Effects of pulsed electric fields on mouse spleen lymphocytes in vitro

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μs, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K⁺. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was i vestigated by measuring [³H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 μs electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 μs pulse of 2.4–3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.     

Measuring single-bond rupture forces using high electric fields in microfluidic channels and DNA oligomers as force tags

The disruption force of specific biotin-streptavidin bonds was determined using DNA oligomers as force tags. Forces were generated by an electric field acting on a biotinylated fluorescently labeled DNA oligomer. DNA oligomers were immobilized via biotin-streptavidin bonds on the walls of microfluidic channels. Channel layout and fluid-based deposition process were designed to enable well-defined localized deposition of the oligomers in a narrow gap of the microchannel. Electric fields of up to 400 V/cm were applied and electric field induced desorption of DNA oligomers was observed. At T approximately 30 degrees C, field-induced desorption of both a 12 mer as well as a 48 mer yielded a streptavidin-biotin disruption force of 75 fN. Streptavidin-functionalized surfaces remained intact and could be reloaded with biotinylated oligomers. At approximately 20 degrees C, however, no field-induced unbinding of the oligomers was observed at electric field strength of up to 400 V/cm, indicating a significant temperature dependence of the bond strength.     

Excitation trapping and charge separation in Photosystem II in the presence of an electrical field

To investigate the effects of a membrane potential on excitation trapping and charge separation in Photosystem II we have studied the chlorophyll fluorescence yield in osmotically swollen chloroplasts subjected to electrical field pulses. Significant effects were observed only in those membrane regions where a large membrane potential opposing the photochemical charge separation was built up. When the fluorescence yield was low, close to F₀, a much higher yield, up to F_max, was observed during the presence of the membrane potential. This is explained by an inhibition by the electrical field of electron transfer to the quinone acceptor Q, resulting in a decreased trapping of excitations. A field pulse applied when the fluorescence yield was high, Q and the donor side being in the reduced state, had the opposite effect: the fluorescence was quenched nearly to F₀. This field-induced fluorescence quenching is ascribed to reversed electron transfer from Q^? to the intermediate acceptor, pheophytin. Its field strength dependence suggests that the midpoint potential difference between pheophytin and Q is at most about 300 mV. Even then it must be assumed that electron transfer between pheophytin and Q spans 90% of the potential difference across the membrane.     

Optical Kerr effect in biopolymer solutions

The electric field in a single mode, YAG laser beam has been used to induce orientational birefringence in solutions of tobacco rattle virus, DNA, heparin, and hyaluronic acid. Using this laser in its “fixed-Q” mode, laser pulses were generated which persisted for up to 200 μsec in which the effective electric field vector rose to 5 kV cm^?1. The birefringence amplitudes so produced had a quadratic dependence on the effective field strength and thus obeyed Kerr's law. From the birefringence decay rates, relaxation times were determined which, by comparison with the birefringence induced by pulsed static electric fields revealed the biopolymer orientational origins of the effects. This indicated how these experiments can lead to the evaluation of particle geometry, the electronic contribution to electrical polarizabilities, and the optical polarizability of biopolymers in solution.     

Effect sizes in experimental pain produced by gender, genetic variants and sensitization procedures

Background

Various effects on pain have been reported with respect to their statistical significance, but a standardized measure of effect size has been rarely added. Such a measure would ease comparison of the magnitude of the effects across studies, for example the effect of gender on heat pain with the effect of a genetic variant on pressure pain.

Methodology/Principal Findings

Effect sizes on pain thresholds to stimuli consisting of heat, cold, blunt pressure, punctuate pressure and electrical current, administered to 125 subjects, were analyzed for 29 common variants in eight human genes reportedly modulating pain, gender and sensitization procedures using capsaicin or menthol. The genotype explained 0–5.9% of the total interindividual variance in pain thresholds to various stimuli and produced mainly small effects (Cohen''s d 0–1.8). The largest effect had the TRPA1 rs13255063T/rs11988795G haplotype explaining >5% of the variance in electrical pain thresholds and conferring lower pain sensitivity to homozygous carriers. Gender produced larger effect sizes than most variant alleles (1–14.8% explained variance, Cohen''s d 0.2–0.8), with higher pain sensitivity in women than in men. Sensitization by capsaicin or menthol explained up to 63% of the total variance (4.7–62.8%) and produced largest effects according to Cohen''s d (0.4–2.6), especially heat sensitization by capsaicin (Cohen''s d = 2.6).

Conclusions

Sensitization, gender and genetic variants produce effects on pain in the mentioned order of effect sizes. The present report may provide a basis for comparative discussions of factors influencing pain.     

Cryosurgery with pulsed electric fields

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion.     

Alignment of microscopic particles in electric fields and its biological implications.

It is well known that electromagnetic fields cause mechanical forces. If one applies an electrical field to a suspension of microscopic particles, these particles realign themselves along the direction of the field and form pearl-chain-like aggregates. These chains are mostly single stranded but they are frequently multistranded. This phenomenon has been investigated by a number of groups. Here we discuss the dependence of threshold field strength on particle size and frequency. Also, pulsed fields have been thought to be more effective than continuous fields of the same average power in evoking biological effects. Our measurement of the threshold power requirement for the pearl-chain formation indicates that pulsed fields require as much power as continuous fields. The biological significance of pearl-chain formation is briefly discussed.     

Newborn neuroblasts feel the field in the adult brain

A paper in this issue of EMBO reports shows that endogenous electric currents exist in the adult mouse brain and that they may guide neuroblast migration. These findings and their implications are discussed here. EMBO reports (2013) 14 2, 184–190 doi:10.1038/embor.2012.215 In the adult mammalian brain, new neurons are continuously generated from a small supply of neural stem cells in two regions—the dentate gyrus of the hippocampus and the subventricular zone (SVZ)—in a manner that modulates numerous learning and memory processes. In the latter region, immature neuroblasts must traverse millimetres of cortical tissue to reach their final destination in the olfactory bulb [1]. How do these cells navigate along this route, termed the ‘rostral migratory stream'' (RMS)? Our understanding is that RMS migration is guided by molecular and cellular mechanisms including chemoattractive and chemorepulsive factor gradients—some established with the aid of cerebrospinal fluid flow [1, 2]. A new study published in this issue of EMBO reports raises the intriguing possibility that an endogenous electric field lying along the RMS might also be important in guiding directional cell migration [3].In 1974, the development of the vibrating probe technique enabled highly sensitive measurements of small endogenous currents within living tissues to be taken [4]. The presence of electric currents and their associated direct current fields has since been established in a variety of adult and developing tissues. As one example, polarized ionic transport through Na⁺/K⁺-ATPases can establish a large potential difference (40–200 mV mm⁻¹) from the apical to basolateral surface of an epithelial cell layer, aided by the high ionic resistance of tight junctions. Furthermore, injury to such epithelial sheets triggers an electrical current that guides epithelial migration during subsequent wound closure [5].Although electrophysiological investigations of neurons and their synaptic connections within the brain have been conducted for over a century, comparatively less is known about whether long distance, macroscopic electric fields are generated as a natural consequence of cellular membrane depolarization or currents. The work by Zhao and colleagues published in this issue presents, for the first time to our knowledge, evidence that endogenous electric currents exist along the RMS and that neuroblasts might migrate in the direction of the associated electric field, a process generally known as galvanotaxis.By using the vibrating probe method, an endogenous electric potential gradient of 3.3 mV mm⁻¹ was measured along the RMS; a separate determination based on current and resistance measurements arrived at a slightly smaller value of 2 mV mm⁻¹. As in the case of transepithelial potentials, this field might be generated by the spatial organization of Na⁺/K⁺-ATPases in the SVZ and olfactory bulb. Specifically, the authors found that epithelia lining the lateral ventricular wall, at the beginning of the RMS, had a high concentration of ATPases on the basal side, which might thus pump excess Na⁺ ions into the brain. On the other end of the RMS, in the olfactory bulb, they found Na⁺/K⁺-ATPases primarily on the apical side of the epithelia, which might pump Na⁺ ions outward from the olfactory bulb and thus create an ionic sink. The authors suggest that a resulting flow of Na⁺ cations from the SVZ to the olfactory bulb is responsible for the low level direct current electrical field along the length of the RMS, which is supported by their finding that inhibition of the ATPase by ouabain significantly reduced the field strength.In vitro and in vivo time-lapse data from this study and previous work [6] demonstrated that electric fields direct neural stem cell migration. Cao and colleagues found that high field strengths (>10 mV mm⁻¹) promoted clear and sustained directional migration towards the cathode. At lower strengths, closer to those measured between the SVZ and olfactory bulb (approximately 3.5 mV mm⁻¹), the in vitro migratory bias was slight yet statistically significant. Additionally, time-lapse analysis of labelled neuroblasts in live explant slices showed that migration towards the olfactory bulb was strongly enhanced by exogenous fields as low as 10 mV mm⁻¹—approximately three times the endogenous potential. Furthermore, reversing the field direction with a high exogenous potential (50 mV mm⁻¹) caused cells to steer off course and in the direction of the imposed field. Finally, by using pharmacological inhibition as well as RNA interference knockdown, Cao and colleagues implicate the P2Y1 purinergic receptor, which is expressed specifically in migrating neuroblasts, as a mediator of the galvanotaxis.…endogenous electric currents exist along the RMS and [that] neuroblasts might migrate in the direction of the associated electric fieldThe field of stem cell biology is increasingly recognizing the importance of not only biochemical but also biophysical regulatory cues, and this work provides further support for investigating the role of electrostatics in controlling cellular function. Naturally, it also raises several interesting and open questions. It is clear in this study that neuroblasts migrate in response to strong imposed electrical fields in vitro and in vivo, and weaker fields in vitro, although future work is necessary to establish definitively that the low electrical field measured in vivo (approximately 2–3.5 mV mm⁻¹) is sufficient to influence directional cell migration within the RMS.In addition, these results raise interesting questions about possible relationships between electrostatic and biochemical cues in regulating neuroblast migration. Such migration depends on gradients of the chemorepulsive factor Slit along the RMS [2], as well as the cell adhesion molecule PSA-NCAM, which enables cells to migrate as chains within the rodent RMS [7]. Investigating the relative importance of galvanotaxis compared with chemotaxis in guiding neuroblasts might benefit from inducible genetic manipulation of neural stem cells and their progeny in situ to establish further underlying molecular mechanisms. In addition, future work might address whether electric fields have any role in regulating neuroblasts that do not undergo RMS migration within the human brain [8].Although the phenomenon of galvanotaxis in weak direct current fields is well established across cell types, and Cao and colleagues suggest a role for the P2Y1 receptor, in general the cellular and molecular mechanisms that underlie this process are not well understood. Many cells—including these SVZ neuroblasts—respond robustly to electrical fields of 10 mV mm⁻¹ or more, yet these fields correspond to small potential differences (roughly 0.1 mV) across the dimensions of a cell [5]. One hypothesis is that small direct current fields drive ionic flow of free cations—namely Na⁺—the hydration shell of which drags concordantly along charged membrane-associated proteins towards the anode (Fig 1). The result might be a cell surface gradient of key receptors that in turn direct migration. Another theory is that small potential differences may differentially bias voltage-gated ion channels, although the activation voltages for such gated channels typically range from 50–100 mV [5]. A third explanation is that electrical fields can generate forces on negatively charged cell surface adhesion molecules (such as integrins), leading to differences in cell–extracellular matrix interactions and migratory properties across the length of the cell [9]. Finally, the lipid phosphatase PTEN—a repressor of phosphatidylinositide 3-kinase signalling—mediates an electric field response during wound healing [10], and a structurally related phosphatase (Ci-VSP) that regulates the activity of phosphoinositide-sensitive ion channels was discovered to be voltage sensitive [11]. Given the established importance of PTEN in neural stem cells and glioblastomas, these factors might also offer potential mechanisms.Open in a separate windowFigure 1There are numerous hypothesized mechanisms by which cells might sense an electrical field. A weak electrical field could impose a force on negatively charged cell surface receptors, or alternatively the electric force imposed on positive ions (Na⁺) could result in the flow of their associated hydration shell, which exerts a drag force on cell surface membranes. The resulting asymmetrical redistribution of cell surface receptors, such as the ones involved in sensing chemokines or motogens, could affect cell migration. Alternatively, the electric field could conceivably trigger voltage-gated ion channels or exert forces on adhesion receptors, such as integrins, which result in asymmetrical binding to extracellular matrix (ECM) proteins. Finally, phosphatases, such as Ci-VSP or PTEN, mediate cellular responses to electric fields.In summary, Cao and colleagues establish that electrical currents and field gradients exist along the RMS, provide further support for the observation that adult neuroblasts migrate directionally within electrical fields and provide evidence that an endogenous potential gradient along the RMS might help guide these immature neurons to the olfactory bulb. This study thus lays stimulating groundwork for future investigations to explore the roles of biophysical cues in guiding the fate and flow of stem cells and their progeny in the nervous system.     

Electric-field dependence of the quantum yield in reaction centers of photosynthetic bacteria

Multilayer Langmuir-Blodgett films of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been fabricated with partial net orientation. The films showed substantial electrical response under pulsed illumination. From measurements of the light-induced voltage generated across the Langmuir-Blodgett film, we have succeeded in quantitating the electric-field dependence of the quantum yield of charge separation in photosynthesis. The results presented here are compared with our previous determination of the field effect on quantum yield, in which flash-activated charge separation as a function of the applied field was assayed by the extent of bacteriochlorophyll dimer, (BChl)₂, oxidation measured optically at 860 nm. The two methods provided consistent dependencies of quantum yield on applied electric field. Analysis of the data reveals that the quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation decreases from a value of 0.96 at zero applied field to about 0.75 for a field of 120 mV/nm vectorially directed to hinder light-activated electron transfer. For oppositely applied fields, the quantum yield saturates at unity. The source of the effects is considered to reside in the electric field dependence of the free-energy difference between the energy levels that are involved in the initial charge separation between the (BChl)₂ in the first singlet excited state, (BChl)1₂, through the bacteriopheophytin, BPh, to the primary ubiquinone, Q_A. Possible contributions to the field-induced loss of quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation are: (1) a decrease in the free-energy gap between the states (BChl)1₂ and (BChl)^⨥₂BPh^⨪Q_A, leading to an increased rate of decay via the excited singlet state back to the ground state; (2) a stimulated return from (BChl)^⨥₂BPh^⨪Q_A directly or via the (BChl)₂ triplet state to the ground state and (3) an impeded electron transfer from (BChl)^⨥₂BPh^⨪Q_A to (BChl)^⨥₂BPhQ^⨪_A. These possibilities are discussed. Correlation of the electrical response with measurements of the photo-induced absorbance change allows determination of the projection of the electron-transfer distance on the normal to the plane of the film, which is in good agreement with previous measurements using different techniques.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens^1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region^4,6,7. There are also reports of biocidal effect of red and near infra red⁸ as well as green light⁹.In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems¹⁰.The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log''s.The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.     

Thermodynamics of phospholipid self-assembly

Negatively charged phospholipids are an important component of biological membranes. The thermodynamic parameters governing self-assembly of anionic phospholipids are deduced here from isothermal titration calorimetry. Heats of demicellization were determined for dioctanoyl phosphatidylglycerol (PG) and phosphatidylserine (PS) at different ionic strengths, and for dioctanoyl phosphatidic acid at different pH values. The large heat capacity (ΔC^o_P ∼ −400 J.mol⁻¹ K⁻¹ for PG and PS), and zero enthalpy at a characteristic temperature near the physiological range (T^∗ ∼ 300 K for PG and PS), demonstrate that the driving force for self-assembly is the hydrophobic effect. The pH and ionic-strength dependences indicate that the principal electrostatic contribution to self-assembly comes from the entropy associated with the electrostatic double layer, in agreement with theoretical predictions. These measurements help define the thermodynamic effects of anionic lipids on biomembrane stability.     

Membrane potential perturbations induced in tissue cells by pulsed electric fields

Pulsed electric fields directly influence the electrophysiology of tissue cells by transiently perturbing their transmembrane potential. To determine the magnitude and time course of this interaction, electrotonic cable theory was used to calculate the membrane potential perturbations induced in tissue cells by a spatially uniform, pulsed electric field. Analytic solutions were obtained that predict shifts in membrane potential along the length of cells as a function of time in response to an electrical pulse. For elongated tissue cells, or groups of tissue cells that are coupled electrotonically by gap junctions, significant hyperpolarizations and depolarizations can result from millisecond applications of electric fields with strengths on the order of 10–100 mV/cm. The results illustrate the importance of considering cellular cable parameters in assessing the effects of transient electric fields on biological systems, as well as in predicting the efficacy of pulsed electric fields in medical treatments. © 1995 Wiley-Liss, Inc.     

Suppressing the excitability of spinal motoneurons by extracellularly applied electrical fields: insights from computer simulations.

The effect of extracellularly applied electrical fields on neuronal excitability and firing behavior is attributed to the interaction between neuronal morphology and the spatial distribution and level of differential polarization induced by the applied field in different elements of the neuron. The presence of voltage-gated ion channels that mediate persistent inward currents (PICs) on the dendrites of spinal motoneurons enhances the influence of electrical fields on the motoneuronal firing behavior. The goal of the present study was to investigate, with a realistic motoneuron computer model, the effects of extracellularly applied electrical fields on the excitability of spinal motoneurons with the aim of reducing the increased motoneuronal excitability after spinal cord injury (SCI). Our results suggest that electrical fields could suppress the excitability of motoneurons and reduce their firing rate significantly by modulating the magnitude of their dendritic PIC. This effect was achieved at different field directions, intensities, and polarities. The reduction in motoneuronal firing rate resulted from the reduction in the magnitude of the dendritic PIC reaching the soma by the effect of the applied electrical field. This reduction in PIC was attributed to the dendritic field-induced differential polarization and the nonlinear current-voltage relationship of the dendritic PIC-mediating channels. Because of the location of the motoneuronal somata and initial segment with respect to the dendrites, these structures were minimally polarized by the applied field compared with the extended dendrites. In conclusion, electrical fields could be used for suppressing the hyperexcitability of spinal motoneurons after SCI and reducing the level of spasticity.     

Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury

Background

The factors contributing to chronic Chagas'' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2 ^−/−) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection.

Methodology

Rhesus monkeys and C57BL/6 and Nos2 ^−/− mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2⁺ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue.

Results

Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2⁺ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2 ^−/− mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue.

Conclusion

T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas'' heart disease.     

Inclusion of Many-Body Effects in the Additive CHARMM Protein CMAP Potential Results in Enhanced Cooperativity of α-Helix and β-Hairpin Formation

Folding simulations on peptides and proteins using empirical force fields have demonstrated the sensitivity of the results to details of the backbone potential. A recently revised version of the additive CHARMM protein force field, which includes optimization of the backbone CMAP potential to achieve good balance between different types of secondary structure, correcting the α-helical bias present in the former CHARMM22/CMAP energy function, is shown to result in improved cooperativity for the helix-coil transition. This is due to retention of the empirical corrections introduced in the original CMAP to reproduce folded protein structures—corrections that capture many-body effects missing from an energy surface fitted to gas phase calculations on dipeptides. The experimental temperature dependence of helix formation in (AAQAA)₃ and parameters for helix nucleation and elongation are in much better agreement with experiment than those obtained with other recent force fields. In contrast, CMAP parameters derived by fitting to a vacuum quantum mechanical surface for the alanine dipeptide do not reproduce the enhanced cooperativity, showing that the empirical backbone corrections, and not some other feature of the force field, are responsible. We also find that the cooperativity of β-hairpin formation is much improved relative to other force fields we have studied. Comparison with (ϕ,ψ) distributions from the Protein Data Bank further justifies the inclusion of many-body effects in the CMAP. These results suggest that the revised energy function will be suitable for both simulations of unfolded or intrinsically disordered proteins and for investigating protein-folding mechanisms.     

Modulatory actions of light on the behavioural responses to magnetic fields by land snails probably occur at the magnetic field detection stage

The attenuation of opioid peptide-mediated antinociception or analgaesia is a well-established effect of extremely low frequency (ELF) magnetic fields. Results of prior studies indicated a modulatory role for light such that when the ELF exposures were carried out in the absence of light, the inhibitory effect on analgaesia was reduced. Here, we investigated whether this modulatory effect of light occurs at either the magnetic field detection stage or is associated with a post-detection mechanism. We compared the effects of the presence and absence of light on the attenuation of opioid-induced analgaesia in the land snail,Cepaea nemoralis, by (i) an ELF magnetic field (15 min, 60 Hz, 141 μT peak), and (ii) the prototypic opiate antagonist, naloxone. Determinations were performed during the subjective ''day'' and ''night'' in the presence (1.9 W m^-2 and 1.0 mW m^-2, respectively) and absence of light (less than 10^-6W m^-2). The inhibitory effects of the ELF magnetic fields and naloxone on opioid-induced analgaesia were similar in the presence of light; whereas in the absence of light the inhibitory effects of the ELF magnetic fields as a percentage of sham were markedly reduced, while those of naloxone were unaffected. This indicates that the modulatory effects of light on the actions of the ELF magnetic fields probably affect the detection mechanism prior to its coupling to the opioid system.     

Partial-body exposure of human volunteers to 2450 MHz pulsed or CW fields provokes similar thermoregulatory responses

Many reports describe data showing that continuous wave (CW) and pulsed (PW) radiofrequency (RF) fields, at the same frequency and average power density (PD), yield similar response changes in the exposed organism. During whole-body exposure of squirrel monkeys at 2450 MHz CW and PW fields, heat production and heat loss responses were nearly identical. To explore this question in humans, we exposed two different groups of volunteers to 2450 MHz CW (two females, five males) and PW (65 micros pulse width, 10(4) pps; three females, three males) RF fields. We measured thermophysiological responses of heat production and heat loss (esophageal and six skin temperatures, metabolic heat production, local skin blood flow, and local sweat rate) under a standardized protocol (30 min baseline, 45 min RF or sham exposure, 10 min baseline), conducted in three ambient temperatures (T(a) = 24, 28, and 31 degrees C). At each T(a), average PDs studied were 0, 27, and 35 mW/cm2 (Specific absorption rate (SAR) = 0, 5.94, and 7.7 W/kg). Mean data for each group showed minimal changes in core temperature and metabolic heat production for all test conditions and no reliable differences between CW and PW exposure. Local skin temperatures showed similar trends for CW and PW exposure that were PD-dependent; only the skin temperature of the upper back (facing the antenna) showed a reliably greater increase (P =.005) during PW exposure than during CW exposure. Local sweat rate and skin blood flow were both T(a)- and PD-dependent and showed greater variability than other measures between CW and PW exposures; this variability was attributable primarily to the characteristics of the two subject groups. With one noted exception, no clear evidence for a differential response to CW and PW fields was found.