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The interaction of cytochrome c with cardiolipin (CL) is a critical step in the initial stages of apoptosis and is mediated by a positively charged region on the protein surface comprising several lysine residues (site A). Here, the interaction of wt S. cerevisiae cytochrome c (ycc) and its K72A/K73A, K72A/K79A, K73A/K79A and K72A/K73A/K79A variants with CL was studied through UV-Vis and MCD spectroscopies at pH 7 and molecular dynamics (MD) simulations, to clarify the role of the mutated lysines. Moreover, the influence of the lipid to protein ratio on the interaction mechanism was investigated using low (0.5-10) and high (5-60) CL/ycc molar ratios, obtained with small and gradual or large and abrupt CL additions, respectively. Although all proteins bind to CL, switching from the native low-spin His/Met-ligated form to a low-spin bis-His conformer and to a high-spin species at larger CL concentrations, the two schemes of CL addition show relevant differences in the CL/ycc molar ratios at which the various conformers appear, due to differences in the interaction mechanism. Extended lipid anchorage and peripheral binding appear to prevail at low and high CL/ycc molar ratios, respectively. Simultaneous deletion of two or three surface positive charges from Site A does not abolish CL binding, but instead increases protein affinity for CL. MD calculations suggest this unexpected behavior results from the mutation-induced severe weakening of the H-bond connecting the N ε of His26 with the backbone oxygen of Glu44, which lowers the conformational stability compared to the wt species, overcoming the decreased surface electrostatic interaction.

Stem cells drive embryonic and fetal development. In several adult tissues, they retain the ability to self-renew and differentiate into a variety of specialized cells, thus contributing to tissue homeostasis and repair throughout life span. Alcohol consumption is associated with an increased risk for several diseases and conditions. Growing and developing tissues are particularly vulnerable to alcohol's influence, suggesting that stem- and progenitor-cell function could be affected. Accordingly, recent studies have revealed the possible relevance of alcohol exposure in impairing stem-cell properties, consequently affecting organ development and injury response in different tissues. Here, we review the main studies describing the effects of alcohol on different types of progenitor/stem cells including neuronal, hepatic, intestinal and adventitial progenitor cells, bone-marrow-derived stromal cell, dental pulp, embryonic and hematopoietic stem cells, and tumor-initiating cells. A better understanding of the nature of the cellular damage induced by chronic and episodic heavy (binge) drinking is critical for the improvement of current therapeutic strategies designed to treat patients suffering from alcohol-related disorders.

Neuroglobin (Ngb) is a recently identified hexa-coordinated globin, expressed in the nervous system of humans. Its physiological role is still debated: one hypothesis is that Ngb serves as an electron transfer (ET) species, possibly by reducing cytochrome c and preventing it to initiate the apoptotic cascade. Here, we use the perturbed matrix method (PMM), a mixed quantum mechanics/molecular dynamics approach, to investigate the redox thermodynamics of two neuroglobins, namely the human Ngb and GLB-6 from invertebrate Caenorhabditis elegans. In particular, we calculate the reduction potential of the two globins, resulting in an excellent agreement with the experimental values, and we predict the reorganization energies, λ, which have not been determined experimentally yet. The calculated λ values match well those reported for known ET proteins and thereby support a potential involvement in vivo of the two globins in ET processes.

Background: Obesity is a major risk factor for several chronic diseases. The BioEnterics(®) Intragastric Balloon (BIB(®)) is used worldwide as a short-term (6 months) treatment in morbid obese patients. However, removal of BIB(®) past 6 months is a common occurrence in clinical practice often as a result of patient factors. The aim of the present Italian multicentre cohort study was to evaluate the safety and efficacy of the BIB(®) left in situ for more than 6 months., Materials and Methods: A total of eight Italian centres participated in the study. Participating centres received a standardised questionnaire designed to capture safety and efficacy data. Weight loss data, as well as date, and reason for removal of the BIB(®) after 6 months were recorded. Adverse events, including mortality and complications, operators' subjective technical notes and findings, and difficulties during removal procedure were also collected., Results: Six hundred and eleven patients were included. In the majority of cases, patient extended BIB(®) treatment due to satisfactory weight loss (44 %). At 6 months, all patients achieved a BMI statistically lower than the initial BMI (p < 0.001). There was a non-significant trend towards greater BMIL % in patients who underwent removal up to 15 months versus the results achieved up to 6 months. No major complications were recorded., Conclusion: Extending the duration of BIB(®) use up to 14 months safely maintains weight loss and satiety with greater results than that up to 6 months, without complications.

Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc-CL) immobilized at IMM. In this work, we have studied electrochemically cytc-CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc-CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc-CL in solution. In particular, immobilized cytc-CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/- coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH(-) states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc-CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade.

Purpose: The aim of this study is to assess the diagnostic efficacy and interobserver agreement of acoustic radiation force impulse (ARFI) elasticity imaging in differentiating thyroid nodules., Methods: In our study, 74 consecutive patients (52 females, 22 males; age range 27-77 years, mean: 41 years) with 82 thyroid nodules (60 benign nodules, and 22 malignant) were examined by two radiologists with different experience. Patients underwent either cytology using fine needle aspiration cytology or thyroid surgery. The diagnostic performance of the two operators at ARFI with sensitivity, specificity, positive predictive and negative predictive value, and ROC curves was estimated. Inter-reader variability between the two operators was defined using Cohen's k., Results: According to receiver operating characteristics ROC curves (AUROC = 0.86 for observer 1; 0.81 for observer 2) sensitivity, specificity, PPV and NPV of reader 1 and 2 were respectively: 90, 75, 90.91 and 96.55 %; (cut-off value of shear wave: 2.455 m/s); 90, 72, 90 and 96.90 % (cut-off value shear wave: 2.365 m/s). Concordance between the two operators was good (k = 0.755)., Conclusions: This work is a feasibility study evaluating ARFI imaging. Its results suggest that ARFI imaging is a reproducible method which can be utilized with good diagnostic performance in the thyroid for discriminating benign and malignant nodules using the cut-off value of 2.455 m/s. However, larger studies are needed to validate this method.

Background: Construct validity of virtual laparoscopic simulators for basic laparoscopic skills has been proposed; however, it is not yet clear whether the simulators can identify the actual experience of surgeons in more complex procedures such as laparoscopic Roux-en-Y gastric bypass. This study tested the ability of the Lap Mentor simulator to recognize the experience in advanced laparoscopic procedures and to assess its role in the certification of bariatric surgeons., Methods: Twenty surgeons were divided into two groups according to their experience in laparoscopic and bariatric surgery. The general group included 10 general surgeons performing between 75 and 100 nonbariatric laparoscopic procedures. The bariatric group included 10 bariatric surgeons performing between 50 and 100 laparoscopic bariatric procedures. Participants were tested on the simulator in one basic task (task 1: eye-hand coordination) and in two tasks of the gastric bypass module (task 2: creation of the gastric pouch; task 3: gastrojejunal anastomosis)., Results: Comparing the groups, no significant differences were found in task 1. Analyzing the results from the gastric bypass module (bariatric vs. general), in task 2, significant differences (p < 0.05) were found in the median volume of the gastric pouch (21 vs. 48 cm(3)), in the percentage of fundus included in the pouch (8.4 vs. 29.4 %), in the complete dissection at the angle of His (10 vs. 3), and in safety parameters. In task 3, significant differences were found in the size and position of enterotomies., Conclusions: The Lap Mentor may be proposed as a certification tool for bariatric surgeons because it also recognizes their specific skills in the technical details of the procedure that affect long-term results. Furthermore, the possibility of analyzing the performance in detail can help define areas where the surgeon is lacking. These findings indicate a potential role of the Lap Mentor in tailoring the training to maximize improvement.

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV-vis, magnetic circular dichroism, and (1)H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pK (a) values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of -0.144 and -0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°'.

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.

The 20-kDa di-heme cytochrome c (4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned and expressed in Escherichia coli and investigated through UV-vis and (1)H NMR spectroscopies and protein voltammetry. The model structure was computed using the X-ray structure of Pseudomonas stutzeri cytochrome c (4) as a template. The protein shows unprecedented properties within the cytochrome c (4) family, including (1) an almost nonpolar surface charge distribution, (2) the absence of high-spin heme Fe(III) states, indicative of a thermodynamically stable and kinetically inert axial heme His,Met coordination, and (3) identical E degrees ' values for the two heme centers (+0.322 V vs the standard hydrogen elecrode). At pH extremes, both heme groups undergo the "acid" and "alkaline" conformational transitions typical of class I cytochromes c, involving ligand-exchange equilibria, whereas at intermediate pH values their electronic properties are sensitive to several residue ionizations.

The thermodynamics of the alkaline transition which influences the spectral and redox properties of the type 1 copper center in phytocyanins has been determined spectroscopically. The proteins investigated include Rhus vernicifera stellacyanin, cucumber basic protein and its Met89Gln variant, and umecyanin, the stellacyanin from horseradish roots, along with its Gln95Met variant. The changes in reaction enthalpy and entropy within the protein series show partial compensatory behavior. Thus, the reaction free energy change (hence the pK (a) value) is rather variable. This indicates that species-dependent differences in reaction thermodynamics, although containing an important contribution from changes in the hydrogen-bonding network of water molecules in the hydration sphere of the protein (which feature enthalpy-entropy compensation), are to a large extent protein-based. The data for axial ligand variants are consistent with the hypothesis of a copper-binding His as the deprotonating residue responsible for this transition.

The enthalpic and entropic changes accompanying the reduction reaction of the six-coordinate cyanide adducts of cytochrome c, microperoxidase-11 and a few plant peroxidases were measured electrochemically. Once the compensating changes in reduction enthalpy and entropy due to solvent reorganization effects are factorized out, it is found that cyanide binding stabilizes enthalpically the ferriheme following the order: cyochrome c > peroxidase > microperoxidase-11. The effect is inversely correlated to the solvent accessibility of the heme. Comparison of the reduction thermodynamics for the cyanide adducts of cytochrome c and plant peroxidases with those for microperoxidase-11 and myoglobin, respectively, yielded an estimate of the consequences of protein encapsulation and of the anionic character of the proximal histidine on the reduction potential of the heme-cyanide group. Insertion of the heme-CN group into the folded peptide chain of cyt c induces an enthalpy-based decrease in E degrees ' of approximately 100 mV, consistent with the lower net charge of the oxidized as compared to the reduced iron center, whereas a full imidazolate character of the proximal histidine stabilizes enthalpically the ferriheme by approximately 400 mV. The latter value should be best considered as an upper limit since it also includes some solvation effects arising from the nature of the protein systems being compared.

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwald's approach for the interpretation of H/ S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E degrees values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.