An innovative stand-alone bioreactor for the highly reproducible transfer of cyclic mechanical stretch to stem cells cultured in a 3D scaffold.

Much evidence in the literature demonstrates the effect of cyclic mechanical stretch in maintaining, or addressing, a muscle phenotype. Such results were obtained using several technical approaches, useful for the experimental collection of proofs of principle but probably unsuitable for application in clinical regenerative medicine. Here we aimed to design a reliable innovative bioreactor, acting as a stand-alone cell culture incubator, easy to operate and effective in addressing mesenchymal stem cells (MSCs) seeded onto a 3D bioreabsorbable scaffold, towards a muscle phenotype via the transfer of a controlled and highly-reproducible cyclic deformation. Electron microscopy, immunohistochemistry and biochemical analysis of the obtained pseudotissue constructs showed that cells 'trained' over 1 week: (a) displayed multilayer organization and invaded the 3D mesh of the scaffold; and (b) expressed typical markers of muscle cells. This effect was due only to physical stimulation of the cells, without the need of any other chemical or genetic manipulation. This device is thus proposed as a prototypal instrument to obtain pseudotissue constructs to test in cardiovascular regenerative medicine, using good manufacturing procedures.

Telemedicine and pediatric radiology: a new environment for training, learning, and interactive discussions.

OBJECTIVE: To report the experience of the Brazilian Program of Pediatric Teleradiology in combining teleconferencing and a virtual learning environment for services integration, collaborative research, and continuing education in pediatric radiology. MATERIALS AND METHODS: We performed virtual meetings from March 2005 to October 2010 on pediatric radiology-related themes, using a combination of videoconferences and Web conferences, which were recorded and made available in an open-source software (Moodle) for reuse. RESULTS: We performed 58 virtual sessions: 29 anatomical-clinical-radiological sessions, 28 on upgrading themes, and 1 virtual symposium. The average of connected points was 12 by videoconference and 39 by Web conference, and of 450 participants per event. At the time of this writing, 318 physicians and students are registered in the virtual learning environment, with a total of 14,678 accesses. CONCLUSIONS: Telemedicine is being included in pediatric radiology practice, as a means for distance education, training, and continuing integration between groups.

Rational design of modular circuits for gene transcription: A test of the bottom-up approach.

BACKGROUND: Most of synthetic circuits developed so far have been designed by an ad hoc approach, using a small number of components (i.e. LacI, TetR) and a trial and error strategy. We are at the point where an increasing number of modular, inter-changeable and well-characterized components is needed to expand the construction of synthetic devices and to allow a rational approach to the design. RESULTS: We used interchangeable modular biological parts to create a set of novel synthetic devices for controlling gene transcription, and we developed a mathematical model of the modular circuits. Model parameters were identified by experimental measurements from a subset of modular combinations. The model revealed an unexpected feature of the lactose repressor system, i.e. a residual binding affinity for the operator site by induced lactose repressor molecules. Once this residual affinity was taken into account, the model properly reproduced the experimental data from the training set. The parameters identified in the training set allowed the prediction of the behavior of networks not included in the identification procedure. CONCLUSIONS: This study provides new quantitative evidences that the use of independent and well-characterized biological parts and mathematical modeling, what is called a bottom-up approach to the construction of gene networks, can allow the design of new and different devices re-using the same modular parts.

Insights into the sliding movement of the lac repressor nonspecifically bound to DNA.

The Lac repressor finds its DNA binding sequences with an association rate 2 orders of magnitude higher than what is expected for a random diffusive process. This experimental data stimulated numerous theoretical and experimental studies, which led to the facilitated diffusion model. In facilitated diffusion, the Lac repressor binds nonspecifically to DNA. This nonspecific binding is followed by an exploration of the DNA molecule in a reduced space. Single-molecule imaging confirmed that the Lac repressor may move along the DNA molecule; however, it is still under debate whether the LacI movement proceeds through sliding, with a continuous close contact between the protein and DNA, or through hopping between adjacent binding sites. We have investigated the one-dimensional sliding movement of the Lac repressor along nonspecific DNA by full-atomistic molecular dynamics simulations and free-energy calculations based on the umbrella sampling technique. The computed free-energy profile along a helical trajectory was periodic, with periodicity equal to the distance between successive nucleotides and an energy barrier between successive minima of 8.7 +/- 0.7 kcal/mol. The results from the molecular simulations were subsequently used in a Langevin dynamics framework to estimate the diffusion coefficient of the Lac repressor sliding along nonspecific DNA. The computed diffusion coefficient is close to the lower limit of the experimental range.

A computational model of gene expression in an inducible synthetic circuit.

Synthetic biology aims to the rational design of gene circuits with predictable behaviours. Great efforts have been done so far to introduce in the field mathematical models that could facilitate the design of synthetic networks. Here we present a mathematical model of a synthetic gene-circuit with a negative feedback. The closed loop configuration allows the control of transcription by an inducer molecule (IPTG). Escherichia coli bacterial cells were transformed and expression of a fluorescent reporter (GFP) was measured for different inducer levels. Computer model simulations well reproduced the experimental induction data, using a single fitting parameter. Independent genetic components were used to assemble the synthetic circuit. The mathematical model here presented could be useful to predict how changes in these genetic components affect the behaviour of the synthetic circuit.

Model-based analysis of potassium removal during hemodialysis.

Potassium ion (K(+)) kinetics in intra- and extracellular compartments during dialysis was studied by means of a double-pool computer model, which included potassium-dependent active transport (Na-K-ATPase pump) in 38 patients undergoing chronic hemodialysis. Each patient was treated for 2 weeks with a constant K(+) dialysate concentration (K(+)(CONST) therapy) and afterward for 2 weeks with a time-varying (profiled) K(+) dialysate concentration (K(+)(PROF) therapy). The two therapies induced different levels of K(+) plasma concentration (K(+)(CONST): 3.71 +/- 0.88 mmol/L vs. K(+)(PROF): 3.97 +/- 0.64 mmol/L, time-averaged values, P < 0.01). The computer model was tuned to accurately fit plasmatic K(+) measured in the course and 1 h after K(+)(CONST) and K(+)(PROF) therapies and was then used to simulate the kinetics of intra- and extracellular K(+). Model-based analysis showed that almost all the K(+) removal in the first 90 min of dialysis was derived from the extracellular compartment. The different K(+) time course in the dialysate and the consequently different Na-K pump activity resulted in a different sharing of removed potassium mass at the end of dialysis: 56% +/- 17% from the extracellular compartment in K(+)(PROF) versus 41% +/- 14% in K(+)(CONST). At the end of both therapies, the K(+) distribution was largely unbalanced, and, in the next 3 h, K(+) continued to flow in the extracellular space (about 24 mmol). After rebalancing, about 80% of the K(+) mass that was removed derived from the intracellular compartment. In conclusion, the Na-K pump plays a major role in K(+) apportionment between extracellular and intracellular compartments, and potassium dialysate concentration strongly influences pump activity.

A method for monitoring vascular access function during hemodialysis.

We tested a new bedside method to determine the function of native arteriovenous fistula in 16 patients performed during hemodialysis without stopping the treatment. We initially measured vascular access flow (Q(a)) in each patient using the Transonic HD01(plus) device. We then measured the pressure in arterial and venous drip chambers at different blood pump flow rates (Q(bset)=0, 50, 100, 250, 300, 350 ml/min). The intravascular blood pressure gradient (P(f)) between arterial and venous puncture sites was estimated by a mathematical model. P(f) was positive for low Q(bset), but became negative when Q(bset) overcame the threshold value (Q(Inv)). Such critical flow showed a high correlation with Q(a), even if it was systemically lower. Computer analysis of fluid dynamics showed that when the blood pump flow overcame the Q(Inv) threshold, a critical transition from laminar flow to vortex circulation took place downstream of the venous needle, causing a dangerous shearstress on the vessel wall. Our results show that Q(Inv) provides an indication of the maximal blood pump flow rate needed to be reached to maximize blood flow supply in order to limit hemodynamic stress on the vascular access.

Model-based prediction of the alpha-hemolysin structure in the hexameric state.

The alpha-hemolysin toxin self-assembles in lipid bilayers to form water-filled pores. In recent years, alpha-hemolysin has received great attention, mainly due to its possible usage as a sensing element. We measured the ion currents through single alpha-hemolysin channels and confirmed the presence of two different subpopulations of channels with conductance levels of 465 +/- 30 pS and 280 +/- 30 pS. Different oligomerization states could be responsible for these two conductances. In fact, a heptameric structure of the channel was revealed by x-ray crystallography, whereas atomic force microscopy revealed a hexameric structure. Due to the low resolution of atomic force microscopy the atomic details of the hexameric structure are still unknown, and are here predicted by computational methods. Several possible structures of the hexameric channel were defined, and were simulated by molecular dynamics. The conductances of these channel models were computed by a numerical method based on the Poisson-Nernst-Planck electrodiffusion theory, and the values were compared to experimental data. In this way, we identified a model of the alpha-hemolysin hexameric state with conductance characteristics consistent with the experimental data. Since the oligomerization state of the channel may affect its behavior as a molecular sensor, knowing the atomic structure of the hexameric state will be useful for biotechnological applications of alpha-hemolysin.

Induction of NO synthase 2 in ventricular cardiomyocytes incubated with a conventional bicarbonate dialysis bath.

BACKGROUND: In hypotension-prone patients, conventional bicarbonate dialysis (BD) causes a reduced cardiovascular tolerance to the treatment with respect to acetate-free biofiltration (AFB). One possible explanation is an overproduction of endogenous NO (nitric oxide) due to the residual quote of acetate (4 mM) in the BD dialysate formulation. NO overload might cause the impairment of cardiovascular reactivity observed during BD. In this study, a potential direct impact of the residual quote of acetate on the cardiac cells was investigated. METHODS: Ventricular cardiac myocytes isolated from adult rat hearts were treated with three different dialysis baths with or without acetate: BD, AFB and AFB + 4 mM of acetate (AFB(+)). Corresponding levels of expression of the inducible NO synthase 2 (NOS2) were assessed after the treatments along with the measurement of single-cell action potential (AP). RESULTS: Incubation with acetate-containing dialysis solutions significantly enhanced (P < 0.05) the expression of NOS2 mRNA (BD: 1.11 +/- 0.31; AFB(+): 0.73 +/- 0.04, NOS2/GAPDH intensitometric ratio) with respect to the acetate-free bath (AFB: 0.03 +/- 0.01). Accordingly, protein translation was also enhanced (BD: 0.176 +/- 0.021; AFB(+): 0.135 +/- 0.009, NOS2/alpha-tubuline intensitometric ratio) with respect to AFB (0.002 +/- 0.001, P < 0.05). Measurement of the AP indicates that acetate-containing solutions determine a shortening of the repolarization phase as compared to treatment with AFB (BD: 95 +/- 13; AFB(+): 76 +/- 10; AFB: 162 +/- 16 ms). CONCLUSION: These findings show that the residual quote of acetate of the BD bath formulation affects the expression of NOS2 and the duration of AP in cardiac cells. This might cause the cardiac contractile impairment in unstable patients during BD treatment.

Patients with complex arrhythmias during and after haemodialysis suffer from different regimens of potassium removal.

BACKGROUND: Although sudden death is one of the most frequent causes of death in haemodialysis (HD) patients, the problem of cardiac arrhythmias, the major cause of these outcomes, has been little discussed. METHODS: In 30 arrhythmia-prone HD patients, we compared the arrhythmogenic effects of two dialysis techniques differing in dialysate potassium (K) content. Each patient underwent Acetate-Free Biofiltration sessions with constant (2.5 mEq/l) K (AFB) and sessions with decreasing intra-HD K (AFBK), according to a crossover single blind design. Holter ECG recording and plasma electrolyte measurements were performed during each dialysis session. RESULTS: There was a tendency in the whole sample for arrhythmia appearance in AFBK to be reduced as compared to AFB throughout the 24 hr period, although this reduction was not statistically significant. In the subset of patients sensitive to dialysis as far as arrhythmia onset is concerned, AFBK was systematically less arrhythmogenic than AFB (P < 0.01). The highest difference was achieved around the 14th hour after the end of dialysis, when the premature ventricular contractions in AFB were 3.9 times higher than in AFBK (P < 0.05). Potassium kinetics differed between the two procedures. At the first hour of treatment, the plasma K concentration was lower in AFB than in AFBK (3.67 +/- 0.15 mEq/l in AFB vs 4.06 +/- 0.13 mEq/l in AFBK, P = 0.05). CONCLUSIONS: Our study shows a greater arrhythmogenic activity with the use of a constant and relatively low K concentration as compared to decreasing K profiling in dialysis-sensitive arrhythmic patients. Smoother K removal may well engender a kind of protective effect.

Role of the intracellular cavity in potassium channel conductivity.

The role of several fragments of the potassium channel KcsA has been examined by the Poisson-Nernst-Planck (PNP) theory. The efficiency of the computational method allowed comparing a large number of channel models, with different intracellular gate openings, partial atomic charges, and amino acid sequences. Perhaps counter-intuitively, the calculated ion current decreases when the mean radius of the entrance cavity increases. Widening of the vestibule, in fact, increases the volume accessible to water, which is the volume with a high dielectric constant. In turn, water screens the attractive charges of the P-loop backbone. The backbone charges of the M2 helixes instead oppose the entrance of potassium ions through a complicated mechanism that can be separated in the activity of two interfering dipoles. The conductance of the KcsA models increased when two neutral residues in M2 were mutated to glutamic acid, in agreement with experimental results (Brelidze, T. I.; Niu, X.; Magleby, K. L. PNAS 2003, 100, 9017-9022). As a general conclusion, a relation between channel conductance and potassium concentration in the intracellular cavity emerged. Although the ion transport is the result of the fine balance of a number of different effects, the experimental results can be reproduced quantitatively only on the basis of electrostatic forces, which are the only driving forces modeled by the PNP theory.

In silico assessment of Y1795C and Y1795H SCN5A mutations: implication for inherited arrhythmogenic syndromes.

The effects of two SCN5A mutations (Y1795C, Y1795H), previously identified in one Long QT syndrome type 3 (LQT3) and one Brugada syndrome (BrS) families, were investigated by means of numerical modeling of ventricular action potential (AP). A Markov model capable of reproducing a wild-type as well as a mutant sodium current (I(Na)) was identified and was included into the Luo-Rudy ventricular cell model for action potential (AP) simulation. The characteristics of endocardial, midmyocardial, and epicardial cells were reproduced by differentiating the transient outward current (I(TO)) and the ratio of slow delayed rectifier potassium (I(Ks)) to rapid delayed rectifier current (I(Kr)). Administration of flecainide and mexiletine was simulated by appropriately modifying I(Na), calcium current (I(Ca)), I(TO), and I(Kr). Y1795C prolonged AP in a rate-dependent manner, and early afterdepolarizations (EADs) appeared during bradycardia in epicardial and midmyocardial cells; flecainide and mexiletine shortened AP and abolished EADs. Y1795H resulted in minimal changes in the APs; flecainide but not mexiletine induced APs heterogeneity across the ventricular wall that accounts for the ST segment elevation induced by flecainide in Y1795H carriers. The AP abnormalities induced by Y1795H and Y1795C can explain the clinically observed surface ECG phenotype. For the first time by modeling the effects of flecainide and mexiletine, we are able to gather mechanistic insights on the response to drugs administration observed in affected patients.

Application of the Poisson-Nernst-Planck theory with space-dependent diffusion coefficients to KcsA.

The Poisson-Nernst-Planck electrodiffusion theory serves to compute charge fluxes and is here applied to the ion current through a protein channel. KcsA was selected as an example because of the abundance of experimental and theoretical data. The potassium channels MthK and KvAP were used as templates to define two open channel models for KcsA. Channel boundary surfaces and protein charge distributions were defined according to atomic radii and partial atomic charges. To establish the sensitivity of the results to these parameters, two different sets were used. Assigning the potassium diffusion coefficients equal to the value for free-diffusion in water (1.96 x 10(-9) m(2)/s), the computed currents overestimated the experimental data. Ion distributions inside the channel suggest that the overestimate is not due to an excess of charge shielding. A good agreement with the experimental data was achieved by reducing the potassium diffusion coefficient inside the channel to 1.96 x 10(-10) m(2)/s, a value of substantial motility but nonetheless in accord with the intuitive notion that the channel has a high affinity for the ions and therefore slows them down. These results are independent of the open channel model and the parameterization adopted for atomic radii and partial atomic charges. The method offers a reliable estimate of the channel current with low computational effort.

Computer simulation of wild-type and mutant human cardiac Na+ current.

Long QT syndrome (LQTS) and Brugada syndrome (BrS) are inherited diseases predisposing to ventricular arrhythmias and sudden death. Genetic studies linked LQTS and BrS to mutations in genes encoding for cardiac ion channels. Recently, two novel missense mutations at the same codon in the gene encoding the cardiac Na+ channel (SCN5A) have been identified: Y1795C (causing the LQTS phenotype) and Y1795H (causing the BrS phenotype). Functional studies in HEK293 cells showed that both mutations alter the inactivation of Na+ current and cause a sustained Na+ current upon depolarisation. In this paper, a nine state Markov model was used to simulate the Na+ current in wild-type Na+ cardiac channel and the current alterations observed in Y1795C and Y1795H mutant channels. The model includes three distinct closed states, a conducting open state and five inactivation states (one fast-, two intermediate- and two closed-inactivation). Transition rates between these states were identified on the basis of previously published voltage-clamp experiments. The model was able to reproduce the experimental Na+ current in mutant channels just by altering the assignment of model parameters with respect to wild-type case. Parameter assignment was validated by performing action potential clamp experiments and comparing experimental and simulated I(Na) current. The Markov model was subsequently introduced in the Luo-Rudy model of ventricular myocyte to investigate "in silico" the consequences on the ventricular cell action potential of the two mutations. Coherently with their phenotypes, the Y1795C mutation prolongs the action potential, while the Y1795H mutation causes only negligible changes in action potential morphology.

Cardiac response to hemodialysis with different cardiovascular tolerance: heart rate variability and QT interval analysis.

A therapy-specific worsening of cardiovascular stability during bicarbonate dialysis (BD) with respect to acetate-free biofiltration (AFB) have been previously reported. We further investigated the impact of the 2 therapies on electrocardiographic parameters in order to gain novel insight into the cardiac responses. Holter ECG acquired during hypotension-free sessions (12 BD + 12 AFB) were retrospectively analyzed. R-R intervals were extracted from ECG recordings. An autoregressive spectral technique was used to compute low- and high-frequency (LF and HF) components of heart rate variability (HRV). QT interval duration was measured with a computer-assisted technique and corrected for HR. In BD the LF component of HRV after an initial increase was slowly depressed with respect to AFB (p < 0.05). QT duration showed a significant (p < 0.01) hemodialysis-induced reduction. QT shortening was more pronounced (p < 0.05) in BD than in AFB (-31 vs. -10 ms), even after correction for HR (p < 0.05). Cardiac electrical activity is significantly affected by the hemodialysis technique. The decrease in the LF component of HRV and the QT shortening are coherent with the worse cardiovascular tolerance observed in BD and with the hypothesis of an enhanced production of endogenous nitric oxide.

Mathematical modeling of arterial pressure response to hemodialysis-induced hypovolemia.

A computer model of pressure response to hemodialysis-induced hypovolemia is reported. Heart rate and hematocrit, measured in the course of hemodialysis, are imposed as computer model inputs and the model computes the arterial pressure response after tuning model parameters representative of patient's cardiovascular reactivity. Computer model reproduced with good accuracy experimental data (arterial pressure, cardiac output and total peripherical resistance). Parameter identification over successive sessions of the same patients revealed satisfactory reliability, providing a physiological interpretative key to patient's hemodynamic behavior during hemodialysis.

Effect of premature ventricular beats on manual and automatic repolarization measurements.

OBJECTIVE: To compare QT interval and QT dispersion in ventricular ectopic beats with measurements from the preceding and the immediately following sinus beats, and investigate differences between manual and automatic measurements. PATIENTS: Eleven chronic uremic patients. MAIN OUTCOME MEASURES: ECGs were recorded during hemodialysis treatment and 12-lead sections containing five consecutive beats were extracted, each containing four sinus beats and one centrally-positioned premature ventricular beat. QT measurements were performed both manually and with a computer-automated technique. RESULTS: T wave amplitude was greater in the ectopic beats compared to the sinus beats (0.61 +/- 0.18 vs. 0.23 +/- 0.06 mV, P <.001). The ectopic beats had a greater QT than the sinus beats when measured manually (415 +/- 35 ms vs. 386 +/- 28 ms, P <.001), or automatically (375 +/- 30 vs. 366 +/- 27 ms, P<.01). The sinus beats following the ectopics had a greater QT than the preceding sinus beats (400 +/- 27 vs. 386 +/- 28 ms, P<.001, manual; 382 +/- 24 vs. 366 +/- 27 ms, P<.001, automatic). Differences in QT dispersion were seen only between the ectopic and sinus beats (91 +/- 31 vs. 58 +/- 27 ms, P <.001, manual; 68 +/- 33 vs. 49 +/- 35 ms, P <.001, automatic). CONCLUSIONS: Manual measurement resulted in greater QT values than automatic measurement. Both techniques identified differences between sinus and ectopic beats. The ventricular ectopic beats resulted in an increase in the QT of the immediately following sinus beats. These results confirm the need to interpret QT measurements with care in the presence of ectopic beats.

Model-based study of the effects of the hemodialysis technique on the compensatory response to hypovolemia.

BACKGROUND: Hemodialysis technique (dialysate composition, filter, convection/diffusion ratio, etc.) can have an impact on the patient's tendency to acute hypotension. We have examined the hypothesis that the dialysis technique affects the hypotension risk by altering the cardiovascular compensatory response to hemodialysis-induced hypovolemia. METHODS: Twelve hypotension-prone subjects were studied during six sessions of conventional bicarbonate dialysis (BD) and six sessions of acetate-free biofiltration (AFB). Blood volume (BV) control system was used in AFB to provide a BV change equivalent to the BV change observed in BD. The efficacy of reflex compensatory mechanisms was assessed by a model-based computer analysis of the BD and AFB sessions. RESULTS: BD sessions were complicated by hypotension more frequently than the AFB ones (34/66 BD vs. 18/66 AFB). Hypotension arose about 60 minutes earlier in BD (123 +/- 41 minutes in BD vs. 183 +/- 25 minutes in AFB, P < 0.01), and after a smaller BV reduction (hypotension BV 7.9%+/- 2.0% in BD vs. 10.9%+/- 2.6% in AFB, P < 0.05). Model-based computer analysis of the sessions without hypotension revealed differences in peripheral resistance adaptation (9%+/- 9% BD vs. 19%+/- 7% AFB, P < 0.05) as well as in the stroke volume reduction (19%+/- 8% BD vs. 10%+/- 8% AFB, P < 0.001). Model analysis of sessions with hypotension indicated that compensatory mechanisms were almost inoperative in BD, whereas a residual capacity to control peripheral resistance and cardiac contractility was present in AFB. Model simulations demonstrated that hypotension occurred later in AFB since the residual compensatory capacity in AFB was able to sustain the arterial pressure for larger BV reductions (8.3% BD vs. 11.2% AFB). CONCLUSION: The increased risk of acute hypotension in BD compared to AFB is caused by a therapy-induced inhibition of reflex compensatory response to hypovolemia.

Altered excitability of motor neurons in a transgenic mouse model of familial amyotrophic lateral sclerosis.

Various evidence suggests that amyotrophic lateral sclerosis (ALS) selectively affects motor neuron functioning, but electrophysiological alterations of single motor neurons in ALS remains to be documented. In the present work, the excitability of motor neurons has been tested in a transgenic mouse model of a familial form of ALS, associated with a mutation in Cu,Zn superoxide dismutase (Gly(93)-->Ala). Patch-clamp recordings of membrane potential in transgenic mice motor neurons showed that they fire with increased frequency and shorter duration compared to motor neurons from control mice. The passive membrane properties of these neurons were equivalent however. Such results suggest that an altered motor neuron excitability accompanies an ALS associated mutation and that may contribute to the pathogenesis of the disease.