Functional Characterization of Two Novel Mutations in SCN5A Associated with Brugada Syndrome Identified in Italian Patients

Background. Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac disease characterized by “coved type” ST-segment elevation in the right precordial leads, high susceptibility to ventricular arrhythmia and a family history of sudden cardiac death. The SCN5A gene, encoding for the cardiac voltage-gated sodium channel Nav1.5, accounts for ~20–30% of BrS cases and is considered clinically relevant. Methods. Here, we describe the clinical findings of two Italian families affected by BrS and provide the functional characterization of two novel SCN5A mutations, the missense variant Pro1310Leu and the in-frame insertion Gly1687_Ile1688insGlyArg. Results. Despite being clinically different, both patients have a family history of sudden cardiac death and had history of arrhythmic events. The Pro1310Leu mutation significantly reduced peak sodium current density without affecting channel membrane localization. Changes in the gating properties of expressed Pro1310Leu channel likely account for the loss-of-function phenotype. On the other hand, Gly1687_Ile1688insGlyArg channel, identified in a female patient, yielded a nearly undetectable sodium current. Following mexiletine incubation, the Gly1687_Ile1688insGlyArg channel showed detectable, albeit very small, currents and biophysical properties similar to those of the Nav1.5 wild-type channel. Conclusions. Overall, our results suggest that the degree of loss-of-function shown by the two Nav1.5 mutant channels correlates with the aggressive clinical phenotype of the two probands. This genotype-phenotype correlation is fundamental to set out appropriate therapeutical intervention.     

Acute neonatal respiratory distress in Italy: a one-year prospective study. Italian Group of Neonatal Pneumology

We treated two cases of primary pulmonary cryptococcosis with fluconazole (FLCZ), the clinical usefulness of FLCZ was evaluated. FLCZ was administered orally in doses of 300 mg daily for about six months. Concentrations of FLCZ were measured in the serum of the two cases and in the bronchoalveolar lavage (BAL) fluid in one case. The following results were obtained: 1. Clinical cures were obtained in the two cases. 2. The serum levels of FLCZ was 15.1 microliters/ml, 13.6 micrograms/ml two hours after administration of 100 mg in case 1, that of levels were 11.1 micrograms/ml, 8.9 micrograms/ml one hour and 4.5 hours, respectively, after administration of 100 mg in case 2. BAL was performed 4.5 hours after administration of 100 mg in case 2, the BAL fluid level of FLCZ was 0.7 microgram/ml. 3. The minimal inhibitory concentration of FLCZ against one strain obtained from the cytology brush in case 1 was 4.0 micrograms/ml. 4. The cryptococcal antigen titer decreased with the improvement of clinical signs and the resolution of chest X-ray abnormalities within about six months, and there was no relapse. From these results, we consider that FLCZ is a useful antifungal agent for primary pulmonary cryptococcosis, and we therefore recommend a six month treatment.     

Procedure to Perform a Validated Incremental Hole Drilling Measurement: Application to Shot Peening Residual Stresses

Abstract: The paper describes a test rig designed to check and assess the accuracy of the incremental hole drilling (IHD) method. An external load produces a controlled linearly through thickness variable uniaxial stress field (reference bending stress), known with good accuracy, that can be applied and removed at each hole increment. After the separation between the bending relaxed strain from the residual stress relaxed strain, it is possible to reproduce the bending stress distribution in order to have complete confidence of the residual stress measurement. The bending verification of the IHD method was already proposed by other investigators before, but residual stress measurements were then performed on independent configurations. The proposed testing procedure gives a ‘real time’ verification of the residual stress measurement. Any experimental malfunctioning due to the operator inexperience, or any error during the stress calculation from the relaxed strain would produce an evident difference between the expected reference bending stress and the IHD bending stress output. Moreover, the reference bending stress helped for understanding that the not perfect hole cylindrical shape causes an underestimate of the predicted stress near the surface. A zero depth offset correction was proposed. This correction was tested on the reference bending stress, and then applied to the residual stress prediction. Three shot peening residual stresses IHD measurements were successfully validated by means of the bending stress; moreover, they were in good agreement with independent X‐ray diffraction measures also proposed in the paper.     

Combined experimental and numerical investigations on the roughness effects on the aerodynamic performances of LPT blades

The aerodynamic performance of a high-load low-pressure turbine blade cascade has been analyzed for three different distributed surface roughness levels (Ra) for steady and unsteady inflows. Results from CFD simulations and experiments are presented for two different Reynolds numbers (300000 and 70000 representative of take-off and cruise conditions, respectively) in order to evaluate the roughness effects for two typical operating conditions.     

High Load Ratio Fatigue Strength and Mean Stress Evolution of Quenched and Tempered 42CrMo4 Steel

The fatigue strength at a high number of cycles with initial elastic–plastic behavior was experimentally investigated on quenched and tempered 42CrMo4 steel. Fatigue tests on unnotched specimens were performed both under load and strain controls, by imposing various levels of amplitude and with several high load ratios. Different ratcheting and relaxation trends, with significant effects on fatigue, are observed and discussed, and then reported in the Haigh diagram, highlighting a clear correlation with the Smith–Watson–Topper model. High load ratio tests were also conducted on notched specimens with C (blunt) and V (sharp) geometries. A Chaboche model with three parameter couples was proposed by fitting plain specimen cyclic and relaxation tests, and then finite element analyses were performed to simulate the notched specimen test results. A significant stress relaxation at the notch root became clearly evident by reporting the numerical results in the Haigh diagram, thus explaining the low mean stress sensitivity of the notched specimens.     

Probing the backbone dynamics of oxidized and reduced rat microsomal cytochrome b5 via 15N rotating frame NMR relaxation measurements: biological implications

Rotating frame 15N relaxation NMR experiments have been performed to study the local mobility of the oxidized and reduced forms of rat microsomal cytochrome b5, in the microsecond to millisecond time range. Measurements of rotating frame relaxation rates (R1rho) were performed as a function of the effective magnetic field amplitude by using off-resonance radio frequency irradiation. Detailed analysis of the two data sets resulted in the identification of slow motions along the backbone nitrogens for both oxidation states of the protein. The local mobility of reduced and oxidized cytochrome b5 turned out to be significantly different; 28 backbone nitrogens of the oxidized form were shown to participate in a conformational exchange process, while this number dropped to 12 in the reduced form. The correlation time, tauex, for the exchange processes could be determined for 21 and 9 backbone nitrogens for oxidized and reduced cytochrome b5, respectively, with their values ranging between 70 and 280 microseconds. The direct experimental evidence provided in this study for the larger mobility of the oxidized form of the protein is consistent with the different backbone NH solvent exchangeability recently documented for the two oxidation states [Arnesano, F., et al. (1998) Biochemistry 37, 173-184]. Our experimental observations may have significant biological implications. The differential local mobility between the two oxidation states is proposed to be an important factor controlling the molecular recognition processes in which cytochrome b5 is involved.     

Towards a protocol for solution structure determination of copper(II) proteins: the case of Cu(II)Zn(II) superoxide dismutase

We have developed an optimized protocol to solve the solution structure of copper(II) proteins. After assignment, proton-proton NOEs are used for the shell where 1H spectra are conveniently observed. In a shell closer to the metal ion, 13C NMR spectra with band-selective homonuclear decoupling provide the assignment of all nuclei except for those of the metal ligands. A convenient method for the measurement of 13C longitudinal-relaxation rates (R1) of carbonyls and carboxylate moieties is proposed. 1H NOEs and 1H and 13C R1 data are sufficient to produce a good/reasonable solution structure, as demonstrated for a monomeric species of superoxide dismutase, a 153-residue protein.