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Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. However, basigin is not alone on the erythrocyte surface. Instead, we show that it is exclusively found in one of two macromolecular complexes, bound predominantly to either plasma membrane Ca2+-ATPase 1/4, PMCA1/4, or monocarboxylate transporter 1, MCT1. PfRH5 binds to either of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, ruling this out as the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context.

The fungus Fusarium oxysporum (Fo) is widely known for causing wilt disease in over 100 different plant species. Endophytic interactions of Fo with plants are much more common, and strains pathogenic on one plant species can even be beneficial endophytes on another species. However, endophytic and beneficial interactions have been much less investigated at the molecular level, and the genetic basis that underlies endophytic versus pathogenic behavior is unknown. To investigate this, 44 Fo strains from non-cultivated Australian soils, grass roots from Spain, and tomato stems from United States were characterized genotypically by whole genome sequencing, and phenotypically by examining their ability to symptomlessly colonize tomato plants and to confer resistance against Fusarium Wilt. Comparison of the genomes of the validated endophytic Fo strains with those of 102 pathogenic strains revealed that both groups have similar genomes sizes, with similar amount of accessory DNA. However, although endophytic strains can harbor homologs of known effector genes, they have typically fewer effector gene candidates and associated non-autonomous transposons (mimps) than pathogenic strains. A pathogenic ‘lifestyle’ is associated with extended effector gene catalogs and a set of “host specific” effectors. No candidate effector genes unique to endophytic strains isolated from the same plant species were found, implying little or no host-specific adaptation. As plant-beneficial interactions were observed to be common for the tested Fo isolates, the propensity for endophytism and the ability to confer biocontrol appears to be a predominant feature of this organism. These findings allow prediction of the lifestyle of a Fo strain based on its genome sequence as a potential pathogen or as a harmless or even beneficial endophyte by determining its effectorome and mimp number.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.

Plasma membrane Ca2+-ATPases (PMCA), or Ca2+-pumps, terminate Ca2+-signals in any type of cell by extruding Ca2+ from the cytosol to the extracellular space. In neurons and epithelial cells, Ca2+-extrusion occurs within tens of milliseconds, a speed that is not compatible with current knowledge on PMCA activity. Here we investigated the transport velocity of Ca2+ -pumps assembled from PMCA2 and the recently identified auxiliary subunit Neuroplastin in intact cells. Using Ca2+-activated K+ channels as fast reporters, we show that PMCA2-Neuroplastin complexes, the most abundant Ca2+-transporters in the mammalian brain, provide Ca2+-clearing in the low millisecond-range. Freeze-fracture derived immuno-EM data on densities of Ca2+-source(s) and Ca2+-transporters translated these kinetics into transport rates for PMCA2-Neuroplastin complexes of more than 6000 cycles/s. Direct comparison with the Na+/Ca2+-exchanger NCX2, an alternate-access transporter with fast kinetics, indicated similar efficiencies in Ca2+-transport. Our results establish PMCA-Neuroplastin complexes as Ca2+-transporters with unanticipated high transport rates and demonstrate that under cellular conditions ATPases may operate in the kHz range.

Endophytic microbes conferring biocontrol are an eco-friendly alternative to control diseases in crops. Unfortunately, the use of endophytes to control diseases is not yet widespread as their application in agricultural settings is challenging and the outcome variable. Translating strains that perform well under laboratory conditions to the field poses several challenges. One is large scale inoculum production in a cost-effective manner. Here, we developed a framework to scale up inoculum production of Fusarium oxysporum 47 (Fo47), assess inoculum viability and its performance in the field and effects on potato yield and performance. The Fo47 endophyte is a well-described biocontrol agent, isolated from disease suppressive soils in the 1980’s. Using mung bean medium, we could routinely produce ≈7 × 108 spores/mL. Using 60 mL of 107 spores/mL per tuber we could re-isolate the fungus 79 days after application from 60 to 70% of the inoculated plants in a large-scale potato field trial (Clenze, Germany). Furthermore, this protocol can be used to assess Fo47 biocontrol potential under field conditions. The presence of the fungus did not negatively affect plant yield or starch production and did not increase susceptibility to endemic pathogens.

The generation of aryl radicals from the corresponding halides by redox chemistry is generally considered a difficult task due to their highly negative reduction potentials. Here we demonstrate that α-aminoalkyl radicals can be used as both initiators and chain-carriers for the radical coupling of aryl halides with pyrrole derivatives, a transformation often employed to evaluate new highly reducing photocatalysts. This mode of reactivity obviates for the use of strong reducing species and was also competent in the formation of sp2 C–P bonds. Mechanistic studies have delineated some of the key features operating that trigger aryl radical generation and also propagate the chain process., Aminoalkyl radicals can be used as both initiators and chain-carriers for the conversion of aryl halides into the corresponding radicals. This approach by-passes the requirement for strongly reducing photocatalysts.

The plant immune system employs resistance (R) genes to detect the presence of pathogenic microbes by the avirulence (Avr) factors they produce. Whereas some R-genes confer extreme resistance, completely blocking pathogen proliferation, others act later during infection and allow initial microbial multiplication in the host. We hypothesized that transmembrane R-proteins – as opposed to intracellular R-proteins - may recognize and arrest pathogen entry earlier in the infection process. In tomato, two transmembrane R-proteins (I and I-3) and one intracellular R-protein (I-2) have been identified conferring resistance against the same pathogen: the soil borne fungus Fusarium oxysporum f.sp. lycopersici (Fol). We found that in all root inoculations with Fol on resistant tomato plants, the fungus was able to reach the vasculature of the stem. However, the extent of host vasculature colonization was less than that in susceptible plants. This indicates that a complete blockade of fungal ingress does not occur in any of the incompatible interactions. However, resistance mediated by the intracellular R-protein I-2 allowed more extensive fungal colonization than resistance mediated by the transmembrane R-proteins I or I-3. The first phases of invasion – penetration of the root epidermis, cortex colonization and early stage xylem colonization – are unaffected by R-gene mediated resistance of the host. We suggest that all 3 R-proteins only limit Fol colonization after the fungus has reached the xylem tissues, and that I and I-3 act prior to and/or are more effective than I-2 in reducing pathogen proliferation and spread.

Voltage-activated calcium (Cav) channels couple intracellular signaling pathways to membrane potential by providing Ca2+ ions as second messengers at sufficiently high concentrations to modulate effector proteins located in the intimate vicinity of those channels. Here we show that protein kinase Cβ (PKCβ) and brain nitric oxide synthase (NOS1), both identified by proteomic analysis as constituents of the protein nano-environment of Cav2 channels in the brain, directly coassemble with Cav2.2 channels upon heterologous coexpression. Within Cav2.2-PKCβ and Cav2.2-NOS1 complexes voltage-triggered Ca2+ influx through the Cav channels reliably initiates enzymatic activity within milliseconds. Using BKCa channels as target sensors for nitric oxide and protein phosphorylation together with high concentrations of Ca2+ buffers showed that the complex-mediated Ca2+ signaling occurs in local signaling domains at the plasma membrane. Our results establish Cav2-enzyme complexes as molecular entities for fast electrochemical coupling that reliably convert brief membrane depolarization into precisely timed intracellular signaling events in the mammalian brain.

Infant complementary feeding is important for establishing food preferences. Few studies exist on the effects of infant complementary feeding choices (food preparation methods) on dietary intake, growth or adiposity. We examined whether provision of homemade complementary food is associated with the development of dietary diversity, nutrient intakes and quality of infant growth.Secondary analysis of feeding practices from a randomized trial of vitamin D supplementation in 132 healthy breastfed 1-month-old infants from Montréal, Canada. This longitudinal study used diet records, anthropometric and body composition data (dual-energy X-ray absorptiometry) from assessments that occurred when infants were 6, 9, 12 and 36 months of age. Infants were grouped into three categories of food preparation method on the basis of whether or not they had consumed homemade or commercial meat or fruit and vegetable by 9 months (homemade, commercial and both). Multivariable regression controlled for family income, maternal education and infant sex.Dietary data were available for 65 infants. By 9 months, 22% of infants had exclusively received homemade (n=14), 14 infants had exclusively received commercial and 37 infants had received both. The development of dietary diversity (number of World Health Organization-recommended food groups) was higher (0.76 (95% confidence interval (CI): 0.14, 1.38); P0.05) in the homemade group versus commercial. Energy and nutrient intakes did not differ by group over time. The homemade group had 773 g (-1364, -182; P0.01) lower whole-body fat mass and 7.1% (-12.6, -1.6; P0.05) lower % body fat at 12 months compared with the reference group (both homemade and commercial). Reduced whole-body fat mass in the homemade group persisted at 36 months (-696 g (95% CI: -1341, -52); P0.05). There were no differences between groups for changes in growth Z-scores (length-for-age, weight-for-age and body mass index-for-age).Provision of homemade complementary food is associated with increased dietary diversity during the first year of life and reduced adiposity.

In the original article, there was an error. The amount of xylem sap protein used for nLC-MS/MS analysis was incorrectly depicted; instead of 540 µg of protein 60 µg of protein was TCA precipitated and used for SDS-polyacrylamide gel electrophoresis. A correction has been made to the MATERIALS AND METHODS section, in the sub-section Sample Preparation for nLC-MS/MS: Potential fungal spores were removed from the sap by centrifugation at 800 ×g for 10 min. Xylem sap proteins were concentrated by passing 12 ml of cleared sap through Amicon Ultra-15 Filter Units (Millipore). After centrifugation at 2500 ×g for 15–30 min retentates containing the proteins were recovered. A BCA (bicinchoninic acid) assay (ThermoFischer) was performed to determine the protein concentration. Based on BCA quantification, a volume containing 60 µg of protein was trichloroacetic acid/aceton-precipitated and the pellet was resuspended in SDS loading buffer (2% SDS, 10% glycerol, 60 mM TRIS-HCl pH 6.8, 5% β-mercaptoethanol, 0.01% bromophenol blue), heated at 98◦ C for 5 min and loaded on a 12% SDS-polyacrylamide gel. Following a short electrophoresis, the proteins were stained overnight at 4◦ C with Commassie PageBlue (ThermoFischer). The bands containing the proteins were excised and cysteine reduction and alkylation of the proteins was performed by adding 10 mM DTT pH 8 (incubation at 60◦ C for 1 h) and 20 mM iodoacetamide pH 8 (incubation at room temperature in the dark for 30 min). Protein-containing gel slices were chopped into pieces of approximately 1 mm2 and transferred to 1.5 ml low-binding tubes (Protein LoBind microcentrifuge tubes, Eppendorf). Tryptic in-gel digestion was performed overnight by adding 50 µl of 5 ng/µl Trypsin Sequencing Grade (Sigma-Aldrich). In-house prepared µcolumns were set up by adding C18 Empore disk and LichroprepC18 column material into a 200 µl pipette tip and the tryptic peptides were eluted from the µcolumn with 50 µl of 50% acetonitrile. Acetonitrile content was reduced to

Root endophytes can confer resistance against plant pathogens by direct antagonism or via the host by triggering induced resistance. The latter response typically relies on jasmonic acid (JA)/ethylene (ET)-depended signaling pathways, but can also be triggered via salicylic acid (SA)-dependent signaling pathways. Here, we set out to determine if endophyte-mediated resistance (EMR), conferred by the Fusarium endophyte Fo47, against Fusarium wilt disease in tomato is mediated via SA, ET or JA. To test the contribution of SA, ET, and JA in EMR we performed bioassays with Fo47 and Fusarium oxysporum f. sp. lycopersici in tomato plants impaired in SA accumulation (NahG), JA biosynthesis (def1) or ET-production (ACD) and -sensing (Nr). We observed that the colonization pattern of Fo47 in stems of wildtype plants was indistinguishable from that of the hormone mutants. Surprisingly, EMR was not compromised in the lines affected in JA, ET, or SA signaling. The independence of EMR on SA, JA, and ET implies that this induced resistance-response against Fusarium wilt disease is distinct from the classical Induced Systemic Resistance (ISR) response, providing exciting possibilities for control of wilt diseases independent of conventional defense pathways.

Root colonization by Fusarium oxysporum (Fo) endophytes reduces wilt disease symptoms caused by pathogenic Fo strains. The endophytic strain Fo47, isolated from wilt suppressive soils, reduces Fusarium wilt in various crop species such as tomato, flax, and asparagus. How endophyte-mediated resistance (EMR) against Fusarium wilt is achieved is unclear. Here, nonpathogenic colonization by Fo47 and pathogenic colonization by Fo f.sp. lycopersici (Fol) strains were assessed in tomato roots and stems when inoculated separately or coinoculated. It is shown that Fo47 reduces Fol colonization in stems of both noncultivated and cultivated tomato species. Conversely, Fo47 colonization of coinoculated tomato stems was increased compared to single inoculated plants. Quantitative PCR of fungal colonization of roots (co)inoculated with Fo47 and/or Fol showed that pathogen colonization was drastically reduced when coinoculated with Fo47, compared with single inoculated roots. Endophytic colonization of tomato roots remained unchanged upon coinoculation with Fol. In conclusion, EMR against Fusarium wilt is correlated with a reduction of root and stem colonization by the pathogen. In addition, the endophyte may take advantage of the pathogen-induced suppression of plant defences as it colonizes tomato stems more extensively.

Plasma membrane Ca

The transfer price is basically the price used for transfer of tangible and intangible assets between related parties and it should be determined on the basis of market value without being influenced by the relationship of affiliation. However, if for services or tangible assets, the comparison of the transfer price to the market one is relatively easy to achieve, for the intangible ones, quantifying future benefits waived by the affiliated person compared to the situation in which it would be independent is harder to be established.

In the current global economic situation, when multinationals companies are no longer a rarity and the increase of volume and frequency of intra-group transactions are a reality, the transfer prices issue has become a problem for big companies because it significantly affects the fiscal task of the groups. The transfer price is basically the price used for transfer of tangible and intangible assets between related parties and it should be determined on the basis of market value without being influenced by the relationship of affiliation. However, if for services or tangible assets, the comparison of the transfer price to the market one is relatively easy to achieve, for the intangible ones, quantifying future benefits waived by the affiliated person compared to the situation in which it would be independent is harder to be established.

HL-1 cells are currently the only cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining a differentiated cardiac phenotype. Extensive characterization using microscopic, genetic, immunohistochemical, electrophysiological, and pharmacological techniques has demonstrated how similar HL-1 cells are to primary cardiomyocytes. In the few years that HL-1 cells have been available, they have been used in a variety of model systems designed to answer important questions regarding cardiac biology at the cellular and molecular levels. Whereas HL-1 cells have been used to study normal cardiomyocyte function with regard to signaling, electrical, metabolic, and transcriptional regulation, they have also been used to address pathological conditions such as hypoxia, hyperglycemia-hyperinsulinemia, apoptosis, and ischemia-reperfusion. The availability of an immortalized, contractile cardiac cell line has provided investigators with a tool for probing the intricacies of cardiomyocyte function. In this review, we describe the culture and characterization of HL-1 cardiomyocytes as well as various model systems that have been developed using these cells to gain a better understanding of cardiac biology at the cellular and molecular levels.

This work introduces a new electron gun geometry capable of robust functioning in the presence of a high strength external magnetic field for axisymmetric magnetic resonance imaging (MRI)-linac configurations. This allows an inline MRI-linac to operate without the need to isolate the linear accelerator (linac) using a magnetic shield. This MRI-linac integration approach not only leaves the magnet homogeneity unchanged but also provides the linac flexibility to move along the magnet axis of symmetry if the source to target distance needs to be adjusted.Simple electron gun geometry modifications of a Varian 600 C electron gun are considered and solved in the presence of an external magnetic field in order to determine a set of design principles for the new geometry. Based on these results, a new gun geometry is proposed and optimized in the fringe field of a 0.5 T open bore MRI magnet (GE Signa SP). A computer model for the 6 MeV Varian 600 C linac is used to determine the capture efficiency of the new electron gun-linac system in the presence of the fringe field of the same MRI scanner. The behavior of the new electron gun plus the linac system is also studied in the fringe fields of two other magnets, a 1.0 T prototype open bore magnet and a 1.5 T GE Conquest scanner.Simple geometrical modifications of the original electron gun geometry do not provide feasible solutions. However, these tests show that a smaller transverse cathode diameter with a flat surface and a slightly larger anode diameter could alleviate the current loss due to beam interactions with the anode in the presence of magnetic fields. Based on these findings, an initial geometry resembling a parallel plate capacitor with a hole in the anode is proposed. The optimization procedure finds a cathode-anode distance of 5 mm, a focusing electrode angle of 5°, and an anode drift tube length of 17.1 mm. Also, the linac can be displaced with ± 15 cm along the axis of the 0.5 T magnet without capture efficiency reduction below the experimental value in zero field. In this range of linac displacements, the electron beam generated by the new gun geometry is more effectively injected into the linac in the presence of an external magnetic field, resulting in approximately 20% increase of the target current compared to the original gun geometry behavior at zero field. The new gun geometry can generate and accelerate electron beams in external magnetic fields without current loss for fields higher than 0.11 T. The new electron-gun geometry is robust enough to function in the fringe fields of the other two magnets with a target current loss of no more than 16% with respect to the current obtained with no external magnetic fields.In this work, a specially designed electron gun was presented which can operate in the presence of axisymmetric strong magnetic fringe fields of MRI magnets. Computer simulations show that the electron gun can produce high quality beams which can be injected into a straight through linac such as Varian 600 C and accelerated with more efficiency in the presence of the external magnetic fields. Also, the new configuration allows linac displacements along the magnet axis in a range equal to the diameter of the imaging spherical volume of the magnet under consideration. The new electron gun-linac system can function in the fringe field of a MRI magnet if the field strength at the cathode position is higher than 0.11 T. The capture efficiency of the linac depends on the magnetic field strength and the field gradient. The higher the gradient the better the capture efficiency. The capture efficiency does not degrade more than 16%.

The Greig Health Record is an evidence-based health promotion guide for clinicians caring for children and adolescents aged six to 17 years. It is meant to provide a template for periodic health visits that is easy to use and is easily adaptable for electronic medical records. On the Greig Health Record, where possible, evidence-based information is displayed, and levels of evidence are indicated in boldface type for good evidence and italics for fair evidence. Checklist templates include sections for weight, height and body mass index; psychosocial history and development; nutrition; education and advice; specific concerns; examination; and assessment, immunization and medications. Included with the checklist tables are three pages of selected guidelines and resources. Regular updates to the statement and tool are planned. The Greig Health Record is available in English only at www.cps.ca/english/CP/PreventiveCare.htm.

The stereochemistry of the reaction catalysed by Serratia marcescens chitobiase was determined by HPLC separation of the anomers of N-acetylglucosamine produced during the hydrolysis of p-nitrophenyl N-acetyl-β-D-glucosaminide (PNP-GlcNAc). In the early stages of the reaction, the β-anomer was found to prevail, whereas the α-anomer dominated at mutarotation equilibrium. This established that chitobiase hydrolyses glycosidic bonds with overall retention of the anomeric configuration. Chitobiase-catalysed hydrolysis of PNP-GlcNAc was competitively inhibited by a series of chito-oligosaccharides (degree of polymerization 2-5) that were selectively de-N-acetylated at their non-reducing end. The results are in accord with the participation of the acetamido group at C-2 of the substrate in the catalytic mechanism of chitobiase and related enzymes.

Obstructive sleep apnea (OSA) is a common disorder of breathing in children characterized by partial and/or complete upper airway obstruction that disrupts sleep and normal gas exchange. The accepted gold standard for diagnosis of OSA is polysomnography performed in a sleep laboratory. Alternative diagnostic measures include nocturnal home pulse oximetry. The most common cause of OSA in children is adenotonsillar hypertrophy and the most common treatment is adenotonsillectomy. Other treatments include noninvasive ventilation and anti-inflammatory medication. If left untreated, OSA may lead to serious sequelae, including cardiovascular, metabolic, and neurobehavioral conditions, growth failure, learning deficits, and poor quality of life.

Oncostatin M (OSM) is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130-/-) and gp130 floxed (gp130fl/fl) mice. Subcutaneous injection of pathophysiologically relevant concentrations of OSM into the hind-paw of C57BL6J wild type mice significantly reduced paw withdrawal latencies to heat stimulation. In contrast to gp130fl/fl mice, OSM did not induce heat hypersensitivity in vivo in SNS-gp130-/- mice. OSM applied at the receptive fields of sensory neurons in in vitro skin-nerve preparations showed that OSM significantly increased the discharge rate during a standard ramp-shaped heat stimulus. The capsaicin- and heat-sensitive ion channel TRPV1, expressed on a subpopulation of nociceptive neurons, has been shown to play an important role in inflammation-induced heat hypersensitivity. Stimulation of cultured dorsal root ganglion neurons with OSM resulted in potentiation of capsaicin induced ionic currents. In line with these recordings, mice with a null mutation of the TRPV1 gene did not show any signs of OSM-induced heat hypersensitivity in vivo. The present data suggest that OSM induces thermal hypersensitivity by directly sensitizing nociceptors via OSMR-gp130 receptor mediated potentiation of TRPV1.

Background Cancer pain is a significant clinical problem being one of the first symptoms of disease with 75–90% of the patients experiencing chronic pain syndromes in advanced stages [1]. The management of cancer pain is mainly based on the use of opioid drugs; however their clinical use is limited by high incidence of adverse effects. There is a continued search for highly efficacious opioid analgesics with reduced complications and improved patient compliance. An analogue of the clinically used oxymorphone, 5-methyl-substituted 14-O-methyloxymorphone (HS198), is a selective μ opioid agonist and a potent antinociceptive agent in animal models of nociceptive and inflammatory pain, while exhibiting a favourable dissociation between analgesia and the occurrence of side effects [2]. We report data on efficacy of this opioid agonist after subcutaneous administration (s.c.) in a murine model of cancer pain. The opioid receptor-mechanistic basis of the antinociceptive action was also investigated.

Using magnetic resonance imaging (MRI) for real-time guidance during radiotherapy is an active area of research and development. One aspect of the problem is the influence of the MRI scanner, modeled here as an external magnetic field, on the medical linear accelerator (linac) components. The present work characterizes the behavior of two medical linac electron guns with external magnetic fields for in-line and perpendicular orientations of the linac with respect to the MRI scanner.Two electron guns, Litton L-2087 and Varian VTC6364, are considered as representative models for this study. Emphasis was placed on the in-line design approach in which case the MRI scanner and the linac axes of symmetry coincide and assumes no magnetic shielding of the linac. For the in-line case, the magnetic field from a 0.5 T open MRI (GE Signa SP) magnet with a 60 cm gap between its poles was computed and used in full three dimensional (3D) space charge simulations, whereas for the perpendicular case the magnetic field was constant.For the in-line configuration, it is shown that the electron beam is not deflected from the axis of symmetry of the gun and the primary beam current does not vanish even at very high values of the magnetic field, e.g., 0.16 T. As the field strength increases, the primary beam current has an initial plateau of constant value after which its value decreases to a minimum corresponding to a field strength of approximately 0.06 T. After the minimum is reached, the current starts to increase slowly. For the case when the beam current computation is performed at the beam waist position the initial plateau ends at 0.016 T for Litton L-2087 and at 0.012 T for Varian VTC6364. The minimum value of the primary beam current is 27.5% of the initial value for Litton L-2087 and 22.9% of the initial value for Varian VTC6364. The minimum current is reached at 0.06 and 0.062 T for Litton L-2087 and Varian VTC6364, respectively. At 0.16 T the beam current increases to 40.2 and 31.4% from the original value of the current for Litton L-2087 and Varian VTC6364, respectively. In contrast, for the case when the electron gun is perpendicular to the magnetic field, the electron beam is deflected from the axis of symmetry even at small values of the magnetic field. As the strength of the magnetic field increases, so does the beam deflection, leading to a sharp decrease of the primary beam current which vanishes at about 0.007 T for Litton L-2087 and at 0.006 T for Varian VTC6364, respectively. At zero external field, the beam rms emittance computed at beam waist is 1.54 and 1.29n-mm-mrad for Litton L-2087 and Varian VTC6364, respectively. For the inline configuration, there are two particular values of the external field where the beam rms emittance reaches a minimum. Litton L-2087 rms emittance reaches a minimum of 0.72n and 2.01 n-mm-mrad at 0.026 and 0.132 T, respectively. Varian VTC6364 rms emittance reaches a minimum of 0.34n and 0.35n-mm-mrad at 0.028 and 0.14 T, respectively. Beam radius dependence on the external field is shown for the in-line configuration for both electron guns.3D space charge simulation of two electron guns, Litton L-2087 and Varian VTC6364, were performed for in-line and perpendicular external magnetic fields. A consistent behavior of Pierce guns in external magnetic fields was proven. For the in-line configuration, the primary beam current does not vanish but a large reduction of beam current (up to 77.1%) is observed at higher field strengths; the beam directionality remains unchanged. It was shown that for a perpendicular configuration the current vanishes due to beam bending under the action of the Lorentz force. For in-line configuration it was determined that the rms beam emittance reaches two minima for relatively high values of the external magnetic field.

Peripheral injury or inflammation leads to a release of mediators capable of binding to a variety of ion channels and receptors. Among these are the 7-transmembrane receptors (G protein-coupled receptors) coupling to G(s), G(i/o), G₁₂/₁₃, or G(q/11) G proteins. Each of the G protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of individual signaling pathways in vivo has not yet been worked out. The G(q)/G₁₁ signaling branch is of particular interest because it leads to the activation of phospholipase C-β, protein kinase C, the release of calcium from intracellular stores, and it modulates extracellular regulated kinases. To investigate the contribution of the entire G(q/11)-signaling pathway in nociceptors towards regulation of pain, we generated double-deficient mice lacking G(q/11) selectively in nociceptors using a conditional gene-targeting approach. We observed that nociceptor-specific loss of G(q) and G₁₁ results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. Surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in G(q/11) mutant mice, suggesting a novel function for G(q/11) in tonic modulation of acute nociception. Patch-clamp recordings revealed changes in voltage-dependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of G(q/11), whereas potassium currents remained unchanged. Our results indicate that the functional role of the G(q)/G₁₁ branch of G-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain.

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P₁ receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P₁ receptor. Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

The collision-induced decomposition pathways of phenothiazine were studied in detail by ion trap mass spectrometry. With this approach, different amounts of energy can be deposited into the fragment ions, thus favouring particular structures. Different relative abundances in the fragmentation patterns of selected ions are observed, depending on whether they are produced by electron impact or multiple mass spectrometry [(MS)n] methods.

Interleukin-6 (IL-6) is a key mediator of inflammation. Inhibitors of IL-6 or of its signal transducing receptor gp130 constitute a novel class of anti-inflammatory drugs, which raise great hopes for improved treatments of painful inflammatory diseases such as rheumatoid arthritis. IL-6 and gp130 may enhance pain not only indirectly through their proinflammatory actions but also through a direct action on nociceptors (i.e., on neurons activated by painful stimuli). We found indeed that the IL-6/gp130 ligand-receptor complex induced heat hypersensitivity bothin vitroandin vivo. This process was mediated by activation of PKC-δ via Gab1/2/PI3K and subsequent regulation of TRPV1, a member of the transient receptor potential (TRP) family of ion channels. To assess the relevance of this direct pain promoting effect of IL-6, we generated conditional knock-out mice, which lackgp130specifically in nociceptors, and tested them in models of inflammatory and tumor-induced pain. These mice showed significantly reduced levels of inflammatory and tumor-induced pain but no changes in immune reactions or tumor growth. Our results uncover the significance of gp130 expressed in peripheral pain sensing neurons in the pathophysiology of major clinical pain disorders and suggest their use as novel pain relieving agents in inflammatory and tumor pain.

Inhibitor κB kinase (IKK) regulates the activity of the transcription factor nuclear factor-κ B that normally protects neurons against excitotoxicity. Constitutively active IKK is enriched at axon initial segments and nodes of Ranvier (NR). We used mice with a Cre–loxP-mediated specific deletion of IKKβ in sensory neurons of the dorsal root ganglion (SNS–IKKβ−/−) to evaluate whether IKK plays a role in sensory neuron excitability and nociception. We observed increased sensitivity to mechanical, cold, noxious heat and chemical stimulation inSNS–IKKβ−/−mice, with normal proprioceptive and motor functions as revealed by gait analysis. This was associated with increased calcium influx and increased inward currents in small- and medium-sized primary sensory neurons ofSNS–IKKβ−/−mice during stimulation with capsaicin or Formalin, specific activators of transient receptor potentials TRPV1 and TRPA1 calcium channels, respectively.In vitrostimulation of saphenous nerve preparations ofSNS–IKKβ−/−mice showed increased neuronal excitability of A- and C-fibers but unchanged A- and C-fiber conduction velocities, normal voltage-gated sodium channel currents, and normal accumulation of ankyrin G and the sodium channels Nav1.6 at NR. The results suggest that IKKβ functions as a negative modulator of sensory neuron excitability, mediated at least in part by modulation of TRP channel sensitivity.

To provide a tool to investigate the mechanisms inducing and maintaining cancer-related pain and hyperalgesia, a soft tissue tumor/metastasis model was developed that is applicable in C57BL/6J wild-type and transgenic mice. We show that the experimental tumor-induced heat hyperalgesia and nociceptor sensitization were prevented by systemic treatment with the tumor necrosis factor α (TNFα) antagonist etanercept. In naive mice, exogenous TNFα evoked heat hyperalgesiain vivoand sensitized nociceptive nerve fibers to heatin vitro. TNFα enhanced the expression of the nociceptor-specific heat transducer ion channel transient receptor potential vanilloid 1 (TRPV1) and increased the amplitudes of capsaicin and heat-activated ionic currents via p38/MAP (mitogen-activated protein) kinase and PKC (protein kinase C). Deletion of the tumor necrosis factor receptor type 2 (TNFR2) gene attenuated heat hyperalgesia and prevented TRPV1 upregulation in tumor-bearing mice, whereas TNFR1 gene deletion played a minor role. We propose endogenous TNFα as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2.

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

Depuis le milieu des annees 80, beaucoup d’acteurs economiques de la foret de montagne (proprietaires, gestionnaires, exploitants) parlent du “handicap” de cette foret et reclament des aides specifiques en s’appuyant sur la grave crise que connait le secteur. Un modele d’aide frequemment evoque est celui de l’indemnite speciale de montagne accordee aux exploitants agricoles de montagne, pour lesquels ce soutien public est le reflet de « l’expression de la solidarite nationale, en compensation des handicaps naturels et de la prise en compte des specificites montagnardes ». Pourquoi la filiere foret-bois, qui participe egalement a l’amenagement du territoire en montagne, ne profiterait-elle pas de la meme solidarite nationale ?

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P1 receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P1 receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P1 receptor. Our data show that neuronally expressed S1P1 receptors play a significant role in regulating nociceptor function and that S1P/S1P1 signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.