metastasis proposal for foregut NETs of the stomach, duodenum, and pancreas that was designed, discussed, and consensually approved at this conference. In addition, we report the proposal for a working formulation for the grading of digestive NETs based on mitotic count and Ki-67 index. This proposal, which needs to be validated, is meant to help clinicians in the stratification, treatment, and follow-up of patients.

Biomarkers, Tumor↗

A case of intraductal papillary mucinous tumour following recurrent attacks of pancreatitis lasting 26 years.

We report an interesting case of a patient with neither family nor personal history for pancreatic diseases that was admitted to our department in 1982, at the age of 25 years. At that time, medical history, absence of alcohol abuse, and radiological imaging suggested a diagnosis of idiopathic chronic pancreatitis. The patient underwent a left-pancreatectomy, with histological confirmation of chronic pancreatitis. He was asymptomatic until 1988, when episodes of pain arose, requiring a pancreatico-jejunostomy. No further problems ensued until 2004 when radiological investigation following pain-related symptoms revealed enormous dilation of the pancreatic duct. A pylorus-preserving pancreaticoduodenectomy resulting in total pancreatectomy was performed. Histological examination revealed an intraductal papillary mucinous non-invasive carcinoma. Review of the previously resected specimen revealed former misdiagnosis. This tumour usually affects an elderly population and nowadays is recognised as a possible cause of chronic obstructive pancreatitis. This report represents a slippery case of misdiagnosis and demonstrates that follow-up is always mandatory following a diagnosis of idiopathic chronic pancreatitis.

Adenocarcinoma, Mucinous↗

Development of a disease-specific Quality of Life questionnaire module for patients with gastrointestinal neuroendocrine tumours.

Quality of life (QoL) measurements are increasingly being used as an end point in cancer clinical trials. Standard generic QoL questionnaires may not assess symptoms produced by neuroendocrine tumours. Here we report the development of a disease-specific quality of life score questionnaire for patients with neuroendocrine tumours of the gut to supplement the EORTC core cancer questionnaire, the QLQ-C30. Phases 1-3 of the EORTC quality of life group guidelines for module development were used to design the new questionnaire. Forty-one relevant issues (questions) were generated after an extensive literature search. Following interviews of 15 health care workers and 35 patients, a 35 question provisional questionnaire was constructed. This was translated into seven European languages and pre-tested in 180 patients resulting in a 21-item module that will be validated in an international clinical trial. The EORTC QLQ-NET21 provides a site-specific module to supplement the QLQ-C30 for patients with neuroendocrine tumours.

Adolescent↗

Carcinoid crisis induced by receptor radionuclide therapy with 90Y-DOTATOC in a case of liver metastases from bronchial neuroendocrine tumor (atypical carcinoid).

SS receptors are overexpressed in many tumors, mainly of neuroendocrine origin, thus enabling the treatment with SS analogs. The clinical experience of receptor radionuclide therapy with the new analog [90Y-DOTA0-Tyr3 ]-octreotide [90Y-DOTATOC] has been developed over the last decade and is gaining a pivotal role in the therapeutic workout of these tumors. It is well known that some procedures performed in diagnostic and therapeutic management of endocrine tumors, such as agobiopsy and hepatic chemoembolization, can be associated with the occurrence of symptoms related to the release of vasoactive amines and/or hormonal peptides from tumor cell lysis. This is the first report of a severe carcinoid crisis developed after receptor radionuclide therapy with 90Y-DOTATOC administered in a patient affected by liver metastases from bronchial neuroendocrine tumor (atypical carcinoid). Despite protection with H1 receptor antagonists, octreotide and corticosteroids, few days after the therapy the patient complained of persistent flushing of the face and upper trunk, severe labial and periocular oedema, diarrhoea and loss of appetite. These symptoms increased and required new hospitalisation. The patient received iv infusion of octreotide associated with H1 and H2 receptor antagonists and corticosteroid therapy, which induced symptom remission within few days. The case here reported confirms that radionuclide therapy is highly effective in determining early rupture of metastatic tissue and also suggests that pre-medication should be implemented before the radiopeptide administration associated with a close monitoring of the patient in the following days.

Aged↗

Reduced antigenicity of type I collagen and proteoglycans in sclerotic dentin.

Antigenic alterations to the dentin organic matrix may be detected by an immunohistochemical approach. We hypothesized that alterations in the antigenicity of type I collagen and proteoglycans occur in sclerotic dentin under caries lesions. Transverse sections were prepared from carious teeth in the sclerotic zone and normal hard dentin. A double-immunolabeling technique was performed on these sections, with anti-type I collagen and anti-chondroitin 4/6 sulfate monoclonal primary antibodies. We used gold-conjugated secondary antibodies to visualize the distribution of intact collagen fibrils and proteoglycans by high-resolution SEM. For sclerotic dentin, labeling densities were 19.57 +/- 3.01/microm2 for collagen and 9.84 +/- 2.62/microm2 for proteoglycans. For normal hard dentin, values were 35.20 +/- 2.73/microm2 and 17.03 +/- 1.98/microm2, respectively. Distribution of intact collagen fibrils and proteoglycans in sclerotic dentin was significantly lower than in normal hard dentin. Reductions in antigenicity from the organic matrix of sclerotic dentin under caries lesions raise concern about the potential of intrafibrillar remineralization.

Collagen Type I↗

Predictive factors of efficacy of the somatostatin analogue octreotide as first line therapy for advanced pancreatic endocrine carcinoma.

About 40% of nonfunctioning pancreatic endocrine carcinomas (NF-PEC) cannot be cured by surgery due to advanced stage disease. Somatostatin analogues have been proposed as first line therapy in these cases. We performed a prospective phase IV study to assess the efficacy of octreotide in advanced NF-PEC and identify factors predictive of response to therapy. Twenty-one consecutive patients with octreoscan-positive advanced-stage well-differentiated NF-PEC were treated with long-acting release octreotide 20 mg i.m. at diagnosis. The immunohistochemical expression of somatostatin receptor 2 (SSTR2) and the quantitative mRNA analysis of SSTR2 and SSTR5 were assessed in 12 tumours. The tumour proliferative fraction was assessed by immunohistochemistry for Ki-67. Eight patients (38%) had stable disease (SD) after a median follow-up of 49.5 months. Thirteen patients (62%) developed progression after a median of 18 months. Tumour progression correlated with a proliferative index>or=5% (P=0.016), weight loss (P=0.006) and absence of abdominal pain (P=0.003) at diagnosis. Other clinical (age, gender and primary tumour resection) or pathological parameters (site, size and liver metastasis) lacked significant correlation with tumour progression. No difference in the amount of SSTR2 mRNA and protein or SSTR5 mRNA was found between tumours that were stable (n=5) and seven tumours that progressed (n=7). Treatment with long-acting release octreotide was associated with stabilization of disease and a good quality of life in 38% of patients. A Ki-67 index>or=5% and/or the presence of weight loss may justify more aggressive therapy without waiting for radiologically proven progression of disease.

Aged↗

Long-term clinical outcome of somatostatin analogues for treatment of progressive, metastatic, well-differentiated entero-pancreatic endocrine carcinoma.

BACKGROUND: Knowledge of factors able to predict the clinical outcome of homogenous series of entero-pancreatic endocrine tumours treated with somatostatin analogues is poor. This study was aimed at identifying predictors for efficacy of somatostatin analogues at inhibiting tumour growth and modifying patients' survival during long-term follow-up. PATIENTS AND METHODS: 31 patients with entero-pancreatic well-differentiated endocrine carcinoma received long-acting somatostatin analogues. All had progressive, metastatic disease (87% liver metastases, 38.7% distant extra-hepatic metastases). RESULTS: Response rate after 6 months of treatment was 45.2% (all disease stabilisation: 27.8% of pancreatic vs. 81.8% of intestinal tumours, P = 0.007). The predictors for non-response were: pancreatic tumour (OR 5.8), no previous surgery (OR 6.7), and the presence of distant extra-hepatic metastases, the latter being also confirmed by multivariate analysis (OR 10.0). Responders maintained stabilisation for 26.5 months, and none died during follow-up. Different survival curves were observed for patients, responding at 6 months compared to non-responders (P = 0.004), 3-year survival rate being 100% and 52.3%, respectively. CONCLUSIONS: Distant extra-hepatic metastases are the major predictor of poor efficacy of somatostatin analogues in progressive, metastatic, well-differentiated entero-pancreatic endocrine carcinomas. Patients achieving response after 6 months of treatment, maintain it throughout a long-term follow-up. Non-responders after 6 months of treatment, have a worse survival, and should be considered for alternative treatments.

Adult↗

High resolution morphological analysis of in situ human chromosomes.

The purpose of this study was to analyze the inner structure of chromosomes in cells arrested, fixed and cryosectioned in metaphase. The chromosomes in metaphase maps prepared using standard cytogenetic protocols, are usually covered by cellular debris, which obscures the structural details on the surface and limits analysis by techniques when using nanometric resolution. By using cryosectioning, the debris is removed and it is possible to analyze the internal structure of the chromosomes. We described the ultrastructure of chromosome sections fixed with either acetic acid, methanol or glutaraldehyde, evaluating the effect and the influence of the fixative on the morphology. Furthermore, we subjected those cells previously fixed with glutaraldehyde to osmic maceration in order to better visualize the intracellular structure. All samples were examined with a Field Emission In Lens Scanning Electron Microscope (FEISEM), which allows high-resolution analysis of biological samples without any metal coating. The results showed a package morphology in samples fixed with glutaraldehyde, mainly due to the high capacity of the fixative to strongly crosslink the proteins. In contrast, the fibrillar structure seen in cryosections fixed with acetic acid/methanol is due to the propensity of the fixatives to extract and remove proteins. We propose that in situ chromosomes fixed with glutaraldehyde and then osmicated are a good model for studying the inner structure of chromosomes by using high resolution scanning electron microscopy.

Chromosomes, Human↗

Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface.

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.

Amino Acids↗

Utility of combined use of plasma levels of chromogranin A and pancreatic polypeptide in the diagnosis of gastrointestinal and pancreatic endocrine tumors.

BACKGROUND: Chromogranin A (CgA) is considered the most accurate marker in the diagnosis of gastro-entero-pancreatic (GEP) endocrine tumors. Pancreatic polypeptide (PP) has also been proposed to play this role, but then not used due to its low sensitivity. The aim of the present study was to determine whether the assessment of PP would improve the diagnostic reliability of CgA in patients with GEP tumors. PATIENTS AND METHODS: Both markers were assessed in 68 patients [28 functioning (F), 40 non functioning (NF)]. Twenty-seven patients disease-free (DF) after surgery, and 24 with non-endocrine tumors (non-ETs) were used as control groups. RESULTS: CgA sensitivity was: 96% in F, 75% in NF, 74% in pancreatic, and 91% in gastrointestinal (GI) tumors. Specificity was 89% vs DF, and 63% vs non-ETs. PP sensitivity was: 54% in F, 57% in NF, 63% in pancreatic, and 53% in GI tumors. Specificity was 81% vs DF, and 67% vs non-ETs. By combining the two markers a significant gain in sensitivity vs CgA alone was obtained: overall in GEP tumors (96% vs 84%, p = 0.04), in NF (95% vs 75%, p = 0.02), and in pancreatic (94% vs 74%, p = 0.04). More specifically, a 25% gain of sensitivity was obtained in the subgroup of NF pancreatic tumors (93% vs 68%, p = 0.04). CONCLUSION: The combined assessment of PP and CgA leads to a significant increase in sensitivity in the diagnosis of GEP tumors, particularly in pancreatic NF.

Adult↗

Role of unlabelled somatostatin analogues in the prevention of complications after elective pancreatic and peripancreatic surgery: a critical review.

Although studies on the use of the somatostatin analogues in the elective pancreatic surgery are mostly prospective, double blind and randomised, the results are contradictory and not univocally interpretable. Through the examination of all randomised perspective works published on this subject, a critical interpretation is attempted which may give relevant suggestions for further studies. A new clinical, randomised, double blind and multicentric prospective trial should take into proper consideration even the changes which have occurred in the care of the patients. Over the years a significant decrease of postoperative hospital stay and a deeper awareness of the medical expenses have been observed. Moreover, since the drug has a potential advantage on specific pancreatic complications, only these must be considered among the end points of the study and the population studied will be limited exclusively to patients who underwent resection of the pancreatic head or of the periampullar region because of neoplastic disease. Finally, the selection of the centres that enrol the patients must be considered, since the expertise of each operator or of the team, affects, as an independent variable, both morbidity and mortality.

Double-Blind Method↗

Prospective multicentre survey on acute pancreatitis in Italy (ProInf-AISP): results on 1005 patients.

BACKGROUND AND AIM: Till now, no Italian studies providing information on acute pancreatitis have been published. The aim of this study was to evaluate the epidemiological and clinical characteristics of acute pancreatitis in Italy. MATERIALS AND METHODS: The study involved 37 Italian centres distributed homogeneously throughout the entire national territory and prospectively collected epidemiological, anamnestic, laboratory, radiological, therapeutic (pharmacological, endoscopic and surgical) data, relevant to each individual case of acute pancreatitis consecutively observed during the period from September 1996 to June 2000. RESULTS: One thousand two hundred and six case report forms were collected, but 201 patients (16.6%) were subsequently eliminated from the final analysis. We therefore studied 1005 patients, 533 (53%) males and 472 (47%) females, mean age 59.6 +/- 20 years. On the basis of the Atlanta classification of acute pancreatitis, 753 patients of the 1005 cases analysed (75%) were mild and 252 patients (25%) severe. The aetiology was biliary in 60% of the patients, related to alcohol abuse in 8.5%, while in 21% of the cases it could not be identified. Over 80% of the patients (83%) were admitted to hospital within 24 h from the onset of clinical symptoms, while only 6% were admitted after 48 h. In particular, 65% of the patients were admitted to hospital within the first 12 h. Antibiotics were used in 85% of the severe and 75% of mild forms. Endoscopic therapy was carried out in 65% of the severe cases, but only in 40% it was carried out prior to 72 h. Eighty-five patients (8.5% of the total, 34% of the severe forms) underwent surgical intervention: 20% on the first day, 38.5% within the fourth day, and the remaining (41.5% of the cases) later on for infected necrosis. The mean duration of hospitalisation for patients with mild pancreatitis was 13 +/- 8 days, while for the severe disease it was of 30 +/- 14 days. The overall mortality rate was 5%, 17% in severe and 1.5% in mild pancreatitis. CONCLUSIONS: Acute pancreatitis in Italy is more commonly a mild disease with a biliary aetiology. The treatment of the disease is not optimal and, on the basis of these data, needs to be standardised. Despite this, the overall mortality rate is low (5%).

Acute Disease↗

Thermophilicity of wild type and mutant cold shock proteins by molecular dynamics simulation.

In the attempt to clarify possible mechanisms underlying thermal stability of proteins, we study through molecular dynamics thermophile Bc-Csp, mesophile Bs-CspB, and selected mutants. These proteins have been extensively characterized experimentally; researchers showed that differential thermostability among the wild type proteins is fundamentally linked to one or two mutated amino acids, and that the nature of the effect is electrostatic. They also inferred an atomistic mechanism related to removal of unfavorable interactions, rather than to the formation of salt bridges. Molecular dynamics allows us to confirm and support both hypotheses. Several other collective parameters have also been monitored in relation to thermophilicity, such as global and local rigidity, permanence and number of hydrogen bonds, or of salt links. None of these clearly correlates with the thermal stability of the presently studied proteins.

Bacillus↗

Immunocytochemical analysis of dentin: a double-labeling technique.

Immunocytochemical analysis is a fundamental and selective technique for identifying different molecular components of human dental structure. The hypothesis tested here is that the application of different etching solutions on dentin does not hinder collagen fibrils and proteoglycans from maintaining their immunochemical antigenicity. Human dentin disks were treated with 0.5M of EDTA, citric acid, maleic acid, or phosphoric acid (for 15 or 30 s). A double-immunolabeling technique was performed to identify, simultaneously, collagen fibrils and chondroitin sulfate. The use of different acids resulted in different degrees of labeling. Maleic and citric acids revealed a diffuse and intense labeling for both collagen fibrils and proteoglycans. The use of phosphoric acid on dentin showed a massive coagulation of the proteoglycans (15 s) or very low labeling (30 s). These data clarify that the use of acids on dentin components is able to modify their antigenicity. Moreover, the double-labeling immunocytochemical technique allows understanding of the spatial relationships between the collagen fibrils and proteoglycans of the dentin matrix.

Animals↗

Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy).

Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI-HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated.

Complementarity Determining Regions↗

Static and dynamic water molecules in Cu,Zn superoxide dismutase.

Understanding protein hydration is a crucial, and often underestimated issue, in unraveling protein function. Molecular dynamics (MD) computer simulation can provide a microscopic description of the water behavior. We have applied such a simulative approach to dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase, comparing the water molecule sites determined using 1.0 ns MD simulation with those detected by X-ray crystallography. Of the water molecules detected by the two techniques, 20% fall at common sites. These are evenly distributed over the protein surface and located around crevices, which represent the preferred hydration sites. The water mean residence time, estimated by means of a survival probability function on a given protein hydration shell, is relatively short and increases for low accessibility sites constituted by polar atoms. Water molecules trapped in the dimeric protein intersubunit cavity, as identified in the crystal structure, display a trajectory mainly confined within the cavity. The simulation shows that these water molecules are characterized by relatively short residence times, because they continuously change from one site to another within the cavity, thus hinting at the absence of any relationship between spatial and temporal order for solvent molecules in proximity of protein surface.

Algorithms↗

Active-site copper and zinc ions modulate the quaternary structure of prokaryotic Cu,Zn superoxide dismutase.

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, Delta V, and the dissociation constant K(d0), at atmospheric pressure, (25 ml/mol and 3 x 10(-7)M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower Delta G degrees limit of 11.5 kcal/mol, corresponding to a dissociation constant K(d0)<10(-9)M.

Binding Sites↗