Computational methods and theory for ion channel research.

Ion channels are fundamental biological devices that act as gates in order to ensure selective ion transport across cellular membranes; their operation constitutes the molecular mechanism through which basic biological functions, such as nerve signal transmission and muscle contraction, are carried out. Here, we review recent results in the field of computational research on ion channels, covering theoretical advances, state-of-the-art simulation approaches, and frontline modeling techniques. We also report on few selected applications of continuum and atomistic methods to characterize the mechanisms of permeation, selectivity, and gating in biological and model channels.

Management of large perforations of the sinus mucosa with PRGF-Endoret® platelet concentrate.

The aim of the present study is to describe a new technique through which it is possible to complete the maxillary sinus lift procedure even in case of severe damage or complete removal of the sinus mucosa using the PRGF-Endoret® platelet concentrate. Eighteen patients (ratio F:M=4:5; average age: 58.2 years; DS: 8.85 years) with severe perforation (more than 10 millimetres of diameter) of the sinus mucosa during the maxillary sinus lift procedure were selected. Normally the procedure is interrupted due to impossible stabilization of the graft material inside the subantral cavity. On the contrary, our protocol foreseen the sealing of the perforation using the PRGF autologous gel membranes or the creation of a new sinus pseudomembrane through which the graft material was covered. The PRGF-Endoret were obtained according to the protocol developed by BTI (Biotechnology Institute - Vitoria, Spain). In 14 cases out of 18 implant fixtures were concurrently inserted while in 4 cases the fixture insertion was postponed after 6 months: 37 fixtures were inserted (27 at the same time and 10 after 6 months). 2 months after surgery the CBCT showed a correct pneumatization of the maxillary sinus in 16 patients out of 18 (89% of cases), while after 12 months the radiological normalization of the maxillary sinus was present in 17 patients out of 18, bringing the healing rate to 94% of cases. Regarding implant healing, 2 out of 37 implants inserted were lost in the first month after the surgical phase, whereas 12 months after prosthesis application the other 35 implants were perfectly osteointegrated with a healing rate equal to 94.6% of the fixtures. 36 months after the surgery all the fixtures were osteointegrated (35 of 37 implants with a percentage of 94.6% of success). We may conclude that the use of PRGF allowed to complete the sinus lift even in case of severe perforation of the sinus mucosa or its total removal thanks to its capability to stabilize the graft, its antibacterial and antifungal activity and its anabolic effect and favouring bone regeneration.

Analysis of the foot load in subjects with prevalent unilateral chewing.

The relationship between occlusion and posture has been and is still strongly debated. This study examines 40 male and female healthy subjects, (11 males and 29 females, average age: 26.27 years, st dv: 3.03) aged between 21 and 32. The baropodometric evaluation was performed with the subject in rest position and in usual centric occlusion. The results obtained were analyzed using a baropodometric platform and Freesteps software (Sensor Medica srl, Rome - Italy) analyzing the podalic load, the foot axis and the foot angles. The values reported show that 80% of subjects in rest position (p-value 0.01) and 70% of subjects in centric occlusion (p-value 0.05) have a greater foot load in the contralateral foot compared to the chewing side; moreover, the foot axis values are statistically significant because 77% of subjects in rest position and 72% in centric occlusion have a foot axis more open on the same side than the chewing one. The foot angles values are not significant. These results could be understood analyzing the activation of the body muscular chains: on the chewing side there is an increase of the activity of the flexion chain with side bending of the trunk. This induces a change of the body barycenter compensated by an outer rotation of the homolateral leg and foot; moreover, the body bending creates a false short leg on the same side, explaining the excess of podalic load on the other side. These values show that the hypothesis of a correlation appears to be likely, although obviously it still needs confirmation and further analysis.

Pressure control in interfacial systems: Atomistic simulations of vapor nucleation.

A large number of phenomena of scientific and technological interest involve multiple phases and occur at constant pressure of one of the two phases, e.g., the liquid phase in vapor nucleation. It is therefore of great interest to be able to reproduce such conditions in atomistic simulations. Here we study how popular barostats, originally devised for homogeneous systems, behave when applied straightforwardly to heterogeneous systems. We focus on vapor nucleation from a super-heated Lennard-Jones liquid, studied via hybrid restrained Monte Carlo simulations. The results show a departure from the trends predicted for the case of constant liquid pressure, i.e., from the conditions of classical nucleation theory. Artifacts deriving from standard (global) barostats are shown to depend on the size of the simulation box. In particular, for Lennard-Jones liquid systems of 7000 and 13 500 atoms, at conditions typically found in the literature, we have estimated an error of 10-15 kBT on the free-energy barrier, corresponding to an error of 104-106 s-1σ-3 on the nucleation rate. A mechanical (local) barostat is proposed which heals the artifacts for the considered case of vapor nucleation.

Activated Wetting of Nanostructured Surfaces: Reaction Coordinates, Finite Size Effects, and Simulation Pitfalls.

A liquid in contact with a textured surface can be found in two states, Wenzel and Cassie. In the Wenzel state the liquid completely wets the corrugations while in the Cassie state the liquid is suspended over the corrugations with air or vapor trapped below. The superhydrophobic properties of the Cassie state are exploited for self-cleaning, drag reduction, drug delivery, etc., while in the Wenzel state most of these properties are lost; it is therefore of great fundamental and technological interest to investigate the kinetics and mechanism of the Cassie-Wenzel transition. Computationally, the Cassie-Wenzel transition is often investigated using enhanced sampling ("rare events") techniques based on the use of collective variables (CVs). The choice of the CVs is a crucial task because it affects the free-energy profile, the estimation of the free-energy barriers, and the evaluation of the mechanism of the process. Here we investigate possible simulation artifacts introduced by common CVs adopted for the study of the Cassie-Wenzel transition: the average particle density in the corrugation of a textured surface and the coarse-grained density field at various levels of coarse graining. We also investigate possible additional artifacts associated with finite size effects. We focus on a pillared surface, a system often used in technological applications. We show that the use of a highly coarse-grained density (a single CV) of the fluid in the interpillar region brings to severe artifacts: errors of hundreds of kBT in the difference of free energy between the Cassie and Wenzel states, of tens of kBT in the estimate of the free-energy barriers, and erroneous wetting mechanisms. A proper description of the wetting mechanism and its energetics apparently requires a fine discretization of the density field. Concerning the finite-size effects, we have found that the typical systems employed in simulations of the Cassie-Wenzel transition, containing a single pillar within periodic boundary conditions, prevent the complete break of translational symmetry of the liquid-vapor meniscus during the process. Capturing this break of symmetry is crucial for describing the transition state along the wetting process and the early stage of the opposite process, the Wenzel-Cassie transition.

Intrusion and extrusion of a liquid on nanostructured surfaces.

Superhydrophobicity is connected to the presence of gas pockets within surface asperities. Upon increasing the pressure this 'suspended' state may collapse, causing the complete wetting of the rough surface. In order to quantitatively characterize this process on nanostructured surfaces, we perform rare-event atomistic simulations at different pressures and for several texture geometries. Such an approach allows us to identify for each pressure the stable and metastable states and the free energy barriers separating them. Results show that, by starting from the superhydrophobic state and increasing the pressure, the suspended state abruptly collapses at a critical intrusion pressure. If the pressure is subsequently decreased, the system remains trapped in the metastable state corresponding to the wet surface. The liquid can be extruded from the nanostructures only at very negative pressures, by reaching the critical extrusion pressure (spinodal for the confined liquid). The intrusion and extrusion curves form a hysteresis cycle determined by the large free energy barriers separating the suspended and wet states. These barriers, which grow very quickly for pressures departing from the intrusion/extrusion pressure, are shown to strongly depend on the texture geometry.

Thrombin and thrombin-derived peptides promote proliferation of cardiac progenitor cells in the form of cardiospheres without affecting their differentiation potential.

Many studies demonstrated that human adult cardiac progenitor cells in the form of cardiospheres (CSps) could represent a powerful candidate for cardiac cell therapy. To achieve the clinical translation of this biotechnological product, the development of well-defined culture conditions is required to optimize their proliferation and differentiation. Thrombin, a serine protease acting through the protease-activated receptor 1 (PAR-1) signalling to modulate many cellular functions such as proliferation and differentiation in several cell types, is one of the factors included in the CSps medium. Therefore, the assessment of the effective dependence of the thrombin related cellular effects from PAR-signalling is strategic both for understanding the biological potential of these cells and for the GMP translation of the medium formulation, using synthesised analogs. In this study the effects of thrombin on human CSps and their potential relationship with the specific proteolytic activation of PAR-1 have been investigated in different culture conditions, including thrombin inhibitor hirudin and PAR-1 agonist/ antagonist peptides TFLLR and MUMB2. In this study we show that, in the presence of thrombin and TFLLR, CSps, in which PAR-1 expression was evidenced by immunofluorescence and western blot analysis, increase their proliferation activity (BrdU assay). Such increased proliferative rate was consistently associated with a higher phosphorylation level of the cell cycle inhibitor GSK3. Concerning the assessment of the potential effects of thrombin and its agonist on differentiation, both western blot and real-time PCR analysis for stemness, cardiac and vascular markers (such as cKit, cx43 and KDR) showed that CSps commitment was substantially unaffected, except for GATA4 mRNA, whose transcription was down-regulated in the presence of the natural protease, but not after treatment with TFLLR. In conclusion, activation of PAR-1-dependent signalling is important to support CSps proliferative potential, keeping unaltered or at best stable their differentiation properties. The availability of thrombin agonists, such as TFLLR, able to guarantee the required growth effect without affecting CSps lineage commitment, could represent a technological improvement for cost-effective, easy-to-handle and GMPtranslatable synthetic media.

Caffeine-induced Ca(2+) signaling as an index of cardiac progenitor cells differentiation.

Cardiac progenitor cells (CPCs), migrating from heart tissue, in culture aggregate to form cardiospheres (CSs) in which replication and cardiogenic differentiation occur. However, the frequency of functional differentiation in CSs and the role of cell clustering in supporting it remain to be established. The aim of our study is to quantify differentiation of a muscle-type Ca(2+) release mechanism in CS-derived cells, correlate it with cardiac differentiation markers and test its dependency on CS formation. CPCs migrating from murine cardiac explants were studied prior and after CSs formation (Pre-CS and Post-CS). Inducibility of RyR- and IP3-R-mediated Ca(2+) transients in individual cells was tested by exposure to caffeine and ATP, respectively; expression of cardiac and non-cardiac lineage markers was assessed. Caffeine responsiveness was negligible in Pre-CS cells and increased by 7.5 fold in Post-CS cells (3.6 vs. 26.9%; p < 0.05), and was closely correlated with activation of the cardiac TnI gene promoter. ATP-induced responses, frequent in Pre-CS (86%), were slightly increased in Post-CS cells (94%; p < 0.05). Expression of cardiac-specific Ca(2+)-handling proteins (Cav1.2, NCX1, RyR2, SERCA2a) was either limited to the Post-CS stage, or markedly enhanced. CS beating was infrequent, but its pharmacology was compatible with cardiac excitation-contraction coupling. Expression of non-cardiac lineage was low in general, and similar between Pre- and Post-CS cells. Culture conditions inhibiting CSs formation prevented the increase in caffeine responders. In conclusion, clustering in CSs leads to the induction of a muscle-specific functional response in about 30% of CPCs; this is accompanied by development of a cardiac-specific expression pattern.

New perspectives to repair a broken heart.

The aim of cardiac cell therapy is to restore at least in part the functionality of the diseased or injured myocardium by the use of stem/progenitor cells. Recent clinical trials have shown the safety of cardiac cell therapy and encouraging efficacy results. A surprisingly wide range of non-myogenic cell types improves ventricular function, suggesting that benefits may result in part from mechanisms that are distinct from true myocardial regeneration. While clinical trials explore cells derived from skeletal muscle and bone marrow, basic researchers are investigating sources of new cardiomyogenic cells, such as resident myocardial progenitors and embryonic stem cells. In this commentary we briefly review the evolution of cell-based cardiac repair, some progress that has been made toward this goal, and future perspectives in the regeneration of cardiac tissue.

Potential role of mycophenolate mofetil in the management of neuroblastoma patients.

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA) at concentrations (10(-7)-10(-6) M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up-regulation of p53, p21 and bax and down-regulation of bcl-2 and survivin). MPA-induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA-induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 microM) leading to competition on HPRT-mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 microM (up to 100 microM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol-mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The apoptotic effects of MPA do not appear to be significantly increased by the UDP-glucuronosyltransferase inhibitor niflumic acid.

Cyclic nucleotides and neuroblastoma differentiation.

We have shown that intracellular cGMP levels increase during retinoic acid- and mycophenolic acid-induced neuroblastoma differentiation and that a 6 days treatment with 1 mM dbcGMP lead LAN5 cell to elaborate a network of neuritic processes suggesting an involvement of cGMP in neuroblastoma differentiation. We have also investigated the effects of some specific inhibitors of phosphodiesterases (PDE1, PDE3, PDE4 and PDE5) on human neuroblastoma (LAN5 and SHEP) growth and differentiation. After six days of incubation in the presence of each specific inhibitor at 10 x IC50 levels a cytostatic and differentiating effect was only observed with the PDE5 inhibitors Zaprinast and MY-5445. The cytostatic effect of these compounds increased increasing their concentrations far above their IC50 levels for PDE5, suggesting that these compounds could act by interfering with other molecular events than direct cGMP-PDE inhibition. No appreciable effect was observed using Dipyridamole, another specific PDE5 inhibitor.

Relation between serum triglyceride level, serum urate concentration, and fractional urate excretion.

The relations between serum urate levels and age, serum cholesterol, creatinine, glucose, and triglyceride concentrations were studied in 930 men and 298 premenopausal and 478 postmenopausal women, taking into account the renal handling of uric acid. All subjects were outpatients, and statistical evaluation was performed on cases corresponding to the central 95th percentile of the biochemical variables measured. An alternative analysis excluding subjects with serum glucose or creatinine levels outside the normal range was also performed. In men, premenopausal and postmenopausal women, and their normoglycemic normocreatininemic subsets, serum triglyceride level was positively correlated with serum urate concentration and inversely correlated with fractional urate excretion in both simple and multiple linear regression analysis. In men and postmenopausal women, there was also a strong positive independent association between serum urate and creatinine levels. This association was significant also in the normoglycemic normocreatininemic subset of men, but it was insignificant in normoglycemic normocreatininemic postmenopausal women. In this large cross-sectional study, a negative correlation between serum triglyceride concentration and renal uric acid excretion has been demonstrated.

Hypoxanthine and xanthine levels in human aqueous humor from cataractous eyes.

Purine nucleotide degradation products have been determined by HPLC in aqueous humor obtained during cataract surgery and from plasma of 22 patients (12 women). Uric acid, cytosine, guanosine monophosphate, uracyl, guanine, adenosine, adenosine monophosphate, thymine, adenine, inosine, cyclic guanosine monophosphate, hypoxanthine and xanthine were evaluated. Uric acid and the last two were the only compounds detectable in measurable amounts in aqueous humor and in plasma of all patients. Aqueous humor xanthine levels were not significantly different from plasma; aqueous humor hypoxanthine concentrations were lower than those of xanthine and than plasma oxypurine levels. In 8 patients, treated with allopurinol, oxypurinol concentrations in aqueous humor and in plasma were comparable suggesting that oxypurines are transported through the blood-aqueous humor barrier.