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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 . 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 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 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 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 , 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 . 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 .Specimens obtained during the course of surgery, referred to in this paper as "osteoma", were immediately placed in sterile Gibco Hanks' balanced salts solution 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 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 . 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 , OCN , anti-human polyclonal goat antibody against type I collagen , anti-rat polyclonal rabbit antibody against type X collagen and anti-human polyclonal rabbit antibody against VEGF were used at 1 mg/ml. Peroxidase-conjugated goat anti-mouse was purchased from Immunovision Technologies , peroxidase-conjugated goat anti-rabbit from Dako and peroxidase-conjugated donkey anti-goat from Santacruz Biotechnology . 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 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 -ethylamino}-nicotinonitrile) was synthesized in 7% overall yield using a convergent approach from 2,4-dichlorobenzoyl chloride and 6-chloro nicotinonitrile respectively -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 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 , 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 . 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 . The BP- insulin . The FOXThe TOPflash reporter plasmid kit were obtained from Upstate Biotechnology . 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 . Betweenribed in .6 cells/75-cm2 tissue culture flask) in DEM/F12 supplemented with EGF 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 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 , or Tuj1 , or O4 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 for 1 hour at room temperature and in the dark. The nuclei were stained with ethidium homodimer . 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 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 which is a S-phase marker, or propidium iodide 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 and finally incubated with an anti-BrdU antibody for 1 hour at room temperature 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 . 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 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" 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 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 or actin, as control for protein loading 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 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 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 .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 = 22.33; p < .001) and Flanker Type = 7.08; p < .05) were significant, whereas the Group × Flanker Type interaction was marginal = 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 . 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 = 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.

Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-κB is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-κB during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-κB activation in all the cells; invasion of cells by the bacteria is not required to activate NF-κB.Infection of intestinal epithelial cells by pathogenic Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-κB via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-κB activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-κB activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both.Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and Ikappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella mediated NF-κB and proinflammatory signaling and gene activation by this flagellated pathogen. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Lastly, TLR5 expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition.These collective results provide the evidence that flagellin acts as the main determinant of Salmonella and other enteroinvasive pathogenic bacteria such as enteroinvasive E. Coli, Shigella and Yersinia upon infection of IECs leads to the up-regulation of the expression of host genes, the products of which activate mucosal inflammatory and immune responses and alter epithelial cell functions aminomethane (Tris) was purchased from Fisher Scientific . Fetal calf serum was purchased from US Biotechnologies Inc. . Para-nitro-phenylphosphate (PNPP) was purchased from Aldrich Chemical . The Polyacrylamide gel electrophoresis (PAGE) supplies: acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), TEMED, and ammonium persulfate were purchased from Bio-Rad Laboratories . Dulbecco's modified essential medium (DMEM), DMEM:F12, phosphate buffered saline (PBS), glutamine, penicillin G, streptomycin, amphotericin B, and Grace's Insect medium were purchased from Invitrogen . Luria Broth (LB) was purchased from Becton Dickson and Co . The protease inhibitors: aprotinin, bestatin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Cal Biochem . Protease inhibitor cocktail contained 10 μg/ml aprotinin, 2.5 μg/ml leupeptin, 8.3 μg/ml bestatin, and 1.7 μg/ml pepstatin A. Phorbol 12-myristate 13 acetate (PMA), N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes), anisomycin, and 2-[N-morpholino]ethanesulfonic acid (MES) were purchased from Sigma Chemical . All other reagents were purchased from Sigma Chemical or Fisher Scientific unless stated otherwise.2 atmosphere. T84 colorectal carcinoma cells (ATCC CCL-248) were cultured in DMEM:F12 with 2 mM glutamine, 5% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2 atmosphere. H5 insect cells (Invitrogen) were cultured in Grace's medium with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, 100 μg/ml Streptomycin, and 0.25 μg/ml amphotericin B at 28°C. MyD88-/- & TLR2-/-/TLR4-/- double knockout cells were obtained from Shizuo Akira and Osamu Takeuchi and grown in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2 atmosphere.HT29 human intestinal epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% COSalmonella typhimurium strain SJW1103 [Salmonella typhimurium and can only express the Phase I fliC flagellin, SJW86 (SJW1103 FliC::TN10), and SJW134 (SJW1103 FliC and FljB deletions) were obtained from Robert Macnab and have been described [Salmonella serovar dublin strain 2229, strain SE1 (2229 SopE mutant), strain SB2 (2229 SopB mutant), and SE1SB2 (2229 SopE and SopB mutant) were obtained from Edward Galyov and have been described [Salmonella strains for stimulation were grown in LB at 37°C without agitation for 16 hours, centrifuged at 6,000 × g for 1 minute, gently washed with PBS, and gently suspended in DMEM to maintain cells with attached flagella.bilized) is a wilescribed . Salmoneescribed ,15. SalmSalmonella host is internalized by mammalian cells, obtained from Stanley Falkow [Plasmid pFM10.1 (ampicillin resistance), encodes a green fluorescent protein (GFP) expressed after the ord, CA) and was S. dublin 2229 or S. typhimurium SJW1103. Starter cultures were grown in Luria broth (LB) for 18 hours at 37°C with aeration, diluted 1:5000 in fresh LB, and grown for 12 hours under the same conditions. All subsequent procedures were performed at 4°C. Cells were removed from the medium by centrifugation at 10,000 × g for 5 min and discarded. The supernatant containing flagellin was filtered through a 0.8 micron filter to remove residual cells. Supernatant was concentrated 100 fold using an Amicon 100 kiloDalton (kDa) cutoff membrane (Millipore). Initial studies used concentrated culture supernatant from S. dublin strain 2229 that was treated with DNase, RNase, Protease K, boiled for 20 min or 100 mM DTT at 37°C for 2 hours and used for stimulation of cultured cells.Native flagellin was harvested from S. typhimurium 1103 bacterial culture supernatant was washed 4 times by 1:10 dilution with 50 mM MES, pH 6.0, 50 mM NaCl and re-concentrated. Material not retained by the 100 kDa membrane was discarded. Washed culture supernatant was fractionated by gel permeation or anion exchange chromatography for analysis. For long-term storage, washed culture supernatant was supplemented with protease cocktail and stored at -20°C.Concentrated Fractionation by gel permeation chromatography was performed with a Superose 12HR column (Pharmacia) on a Bio-Logic system (Bio-Rad). One-half mililiter of 100× washed supernatant was separated on the column at 0.4 ml/minute in 50 mM Hepes, pH 7.4, 200 mM NaCl. Fractions (0.5 ml) were collected, and 50 μl was fractionated by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Thirty microliters of each fraction was used for stimulation of HT29 cells (60 mm dishes) for 45 min and NF-κB DNA binding activity in the resulting whole cell extracts extracts were assayed by EMSA. The column was standardized with catalase (232 kDa), aldolase (158 kDa), abumin (67 kDa), ovalbumin (43 kDa), and Chymotripsinogen A (25 kDa), all obtained from Amersham-Pharmacia.Fractionation by anion exchange chromatography was performed with Poros HQ matrix on a Bio-Logic system. Five mililiters of 100× washed supernatant was separated at 1 ml/minute in 50 mM Hepes, pH 7.4, and a NaCl gradient from 50–500 mM. Fractions were collected and 5 μl of each fraction was examined by 10% SDS-PAGE. Proteins were fractionated on duplicate 10% SDS-PAGE precast gels (BioRad). One gel was stained with Bio-Safe Coomassie (Bio-Rad) and the protein bands were isolated for Mass Spectroscopy analysis (CCF Mass spectroscopy core facility) from the other identical non-stained gel, by electro-elution with a whole gel eluter (Bio-Rad) and SDS was removed with SDS-Out per the manufacturers directions. Proteins isolated from bands B1 to B6 were acetone precipitated by addition of 20 μg Aprotinin and 5 μg of BSA to each eluted fraction, ice-cold acetone (-20°C) was added to 80%, mixed well and precipitated overnight at -20°C. Proteins were pelleted by centrifugation at 14,000 × g in the cold for 30 min, acetone/liquid was removed and the pellets washed 2× with 1 ml acetone (-20°C). After removal of the acetone, protein pellets were air dried and then resuspended and denatured in 5 μl of 6 M guanidinium hydrochloride (Gu-HCl) at room temperature for 30 min. Resuspended proteins were two-fold serially diluted in DMEM to a final Gu-HCl concentration of 55 mM to renature the proteins. Two hundred fifty microliters of individual renatured proteins/DMEM were added per ml to HT29 cells (60 mm dishes) and whole cell extracts were prepared 45 min after stimulation and were assayed for NF-κB DNA binding activity by EMSA.S. typhimurium 1103 containing flagellin was boiled for 20 minutes and precipitants removed by centrifugation at 15,000 × g. The supernatant containing flagellin was diluted 1:2 with 50 mM MES, pH 6.0, 50 mM NaCl and mixed with 2 ml Poros SP cation exchange matrix (PerSeptive Biosystems) per 1 liter of original culture. The Poros SP matrix was prepared as a 50% slurry and equilibrated with 50 mM MES, pH 6.0. The flagellin preparation and matrix were mixed on a roller at 12 to 14 RPM for 2 hours. The matrix along with bound contaminants was removed by filtration through a 0.85 micron filter and discarded, flagellin failed to bind to the cation exchange matrix at pH 6.0 and eluted in the flowthrough and was collected.The washed and concentrated culture supernatant from The pH of the flowthrough was adjusted by five-fold dilution of the sample with 50 mM Hepes, pH 7.8, 50 mM NaCl, and loaded onto a Poros HQ anion exchange column equilibrated with 50 mM Hepes, pH 7.4, 50 mM NaCl. The column was washed with 2 volumes 50 mM Hepes, pH, 7.4, 50 mM NaCl, and eluted with a 10 column volume linear gradient of 50–500 mM NaCl in 50 mM Hepes, pH 7.4. Flagellin eluted from the column between 200–275 mM NaCl. Fractions containing flagellin were pooled and concentrated. The preparation was determined to be pure by electrophoresis of 5 μg protein by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Samples were stored at -80°C in 50 mM Hepes, pH 7.4, approx 225 mM NaCl, 10% glycerol and protease cocktail. A 4 liter preparation of culture supernatant yielded 2 mg purified flagellin.Gels were fixed and stained . All of the following procedures were performed by the CCF Mass spectroscopy core facility. Excised gel bands were reduced (100 mM DTT), and alkylated (100 mM iodoacetamide). Proteins in the gel bands were digested with modified trypsin with an overnight incubation at 37°C. Tryptic peptides were extracted from the gel with 50% acetonitrile, 0.1% acetic acid, concentrated in a SpeedVac (Thermo Savant) to remove acetonitrile, and reconstituted to 20 uL with 0.1% acetic acid. Extracted peptides were subjected to reversed phase liquid chromatography , coupled to a Finnigan LCQ DECA ion trap mass spectrometer for peptide sequencing, as described .IκBα amino acids 1 to 54 fused to GST or cJUN amino acids 1–79 fused to GST were prepared as previously described -39 and sSalmonella invasion. Cover slips were mounted with Vectashield mounting medium with DAPI , and cover slips sealed to slides.HT29 cells for microscopic examination were grown in 6 well plates on sterile cover slips to a density of 50–75%. Cells were stimulated as described above. After stimulation, cover slips with HT29 cells were washed 2 times with ice cold PBS and fixed with 4% w/v formalin at room temperature for 20 minutes. Cells were washed 4 times with PBS prior to mounting for visualization of Cells for antibody staining were treated with absolute methanol for 20 minutes following formalin fixation, then washed 3 times with PBS supplemented with 0.1% BSA (PBSB) and used directly or stored in the cold after azide was added to 0.02%. For p65(RelA) localization, cells on coverslips were blocked for 1 h at 37°C with PBS supplemented with 1% BSA. The PBSB was removed, washed once with PBSB and coverslips were placed cell-side down onto 150 μl of p65 antibody diluted 1:1500 in PBSB on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 3 × 5 min with PBSB. Coverslips were then removed and placed cell-side down onto 150 μl of FITC-labeled donkey anti-rabbit secondary antibody (1:300 in PBSB) on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 5 × 5 min with PBSB, removed and placed cell-side down onto slides mounted with Vectashield with DAPI and then sealed. NF-κB localization was determined by indirect immunofluorescence. Samples were observed on a Leica DMR upright microscope at 400× with oil immersion and equipped with FITC and UV filters. Images were collected with a MicroMax RS camera , and Image Pro plus, version 4.5, software . Color enhancements were performed with Image Pro plus software. Visible light plus color overlays for Fig. 8 Salmonella/ml at 37°C for desired times and extracts prepared as below. Cells harvested beyond one hour were washed with warm PBS and supplemented with warm DMEM, 2 mM glutamine, and 200 ug/ml gentamycin after 1 hour and returned to 37°C until extract preparation desired.Mouse embryo fibroblasts (MEFs) or HT29 cells were grown in DMEM as above to a density of 90% prior to stimulation. All cells were washed with warm PBS and supplemented with DMEM without serum or antibiotics in preparation for stimulation. Cells were stimulated with; 10 ng/ml TNFα, 1 μg/ml flagellin unless specified otherwise, 20 μg/ml Anisomycin, 12.5 ng/ml PMA, or 10Cells were washed with ice-cold PBS and all subsequent steps carried out at 4°C or on ice. Cells were scraped from the dish in ice-cold PBS, and collected by centrifugation at 1000 × g for 1 minute. Cells were lysed by suspension in 50 mM Tris-HCl, pH 7.6, 400 mM NaCl, 25 mM beta-glycerol phosphate, 25 mM NaF, 10 mM PNPP, 10 % glycerol, 0.5 mM sodium orthovanadate, 0.5% nonidet-40 (NP-40), 5 mM benzamidine, 2.5 mM metabisulfite, 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail as described .NF-κB DNA binding assays were carried out as previously described ,35,38. AHT29 cells, 90–95% confluent in 35 mm round dishes, were prepared for stimulation as above and treated with a 1 ml suspension of Salmonella SJW1103 or SJW134 or left untreated in triplicate as above. After one hour, HT29 cells were washed 4× with warm PBS, supplemented with warm DMEM, 2 mM glutamine, and 200 μg/ml gentamycin, and incubated at 37°C for 4 hours. Cells were then harvested as above and lysed by suspension in 1 ml sterile distilled water. Ten-fold serial dilutions were prepared in PBS and 100 μl of each dilution was plated on LB agar plates and grown at 37°C for 20 hours. Colonies were counted and averaged.2). Immunopellets were resuspended in 30 μl Kinase buffer with 0.1 mM orthovanadate, 50 μM "cold" ATP, 5 μCi γ-32P-ATP, 2 mM DTT, and 2 μg of soluble GST-IκBα1–54 or GST-cJUN1-79, and incubated at 30°C for 30 minutes. Reactions were stopped by the addition of 15 μl 4× SDS-PAGE loading buffer, heated at 95°C for 5 minutes, and resolved on 10% SDS-PAGE gels by standard procedures. Gels were rinsed, stained with Bio-Safe Coomassie (Bio-Rad) to visualize protein bands, rinsed, photographed then dried and exposed to Kodak X-OMAT AR film to detect substrate phosphorylation.Whole cell extracts (250 μg) were supplemented with 150 μl of Buffer A , and immuno precipitation kinase assays carried out as described using eiProtein samples (40 μg) were resolved by SDS-PAGE on a 10% acrylamide gels by standard procedures, and proteins transferred to PVDF membrane (Millipore) and probed with antibodies as described . MembranAll DN-TLRs were constructed using PCR. The universal 5' primer consisted of a 5'KPN I restriction site followed by sequences encoding the kozak sequence, translational start site, and preprotrypsin leader sequence of pCMV-1 (Sigma) that all the wild-type TLRs were initially cloned into. The 3' anti-sense (AS) primers were human TLRgene-specific primers (sequences available upon request) that created a stop codon immediately after a conserved tryptophan in Box 9 of the TLR TIR homology domain according to Bazan , thus crRenilla luciferase normalization reporter and 1.85 μg pCDNA3.1 plasmid DNA as bulk filler DNA) and fire-fly luciferase expression was normalized to Renilla luciferase expression using the dual-luciferase assay . Fold inductions were calculated and values between experiments did not vary more than 15%, a representative experiment is presented. Transfection of 293T cells was performed with lipofectamine 2000 (Invitrogen) in 6-well dishes in triplicate as per the manufacture's protocol. TLR expression plasmids were added at 2 μg/well, and NF-κB and normalization control plasmids were as above with HT29 cells and pCDNA3.1 plasmid DNA as bulk filler DNA to a final DNA mass of 4 μg/well. Fold inductions were calculated and values between experiments (N of 3) did not vary more than 10%, a representative experiment is presented.HT29 cells were transfected with Lipofectamine Plus (Invitrogen) as previously described . In tran2PCR was performed with an iCycler (Bio-Rad) to quantify TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT2PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) per manufacturers instructions in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 15 min to activate Taq and amplified for 40 cycles . RT2PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Real-time PCR analysis was performed using SYBR-green (Perkin-Elmer) according to manufacture's instructions with the specific primer pairs indicated above and primer pairs for 18S ribosomal RNA as reference RNA (Classic 18S primer pairs – Ambion Inc). Cycle time (Ct) was measured using the iCycler™ and its associated software (Bio-Rad). Relative transcript quantities were calculated by the ΔΔCt method using 18S ribosomal RNA as a reference amplified from samples using the Classic 18S primer pairs from Ambion, Inc . Normalized samples were then expressed relative to the average ΔCt value for untreated controls to obtain relative fold-change in expression levels. Fold change in mRNA expression was expressed as 2ΔΔCt. ΔCt is the difference in threshold cycles for the TLR mRNAs and 18S rRNA. ΔΔCt is the difference between ΔCt non-simulated control and ΔCt stimulated sample. Values for fold-induction varied less than 5% among replicates.Cells N = 3) were stimulated 3 hours at 37°C with TNFα or FliC or left untreated and harvested for total RNA isolation. Total cellular RNA was extracted from cells with Trizol reagent (Invitrogen) [ were stiSalmonella invasion protein; PMA, phorbol 12-myristate 13 acetate; PNPP, para nitrophenyl phosphate; TK, thymidine kinase; BF, bright field; NP-40, nonidet-40; NRS, normal rabbit serum; IN, input; Ct, cycle time.The abbreviations used are: FBS, fetal bovine serum; IL-1, interlukin-1, SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; EMSA, electromobility shift assay; IB, immunoblot; KA, kinase assay; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TNFα, tumor necrosis factor α; NF-κB, nuclear factor kappa B; IKK, Ikappa B kinase; IκB, Ikappa B; PCR, polymerase chain assay; RT-PCR, reverse transcription polymerase chain assay; Gu-HCl, guanidinium hydrochloride ; MAPK, mitogen activated protein kinase; SAPK, stress-activated protein kinase; ERK, extracellular regulated kinase; TLR, toll-like receptor; DN, dominant-negative; JNK, Jun N-terminal kinase; AP-1, activator protein-1; MEF, mouse embryo fibroblast; WCE, whole cell extract; IEC, intestinal epithelial cell; MCP1, macrophage chemoattractant protein 1; TTSS, type III secretion system; Sip, TT and AD initiated the study and performed the majority of the experiments and contributed equally and were assisted by NK and JL. MD constructed a number of DN-TLRs and JD developed the study, provided funding support, oversaw the project and also constructed a number of mutant TLRs.

Profile-based analysis of multiple sequence alignments (MSA) allows for accurate comparison of protein families. Here, we address the problems of detecting statistically confident dissimilarities between (1) MSA position and a set of predicted residue frequencies, and (2) between two MSA positions. These problems are important for (i) evaluation and optimization of methods predicting residue occurrence at protein positions; (ii) detection of potentially misaligned regions in automatically produced alignments and their further refinement; and (iii) detection of sites that determine functional or structural specificity in two related families.For problems (1) and (2), we propose analytical estimates of P-value and apply them to the detection of significant positional dissimilarities in various experimental situations. (a) We compare structure-based predictions of residue propensities at a protein position to the actual residue frequencies in the MSA of homologs. (b) We evaluate our method by the ability to detect erroneous position matches produced by an automatic sequence aligner. (c) We compare MSA positions that correspond to residues aligned by automatic structure aligners. (d) We compare MSA positions that are aligned by high-quality manual superposition of structures. Detected dissimilarities reveal shortcomings of the automatic methods for residue frequency prediction and alignment construction. For the high-quality structural alignments, the dissimilarities suggest sites of potential functional or structural importance.The proposed computational method is of significant potential value for the analysis of protein families. Profile-based methods of sequence analysis use multiple sequence alignments (MSA) to extract information about conserved features of a protein family, which are impossible to decipher from a single sequence. Such methods increase both the sensitivity of homology detection and the quality of produced alignments -10, mainin silico sequence design that generates native-like sequences from a structural template. Detection of discrepancies between the model and the real data would assist the analysis of the model's performance and its further improvement. To our knowledge, such statistical assessment has not been proposed up to date.Statistical analysis at the level of individual MSA positions may be used to compare residue frequencies predicted from some model to the actually observed residue usage at the given position in sequence homologs. The model may represent, for example, a method for Several approaches have been proposed to detect potential regions of low alignment quality in sequence-sequence and sequence-profile alignments. These approaches range from identifying low-scoring regions in pairwise alignment to more When the analyzed alignment is highly reliable, detecting positions of significant dissimilarity may reveal sites that determine functional or structural specificity of otherwise similar proteins. Several approaches have been proposed that use comparison of multiple sequence alignments in order to predict such sites -21. HoweP-value Estimation for Alignment Columns), to the analysis of real MSA.In this work, we consider approximate analytical estimates of P-value in two settings: (1) comparison of an alignment column to an emission vector of residue probabilities, and (2) comparison of two alignment columns. These estimates allow detecting cases where the null hypothesis (assumption of similarity) can be confidently rejected. We performed simulation experiments that show consistency of the estimates with the statistical model, and applied our method, PEAC is generated by given vector of emission probabilities f. If this hypothesis is rejected, then the set of emission probabilities is inadequate for the description of the residue content in this alignment column.given alignment column (vector of residue counts ρ(n | f), which is difficult for analytical consideration. To calculate the P-value, we use the multivariate Gaussian approximation of the multinomial distribution, based on the assumption of large statistical samples :The assumed null model of random columns corresponds to a multinomial form of x = {xi} is a random d-dimensional vector of residue counts of size , f is emission vector of residue frequencies, is the mean vector of residue counts, Σ = ||cov|| is the covariance matrix. This approximation of p.d.f. allows for the analytical expression for the P-value (Appendix 1 [see where ix 1 see ): is a regularized gamma-function, d is dimensionality of vector f. Thus, P-value is described by a χ2 distribution with (d - 1) degrees of freedom.where f = {fi}120 to generate a large number Ω = 107 of random columns of a fixed size N, i.e. Ω sets of N residues drawn randomly according to probabilities fi. For each random column, residue counts n = {ni}120 were derived and the multinomial probability of its generation was calculated as , where is the multinomial coefficient. All Ω generated columns were sorted by ρmult in the ascending order. For a given P-value P*, the column number ΩP0 was chosen from this sorted list. This column corresponded approximately to the multinomial P-value P*. This P-value was compared to our estimate Pestim (formula (2)) calculated for the chosen column in the Gaussian approximation of multinomial distribution. For each value P* we performed 10 independent simulations and plotted average values of Pestim against P*, which showed their general consistency. Figure f derived from real alignment columns, and for three typical column sizes N. The accuracy of estimates becomes poorer for lower column sizes and more skewed frequency sets ) with P-values based on the multinomial model. In particular, we used a set of residue frequencies ets Fig. . Howeverm* and n* are generated by a single vector of emission probabilities. As the prior distribution of emission vectors, we use the maximum likelihood (ML) estimate based on m* and n*. Such prior should produce the conservative upper estimate of the P-value. Rejection of hypothesis H0(2) would mean that the two alignment columns are highly dissimilar.two observed columns The P-value for this hypothesis is calculated in three steps:n, m}, we produce the ML estimate of the p.d.f. for emission vectors f that can generate both columns simultaneously. We assumed a simple form of multivariate Gaussian distribution and calculated ML estimates of its mean and variance values (formulae B5).a). Given the two vectors of residue counts {θ(f) as the prior to calculate the posterior probability ρ) that a pair of random columns {n, m} is produced by any single emission vector f. Similarly to problem 1, we use multivariate Gaussian approximation of the multinomial distribution that assumes large total residue counts in the generated columns. The posterior probability density can be calculated asb). We use this p.d.f. ρ) = ∫ρ θ (f) df     (3)c). Using (3), we calculate P-value as the integral (Appendix 2 see ):θ(f) is a ML estimate based on the observed alignment columns. The partial integral ∫ρ dm dn can be calculated analytically for any emission vector f, but analytical calculation of full integral (4) is problematic. However, an approximate estimate of this value would suffice, since (i) expression (4) already contains approximations introduced by estimates of θ(f), ρ) and ρ); and (ii) we are interested in a conservative estimate of the upper P-value limit. Hence, we calculate an approximate upper estimate of P-value :erf(x) is error function, andwhere f was used to produce a column of size N by random draw according to these frequencies. Having the vector residue counts n in this column, we produced another vector of counts m that made our estimated P-value Pestim equal to the specified value P0. To produce this vector, we considered sets of residue counts as points in multidimensional and randomly chose a straight line passing through the point n. On this randomly directed line, we found the point m as the solution of equation Pestim = P0, where Pestim is defined by formula (5). Thus, we generated a pair of columns that corresponded to the specified P-value according to the PEAC estimate. We compared this estimate to the actual P-value P* calculated for the generation of m and n by the original vector f. As shown by the plot of P* against Pestim : (1) a given alignment column is generated by a given set of emission residue frequencies; and (2) two given alignment columns are generated by a single set of residue frequencies. We applied both types of estimates to the analysis of real multiple alignments, detecting cases of significant dissimilarity where the null hypotheses were confidently rejected.-320 and 1.0, with the median being approximately 0.01.Using our method, we assessed the consistency between predictions of residue frequencies based on structural considerations, and the frequencies in multiple alignments of sequence homologs. Specifically, we prepared a dataset of 1695 PDB structures and made predictions of residue propensities at each position, based on local structural environment. In parallel, the sequences corresponding to these structures were used as queries for PSI-BLAST searches, and profiles of detected confident sequence homologs were constructed (see Methods). The effective residue frequencies at profile positions were compared to the structure-based predictions, and P-values for each position were estimated using PEAC. The histogram of produced P-values for all positions is shown in Fig. P < 10-100). These sites were located mainly in the secondary structure elements, most frequently at their ends, and corresponded to unusual local distortions of 3D conformations. We compared residue content in the corresponding subset of alignment columns to the whole dataset. As shown in Fig. To analyze the cases of most pronounced discrepancy between our structure-based predictions and residue frequencies observed among sequence homologs, we chose ~1000 protein positions (0.3% of the whole dataset) that had lowest P-values of their carboxyl caps (data not shown). When we excluded glutamates and aspartates whose charge could be neutralized by contacts with positively charged arginine, lysine or histidine, the remainig portion of the set was still comprised of mostly buried residues In automatically produced alignments of sequences or structures, consideration of profiles of confident homologs helps to detect inconsistencies. According to our observations, these inconsistencies are caused mainly by alignment errors. (ii) In the high-quality structure based alignments, where structural equivalence of residues is confident, the low P-values may indicate functional specificity of spatially aligned residues.As an example of application (i), we evaluated our method by ability to predict erroneous residue matches produced by an automatic sequence aligner , as com-2.We then sorted all ClustalW positional matches by ascending P-values and classified them as true or false predictions of ClustalW errors. For our purpose, the ClustalW matches different from those in BaliBase were considered true positive predictions; whereas correct matches were considered false positives. Having the ranked list of true and false positive predictions, we generated sensitivity curve collected pairs of protein domains that are structurally similar according to the DALI alignments in the F-2 and the median was approximately 0.1 10-2. For identities around 25% . Using Insight II suit for molecular modeling and simulation (Accelrys), we performed a detailed manual analysis of structural superposition for a portion of the corresponding structural alignments. We found that the majority of inspected positions were apparently misaligned. Approximately 80% of these residues were located within 5 positions from a gap introduced in the structural alignment. Vicinities of gaps generally correspond to less similar fragments of aligned structures, which are more difficult to superimpose and where alignment errors can occur more frequently.We considered residue contents of the MSA columns corresponding to these low-P-value position pairs, and compared these contents to the average residue frequencies in the whole MSA datasets. In the set corresponding to 15 ± 1% sequence identity, the most pronounced difference was a higher frequency of aspartate at the position pairs with low P-values Fig. . In the We further concentrated on the aligned structural positions that showed unusual residue frequencies in the corresponding MSA columns. In the set corresponding to 15 ± 1% sequence identity, we considered positions with highly conserved aspartate, whereas in the set corresponding to 25 ± 1% sequence identity, we considered positions with high combined frequency of methionine, leucine and isoleucine. In an attempt to exclude apparently misaligned positions, we considered only those positions that were distanced more than 5 residues from gaps in the FSSP structural alignment. We selected and manually analyzed 16 of such positional matches. However, even among these selected matches most of the discrepancies were still caused by apparent alignment errors: 10 cases corresponded to structural misalignments , and 3 cases were caused by biased residue frequencies at profile positions, due to errors in PSI-BLAST alignments of sequence homologs. The remaining 3 position pairs did not involve apparent errors of either DALI or PSI-BLAST. These pairs might represent real differences in residue preferences at structurally equivalent positions.b). In glyoxalase II, D134 binds zinc atoms directly [Figure directly , whereasdirectly Fig. 7Cb. In glya illustrate superposition errors as the typical source of low P-values for automatic structural alignments. DALI . However, such alignment corresponds to low positional P-values, indicating a significant difference between structure-based and sequence-based position similarity. The optimal profile-based alignment . These proteins possess the same α/β knot fold but belong to different SCOP families, SpoU-like RNA 2'-O ribose methyltransferase and Ybea-like, respectively. Using manually curated structure-based alignment of the two proteins and MSAs of their homologs detected by PSI-BLAST, we considered structurally equivalent positions that were well aligned in space . We found 24 such positions, the majority being concentrated in the region of dimer interface, which includes the 'knotted' C-terminal helix D detection of potentially misaligned regions in automatically produced alignments and their further refinement; and (iii) detection of sites that determine functional or structural specificity in two related families.We proposed P-value estimates to assess statistical significance for (1) comparison of a single position in a multiple alignment to a set of emission residue frequencies; and (2) comparison of two alignment positions. Computational implementation of these estimates showed its potential value for several important tasks in sequence analysis: (i) evaluation and optimization of methods predicting propensities for residue occurrence at protein positions, such as protocols for neffPSIC for each symbol in the alignment column , and then applied the following transformation [Effective residue counts at alignment positions were calculated based on the PSIC method. ormation :neff corresponds to the number of randomly aligned sequences with the average number of residue types per position equal to neffPSIC .-5. In the resulting multiple alignments of detected homologs, we purged sequences whose identity to the query was less than 25%, so that only confident sequence homologs were used for profile construction. We split query sequence into fragments of fixed length F. For each fragment, we extracted the corresponding segment of the multiple alignment and removed the sequences with deletions (gaps) in this fragment. For a query of length L we produced L-F+1 sub-alignments and derived effective residue counts as described above. In this work, we used the library of profile fragments of length F = 6, which provided accurate results when applied to the prediction of local structural environment from a sequence profile [We applied our method to compare structure-based predictions of residue probabilities to the actual residue frequencies observed among sequence homologs. For such a comparison, we produced sequence profiles that correspond to fragments of known 3D structures. Briefly, we used a non-redundant set of structures from PDB . SCOP ,46. entr profile .φ and ψ dihedral angles) at the given position and the preceding position, and solvent accessibility of the sidechain at the given position. For a given position we used the partition of Ramachandran plot into 15 classes proposed by Shortle [The equilibrium frequency of an amino acid at a position in protein structure reflects the energetic fitness of the sidechain in the local structural environment ,39. To e Shortle For the As the second application, we estimated statistical significance of similarity between pairs of columns in multiple alignments. Namely, we used pairs of structurally similar proteins , proWe chose protein pairs with relatively low sequence identities, where detection of similarity between sequences is not straitforward. We focused on two identity ranges: 25 ± 1% (at the upper bound of twilight zone) and a lower range of 15 ± 1%. From each FSSP family, we extracted the parent sequence and all sequences of a significant structural similarity to the parent (Z-score greater than 5.0), with sequence identity to the parent within a given range. We found totally 494 and 1406 sequence pairs with identities 25 ± 1% and 15 ± 1%, respectively. These numbers were reduced by purging symmetric pairs and manual inspection of the remaining domains for the presence of repeats and low-complexity regions. For further analysis, we used 251 sequence pairs with identity 25 ± 1% and 340 pairs with identity 15 ± 1%, each pair representing a unique FSSP family. For each sequence, we ran 5 iterations of PSI-BLAST 2.2.1 against the NCBI nr database and obtained multiple alignments of detected homologs. We then applied a procedure of the alignment processing similar to that implemented in PSI-BLAST [Solvent accessible surface area (ASA) for the residues of interest was determined using NACCESS package , which wRS carried out the theoretical considerations, computational experiments, analysis of the results and drafted the manuscript. NG conceived of the study, and participated in its design and coordination. Both authors read and approved the final manuscript."P-value for multivariate Gaussian distribution".Click here for file"Upper estimate of P-value for similarity between two alignment columns".Click here for file

The coronary artery calcium (CAC) score is an independent predictor of coronary heart disease. We sought to combine information from the CAC score with information from conventional cardiac risk factors to produce post-test risk estimates, and to determine whether the score may add clinically useful information.We measured the independent cross-sectional associations between conventional cardiac risk factors and the CAC score among asymptomatic persons referred for non-contrast electron beam computed tomography. Using the resulting multivariable models and published CAC score-specific relative risk estimates, we estimated post-test coronary heart disease risk in a number of different scenarios.Among 9341 asymptomatic study participants , we found that conventional coronary heart disease risk factors including age, male sex, self-reported hypertension, diabetes and high cholesterol were independent predictors of the CAC score, and we used the resulting multivariable models for predicting post-test risk in a variety of scenarios. Our models predicted, for example, that a 60-year-old non-smoking non-diabetic women with hypertension and high cholesterol would have a 47% chance of having a CAC score of zero, reducing her 10-year risk estimate from 15% (per Framingham) to 6–9%; if her score were over 100, however (a 17% chance), her risk estimate would be markedly higher (25–51% in 10 years). In low risk scenarios, the CAC score is very likely to be zero or low, and unlikely to change management.Combining information from the CAC score with information from conventional risk factors can change assessment of coronary heart disease risk to an extent that may be clinically important, especially when the pre-test 10-year risk estimate is intermediate. The attached spreadsheet makes these calculations easy. Aggressive primary prevention of coronary heart disease (CHD) is most appropriate in patients at relatively high risk of CHD events ,2. The cTo answer this question, we need to know the effects of age, sex and other CHD risk factors on the expected distribution of CAC scores. Several large cross-sectional studies have described the prevalence and extent of CAC among different age/sex groups ,8-10 witWe identified a large sample of men and women without clinical CHD who presented for electron beam computed tomography scanning. Using questionnaire data collected from these patients about smoking habits and medical history , we determined how conventional CHD risk factors, along with age and sex, affect CAC scores. We then developed a method for combining information from conventional risk factors and the CAC score , and we present several examples illustrating how that method may be applied in common clinical situations.All persons referred by their physician to an electron beam computed tomography (EBCT) scanning center in Nashville, Tennessee for measurement of coronary artery calcification between May 15, 1995 and December 31, 1997 were eligible for inclusion. Subjects with a history of CHD or complaining currently of any chest pain were excluded, as were subjects for whom CHD risk factor data were incomplete or missing. Only the first CAC score was included for those who received more than one EBCT scan.Current age, sex and presence of CHD risk factors were elicited by questionnaire from subjects and referring physicians. Each subject was labeled with hypertension, high cholesterol and/or diabetes mellitus if they answered affirmatively to the question, "Has your physician ever told you that you needed medicine for X?", or if their physician confirmed that such a condition was documented in their medical records. Patients were labeled as smokers if they currently smoked or had quit smoking within the preceding 3 months. No direct measurements of blood pressure, lipids or glucose were taken for the purposes of this study.We estimated the 10-year risk of a first CHD event using published mathematical models based on the Framingham study . For thi2) with density ≥ 130 Hounsfield units. The CAC score was calculated according to the method described by Agatston [Each subject underwent electron beam computed tomography scanning with an Imatron C-100 or C-150 scanner after giving written informed consent. During a single breath hold, 40 consecutive slices of 3 mm thickness were obtained starting at the level of the carina and proceeding to the level of the diaphragm. Scans were obtained within 100 ms and were electrocardiographically triggered at 60–80% of the R-R interval. Coronary calcification was defined as a plaque of at least 3 consecutive pixels age and sex, 2) age, sex, hypertension, high cholesterol, smoking, and diabetes, and 3) the Framingham 10-year CHD risk estimate. We examined whether the effects of age were linear by testing a quadratic term in the model containing only age and sex. We evaluated the ability of each logistic model to discriminate subjects at high and low risk for CAC using the C-statistic, and estimated the proportion of variability in the extent of CAC explained in each linear regression model using the adjusted-R2 tests with 3 degrees of freedom to compare the expected frequencies based on each model with the observed frequencies. Lower p values, in this case, indicate a poorer fit of the model to the observed data. All statistical analyses were performed with Stata 7.0 .Finally, we used coefficients, intercepts and residual variance from logistic and linear models to estimate the probability that the CAC score of an individual with known risk factors would fall into each of four standard CAC score categories . We estimated these probabilities, using models containing the 10-year risk estimate as the only predictor, for a range of 10-year risk estimates. We also estimated these probabilities, using models with all CHD risk factor predictors, for the specific clinical scenario described in the Introduction (a 60-year-old woman with hypertension and high cholesterol) and for several other scenarios. We compared the actual distribution of CAC scores among 58–62-year-old women with hypertension and high cholesterol in our sample (n = 130) with predictions from 1) our two-stage model, 2) a one-stage model using Ln(CAC score + 1) as a continuous outcome in a linear regression model, and 3) a one-stage model using a censored normal distribution of cube-root transformed CAC scores (a Tobit regression model). This comparison was made both graphically and statistically, using XFirst, we calculated the Framingham 10-year CHD risk estimate according to published models . Next, wWe identified 9341 persons without chest pain or a history of CHD presenting for their first EBCT scan between 4/15/95 and 12/31/97. Our sample was mostly middle-aged, but included persons as young as 35 years and as old as 88 years of age. Forty percent were women. The proportion with cardiac risk factors was high, though only 9% were diabetic (Table th–75th percentile: 0 – 87). The prevalence of zero scores ranged from 80% among women younger than 50 years to 5% among men 70 years old or older. After excluding zero scores, log-transformed CAC scores were approximately normally distributed, and appeared to be strongly associated with age and sex was 135 (± 377), and the median was 4 (25Age and sex were strong predictors of the presence of CAC in logistic regression models Table . There w2 = 0.11) than the full model (R2 = 0.17).Among patients with non-zero CAC scores, age and sex remained strong predictors of the extent of coronary artery calcification, as measured by the Ln(CAC score) Table . Again, Using these models, we estimated the probability of measuring a CAC score in each of four standard CAC score categories using the Framingham 10-year CHD risk estimate, a value easily calculated from conventional CHD risk factors using accessible web- or handheld computer-based software. These probabilities ranged widely based on the value of the 10-year risk estimate, with the probability of measuring a zero CAC score going from 75% (at a 10-year risk of 2.5%) to 13% (at a 10-year risk of 25%) and high cholesterol will have a 15% risk of suffering a CHD event in 10 years, according to the Framingham equation. If this women undergoes EBCT scanning, our models predict a 47% chance that her CAC score will be zero, a 36% chance that it will be between 1–100, a 12% chance that it will be between 101–400, and a 5% chance that it will be greater than 400. By integrating this information with previously published relative risk estimates see , we estiOur strategy outperformed two other modeling strategies in predicting the actual CAC distribution among the 58–62-year-old non-smoking non-diabetic women with hypertension and high cholesterol in our study sample n = 127) are independent predictors of coronary artery calcification. This finding is consistent with previous studies -15. We aFinally, our analysis provides a guide for how to use the CAC score as a surrogate outcome when studying causes of coronary artery disease (a widely used study design -27). TheOur analysis has a number of limitations, perhaps the most important being a lack of clinical detail about participants. While we had information about conventional risk factors , the data were only available from a questionnaire, and were not confirmed by direct measurement. Only dichotomous indicators of such conditions were used. Furthermore, other conditions and indicators of high CHD risk such as family history of CHD, obesity, physical activity, income, education, and levels of C-reactive protein, triglycerides and Lp(a), for example, were unavailable. Whether such factors are important predictors of the presence and extent of coronary artery calcification is unknown. On the other hand, CHD risk assessment is often based on the same type of limited information we had available on each of our patients, so the models we present are perhaps more easily applicable to common clinical situations than models based on more detailed clinical data. Furthermore, a historical indicator of past exposure to high blood pressure or high cholesterol, as we had access to in this study, may actually be more useful as a predictor of CAC than treated blood pressure measured at one point in time. Another important limitation of this study is our lack of data on race/ethnicity – our results may not apply to all ethnic groups. Finally, our data are limited in application to CAC scores measured by electron beam computed tomography with 3 mm slice thickness and the described protocol. While CAC scores measured by the latest spiral computed tomography scanners appear to be similar to those generated by electron beam computed tomography , we cannThe Clinical Research Roundtable at the Institute of Medicine has identified translation of clinical research findings into improvements in medical care as the "next scientific frontier" . While oMP has received speaking and consulting fees from Bayer.MJP conceived the idea for the study, performed the analysis and drafted the manuscript. JAT and MP helped design and interpret the analysis. CM provided statistical guidance and interpretation. TQC recruited the patients and collected the data. WSB provided senior guidance in all aspects. All authors reviewed and commented on multiple drafts of the manuscript and approved the final draft.The pre-publication history for this paper can be accessed here:This spreadsheet is used for combining information from conventional risk factors and the coronary artery calcium score to estimate coronary heart disease risk in an individual patient. Step 1: Enter your patient's clinical information (the red numbers). Step 2: Choose an assumption about the coronary artery calcium score relative risks (optimistic or conservative). Step 3: Find the following results: 1) "Pre-test" 10-year risk of coronary heart disease (CHD) based on Framingham equations; 2) The probability of having a coronary artery calcium (CAC) score that falls within 4 standard CAC score categories; and 3) The "post-test" 10-year risk of CHD for each CAC score category. Step 4: Use the results to interpret a CAC score, or to decide whether or not to order a coronary artery calcium scan. If a score that would change your management is unlikely to occur, it may not be worth the money.Click here for file

Previous research indicated that women are more vulnerable than men to adverse psychological consequences of weight gain. Other research has suggested that weight gain experienced during antipsychotic therapy may also psychologically impact women more negatively. This study assessed the impact of acute treatment-emergent weight gain on clinical and functional outcomes of patients with schizophrenia by patient gender and antipsychotic treatment .Data were drawn from the acute phase (first 6-weeks) of a double-blind randomized clinical trial of olanzapine versus haloperidol in the treatment of 1296 men and 700 women with schizophrenia-spectrum disorders. The associations between weight change and change in core schizophrenia symptoms, depressive symptoms, and functional status were examined post-hoc for men and women and for each medication group. Core schizophrenia symptoms (positive and negative) were measured with the Brief Psychiatric Rating Scale (BPRS), depressive symptoms with the BPRS Anxiety/Depression Scale and the Montgomery-Asberg Depression Rating Scale, and functional status with the mental and physical component scores on the Medical Outcome Survey-Short Form 36. Statistical analysis included methods that controlled for treatment duration.Weight gain during 6-week treatment with olanzapine and haloperidol was significantly associated with improvements in core schizophrenia symptoms, depressive symptoms, mental functioning, and physical functioning for men and women alike. The conditional probability of clinical response (20% reduction in core schizophrenia symptom), given a clinically significant weight gain (at least 7% of baseline weight), showed that about half of the patients who lost weight responded to treatment, whereas three-quarters of the patients who had a clinically significant weight gain responded to treatment. The positive associations between therapeutic response and weight gain were similar for the olanzapine and haloperidol treatment groups. Improved outcomes were, however, more pronounced for the olanzapine-treated patients, and more olanzapine-treated patients gained weight.The findings of significant relationships between treatment-emergent weight gain and improvements in clinical and functional status at 6-weeks suggest that patients who have greater treatment-emergent weight gain are more likely to benefit from treatment with olanzapine or haloperidol regardless of gender. Because antipsychotic drugs are considered the core treatment modality for schizophrenia the diffAlthough most of the literature on treatment-emergent weight gain tends to focus on this event as adverse, a growing body of research has demonstrated a significant link between beneficial therapeutic response and treatment-emergent weight gain. With the exception of a few studies that failed to find an association between weight gain and better clinical outcome -9, most The study of gender differences in the relationship between treatment-emergent weight gain and therapeutic response has gained limited attention and provided conflicting results. A brief report on weight gain during clozapine therapy indicated that greater weight gain was associated with clinical improvement among women, but not among men . In contWomen in the general population appear to be vulnerable to the adverse emotional and psychosocial consequences of weight gain. For women, obesity has been linked to lower life satisfaction, increased social isolation , and lowThe primary objective of this study was to expand on prior research and investigate whether the relations between acute weight gain during antipsychotic therapy and treatment outcomes differ based on patient gender and the specific antipsychotic used in the treatment regimen, olanzapine or haloperidol. This study also aimed to broaden the definition of therapeutic response by extending beyond positive and negative symptoms of schizophrenia to depressive symptoms and levels of mental and physical functioning, because these domains tend to deteriorate with weight gain among women in the general population.We used data of 1296 men and 700 women who participated in a randomized, double-blind, multi-center, clinical trial comparing olanzapine to haloperidol . ParticiParticipants were randomly assigned in 2:1 ratio . Although randomization was not stratified on gender or any other patient characteristics, it resulted in a 2:1 ratio for males (870/426) and for females (426/233). The olanzapine group (N = 1337) included 467 women and 870 men, and the haloperidol group (N = 659) was comprised of 233 women and 426 men.Participants were randomly assigned to olanzapine, 5 to 20 mg/day, or haloperidol, 5 to 20 mg/day. The type of antipsychotic medication used prior to enrollment was not assessed in the current study, but the likelihood of previous treatment with an atypical antipsychotic drug was very low, because the study was initiated in 1994 when only clozapine was available in some of the sites. Further, randomization worked for patient and illness characteristics , and theWe used data from the acute phase, the first 6 weeks of the study, for several reasons. First and foremost, this study was a 6-week randomized double blind clinical trial with a 46-week "responder maintenance period", in which only patients who responded to the acute 6-week treatment per predetermined response criteria were eligible to continue. Consequently, the study design did not permit a longer-term analysis on the link between weight gain and improvement because only patients who improved during the first 6-weeks phase were followed-up for a longer time period. Second, the 6-week period represents a relevant time frame often used in clinical practice to determine treatment outcome and decide on treatment discontinuation . For manDuring the 6-week acute phase, the mean modal dose was 13.2 mg/day (SD = 5.8) for olanzapine and 11.8 mg/day (SD = 5.6) for haloperidol. There were no discontinuations due to weight gain as an adverse event for any treatment group during the 6-week study period, and the rate of discontinuation for any cause was similar for women (62.7%) and men (60.8%), with a significantly smaller proportion of the patients in the olanzapine group (33.5%) than in the haloperidol group . In addition, the percentage of patients who discontinued treatment because of an adverse event or a lack of efficacy was significantly higher in the haloperidol group than in the olanzapine group. Further details on the parent study design and primary findings are available elsewhere .2).This investigation used measures of positive and negative symptoms ("core schizophrenia symptoms"), depressive symptoms, functional status, and body weight. Core symptoms of schizophrenia were assessed by the Positive Symptom and the Negative Symptom subscales on the BPRS extracted from the Positive and Negative Syndrome Scale (PANSS) . Levels To enhance comparability of findings on different measures, the clinical measures were all standardized to z-scores. For the BPRS Core Symptoms, MADRS, and BPRS Anxiety and Depressive Subscale, this was done by subtracting the measure's overall mean and dividing by the measure's standard deviation at baseline. A single measure of depressive symptoms was calculated as the average of the standardized MADRS and BPRS Anxiety and Depressive Subscale. If a score was missing on either depression measure, the score on the available measure was used. The two depression measures were pooled because each is an independent and valid estimate of patients' level of depressive symptoms, and aggregating them should provide the best and most comprehensive estimate of depressive symptoms. Additionally, the pooling helped minimize loss of data, which are assumed not to be missing at random. The SF-36 Physical and Mental Component scores were converted from T-Scores to z-scores.Baseline comparisons used independent samples t-tests for continuous variables and chi-square tests for categorical variables. Effects of treatment and gender on independent variables were assessed using ANCOVA, with the baseline score as well as the number of weeks in the study as covariates. The relationship between change in weight and change in each outcome variable was assessed using separate multiple linear regression analyses, each with corresponding clinical change score as a dependent variable, the corresponding baseline score and number of weeks in the study as covariates, and the following independent variables: weight change, treatment group assignment, and gender. In an additional analysis, the interactions of these three independent variables were added to the regression models.The analyses included measures from baseline and the 6-week visit. Missing data were handled by carrying forward the last observation for all patients with at least one post-baseline assessment. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 11.0.Relative to men, women were older, more likely to be Caucasians, were more likely to be overweight or obese, had less severe positive symptoms, lower levels of physical functioning, and had higher levels of depressive symptoms Table . Women w2 = 51.8, p < 0.001).In order to illustrate the differences in weight gain by treatment group and gender, the patients were grouped into thirds based on their percentage of change in weight from baseline. Approximately one third of all patients (29.8%) lost weight (any decrease), one third (36.6%) had relatively stable weight (0% to <3% increase), and one-third (33.6%) gained weight (≥ 3% increase). The corresponding mean weight changes in kilograms were -2.1 kg, 0.9 kg, and 4.6 kg, for lost, stable, and increased weight groups, respectively. Figure 2. Within the olanzapine treatment group, but not the haloperidol treatment group, significantly more men than women experienced a potentially clinically meaningful weight gain = 4.0, p = 0.045 for women and men in the olanzapine treatment group, and 2.3% vs. 3.5%; χ2 = 0.7, p = 0.40, for women and men in the haloperidol treatment group).Compared to women, men experienced greater increases in absolute weight = 17.3, p < 0.001), were more likely to experience greater increases in BMI = 5.8, p = 0.016), and were more likely to have an increase of at least 7% from baseline body weight . The results indicated that gender was not a significant variable . Therefore, gender was dropped from subsequent analyses.B = -0.038, t(1899) = 5.6, p < 0.001), in depressive symptoms = 5.3, p < 0.001), in mental functioning = 2.0, p = 0.047), and in physical functioning = 2.3, p = 0.021). Because level of depressive symptoms was based on two depression measures, the MADRS and the BPRS depression/anxiety subscale, we repeated the analysis using each of these measure separately. Results were unchanged.Since men and women were not found to significantly differ on any of the clinical outcome measures and had a similar pattern of weight gain within each treatment group, we examined the association between weight change and change in treatment outcomes for all patients within each treatment group. Regression analyses demonstrated that for both olanzapine and haloperidol-treated patients, increases in weight were significantly associated with improvements in core schizophrenia symptoms, (B's) indicated that every one- kilogram increase in weight at 6-weeks was associated with approximately 0.03 standard deviations improvement in each clinical outcome parameter, when controlling for the effects of treatment group, gender, treatment group-gender interaction, baseline weight, the corresponding baseline outcome measure, and the number of weeks in the study.The regression coefficients was about a third of 6.26 kg mean weight gain found at 39 weeks, when weight gain tends to plateau on olanzapine . SimilarAlthough weight gain appears to be greatest and most rapid during the first 6 weeks of treatment with clozapine , and durIt is of interest to note, however, that despite rapid weight gain during the 6-week period in our study, when weight gain is more likely to be noticed by the patients and their clinicians and thus may elicit a negative emotional response, the weight gain in this study was not only linked to improved clinical and functional status but also to reduced emotional distress as measured by the depression scales.Another limitation of the study is its lack of assessment of patients' adherence with medication. It is possible that weight gain and improvement occurred together because improvements occur mostly in patients who are medication adherent. Although this was not assessed in the present study, this possibility was previously studied by Meltzer et al. , who fouAnother study limitation is the correlational nature of the analyses, which precludes cause-effect relationship and allows for the possibility that the observed associations might be due to an unobserved variable or set of variables. Further, the relatively low correlations suggest that the association explains only a small proportion of the variance in treatment outcomes. Response to antipsychotic medications is a complex phenomenon that is associated by numerous relatively independent components and weigNext, because the study included patients with a moderately severe level of symptomatology, the current findings may not generalize to patients with milder or residual symptoms of schizophrenia. However, the relationships among the severity of patients' baseline symptomatology, treatment-emergent weight gain and therapeutic response is currently unclear. And lastly, this study used the SF-36, a self-report measure of functional status, which was not designed to assess the potential impact of weight gain on patients' functional status or quality of life. Preliminary information on the first measure designed to specifically capture the impact of antipsychotic-emergent weight gain on patients' psychosocial functioning was only recently published . One wouWomen (and men) with schizophrenia who gained weight during treatment with olanzapine or haloperidol did not experience worsening of clinical or functional status. To the contrary, they had significant improvements in core symptoms of schizophrenia, depressive symptoms, and mental and physical level of functioning. Although excessive weight gain, regardless of origin, is of concern due to its association with physical health problems, the current findings suggest that patients who have greater treatment-emergent weight gain are more likely to benefit from treatment with olanzapine or haloperidol. Findings highlight the complexity inherent in medication management of schizophrenia patients and the need to balance treatment risks and benefits for each patient. In addition, further prospective studies will be required to assess the effects of weight gain, in both psychiatric and medical terms, on individuals treated for schizophrenia with various antipsychotic medications.The authors are employees of Eli Lilly and Company, Indianapolis, Indiana• HAS conceived of the study, participated in its design, the analytical plan, the interpretation of the results, and drafted the manuscript• MS participated in the design of the study, the analytical plan, the interpretation of the results, and performed the statistical analysis• ZZ and BK participated in the design of the study, the interpretation of the results, and the drafting of the manuscript.The pre-publication history for this paper can be accessed here:

Virtual environments (VE) are a powerful tool for various forms of rehabilitation. Coupling VE with high-speed networking [Tele-Immersion] that approaches speeds of 100 Gb/sec can greatly expand its influence in rehabilitation. Accordingly, these new networks will permit various peripherals attached to computers on this network to be connected and to act as fast as if connected to a local PC. This innovation may soon allow the development of previously unheard of networked rehabilitation systems. Rapid advances in this technology need to be coupled with an understanding of how human behavior is affected when immersed in the VE.This paper will discuss various forms of VE that are currently available for rehabilitation. The characteristic of these new networks and examine how such networks might be used for extending the rehabilitation clinic to remote areas will be explained. In addition, we will present data from an immersive dynamic virtual environment united with motion of a posture platform to record biomechanical and physiological responses to combined visual, vestibular, and proprioceptive inputs. A 6 degree-of-freedom force plate provides measurements of moments exerted on the base of support. Kinematic data from the head, trunk, and lower limb was collected using 3-D video motion analysis.Our data suggest that when there is a confluence of meaningful inputs, neither vision, vestibular, or proprioceptive inputs are suppressed in healthy adults; the postural response is modulated by all existing sensory signals in a non-additive fashion. Individual perception of the sensory structure appears to be a significant component of the response to these protocols and underlies much of the observed response variability.The ability to provide new technology for rehabilitation services is emerging as an important option for clinicians and patients. The use of data mining software would help analyze the incoming data to provide both the patient and the therapist with evaluation of the current treatment and modifications needed for future therapies. Quantification of individual perceptual styles in the VE will support development of individualized treatment programs. The virtual environment can be a valuable tool for therapeutic interventions that require adaptation to complex, multimodal environments. Visual imaging is one of the major technological advances of the last decade. Although its impact in medicine and research is most strongly observed in the explosion of PET and fMRI studies in recent years , there hLet us first define what we consider a VE and consider the signals that need to be transmitted for such a system to operate remotely (TeleImmersion). VE is immersion of a person in a computer generated environment such that the person experiences stereovision, correct perspective for all objects regardless of their motion, and objects in the environment move in a natural fashion with subject motion. To achieve theses characteristics, certain technology must be utilized. To provide stereovision, slightly different images must be presented to the right and left eyes with little if any cross talk between the two images. In some systems this is provided by using field sequential stereo in combination with liquid crystal shutter glasses . In this system the right liquid crystal lens is clear while the left is opaque and the perspective scene generated on the screen is that for the right eye. Then the left eye lens is clear and the right is opaque and the left eye's view is displayed. This method of producing stereo has found its way into projection based systems ,5 and deRegardless of the system used, to keep all the stereo objects in the correct perspective and to keep them from being distorted when the person moves in the environment, it is necessary to track the movements of the person so that the computer can calculate a new perspective image given the reported location of the person's head/eyes. The tracking systems that are used to do this are varied. The most commonly used of these are the 6-degrees of freedom (DOF) magnetic tracking systems . With these systems a small sensor cube is placed on the subject and the location of the sensor within the magnetic field is detected. When the sensor is place on the head or glasses of the person the orientation of the head and therefore the location of the eyes can be presumed. Other non-magnetically based systems use a combination of acoustic location to delineate position and acceleration detection to obtain body coordinates in space. The combination results in 6 DOF for the location information . Other systems use cameras to track the person and then transform this information to the 6-DOF needed to maintain a proper image in the VE .So far we have confined our discussion to visual objects and have not considered the use of haptic or other forms of information to be integrated into the VE system . To provIn networked VEs several types of data need to be transmitted between collaborating sites: 1. the main data-set itself (this often consists of 3D geometry); 2. the changes to the data-set (these occur when collaborating users modify the geometry in some way – perhaps by moving the object or deforming it); 3. the virtual representation of the remote collaborator (this often is referred to as an avatar); 4. the video and/or audio channel (that facilitates face-to-face conversation.) Video has limited use in stereoscopic projection-based VEs because the large shutter glasses that the viewer uses to resolve the stereo tends to hide the viewers face from the camera. Furthermore most stereoscopic projection systems operate in dimly lit rooms which are usually too dark for effective use of video.The common model for data sharing in networked VEs is to have most of the main data-set replicated across all the sites and transmit only incremental changes. Furthermore the main data-set is often cached locally at each of the collaborating sites to reduce the need for having to retransmit the entire data-set each time the application is started. Classically TCP (Transmission Control Protocol – the protocol that is widely used on the Internet for reliable data delivery) has been the default protocol used to distribute the data-sets. TCP works well in low-bandwidth below 10 Mb/s) or short distance networks. However for high-bandwidth long-distance networks, TCP's conservative transmission policy thwarts an application's attempt to move data expediently, regardless of the amount of bandwidth available on the network. This problem is known as the Long Fat Network (LFN) problem Mb/s or , howeverChanges made to the 3D environment need to be propagated with absolute reliability and with minimal latency and jitter. Latency is the time it takes for a transmitted message to reach its destination. Jitter is the variation in the latency. Fully reliable protocols like TCP have too much latency and jitter because the protocol requires an acknowledgment to verify delivery. Park and Kenyon have shoth of a second) by a more recent update.The virtual representation of a remote collaborator (avatar) is often captured as the position and orientation of the 3D tracking devices that are attached to the stereoscopic glasses and/or 3D input device (e.g. a wand). With simple inverse kinematics one is able to map this position and orientation information onto a 3D geometric puppet, creating lifelike movements . The 3D Audio and video data are similar in property to the avatar data in that they usually comprise an unending stream that is best transmitted via UDP to minimize latency and jitter. Often video and audio packets are time stamped so that they can be synchronized on the receiving end. When more than two sites are involved in collaboration it is more economical to send audio/video via multicast. In multicast the sender sends the data to a specific device or machine that then copies the data to the various people that are subscribers to the data. For example, a user send their data to a multicast address and the routers that receive the data send copies of the data to remote sites that are subscribed to the multicast address. One drawback of multicast is that it is often disabled on routers on the Internet as one can potentially inundate the entire Internet. An alternative approach is to use dedicated computers as "repeaters" that intercept packets and transmit copies only to receivers that are specifically registered with the repeater. This broadcast method tends to increase the latency and jitter of packets, especially as the number of collaborators increases.QoS refers to a network's ability to provide bandwidth and/or latency guarantees. QoS is crucial for applications such as networked VE, especially those involving haptics or tele-surgery, which are highly intolerant of latency and jitter. Early attempts to provide QoS (such as Integrated Services and Differentiated Services) have been good research prototypes but have completely failed to deploy across the wider Internet because telecommunications companies are not motivated to abide by each others QoS policies. It has been argued that QoS is unnecessary because in the future all the networks will be over-provisioned so that congestion or data loss that result in latency and jitter, will never occur. This has been found to be untrue in practice. Even with the enormous increase in bandwidth accrued during the dot-com explosion, the networks are still as unpredictable as they were a decade ago. Ample evidence is available from the online gaming community which often remarks about problems with bandwidth, latency and jitter during game sessions . These gFrustrated by the lack of QoS on the Internet, there is growing interest in bypassing the traditional routed Internet by using the available dark fiber in the ground. Dark fiber is optical fiber that has not yet been lit. Currently it is estimated that only about 5–10% of the available fiber has been lit, and each fiber has several terabits/s of capacity. The dot-com implosion has made this dark fiber and wavelengths of light in the fiber, very affordable. The newly emerging model is to construct a separate customer-owned network by purchasing or leasing the fiber from a telecommunications company, and installing one's own networking equipment at the endpoints. A number of federally supported national and international initiatives have been underway for the last few years to create customer-controlled networks explicitly for the scientific community. These include the National Lambda rail , StarLigThe ability to use virtual technology for rehabilitation is a function of cost, availability, and the kind of applications that can best utilize the network and provide rehabilitation services. Thus far, tele-rehabilitation research has focused on the use of low speed and inexpensive communication networks. While this work is important, the potential of new high-speed networks has not gathered as much attention. Consequently, we have little but imagined scenarios of how such networks might be utilized. Let us consider the case where a high-speed network connects a rehabilitation center and a remote clinic. The question is what kind of services can be provided remotely.The scenario that we envision is one where patients are required to appear at a rehabilitation center to receive therapy. Our scenario could work in several conditions. For example, a therapist at one location may want an opinion about the patient from a colleague at another location or, perhaps, the therapist can only visit the remote location once per week and with virtual technology the daily therapy could still be monitored by the therapist remotely. In our imagined condition we have a therapist at a rehabilitation center with VE, haptic and video devices and software to help analyze the incoming data feeding to a remote clinic with identical equipment connected together through a dedicated high speed network. As displayed in Fig. A system as described above is possible today although expensive. The network characteristics that would be needed for each information channel would be as follows. A high-bandwidth connection would be needed for video and audio streamed to the plasma displays at each location, in addition to the high bandwidth a low latency and jitter connection would be needed for the Varrier Display system (VE). For a force feedback haptic device communicating between the patient and the therapist, a low network bandwidth could be used but the latency and jitter need to be low.After all possible consideration of how to best construct the virtual system, the next concern is how to associate the complex stimuli with the behavior of interest. The relative influence of particular scene characteristics, namely field of view (FOV), scene resolution, and scene content, are critical to our understanding of the effects of the VE on our response behaviors and the Expectation of the visual scene characteristics will also influence responses in a VE. When subjects had some knowledge of the characteristics of a forthcoming visual displacement most reduced their postural readjustments, even when they did not exert active control over the visual motion . Thus weThe challenge is to determine whether the subject has become immersed in the environment, i.e., has established a sense of presence in the environment (see paper by Riva in this issue), and then to establish the correlation between the stimulus and response properties. The experience within the VE is multimodal, requiring participation of all sensory pathways as well as anticipatory processing and higher order decision making. Consequently, it is difficult to attribute resultant behaviors to any single event in the environment and responses across participants may be very variable. We have united an immersive dynamic virtual environment with motion of a posture platform to recorIn our laboratory, a linear accelerator (sled) that could be translated in the anterior-posterior direction was controlled by D/A outputs from an on-line PC. The sled was placed 40 cm in front of a screen on which a virtual image was projected via a stereo-capable projector (Electrohome Marquis 8500) mounted behind the back-projection screen. The wall in our system consisted of back projection material measuring 1.2 m × 1.6 m. An Electrohome Marquis 8500 projector throws a full-color stereo workstation field (1024 × 768 stereo) at 200 Hz [maximum] onto the screen. A dual Pentum IV PC with a nVidia 900 graphics card created the imagery projected onto the wall. The field sequential stereo images generated by the PC were separated into right and left eye images using liquid crystal stereo shutter glasses worn by the subject . The shutter glasses limited the subject's horizontal FOV to 100° of binocular vision and 55° for the vertical direction. The correct perspective and stereo projections for the scene were computed using values for the current orientation of the head supplied by a position sensor attached to the stereo shutter glasses (head). Consequently, virtual objects retained their true perspective and position in space regardless of the subjects' movement. The total display system latency from the time a subject moved to the time the new stereo image was displayed in the environment was 20–35 ms. The stereo update rate of the scene (how quickly a new image is generated by the graphics computer in the frame buffer) was 60 stereo frames/sec. Flock of birds data was sampled at 120 Hz.The scene consisted of a room containing round columns with patterned rugs and painted ceiling Fig. . The colSubjects gave informed consent according to the guidelines of the Institutional Review Board of Northwestern University Medical School to participate in this study. Subjects had no history of central or peripheral neurological disorders or problems related to movements of the spinal column and a minimum of 20/40 corrected vision. All subjects were naive to the VE.We have tested 7 healthy young adults (aged 25–38 yrs) standing on the force platform (sled) with their hands crossed over their chest and their feet together in front of a screen on which a virtual image was projected. Either the support surface translated ± 15.7 cm/sec (± 10 cm displacement) in the a-p direction at 0.25 Hz, or the scene moved ± 3.8 m/sec (± 6.1 m displacement) fore-aft at 0.1 Hz, or both were translated at the same time for 205 sec. Trials were randomized for order. In all trials, 20 sec of data was collected before scene or sled motion began (pre-perturbation period). When only the sled was translated, the visual scene was visible but stationary, thus providing appropriate visual feedback equivalent to a stationary environment.Three-dimensional kinematic data from the head, trunk, and lower limb were collected at 120 Hz using video motion analysis . Infrared markers placed near the lower border of the left eye socket and the external auditory meatus of the ear were used to define the Frankfort plane and to calculate head position. Other markers were placed on the back of the neck at the level of C7, the left greater trochanter, the left lateral femoral condyle, the left lateral malleolus, and on the translated surface. Markers placed at C7 and the greater trocanter were used to calculate trunk position, and shank position was the calculated from the markers on the lateral femoral condyle and the lateral malleolus.For trials where the sled moved, sled motion was subtracted from the linear motion of each segment prior to calculating segmental motion. Motion of the three segments was presented as relative segmental angles where motion of the trunk was removed from motion of the head to determine the motion of the head with respect to the trunk. Motion of the shank was removed from motion of the trunk to reveal motion of the trunk with respect to the shank. Motion of the shank was calculated with respect to the sled.The response to visual information was strongly potentiated by the presence of physical motion. Either stimulus alone produced marginal responses in most subjects. When combined, the response to visual stimulation was dramatically enhanced Fig. , perhapsUsing Principal Component Analysis we have determined the overall weighting of the input variables. In healthy young adults, some subjects consistently responded more robustly when receiving a single input, suggesting a proprioceptive (see S3 in Fig. Results from experiments in our laboratory using this sophisticated technology revealed a non-additive effect in the energy of the response with combined inputs. With single inputs, some subjects consistently selected a single segmental strategy. With multiple inputs, most produced fluctuating behaviors. Thus, individual perception of the sensory structure was a significant component of the postural response in the VE. By quantifying the relative sensory weighting of each individual's behavior in the VE, we should be better able to design individualized treatment plans to match their particular motor learning style. Developing treatment interventions in the virtual environment should carry over into the physical world so that functional independence will be increased for many individuals with physical limitations. In fact, there is evidence that the knowledge and skills acquired by disabled individuals in simulated environments can transfer to the real world -31.The ability for us to use this technology outside the area of research labs and bring these systems to clinics is just starting. However, the cost is high and the applications that can best be applied to rehabilitation are limited. The cost of such systems might be mitigated if this technology allowed therapists and patients to interact more frequently and/or resulted in better patient outcomes. Such issues are under study now at several institutions. This brings us to the idea of tele-rehabilitation, which would allow therapy to transcend the physical boundaries of the clinic and go wherever the communication system and the technology would allow . For exaThe ability to provide rehabilitation services to locations outside the clinic will be an important option for clinicians and patients in the near future. Effective therapy may best be supplied by the use of high technology systems such as VE and video, coupled to robots, and linked between locations by high-speed, low-latency, high-bandwidth networks. The use of data mining software would help analyze the incoming data to provide both the patient and the therapist with evaluation of the current treatment and modifications needed for future therapies.The ability to provide rehabilitation services to locations outside the clinic is emerging as an important option for clinicians and patients. Effective therapy may best be supplied by the use of high technology systems such as VE and video, coupled to robots, and linked between locations by high-speed, low-latency, high-bandwidth networks. The use of data mining software would help analyze the incoming data to provide both the patient and the therapist with evaluation of the current treatment and modifications needed for future therapies. Although responses in the VE can vary significantly between individuals, these results can actually be used to benefit patients through the development of individualized treatments programs that will raise the level of successful rehabilitative outcomes. Further funding for research in this area will be needed to answer the questions that arise from the use of these technologies.

The Proteome section contains extensive data on each of 19 HIV-1 proteins, including their functional properties, a sample analysis of HIV-1HXB2, structural models and links to other online resources. The HIV-1 Protease Cleavage Sites section provides information on the position, subtype variation and genetic evolution of Gag, Gag-Pol and Nef cleavage sites. The HIV-1 Protein Data-mining Tool includes a set of 27 group M (subtypes A through K) reference sequences that can be used to assess the influence of genetic variation on immunological and functional domains of the protein. The BLAST Structure Tool identifies proteins with similar, experimentally determined topologies, and the Tools Directory provides a categorized list of websites and relevant software programs. This combined database and software repository is designed to facilitate the capture, retrieval and analysis of HIV-1 protein data, and to convert it into clinically useful information relating to the pathogenesis, transmission and therapeutic response of different HIV-1 variants. The HIV-1 Proteomics Resource is readily accessible through the BioAfrica website at: Most Internet online resources for investigating HIV biology contain either bioinformatics tools, protein information or sequence data. The objective of this study was to develop a comprehensive online proteomics resource that integrates bioinformatics with the latest information on HIV-1 protein structure, gene expression, post-transcriptional/post-translational modification, functional activity, and protein-macromolecule interactions. The BioAfrica HIV-1 Proteomics Resource Although the HIV-1 genome contains only 9 genes, it is capable of generating more than 19 gene products. These products can be divided into three major categories: structural and enzymatic ; immediate-early regulatory , and late regulatory proteins. Tat, Rev and Nef are synthesized from small multiply-spliced mRNAs; Env, Vif, Vpu and Vpr are generated from singly-spliced mRNAs, the Gag and Gag-Pol precursor polyproteins are synthesized from full-length mRNA. The matrix (p17), capsid (p24) and nucleocapsid (p7) proteins are produced by protease cleavage of Gag and Gag-Pol, a fusion protein derived by ribosomal frame-shifting. Cleavage of Nef generates two different protein isoforms; one myristylated, the other non-myristylated. The viral enzymes are formed by protease cleavage of Gag-Pol. Alternative splicing, together with co-translational and post-translational modification, leads to additional protein variability [Phylogenetic analysis, on its own, provides little information about the conformational, immunological and functional properties of HIV-1 proteins, but instead, focuses on the evolution and historical significance of sequence variants. To understand the clinical significance of genetic variation, sequence analysis needs to be combined with methods that assess change in the structural and biological properties of HIV-1 proteins. At present, information and tools for the systematic analysis of HIV-1 proteins are limited, and are scattered across a wide-range of online resources ,3. To faWe have categorized the Proteomics Resource into the following main subject headings Figure &3:HIV Proteome – Information about structure and sequence, as well as references and tutorials, for each of the HIV-1 proteins proteins using information available in public databases and tools ; the other from singly-spliced mRNA (p16) [MEPVDPRLEPWKHPGSQPKTA-21; the hydrophobic residues are highlighted in bold, and polar residues are italicized) at the N-terminus of the protein; the cysteine-rich disulphide bond region (22-CTNCYCKKCCFHCQVC-37); the core, basic and glutamine-rich region (49-RKKRRQRRRAHQNSQTHQASLSKQ-72) that is important for nuclear localization and TAR-binding activity, and the RGD cell-attachment site that binds to cellular integrins. In addition to being expressed in HIV-1-infected cells, Tat is also released into the extracellular fluid where it acts as a growth factor for the development of Kaposi's Sarcoma. Additional information about Tat and its protein-protein interactions can be found on the proteome page of the BioAfrica website located at .In the HIV-1 Proteome section, each of the 19 HIV-1 proteins has a webpage that is divided into six parts: "general overview", "genomic location", "domains/folds/motifs", "protein-macromolecule interactions", "primary and secondary database entries", and "references and recommended readings" Figure . The oveHXB2 , sequencB2 [HXB2 , and proB2 [HXB2 ,6 . ImportaPost-translational cleavage of the Gag, Gag-Pol and Nef precursor proteins occurs at the cell membrane during virion packaging, and is essential to the production of infectious viral particles. Drugs that inhibit this process, the protease inhibitors (PIs), are the most potent antiretroviral agents currently available. Thus it is important to collect information, not only on the sequence of protease enzymes from different HIV-1 subtypes, but also on the natural polymorphisms and resistance mutations that may effect their catalytic activities, drug responsiveness, substrate specificities, and cleavage site characteristics. Studies have shown that resistance mutations in the protease of subtype B are associated with impaired proteolytic processing and decreased enzymatic activity, and that compensatory mutations at Gag and Gag-Pol cleavage sites can partially overcome these defects . These fThe cleavage site section of the BioAfrica webpage is the direct extension of a recent publication in the Journal of Virology describing the location and variability of protease cleavage sites Figure . TogetheHXB2 isolate, which, in addition to a premature stop codon, contained no cAMP/cGMP, PKC or CKII phosphorylation sites.The HIV-1 Protein Data-Mining Tool contains twelve sequence analysis techniques for assessing protein variability among different strains of HIV-1 Figure . These tThe HIV-1 BLAST Structure Tool facilitates the analysis of HIV-1 protein structure by allowing for rapid retrieval of archived structural data stored in the public databases Figure . Users mThe HIV-1 Proteomics Tools Directory is divided into two web pages. The initial webpage is a concise compilation of some of the most commonly used protein-specific Internet resources Figure . This "bThe impending rollout of antiretroviral therapy to millions of HIV-1-infected people in sub-Saharan Africa provides a unique opportunity to monitor the efficacy of non-B treatment programs from their very inception, and to obtain critical new information for the optimization of treatment strategies that are safe, affordable and appropriate for the developing world. An integral part of this massive humanitarian effort will be the collection of large amounts of clinical and laboratory data, including genetic information on viral subtype and resistance mutations, as well as routine CD4+ T-cell counts and viral load measurements. The mere collection of this data, however, does not ensure that it will be used to its maximum potential. To achieve full benefit from this explosive source of new information, the data will need to be appropriately collated, stored, analyzed and interpreted.[The rapidly emerging field of Bioinformatics has the capacity to greatly enhance treatment (and vaccine) efforts by serving as a bridge between Medical Informatics and Experimental Science. By correlating genetic variation and potential changes in protein structure with clinical risk factors, disease presentation, and differential response to treatment and vaccine candidates, it may be possible to obtain valuable new insights that can be used to support and guide rationale decision-making, both at the clinical and public health levels. The HIV-1 Proteomics Resource, described in this report, is an initial first step in the development of improved methods for extracting and analyzing genomics data, converting it into biologically useful information related to the structure, function and physiology of HIV-1 proteins, and for assessing the role these proteins play in disease progression and response to therapy. The Resource, developed at the Molecular Virology and Bioinformatics Unit of the Africa Centre of Health and Population Studies, is a centralized user-friendly database that is easily accessed through the BioAfrica website at .AA – Amino AcidBLAST – Basic Local Alignment Search ToolCKII – casein kinase IICTLs – cytotoxic T-lymphocytesDIP – Database of Interacting ProteinsDNA – deoxyribonucleic acidEnv – envelope glycoproteinGag – group-specific antigen polyproteinGIF – Graphics Interchange FormatHIV – Human Immunodeficiency VirusHIV-1 – Human Immunodeficiency Virus Type-1HTTP – Hypertext Transfer ProtocolLTR – long-terminal repeatmRNA – messenger RNANCBI – National Center for Biotechnology InformationNef – negative factorPDB – Protein Data BankpI – isoelectric pointPIs – protease inhibitorsPKC – protein kinase CPol – polymerase polyproteinRev – ART/TRS anti-repression transactivator proteinRNA – ribonucleic acidRNase H – ribonuclease HTat – transactivating regulatory proteinVif – virion infectivity factorVpr – viral protein RVpu – viral protein UThe author(s) declare that they have no competing interests.RSD created and maintains BioAfrica's HIV proteomics resource, HIV proteome section, proteomics tools directory, HIV-1 protein data-mining tool and HIV structure BLAST tool; performed protein sequence and structural model analyses; and wrote the manuscript.TDO conceived and maintains the BioAfrica website, and continues to oversee its rapid expansion; created the cleavage sites section; and participated in the design and implementation of the HIV proteomics resource.CS participated in the design of the HIV proteomics resource, with an emphasis on the proteomics tools directory.SD participated in the design and creation of the HIV proteome section, with an emphasis on the HIV-1 Tat protein.MG participated in the design of the HIV proteomics resource, with an emphasis on the HIV proteome section.SC supervised the project, and participated in the design and implementation of the HIV proteomics resource.All authors read and approved the final manuscript.A table containing a comparative summary of potential functional motifs in the HIV-1 Tat proteins of subtypes B and C, as identified using PROSITE.Click here for file

This paper analyzes the effect of the mean-square error principle on the optimization process using a Special Case of Hopfield Neural Network (SCHNN).The segmentation of multidimensional medical and colour images can be formulated as an energy function composed of two terms: the sum of squared errors, and a noise term used to avoid the network to be stacked in early local minimum points of the energy landscape.Here, we show that the sum of weighted error, higher than simple squared error, leads the SCHNN classifier to reach faster a local minimum closer to the global minimum with the assurance of acceptable segmentation results.The proposed segmentation method is used to segment 20 pathological liver colour images, and is shown to be efficient and very effective to be implemented for use in clinics. Segmentation is an important step in most applications that use medical image data. For example, segmentation is a prerequisite for quantification of morphological disease manifestations and for radiation treatment planning ,2, for cA Number of algorithms based on approaches such as histogram analysis, regional growth, edge detection and pixel classification have been proposed in other articles of medical image segmentation. In recent years, Artificial Neural Networks (ANNs) have been proposed as an attractive alternative solution to a number of pattern recognition problems. In our previous works , we haveHopfield network for the optimization applications consists of many interconnected neuron elements. The network minimizes an energy function of the form:Vk is the output of the kth neuron, Ik is the bias term, and Tkl is the interconnection weight between the kth and lth neurons. The energy function used in the segmentation problem is slightly different from the one defined by Hopfield and the arguments are given in [where N is the number of neurons, given in .The results that have been obtained in were preThe segmentation problem of an image of N pixels is formulated in as a parRkl is the Mahalanobis distance measure between the kth pixel and the centroid of class l, Rkl is also equivalent to the error committed when a pixel k is assigned to a class l. The index n in is the power or weight of the considered error in the energy function of the segmentation problem, and Vkl is the output of the klth neuron. Nkl is a N × M vector of independent high frequency white noise source used to avoid the network being trapped in early local minimums. The term c(t) is a parameter controlling the magnitude of noise which is selected in a way to provide zero as the network reaches convergence. The minimization is achieved by using SCHNN and by solving the motion equations satisfying:where Ukl is the input of the kth neuron, and μ(t) is a scalar positive function of time, used as heuristically motivated stopping criterion of SCHNN, and is defined as in [where ed as in by:β(t) = t(Ts - t)     (4)t is the iteration step, and Ts is the pre-specified convergence time of the network which has been found to be 120 iterations [kth pixel and the centroid of class l given by:where erations . The netXk is the P-dimensional feature vector of the kth pixel (here P = 3 with respect to the RGB color space components), is the P-dimensional centroid vector of class l, and Σl is the covariance matrix of class l. The segmentation algorithm is described as follows [where follows .Step 1 Initialize the input of the neurons to random values.Step 2 Apply the following input-output relation, establishing the assignment of each pixel to only and only one class.Step 3 Compute the centroid and the covariance matrix Σl of each class l as follows:nl is the number of pixels in class l, and the covariance matrix is then normalized by dividing each of its elements by .where Step 4 Update the inputs of each neuron by solving the set of differential equations in (2) using Eulers approximation:Step 5 if t <Ts, repeats from Step 2, else terminated.For this study, a total of 20 liver tissue sections were provided by the pathological division of National Cancer Center in Tokyo. These sections were taken using needle biopsy, stained with hematoxylin and then magnified with an optical microscope. Figure Ts values between 30 and 120 iterations. Similar curves were obtained for the rest of the images of the dataset. As it is illustrated in Figure Ts = 120 iterations gives the optimal solution, the same as it is with MRI data [Figure MRI data .Rkl in the cost function (2), we have provided a simple modification to the above algorithm as follows:In order to study the effect of the weight of the Mahalanobis distance Step 1 Use the same random initialization N × M matrix, as input of the neurons, when minimizing the energy function (1) with different error's weight n.This condition is added to the algorithm in order to make sure that the random field does not have any effect on the generated results.Step 2 trough Step 5 remain the same.n in equation (2). As aforementioned, the pre-specified convergence time of SCHNN is fixed to Ts = 120 iterations. However, we can clearly see from Figure n in Equation (2), the same convergence point or a close position is reached in half the time of the one reached with n = 2 and Ts = 120 iterations. So, this raises the following question: what is the type of relation between the variable n in (2) and the pre-specified convergence time Ts?Figure n that corresponds to the optimum solution with Ts = 120 iterations. From Figure n = 6 gives the optimum solution with Ts = 120. Similar figures to Figure Before answering this question, it is essential to know at this level what is the best value of n, in Equation (2). When both (Ts and n) used together they give a local optima in the energy landscape of SCHNN. Figure n in Equation (2) that are obtained with Ts values 120, 60, and 30. We realized that the curves corresponding to Ts = 120 and Ts = 60 intersect in their optimum solutions obtained with n = 6, and the two curves are similar when n is in the range 5–10. However, the curve corresponding to Ts = 30, shows higher error at convergence of all values of n.In order to study the effect of the pre-specified time, we repeated the above experiments with different Ts values. We realized that each value of Ts corresponds to a value of n in (2) when it takes the value of six where SCHNN gives an optimum and acceptable results that agree with the pathological experts point of views. However, with other values of n, the random initialization may affect the solution of the problem, or in other words, may affect the error of the SCHNN at convergence as shown in Figure In order to see the effect of the random initialization on the results of the algorithm described in section 3, we have executed the same algorithm with different initialization matrices and the curves of the convergence values of SCHNN corresponding to these initializations are shown in Figure We analyzed the effect of considering the mean-square error in formulating the segmentation problem of multidimensional medical images. We have shown, empirically, that considering an integer power equal to six, of the error in the energy function of the problem, helped SCHNN to converge twice as fast as the same optimal solution obtained with the mean-square error algorithm. This result is promising to make our segmentation method useful for a Computer Aided Diagnosis (CAD) system for liver cancer and the like. In our future work, we will study deeply the effect of the random initialization and its effect on the segmentation result and on the SCHNN classifier.The author(s) declare that they have no competing interests.Rachid carried out the theoretical study, the sequence alignment and drafted the manuscript. Mohammed participated in the design of the study and performed the analysis and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: