Previous reports indicate altered metabolism and enzyme kinetics for various organisms, as well as changes of neuronal functions and behaviour of higher animals, when they were exposed to specific combinations of weak static and alternating low frequency electromagnetic fields. Field strengths and frequencies, as well as properties of involved ions were related by a linear equation, known as the formula of ion cyclotron resonance (ICR, abbreviation mentioned first by Liboff). Under certain conditions already a aqueous solution of the amino acid and neurotransmitter glutamate shows this effect.An aqueous solution of glutamate was exposed to a combination of a static magnetic field of 40 μT and a sinusoidal electromagnetic magnetic field (EMF) with variable frequency (2–7 Hz) and an amplitude of 50 nT. The electric conductivity and dielectric properties of the solution were investigated by voltammetric techniques in combination with non linear dielectric spectroscopy (NLDS), which allow the examination of the dielectric properties of macromolecules and molecular aggregates in water. The experiments target to elucidate the biological relevance of the observed EMF effect on molecular level.An ion cyclotron resonance (ICR) effect of glutamate previously reported by the Fesenko laboratory 1998 could be confirmed. Frequency resolution of the sample currents was possible by NLDS techniques. The spectrum peaks when the conditions for ion cyclotron resonance (ICR) of glutamate are matched. Furthermore, the NLDS spectra are different under ICR- and non-ICR conditions: NLDS measurements with rising control voltages from 100–1100 mV show different courses of the intensities of the low order harmonics, which could possibly indicate "intensity windows". Furthermore, the observed magnetic field effects are pH dependent with a narrow optimum around pH 2.85.Data will be discussed in the context with recent published models for the interaction of weak EMF with biological matter including ICR. A medical and health relevant aspect of such sensitive effects might be given insofar, because electromagnetic conditions for it occur at many occasions in our electromagnetic all day environment, concerning ion involvement of different biochemical pathways. A possible influence on life processes was already mentioned in the late 19 century , singlet century , and the century .et al. [B of the static component and the frequency f of the alternating EMF relate to the "ion cyclotron resonance (ICR) formula":Ferromagnetism has been implicated in animal navigation  distort the field. This induces alternating voltages over and currents through the solution, which are detected by 2 auxiliary electrodes in order to avoid polarisation effects. Phase shifts and distortions of the obtained signals, as compared to the input signal, contain information on damping and relaxation kinetics. Therefore, the signals are y domain -24. Usuasample is the signal output intensity of the nth harmonic from the sample measuring channel, and U(n)ref the corresponding value from the reference channel.Where U(n)et al. [Zhadin et al.  reportedet al. . By recoAll preparations were performed with doubly de-ionized water. The solutions were degassed and stored under Argon, in order to avoid oxidation of the solute and increased electrode fouling during the subsequent measurements. An acidic solution of 2.24 mM Glu was adjusted to pH = 2.85 ± 0.03 with a stock solution of 5 mM HCl. Equilibration was assumed, when the pH varied less than ± 0.03 for at least one minute. All procedures were performed at 20°C. For yielding a reference signal, an aqueous solution of HCl was provided by diluting the HCl stock solution with water to pH = 2.85. All solutions were stored at 4°C under Argon.1, E2) consisting each of 4 gold wires (Au 99.9%, Johnson Matthey, Karlsruhe) with a diameter of 0.25 mm, mounted parallel at a distance of 2 mm on a teflon frame. The required sample volume was 1 ml. These electrode carriers are mounted on a stable socket for electric connection and mechanical adjustment (not shown). The cuvettes are enclosed by a hermetically sealable plastic tank (T) with a copper bottom, which is filled at a height of 2 cm around the cuvettes with water for thermal coupling to an outer temperature controlled water bath. The setup is kept under Ar atmosphere throughout the experiment. Thermic control (20 ± 0.1 °C) of the cuvettes is provided by a water thermostat (Haake "G", Karlsruhe-Berlin, Germany) with a sequential home built temperature fine controller, ensuring highly stable working conditions for the electrodes. Once assembled, these components form a mechanically stable unit, with in- and outlets for gas and samples by small teflon hoses (not shown). The assembly is placed in the center of a solenoid (S), consisting of two cylindrical coils with a inner diameter of 16 cm and a height of 7 cm for applying the vertically orientated EMF (B). The coil for the static field component consisted of 300 turns of coated copper wire (diameter 0.5 mm), the other coil was winded above and had 50 turns.The experimental arrangement for differential non-linear dielectric spectroscopy (DNLDS) is shown in Figure For electric and magnetic shielding the complete setup resides in a grounded double-walled Permalloy box with a total wall thickness of 1 mm. A overall inhomogeneity ≤ 0.3 % of the generated fields was determined inside the box with a triaxial CXM539 magnetometer (CMT GmbH, Herrsching, Germany) over the cuvette locations. For coil calibration the relation of field strength to coil current could be ascertained directly in measurement series with the magnetometer for 0.1–100 μT, showing a overall deviation from linearity ≤ 0.2 % (DC and AC), so currents corresponding to even lower field strengths were obtained by extrapolation.Signal processing was mostly done as previously described . Figure et al. [et al. [2 (0.5 M in water) in a ultrasonic bath (Bachhofer, Reutlingen), and finally rinsed with de-ionized water (<2 μS). This treatment resulted in amplitude deviations ≤ 5% over an experimental session of up to 2 h. If electrodes were not used for DC measurements, but for NLDS, they were additionally coated with a thin polymer film in order to improve noise reduction and stability [For cleaning, the electrodes were first treated with chromosulfuric acid for 1 h at room temperature and intensively rinsed with de-ionized water. This procedure was repeated approximately once per week. An improved long-term electric stability was obtained by slight modifications of the treatments described by Woodward et al.  and Yard [et al. : The eletability .2 from the solutions, avoid oxidation reactions and subsequent arising of reactive oxygen species (ROS) in the solute, then the hoses were sealed with rubber caps. After reaching a stable temperature of 20 ± 0.2°C, measurements were started. First 10 "dummy" scans were performed, in order to obtain a dynamic equilibration of the electrodes. dc B= 40 μT was selected as static magnetic field component for the ICR condition, because it is of comparable intensity as the natural geomagnetic field of the earth. A new sample was used for every experiment, an "aging effect" of the test solutions was observed, similar to an earlier seen effect, which resulted in a decreasing reproducibility for experiments with magnetic field exposed lipid vesicles [The cuvettes could be charged with the test solutions, discharged and rinsed through the teflon hoses by a syringe. A sample volume of 1 ml was used. Device specific, systematic errors were routinely checked by exchanging the electrode arrays used for sample and reference measurements and testing several cuvettes of the same type. After loading they were flooded with Argon for about 10 min. in order to remove Ovesicles .Three types of techniques for measuring the electric currents in the solutions were applied, always using the gold electrode array described above:et al. [dc B= 40 μT or 50 μT and a frequency sweep of the alternating magnetic field ac B= 50 nT  from 2 to 7 Hz with 0.025 Hz/s and a resolution of 0.05 Hz were used.1) For the validation of the ICR parameters of the Glu-HCl solution, the experiment of Zhadin et al.  was repeet al. , superimdc B= 40 μT was used.2) For the investigation of the ICR transition with NLDS the same magnetic field setup is used like described under 1), the NLDS sine wave was applied on the electrodes (instead of the DC-voltage) and a constant magnetic field dc B= 40 μT and ac B= 50 nT, 4.14 Hz fixed), for reference experiments only the static component (dc B= 40 μT) was applied with dc Bswitched off. The amplitude of the sinusoidal scanning voltage was increased in each experiment from 100–1100 mV in steps of 10 mV, record by record, the duration of each cycle was 4 s. The two data sets (from Glu-HCl and HCl sample) yielded by every single record were seperately Fourier transformed in order to get the spectra, these two spectra were divided by themselves (Glu-HCL spectrum by HCl spectrum) and the ratio spectrum was subsequently attached to a data file on a harddisc for later evaluation.3) Finally the FRV setup allowed the frequency analysis of the electric signals with variable amplitudes using the DNLDS technique described above. Glu-HCl samples were exposed to constant ICR conditions . Subsequently, the pH-dependence was determined under identical magnetic field and scanning conditions mentioned above. Resonance effects are only seen in a narrow pH range of Glu-HCl (pH 2.75 – 2.90), with an maximum at 2.85, and vanishes outside this range.Applying a constant magnetic field of n et al. , and theet al. [After this verification of the experiment of Zhadin et al. , these edc B= 40 μT (no acB). A voltage range of 100–1000 mV was selected to allow a comparison with the voltammetric information out of the frequency resolved voltammetry (FRV). Again, maxima of conductivity were obtained, they lie at 250 ± 10 mV for Glu-HCl and 280 ± 10 mV for water/HCl pH 2.85 (data not shown).Further some DC voltage scans were performed with the gold electrode array for Glu at pH 2.85, and for dilute HCl adjusted to pH 2.85, applying only a static magnetic field dc B= 40 μT and a ac Bwith f = 4.15 Hz). 15 experiments were performed and averaged. Figure nd harmonic (referenced against dilute HCl, pH 2.85). The full dataset is shown in Figure st harmonic is split up into 2 closely spaced peaks around the ICR frequency. This is also well seen in Figure Next, the solutions were investigated by NLDS spectroscopy, in order to investigate in which way the frequency composition of the current spectra will change, when the predicted ICR condition for Glu-HCl is matched  were normalized to ± 1, and all 12 experiments were averaged, Figure 100 DNLDS spectra were recorded with single 2 Hz sinus signals with 100 mV amplitude. t + 0.5015). The course seems to reach a new steady value after on/off switching of the alternating magnetic field with a time delay, which seems larger, when ICR is switched off. The average current change after the switching processes is -0.2 nA/s, the negative values result from comparison with a reference.Changes of the signal intensity become obvious, when switching the alternating magnetic field on or off. Over the entire experiment there seems to be a constant drift which we take as an indication for irreversible processes. This drift is indicated by the dotted line, which results from a linear regression of the entire dataset (-0.0039dc B= 40 μT), but not acB. Each of the two groups of experiments were averaged separately. Then the two resulting datasets were subtracted . This differential dataset had a total amplitude of 2.03 ± 0.38 dB, presenting just the contribution of Glu, because the voltammetric background from HCl was subtracted.Finally, the FVR method should show the intensity distributions of the harmonics of the DNLDS spectra and their dependence from the used amplitude of the electrode input voltage. Again Glu-at pH = 2.85 was investigated, using diluted HCl (pH 2.85) as the reference. The ratio of the resulting two NLDS power spectra was calculated according to equation (2), resulting in a logarithmic DNLDS spectrum. 101 such scans (4 s each) were performed for every single experiment, during which the amplitude of the applied course of 4 periods of a 2 Hz sine voltage increased from 100 to 1100 mV in 10 mV steps. Corresponding datapoints of the successive single DNLDS spectra generated one AC voltammogram each, for the respective frequency. Altogether, a set of 201 frequency resolved voltamogramms was obtained, because every spectrum contains 201 data values. Subsequently 20 such experiments were performed in which the solution was exposed to ICR conditions, alternating with 20 experiments, were only the static field was applied  frequency of the ions. Interactions of the internal electric and external magnetic field could probably cause side band modulations. They are probably responsible for the seen splitting up of the ICR resonance peak  with variable amplitudes. Two explanations for this effect would be possible. The additional electric field caused by the AC signal of the NLDS could modulate the charged particles inside a "quantum box", whatever will be the reason for its existence. An indication for such a mechanism could be the more or less ordered local maxima of conductivity in spectral as well as in the voltammetric domain of the data. But the frequency dependent conductivity band shifts of the FRV experiments Figure  could eik figure  when usiFor progressed investigation of the observed effects some additional properties of the electric charge environment of the Glu ion should be known. The isoelectric point of Glu is at pH 3.22, the pK of the α-COOH-group is 2.19, that of the β-COOH at 4.25, the small optimum for the EMF effect around pH 2.85 does not coincide with any of these points. The Debye-Hueckel radii for Glu are about 5 nm, they determine the free ion movement, and influence consequently the current. Moreover, they could be responsible for a proper fit of the spatial ion distribution to the environing structure whatever, which enables a resonant EMF effect.The results strengthen the idea, that weak electromagnetic fields can cause an resonance effect on molecular or even supramolecular scale in electrolyte solutions ,35, and In general any kind of a suitable coherence mechanism should be essential for the observed effects in a dense medium like water, which had to support an energy gap against the thermal fluctuations of the environment, and enable a movement of charged particles which are only magnetically coupled to their outer environment. Not at least, the high sensitivity of the ICR to weak electromagnetic fields should be regarded. It makes the modulation of biological processes by the weak EMF of our everyday environment conceivable , possiblThe geomagnetic field, with all its anomalies and regional differences , in combEMF: (low frequency) electromagnetic fieldICR: ion cyclotron resonanceNLDS: non linear dielectric spectroscopyDNLDS: differential non linear dielectric spectroscopy.FRV: frequency resolved voltammetryGlu-HCl: A glutamate solution adjusted to pH 2.85 with hydrochloric acid (HCl).The author itself carried out all experiments and drafted the manuscript.


Neurogenic Para-Osteo-Arthropathy (NPOA) occurs as a consequence of central nervous system injuries or some systemic conditions. They are characterized by bone formation around the main joints.In order to define some biological features of NPOAs, histological and immunohistological studies of the soft tissue surrounding osteoma and Ultrasound examination (US) of NPOA before the appearance of abnormal ossification on plain radiographs were performed.We have observed a great number of ossifying areas scattered in soft tissues. US examination have also shown scattered ossifying areas at the early stage of ossification. A high osteogenic activity was detected in these tissues and all the stages of the endochondral process were observed. Mesenchymal cells undergo chondrocytic differentiation to further terminal maturation with hypertrophy, which sustains mineralization followed by endochondral ossification process.We suggest that periosteoma soft tissue reflect early stage of osteoma formation and could be a model to study the mechanism of osteoma formation and we propose a mechanism of the NPOA formation in which sympathetic dystony and altered mechanical loading induce changes which could be responsible for the cascade of cellular events leading to cartilage and bone formation. Neurogenic Para Osteo-Arthropathies (NPOA) occurs in patients with brain or spinal cord injury, hemiplegias, various encephalopathies, tetanus  or neuroThe first clinical manifestations are local inflammatory signs, tumefaction and progressively limited range of motion of the involved joint region. Those appear between the second and tenth weeks after the onset of the pathological condition . DespiteAs shown by radiographic and scintigraphic observations, heterotopic bone formation evolves from an early appearance of soft tissue densification and attenuation of the muscle signal to a mineral signal . After 6Very little is known about the pathophysiology of NPOA formation. Assuming such a freezing of the process of NPOA formation and an involvement of the periosteoma tissues in the reported relapses following surgery, we postulated that the periosteoma soft tissues could show some of the very early stages of the NPOA formation. We performed histological, histochemical and immunohistochemical studies of soft tissues dissected from the periphery of osteomas. We used samples of varying age lesions and searched for the main osteogenic and chondrogenic markers: alkaline phosphatase (ALP) activity, type I collagen and osteocalcin (OCN) for the bone -13, and The 28 specimens were obtained from 27 patients undergoing orthopedic surgery for osteoma excision. NPOA's were localized on: elbows (7), hips (18) and knees (3). The time from the neurologic insult ranged from 5 months to 216 months. The initial conditions were: 11 Brain Injuries (BI), 3 Spinal Cord Injury (SCI), 1 BI plus SCI, 4 strokes, and 9 patients sustained coma of various etiology (legionellose, anoxia, toxic condition, pneumonia, suicide attempt using neuroplegic).Specimens obtained during the course of surgery, referred to in this paper as "osteoma", were immediately placed in sterile Gibco Hanks' balanced salts solution (Invitrogen, Cergy-Pontoise, France) at 4°C for transportation. The soft connective tissue was easily dissected off from the osteoma in order to exclude any part of the bony mass Fig . The spe2O2 until total clearing of oxygen bubbles. Non-specific protein binding was performed with 10% non-immune serum, same host as secondary antibody, in PBS with 1% Bovin Serum Albumin . Sections were then incubated with primary antibodies for 1 hour at room temperature, or 24 hours at 4°C with anti-type I collagen. Excess antibody was removed by washing the sections with PBS. Sections were incubated 1 hour with horseradish peroxydase-labeled secondary antibody diluted in PBS. 3-3'diaminobenzidine (DAB) solution (Dako Corporation, CA, USA) was then added in order to obtain staining. Sections were counter-stained with hematoxylin-eosin or nuclear red/eosin, dehydrated, and mounted with Mountex medium (Microm, France). Controls were systematically performed omitting the primary antibody. Slides were examined by light microscopy using a Leica DMR microscope .The anti-human monoclonal mouse antibodies against type II collagen (NeoMarkers Inc., CA, USA), OCN (Interchim, Montlucon, France), anti-human polyclonal goat antibody against type I collagen (Santacruz Biotechnology, CA, USA), anti-rat polyclonal rabbit antibody against type X collagen  and anti-human polyclonal rabbit antibody against VEGF (Santacruz Biotechnology, CA, USA) were used at 1 mg/ml. Peroxidase-conjugated goat anti-mouse was purchased from Immunovision Technologies (CA, USA), peroxidase-conjugated goat anti-rabbit from Dako (Dako Corporation, CA, USA) and peroxidase-conjugated donkey anti-goat from Santacruz Biotechnology (CA, USA). They were used at a 1/20, 1/50 and 1/100 dilution, respectively. Frozen sections post-fixed with acetone were treated with hyaluronidase  one hour at 37°C. For type X collagen immunostaining slides were treated first 15 minutes at 37°C with hyaluronidase (5 mg/ml in PBS), washed two times in PBS, then 15 minutes with chondroitinase (2 U/ml in PBS), and washed two times in PBS. Immunohistochemistry was performed using ABC method. Briefly, endogenous peroxydase activity was eliminated with 0.3% HMost of the patients were referred to Raymond Poincaré teaching hospital, at the time of surgery. US examination was performed when NPOA was clinically suspected in five patients whose rehabilitation has begun. Five hips were explored by US in two BI and three SCI. Linear 8 to 15 MHz and sectorial 4 MHz transducers (Sequoia Acuson-Siemens Erlangen) were used. A sectorial low frequency transducer was used because in the hip area NPOA can be very large and very deep especially in the gluteus area compared to the subcutaneous plane. US examination was combined with color and energy Doppler. In all cases a plain film was obtained the same day as the US examination.Non mineralized connective tissues from the periphery of the osteoma were examined by light microscopy on Erlich's hematoxylin-eosin-stained sections. Several kinds of tissues appeared on the slides so the diversity of these figures deserves a systematic analysis which will be completed in the next sections. Briefly, the ground basis of our preparations was a more or less fibro-cellular connective tissue displaying sometimes edema and/or necrosis. Suffering and degenerating tissues with vacuolized myofibers, thrombotic vessels and adipose tissue were often observed Fig . MusculaMorphologically normal vessels were rarely observed and winding of the vasculature was an almost constant finding. Many of the vessels were thrombotic, sometimes with hyperplastic intimae or media Fig . Some peThese findings point to a chondrogenic or osteogenic differentiation of formerly undifferentiated mesenchymal cells from the stroma and the vessel walls. More advanced stages of cartilaginous differentiation were frequently observed in avascular areas with varying degrees of chondrocyte maturation. Morphologically recognizable columns of chondrocyte-like cells presenting a high ALP activity were observed. Moreover, we could observe all the stages of progressive chondrocyte differentiation from quiescence to chondrocyte hypertrophy/matrix mineralization and endochondral ossification Fig .A high ALP activity was also found in cells surrounding the cartilage areas undergoing mineralization and embedded in a slight envelope of woven bone with ALP positive cells Fig . On the Bands of eosinophilic material stained partially by Von Kossa underlined some borders of the mineralized cartilage. These osteoid-like bands were lined by cells which morphologically appeared to be osteoblast-like cells.To confirm the osteoblastic nature of these cells, immunolabelling for OCN was performed Fig . In fronIn order to document the collagenic composition of these tissues and confirm their osseous or cartilaginous nature, immunolabelling for type I, II and X collagens were performed.Conspicuous immunolabelling for type X collagen was observed in most of the hypertrophic chondrocytes and in their matrix. The immunoreactivity became more intense nearby the mineralization front. In addition the matrix of the fibrous and non cartilage-like tissue around some mineralized areas was unexpectedly labelled Fig . ControlDistribution of type II collagen was limited to the matrix of non hypertrophic and prehypertrophic chondrocytes with high ALP activity Fig . NeverthBone deposition was frequently observed by polarized light and confirmed by type I collagen immunostaining Fig . Type I In order to determine the chronology of events at work in the described endochondral ossification, we performed serial cryosectioning of samples in which a cortical bone followed soft tissue. These samples seem to be appropriate to have all stages of osteogenesisOne of the specimens appeared to contain an aponeurotic tissue which showed signs of bursitis. In a highly cellular tissue we observed a high angiogenic activity. Bundles of vessels surrounded amorphous and avascular zones Fig . Some ofAt this stage a strong ALP activity was observed in the cells surrounding the cartilage zone as well as in non mineralized hypertrophic areas Fig . FinallyTo further confirm the process of endochondral ossification, we decided to search for VEGF expression. Immunolabelling of these tissues with VEGF monoclonal antibody, showed a labelling of the hypertrophic chondrocytes as well as an intense labelling of activated osteoblats lining the osteoid. Some clusters of rounded cells also expressed VEGF in the fibrous part of these preparations Fig .US examination showed a huge focal disorganization of the muscles in the area of the suspected NPOA. Normal longitudinal muscular striation disappeared and was replaced by a relatively well defined mass with a very heterogeneous echostructure. The masses ranged from six to eleven centimeters of long axis. No scattered ossified areas were detected by US at this stage. Hypervascularization was detected on Doppler examination inside and outside the NPOA tumors Fig .Classical zone phenomenon previously described in the literature ,16 was vThe zone phenomenon became visible on the second US examination performed one week later Fig .The opacity of the early ossification became slightly visible on plain films only two weeks after the US detection of zone phenomenon Fig .NPOA pathogenesis is still poorly understood, and the exact environmental and humoral conditions underlying the ossifying process are not clear. In this study we postulated that periosteoma soft tissues display interrupted early stages of osteoma formation, which could help us to understand the chronology as well as the mechanism of osteoma formation. Thus, histological and immunohistological experiments were performed on 28 specimens. Moreover, ultrasound examination of suspected NPOA tumor on five patients permitted to follow osteoma formation in the early stages before ossification.Histological studies have shown varying amount of muscle and connective tissue degeneration in which some areas underwent reorganization. Islands of cartilage, woven bone, and mature lamellar bone were a constant finding in most of specimens, whatever the estimated age of the studied lesion. The spatial organization of chondrocytes was reminiscent of the epiphyseal growth plate or of the fracture callus organization . In the Some sections showed a high expression of type X collagen in the hypertrophic chondrocytes areas, and unexpectedly in non differentiated mesenchymal tissue. As this labelling did not occur in other fibrocellular areas it was unlikely to be produced by a binding of the antibody to some other matricial component of the extra cellular matrix. It was shown, that type X collagen is not only associated with chondrocyte proliferation and hypertrophy, but also with resting chondrocytes, cells at the border of the perichondrium and resting cartilage of the fetal femoral head .The finding of still degenerating muscular fibers and early chondro-osteogenesis accompanied by heavy ALP activity in large parts of the soft tissues in old lesions (till 8 years) is singular. Except when serial sectioning was performed the studied specimens were carefully dissected from the osteoma during the surgery or at the fixation time. Therefore the extra-osteoma localization of these tissues can without any doubt be assumed. In one study  "recent It was claimed that osteoma develops in the periphery of a muscle in which some myofibers undergo degeneration ,26, and On the other hand it has been shown that hypoxia promotes chondrocyte differentiation ,35. In oThe volume of the tumor is acquired during the very early stages of NPOA. After tumefaction there is no real change in the tumor size . Moreovede novo cartilaginous differentiation. US examination of the same NPOA tumor one week later showed scattered ossifying nodules. This nodular activity is in accordance with our histological finding in periosteoma soft tissue. Extense of the hypoxic area determines the size of each nodule. Areas with important hypoxia induce larger cartilage zones which could join together after ossification but small nodules remain scattered in periosteoma soft tissue. These results confirm that, periosteoma soft tissue has the same pattern of early stage of osteoma ossification, and could be a model of ossification for further studies.On tissue section, VEGF immunolabelling revealed a more intense expression by osteoblasts and osteoblast-like cells, in addition to its expected expression by hypertrophic chondrocytes. VEGF induces neoangiogenesis and then endochondral ossification occurs. The restoration of normoxic conditions promote the onset of lamellar ossification and hamper any other Moreover, NPOA occurs in neurologically deficient patients with altered mechanical loading. Mechanical loading is of pivotal importance to the development, function and repair of all tissues in the musculoskeletal system. In nonfunctional joints, as it is the case of these patients, the absence or reduction of intermittent hydrostatic pressure in the articular cartilage could permit cartilage degeneration and the progressive advance of the ossification These mechanical influence could indeed shed light on the finding that osteomas only occur near the main joints.Moreover, Carter and associates have also shown that intermittently applied shear stresses (or strain energy) promote endochondral ossification and that intermittently applied hydrostatic compression inhibits or prevents cartilage degeneration and ossification. Thus, the imbalance of these forces among these patients can promote endochondral ossification of the cartilage nodules in areas of high shear (deviatoric) stresses.Urist demonstrated that the induced endochondral bone is resorbed once the inductive agent has disappeared . We haveWe therefore propose a model of the NPOA lesion formation. The sympathetic hyperactivity causes major changes in the peripheral vascular dynamics. As related some of these changes end in vascular stasis and/or thrombosis . Edema fSome questions remain which deserve further studies. Why do NPOAs form only around the main joints? Why are they not resorbed as does any intramuscularly implanted bone graft . What frIn conclusion, our results demonstrate that periosteoma soft tissues are a replica of the early stages of osteoma formation, and could be used as a model for NPOA formation. We propose also a mechanism for osteoma formation in which hypoxia is a major cause of nodular osteoinduction and chondrocyte differentiation. Combination of hypoxia and applied shear stresses induce endochodral ossification. Finally our results indicate implication of different types of mesenchymal cells in NPOA formation but US examination support specially muscular origin hypothesis.The pre-publication history for this paper can be accessed here:


The purpose of the study was to compare the knowledge scores of medical students in Problem-based Learning and traditional curriculum on public health topics.We planned a cross-sectional study including the fifth and sixth year medical students of Dokuz Eylul University in Turkey. The fifth year students (PBL group, n = 56) were the pioneers educated with PBL curriculum since the 1997–1998 academic year. The sixth year students  were the last students educated with traditional education methods. We prepared 25 multiple-choice questions in order to assess knowledge scores of students on selected subjects of Public Health. Our data were collected in year 2002.Mean test scores achieved in PBL and traditional groups were 65.0 and 60.5 respectively. PBL students were significantly more successful in the knowledge test (p = 0.01). The knowledge scores of two topics were statistically higher among PBL students. These topics were health management and chronic diseases.We found that mean total evaluation score in the PBL group was 4.5 points higher than in the traditional group in our study. Focusing only on the knowledge scores of students is the main limitation of our study. Upon the graduation of the first PBL students in the 2002–2003 academic year, we are planning additional studies regarding the other functions of a physician such as skill, behaviour and attitude. During the last 25 years, ideas concerning the aim, structure and system of medical education have been discussed. Debates generally have arisen from the perception that medical education couldn't serve the purpose of improving health standards of the communities ."Health for All" was adopted in 1977 and launched at the Alma Ata Conference in 1978 to underline the fact that large numbers of people and even whole countries were not enjoying an acceptable standard of health . In ordeIn the Edinburgh Declaration of the World Medical Association in 1988, similar problems were mentioned and the purpose of the medical education was declared as training physicians capable of improving communities' health standards. This declaration suggested that medical education should be focused on common health problems of the large communities, and the medical school curriculum should be restructured according to the health requirements of the community. According to the declaration, medical students must gain professional skills and social values in addition to theoretical knowledge and the principle of lifelong medical education should be adopted .The ideas and suggestions mentioned above have aroused strong winds of change in the medical education arena. Mc Donald et al. from Mc Master University determined an approach based on the community's main health problems and stressed the importance of focusing on these problems while designing their medical school's curriculum .Since then, this approach has been adopted by many medical schools all over the world. The schools which designed their curriculum according to the priority health problems of the community, managed to raise the physicians' awareness of their community and the preventive measures and solutions of their main health problems.In Turkey, problems of medical education have been discussed since early 1970s. Several studies showed that the goals of medical education did not overlap with the health requirements of the Turkish community. The education of health professionals was abstracted from the realities of the country. In 1990s Turkish Parliament and Turkish Medical Association determined and reported the difficulties of medical education. In a 1991 report of the Turkish Parliament, the facts that the number of qualified physicians who were trained according to the health needs of the country was limited and that this number was not sufficient to improve its health standards were underlined. Several deans from different medical schools of the country contributed to Turkish Parliament's study and reported that a greater importance should be given to the health problems of the population while planning the educational programs and the medical education should not be restricted to the university hospitals .In The Turkish Medical Association's report the fact that medical education was not relevant to health needs of the country was emphasized. New medical graduates were not fully aware of common national health problems. The recommendations of the Turkish Medical Association to improve the health standards of the Turkish population were; training the general practitioners capable of working effectively in the primary health care and restructuring the medical education on a community basis and implementing Problem-based Learning methods .International developments and the reports of Turkish Parliament and Turkish Medical Association led the faculty of Dokuz Eylül University School of Medicine (DEUSM) to seek solutions to the problems mentioned in the reports. As a result, Problem-based Learning (PBL) a more active and student-centred learning- was adopted and launched in the 1997–98 academic year. One of the main features of the education program was its relevancy to the philosophy of community-based medical education .The curriculum of DEUSM was structured considering social, biological, behavioural and ethics objectives of medical education. The curriculum was structured in a modular system and adopted to a spiral configuration providing horizontal and vertical integration. During the first three years of undergraduate education, PBL sessions are the main focus of a modular structure. The weekly schedule of a module allowed for all the educational activities such as PBL sessions, lectures, field studies, communication skills and clinical skills courses lectures existing one hour a day in the weekly program support the PBL sessions and independent learning .PBL sessions were based on written problems, which are likely to happen in real life. Special emphasis was also given to the integration of knowledge, acquisition of professional and moral values and to the development of communication skills.Medical knowledge and practical skills that a physician is supposed to have were on the basis of the advice of Turkish Medical Association and the faculty departments. The Department of Public Health also contributed to the education program by setting social standards and determining the most important health problems of the community.PBL Curriculum of DEUSM aimed to teach the students the main health problems of the community, their prevention and ways of treatment.Public Health topics of Dokuz Eylül University School of Medicine consists of;• Holistic approach in health,• Basic principles of Public Health,• Personal and social points of view on health events,• Bio-psychosocial (holistic) approach to any individual,• Principles of preventive medicine,• Structure and mechanisms of national health organization,• Demographic structure and trends, factors affecting them,• Basic principles of planning and conducting a scientific research on health,• Sound knowledge on leading health problems of the country, personal and social approaches for their solutions,• Environmental and occupational factors threatening community health and their prevention.Cases in the scenarios of the PBL modules were selected among common and important health problems, for which early diagnosis or prevention is possible. Lectures and small group studies with students were also organized to contribute to the educational effectiveness of the modules. Public Health topics of the medical education may be achieved more easily when theoretical knowledge and practical skills are complemented by field studies . It is rPrior to the implementation of PBL curriculum in the 1997–1998 academic year, lectures on Public Health were presented to the first, the third and the last year students by the faculty members of the Department of Public Health. Lectures on bio-statistics and research methods were given weekly throughout the first year. The other topics of Public Health were held in 72-hour Public Health Courses at the end of the third year . In the Comparison of old and new curriculum using some measurement tools is mandatory to observe the effects of innovations. In the literature, the determination of students' performances in scientific or licensing examinations was used to compare the efficiency of traditional education and PBL. Nandi P. et al. reviewed the studies and meta-analyses comparing PBL and traditional lecture-based education methods. In meta-analysis of the data published between 1980–1999, they concluded that PBL helped students show slightly but not significantly better performance than the others on clinical examinations . SimilarBlake et al. compared formerly lecture-based educated and recently Problem-based educated graduates of Missouri-Columbia School of Medicine concerning their performances on medical licensing examinations. They reported that mean scores achieved on these examinations were better among graduates of PBL, but the difference between old and new graduates' scores was not statistically significant .Some other studies have attempted to compare students' performances on special areas of medicine instead of general evaluation. Antepohl and Herzig conducted a randomized controlled study among the students who enrolled for the course of basic pharmacology at the University of Cologne. They randomly divided the students into two groups of PBL and traditional lecture based learning in order to compare their final examination scores. They could not find any significant difference between the two groups. However, in short essay questions there was a tendency towards higher scores among the students in the PBL group. The authors also found that the PBL students reached almost identical scores in their multiple choice questions and their short essay questions whereas the students who had been in the lecture based group scored significantly lower scores in their short essays than in their multiple choice questions .In a multi-centric study conducted by Schmidt et al., comparison of PBL and lecture based learning students showed that PBL students had higher knowledge scores on the areas of primary care services, psychological health, collaboration of different sectors on health and occupational ethics .The purpose of our study was to compare the knowledge scores of medical students in PBL and traditional curriculum on public health topics.We planned a cross-sectional study including the fifth and sixth year medical students of DEUSM. The fifth year students (PBL students) were the pioneers educated with PBL curriculum since the 1997–1998 academic year. The sixth year students  were the last students educated with traditional education methods. The knowledge scores of students on Public Health topics were evaluated. In both of the PBL and Traditional curriculum, all the knowledge acquired in the first five years of the school was reviewed during the two-month Public Health internship period in the sixth year. Since this period may remind the students of some issues which may have been previously forgotten, we decided to exclude the sixth year students who have completed their internship period. 56 fifth year students and 78 sixth year students who have not so far completed their internship period in the Department of Public Health were included in our study. Participation rates were 96.4% (54 out of 56 students) in the fifth year and 100%  in the sixth.Before the application of the inquiry form, the purpose of the study was explained to the students and their oral consents were obtained.We analyzed the knowledge scores of the two groups of students' on Public Health issues. PBL and traditional programs were the independent variables. Descriptive variables were age and gender.By reviewing a five yearlong section of educational programs, we determined that nine Public Health main topics were common to both PBL and traditional programs. The main topics were communicable diseases, epidemiology, mother and child health, health management, chronic diseases, occupational health, nutritional principles in community, demography and environmental health.We prepared 25 multiple-choice questions in order to assess knowledge scores of students on selected subjects. The number of questions related to each topic was proportional to the time allocated for each of the topic in the curriculum. The content validity of the questions was tested by consulting experts in relevant fields. All the data were collected between February and March 2002. Scoring procedure was implemented over "100 points" where each correct answer was scored "four points" and each wrong answer was scored "zero point".Data were subjected to statistical analysis by the chi-square test and the t-test in SPSS 10.0.Overall mean age was 23.6 ± 2.1 (21–45) years. The rates of male and female students were 55.4 % and 44.6 % respectively. There were no statistically significant differences between the two groups regarding mean ages, gender distribution or other personal variables.Mean scores achieved at the 25 question-test were 65.0 in PBL group and 60.5 in the traditional group. Students in the PBL group were significantly more successful in the knowledge test Table-.The knowledge scores of seven topics were higher among students in PBL curriculum. These topics were communicable diseases, epidemiology, health management, chronic diseases, occupational health, demography and environmental health. Traditional curriculum students were found to be more knowledgeable on two topics; mother and child health and nutritional principles in the community. However, the differences between PBL and traditional students' knowledge scores in only two topics, chronic diseases and health management, were statistically significant Table-.In our study, we found a statistically significant difference between knowledge scores of PBL and Traditional education groups in favour of the PBL group Table .The students of the PBL group had higher knowledge scores on 7 of the 9 identified topics. But the difference between mean scores of the groups was statistically significant in only two topics, "health management" and "chronic diseases". The reason of significantly higher knowledge scores among the students in PBL group may be that these students have more opportunities such as observations during field studies, work-shops or presentations to study on these two topics than those in the other group. They experienced a two week training period in a "community health center" at the end of the first year and observed the health center services and prepared a structured form concerning the procedures of health centers. They also studied in "community health centers" as small groups including two students in each fortnightly during their third year in the school and completed comprehensive forms about the topics on which they studied. The reason of better knowledge scores of PBL group on "chronic diseases" may result from the special educational efforts improving the effects of relevant modules on this topic. Actually special learning opportunities were provided for all topics and we were expecting to find a difference on remaining 7 topics too. On the other hand, the students in the traditional education group had slightly higher mean scores about the topics of "mother and child health" and "nutritional principles in community" although the differences between the groups' mean scores were not statistically significant. These knowledge deficiencies among PBL students were already revealed and an additional module was implemented in the curriculum to compensate them. Curriculum of DEUSM is being looked over by curriculum committee continuously and the departments try to make interventions for problematic parts.We found that the mean total evaluation score in the PBL group was 4.5 points higher than in the traditional group in our study. Actually, we expected a much larger difference between the two groups in favour of PBL students for their education was supported by lectures, small group studies and field studies in addition to the PBL sessions. They also had the advantage of studying on Public Health issues in each year of the school by means of homogenous allocation of the modules and blocks in the first five years instead of accumulation in a short period of time as it was in the traditional curriculum. Therefore, the difference between the evaluation scores of the groups did not meet our expectations although it was statistically significant. The reason for this underachievement of Public Health objectives among our PBL students may be related to both students and PBL tutors. The common perception among the students that they have enough knowledge to say something about social and behavioural aspects of PBL modules lead them to focus on biological objectives more and they do not need to study on social issues in depth. Furthermore, a common misunderstanding among faculty members that achieving the Public Health objectives in PBL is just the responsibility of the Department of Public Health may have led the PBL tutors to withdraw from the responsibility of focusing on these subjects sufficiently. Additionally, when they are less informed or less equipped with supporting material about Public Health objectives, they may not have felt very competent while facilitating their groups by asking appropriate questions.One assumption of curricular comparison studies, included this one, is that students will do better either in one or the other type of curriculum. However, each curriculum demands different skills and deployment of learning strategies from the students. This is important because, it is well known in the educational literature that not all students do well in one particular learning program and that they do better when the program adapts to their preferred way of learning. The studies of learning styles may shed light in why the differences between performance scores are always so close when medical curricula are compared.As we mentioned before, in DEUSM, the written problem used in PBL sessions are oriented to biological as well as social and behavioural objectives. In order to achieve all these three objectives the tutors must attach the same importance to each subject and ensure that their groups give enough time and effort for each objective. But when the tutors get inadequate information and support from the experts of the related subjects, they generally focus only on biological objectives and their groups can't manage to integrate all objectives. If the tutors are less sensitive to objectives other than biological ones, then their students will be less motivated to learn and, like their educators, will be equally insensitive to Public Health topics. In order to prevent this, faculty members of the department of Public Health who take place in the scenario committees review the PBL problems regarding Public Health objectives. They make every effort to insure that the Public Health objectives are included while writing the problems and that the tutors are sufficiently informed on these objectives before their sessions. Field Work Committee has been trying to increase students' motivation and raise their awareness on Public Health issues to increase the effectiveness of field studies.Focusing only on the knowledge scores of students is the main limitation of our study. Upon the graduation of the first PBL students in the 2002–2003 academic year, we are planning additional studies regarding the other functions of a physician such as skill, behavior and attitude.The authors declare that they have no competing interests.EG conceived of the study, participated in the design of the study and drafted the manuscript, BM conceived of the study, participated in the design of the study and coordination, performed the statistical analysis, GA participated in the design of the study and performed the statistical analysis, RU conceived of the study, participated in the design of the study. All authors read and approved the final manuscript.primary prevention method?While working in a health center as a general practitioner, you have noticed that hypertension prevalence is high among the people living in the region under your responsibility. Which of the following would be your choice as a) I would educate the hypertensive patients on their disease.b) I would treat the hypertensive patients with antihypertensive drugs.c) I would send the hypertensive patients to a secondary care hospital for further investigation and treatment.d) I would educate healthy individuals on risk factors associated with hypertension and prevention methods.most common childhood nutritional disorder in Turkey?Which of the followings is the a) Protein calorie deficiencyb) Marasmusc) Iron deficiency anaemiad) Ricketswrong?Which of the following is a) Demography is a science that analyse the body, structure and differentiations of human populations.b) The goal of family planning is to decrease current number of population.c) Dependent population ratio is found by dividing the total number of population younger than fifteen years and older than 65 years of age by the total number of population between 15–65 years of age.d) Principal of pronatalist population policy is to increase the total number of population.is not one of the basic records kept in a health center?Which of the followings a) Household determination card.b) Follow-up card for the females between 15–49 years old.c) Follow-up card for aged individuals.d) Antenatal and postnatal follow up card.e) Infant and child follow-up card.Which of the followings is not among the responsibilities of an occupational health unit?a) Health prevention services in work settingsb) Work safety preventionsc) Following up the health and safety conditions in work settingsd) Preventing any interruption in productione) Giving outpatient clinic services in work setting.not required as an immediate intervention?An 11 year old girl was bitten by a neighbour-dog while she was playing in her house-garden. Which of the followings is a) To investigate if the dog is vaccinated.b) To vaccinate the girl for rabies prevention.c) To clean the wound by soap and water.d) To apply one dose of tetanus vaccine.e) To try to understand how the dog bit the girl.most common used effective family planning (contraception) methods?Which of the followings is the a) Intrauterine deviceb) Withdrawal (coitus interruptus)c) Combined oral contraceptivesd) Condome) Subcutaneous implantsbest represents the environmental health related responsibilities of a general practitioner who works in a health centre?Which of the followings a) Waste control and giving education to correct misapplicationsb) Analyzing and chlorinating drinking water, control of potable waterc) Controlling and improving the condition of toilets,d) Coordination of conduction of above mentioned services by auxiliary personnel of health centre, although these services are among the tasks of municipality.e) All of the statements above are true.After looking over one-year medical records of an internal medicine outpatient clinic, it was found that 25 % of the diagnoses were Diabetes mellitus. Regarding this result a screening procedure was conducted in the field and Diabetes mellitus prevalance was found 5 %.can not be the conclusion of above mentioned situation?Which of the followings I) Outpatient clinic may admit people coming from other regions.II) Outpatient clinic records represent the health status of the community.III) One-fourth of the patients have Diabetes mellitus diagnosis.IV) Field studies are needed to determine the real prevalance of a disease.a) I, IIb) I, IIIc) II, IIId) I, II, IVe) II, III, IVThe pre-publication history for this paper can be accessed here:


Thymine-rich Insulin Response Element (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis.Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression.These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents. Insulin-like growth factors (IGF-I and II) have a broad range of biological activities that include the stimulation of mitogenesis and differentiation, and insulin-like effects on glucose uptake and lipogenesis . These aStudies using inhibitors of PI 3-kinase have demonstrated a requirement for this enzyme in insulin regulation of IGFBP-1 . Indeed,In the present study, we have examined the role of GSK-3 in the regulation of a third TIRE-containing gene promoter, namely IGFBP-1. We demonstrate that four different classes of inhibitors of GSK-3 can mimic the action of insulin and reduce IGFBP-1 gene expression. Furthermore, we find that inhibition of GSK-3 reduces the activity of a heterologous promoter containing the IGFBP-1 TIRE, and propose that this mechanism underlies the repression of all three promoters by inhibitors of GSK-3. Finally, we demonstrate for the first time a requirement for inhibition of GSK-3 in the insulin regulation of the TIRE, and hence IGFBP-1 expression.in vivo, reduces both basal and glucocorticoid-induced IGFBP-1 gene expression . Importin vitro [Paullones are a family of benzazepinones that are potent , ATP-competitive inhibitors of cyclin-dependent kinases (CDKs) and the closely related neuronal CDK5/p25 -32. Subsin vitro . AlsterpAlthough alsterpaullone, kenpaullone, SB214763, and SB415286 are potent inhibitors of GSK-3, they also exhibit activity against CDKs. However, the aminopyrimidine CHIR99021 shows 350-fold selectivity toward GSK-3 compared to CDKs , and exhibits a Ki of < 10 nM in vitro . It is tH4IIE cells were transiently transfected with a luciferase-reporter construct containing TCF/LEF binding sites, whose activity is regulated by the GSK-3 substrate, β-catenin. Inhibition of GSK-3 results in the accumulation of β-catenin in the cytoplasm where it can form complexes with TCF/LEF. The complex translocates to the nucleus and activates transcription of target genes. Treatment of transfected H4IIE cells with CHIR99021 results in a dose-dependent increase in luciferase activity, regulated by the β-catenin/TCF complex Fig . The β-cMeanwhile, insulin treatment of H4IIE cells previously transfected with a luciferase reporter construct under the control of a thymidine kinase promoter containing the IGFBP-1 TIRE (BP-1WT), reduces luciferase expression by 60% Fig . This efIn order to assess the requirement for inhibition of GSK-3 in insulin regulation of the IGFBP-1 TIRE we over expressed wild-type GSK-3 (GSK-3β-WT), insulin-insensitive GSK-3 (GSK-3β-S9A) or control protein  in H4IIE cells using adenoviral vectors. Infected cells were subsequently transfected with BP-1-WT and treated with or without insulin Fig . The inhBP-1 TIRE activity can be regulated by co-expression of FOXO-1 ,36. TherThis study demonstrates that six agents, of four different chemical classes, which share an ability to inhibit GSK-3 mimic the effect of insulin on IGFBP-1 gene expression. This is reminiscent of the effect of lithium ions, SB216763 and SB415286 on two other insulin repressed gene promoters, PEPCK and G6Pase , aspartaInsulin induces PKB activity, promoting phosphorylation of Ser-21 of GSK-3α and Ser-9 of GSK-3β, thereby reducing total GSK-3 activity by between 20 and 80%, dependent on cell type. Therefore, expression of a mutant GSK-3β with Ser-9 replaced by alanine renders cellular GSK-3 activity insensitive to insulin . Indeed,in vitro and in some cases in vivo (for review see [in vitro. The only known GSK-3 substrates that have been demonstrated to bind to or regulate the TIRE are members of the CAAT-enhancer binding protein (C/EBP) family of transcription factors. GSK-3 phosphorylates C/EBPα at Thr-222/Thr-226 [The GSK-3 inhibitors regulate IGFBP-1 gene expression in the absence of regulation of PKB, MAP kinase, FOXO-1 or mTOR  and Figs, known rview see ). These view see ,44, c-juview see ,46, GR [view see  and c-myview see  by GSK-3view see , althougview see . Since i/Thr-226  while C//Thr-226 . The rep/Thr-226 . We are /Thr-226 . LIP sub/Thr-226 . FinallyAgents that mimic the physiological processes that are regulated by insulin have the potential to be of therapeutic value for the treatment of insulin resistant states such as diabetes. Lithium chloride, SB216763, SB415286 and CHIR99021 inhibit GSK-3 and therefore mimic many of the actions of insulin. For instance, lithium chloride stimulates glucose transport and glycogen synthesis in adipocyte and muscle cell lines -56, whilThe work presented herein demonstrates for the first time that inhibition of GSK-3 is required for complete insulin regulation of IGFBP-1, while we have identified the DNA element by which GSK3 targets this gene promoter. As such, GSK-3 inhibition will mimic the insulin regulation of IGF1 bio-availability, as well as reducing the expression of hepatic gluconeogenic genes. It remains to be seen how many other insulin-regulated (and/or TIRE-containing) gene promoters are sensitive to these inhibitors.32P}-ATP) and ICN, Thame, Oxfordshire, UK ({α32P}-UTP). Insulin was purchased from Novo Nordisk, (Crawley, West Sussex, UK), kenpaullone and alsterpaullone from Calbiochem (La Jolla, CA) and the RNase Protection Assay Kit II from AMS Biotech/Ambion, (Austin, Texas). All other chemicals were of the highest grade available.Radioisotopes were obtained from Amersham, Bucks, UK ({γH-imidazol-2-yl)-pyrimidin-2-ylamino]-ethylamino}-nicotinonitrile) was synthesized in 7% overall yield using a convergent approach from 2,4-dichlorobenzoyl chloride and 6-chloro nicotinonitrile respectively ([CHIR99021 (6-{2-[4-(2,4-Dichloro-phenyl)-5-(4-methyl-1ctively ( and refsThe rat hepatoma cell line H4IIE was maintained in Dulbecco's Modified Eagle's medium (DMEM) containing 1000 mg/l glucose, 5% (v/v) foetal calf serum, as described previously . Cells wH4IIE cells were serum-starved overnight and treated with hormone/inhibitor for the times and at the concentrations indicated in the figure legends. Total cellular RNA was isolated using TriReagent™ (Sigma) following the manufacturer's instructions. An RNase Protection Assay (RPA) was performed to determine the relative amounts of IGFBP-1 and cyclophilin mRNA in each sample . Band inH4IIE cells were incubated in serum-free medium with hormones and inhibitors for the times and at the concentrations indicated in the figure legends. Cells were then scraped into ice-cold lysis buffer (25 mM Tris/HCl, pH 7.4, 50 mM NaF, 100 mM NaCl, 1 mM sodium vanadate, 5 mM EGTA, 1 mM EDTA, 1% (v/v) Triton X-100, 10 mM sodium pyrophosphate, 1 mM benzamidine, 0.1 mM PMSF, 0.27 M sucrose, 2 μM microcystin and 0.1% (v/v) 2-mercaptoethanol). Cell debris was removed by centrifugation at 13000 × g for 5 min and the protein concentration determined by the method of Bradford, using BSA as an internal standard.Antibodies to phospho ribosomal protein S6 (Ser-235), phospho-FKHR-L1 (Thr-32) and GSK-3β were purchased from Upstate (Lake Placid, USA), while the phospho-specific Ser9/Ser21 GSK-3, Thr-308 PKB, Thr389-S6K1, and Thr-183/Tyr185 p42/p44 MAPK antibodies were purchased from Cell Signalling Technologies (Hertfordshire, UK). H4IIE cell lysates were prepared following incubation with hormones as described in figure legends and analysed by Western blot analysis.GAAACTTCTTTTG) produces a mutant promoter (BP-1 DM5) that is no longer responsive to insulin [The plasmids BP-1 WT and BP-1 DM5 were a gift from Dr Robert Hall and Professor Daryl K. Granner (Vanderbilt University, TN, USA) . The BP- insulin . The FOXThe TOPflash reporter plasmid kit were obtained from Upstate Biotechnology (Lake Placid, USA). TOPflash has Tcf binding sites driving luciferase expression. Tranfections were performed using the calcium phosphate procedure as described previously . H4IIE c8 and 109 plaque forming units per ml, incubated at 37°C for 16 hr. Cells were then transfected with 10 μg of BP-1 WT as described above and incubated for a further 24 hr in the presence or absence of 10 nM insulin. Luciferase was harvested and assayed or cell extracts were prepared for western blot analysis, as described earlier.H4IIE cells were infected with virus between a titre of 10As a measure of statistical significance of differences in experimental groups, student t-tests were performed and 5% confidence limits applied.G6Pase, glucose 6-phosphatase; IGFBP-1, IGF-binding protein-1; phosphatidyl inositol 3, kinase, PI 3-kinase; TIRE, thymine rich insulin response element; PKB, protein kinase B; PEPCK phosphoenolpyruvate carboxykinase; GSK-3, Glycogen synthase kinase 3The majority of the data was obtained in equal measure by D.F. and S.P, the CHIR99021 was synthesised, purified and analysed by N.S. and R.M., the adenoviral vectors were produced and characterised by L.M.D. and C.J.R., while the project was conceived and supervised by C.S.


Spontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies.In this study, we addressed the question of the possible influence of mesenchymal stem cells on neural stem cell fate. We have previously reported that adult rat mesenchymal stem cells are able to express nestin in defined culture conditions (in the absence of serum and after 25 cell population doublings) and we report here that nestin-positive (but not nestin-negative) mesenchymal stem cells are able to favour the astroglial lineage in neural progenitors and stem cells cultivated from embryonic striatum. The increase of the number of GFAP-positive cells is associated with a significant decrease of the number of Tuj1- and O4-positive cells. Using quantitative RT-PCR, we demonstrate that mesenchymal stem cells express LIF, CNTF, BMP2 and BMP4 mRNAs, four cytokines known to play a role in astroglial fate decision. In this model, BMP4 is responsible for the astroglial stimulation and oligodendroglial inhibition, as 1) this cytokine is present in a biologically-active form only in nestin-positive mesenchymal stem cells conditioned medium and 2) anti-BMP4 antibodies inhibit the nestin-positive mesenchymal stem cells conditioned medium inducing effect on astrogliogenesis.When thinking carefully about mesenchymal stem cells as candidates for cellular therapy in neurological diseases, their effects on resident neural cell fate have to be considered. During development of the central nervous system (CNS), all types of neuronal and macroglial cells derive from neuroepithelial neural stem cells (NSCs) ,2. NSCs BMPs (Bone Morphogenetic Proteins) are secreted members of the TGF-β superfamily. Together with their receptors, they are abundantly expressed in the brain both during embryogenesis and in the adult -9. More in vivo studies demonstrated that MSCs fuse with host neuronal cells [Cellular therapies using stem cells are promising approaches for the treatment of several chronic or acute neurological diseases such as Parkinson's  or Huntial cells ,31. Thesin vitro and demonstrate that MSCs favour astroglial lineage. We observe that npMSCs are able to stimulate astroglial fate in striatal progenitor cultures and to repress neuronal and oligodendroglial fate through the release of diffusible factor(s). Using BrdU incorporation, we demonstrate that this npMSC conditioned medium has no effect on the astrocytic or oligodendrocytic progenitor proliferation. Propidium iodide incorporations suggest that the npMSCs conditioned medium protect GFAP-positive cells from cell death in comparison to the effect of nnMSCs conditioned medium or to the control condition. Meanwhile, no increase of cell death is observed in neuronal and oligodendroglial populations. We then demonstrated that BMP4 is present in a biologically-active form in the npMSCs but not in nnMSCs conditioned medium and is responsible for both the increase of astroglial numbers and the inhibition of oligodendrocyte differentiation in striatal NSC cultures.Recently, we demonstrated that two phenotypes of MSCs could be obtained in culture: nestin-positive MSCs (npMSCs) which are able to integrate some extrinsic signals when co-cultured with neurons leading to a differentiation into astrocyte-like cells and nestin-negative MSCs (nnMSCs) which are unable to adopt a neural phenotype but remain able to differentiate into adipocytes, chondrocytes or osteocytes . When coNeurosphere-derived cells from GFP-positive E16 green mouse striata include neural stem cells and progenitors which are still proliferating but already committed to a given cell fate. Upon transfer on adherent surfaces (poly-ornithine coated dishes), these cells spread and spontaneously differentiate as follows after 5 days of culture: 44.2 ± 2.5% GFAP-positive cells , 15 ± 2.1% Tuj1-positive cells  and 5.86 ± 0.6% 04-positive cells . When co-cultivated with nestin-positive MSCs (npMSCs), phenotypic allocation of neurosphere-derived cells (identified as GFP-positive cells) become strikingly different: GFAP labelling is increased to 78.5 ± 3.9% Fig.  n = 12, n = 12, n = 8, .To further characterize the mechanism (soluble factor versus membrane-bound factor) responsible for such an increase in astrocytes number in presence of npMSCs, we tried to induce the differentiation of neurospheres with npMSC conditioned medium. The following data were obtained: 77.5 ± 2.5% of the neurosphere-derived cells became GFAP-positive, 4.3 ± 0.9% were Tuj1-positive and 1.5 ± 0.6% were 04-positive  medium (DEM/F12 + B27) did not show any significant differences  increase the astrocytes numbers while inhibiting neuronal and oligodendroglial numbers. Conversely, nnMSCs (with a maximum of 5 passages in vitro) only decrease the neuronal differentiation. Since similar results were obtained using MSC conditioned media, we concluded that these effects could be mediated by diffusible factor(s). We then analysed the effect of various conditioned media on the cell proliferation (using BrdU incorporation) as well as the cell death (using propidium iodide incorporation) in each neurosphere-derived cell types and at two different differentiation period (48 or 96 hours). We only observed a significant decrease of propidium iodide incorporation in GFAP-positive cell population at 48 hours of differentiation suggesting that soluble factor(s) select astroglial lineage by protecting those cells from cell death.As we mentioned above, environment is able to modify the cellular differentiation capacity. In this study, we addressed the question of a possible effect of MSCs on neural progenitor cell fate and we choose an The activity which drives neurosphere-derived cells into astrocytes only occurs in npMSC conditioned medium while the neuronal differentiation-inhibiting activity is present both in np- and nnMSC conditioned media. We tried to identify the astrogliogenetic factor(s) by measuring the expression by nn- and npMSCs of cytokines known to promote an astroglial fate -41. Our The release by npMSCs of biologically-active form of BMP4 which promotes astroglial differentiation and inhibits oligodendroglial differentiation is consistent with previous studies demonstrating that BMPs play multiple roles in development . Other sWhen considering the use of MSCs in cell replacement strategies for the treatment of various neurological diseases, it should be taken into account that those cells could also influence the development host neural precursors.6 marrow cells were plated on 175-cm2 tissue culture flask in DEM/10% foetal bovine serum (Invitrogen). After 24 hours, the non-adherent cells were removed. When the rMSCs became confluent, they were resuspended with 0.25% Trypsin and 1 mM EDTA and then sub-cultured. Nestin expression by MSCs was induced as described in [Adult rat bone marrow was obtained from femoral and tibial bones by aspiration and was resuspended into 5 ml of DEM (Invitrogen, Merelbeke, Belgium) . Betweenribed in .6 cells/75-cm2 tissue culture flask) in DEM/F12 supplemented with EGF (20 ng/ml, Sigma) and B27 (Invitrogen), a multi-component cell culture supplement devoid of any growth factor. When the size of neurospheres reached approximately 50 cells, they were dissociated into a single cell suspension by trituration and replated in fresh culture medium. Neurospheres with a maximum of 3 passages were used in this study.Green C57BL/6 mice embryos (Jackson Immunoresearch Laboratory, Inc., West Grove, USA) or NMRI mice were used as a source of striatal neural progenitor and stem cells. Green mouse express GFP under control of the β-actin promoter [MSCs and neurospheres were plated on polyornithine coated dishes for 5 days, in DEM/F12 containing only B27 supplement and were then processed for immunocytochemical analysis as described below.MSCs were placed in DEM/F12 medium supplemented with B27 (Invitrogen), during 3 days. The conditioned media were then filtered with 0.22 μm-pore filter before being replaced on plated neurospheres during 5 days.The cultures were fixed with 4% (v/v) paraformaldehyde for 15 minutes at room temperature and washed 3 times in TBS buffer. They were then permeabilized in 1% Triton-X100 (v/v) for 15 minutes and washed 3 times in TBS buffer. Non-specific binding was blocked by a 1 hour treatment in TBST (TBS buffer with 0.1% Tween) containing defatted milk powder (30 mg/ml). The cells were then incubated for 1 hour at room temperature with either anti-glial fibrillary acidic protein (GFAP, Dako, mouse IgG, dilution 1:500), or Tuj1 (Molecular Probes, mouse IgG, dilution 1:1000), or O4 (Chemicon, mouse IgM, dilution 1:100) primary antibodies (diluted in blocking buffer). After 3 washes in TBS, cells were then incubated in FITC- or Cy5-conjugated anti-mouse IgG or IgM (Jackson Immunoresearch, 1:500) for 1 hour at room temperature and in the dark. The nuclei were stained with ethidium homodimer (0.2 μM, Sigma). The preparations were then mounted in Fluoprep™  and observed using a Bio-Rad MRC1024 laser scanning confocal microscope. The fraction of positive cells was determined by counting 10 non-overlapping microscopic fields (±50 cells/field, ±3500 cells/experiments) for each coverslip in at least three separate experiments (n then corresponds to the number of coverslips).After 24 hours or 3 days of culture, BrdU (20 μM, Sigma) which is a S-phase marker, or propidium iodide (400 mg/ml, Sigma) was added to the differentiating neurosphere cultures for 18 hours before fixation and staining. Tuj1, O4 and GFAP immuno-labellings were performed as described above. For BrdU labelling, coverslips were then post-fixed for 10 minutes in 4% (v:v) paraformaldehyde, incubated in HCl 1 N for 20 minutes at 37°C, washed with sodium perborate solution (50 mM, pH 8.5) and finally incubated with an anti-BrdU antibody for 1 hour at room temperature (Oxford, rat IgG, dilution 1:200) and Cy-5-conjugated anti-rat antibody, 1 hour at room temperature. The preparations were analysed as describe above.Total RNA was prepared using the RNeasy total RNA purification kit (Qiagen, Westburg). For cDNA synthesis, random hexamer primers (Invitrogen) were used to prime reverse transcriptase reactions. The cDNA synthesis was carried out using Moloney-murine leukemia virus (M-MLV) Superscript II Reverse transcriptase (Invitrogen) following the manufacturer's instructions. Quantitative PCR was carried out using standard protocols with Quantitec SYBR Green PCR Kit (Qiagen). The PCR mix contained SYBR Greeen Mix, 0.5 μM primers Table , 1 ng DNAfter 72 hours of culture, the conditioned medium (1.5 ml) from cultures of neural progenitor cells, npMSCs and nnMSCs were incubated with heparin sepharose CL-6B (Amersham Biosciences, Belgium) at 4°C for 24 hours. After centrifugation (700 × g), the supernatant was removed and bound proteins were eluted in loading buffer  by heating at 70°C for 10 minutes. The protein concentrations of various samples were quantified using the "RC DC Protein Assay" (Bio-Rad, Belgium) and equal protein quantities were loaded in each lane in a 15% sodium dodecyl sulfate polyacrylamide gel and electrophoresed. Then the proteins were transferred to PVDF membrane (Amersham Biosciences, Belgium) using a Trans-Blot Semi-Dry Transfer apparatus (Bio-Rad). The membranes were saturated with 3% gelatin (BioRad) during 1 hour at 37°C, then incubated for 1 hour with a monoclonal antibody against BMP4 (R&D, goat IgG, 0.1 μg/ml) or actin, as control for protein loading (Sigma, mouse IgG 1:5 000) at room temperature and then washed several times with PBS-0.1% Tween. The membrane was then incubated in Cy5-conjugated anti-goat IgG or anti-mouse IgG (Jackson Immunoresearch, 1:2500) for 1 hour at room temperature, in the dark. After several washes in PBS, the membranes were scanned using a Typhoon 9200 Scanner (Amersham Biosciences) and subsequent analyses were performed with ImageQuant Software (Amersham Biosciences).Neurospheres were plated on polyornithine-coated dishes and incubated during 5 days in DEM/F12 medium supplemented with B27 (Invitrogen) and previously conditioned by npMSCs during 3 days or not. Anti-BMP4 antibody (R&D, 2 μg/ml) was added on day 1 of this incubation.SW performed most of the work and wrote a first draft of the manuscript; FB performed quantitative RT-PCR; GH, GM and PL were involved in the writing of the manuscript; BR conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.


Tasks involving conflict are widely used to study executive attention. In the flanker task, a target stimulus is surrounded by distracting information that can be congruent or incongruent with the correct response. Developmental differences in the time course of brain activations involved in conflict processing were examined for 22 four year old children and 18 adults. Subjects performed a child-friendly flanker task while their brain activity was registered using a high-density electroencephalography system.General differences were found in the amplitude and time course of event-related potentials (ERPs) between children and adults that are consistent with their differences in reaction time. In addition, the congruency of flankers affected both the amplitude and latency of some of the ERP components. These effects were delayed and sustained for longer periods of time in the children compared to the adults.These differences constitute neural correlates of children's greater difficulty in monitoring and resolving conflict in this and similar tasks. Conflict tasks involve the selection of a sub-dominant object or response in the presence of a competing dominant object or response. One of the most common tasks used in the literature to measure conflict is the flanker task. In this task, a target surrounded by stimuli suggesting either the same (congruent) or the opposite (incongruent) response is presented. Conflict is induced by incongruent flankers which, compared to congruent ones, produce larger reaction times and reduced response accuracy .conflict monitoring to describe these operations. Once conflict is detected, it is necessary to determine the appropriate action in a goal-directed manner. Depending on the task, the resolution of conflict might involve different processes (e.g. inhibition, rule-holding, set switching, planning, etc.).Different cognitive operations appear to be involved in processing conflict . First, Monitoring and resolving conflict is a function of executive attention . A netwoRecent studies have dissociated the brain areas within the executive network that are responsible for monitoring and resolving of conflict . In a fMYoung children have more difficulty than older children and adults performing tasks that involve conflict. Using conflict tasks adapted to children, we have reported a considerable reduction in the amount of interference produced by distracting information in children from 2 to 3 years of age . This reSome developmental studies have been carried out using neuroimaging aimed at understanding the brain mechanisms that underlie the development of executive functions. For instance, Casey and her colleagues  conducteA number of studies have used the high temporal resolution of event related potentials (ERPs) to assay the timing of action-monitoring processes with adults. The N2 is one of the ERP indexes that have been associated with executive attention. The N2 is a pre-response negative deflection in the ERP at around 300 ms post-stimulus, which appears to be larger (more negative) for trials that involve more conflict. The N2 is observed over parietal and frontal leads and has been obtained with both flanker ,20 and GOnly a few ERP studies have been conducted with children using conflict tasks. In one of these studies, a flanker task was used to compare conflict resolution in three groups of children aged 5 to 6, 7 to 9 and 10 to 12, and a group of adults  while P3Recently, Davis et al.  conducteSo far, the literature suggests that monitoring and resolution of conflict involve separate brain areas in adults, and that children activate similar, but somewhat larger areas. Moreover, the N2 component of the ERPs appears to be related to activation coming from the ACC and to be mainly associated with conflict monitoring, whereas later components (e.g. LPC) might result from prefrontal sources of activation and could be related to conflict resolution.The flanker task is an appropriate experimental paradigm for assessing conflict processing. The aim of this study was to use a version of this task with children and adults to assess developmental differences in the time course of the different operations involved in conflict processing. We have recently developed a flanker task appropriated for use with children as young as 4 years . In thisTo study the time course of conflict processing, we examined the latency to significant differences between the ERPs for congruent and incongruent trials, and the sustainability over time of these differences. In the adult literature, the amplitude of the N2 component has been shown to be modulated by the congruency of distracting information in flanker tasks and has been related to conflict monitoring, but this component was not assessed in the study by Ridderinkhof and van der Molen. We aim to replicate the effect with adults using our child-friendly flanker task as well as analyzing this ERP component in 4 year olds. While there may be subcomponents of the N2 sensitive to other types of manipulations such as the degree to which the predicted identity of a display is violated , in our Means of the median RT and percentage of errors are shown in Table A similar 2 (Group) × 2 (Flanker Type) ANOVA was conducted using percentage of errors as the dependent measure. Again, the main effects of Group (F(1,38) = 22.33; p < .001) and Flanker Type (F(1,38) = 7.08; p < .05) were significant, whereas the Group × Flanker Type interaction was marginal (F(1,38) = 3.28; p = .078).Figure We hypothesized that particular ERP components would be sensitive to the presence of conflict in the display. To examine these predicted effects, we computed the latency and amplitude of the peaks of the N1, N2 and P3 components in both groups and of the LPC in the group of children separately for congruent and incongruent conditions in a selection of frontal and parietal leads (see Table Because of the greater presence of artifacts in the children's ERPs, a significantly larger number of segments were used to compute the averaged ERPs in the adult data (see Method section). To control for possible influence of this difference on the amplitude of the ERPs , the numFor the peak latency, the main effect of Group was significant in all the ERP components. The main effect of Flanker Type was significant for the N1, P3 and LPC. No significant interactions were found for any of the ERP components for the latency data.For the peak amplitude values, the main effect of Group was significant for the N1 and N2. The main effect of Flanker Type was not significant for any of the components although it was marginally significant for the N1 and for the LPC in the children. The main effect of Channel was marginal for the N2 and highly significant for the P3. Interestingly, there was a significant Group × Flanker interaction for the P3, indicating a significant effect of the type of flankers in the peak amplitude of this component for children (F(1,30) = 6.0; p < .05) but not for adults (F < 1).Although the peak amplitude of ERP components is a widely used measure to look at effects of the variables of interest in the patterns of brain activation, these effects can certainly occur along the entire epoch and not only in the peaks of the components. To examine the effect of congruency in the amplitude of the registered activity in the entire epoch, we computed amplitude differences between congruent and incongruent conditions sample by sample along the ERP segment in all channels for children, and a selection of channels around the Fz, Fcz and Pz positions in the adult data. T-tests were carried out to assess the significance of these differences along the epoch. In Figure In order to explore possible associations between the amplitude and latency dimensions of the ERP components and the particular cognitive processes measured by reaction time (RT), we examined correlations between RT measures and patterns of brain activity at Fz, Fcz and Pz positions and their left and right equivalents. Correlations were computed independently for adults and children. The first set of correlations involved the conflict effect as measured by subtracting the RT for congruent trials from the RT for incongruent trials (conflict score) and the overall RT as behavioral measures, and the overall (across flanker conditions) latency and amplitude of the ERP components as electrophysiological measures. For the adults, the overall RT correlated negatively with the amplitude of the N1 at channel F3 , and positively with the latency of the N2 component at channel Fc4 . For the children, the overall RT correlated negatively with the amplitude of the P3 at channel P4 , and positively with the latency of the N1 at channel F4  and the N2 at channel Fcz . No significant correlations were established between any of the overall ERP components and the conflict score in either adults nor children.Finally, correlations were calculated between the behavioral measures of overall RT and conflict score, on the one hand, and the effect of flankers on the amplitude and latency of the peaks of the ERP components on the other. For the adults, overall RT correlated positively with the N2 latency effect at Fcz  and the P3 amplitude effect at P4 , whereas the correlation was negative with the N2 latency effect at Fc4 . On the other hand, the conflict score correlated positively with the amplitude effect on the N2 at Fc4  and Fcz , although the last effect was only marginal. In the children, the overall RT correlated negatively with the N2 latency effect at Fc3 , and marginally with the N1 amplitude effect at Fz and F4 . However, the conflict score correlated negatively with the P3 latency effect at channel P4 .As expected, young children showed increased difficulty compared to adults in both processing the target and dealing with distracting information incongruent with the correct response. The greater difficulty of the task for children was reflected in children's much longer overall RT and conflict scores. The main goal of the current study was to analyze the differences in brain activation between children and adults underpinning their behavioral differences. Our results show differences among children and adults in both the time course of brain activations overall and across flanker conditions.Significantly larger N1 and N2 amplitudes were found for children than for adults, whereas the P3 showed equivalent amplitudes in the two groups. Children usually show larger event related potentials and often with delayed latency compared to adults ,24. ThesDifferences in latency of the ERPs components can be of special interest when it comes to accounting for differences in RT. Accordingly, children showed significant delays in the latency of all components compared to adults. The difference between children and adults was greater in the later components, suggesting that children's delay in target processing is more pronounced in later stages of processing. An objection to this conclusion is that latency and amplitude of the waveforms deflections are not independent, given the fact that greater peak latencies can be expected with more pronounced differences in amplitude. However, two pieces of information in our data point to the fact that differences in amplitude cannot account for all differences in latency. First, the P3 component shows a large difference in latency despite no overall differences in amplitude. Second, the overall RT appears to correlate negatively with the amplitude of some of the components in both children and adults, whereas it correlates positively with latency measures.In addition, overall RT appear to correlate with the overall amplitude and latency of some ERP components, while no significant correlations are established between these and the conflict score, suggesting that the general speed of processing but not the ability to manage conflict might be related to the general form of the ERP.It should be borne in mind in comparing the adult data with previous studies conducted with other flanker tasks that the child-friendly version of this task used in our study was very easy for adults. This could account for the modest amplitude differences between congruent and incongruent trials in this study compared to what has been found with other versions of the flanker task . From whAs shown in Table On the other hand, 4 year old children do not show differences in brain activity among the two flanker conditions until approximately 500 ms post target. Therefore, the effect of flankers is not observed at this age in the relatively early N1 component, and only very weakly at the N2  and twenty-two children  participated in the study. All participants were right-handed. The adult participants and the parents of children involved in the study gave written consent prior to the experimental session. Both children and adults were paid for participating in the study.The stimulus sequence for each trial was controlled using E-Prime . Each trial began with a sound to alert participants about the start of the trial. One second after the sound, a line with five drawn fish was presented in the center of the screen Figure . The cenEEG was recorded using a 128-channel Geodesic Sensor Net  for chilThe EEG signal was digitized at 250 Hz. Impedances for each channel were measured prior to recording and kept below 80 kΩ during testing. Recording in every channel was vertex-referenced and the time-constant value was 0.01 Hz for both children and adults. Data were recorded using Net Station 2.0 (EGI Software) and processed using Net Station 3.0.vs adults difference was significant (t(23.4) = -3.66; p < .001).Once acquired, data were filtered using a FIR bandpass filter with 12 Hz low-pass and 1 Hz high-pass cutoffs. Continuous EEG data was segmented into target-locked epochs. The epochs were 1 sec. long for adults (-200 ms to 800 ms around target) and 1.7 sec. long for children (-200 ms to 1500 ms around target). Segmented files were scanned for artifacts with the Artifact Detection NS tool using a threshold of 70 μV (adults) or 100 μV (children) for eye blinks and eye movements. Segments containing eye blinks or movements as well as segments with more than 25 bad channels were rejected. Within each segment, channels with an average amplitude of more than 200 μV or a difference average amplitude of 100 μV were also discarded from further processing. Finally, particular channels were rejected if they contained artifacts of any kind in more than 50% of the segments. Children's data were also visually inspected trial by trial to make sure the parameters of the artifact detection tool were appropriate for each child. As a consequence of the artifact detection procedure, an average of 36% of the ERP segments in the children data and an average of 18.5% of the ERP segments in the adults were rejected. The larger number of rejected segments for the children was due to a higher frequency of blinks, mouth and/or head movements, speaking, and other behaviors that generate artifacts on the EEG signal during the experimental procedure. Thus, we decided to have a criterion of a minimum of 12 clean segments per flanker condition among the correctly responded trials for further processing individual data. All adults participants and a total of 14 children reached this selection criterion. The average number of segments included in the averaged ERPs was 53.2 (SD: 23.1) for the children , and 80.3 (SD: 17.3) for adults , and this children Artifact-free segments for correct responses were averaged across conditions and subjects and re-referenced against the average of all channels. The 200 ms preceding the target served as baseline.MRR, MIP & MKR designed the study and participated in the theoretical elaboration of the paper. MRR was responsible for the data collection and data analysis processes. CPD designed and performed part of the statistical analysis on the EEG data.