Artesunate plus amodiaquine is a coblistered ACT, given as a single daily intake. It has been suggested that, in view of the number of tablets to be taken (particularly in adults), it may be possible to improve compliance by allowing patients to divide the daily dose. The objectives of this randomized, comparative, open-label, multicentre study, conducted in Senegal and in Cameroon in 2005, was to demonstrate the non-inferiority and to compare the safety of artesunate plus amodiaquine, as a single daily intake versus two daily intakes.A three-day treatment period and 14-day follow-up period was performed in any subject weighting more than 10 kg, presenting with a malaria paroxysm confirmed by parasitaemia ≥ 1,000/μl, after informed consent. Patients were randomly allocated into one of the two regimens, with dosage according to bodyweight range. All products were administered by an authorized person, blinded to both the investigating physician and the biologist. The primary endpoint was an adequate response to treatment on D14 (WHO definition). The two-sided 90% confidence interval of the difference was calculated on intent to treat (ITT) population; the acceptance limit for non-inferiority was 3%. The safety was evaluated by incidence of adverse events.Three-hundred and sixteen patients were included in the study. The two patient groups were strictly comparable on D0. The adequate responses to treatment were similar for the two treatment regimens on D14, PCR-corrected . The statistical analyses demonstrated the non-inferiority of administering artesunate/amodiaquine as two intakes. The drug was well tolerated. The main adverse events were gastrointestinal disorders (2.5%) and pruritus (2.5%); safety profiles were similar in the two groups.This pilot study confirms the efficacy and good tolerability of artesunate plus amodiaquine, administrated either in one or in two daily intakes. Plasmodium falciparum attacks.Nearly all currently available antimalarial monotherapies are facing resistance, the severity and incidence of which may vary according to endemic areas. Only one class of antimalarials, artemisinin derivatives and artesunate in particular, has been able to escape this phenomenon so far, most probably owing to its recent introduction, its mechanism of action and its rapid elimination from the body. National and international experts, in agreement with the WHO , have prThe onset of resistance to this type of treatment combination is unlikely, partly because the combined agents have different mechanisms of action, and also because the artemisinin derivative very rapidly reduces the parasite biomass which enables lasting exposure of the few remaining parasites to a high concentration of the combined drug.A number of controlled phase III studies under the auspices of the TDR , togethe® trademark.Bitherapy with artesunate and amodiaquine has obtained a marketing authorization in several African countries, including Senegal and Cameroon, under the ArsucamArtesunate plus amodiaquine was evaluated as a single daily intake in all of the studies already performed. The number of tablets to be taken for each intake (particularly in adults: eight tablets per intake) suggests that compliance may be improved by allowing patients to divide tablet intake into two daily intakes. The objective of this multicentre, randomized, open-label phase IV study, was to demonstrate the non-inferiority, in terms of clinical and parasitological efficacy on D14, of the administration of artesunate/amodiaquine as a single daily intake versus two daily intakes. The safety and tolerability of treatment were also studied.The study took place in three sites, two in south Senegal, where the climate is soudanian and the rainy season concentrated between June and October and one site in central Cameroon with a sub-equatorial climate, the rainy season is from April to November.Any adults or children weighing ≥ 10 kg, able to receive oral treatment, suffering from uncomplicated malaria attack with parasitaemia ranging from 1,000 to 200,000/μl (moderate and high transmission area) and axillary temperature ≥ 37.5°C or history of fever within the previous 24 hours, was enrolled after signed informed consent. Patients could not be included in the study if at least one sign of severe malaria or clinical danger (WHO definition) was present: serious concomitant disease, allergy to one of the investigational medicinal products, pregnancy, or documented intake of an antimalarial at a suitable dosage within seven days prior to inclusion.Conseil National de Recherche en Santé in Senegal and the Comité National d'Ethique in Cameroon on 17 June 2005 and 23 May 2005, respectively.An ethical approval was given by the 1 = 99% as the envisaged response rate for artesunate plus amodiaquine single dose (reference), a non-inferiority limit of 3%, a type I (α) error probability of 5% and a type II (β) error probability of 20%, the number of patients per group corresponded to 136. In order to anticipate premature withdrawals, a decision was made to include 150 patients per group, i.e. a total of 300 patients. Each site was, therefore, required to recruit 100 patients. In order to avoid any imbalance between the different weight categories, the randomization list was drawn up by centre and by weight range.Based on previous studies, with the hypotheses PDuring the inclusion visit (D0), a physical examination was performed with recording of medical history, demographic data , weight and height, and measurement of vital signs , and axillary's temperature, together with evaluation of symptoms. Parasitological tests  together with PCR filter paper were also performed.Follow-up visits took place on D1, D2, D3, D7 and D14 during which a physical examination was performed, with evaluation of clinical safety and symptoms, measurement of vital signs, and parasitological tests (except on D1 and D2). Filter paper blood spot samples were prepared on D14, if the thick blood film result was positive, with a view to PCR analysis.Since the artesunate plus amodiaquine coblisters contain one tablet of 50 mg artesunate and one tablet of 153 mg amodiaquine, dosages had to be adapted to the patient weight range: patients weighing between 10 and 21 kg received one tablet of each product per day, patients weighing ≥ 21 and ≤ 40 kg received two tablets of each product per day and patients weighing >40 kg received 4 tablets of each product per day. According to randomization, tablets were given either once or twice a day. The duration of treatment was three days in the two groups.Each patient was randomly allocated to one of the study groups, according to a pre-defined randomization list. The tablets were administered with a small amount of water. For the younger children, the tablets were crushed and administered with water. All treatments were administered in the presence of nursing staff. The patients stayed at the centre for 30 minutes after administration, for observation. If the patient rejected the treatment during the observation period, the same dose was re-administered. In the event of repeated vomiting, the patient had to be withdrawn from the study and a replacement treatment had to be introduced. All possible steps were implemented in order to avoid losing patients to follow-up: active follow-up, even going as far as the patient's home, was carried out by a member of the principal investigator's team.P. falciparum form after examination of 300 leucocytes; 10% of the slides were re-read at the centre's reference Parasitology Department at Dakar University.Thick films together with thin blood films were prepared at each study visit with the exception of D1. A molecular biology analysis (PCR) was performed on inclusion and on D14 +/- 1d in the event of a positive thick film on D14 +/- 1d. Slide-reading and parasite counts were performed on site and considered as negative in absence of asexual msp-1, msp-2 and microsatellitesThe molecular biology analyses were performed at the parasitology laboratory at the Faculty of Medicine, Université Cheikh Anta Diop Dakar – Senegal using filter paper blood spot samples prepared in the field. The distinction of reinfections or recrudescence cases was carried out using genetic markers for Independent duplicate data entry was performed using the SAS software program, version 8.2, in compliance with the standards stipulated by GCP guidelines. All of the qualitatives and quantitatives variables recorded were described according to treatment group and globally. The 95% confidence intervals were calculated.The non-inferiority of artesunate plus amodiaquine as two intakes versus artesunate plus amodiaquine as a single intake was studied on the basis of the adequate response to treatment on D14 (WHO definition). The two-sided 90% confidence interval of the difference in the proportions of responders on D14 between the two treatment groups was calculated. If the upper limit of the confidence interval was below the acceptance limit (d = 3%), the non-inferiority of artesunate plus amodiaquine in two intakes versus a single intake would be demonstrated. This analysis was conducted on the ITT and per protocol (PP) populations. The main analysis corresponded to the ITT population.The safety of artesunate plus amodiaquine was evaluated by the presence or absence of adverse events, type of adverse event, intensity and the causal relationship with the investigational product in the safety population.From June to October 2005, 316 patients were included in the study. 161 patients were assigned to the artesunate plus amodiaquine single-intake treatment group (reference) and 155 to the artesunate plus amodiaquine two-intake treatment group. Seven patients were withdrawn from the study: five patients in the single-intake group and two in the two-intake groups. Only one patient (in the single-intake group) was withdrawn due to an adverse event, five patients were withdrawn because of loss to follow up and one because of consent withdrawn . Irrespective of treatment regimen, more than 99% of patients showed an adequate clinical and parasitological response.The primary efficacy endpoint was the adequate response to treatment after PCR analysis (ACPRc) between the two treatment groups was:  was below the acceptance limit, d = 3%., assessing that the two intakes treatment regimen is non-inferior to the single intake treatment regimen on the ITT population. These results were confirmed on the PP population.The results of the evaluation of efficacy on D14 before PCR and after PCR on the ITT population are presented on Table The changes in mean temperature during the study were similar in the two treatment groups, with values for mean temperature below 37.5°C as from D1. The percentage of apyretic patients between D1 and D14 is shown in Figure Gametocyte-carrier patients were 12 on inclusion, D7 and D14, and 14 on D3. The mean values for gametocytaemia were halved between D1 and D7 in both groups. With the exception of anorexia wich mainly affected patients under seven years of age in the two treatment groups, and was on D1 statistically higher in the single-intake group . A similar and favourable outcome, with regard to the symptoms of malaria, was observed for the two patient groups during the study. The changes in the percentage of patients in each group presenting these symptoms are shown in the Table Certain symptoms attributable to malaria, but not present on inclusion, appeared during treatment: asthenia (5.7%), vomiting (4.7%), anorexia (4.7%), diarrhoea (2.8%), abdominal pain (1.9%) and dizziness (2.2%), without any differences between the treatment groups. The influence of the number of tablets per intake, or the total quantity of drug substance to be ingested, on the onset of these symptoms was the subject of a descriptive analysis only, owing to the limited sample size.With the exception of two patients prematurely withdrawn from the study in the single-intake group , who only received treatment for one day, all of the other patients received treatment for three days, as provided for in the protocol. The adverse events (AE) recorded during the study were coded in compliance with the MedDRA dictionary, and described for the safety population.Among the population included, the AEs reported during the study mainly corresponded to gastrointestinal disorders (2.5%) or pruritus (2.5%), irrespective of the treatment regimen. Three patients in the two-intake group reported AEs related to nervous system disorders (two cases of drowsiness and one case of dysgeusia – bitter taste) versus one patient in the single-intake group (one case of seizure). All of the gastrointestinal or nervous system disorders and cases of pruritus reported during the study were perceived as related to the investigational product, irrespective of treatment regimen. Intensity of adverse events reported as being related to the investigational product is shown in Table Two serious adverse events occurred during the study, one in each treatment regimen; they were both perceived as unrelated to the investigational product. No death occurred during this study.Rejection of treatment during the first half-hour after administration was observed in 10 patients, six in the artesunate plus amodiaquine single-intake group and four in the two-intake group, mainly occurring on D0 Table . Out of Fifteen patients presented episodes of vomiting during the treatment period although this symptom was not observed on inclusion: seven patients (4.3%) in the single-intake group and eight patients (5.2%) in the two-intake group. It should be noted that this description does not take early treatment rejection into account. All these patients presented at least one of vomiting in the previous 24 hours. The number of tablets taken by the 15 patients concerned is shown in the Table The episodes of vomiting during treatment appear to have been influenced by the number of tablets taken each day. Dose fractionation does not appear to have any impact on the number of patients having presented this symptom during treatment. However, no conclusions may be drawn owning to the limited sample size in these subgroups.The correct prescription of ACT by healthcare staff and the compliance of patients are essential for an effective management of malaria in Africa. Different previous studies had already shown the efficacy and tolerability of artesunate plus amodiaquine in sub-Saharan Africa -7.The objective of this pilot study was to verify the non-inferiority of two daily intakes versus a single intake (which can permit to decrease the number of tablet to be taken per dose) and to evaluate the impact of dose fractionation on clinical safetyThis study showed that adequate responses to treatment were similar for the two treatment regimens, and approaching 100% before and after PCR analysis on D14. The statistical analyses conducted on the ITT and PP populations demonstrated the non-inferiority of administering Artesunate plus amodiaquine as two intakes versus a single daily intake, in terms of clinical and parasitological efficacy on D14.It was also demonstrated that the two tested treatment regimen were well tolerated; the observed AEs were similar in terms of type and incidence in the two treatment groups. No changes were observed in the safety profile when dividing Artesunate plus amodiaquine treatment into two daily intakes. The events reported during the study were expected and have already been described with this type of antimalarial.Numerous papers described an impact of artemisinin derivatives on gametocyte carriage, by killing immature stages of gametocytes. During this study, the number of gametocyte-carrier patients remained stable during follow-up (from D0 to D14), but the mean values for gametocytaemia were halved between D1 and D7 in both groups. This can possibly be explained by the presence of different stage of gametocytes in the study populations.The duration of follow-up of this study was only 14 days, but the efficacy has been demonstrated in numerous other studies within a D28 follow-up , and theThe combination artesunate plus amodiaquine has already been adopted as first line treatment of uncomplicated malaria attack in 18 countries in sub-Saharan Africa, with a regimen of one daily intake for three days. Even if the tolerability profile remains good in the clinical trials, in real life, some patients (mainly adults) complain about the too important number of tablets to be ingested at once. The results of this study will permit to divide the daily intake into two administrations.A fixed dose combination of these two active components has already been developed and is currently being evaluated in sub-Saharan Africa. This new formulation, with a limited number of tablets per intake may allow a better adherence to an effective treatment for African communities.The author(s) declare that they have no competing interests.JLAN: designed the study, collected data, and prepared the manuscriptBF : collected data and contributed in the preparation of the manuscriptVL: designed the study and prepared the manuscriptPh B and ASE : participated in the design of the study and collected dataAMD, PAS, M C and T K ; collected data on fieldOG : designed the study and prepared the manuscript


In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo.


Recently, a novel function of PGE2 in the brain has shed light on aspects of synaptic plasticity such as long-term potentiation (LTP). More recently, we have proposed a hypothesis for the mechanisms of this PGE2-related form of synaptic plasticity in the visual cortex. This involves the dynamics of two subtypes of PGE2 receptors that have opposing functions in intracellular signal transduction. Consequently, mechanisms that increase the level of cyclic AMP in the cytosol may explain for the mechanisms of LTP in the visual cortex. The current notion of bidirectional trafficking of PGE2 receptors under this hypothesis is reminiscent of the “silent synapse” mechanism of LTP on the trafficking of the AMPA receptors between the membrane and cytosol. Moreover, we propose the hypothesis that PGE2 acts as a “post-to-postsynaptic messenger” for the induction of LTP in the visual cortex. This review describes a complex mode of action of PGE2 receptors in synaptic plasticity in the brain.Prostanoids have a broad spectrum of biological activities in a variety of organs including the brain. However, their effects on synaptic plasticity in the brain, which have been recently revealed, are ambiguous in comparison to those in the other organs. Prostaglandin E All of these products are metabolites of arachidonic acid, which originates from the phospholipid components of the cell membrane. Arachidonic acid is converted to PGG2 by the cyclooxygenases, which comprise cyclooxygenase(COX)-1, -2, and 3 D2, PGH2, PGI2 and TxA2 are degraded into inactive products within several minutes even under physiological conditions, whereas in in vivo the other prostaglandins are immediately inactivated through circulation in the lungs, where oxidative stress is strong. It is believed that because of the chemical and biological instability of prostanoids, the area, where the activity of prostanoids is essential for performing and maintaining the physiological conditions is believed to be restricted around the region where they prostanoids are produced. Prostanoids are hydrophobic, because they are synthesized from fatty acids in the cell membrane. This property allows them to pass through the membrane and reach neighboring cells. Thus, prostanoids are able to exert their functions approximately in spite of their short lifetime. This is consistent with the fact that the prostanoids which are produced in the brain do not circulate in the blood, and yet can act in the neighboring subcellular regions, such as synapses.In the prostanoids, PGG2 have been identified: EP1, EP2, EP3, and EP4 (EP1–4)  and gonadotropin  . Howeverdotropin . A studyhe brain . EP3 mRNhe brain .2+ , in a G protein-coupled manner. The activation of EP1 elevates the levels of intracellular inositol phosphate and Ca2+ , whereas2+ . Convers2+ . Recentl2 is a mediator of fever induction (in situ hybridization study showed that EP3 mRNA is expressed at a particularly high level in the regions surrounding the OVLT (2 have an easy access to the OVLT because it lacks blood-brain barrier. There have been increasing numbers of knockout (KO) studies on the four receptor subtypes, EP1–4, and in one of these studies EP3-deficient mice showed no febrile responses to any of the pyrogens tested , bone resorption and formation  the unpredictable effects of the knocked-out protein on other proteins that might in turn influence some other proteins especially during development. This makes it difficult to know whether the results obtained are caused because of the absence of the target protein or other proteins; (2) the high frequency of death before birth or during early postnatal development, resulting in reduced opportunity to conduct experiments; (3) the limitation of the knocked-out region . The effect of ordinary KO manipulation is systemic and begins at fertilization in almost all cases. It is difficult to predict the location and timing of the effect of the KO desired; (4) the fact that in most cases KO experiments are limited to mice, resulting in the restriction on the use of antibodies, DNA probes and so on; and (5) the large expenditure, labor, and time required to establish the KO model. With respect to reason (1), it is difficult to know whether the results obtained are caused by the absence of the target protein or by the absence or lack of other proteins. With respect to the reason (3), the effect of ordinary KO manipulation is systemic and begins at fertilization in almost all cases. It is difficult to predict exactly the location and timing for the effects of the KO desired. Nonetheless, KO manipulations have been used to analyze the function of the target proteins by many researchers. This is mainly because of the specificity of targeting, which presumably avoids none-specific actions, such as pharmacological actions.de novo RNA synthesis by an RNA-dependent RNA polymerase, resulting in the amplification of RNA. With respect to target specificity and the efficacy of maintenance of RNAi, the method using siRNA has great advantages over the use of the conventional antisense oligonucleotides to knock down the target.An alternative way to inhibit the target expression is by RNA interference (RNAi), in which stretches of 21–23 nucleotides of RNA, termed short  interfering RNA (siRNA), are used to inhibit the expression of the target mRNA, resulting in the inhibition of the target protein . The mecin vivo electroporation are whole organs, such as the liver and brain in the embryonic stages of the chick and mouse. Electroporation at the embryonic stage offers the possibility of influencing the destiny of the proteins other than the target as mentioned above.For introducing exogenous molecules such as proteins, RNA, and DNA into the cells, electroporation is a convenient and efficient method  in hippocampal slices and frequency, and that this action is via the EP2 which is located at the presynaptic sites  induced LTP in the slices of rat visual cortex. Glutamate released from presynaptic sites by TBS activates N-methyl-D-aspartate receptor (NMDAR) at postsynaptic sites, followed by a Ca2+ influx. This activates Ca2+-dependent cPLA2, which then produces arachidonic acid from the membrane lipid substrate, in addition to the activation of Ca2+/calmudulin-dependent protein kinase II (CaMKII). Arachidonic acid is metabolized to PGH2 by COX-2 that has been activated by TBS concomitantly  in hippocampal neurons in culture , but less colocalized with synaptophysin   . The dif2 increases the magnitude of both E- and L-LTP in the visual cortex , which may in turn activate cAMP response element binding protein (CREB), a transcription factor, in the nucleus. The maintenance of L-LTP depends on protein synthesis  . E-LTP ll cortex . This inectively . The TBS cyclase . This shynthesis . This neA pharmacological analysis revealed that CaMKII may be involved in the trafficking of EP2 from the cytosol to the membrane, but the mechanism of EP3-trafficking is unknown . The dyn2 has a direct action on E-LTP and an indirect action on L-LTP via the production of BDNF.A candidate for the product formed as a consequence of the activation of CREB is brain-derived neurotrophic factor (BDNF). Our previous work has shown that BDNF increases the efficacy of synaptic transmission and TBS-induced LTP in the visual cortex . Moreove2 works as a retrograde messenger, because PGE2 is produced at the postsynaptic site and then diffuses across the postsynaptic membrane to the presynaptic site where EP2 is distributed (2 works as a “post-to-postsynaptic messenger,” because PGE2 is produced at the postsynaptic site and then passes across the membrane into the synaptic cleft where PGE2 activates EP2 at the postsynaptic membrane (The induction of LTP requires the activation of NMDA receptor at the postsynaptic cells. The maintenance of LTP requires, at least in part, a change in presynaptic function. In other words, some known or unknown factors, the so-called retrograde messengers, must be transmitted from the postsynaptic to the presynaptic cells, where they must affect presynaptic functions. Many candidates for these retrograde messengers have been proposed (for review, see tributed . In the membrane .2 is generated from membrane phospholipids, which is ubiquitous in the central nervous systems. The prostanoids that is produced from this source is not only PGE2. Therefore, it is important to regulate the production of PGE2 for the maintenance and successful accomplishment of function. Here I want to propose “an intricate machinery” of neurons to yield the energy that is necessary for ordinary synaptic functions. Bidirectional trafficking of the two subtypes, EP2 and EP3, which have reciprocal actions is one of the ways by which synaptic functions may be regulated. In future, further elucidation of the mechanisms of action of PGE2 in the synaptic plasticity, in combination with new methods for introducing agents into the restricted brain regions, such as RISLE, may come to be useful for developing new therapies for psychoneurological diseases such as dementia and cerebrovascular diseases.PGE


In addition to these, there were also changes in the common wood minerals. Statistical evaluation of 12 minerals by discriminant analysis revealed: a. a difference among all four Cremona instruments, b. the difference of the Cremonese instruments from the French and English antiques, and c. only the Cremonese instruments differed from all commercial woods. These findings may provide the answer why all attempts to recreate the Stradivarius from natural wood have failed. There are many obvious implications with regard to how the green tone wood should be treated, which chould lead to changes in the practice of violin-making. This research should inspire others to analyze more antique violins for their chemical contents.Following the futile efforts of generations to reach the high standard of excellence achieved by the luthiers in Cremona, Italy, by variations of design and plate tuning, current interest is being focused on differences in material properties. The long-standing question whether the wood of Stradivari and Guarneri were treated with wood preservative materials could be answered only by the examination of wood specimens from the precious antique instruments. In a recent communication , we reported about the degradation of the wood polymers in instruments of Stradivari and Guarneri, which could be explained only by chemical manipulations, possibly by preservatives. The aim of the current work was to identify the minerals from the small samples of the maple wood which were available to us from the antique instruments. The ashes of wood from one violin and one cello by Stradivari, two violins by Guarneri, one viola by H. Jay, one violin by Gand-Bernardel were analyzed and compared with a variety of commercial tone woods. The methods of analysis were the following: back-scattered electron imaging, X-ray fluorescence maps for individual elements, wave-length dispersive spectroscopy, energy dispersive X-ray spectroscopy and quantitative microprobe analysis. All four Cremonese instruments showed the unmistakable signs of chemical treatments in the form of chemicals which are not present in natural woods, such as BaSO For centuries, violin-makers have tried in vain to match the high standards of excellence set in the first half of the 18th century by the two legendary masters of the craft, Antonio Stradivari and Joseph Guarneri del Gesù in Cremona, Italy. Since craftsmen of our age have employed all the traditional know-how of the art and have also been aided increasingly by volumes of acoustical research, their failure is hard to explain. The mechanical properties of naturally seasoned spruce and maple were thoroughly studied by several investigators The proposition that the answer may lie in the material differences caused by an ancient and forgotten practice of wood preservation has surfaced many times but it received less than due attention. The 1987 exhibit of documents from the State Archives of the Serenissima Republic of Venice The wood of the great masters received only little attention by scientists in the past, and the focus was on the area penetrated by the ground layer of the finishing materials et al.More recently, the powerful methods of solid-state NMR and FTIR spectroscopy were employed by Nagyvary In the present study we report on the mineral analysis of the ashes from the previously used maple samples 2; the outer discolored layers were excluded. The samples were derived from the maple backs of the following provenance: (a) a violin by Stradivari, 1717 CE, (b) a cello of Stradivari, 1731 CE, (c) a violin by Guarneri del Gesù, 1741 CE, (d) a violin by Guarneri del Gesù from the mid-1730s, (e) a violin by Gand-Bernardel of Paris, mid-1840s, (f) a viola by Henry Jay from London, 1769 CE. The last two instruments were salvaged from a house fire and were exposed to a brief period of heat. For comparisons, we used maple tone wood from all important European regions such as Bosnia, Slovenia, Slovakia, Germany and also from southwest China. Such samples were either purchased from commercial companies such as International Violin Co., Baltimore, MD; David Morse Violins, Soquel, CA; Rivolta Co., Desio, Italy; ViolinMaple Co., Bratislava, Slovakia, or they were donated by private individuals who acquired them on locations. Although we asked for naturally seasoned wood, some contamination could not be excluded since the practice in some locations includes spraying of the logs with fungicides. Unusual Na values found in one Bosnian and one Slovenian samples could be due to such contamination.Wood samples from the instruments of Stradivari and Guarneri del Gesù were taken from the interior of the backs during the repair of cracks when the restorer removed thin shavings of old wood several millimeter deep from an area approx. 6 to 8 cmAsh samples were prepared from the wood shavings using the following technique. Between 20 and 80 mg of wood shavings were weighed and transferred to Coors porcelain covered crucibles; only 13 mg of Cremona soot was used because of the smaller expected loss of volatiles. The crucibles were inserted into a cold Lindberg muffle furnace and the furnace was set to 500°C and turned on. After 2 hours, crucibles were removed from the furnace and allowed to cool. Ash was removed from the crucibles and pressed between 2 highly-polished (0.25 µm surface finish) stainless steel cylinders to form a 1–3 mm diameter pellet. Each pellet was transferred onto the surface of conductive carbon tape and coated with 15 nm of spectroscopically pure amorphous carbon in a Ladd carbon evaporator to make the pellet conductive under an electron beam.Analyses were carried out on a Cameca sx50 electron microprobe equipped with four wavelength dispersive X-ray spectrometers (WDS) and an energy dispersive X-ray spectrometer (EDS) using standard microprobe imaging and microanalytical methods EDS qualitative analyses in the form of X-ray spectra were acquired on the pressed powder pellets. The sizes of the areas analyzed varied from about 1 µm up to 1 mm. The smaller sizes were used to examine different BSE heterogeneous areas, while the larger sizes were used to average as large an area as possible on the pellets. EDS quantitative analyses were not attempted because of the larger errors associated with EDS analyses compared to WDS analyses.WDS quantitative analyses were performed on the samples using standard methods after careful calibration using well characterized Smithsonian Boron was detected semi-quantitatively using WDS spectral scans centered on the boron K alpha X-ray emission line and carried out under identical conditions on the pressed powder pellets.2O, K2O, CaO, MgO, SiO2, Al2O3, P2O5, SO3, FeO, MnO, TiO2. The full set of data, compiled in Supplemental Information  imaging targeting an area of 1 mm2 for the anticipated elements Al, B, Ba, Ca, Cl, Cu, F, Fe, K, Mg, Na, P, S, Si and Zn. These images were useful to show the uneven distribution of elements within the ash pellet, and they also helped in identifying some chemical compounds that were originally applied to the wood, such as CaSO4, BaSO4, Na-borate and CaF2. In general, the distribution of the major elements Ca, K, and Na is more even than those of the minor elements. 4 is also indicated by the coincidence of the Ba and S images. It is not a constituent of wood, and its presence must be due to a deliberate chemical manipulation.The use of WDS detectors allowed for the imaging of individual elements. We acquired such X-ray maps covering an area of 1 mm2, a compound otherwise not found in wood  seem to have a strong bearing on our conclusions concerning the Cremonese wood treatment. Schwarze The limitations of our study are obvious: our measurements and statistics encompassed only materials from a square inch of surface from a few musical instruments. In an ideal world of scientist-collector cooperation, one would like to have several areas of the same instrument sampled, and one would like to have several samples from all creative periods of Stradivari and Guarneri. It is our hope that others would now be attracted to this promising field of research and would be more successful than we were in acquiring precious samples.sine-qua-non towards the successful reproduction of the old standards. It follows then that natural wood may not be a suitable material if the goal is to make violins with the old Cremona-type tone. This recognition may instigate a major change in how the commercial wood should be processed and also in the state of the art of violin-making.In summary, several lines of evidence suggest that the wood of the great Italian masters of Cremona was chemically and physically different from those of the later makers. These new insights strengthen the case for the postulated chemical/material paradigm of the Cremona violin Table S1Data for multivariate discriminant analysis. The full set of data included 95×12 values for the Stradivarius violin, 75×12 for the early Guarneri and 30×12 for the rest, with the exception of the German maple which had only 15×12 data points. The data set from the pellets from each musical instrument was analyzed in its entirety as one group, while the commercial woods were analyzed as sets of 15. Abbreviations: Guarn: the early Guarneri violin; Strad: Stradivari; StrCello: Stradivari cello; Gand: Gand-Bernardel violin. The following commercial wood ashes were analyzed: Bosn2M1 and Bosn2M2 are 2 groups of 15 sites each from the Bosnian tree no. 2; similarly, Bosn3M1, Bosn3M2 are 2 groups of 15 from the Bosnian tree no. 3 tree and so on up to Bosn6M1 and Bosn6M2; ChinaM1 and ChinaM2 are 2 groups of 15 sites from the ash pellet of one Chinese maple; GerMaple has only15 sites from the pellet of the German maple; slven1-ht and slven1-sp are groups of 15 sites from the heartwood and sapwood of a Slovenian maple, and slven2-ht and slven2-sp are 2 groups of 15 from the same board but a deeper layer of wood; slvk1-ht and slvk1-sp are 15 sites each from the heartwood and sapwood of a Slovakian maple, and slvk2-ht and slvk2-sp represent sites from the same board but a deeper layer; slvk1995-1 and slvk1995-2 designate 2 groups of 15 sites from a different Slovakian maple board obtained in 1995.(1.90 MB DOC)Click here for additional data file.


Myotonic dystrophy type 1 (DM1) is a multisystem disorder that demonstrates variable symptoms and rates of progression. Muscle weakness is considered one of the main problems with a clinical picture that is characterized by distal weakness of the limbs progressing to proximal weakness. The main objective of this study was to characterize the maximal strength of ankle eversion and dorsiflexion in DM1 patients. Manual and handheld dynamometer (HHD) muscle testing were also compared.The maximal strength of 22 patients from Quebec  and 24 from Lyon  were compared to 16 matched controls.2 = 0,94/Quebec; 0,89/Lyon) and dorsiflexion . The differences between 3 groups of DM1  and between them and controls were all statistically significant . No statistical differences between sites were observed (p > 0.05). The degree of muscle strength decline in dorsiflexion (eversion) were 60% (47%), 77% (71%), and 87% (83%) for DM1 with mild, moderate, and severe impairments, respectively. The smallest mean difference between all DM1 patients taking together was 2.3 Nm, a difference about twice than the standard error of measurement. There was a strong relationship between eversion and dorsiflexion strength profiles . Using a 10-point scale, manual muscle testing could not discriminate between the 3 groups of DM1 patients.With the use of HHD, an excellent reproducibility of the torque measurements was obtained for both centers in eversion (RThe HHD protocol showed discriminative properties suitable for multicentre therapeutic trial. The present results confirmed the capacity of quantitative muscle testing to discriminate between healthy and DM1 patients with different levels of impairments. This study is a preliminary step for the implementation of a valid, reliable and responsive clinical outcome for the measurement of muscle impairments with this population. Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder that is the most common inherited myopathy in adults. DM1 is Previous studies with myotrophic dystrophy have characterized muscle strength deficits, established baseline data and compared the rate of change in strength of proximal and distal muscles. Different tools were used to assess the muscular impairments including manual muscle testing, timed functional tests, quantitative motor evaluation, isokinetic systems, Jamar dynamometer, and hand held dynamometers -7. From Hand held dynamometer (HHD) protocols represent a good option for quantitative muscle testing in a clinical setting. HHDs are portable, economic, and user-friendly. The use of HHD provides a quick, simple, valid, reliable and sensitive outcome measurement of the human muscle strength and a high level of agreement can be obtained with this type of quantified muscle testing -15. MathIn view of future therapeutic trials for DM-1 patients, ankle dorsiflexor and evertor muscles could be used to monitoring the efficacy of treatment. These muscles are affected early in the disease progression and are easy to inject and analyze. Also, as the evertors are the best protection for a near-maximally inverted ankle at footstrike, they act in synergy with the ankle dorsiflexors to stabilise the foot-ankle complex during weight bearing activities and they may compensate for a lack of dorsiflexion . In viewThe objectives of this study were to characterize the maximal isometric strength of ankle evertors and dorsiflexors with HHD quantitative muscle testing in DM1 patients to establish the intra- and inter reliability, which is of major importance for future multicentric therapeutic trials. Manual and quantitative muscle testing were also compared to provide data to validate the most appropriate method for monitoring muscular changes in patients with DM1 presenting with a large spectrum of impairments.Inclusion criteria were limited to DM1 patients older than 18 years old, weakness of the tibialis anterior (TA) as assessed by manual muscle testing, DM1 diagnosis confirmed by molecular testing, and to the patient' ability to take part in manual and quantified muscle strength assessment. Patients with congenital form of the disease, cognitive impairments, Body Mass Index (BMI) > 30, pregnant women, and severe pulmonary deficiencies were excluded from the study. Patients with congenital or juvenile DM, severe pulmonary deficiencies (FVC < 50%) and severe sleepiness were excluded: the latter two groups were excluded because they would not have been able to undergo the full research protocol. Patients were recruited from the medical chart based on the manual testing scores of the TA to ensure a wide distribution of muscle impairment from mild (≥ 4 et < 5), to moderate (≥ 3 et < 4), and severe (< 3). At the inclusion in the study, all DM1 patients and controls recruited were tested using the conventional 0-5 manual muscle testing scale. Thereafter, using the Modified Research Council Scale (MRCS) with a 0 to 10 point scoring system, all patients were assessed by the physiotherapist and classified according to the score obtained in: Group 1  for a score from 7 to10, Group 2  for a score from 4 to 6, and Group 3  for a score from 0 to 3 [A sample of convenience of 22 patients (M/F 11/11) in Quebec and 24 patients in Lyon (France) (M/F 11/13) with different levels of muscle impairments participated in the study. A random probabilistic sample of 16 control subjects (G0) was established using the members of the patient family as paired subjects for age and gender. For all controls, the molecular testing was negative, they were not affected by any muscular pathology.The project was approved by the ethic committees of the IRDPQ for Quebec and of the Cardio-neurology Hospital for Lyon. Written informed consent was obtained from all patients and controls prior to the examination.A physiotherapist using manual muscle testing assessed the maximum isometric muscle strength of the ankle dorsiflexors and evertors. Both muscle groups were assessed with the subject sitting on the edge of a table, both legs hanging, the ankle at 90° of flexion. The subject was first asked to do a full dorsiflexion or an eversion of the ankle as far as possible. At the end of the movement, the examiner was doing a break test trying to break the resistance toward the plantar flexion (to assess the dorsiflexors) or inversion (to assess the evertors). For both muscle groups, a modified version of the modified MRCS (MM-MRSC) with a 0 to 10 point scoring system was used to quote the muscle group . The lowThe new generation of push-pull HHDs are more accurate and sensitive and they offer a better ergonomic design for the examiner to resist different muscle groups in several alternative positions. The maximum isometric muscle strength of the ankle dorsiflexors and evertors was assessed with a Chatillon push-pull hand-held dynamometer . All physiotherapists received an extensive training on the use of the standardized HHD protocol. The Figure Before each trial, the subject was asked to perform two sub-maximal contractions of about 50% to warm-up, ensure the task was well understood and verify that the stabilisations were adequate. Subjects were instructed to avoid explosive contraction and to increase their effort gradually to their maximum immediately after hearing the signal «3,2,1, GO». The tone and words of encouragement used by the tester were also matching a crescendo pattern to make sure the maximum contraction was produced at every trial. Each contraction was held 10 seconds (the dynamometer was recording the peak force) maximal force was followed by a 60 second-rest period. Due to the lower level of endurance and fatigue of some patients, additional and sufficient resting time was given before proceeding to a second trial. Strength measurements were tested bilaterally and values were averaged for the two sides. At baseline, strength was measured at day one (D1) and 21 days later (D21) by one experienced physiotherapist (one in Quebec and one in Lyon). When testing at D21, the tester was blind about the subject's strength values obtained at D1.Even though the HHD protocol used has shown excellent psychometric properties, the specific inter-rater reliability of the evaluators of the current study was verified. Three examiners, the two experienced physiotherapists appointed to the study at each centre and one backup for the Quebec site, assessed 9 healthy subjects with the above HHD protocol. The inter-rater reliability was then calculated using Pearson correlation coefficients. The above protocol was used as described except that 5 trials per muscle group were completed.Descriptive statistics (mean and standard deviations) of maximal torques were calculated for ankle evertors and dosrsiflexors of patients of all groups and controls. Paired t-tests were used to verify significant statistical differences between groups and centres. Simple linear regressions were used to determine the relationships between the maximal strength between evertors and ankle dorsiflexors for both centres (Lyon and Quebec). Maximal torques obtained with the HHD quantitative muscle strength assessment were plotted against the scores obtained with the manual muscle strength assessment from the MMRC to verify the discriminative properties of manual muscle testing. The original alpha level for the statistical analyses was set at 0.05.p) ranging from de 0.70 to 0.93 and 0.72 to 0.94 for ankle dorsiflexion and eversion, respectively. Also, the Standard Error of Measurement (SEM) was 1.0 Nm for the dorsiflexion, and 1.3 Nm for eversion.Prior to the study, preliminary testing using the above-described HHD protocol was done. Three physiotherapists assessed a total of nine healthy subjects over two sessions. Five trials per muscle group per side were completed. Results showed a good to excellent inter-rater reliability between all testers with Pearson correlation coefficients  from Quebec and the 24 from Lyon  who participated to the study had a mean age of 41,1 ± 13,8 (patients from Quebec) and 41,6 ± 10,2 (patients from Lyon). Most of patients had myotonia but did not receive any drugs. The matched control group was composed of 16 healthy subjects including 7 persons  from Quebec with a mean age of 43,1 ± 9.0, and 9 persons  from Lyon with a mean age of 45,7 ± 9,0. In the present study, the mean number of years of disease duration were 19.2 (G1), 30.2 (G2) and 26.6 (G3).2 = 0,94 for Quebec and 0,89 for Lyon) as well as in ankle dorsiflexion .As observed on the Figure The torques values of ankle evertors and dorsiflexors of all groups for both centers are reported in detail in the Table 2) of 0,87 and 0,80 for Quebec and Lyon, respectively.As shown on Figure The Figure The use of a valid and reliable protocol to detect low sub-maximal muscle impairments in DM1 patients is essential to characterize the natural history of muscular involvement and measure the efficacy of short-term therapeutic trials in multicenter studies. The method used to assess muscle strength must be simple, easy to perform for the clinician and it must be sensitive enough to allow the early detection of distal muscle weaknesses. Moreover, the method used must be able to discriminate between different levels of severity of muscle impairments for choosing the best clinical decisions with regard to treatment plan.This study has allowed us to characterize the maximal isometric strength of ankle evertors and dorsiflexors with quantitative muscle testing between three levels of severity of DM1  and control subjects in a multicenter study setup. When averaging the torque values for genders, sides, centers, and days at baseline, the mean peak torque of the control subjects was 26.5 Nm. This is similar to the values reported by Hogrel et al. 2007) that was 30.5 Nm when combining men and women. The sli07 that wOur study suggests that the initial decline of muscle strength in DM1 patients in the first 19 years of the disease (comparing the change of strength between G0 and G1) is about 2.5% (evertors) to 3.2% (dorsiflexors) per year followed by a rate of progression of about 1.5% (dorsiflexors) to 2.2% (evertors) for the subsequent 11 years. Using a Nicholas push HHD, Mathieu et al. (2003) previously reported a strength decline per year of disease duration of 1.2-1.6% for hip flexors, a proximal group, and 2.0-3.0% for the hand grip flexors, a distal group[Because our study design was not longitudinal, it is not possible to know if the progression of the muscle strength decline is linear or not. Also, as the disease duration is assessed essentially through the patient's interview, it remains an estimate which may not be perfectly accurate.In the current study, both the ankle evertors and dorsiflexors were considered. The justification to include the evertors in the current study was based on the premise that, near-maximally inverted ankle at footstrike, the evertors would act in synergy with the ankle dorsiflexors to stabilize the foot-ankle complex during weight bearing activities. Because of the consistent muscle weakness of ankle dorsiflexors reported in the literature in DM1 patients, we hypothesised that the evertors could compensate for a lack of ankle dorsiflexion strength. However, the current findings suggest that there is no added value to measure the strength of both the evertor and dorsiflexor muscles to follow the progression of the disease in DM1 patients.The HHD protocol used showed an excellent reproducibility of the measures at baseline even when the protocol was administered by different centers and testers. This later finding confirms the usefulness of using a HHD protocol for longitudinal follow-up in a future multicentre therapeutic trial aimed at assessing the efficacy of a gene therapy. The present results also confirmed the capacity of QMT to discriminate between healthy and myotonic dystrophy type 1 patients with different levels of impairments. Even when manual muscle testing is used by clinicians who have several years of experience and are utilizing a more sensitive tool such as the MM-MRSC 10 point scale, as shown in the present study, it cannot properly classify DM1 patients. As seen on Figure In addition to verify the feasibility of measuring the isometric muscle strength assessment with DM1 patients using a hand-held dynamometer protocol, this study is a preliminary step for the development of a valid, reliable and responsive outcome for the measurement of muscle impairment with this population. This measure could be used to monitor the major stages of DM1 progression, characterize the natural history of the disease, verify the efficacy of therapies, and document the relationship between impairments and disabilities. However, future research should be done to better characterize the inter-testers reproducibility using our method and to look at systematic differences between sessions for other muscle groups. The present results are very promising and provide us with the basis we need to move forward and proceed with a longitudinal study to determine the rate of muscle strength decline through all stages of the disease and to verify the potential of quantitative measures of muscle impairments as a valid indicator of other biological changes.Manual muscle testing, even when used by experimented clinicians, cannot be used to properly classify DM1 patients. Our results confirm the usefulness of using a standardized HHD protocol to measure muscle strength in DM1 patients. The capacity of an HHD protocol to discriminate between healthy and DM1 patients with different levels of impairments suggests that this method may be used as a biological marker for longitudinal follow-ups.The authors declare that they have no competing interests.LJH participated in the design of the study, carried out the acquisition, the analysis and the interpretation of data, and he drafted the manuscript. JFR participated in the design of the study, in the analysis and interpretation of data, and he has been involved in drafting the manuscript. JS participated in the design of the study, in the analysis and in the interpretation of data, and he has been involved in drafting the manuscript. CV participated in the design of the study, in the analysis of data, and has been involved in drafting the manuscript. JP participated in the design of the study, the acquisition, the analysis, and in the interpretation of the data, and he has also been involved in drafting the manuscript. All authors read and approved the final manuscript.Dr Jack Puymirat (JP) is the Director of the French Canadian program on Myotonic dystrophy supported by AFM.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/11/72/prepubAppendix. Modified-modified medical research council scale (MM-MRCS).Click here for file


The rare displaced fractures of the femoral neck in children need accurate reduction and rigid fixation. The implants commonly used for internal fixation in children are pins or screws. We evaluated the long-term outcome in children who sustained fractures of the proximal femur that were treated by screw fixation.All 22 children (mean age 12 (5–16) years) with fractures of the femoral neck that were treated with screw fixation (mean 2.4 (1–3) screws) at our department between 1990 and 2006 were evaluated. For measurement of outcome, the Harris hip score (HHS) was used and the development of post-traumatic coxa vara was assessed from the difference in the neck-shaft angle postoperatively and at the latest follow-up examination, after mean 4 (2–15) years.A loss of reduction was observed in 12 patients. There was a statistically significant correlation between the HHS and the changes in the neck-shaft angle.Loss of reduction was found in more than half of the children. Screw fixation cannot be recommended for the treatment of femoral neck fractures in children due to a substantial number of post-traumatic coxa vara. In children, fractures of the proximal femur and femoral neck are rare, representing less than 1% of all pediatric fractures . Most ofIn the child, bone remodeling may realign initially malunited fragments, making accurate anatomic reduction less important in other regions of the body. The physis of the proximal femur is, however, responsible for only 13% of the growth of the thigh and therefore it provides little capacity for spontaneous fracture remodeling . Thus, dComplications of this type of fracture are associated with a high long-term morbidity, especially in older children , includiA computerized search of the medical records in our department from the previous 16 years (1990 through 2006) was performed to detect all patients with fractures of the femoral neck. Inclusion criteria for our study were operative fracture treatment with screws, a patient's age of younger than 18 years at the time of injury, and open growth plates of the proximal femur and full radiographic records. Of the 40 children identified, 18 were excluded . Mean age at the time of injury in the 22 children (12 girls) included in the study was 12 (5–16) years. 7 fractures were Delbet type II and there were 15 Delbet type III fractures. According to the classification of All 22 fractures were reduced using an extension table. 12 fractures were stabilized using percutaneous cannulated screws after closed reduction, while in 10 patients open reduction and internal screw fixation was necessary. The diameter of the screws was 4.0 mm in 3 patients and 6.5 mm in the remaining 19 patients. The mean number of implanted screws was 2.4 (1–3). Pauwels type I fractures were stabilized with mean 2.2 screws, type II fractures with mean 2.6 screws, and type III fractures with mean 2.0 screws. No infections occurred. A spica cast was not used in any of our patients. Passive motion exercises started on the second postoperative day, followed by walking with crutches without weight bearing for 6 weeks, after which cycling and swimming was recommended. Contact sports were not allowed for at least 3 months.At our department, the regular follow-up for dislocated fractures of the femoral neck is until the end of growth, including radiographs of the hip in 2 planes. The latest follow-up examination was done on average 4 (2–15) years after surgery.The radiographs were analyzed retrospectively by one of the authors who was not the treating surgeon (RE). For characterization of fracture stability, the fracture angle was classified according to the Pauwels classification (types I to III). Conventional follow-up radiographs of the hip were performed in 2 planes. For measurement of the neck-shaft angle on the antero-posterior radiographs, the patella was positioned properly in neutral position concerning rotation. The postoperative neck-shaft angle was compared to that of the unaffected side and to that in the most recent follow-up examination. The angle was evaluated with a first line drawn through the center of the femoral head and through the middle of the narrowest distance of the two cortices of the femoral neck, and a second line following the femoral shaft. To determine the most accurate neck-shaft angle, a CT scan would be necessary to compare the neck-shaft angles to the results obtained from conventional radiographs. The magnitude of exposure to radiation does not, however, justify this diagnostic strategy.The outcome measurement included the Harris hip score (HHS). In order to reflect the activity of children, the HHS was modified to assess the possibility of participating in sports.For comparing groups, an ANOVA with Scheffe's test was performed and a p-value of < 0.05 was considered statistically significant.Loss of fracture reduction (defined as differences in the neck-shaft angle of more than 4 degrees) was found in 12 patients . Patient2 patients developed osteonecrosis (1 Ratliff type I and 1 type III). At the time of the most recent visit to the clinic, the patient with the Ratliff type III osteonecrosis (4 years after surgery) had normal radiographs. In the other patient, osteonecrosis of the complete femoral head persisted 7 years after surgery.Femoral neck fractures in children are associated with a high incidence of complications , the mosAnother serious problem is post-traumatic coxa vara. Screw fixation is an adequate method for treatment of stable fractures and injuries with a fracture line that is more horizontal, with intact medial cortex . We recoThere are several limitations to our study. Even though we analyzed 22 patients treated over 16 consecutive years, a higher number of patients would be necessary to confirm our results—preferably with a prospective, multicenter study. We measured the outcome with the HHS, which was developed for adult patients with acetabular fractures. To our knowledge, there is no score for evaluation of femoral neck fractures in children and there might be a large ceiling effect in this age group.


Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor.GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found.Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way. Adenine , uracil  and sugar nucleotides  are universal and phylogenetically-ancient signaling molecules involved in a multitude of biological processes, from embryogenesis to adult homeostasis. Actions of extracellular nucleotides on target cells are mediated by specific membrane receptors: the ligand-gated P2X channels, and the G protein-coupled P2Y receptors, which are widely distributed in human tissues .P2Y receptors have recently attracted a lot of interest from the scientific community, since they belong to the 7-transmembrane (TM) rhodopsin family of G-protein-coupled receptors (GPCRs), which are the target of more than 60% of currently marketed drugs GTPgammaS binding, was different from those of already known CysLT and P2Y receptors, with EC50 values in the nMolar and μMolar range, for cysteinyl-LTs and uracil nucleotides, respectively.On this basis, we have recently cloned and deorphanized GPR17; we demonstrated that its heterologous expression in a number of different cell lines results in the appearance of highly specific responses to both uracil nucleotides  and cysteinyl-LTs . Agonist1 selective antagonist 2'-deoxy-N6-methyladenosine 3',5'-biphosphate (MRS2179), the P2Y12/13 antagonist N(6)-(2-methyl-thioethyl)-2--beta, gamma-dichloromethylene-ATP (cangrelor), and the CysLT1 antagonists montelukast and pranlukast were found to be able to counteract GPR17 activation in vitro .As a first step to the rational design of selective GPR17 ligands, a homology model of human GPR17  and at the middle of the EL3 domain (Cys269), respectively . Interestingly, these residues are not present in tivation . Figure eceptors , we inclThe initial structure obtained from homology modeling was topologically close to the template; polar hydrogens were added and optimization of sidechains was run in cycles in which the backbone was kept fixed.The locally minimised structure has been then embedded in a fully hydrated phospholipidic bilayer , as described in Methods.bRh X-ray file derived from the crystal asymmetric unit reports 66 water molecules associated with both chain A and chain B. These molecules are localized in the vicinity of highly conserved residues and in the retinal pocket, and they are probably involved in the regulation of the activity of bRh-like GPCRs GTPgammaS binding studies, we noticed a marked species difference in the potency of some purinergic agonists and antagonists between the rat and human receptor GTPgammaS binding , that is 339 aminoacid-long and lacks the starting Nt 28 residues that are present in the full-length isoform. We chose not to take into account the initial portion of the Nt domain since all the experimental data that are available on GPR17 have been obtained with the pharmacologically active shortest isoform of hGPR17 [hGPR17 and bRh sequences to the NEST [bRh structure at 2.2 Å [bRh, GPR17 has an additional pair of cysteines which is conserved in all P2Y and CysLT receptors, the existence of an additional extracellular disulphide bridge was assumed, and this presumptive bridge was added via computer graphics, by linking the two conserved Cys23 and Cys269 residues in the Nt and EL3, respectively.The aminoacidic sequences of y server . Multiply server ,48. For GPCRDB: Q304-2, ththe NEST  module oat 2.2 Å , depositat 2.2 Å , as a tein vacuo by constraining the backbone of the helices in order to give a first optimization of the rough geometry derived from homology modeling.The 3-D molecule was locally minimized The model of DPPC in the liquid-crystalline phase proposed by Tieleman and Berendsen ,52 was thGPR17 was manually inserted in the centre of the 128 lipid bilayer, in such a way that the principal axes of the helical bundle was parallel to the membrane axis (z) and perpendicular to the membrane plane (xy), and the extracellular and intracellular loops were at the lipid interface. Twenty eight lipid molecules overlapping with the receptor were removed and the system was solvated with the spc216 water model [The structure of er model  provideder model ,56.bRh. Our receptor model was first superimposed to both chain A and chain B, including the already observed 66 water positions; the 36 non-overlapping internal water molecules were kept in their position.Additional solvent molecules were included into the system at the water positions reported in the interior of the 1U19 crystallographic structure of The resulting system consisted of 339 aminoacid residues, 100 DPPC molecules, 6384 water molecules , for a total of 27544 atoms in a rectangular box of 61 × 61 × 67 Å.The final ensemble was submitted to energy minimization cycles, followed by simulated annealing in order to lead the system to a more favourable energetic condition before starting the pure MD simulation.During all these simulation steps, the backbone of the seven TM helices, the helix 8 domain and the structured EL2 motifs were constrained to maintain the overall arrangement of the helical bundle and the structural conserved organization of EL2.-1nm-2). The analysis of the dynamic behaviour of the assembly was performed on the energetically minimized structure obtained above. The system was reheated up to 310 K in 400 ps and kept at this temperature for further 240 ps by using a force constant of 100 (kJmol-1nm-2). Ten ns of dynamics simulation were then performed without any constraints in order to explore conformational changes of the receptor protein under constant standard conditions. The reliability of the MD simulation methodology was then compared with the results obtained from a "trial" run performed applying NOE distance restraints to the interhelical H-bond distances for 3 ns. In particular, the restraints were applied for the distances between the backbone carbonyl oxygen atom of the residue "n" and the backbone NH-group of the residue "n+4" of TM7, with the exception of the prolines. After the first 3 ns, the restraints were removed and the MD simulation was continued for further 3 ns without any restraints.The protocol by which the assembly was prepared for the MD run was composed of separated cycles as described below. For the earlier minimization steps, the steepest descent algorithm was applied. Only one or two components of the system were allowed to move in each stage, while the remaining components were fixed. The minimization sequence of the various component of the system was the following: first the lipids, then the water, then both lipids and water, and finally the whole system. The latter was further minimized using the conjugate gradient algorithm, and then a first run of 200 ps of MD simulation at 5 K was performed. At the end of this first minimization and relaxation protocol, a simulated annealing procedure was performed as follows. The system was heated from 5 to 500 K in 240 ps, then cooled from 500 to 300 K and reheated to 500 K in 200 ps. The heating-cooling process was repeated six times; after that, the system was brought back to 5 K. The global duration of the simulated annealing protocol was 1660 ps, during which the backbone of all α-helices and EL2 were potentially restrained using a force constant of 1000  were investigated by means of MD simulations.The average structure of the system taken during the time frame from 3 to 6 ns of the MD was used for the ligand-receptor simulations. The three ligands were docked into the spatial region corresponding to the putative nucleotide binding pocket for P2Y receptors. To ensure that the starting ligand/receptor complex configurations were energetically favourable, docking experiments were conducted on the average structure of MD described above. Briefly, the average structure was initially submitted to a binding-cavity search using the Sitefinder tool included in the Delos package . A transt of 0.1 ps. The pressure coupling was set as independent in the x and y directions (semi isotropic coupling), with a constant pressure of 1 bar and a coupling constant τp of 1 ps. A 2 fs time step was used for the integration of the equations of motions and all bond distances involving hydrogen atoms were constrained using LINCS [MD simulations were run on a Linux cluster Blade with Xion processors. All minimization steps, MD simulations and analysis were curried out using the Gromacs 3.3 package ,56. All ng LINCS . CoulombCP carried out the computational study, performed the analysis of the data and drafted the manuscript, GR participated in the drafting and in the design of the study, MPA conceived of the study, provided the biological background for data interpretation and helped to draft, PF participated in its design and coordination. All authors read and approved the final manuscript.hGPR17, P2Y receptors, CysLT1, CysLT2 and bRh; evolution of the energy of the system; rmsd of the C-α of the two compared MD simulations, with or without distance restraints as a function of time; general topology of the receptor protein; superimposition of GPR17 before and after the simulation; number of H-bonds and number of residues with α-helix geometry observed during the two compared MD simulations, with or without distance restraints; time evolution of the interactions occurring between the receptor, internal water molecules and ligands during the simulations of the receptor-membrane system.Additional file Figures content: Multiple alignment of the sequences of Click here for file


We examined the association between the tryptophan hydroxylase 1 (TPH1) gene and eating disorders focusing on obsessionality.The sample included 62 women with a lifetime diagnosis of anorexia nervosa (AN) as well as 50 women with a lifetime diagnosis of bulimia nervosa (BN) recruited from specialist clinics for eating disorders and 131 healthy women in Korea. Blood samples were collected from all participants for the TPH1 genotyping. The patients were ad ministered the Korean version of the Eating Disorders Examination and obsessionality was conceptualized using measures of persistence, harm avoidance, and obsessive-compulsive symptoms.In the case-control comparisons, the frequency of the A/A genotype was increased in the patients with BN, but this difference was not significant after correcting for multiple testing. We found no effect of the TPH A218C polymorphism on obsessionality in the patients with AN or BN.Although the present findings should be regarded as preliminary because of the small size of our sample, they suggest that the TPH1 gene may contribute to the genetic susceptibility to BN and be associated with the other unexplored traits of bulimic case status. There is compelling evidence to suggest that eating disorders are marked by a disturbance in the serotonergic system, in which the synthesis of 5-HT is one of the key processes. Furthermore, individuals at risk of developing bulimia nervosa (BN) have trait abnormalities in the regulation of their brain serotonin function that might increase their vulnerability to dieting-induced decreases in plasma tryptophan.In a large multicentre genetic study, obsessionality was selected as an important trait for linkage analysis in eating disorders.15Therefore, we investigated whether a genetic variant of the TPH1 gene is associated with eating disorders with particular emphasis on obsessionality. In the absence of any available functional polymorphism in the coding region of the TPH1 gene, the intron 7 A218C polymorphism has been one of the most widely studied polymorphisms in the TPH1 gene and might affect the expression of TPH1.16In the present study, the first aim was to investigate whether the TPH1 A218C polymorphism was associated with eating disorders using a case-control design. Another related objective was to explore the association between the psychopathology of obsessionality and eating disorders and the TPH1 A 218C polymorphism using psychological measurements.A total of 243 individuals, comprising women with AN (n=62), women with BN (n=50), and healthy women (n=131), were included in this study. All of the patients were native Koreans, recruited between 2006 and 2008 from two specialist treatment centers for eating disorders in Korea. The patients with eating disorders had a diagnosis of AN or BN according to the Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) criteria.The healthy controls were recruited by advertisement from among the residents of Korea after careful screening for a current or past history of eating disorders using the EDE-Q adapted to capture episodes of eating disorders in their lifetime and for a personal history of neuropsychiatric disorders. For all participants, the measured weight and height were obtained at the time of assessment, and the highest and lowest ever BMI were determined. This study was approved by the Institutional Review Board of Seoul Paik Hospital. Written informed consent was obtained from all participants.The EDE was used to capture the eating disorder pathology in patients using the restraint, eating concern, weight concern, and shape concern subscales. Obsessionality was conceptualized using traits of persistence and harm avoidance, and obsessive-compulsive symptoms. Persistence and harm avoidance were assessed with the Korean version of the Temperament and Character Inventory (TCI).26Genomic DNA was extracted from blood leukocytes using a DNA extraction kit . Genotyping of the TPH polymorphism  was carried out using an ABI PRISM SNaP Shot Multiplex kit and an ABI Prism 3730xl DNA analyzer  according to the manufacturer's recommendations. The primers used for PCR were: sense, 5'-CATGTTCCATGCTCTATATGTGT-3' and antisense, 5'-TGTCTGATTTTTTTCAGTGTTACATT-3'. The PCR conditions were as follows: 10 min at 95℃ for 1 cycle, and 30 cycles each at 95℃ for 30s, 55℃ for 1 min, 72℃ for 1 min followed by 1 cycle at 72℃ for 7 mins. After amplification, the PCR products were treated with 1 unit each of shrimp alkaline phosphatase (Roche) and exonuclease I (USB Corporation) at 37℃ for 60 minutes and 72℃ for 15 minutes. One µL of the polymerase chain reaction (PCR) products was added to a SNaPshot Multiplex Ready reaction mixture containing 0.15 pmols of the genotyping primer of 5'-TTATTAATTGACAACCTATTAGGTG-3' for the primer extension reaction. The results were analyzed using Gene-Scan analysis software .First, case-control genetic comparisons were designed to evaluate the differences in the genotype and allele frequencies by the chi-square test for the healthy controls and patients (AN or BN). Next, a multivariate analysis of variance (MANOVA) was computed for the patients with AN or BN by integrating the four subscales of EDE or the composite measurements of obsessionality which comprise the Y-BOCS and the MOCI, as well as the two subscales of the TCI, persistence and harm avoidance, and the genotype as an independent variable. The MANOVA was followed by a univariate analysis. p values <0.05 were considered to be indicative of statistical significance, and two-tailed tests were used. A Bonferroni correction was applied if multiple testing was used. All statistical tests were performed with SPSS version 11.0.2}, differed between the groups as expected /ht (m)expected . In the 2=2.01, df=2, p=0.16), BN  and AN . As shown in The genotype and allele frequencies of the variants in the participants with AN and BN and controls are shown in A MANOVA was computed for the patients with eating disorders by integrating the four EDE subscales or the composite measurements for obsessionality. We could not find any effect of genotype on the psychopathologies for eating disorder or obsessionality in either the patients with lifetime AN  or those with lifetime BN . In the subsequent detailed univariate analysis designed to test for genotype effects , there wIn this study, a modest association between the A218C genotype of the THP1 gene and BN was found, although the significance was abolished after a correction for multiple testing was applied. This finding shows that the possibility of there being an association between TPH1 and bulimic case status cannot be excluded. There was no significant effect of the A218C genotype of the THP1 gene on the psychopathologies for eating disorders or obsessionality in the analysis focusing on this issue.2A receptor by producing changes in TPH activity, based on their finding that the AA genotype of the A218C SNP was associated with a lower 5-HT2A receptor density, which is of interest in eating disorders, because it has been implicated in the modulation of feeding2A receptor binding potential relative to controls.The increased prevalence of the AA genotype in BN could be explained by interactions of the THP1 gene with other genes responsible for the serotonergic function in this population. While TPH2 is considered to control 5-HT synthesis in the brain,From the finding that there was no significant effect of the A218C genotype of the THP1 gene on the psychopathology for the disorder or obsessionality, it is noteworthy that the A218C polymorphism has an association with other unexplored behavioral traits in this study, such as aggression and anger-related traitsWe need to consider that the A218C SNP is not the sole determinant of the functionality of the TPH1 gene, because it is in LD with putative functional SNPs in the promoter region, such as A-1067G and G-347T,Our findings support those of previous studies involving Caucasian samples,In conclusion, we found a modest increase in the frequency of the AA genotype of the TPH1 gene in the BN group, which supports the hypothesis that the TPH1 gene contributes to predispose individuals to BN.


Colchicine injected 5, 9 and 24 h respectively before initiation  led to a significant increase in skin tumour incidence in the --9-h group, and an increase in percentage malignancy in both the --5- and --9-h groups. These times corresponded to the peak of metaphase arrest by the colchicine. The results are discussed in relation in those of Pound and Withers (1963) and others, who found that mitotic stimulation at the time of urethane initiating action raised the ultimate tumour incidence; and the inference is drawn that initiating action in mouse skin may occur during the M phase, rather than during the G1, S, or G2 phases, as suggested by others.


Safety of administration of a vindesine (VDS)-anti-CEA conjugate and its ability to localise after radiolabelling were investigated in patients with advanced metastatic carcinoma . For imaging, patients received between 230 and 520 micrograms of 131I labelled antibody. In 5, localisation of conjugate was demonstrated, in another it was equivocal and in 2 patients, undetectable. For assessment of safety each patient also received a single dose of conjugate increasing from 1.2 to 42 mg antibody linked to 24 to 1800 micrograms VDS. The in vitro activity of the anti-CEA antibody and its ability to localise in vivo were preserved after conjugation. There was no obvious toxicity or hypersensitivity attributable to either the radiolocalisation or escalated doses of conjugate in any of the patients. The feasibility of the preparation and administration to patients of a vindesine-antibody conjugate has been demonstrated.


Thrombosis following stenosis of arteriovenous fistulae resulting in the loss of vascular access for hemodialysis is an important complication in patients with chronic renal failure. Percutaneous transluminal angioplasty is being used more frequently in the treatment of stenosis aiming at increasing the patency of arteriovenous fistulae.To evaluate the primary patency of arteriovenous fistulae following percutaneous transluminal angioplasty.Patients submitted to percutaneous transluminal angioplasty in the Angiology service of Hospital de Base in 2004 were analyzed over an average follow-up of 10 months (2 to 16 months).A total of 22 angioplasties were performed in 20 fistulae of 19 patients. Of the 19 patients, one did not complete follow-up and one presented with a rupture of the fistulae. The following complications occurred in the remaining 18 fistulae, three deaths with two fistulae patent until death; one exeresis of prosthesis due to infection (53 days after the procedure); two thromboses (3 and 49 days after the procedure) and four restenosis . At the end of the follow-up, 11 fistulae (55%) were patent and with a flow rate in hemodialysis > 300 mL/min. Primary patency was 82.4% over three months; 81.2% over six months; 54.5% over 9 months and 50% over 1 year.Percutaneous transluminal angioplasty is an efficacious method for the correction of stenosis of arteriovenous fistulae for hemodialysis, prolonging the patency of the fistula and enabling new interventions. The increase in the prevalence of patients with chronic renal failure on hemodialysis has made several measures to maintain the vascular access patent and efficacious necessary as, the morbimortality of these patients is directly related to the effectiveness of hemodialysis. This requires repeated punctures of large vessels capable of offering a rapid blood flow of adequate volume -3.Thrombosis is the most frequent complication of arteriovenous fistulae that results in the loss of the access for hemodialysis. Most episodes of thrombosis coincide with the development of stenosis (in more than 85% of cases), generally located in the venous segment proximal to the arteriovenous anastomosis -6. StenoClinically, some findings may be suggestive of stenosis of the arteriovenous fistula such as the presence of segmental dilation along the route of the vein or its disappearance, persistent edema of the limb and the presence of a pulse without fremitis. Other indicators are evidenced by alterations during hemodialysis, such as inefficacious dialysis, low blood flow, increase in the venous pressure, difficulty to obtain another venous access, increase in the time necessary to reach hemostasis after hemodialysis, increase in the intra-access pressure and signs of recirculation. Thus, when diagnosed, intervention is justified to maintain the patency of the vascular access and consequently reduce the incidence of hemodialysis failure due to loss of the fistula ,6,7.Percutaneous transluminal angioplasty is a method that has proved to be efficacious in the treatment of stenosis to prolong the endurance of the fistulae ,2.The objective of the current study was to evaluate the primary patency of arteriovenous fistulae after percutaneous transluminal angioplasty.A total of 22 angioplasties of 20 fistulae in 19 patients (12 men and 7 women) with a mean age of 53.7 years (range 35 to 75 years) and on hemodialysis for chronic renal failure were analyzed. The angioplasties were performed in the angiology service of Hospital de Base in São José do Rio Preto between January 1 and December 31 2004.The indications for angiographic studies of the arteriovenous fistula were based on clinical criteria or alterations detected during hemodialysis. The criteria utilized were an existent pulse but without fremitis, persistent edema of the limb with the fistula, the presence of constrictions and/or dilations on the route of the fistula, difficulty to insert the needles or hemostasis after their removal, blood flow at the access of less than 200 mL/min, increase in venous pressure and following thrombectomy surgery.The angiographic technique employed in all cases was by venous puncture or puncturing the prosthesis using a 21 needle, the injection of contrast and obtaining digital angiographic images from the arteriovenous anastomosis up to the central veins. If significant stenosis was present, angioplasty was performed immediately  and dilated using a balloon catheter  with a diameter 1 mm greater than the normal size of the venous segment (varying from 4 to 8 mm). The balloon was maintained insufflated for 60 seconds and repeated when necessary using the pressure recommended by the manufacturer.The angioplasties were performed in all cases by proximal catheterization of the fistulae with the insertion of introductory sheaths (varying from 5 to 8 Fr) and regional anticoagulation using 2000 IU of heparin. The vessel was opened using a 0.35 mm × 260 cm hydrophilic guide wire  prostheses.Of the treated fistulae, one patient did not continue with the follow-up in the service after the angioplasty and one presented a rupture of the arteriovenous fistula during the procedure and was successfully submitted to surgical correction with implantation of a PTFE prosthesis.The other 18 fistulae were evaluated after the procedure with a mean follow-up of 10 months (Range: 2 to 16 months). Of these, 17 fistulae presented a clinical follow-up of more than 3 months, 16 more than 6 months, 11 more than 9 months and 8 more than one year.In this period, there were three deaths not related to the arteriovenous fistulae or the procedure, of which two were patients up to their deaths (120 and 289 days after the angioplasty) and one presented with thrombosis after 40 days and peritoneal dialysis was initiated.One of the patients, 53 days after the angioplasty, presented with thrombosis of the fistula and was submitted to a thrombectomy with angioplasty, however he presented with an infection in the PTFE prosthesis which needed to be removed. In two other patients, thrombosis occurred with loss of the fistulae (with 3 and 49 post-procedure days).During the follow-up, 4 cases of restenosis were diagnosed. Of these, 3 were submitted to a second angioplasty resulting in patent fistulae with flow rates for hemodialysis greater than 350 mL/min. One restenosis was surgically treated with the implantation of a PTFE prosthesis due to severe stenosis at multiple sites along the route of the vein (basilic).At the end of the follow-up period, 11 fistulae were patent , with flow for hemodialysis greater than 300 mL/min.The primary patency of the 18 fistulae evaluated after angioplasty was 82.35% over 3 months (14/17); 81.2% at 6 months (13/16); 54.5% at 9 months (6/11) and 50% over 1 year (4/8).The current study shows that angioplasty is an efficacious method for the correction of stenosis of arteriovenous fistulae for hemodialysis and constitutes another therapeutic option. In the past, surgical treatment performing correction of the stenotic lesion using a bypass or a patch was the only option .In recent years, several studies have demonstrated that angioplasty is efficacious with some advantages compared to the conventional surgical treatment such as a shorter time needed to perform the procedure and shorter hospitalization, less discomfort for the patient, and lower infection rates. Additionally, it enables dialysis immediately after the procedure without the necessity of using a central venous catheter -8.Complications of angioplasty have been reported in about 2 to 16% of cases, with the most common being immediate venous rupture during the procedure, the formation of pseudoaneurysms, acute thrombosis and periprocedural bacteremia. Reaction to the contrast has not implicated a higher mortality rate of these patients ,5. In thThe literature shows that primary patency at 12 months after angioplasty is equivalent of surgical treatment [Angioplasty is efficacious in the correction of stenosis of fistulae for hemodialysis, increasing the survival rate of the fistula and allowing more interventions.The authors confirm the participation in study in all of the phases, that not of Conflicts of Interest and agree with the content of the manuscript. All authors certify that this manuscript was not published or by sending for publication in any form.DGM was responsible for surgical procedures and data collection and critically reviewed the manuscript. LFR was responsible for surgical procedures and data collection and critically reviewed the manuscript. AAMS was responsible for surgical procedures and data collection and critically reviewed the manuscript. JMPG conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Over expression of receptor tyrosine kinases is responsible for the development of a wide variety of malignancies. Termination of growth factor signaling is primarily determined by the down regulation of active growth factor/receptor complexes. In recent years, considerable insight has been gained in the endocytosis and degradation of growth factor receptors. A crucial player in this process is the EGFR Protein tyrosine kinase Substrate #15, or Eps15. This protein functions as a scaffolding adaptor protein and is involved both in secretion and endocytosis. Eps15 has been shown to bind to AP-1 and AP-2 complexes, to bind to inositol lipids and to several other proteins involved in the regulation of intracellular trafficking. In addition, Eps15 has been detected in the nucleus of mammalian cells. Activation of growth factor receptors induces tyrosine phosphorylation and mono-ubiquitination of Eps15. The role of these post translational modifications of Eps15 is still a mystery. It is proposed that Eps15 and its family members Eps15R and Eps15b are involved in the regulation of membrane morphology, which is required for intracellular vesicle formation and trafficking. Receptor tyrosine kinases (RTK) are a large family of signaling proteins involved in a large number of human diseases. They all have a similar composition: an extracellular domain that binds to a growth factor, a trans-membrane domain, an intracellular tyrosine kinase domain and a stretch of tyrosine residues that serves as substrates for the kinase. Binding of the growth factor results in kinase activation and consequently in the trans-phosphorylation of the receptor, as well as of various effector molecules resulting in the stimulation of a large number of signaling cascades. One of the most studied RTKs is the epidermal growth factor (EGF) receptor (EGFR or ErbB1), which belongs to a family of four related receptor tyrosine kinases (ErbB1-4 or Her1-4)). EGFR and its family members are strongly implicated in the development and progression of different human tumors, including breast-, lung-, prostate-, colorectal-, head and neck- and brain tumors .Attenuation of RTK signaling is governed by several mechanisms. At the receptor level, tyrosine phosphatases reduce the number of phosphorylated tyrosine residues. At the cellular level, inhibition of signaling is accomplished by receptor desensitization or down regulation. This process involves the internalization of active ligand/receptor complexes and subsequent trafficking to lysosomes, where receptors are degraded . UbiquitThe EGFR Protein tyrosine kinase Substrate # 15 (Eps15) was originally identified in 1993 in a pool of proteins that became phosphorylated after stimulation of cells with EGF . In the The localization of Eps15 in coated pits strongly suggested the implication of Eps15 in the internalization of the EGFR. Different experimental approaches have now confirmed this role. Microinjection of antibodies against Eps15 were shown to inhibit the internalization of EGFR and of the transferrin receptor . More reIn addition to the presence and role of Eps15 in the endocytic system, Eps15 and especially Eps15R have also been found in the nucleus . Eps15, The Eps15 gene is located at chromosome 1 at chromosomal locus p31-p32, which displays a high rate of random chromosomal abnormalities such as deletions in oligodendroglioma and neuroblastoma, and translocations in myeloid and lymphoblastic leukemia . Fusion In summary, Eps15 is functioning in the cytoplasm, where it is involved in in the regulation of intracellular trafficking. In addition, its presence in the nucleus suggests a role for Eps15 in transcriptional regulation. To understand the working mechanism of Eps15 in its diverse physiological functions, a detailed analysis of its structure and molecular binding partners is required.2+ binding properties, consisting of two α-helices linked by a short β-strand. The EH domains of Eps15 have been found to bind Ca2+ constitutively with high affinity. Phage display analysis has identified the protein motif this domain binds to, being composed of the three amino acid motif asparagine, proline and Phenylalanine (NPF) for the first two domains ..clathrinThe function of Eps15 can best be described using a model of EGFR internalization. Activation of the EGFR results in cross-phosphorylation of tyrosine residues in the intracellular domain, causing the binding of SH2 containing proteins, such as Grb2 and the E3 ligase Cbl. As Grb2 also binds via its SH3 domain to a proline-rich motif in Cbl, a complex of EGFR/Grb2/Cbl is formed resulting in mono- and poly-ubiquitination of the EGFR . This isAnother suggested function for the Eps15 family members lays in the sorting process of EGFR that occurs at the membrane of the early endosome . In thisA very recent finding is that Eps15 also showed localization at the Golgi complex . This isSince its discovery in 1993, a function for Eps15 has been described in different processes, one related to a nuclear function and one related to intracellular trafficking. In trafficking, Eps15 acts as an ubiquitin- or Ubl-binding adaptor protein, a function mediated by the two UIMs in the C-terminal part of the molecule. Other domains in Eps15 are involved in the recruitment of different components that are critical for trafficking, including α- and γ-adaptin, Epsin1, synaptojanin etc. In these processes, Eps15 functions as a scaffolding protein that recruits and concentrates those proteins and lipids that are required for the process. Post-translational modifications as phosphorylation and mono-ubiquitination might regulate Eps15 functioning, but molecular details of these processes are still unknown. Similarly, details about the function of Eps15b in the ESCRT-0 complex is not clear. Imaging of the internalization processes at high resolution may help to understand the molecular details of these processes in the future. The trafficking role of the Eps15 molecules includes clearly two aspects: they are bound to membranes and the morphology of these membranes are manipulated. This has also been described for EHD1, an EH domain containing protein that induces tubulation of the membrane . This suThe author declares that he has no competing interests.