Plaque-type psoriasis affecting the nails, scalp, hands or feet can often be difficult to treat; for example, topical treatments and phototherapy may not penetrate the nail plate or scalp. The objective of this large, international, multicentre study was to investigate the efficacy of efalizumab in a Latin American population of adult patients with moderate-to-severe chronic plaque psoriasis who were candidates for systemic therapy or phototherapy.Eligible patients were enrolled in a 24-week, open-label, single-arm, Phase IIIb/IV study of continuous treatment with subcutaneous efalizumab, 1.0 mg/kg/wk. Involvement of the nails, scalp, or hands or feet was assessed using the Nail Psoriasis Severity Index (NAPSI), the Psoriasis Scalp Severity Index (PSSI), or the Palmoplantar Pustulosis Psoriasis Area and Severity Index (PPPASI), respectively. Missing data were handled using a last observation carried forward or nonresponder imputation approach.Of the 189 patients who received treatment, 112 patients had nail involvement, 172 had scalp involvement, and 19 had palmoplantar disease at baseline. At Week 24, ≥50% improvement on the NAPSI, PSSI and PPPASI was observed in 31%, 71% and 68% of patients, respectively, whereas ≥75% improvement on these scores was observed in 17%, 52% and 63%, respectively. Descriptive statistics showed lower NAPSI-75 and higher PSSI-75 and -50 response rates among patients with higher baseline scores.This open-label, uncontrolled study provides supportive evidence of the potential of efalizumab as a treatment for nail, scalp and palmoplantar psoriasis. Psoriasis is a chronic, autoimmune, T-cell-mediated inflammatory skin disorder that affects various parts of the body . PsoriasEfalizumab is a recombinant, humanized, monoclonal, immunoglobulin G1 antibody that binds specifically to the CD11a subunit of leucocyte function-associated antigen-1, inhibiting major steps in the immunopathogenesis of psoriasis: T-cell activation, migration and reactivation . The effIn previous studies of efalizumab, efficacy was assessed using the Psoriasis Area and Severity Index (PASI); no previous detailed analyses of the efficacy of this agent in the treatment of nail, scalp and palmoplantar psoriasis have been published to date. This multicentre, open-label, Phase IIIb/IV trial examined the efficacy of efalizumab in a population of patients with psoriasis in Latin America; primary efficacy and safety results were presented by Stengel et al. at the 2008 5th Spring Symposium of the European Academy of Dermatology and Venereology and are reported elsewhere . Here, wThis was a 24-week, multicentre, open-label, single-arm, Phase IIIb/IV study  conducted between October 2004 and May 2006 in 23 centres in Latin America . The reader is referred to Stengel et al. (this volume) for a complete briefing of the methodology.Patients were between 18 and 75 years of age and had moderate-to-severe plaque psoriasis (≥10% body surface area involved and candidates for systemic therapy or phototherapy). Discontinuation of any systemic psoriasis treatment was required prior to commencement of the trial; in the case of biologics, a 3-month washout period was required. For women of childbearing potential and for men whose partners could become pregnant, consent to use an acceptable method of contraception and agreement to continue to practise an acceptable method of contraception for the duration of their participation in the trial and up to 3 months after the last dose of efalizumab, were mandatory for study participation. The dosage of any medications required for treatment of comorbidities must have been stable for at least 28 days before the administration of study drug. All patients underwent initial screening ≤14 days prior to the first efalizumab injection. Discontinuation of any systemic psoriasis treatment was mandatory before starting study medication; no washout period was required. Patients included in the subgroup analyses had baseline scores > 0 on the Nail Psoriasis Severity Index (NAPSI) , indicatAll patients received open-label efalizumab administered subcutaneously, starting with an initial conditioning dose of 0.7 mg/kg at baseline (study day 0), followed by 23 weekly doses of efalizumab 1.0 mg/kg. Injection sites were rotated on a weekly basis and efalizumab was self-administered by patients after the first injection.The primary efficacy endpoint in the parent trial was the proportion of patients with a rating of “excellent” or “cleared” at Week 24 using the dynamic Physician Global Assessment (PGA), a tool that measures the response of all psoriatic lesions to therapy by comparing the subject's present condition to baseline photographs or body diagrams. The assessor classified response by considering erythema, scaling, plaque thickness and percentage of body surface area affected. Ratings of cleared and excellent represented a 100% improvement (remission) and 75% to 99% improvement of all clinical signs and symptoms relative to baseline, respectively. The nail, scalp and palmoplantar endpoints, which were tertiary endpoints in the parent trial, are presented in detail here. The median percentage improvements over time in NAPSI, PSSI and PPPASI scores are reported for the subgroups of patients who had baseline scores > 0 for these indices, as well as the percentages of patients showing at least a 50% and 75% improvement in these scores between baseline and Week 24.Assessment of nail psoriasis was made using the NAPSI , at baseline and at Weeks 12 and 24. Assessment of scalp psoriasis was made using the PSSI . Palmoplantar psoriasis was assessed using the PPPASI . Both the PSSI and PPPASI were measured at baseline and at Weeks 4, 8, 12 and 24 (or at the time of last dose).Analyses of efficacy in the intent-to-treat (ITT) population were performed using the last observation carried forward (LOCF) approach. The primary endpoint was also analysed using the nonresponder imputation, in which all patients with missing data at Week 24 were counted as nonresponders. No formal hypothesis testing or adjusted analyses were performed. Analysis was performed using SAS software .post hoc.The efficacy data reported here for nail, scalp and palmoplantar psoriasis are for the subsets of patients who had scores on the NAPSI, PSSI and PPPASI that were >0 at baseline . Demographic and treatment data are for the entire ITT population. Summary statistics were determined for scores; 95% confidence intervals (CIs) for the median were estimated using the exact method based on the binomial distribution. For proportions, exact CIs were used. The analysis of patients showing 50% and 75% improvement in NAPSI, PSSI and PPPASI scores was Overall, 189 patients were enrolled and received treatment (ITT population), of whom 137 (72.5%) completed the 24-week treatment period . A totalThe mean (SD) time on study (including follow-up) for the entire treatment group was 202 (52.9) days and the mean (SD) time on treatment was 139 (45.1) days. Almost two-thirds of patients (122/189 [64.6%]) received all 24 planned weekly injections; the mean (SD) number of injections received was 20 (6.4).In the ITT population, 48.7%  of patients achieved or maintained a PGA score of “excellent” or “cleared” at Week 24. Using the nonresponder imputation, 46.0%  achieved this endpoint.At baseline, 112 patients had nail psoriasis, with a median (range) score on the NAPSI of 14 (1–80) . At WeekAmong patients with nail involvement at baseline, 31% had a NAPSI score of ≤7, 22% had a score of >7 to ≤14, 14% had a score of >14 to ≤21, 11% had a score of >21 to ≤28, and 21% had a score of >28. The proportions of patients in these categories showing at least a 75% improvement in NAPSI score at endpoint were 26%, 24%, 6%, 8% and 8%, respectively, indicating that rates of ≥75% improvement on the NAPSI were lower when the baseline score was higher. Corresponding proportions of patients in the categories defined by baseline score who showed at least a 50% improvement in NAPSI score at endpoint were 37%, 32%, 19%, 17% and 38%, respectively.A total of 172 patients had scalp disease at baseline, with a median (range) PSSI score of 16 (1–54) . At WeekAt baseline, the proportions of patients with PSSI scores of ≤7, >7 to ≤14, >14 to ≤21, >21 to ≤28, and >28 were 30%, 13%, 17%, 12% and 27%, respectively. The proportions of patients in these categories who showed at least a 75% improvement in PSSI score at Week 24 were 29%, 70%, 57%, 75% and 55%, respectively. The proportions of patients in the categories defined by baseline score who showed a 50% improvement in PSSI were 52%, 83%, 83%, 85% and 72%, respectively. These findings indicate that baseline PSSI scores >7 were associated with higher response rates than scores ≤7.In 19 patients who had palmoplantar disease at baseline, the median (range) baseline PPPASI score was 2 (0–20) . One patAmong these 19 patients, the baseline PPPASI score was <2 for 11 patients, and between 2 and 8 for 5 patients; 3 patients had high scores . Of those with low baseline scores (<2), 6 achieved a score of 0 on the PPPASI, 3 experienced worsening, and 2 withdrew from the study without an on-treatment PPPASI evaluation. Of the 5 patients with a baseline PPPASI score in the range 2–8, 4 had no psoriasis involvement after 24 weeks of treatment, whereas the remaining patient experienced worsening. The 3 patients with high baseline scores on the PPPASI experienced improvements of 51%, 98% and 100%.The safety and tolerability of efalizumab are reported elsewhere .No new safety concerns were identified in this population.To our knowledge, this was the first large, prospective, international, multicentre study of any biological therapy in patients with psoriasis in Latin America. It was also the first study to assess the efficacy of efalizumab in the treatment of nail, scalp and palmoplantar psoriasis using the NAPSI, PSSI, and PPPASI, although these measures were not the primary endpoint of the parent trial.Scalp psoriasis, which is currently difficult to treat in clinical practice, showed particularly promising results with efalizumab treatment during this trial, as measured by change from baseline PSSI score. Although the PSSI results reported were less impressive when using a nonresponder imputation than the more optimistic LOCF approach, we believe that the difference between these approaches was due more to the level of control of the patient's overall psoriasis than missing values for PSSI scores, which improved early and continued to improve throughout the study period. In addition, given that this was one of the largest clinical trials to date reporting the effect of any treatment on nail psoriasis, it could be concluded that the improvement achieved in nail psoriasis was clinically meaningful, as approximately one-third of patients achieved at least a 50% improvement from baseline NAPSI score.The results for nail and palmoplantar psoriasis in the present study are in agreement with those reported from a comparison of efalizumab with placebo in patients with hand and foot plaque psoriasis . In thatde novo manifestation of palmoplantar and scalp psoriasis has been reported during treatment with infliximab [As topical therapies are generally the first-line treatment for nail, scalp and palmoplantar psoriasis, and systemic treatments are generally reserved for refractory disease ,8,21,22,fliximab –30. Clinfliximab –33, and fliximab . In a smfliximab  and thisfliximab ,37 and sfliximab ,39. In afliximab  and, in fliximab . Larger The encouraging results from the current study are limited by the fact that this was an open-label trial with no comparator group, and that it involved patients with a wide range of disease severity affecting the nails, scalp and palmoplantar regions. Many of the patients also had relatively low baseline scores on the NAPSI, PSSI and PPPASI. For example, in other studies a NAPSI score of 15 has been considered to represent moderate-to-severe disease ,42, wher


Retinal ischemia appears to lead to alterations in retinal transcript levels of a group of genes known to be abundantly expressed in the lens. Our purpose is to study whether these alterations are truly the result of retinal ischemia or whether they could be caused by contamination of the retinal tissue with trace amounts of lens tissue.Changes occurring in the retinal gene expression profile after induction of retinal ischemia were assessed by oligonucleotide microarrays and by real-time quantitative PCR.Microarray analysis of the retinal gene expression profile after 5 or 60 min ischemia showed altered transcript levels for a group of genes with functions related to "structural constituent of eye lens" . Subsequent qPCR assays for this set of genes showed extremely high variations in transcript levels between individual animals of both control and ischemia-treated groups. However, the relative transcript levels, or expression profile, of these genes was constant in all samples. The transcript levels of these genes were on average 2624-times higher in tissue samples isolated from the superficial layers of the total lens. Moreover, all 23 genes had high expression levels in lens compared to retina as was shown by microarray.From these data, it appears plausible that during isolation of the retina, trace amounts of lens tissue may end up in the studied retinal samples. This would explain the high level of variability in transcript levels of genes, the strong correlation of relative levels between samples, and the link with lens-specific function of the "altered" genes. Changes in crystallin gene expression in other models of retinal degeneration have been reported and a careful examination of the transcript level of other lens-specific genes is essential to rule out a possible confounding effect of lens-material transfer. The gradual and selective loss of retinal ganglion cells via apoptosis underlies the progression of glaucoma. A variety of causal factors leading to ganglion cell apoptosis has been put forward, but the relative contribution of these factors to the initiation and progression of glaucoma remains unknown. One of the factors implicated in glaucoma as well as in diabetic retinopathy and central retinal artery occlusion, is hypoxia/ischemia -4. The pThe results of these studies will be published elsewhere . In the The focus of this report was to investigate the possibility that a transfer of tissue from the lens to the retinal sample may occur and corrupt the expression data for genes that are abundantly expressed in the lens compared to the retina.Animal handling and experimental procedures were reviewed and approved by the ethical committee for animal care and use of the Royal Netherlands Academy for Sciences, acting in accordance with the European Community Council directive of 24 November 1986 (86/609/EEC) and the ARVO statement for the use of animals in Ophthalmic and Vision Research. Transient retinal ischemia was induced by means of raising the pressure in the anterior chamber of the rat eye via an inserted needle as described in detail previously -18. For The eyes were enucleated and washed in cold phosphate buffered saline. A circumferential cut was made along the ora serrata. The cornea, ciliary body, lens and adhering vitreous were carefully removed from the eyecup using a pair of brushes. The retina was separated from the sclera, frozen on dry ice, and stored at -80 °C until use. Frozen retina was thawed in Trizol (Invitrogen) and homogenized. Total RNA was isolated following the manufacturer's instructions. The total RNA yield was 8-10 μg/retina and quality checks showed sharp ribosomal RNA bands with RNA Integrity Numbers (RIN) >8.7 .Four eye lenses were isolated, placed in Trizol and shaken for 5-10 min. The total RNA sample obtained was used for microarray probe generation and for qPCR assays.Of each animal, 1 μg of isolated total RNA was used to make pooled samples for each of the groups and one for all contralateral control retinas (n=36). From these pooled samples amino allyl-UTP labeled aRNA was prepared  and coupled to Cy3 or Cy5 monoreactive dyes (Amersham). For details see Kamphuis et al. .Agilent. After normalization, the resulting dye ratio represents the transcript ratio between the two hybridized samples.For microarray hybridization, a common reference design was used. aRNA of the control-group (30 μg) was labeled with Cy3. An aliquot of 1 μg of this sample served as the identical common reference sample present on each array and was hybridized together with 1 μg of Cy5-labeled aRNA from one of the experimental groups. Hybridization and washing was carried out according to the protocols described by Agilent. For details see: "Agilent" 60-mer oligo microarray processing protocol v2.1 (SSPE/SureHyb)® at Agilent or Geo).Oligonucleotide arrays were obtained from Agilent  using default settings for all parameters. The Feature Extraction output shows the sequence identification and description of all chip oligonucleotide probes, the signal strength of the treatment (Cy5) and control (Cy3) channels, the relationship between the two channels in terms of logd papers -21.DAVID and Source. Of the 20,280 features representing expressed sequences 18,690 were linked to a UniGene Cluster ID (92%). Gene Ontology (GO) analysis was performed with GOstat (GOstat) [Genomatix) [Generegulation).Gene annotation of the genes on the array was updated using web-based annotation tools (GOstat) . Searchenomatix) , and witt values were converted to absolute amounts of transcript present in the sample (E-Ct) in arbitrary units, multiplied by 1010 for presentation purposes [For qPCR, 2 μg of total RNA from each individual retina was DNaseI treated, reverse transcribed into cDNA with 100 U Superscript III Reverse Transcriptase (Invitrogen) and 50 ng random hexamer primers. The resulting cDNA sample served as a template for SYBR green RT-qPCR analysis . The amplification efficiencies were close to 100% for all primer combinations. The resulting Cpurposes .Rho (rhodopsin), Hprt (hypoxanthine phosphoribosyl transferase), Pde6b (phosphodiesterase 6B), and Prkca  were identified as most stable and were used for normalization. All presented transcript levels, including crystallin levels, are normalized against the transcript levels of these reference genes.To correct for differences in cDNA load between the different samples, we normalized the target PCR to a set of four reference PCRs. For this purpose, qPCR data for a selection of candidate reference genes on experimental and control samples was used as input for a geNorm analysis ,25. Rho Three different conditions of retinal ischemia were studied: ischemic preconditioning (IPC), ischemia-reperfusion (I/R), and IPC followed by ischemia-reperfusion 24 h later (IPC-I/R). Applying a feature extraction assigned p value <0.001 cutoff on the microarray-derived changes showed that several members of the crystallin-gene family were highly regulated under all three conditions with 2- to 10 fold changes. Gene ontology (GO) analysis , revealeCryaa, Cryab, Cryba1/ba3, Cryba2, Cryba4, Crybb1, Crybb2, Crybb3, Crygd, Crygn) and six genes with a known lens-association: Bfsp1 , Bfsp2 , Gluld1 (Glutamate-ammonia ligase domain containing 1/lens glutamine synthase-like (lengsin)), Grifin , Lenep , and Lim2 (Lens intrinsic membrane protein 2). The transcript levels of these genes were transiently upregulated after IPC by 4- to 7 fold at 1, 3 and 6 h. Following I/R in naive animals, transcript levels were decreased 3- to 10 fold. In contrast, in preconditioned animals, ischemia resulted in a persistent increase of 2- to 6 fold compared to controls. The crystallin features Crym, Cryac, and Cryz were not associated with the cluster.Clustering was carried out on the changes observed in the three different experimental conditions separately and on the combined total data set. Various clustering algorithms consistently identified a set of 25 features, representing 23 different genes, with a parallel response to the different ischemic conditions . This se2=0.61; Gapd, Hprt, Pde6b, Prkca), genes known to give an early response to ischemia  and genes known to show a more delayed response . The correlation coefficient R2 for this group was at 0.73 similar to the set of crystallin genes. However, the two groups of genes differed in the level of variance among the individual samples. For the crystallins the variance was 120% (SD/mean * 100%), while the variance for the second group of genes was around 40% comparable to our earlier findings [c-fos, c-jun, Hmox1, Gfap, Vim, Gria1, Gria2, Pvalb, Thy1, nestin, VIP, and Thy1 were all highly significant [Cryab and Cryac expression levels at 2, 6, and 12 h after I/R and after IPC+I/R .The transcript levels of the 14 crystallin genes were investigated by qPCR assays on all individual RNA-samples that contributed to the sample pools. Linear regression analysis of the fold change as measured on microarray against the average change derived from the qPCR results showed a significant correlation R2=0.61; . qPCR dafindings ,24. The nificant ,24. The Cryaa and Cryba1/3 is presented in Crym, Cryac, and Cryz) did not show this correlation.In contrast to the high inter-individual variance, the transcript levels between different cluster genes showed strong correlations. An example showing the close correlation between Pax6, one of the transcription factors inferred in the regulation of crystallin gene expression may be a candidate for such a role [Pax6 transcript levels were not in line with the pattern presented in c-Maf, Sox-2, Six3, Hsf2, Ap1, Foxd3, Foxj2, Hsf, Oct1, v-Maf), or were not represented on the array . Searches for transcription binding sites in the crystallin genes in rat and mouse genome yielded potential regulation sites for large families of transcription factors ETSF, HOXF, EVE1, GATA, and CREB. Attempts to find a common framework of multiple transcription factor binding sites did not lead to results.These findings may indicate a co-regulation for the expression of this set of genes. However, genes in the cluster are located on different chromosomes thus ruling out a tandem arrangement of these genes under the control of a single promotor region. Expression could also be united by a common transcription factor simultaneously acting on all genes. h a role , howeverS100a4) up to 12,700 (Crybb2). In addition Crygb, a crystallin gene not represented on the array, had a lens/retina ratio of 3273, and the Crygb levels were found to be correlated to the transcript levels found for the cluster genes. Moreover, the crystallins Cryac, Cryz, Crym not assigned to the cluster, had ratios of 0.32, 0.09, and 0.006, respectively, showing a higher expression in the retina compared to expression in the lens.Another explanation for the correlated transcript levels could be the unintentional transfer of lens tissue during the isolation of the retina. Genes that are abundantly expressed in lens and with a low expression level in the retina would display highly correlated levels. To test this hypothesis, 2 μg RNA samples from the lens and retina (n=4 each) were analyzed by qPCR. The results are presented in Gapd, Hprt, 28S and Bfsp1, Lim2, Grifin, Crym, Cryaa, Crybb3. The profile of transcript levels was comparable to that of the retinal samples. However, Rho, Pde6b and Thy1 transcripts were detected in the vitreous samples, indicating a contamination with retinal tissue. The normalized transcript levels of Rho, Pde6b and Thy1 were comparable to those found in retinal samples.We investigated the possibility that transfer of vitreous could be implicated because it is difficult to remove all overlying vitreous from the retina during isolation. The amount of RNA in all of the vitreous obtained from a single rat eye is low . qPCR assays were performed for In order to verify whether the set of 25 genes of the cluster are all genes with a high lens to retina expression ratio, we performed a microarray hybridization of the lens samples against retinal control samples. All 25 features found in the cluster had high expression levels in the lens compared to the retina; the PMT-settings of the scanner had to be reduced to 2% to prevent saturation of these features. The resulting MA-plot is shown in Bfsp1, -2, Gluld1, Grifin, Lenep, and Lim2. These genes showed parallel changes after IPC, I/R, and after IPC-I/R. These findings are based on microarray data of pooled samples. The pooling strategy was prompted by the high cost of the arrays. It must be emphasized that pooling is less favorable from a statistical point of view, in particular for genes with higher variability in expression level, making validation by qPCR on individual samples a necessity [Cryab and Cryac transcript levels was confirmed. This may be of physiological relevance since Cryab confers a cytoprotective effect on various cell types and has been implicated in several neurological diseases like multiple sclerosis, Alzheimer, Parkinson disease [We conducted a series of microarray experiments on rat retina to study alterations in the gene expression profiles after transient ischemia in order to gain insight in molecular pathways that lead to neurodegeneration and neuroprotection ,8,27-30.ecessity . Neverthecessity ,24, and ecessity . The qPCecessity ,24,32,33 disease ,35, and  disease . qPCR reWe therefore explored the possibility that a transfer of lens material into the isolated retinal tissue underlies the coupling in transcript levels of these genes. In accordance with this hypothesis we found that: (i) the transcript levels of the cluster genes were between 215-times and 12,700-times more abundant in RNA isolated from the total lens as compared RNA from the retina, (ii) when comparing equal amounts of lens and retinal mRNA by microarray, the cluster genes had the highest lens/retina ratio. The same cluster genes were not abundantly expressed in samples of the vitreous. In conclusion, a transfer of lens tissue into the isolated retinal sample offers a plausible explanation of our observations.The most likely moment of transfer to take place is during isolation of the retina. The rat lens is relatively large and the lens may be damaged when the circumferential cut along the ora serrata is made. The cornea, ciliary body, lens, and vitreous were removed using a pair of brushes. These brushes were cleaned in buffer and then used to pick up the retina from the eyecup and transfer it to a vial for storage. Following this method, small amounts of lens tissue may contaminate the isolated retinal sample. We tested an alternative method for isolation of the retina on a separate series of 59 eyes; opening the eye with a cut through the cornea and taking out the lens, vitreous and retina simultaneously, to avoid damaging the lens, followed by dissection of the retina in buffer. However, this method also resulted in crystallin transcript levels with a variation coefficient of 133%, indicating that transfer may be difficult to avoid completely. RNA isolation by laser dissection from retinal cryosections may be effective in preventing contamination but this method is labor-intensive and may compromise the quality of RNA (unpublished observations N.Y.).Bfsp1, Bfsp2 [Gja3 [Aqp0 also known as Lim1, another major lens protein, was not represented on our array [Cryba1, -ba2, -ba4, -bb1, -bb3, -gA, -gB, -gC, and -gD), Bfsp1, Bfsp2, Lim2, Lenep, Clic5, and Cd24 in fiber cells compared to epithelial cells. These results support our hypothesis that lens fiber cells from the superficial layers of the lens is most likely source of inadvertently transferred material.The lens contains lens epithelium, stem cells, and lens fiber cells in the transition zone undergoing terminal differentiation into secondary lens fibers. The superficial lens fiber cells are nucleated and have the standard complement of intracellular membrane compartments. Crystallins α, β, and γ are ubiquitously expressed in the more superficial cortical fiber cells positioned at the equatorial region -38, toge1, Bfsp2  and Gja3p2 [Gja3 . Aqp0 alur array . Recentlur array  comparedCrym, Cryz and Cryac, which were not in the cluster, are more abundant in the retina. Localization studies on crystallins have confirmed strong Crym-immunostaining in photoreceptors but not in the lens [Our data show that the transcript levels of the genes of the cluster are low in the retina. In contrast, expression of the the lens . In mousthe lens . MoreoveGryab, Cryz, Crym, and Crygs were strongest, which is in good agreement with our findings in the rat. In conclusion, there is ample evidence for retinal gene expression of crystallins but transcript levels are low. For that reason, due to high transcript levels found in the lens, contamination of retinal tissue with traces of lens tissue will greatly bias these transcript levels.To gain insight into retinal transcript levels in samples not corrupted by lens material, we analyzed microarray data collected from human photoreceptor RNA isolated by laser dissection from cryosections (unpublished observations NY). Detectable hybridization was found for all cluster members but the signal strength was lower than 4% of the maximum intensity on the array, indicating low transcript levels. The hybridization signals for Cryaa, Cryab, Cryba1/3, Crybb2, Crygc, and Grifin transcript levels after 12 h of reperfusion, but no qPCR validation was presented [Cryaa, Crybb2, and Cryab at 8 days after episcleral vein injection but the effect disappeared after 35 days and was not confirmed by qPCR [Lim2, Bfsp1, and Grifin [Cryab and Crygd was confirmed by qPCR and the injury was accompanied by an increased immunoreactivity for crystallin-β,-α, and γ near the scar [Cryaa, Cryba1, Cryba2, Cryba4, Crybb1, Crybb3, Crygb, Cryd, Cryn, Cd24, Grifin, Lim2, and Mip (Lim1), of which Cryba4 and Mip were confirmed by qPCR [An intriguing question is whether our findings have any relevance for the changes in retinal crystallin gene expression that have been reported for several experimental models involving retinal injury or degeneration. As can be concluded from our data, a proper statistical analysis is essential to evaluate the observed changes. For instance, light-induced retinal damage was claimed to lead to a 2- to 3 fold increase in spot intensity of all identified crystallins in 2D-gels, however statistical information was not given . A microresented . Chronic by qPCR . Two stu by qPCR ,13. Loca by qPCR .In conclusion, studies on changes in the retinal transcriptome have often reported alterations of crystallins and several other lens-associated genes. Our observations show that the data on these genes have the risk of being corrupted by transfer of lens material into the isolated retinal tissue. Avoiding pooling strategies in microarray designs, a careful statistical evaluation of individual samples, and the use of comparative lens cDNA samples as controls, is essential to draw any conclusions on gene expression changes in the retina of genes with an abundant expression in the lens relative to the retina.


Osteoporosis is a major health problem in postmenopausal women. Evidence suggests the importance of oxidative stress in bone metabolism and bone loss. Tea consumption may be beneficial to osteoporosis due to its antioxidant capability. However, lack of objective data characterizing tea consumption has hindered the precise evaluation of the association between tea ingestion and bone mineral density in previous questionnaire-based epidemiological studies. On the other hand, although published studies suggest that Tai Chi (TC) exercise can benefit bone health and may reduce oxidative stress, all studies were conducted using a relatively healthy older population, instead of a high-risk one such as osteopenic postmenopausal women. Therefore, this study was designed to test an intervention including green tea polyphenol (GTP) and TC exercise for feasibility, and to quantitatively assess their individual and interactive effects on postmenopausal women with osteopenia.One hundred and forty postmenopausal women with osteopenia  were randomly assigned to 4 treatment arms: (1) placebo group receiving 500 mg medicinal starch daily, (2) GTP group receiving 500 mg of GTP per day, (3) placebo+TC group receiving both placebo treatment and TC training , and (4) GTP+TC group receiving both GTP and TC training for 24 weeks. The outcome measures were bone formation biomarker , bone resorption biomarker (serum tartrate resistant acid phosphatase), and oxidative DNA damage biomarker (urinary 8-hydroxy-2'-deoxyguanosine). All outcome measures were determined at baseline, 4, 12, and 24 weeks. Urinary and serum GTP concentrations were also determined at baseline, 4, 12, and 24 weeks for bioavailability. Liver function was monitored monthly for safety. A model of repeated measurements with random effect error terms was applied. Traditional procedures such as ANCOVA, chi-squared analysis, and regression were used for comparisons.We present the rationale, design, and methodology of a placebo-controlled randomized trial to investigate a new complementary and alternative medicine strategy featuring a dietary supplement and a mind-body exercise for alleviating bone loss in osteopenic postmenopausal women.ClinicalTrials.gov identifier: NCT00625391 Osteoporosis is a degenerative bone disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures, especially of the hip, spine, and wrist . Women aOsteoporosis is associated with many etiological causes, such as poor nutrition, cytokines, hormones, and aging. Reactive oxygen species (ROS) are considered to be responsible for the aging process  and contThere is evidence that ROS are involved in bone resorption with a direct contribution of osteoclast-generated superoxide to bone degradation ,10, and A possible relationship between tea drinking and osteoporosis was suggested by a number of studies . The bioThe most widely recognized properties of tea polyphenols, extract forms of tea, are their antioxidant activities, arising from their ability to scavenge ROS . Tea polHowever, no study has ever been conducted to show the antioxidant role of GTP in bone metabolism of postmenopausal women with low bone mass.Exercise has been shown to benefit osteoporosis by reducing bone loss. Numerous studies have reported the effect of exercise on BMD, particularly in postmenopausal women with or without osteopenia/osteoporosis -32. Tai Exercise can have positive or negative effects on oxidative stress depending on training load, training specificity and the basal level of training . High-inTherefore, the objective of the present study was to test complementary and alternative medicine interventions including GTP and TC exercise for feasibility, and to quantitatively assess their individual and interactive effects on osteopenic postmenopausal women. We hypothesized that 24 weeks of GTP supplement, TC exercise, and their combination would benefit bone remodeling, as measured by bone biomarkers in postmenopausal women with osteopenia, compared to those receiving placebo only, and the changes in bone biomarkers associated with bone remodeling would be correlated with the changes in oxidative stress. This hypothesis was based on the premise that both TC and GTP may reduce oxidative stress, and that oxidative stress has been demonstrated to adversely affect bone health by both increasing differentiation and function of osteoclasts  and inhiIn this paper, we present the design and detailed protocol of a placebo-controlled and randomized trial, as well as a discussion of the overall challenges of conducting this trial. The results from this trial will be reported at the completion of the study in accordance with the Consolidation of Standards for Reporting Trials guidelines .The combination of dietary supplement and moderate intensity exercise now becomes a new strategy in alleviating bone loss in postmenopausal women with low bone mass, due to the possible synergistic effects of the combination.This was a 24-week placebo-controlled and randomized intervention trial to investigate the effects of GTP and TC on relevant primary and secondary endpoints in postmenopausal women with osteopenia. Women at least 2 years after menopause, with osteopenia, were recruited primarily from local senior independent/assisted living facilities, municipal senior community centers, and obstetrics and gynecology clinics. An initial sample size of 140 participants with an expected attrition rate of 15% over 24 weeks of intervention produced a final sample size of 120 participants. This sample size yielded a power of approximately 0.85 to 0.9 at α = 0.05 for detecting differences in primary and secondary outcome measures. After screening, qualified participants were matched for age and were randomly assigned to one of the four treatment groups: placebo, GTP, placebo+TC, and GTP+TC. During the 24-week intervention, all participants were provided with 500 mg elemental calcium and 200 IU vitamin Ddaily. The participants in the placebo group received 250-mg capsules of medicinal starch, 2 per day for 24 weeks. The GTP participants received 250-mg capsules of GTP, 2 per day for 24 weeks. The placebo+TC participants received both placebo and TC intervention  for 24 weeks. The GTP+TC participants received both GTP and TC intervention for 24 weeks. Participants received the primary and secondary outcome measures at baseline, 4, 12, and 24 weeks. The outcome measures were concentrations of bone biomarkers  and a biomarker of oxidative stress DNA damage (urinary 8-OHdG concentration). Urinary and serum GTP concentrations were determined at baseline, 4, 12, and 24 weeks. Liver function was also monitored monthly by assessing the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Investigators evaluating the endpoints were blinded to intervention allocation.  forms, participants completed a detailed questionnaire that was developed to collect demographic, health, and dietary information. Then, the potential participants were invited for bone density screening and fasting blood screening visits.1. Women at least 2 years after menopause with osteopenia . Osteope2. Normal function of thyroid, liver, and kidney.3. Serum 25-hydroxy vitamin D ≥ 20 ng/mL.1. History of metabolic bone diseases or treatments that might influence bone turnover.2. History of cancer except for superficial basal or squamous cell carcinoma of the skin that was treated, and other malignancies treated at least 5 years ago without any evidence of recurrence.3. Uncontrolled intercurrent illness or physical condition that would be a contraindication to exercise.4. Cognitive impairment as defined as Mini-Mental State Examination score of 24 or less .5. Depression as defined as Yesavage self-rated Geriatric Depression Scale score at least 11 .6. Unwilling to accept randomization.Data from previous studies by our team ,48 were All participants who had passed the screening criteria participated in randomization where participants were assigned to one of the 4 treatment arms with a fixed randomized scheme. In order to minimize the consequent bias, a stratified randomization method was employed based on age (>65 or <65 years old), history of green tea consumption, and history of mind-body exercise to assign participants to placebo, GTP, placebo+TC, or GTP+TC group.Placebo and GTP of the same lot, respectively, were purchased from Zhejiang Yuxin Pharmaceutical Co., Ltd., China . Each placebo capsule of 250 mg medicinal starch contained no GTP ingredient at detectable levels. Each GTP capsule contained 250 mg GTP ingredient  and the purity was higher than 98.5%. The use of placebo for masking the control group was desirable in this study to keep respondents blinded to the GTP treatment assignment. Placebo capsules were made of the same size and color as the GTP capsule. The placebo group provided a comparison of blood and urinary outcome measures and served as a basis to assess GTP's effect.Calcium /vitamin D  supplement daily was provided to all participants . Adequate Ca/vitamin D intake is essential for realizing the beneficial effects of dietary supplements and exercise on BMD ,50. Acco[120] for 40 minutes during each session. The cool-down exercise involved whole body relaxation and deep breathing. Make-up sessions were offered to participants if necessary. Participants in the GTP+TC group received both GTP treatment and TC training for 24 weeks.The present study had four intervention groups, placebo, GTP, placebo+TC, and GTP+TC. Provision of placebo to the control and TC only groups increased the accuracy of the interventions, since it kept respondents blinded to the GTP treatment assignment. Participants in the placebo treatment group were provided 500 mg of medicinal starch daily for 24 weeks. Participants were instructed to take 1 capsule containing 250 mg in the morning and another 1 capsule in the evening, after meals. Similar to those in the placebo treatment group, participants in the GTP treatment group were provided 500 mg (250 mg capsule × 2 per day) of GTP per day for 24 weeks. Participants in placebo+ TC group received placebo treatment and TC training for 24 weeks. In addition to 500 mg of medicinal startch daily, the TC training was held at the exercise hall of a local community center, for 60 minutes three times a week (on three nonconsecutive days), for 24 weeks. Each training session consisted of 10 minutes of warm-up exercise, 40 minutes of TC, and 10 minutes of cool-down exercise. The warm-up exercise featured movements of all major body joints that were involved in the subsequent TC exercise. Participants were taught by a qualified instructor the 24-move simplified Yang-style TC A complete and accurate dietary assessment was needed to determine whether drastic dietary changes occurred during the intervention period that could affect outcome measures. A 3-day dietary intake log was collected at the baseline, mid, and final visits and analyzed by Nutritionist Pro  to determine usual dietary intake (including specific macro- and micro-nutrients) over time and deviations from baseline. A physical activity log was collected at the baseline, mid, and final visits to record any deviation from the usual activities. Concomitant medications/therapies (including over the counter and dietary supplements) were documented, including drug name and the reason for the medication/therapies throughout the study period.All investigators, measurement and site personnel were blinded throughout this study. However, it was impossible to keep the TC instructor and the pharmacist, who distributed placebo or GTP capsules, blinded. Investigators evaluating the endpoints were blinded to intervention allocation.Fasting blood and urine samples (morning after first empty of the bladder) were collected for both primary and secondary outcome measures. Blood was drawn from a superficial arm vein with a syringe, transferred to a vacutainer, allowed to clot at room temperature, centrifuged at 1500× g for 10 min within 2 hr of collection, and aliquoted from each collection. During the visit for blood sample collection, urine samples were also collected in acid-washed polyethylene containers. Urine was aliquoted from each collection. All aliquoted blood and urine samples were stored in -80°C freezers prior to biochemical analyses.Adherence to the intervention was evaluated by monitoring the consumption of capsules by pill count and attendance at TC training sessions. Compliance was determined as the percentage of all capsules ingested or possible group classes attended. The TC class attendance record was kept for each participant in the placebo+TC and GTP+TC groups.Adverse effects associated with placebo or GTP treatment were self-reported by the participants if any, and by monitoring liver function monthly in the course of the intervention trial. Participants in the placebo+TC and the GTP+TC groups were also queried about any adverse events during TC training sessions by self-reported phone calls. All observed or volunteered adverse events, regardless of suspected causal relationship to the study treatments, were recorded on the adverse event form throughout the study.Every participant was evaluated at baseline (prior to starting intervention), 4, 12, and 24 weeks of intervention.Biochemical markers of bone turnover have been shown to be promising in predicting fractures in the elderly up to 2 years before the event . AssessiThe concentration of bone formation biomarker, BAP, in serum was measured using Metra™ BAP immunoassay kits . The intra-assay CV of the BAP assay was 5.2%, and the inter-assay CV was 5.0%. Such procedures are routinely performed in our laboratory . The conin situ, or destroyed by apoptotic processes, if not to result in mutations. In humans, 8-OH-Glu glycosylase is the primary enzyme for the repair of 8-OH-Gua in a short-patch base-excision repair [When ROS attack all macromolecules including lipids, proteins, and DNA, the addition of the hydroxyl group (-OH) to the C8-position of guanine produces C8-OH-adduct radical , which in repair . The excn repair . Detecti® HLB 3 cc (60 mg) cartridge. The eluents were dried under ultra-pure N2 stream and reconstituted in buffer  for analysis with the ESA HPLC-CoulArray system. The system consisted of double Solvent Delivery Modules (Model 582 pump), Autosampler (Model 542) with 4°C cool sample tray and column oven, CoulArray Electrochemical Detector (Model 5600A), and an Operating Computer. The HPLC column for 8-OHdG analysis was the Waters YMC basic™ column . The mobile phase consisted of buffer A  and buffer B (methanol). Flow rate was kept at 0.8 ml/min and a linear gradient (0-40% MeOH in 15 min) was applied for chromatographic separation with the peak of 8-OHdG eluted at around 9.5 min. The CoulArray Detector was set at 270, 300, 330, and 360 mv; the highest peak appeared at 330 mv channel. Authentic standard 8-OHdG was used for qualification by retention times and response patterns, and quantification by calibration curves. The limit of detection for 8-OHdG was 1 ng/ml urine. The amount of 8-OHdG was adjusted by urinary creatinine level. Urinary creatinine level was determined colorimetrically with a Diagnostics Creatinine Kit (Sigma Co). Absorbance at 500 nm was recorded by a DU640 VIS/UV spectrophotometer [The concentration of 8-OHdG, DNA damage oxidative stress biomarker, in urine was determined . The uriotometer .The bioavailability of GTP was considered when we quantitatively evaluated the biological effects of GTP intervention. Therefore, we planed to measure the GTP levels in biofluid, such as serum and urine, of participants at baseline, 4, 12, and 24 weeks.The procedures for analyzing serum and urinary GTP conjugates were modified from established protocols ,59. Brie18 . The mobile phase included buffer A  and buffer B . Flow rate was set at 1 ml/min and the gradient started from 4% buffer B, to 24% B at 24 min, to 95% B at 35 min, kept at 95% until 42 min, dropped to 4% at 50 min, and maintained at 4% until 59 min. Authentic standards were prepared with ascorbic acid.The HPLC-CoulArray system consists of double Solvent Delivery Modules (Model 582 pump), Autosampler (Model 542) with 4°C cool sample tray and column oven, CoulArray Electrochemical Detector (Model 5600A), and an Operating Computer. The HPLC column was an Agilent Zorbax reverse-phase column, Eclipse XDB-CCalibration curves for individual GTP components were generated separately, and EGC, EC, EGCG and ECG were eluted at around 14, 21, 24 and 29 min, respectively. The electrochemical detector was set at -90, -10, 70 and 150 mV potentials, with the main peaks appearing at -10 mV (EGC), 70 mV  and 150 mV (ECG). Quality assurance and quality control procedures were taken during analyses, including analysis of authentic standards for every set of five samples and simultaneous analysis of spiked urine sample daily. The limits of detection were 0.5 ng/mL urine for EC, EGC, and methylated-EGC; and 0.5 ng/mL plasma for EGCG, ECG, and dimethylated-EGCG, respectively. The levels of glucuronidated or sulfated GTP or methylated GTP were calculated by subtraction of free GTP levels from corresponding digestions. The four forms of GTP were pooled as a total for calculation of conjugation percentiles.Due to the longitudinal design of the proposed study, a model of repeated measurements with random effect error terms was used. Statistical software SAS was employed to conduct the analyses, controlling for the within subject correlation. First, the measurements were compared across the 4 groups at the baseline. In addition, participant characteristics were compared to detect whether participants in these four groups were different in certain characteristics. Second, the changes in the measurements from baseline to the follow-ups were calculated for each group. We then tested whether these changes were statistically significant. Time-dependent trends, if any, were identified. For between-group differences, a two-way ANCOVA (TC and GTP) was conducted and controlled for within-subject correlation. To control for the effect of age, body mass index, BMD, and other covariates , a multivariate ANCOVA was also performed. Third, the characteristics of participants who dropped out were compared with those of the participants who stayed for the entire study period, in order to detect potential biases.We have presented the rationale, design, and methodology of a placebo-controlled randomized trial, with quantitative studies, to investigate a new complementary and alternative medicine strategy featuring a dietary supplement and a mind-body exercise for alleviating bone loss in postmenopausal women with low bone mass. The results of this research will be presented as soon as they are available.The authors declare that they have no competing interests.CLS obtained funding for the study. CLS, MCC, JKY, CKF, KTX, BCP, and JSW designed this trial. CLS wrote the first draft of the manuscript and the rest of coauthors participated in the revision of subsequent dra. All authors approved the final version of the manuscript. None of the authors declared any conflicts of financial interest.The pre-publication history for this paper can be accessed here:


Danio rerio), an animal model in which social rank and aggressiveness tightly correlate.Aggression is a near-universal behaviour with substantial influence on and implications for human and animal social systems. The neurophysiological basis of aggression is, however, poorly understood in all species and approaches adopted to study this complex behaviour have often been oversimplified. We applied targeted expression profiling on 40 genes, spanning eight neurological pathways and in four distinct regions of the brain, in combination with behavioural observations and pharmacological manipulations, to screen for regulatory pathways of aggression in the zebrafish , previously believed specific to male behaviours, was amongst those genes most associated with aggression, and AVT inhibited dominant female aggression, as in males. However, sex-specific differences in the expression profiles also occurred, including differences in aggression-associated tryptophan hydroxylases and estrogen receptors.Thus, through an integrated approach, combining gene expression profiling, behavioural analyses, and pharmacological manipulations, we identified candidate genes and pathways that appear to play significant roles in regulating aggression in fish. Many of these are novel for non-mammalian systems. We further present a validated system for advancing our understanding of the mechanistic underpinnings of complex behaviours using a fish model. The display of aggression is a near-universal trait in the animal kingdom, of which several forms have been distinguished . In non-Drosophila and mouse . In theP values see Additional File We first determined regions of the brain associated with aggressiveness in zebrafish. This was achieved using males, based on their greater aggression, and greater differential in aggression between social ranks, than observed in females. We determined gene expression profiles in four regions of the brain in dominant compared with subordinate males, identified as such based on behavioural observations of aggression on day 1 of the social interaction experiment. In doing so, we observed marked differences with a total of 34 differentially expressed genes across the different brain regions  of the differentially-expressed genes for different regions of the brain. In the hypothalamus and hindbrain, all of the genes were overexpressed in dominant individuals Fig. .. In conGiven that we identified the hypothalamus and telencephalon as the regions of the brain where the greatest number of differentially-expressed genes occurred relating to aggressiveness (based on our chosen gene set), these regions were selected for our more focused analyses. The genes differentially expressed in these brain regions relative to social rank, together with their associated functional pathways, are shown in Table Interestingly, the differences in expression of the various genes studied was relatively low between the behavioural phenotypes  showed the greatest difference in expression between dominant and subordinate males , and its mammalian homologue arginine vasopressin (AVP), have well established roles in aggression and social position across vertebrate taxa [t-tests: t6 = 4.568 and P = 0.004; t7 = 5.227 and P = 0.001; t8 = 7.286 and P < 0.001; for 0.5, 1, and 5 μg/g.b.w. doses, respectively; Fig.  burtoni  and provate taxa . To testavplr1b was differentially expressed between dominant and subordinate males , which encodes isotocin (the fish homologue of oxytocin), to be overexpressed in dominant males  and one of its receptors (htr1a) were overexpressed in dominant versus subordinate males . This lack of effect of fluoxetine may relate to our choice of a 1 hour exposure duration  rather plendens . Males esst1, encoding the somatostatin peptide, and sstr1, encoding one of its receptors, were higher in dominant males , but this was not the case. Also, expression levels of  burtoni . We are  burtoni ,51.th and slc6a3, which encode the enzyme tyrosine hydroxylase and the dopamine transporter (DAT), responsible for dopamine synthesis and re-uptake, respectively, the latter of which ultimately terminates dopaminergic activity), were associated with higher aggression  in dominant males (Table gnrh3 (the protein product of which stimulates sex steroid production) had 2.2-2.4-fold greater expression in dominant males compared with subordinates  and estrogen receptor (ER) β subtypes  and the H2 receptor (hrh2) in dominant males , and npy expression were associated with aggressiveness. However, these genes had opposite expression profiles in the hypothalamus compared with the telencephalon ,  mammals ,69-71 an mammals , althougFemale zebrafish, as well as male, show territorial aggression . A furthTo probe for the main neurological determinants of aggression in zebrafish, based on expression of the targeted genes, we analysed our entire dataset  using hierarchical clustering and principal component analysis (PCA). The clustering demonstrated that in the hypothalamus, social rank  was a greater determinant of the expression of the chosen gene set than sex Fig. . In teletph1a, npy and avpl, all of which were more highly expressed in dominants  while Manning compound had no effect   Fig. .tph1a, tph2, sst3, slc6a4a, slc6a3, nos1, mao, cyp19a1b, esr1), and some in males only . Our results for the tph genes suggest a sex-related difference in the subtypes of the 5-HT synthesising enzyme TPH that may regulate aggression, with the situation in females being more analogous to that in humans [esr1 was also interesting given that in mice ERα deletion had opposite effects on aggression between sexes [nos1, in mammals neural nitric oxide  inhibitnsmitter  but stimnsmitter ,83, agait14 = 36; P = 0.021). Again these changes in behaviour of the female zebrafish aligned well with the patterns of gene expression with the reduction in differences  observed between female ranks and increased expression of many 'aggression' genes in the hypothalamus in subordinate females.To identify temporal variation in the genes associated with aggression, we compared our data for fish sampled on day 1 of the social interaction experiment with those for equivalent fish sampled after 5 days Fig. . The numavpl and oxtl were specific to dominants in day 1, although 11 genes were specific to dominant males on day 5 . In males . avpl aHierarchical clustering and PCAs of these data on day 5 Fig. , analogoAggression is an evolutionary conserved behaviour with implications for human and animal societies and unravelling its neurophysiological basis has many potential benefits. We identified numerous, including novel, candidate neurological genes and pathways associated with aggression in fish, including those possibly conferring sex related differences. For some of these pathways, we further directly linked the pathways with functional responses, albeit our manipulation experiments were limited and we did not, for example, include any determination of dose/concentration-responses. The conservation of many aspects of aggression neurophysiology in zebrafish and mammals supports the use of this fish model in aggression research generally. Further work on aggression using the zebrafish model should establish whether different forms of aggression, the same forms of aggression in different social contexts, and aggression by dominant/territorial compared to subordinate/non-territorial fish are regulated by similar mechanisms. Additionally, further work needs to investigate the inter linkages between aggression control pathways and develop a greater understanding of sex related differences.Artemia nauplii  and TetraMin tropical flake food . All animal-use procedures were carried out ethically according to UK Home Office guidelines.The zebrafish used were bred in-house from the Wild Indian Karyotype (WIK) strain. Fish were maintained at 28 ± 1°C with a constant photoperiod of 12 h light: 12 h dark (08.30-20.30) and an artificial dawn/dusk transition of 30 min. The tank water was reconstituted reverse osmosis-filtered mains water supplied on a flow-through system (2 L/hour). Prior to the experiments, fish were maintained in large mixed-sex holding tanks. The experimental tanks used were 300 mm × 300 mm × 300 mm with a working volume of 18 L and contained a central spawning site, consisting of glass marbles with an artificial weed. Opaque dividers prevented visual interactions between fish in neighbouring tanks. Fish were fed to satiation twice daily with freshly hatched Immediately prior to the artificial dusk on day zero, 64 fish  were assigned randomly into 16 experimental tanks, with two males and two females per tank, and the fish were allowed to acclimate overnight . The following morning, beginning at 08.30 am, 8 of the tanks were videotaped for 10 min using an Ikegami ICD-848P digital colour-black/white video camera  coupled to a Sony HDV 1080i digital HD videocassette recorder. Dominance/subordinance was assigned for each fish , and the fish were sacrificed by terminal anaesthesia . Four distinct brain regions - the telencephalon (including the olfactory bulbs and POA), hypothalamus, optic tectum, and hindbrain (medulla oblongata and cerebellum) - were dissected from each fish, snap-frozen in liquid nitrogen, and stored at -80°C until processing. For the remaining 8 tanks, the same procedure was conducted on day 5 of the study. Based on observations of behaviour, a dominant-subordinate relationship formed between the two males and between the two females in all, but one of the colonies. In the exception, no obvious hierarchy developed between the two females, and this colony was, therefore, excluded from further analyses.HDV files containing behavioural recordings were streamlined to AV1 files using Adobe Premier, and viewed at half speed with Windows Media Player. Aggressive behaviours  performRNA was extracted separately from each of the four brain regions of each individual fish using the RNeasy Micro Kit , which included a DNase treatment, and RNA quantity/quality was verified by Nanodrop ND-1000 spectrophotometer . For each individual tissue sample , cDNA was synthesized from the total amount of RNA obtained (approximately 20-100 ng) using random hexamers  and M-MLV reverse transcriptase .avplr1b, sstr1, sstr2, sstr3), annotated cDNA sequences were not available for zebrafish. For these genes, cDNA sequences were identified by querying the zebrafish genome database with the sequences for these cDNAs in other fish species using the BLAST algorithm. Hits obtained from the BLASTs were then screened for conserved motifs specific to each transcript via multiple sequence alignment . Primers specific for the target genes were designed with Beacon Designer 3.0  and assays optimised and validated for real-time PCR using SYBR Green chemistry, as described previously [-ΔΔCt method with efficiency correction as previously described [ribosomal protein L8 (rpl8) was used for relative quantification because its expression was unaffected by social rank, sex, or duration of interaction. For further details of the real-time PCR assays and methodologies used, see Additional File A suite of 40 genes involved in HNS, 5-HT, somatostatin, dopamine, histamine, nitric oxide, HPI axis, and HPG axis pathways were selected for study in zebrafish based on the literature for studies on aggression in mammals Fig. . For seveviously  , at a concentration of 16 μg/L, and the SERT inhibitor, fluoxetine HCl (Sigma W108), at concentrations of 3 and 4.5 μg/mL, both by immersion to males only. In these studies, the concentrations/doses and experimental designs we used were based on previous studies with these drugs in fish [Pharmacological agents known to manipulate specific nodes within the HNS and 5-HT pathways in fish/mammals were administered to dominant males or females via i.p. injection or immersion . For all manipulations, a repeated-measures design was used whereby the same fish were tested under both sham and treated conditions. The order of treatments was randomised for each fish within each experiment. On the morning of the day before the start of each experiment, 24 fish were randomly allocated to 12 tanks with 2 fish per tank and a very small section of caudal fin was removed from one of the fish from each tank to permit identification of individual fish . That afternoon, social rank was assigned for each fish and the tanks in which the most stable hierarchies had developed were selected for the drug manipulations . The following morning, the dominant fish from each tank was removed for experimental treatment. When immersion was used as the route of drug delivery, a stock solution of the chemical was prepared in aerated aquarium water and fish exposed to either this (treated) or untreated aquarium water (sham) in a glass beaker (50 mL volume) at 28°C for 1 h. When injection was used as the route of drug delivery, the chemical was dissolved in 0.9% saline and fish injected into the i.p. cavity with either this (treated) or saline alone (sham), using a 0.3 mL 30G needle syringe (BD Micro-Fine U-100 insulin syringe) with a 10 μl injection volume. Following a 40 min recovery period for the fish in their respective tanks, behavioural analyses were conducted blind in duplicate. In these analyses, the total number of aggressive acts performed by the dominant fish towards the subordinate was quantified over a period of 5 min. All fish were given 24-48 hours (based on pilot studies) recovery prior to the subsequent treatment within an experiment. For the HNS axis, we tested the natural AVT peptide, AVT acetate salt (Sigma V0130), which was administered by i.p. injection at doses of 0.5, 1, and 5 μg/g.b.w. to males and 1 μg/g.b.w. to females, and the specific AVT receptor 1a antagonist Manning compound  and mam in fish ).t-test  within Sigma Stat 3.10 (Jandel Scientific Software). Real-time PCR data were imported into GeneSpring GX 7.3 Software . All of the 40 study genes were expressed across the different regions of the brain in all fish, with two exceptions  which we subsequently excluded from the analyses. Based on the entire set of samples for the total of 38 consistently expressed genes, genes and groups of samples were clustered following a hierarchical strategy with average linkage. The similarity measure used was Euclidian distance because this takes into account the magnitude of changes in gene expression. PCA was performed to identify the main trends between the gene expression profiles of individual fish. To identify differentially expressed genes, comparisons between social ranks, sexes and time periods were performed by t-test followed by multiple testing correction . In the pharmacological manipulations, the effect of each drug on aggression was tested by comparing the total number of aggressive acts between sham and experimental treatment for each fish via paired t-test (Sigma Stat 3.10). All differences were considered statistically significant at P < 0.05.In the social interaction experiment, statistical differences in aggression between social ranks and sexes were tested by two-way ANOVA, followed by Holm-Sidak post hoc test, and between days by AF and GP designed and carried out the social interaction experiment. AF carried out the gene expression analyses, data analyses and drafted the manuscript. GP and TH carried out the behavioural analyses. AF, TH and GP carried out the pharmacological manipulations. CT participated as a supervisor in the design of the studies and analyses, and edited the manuscript. All authors read and approved the final manuscript.Genes associated with aggressiveness in different regions of the brain. Genes associated (P < 0.05) with aggressiveness in different regions of the brain in male zebrafish. Analyses were performed using data from dominant and subordinate males sampled on day 1 of aggression.Click here for fileChanges in the expression of individual genes in hypothalamus and telencephalon in males between day 1 and day 5 of the social interaction study. Changes in the expression of individual genes in (A) hypothalamus and (B) telencephalon in males between day 1 and day 5 of the social interaction experiment. Data are represented as means ± SEM and expressed as the ratio of 'gene of interest':rpL8. Significant differences in expression are denoted by an asterisk .Click here for fileChanges in the expression of individual genes in hypothalamus and telencephalon in females between day 1 and day 5 of the social interaction study. Changes in the expression of individual genes in (A) hypothalamus and (B) telencephalon in females between day 1 and day 5 of the social interaction experiment. Data are represented as means ± SEM and expressed as the ratio of 'gene of interest':rpL8. Significant differences in expression are denoted by an asterisk .Click here for fileFurther details of the real-time PCR assays and methodologies used. Word DOC displaying further details of the real-time PCR assays and methodologies used.Click here for fileFurther details of the methodology used in the pharmacological manipulations. Word DOC displaying further details of the methodology used in the pharmacological manipulations.Click here for file


Immune-compromised subjects, especially those with underlying HIV disease, are prone to be infected with Norwegian scabies, where the cutaneous lesions are classically distributed over the extremities.We report the case of an HIV-positive 16-year-old man with severe crusted Norwegian scabies initially misdiagnosed as a dermal fungal infection. The patient had extensive, generalized, thick, hyperkeratotic, crusting, yellowish papule lesions distributed on the entire body from his scalp to his toes.The patient was started with Ivermectin and topical Permethrin, which eventually resulted in complete resolution. Interestingly, despite quarantining efforts, one of the patient's acquaintances and a healthcare worker acquired the symptoms of itching.This atypical presentation of Norwegian scabies emphasizes the need to include scabies in the differential diagnosis when HIV-infected patients present with crusted, generalized cutaneous lesions. Norwegian (crusted) scabies is an opportunistic dermatological manifestation which is seen in HIV-infected individuals and which is probably acquired as a consequence of the immune system's inability to control the mites, thereby facilitating overwhelming reproduction . There iA 16-year-old man with HIV infection was admitted to the inpatient department of the YRG Centre for AIDS Research and Education (YRG CARE) with severe crusted cutaneous lesions all over the body. He had a history of skin lesions that had developed initially over the scalp and forehead, later spreading all over the body over the course of one month with no signs of itching. By the time of admission, the skin condition had worsened rapidly and there was extensive, generalized, thick, hyperkeratotic, crusting, yellowish papule lesions that eventually disseminated across the body including the face, ear lobes, shoulder blades and entire trunk, with squamous lesions not sparing any region  level of 11.4 g/dl , erythrocyte sedimentation rate (ESR) of 20 mm , total thrombocyte count of 144 × 109/l , total leukocyte count of 18.2 × 109/l  and a total lymphocyte count (TLC) of 3.7 × 109/l . His absolute CD4 T-lymphocyte count  was 342 cells/μl  and the CD4 T-lymphocyte percentage was 8%. His liver function test (LFT) revealed low alanine aminotransferase (ALT) at 28 IU/l , normal total bilirubin of 0.4 mg/dl , direct bilirubin of 0.1 mg/dl  and a normal conjugate bilirubin of 0.3 mg/dl . His renal function tests revealed a low urine creatinine 0.5 mg/dl  and blood urea 11 mg/dl .Acarus scabiei infestation, skin crusts were collected and mounted on 10% KOH preparation and observed under low- and high-power objectives. Numerous live and motile, adult A. scabiei mites that measured about 400 μm long and 300 μm wide were seen, which confirmed the diagnosis of Norwegian scabies. The patient was started with Ivermectin (6 mg) for 15 days and topical Permethrin cream with meticulous scrubbing and cleansing of the skin, which eventually resulted in complete resolution 4 weeks later. Intriguingly, in spite of quarantining efforts, one of the patient's acquaintances and a healthcare worker acquired the symptoms of itching, and had to be treated with topical Permethrin cream for a week. However, the diagnosis of scabies was not confirmed in either the acquaintance or the healthcare worker.The differential diagnosis was initially either an adverse drug reaction, atopic dermatitis, dermatitis herpetiformis, psoriasis, ichthyosis, seborrheic dermatitis, erythroderma or Langerhans cell histiocytosis. However, following the suspicions of dermatologists of possible scabies incognito is reported to alter the presentation of lesions, obscuring the clinical diagnosis [Norwegian scabies is reported to be extremely infectious. This case report is of a man with severe crusted scabies with lesions not sparing any region in the body, and which was initially misdiagnosed as a fungal skin infection. This is a very unusual presentation of Norwegian scabies in an HIV-positive patient. The laboratory diagnosis of Norwegian scabies is simple, but clinical suspicion is required on the part of attending healthcare workers. Infrequently, scabies is mistakenly reported initially to be an adverse drug reaction, psoriasis, systemic lupus erythematosus or bullous pemphigoid -7. A coniagnosis . Cliniciiagnosis ,9. In spThe author(s) declare that they have no competing interests.NK, SS, SMT and BD were involved in the case directly and drafted part of the manuscript. RV, RS, PB and EMS were involved in diagnosis, literature review and helped draft the manuscript. All the authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The aim of this double-blind randomized study is to test the efficacy of a radio electric stimulator device using an auricular reflex therapy protocol for stress-related symptoms.The study has been carried out on 200 subjects  that voluntarily came to our Institute declaring to "feel stressed".The participants were randomly allocated with a computerized procedure: 150 were treated with auricular therapeutic protocol with radio electric stimulator device (REAC) and 50 were treated with an inactivated, placebo REAC. Psychological stress was evaluated trough the self-administered questionnaire Psychological Stress Measure (PSM). Assessment data were collected at 2 time points: before the treatment (T0) and immediately after the therapy cycle of 18 sessions about 4 weeks later (T1).In the group treated with REAC, the psychometric evaluation after the therapy's cycle showed a significant reduction of PSM total scores, from 107.8 ± 23,13 at T0 to 87.1 ± 16,21 at T1 (p < 0.5), while in the control group no significant variation in decreasing stress-related symptomatology has been noted .The protocol of the auricular treatment with REAC seems to reduce the subjective perception of stress, as "psychometrically" demonstrated by the significant reduction in PSM test total score. This therapeutical procedure also provides a non invasive, not painful and very simple innovative approach to treat the widely diffused stress related disorders.This trial has been registered in the Australian New Zealand Clinical Trials Registry (ANZCTR) with the number: ACTRN12607000529448 Environmental stress produces important effects on the individual health. Chronic social stress is one of the most important factors -3 responThe Central Nervous System (CNS), as the main biological target of stressful events ,7, has cAllostasis represents the adaptation process of the complex physiological system to physical, psychosocial and environmental changes or social stress -15. The The purpose of this study is to verify if the use of REAC could reduce the subjective perception of stress and stress-related symptoms, evaluated by PSM at T0 and T1 -41. PSM The study was performed in accordance with the Declaration of Helsinki, with the Societa di Ottimizzazione Neuro Psico Fisica e CRM Terapia institutional ethics committee approval, and all subjects provided written informed consent. A double-blind, randomized and placebo-controlled trial was performed to investigate the effectiveness of REAC auricular treatment in reducing of self perception of stress and stress-related symptoms and, consequently, the PSM total score.The study has been carried out on 200 subjects , that voluntarily came to our Institute suffering from a broad stress-related range of psychological symptoms such as affective-emotional reactions i.e. sadness, irritability, anger, depression, poor concentration and attention, insecurity, anxious and apprehensive condition, depression, sleep disorders, generalized muscle tension, gastro intestinal disorders, headaches. Participants were properly informed about significance and objectives of the study and then they completed, in association with the Project Director prior to enrolment, a written consent form. For all participants were properly collected and detailed: general demographic information, background characteristics, treatments history, drug history survey and medical examination.The participants, selected after a clinical screening, have been allocated randomly into 2 socially and demographically matched groups with a computerized procedure: Group A, treated with "active" REAC , and Group B, treated with deactivated-placebo REAC . The 3:1 proportion between cases and controls has been selected only on the base of the main "typology" of the people who require a clinical treatment in our Institute: "stressed" subjects are more numerous than "non-stressed" subjects.REAC protocol consists in 18 session each one lasting 3,5 seconds, administered in about 4 weeks.All subjects were over 18 years old, declaring to suffer from stress-correlated psycho-physical conditions, as demonstrated by a clinical evaluation and the PSM total score >45. No history of drug abuse, nor severe mental illness or personality disorder according to DSM-IVTR criteria  and no psychopharmacological-psychological interventions has been reported.Exclusion criteria for both groups: diagnosis of axis I-II psychiatric disorders, organic CNS pathology, preceding skull traumas with loss of conscience longer than 5 minutes, dementia, alcohol dependence, current therapy with medications known to affect cognitive functioning.The study has been setted in 4 rooms. In 3 of the 4 rooms has been activate REAC and in the other 1 the placebo-REAC. The physicians that have participated in the treatment's administration turned in the different rooms, using both "active" and "inactive" REAC but they didn't know if they used an active or placebo REAC. The computerized randomization process, managed by an external operator to the study, foresaw the assignment of the subjects to each room. The target sample size 200, divided in the 4 rooms, has produced 4 subgroups, each composed by 50 people .PSM, a standardized and validated self-reporting test for the measurement of psychological stress was administered to all subjects, before (T0) and after REAC treatment (T1) -41. PSM The REAC is an innovative medical device  aimed toThe auricular treatment protocol has been conceived by the authors and called Neuro Psycho Physical Optimization (NPPO). The REAC probe was applied to 7 specific points of the auricular pinna: "shen men", kidney, stomach, heart, occiput, ipotalamus, prefrontal cortex. Eighteen sessions of NPPO with REAC were administered on alternate days, in about four weeks, to each patient. Each therapeutic session lasted approximately 3 seconds. The protocol is painless, non-invasive, and without known side effects.Statistical analysis were performed using Statistical Package for Social Science (version 13). The McNemar test is used to compare the relevant frequencies for data resulting from PSM test in patients belonging to group A and to group B before and after NPPO with REAC. Also Wilcoxon Signed Ranks Test and Sign test are used to evaluate the related samples of Total points resulting from PSM test of both groups. All results of p < 0.05 were considered statistically significant.In both groups the total stress evaluation has been measured by PSM administered before (T0) and after a cycle of NPPO. In the group A, PSM total score progressively decreases from 107.8 ± 23.1 at T0 to 87.1 ± 16.2 at T1 . On the contrary, no significant difference has been noted in the group B: in fact, PSM total score ranged from 107.9 ± 25.8 at T0 to 106.3 ± 25.9 at T1 .In the Group A, the greater improvement between T0 and T1 has been reported on PSM items loss of control, irritability, psycho-physiological sensations, confusion, anxiety, depression, physical pain, hyperactivity and agitation table .The use of electricity and magnetic fields in the biomedical studies, and in particular in the treatment of disturbances of the nervous system, is not a new idea -47. NeveIn the present study we considered a group of patients treated with REAC and a group with placebo-REAC. The results suggest that a cycle of NPPO protocol with REAC reduces the total score of the PSM test Figure , 4 in paA growing number of studies suggests that the stress-related symptoms are the result of allostatic processes -27,29-31Besides, this treatment has the advantage of being painless, non-invasive and free of side-effects not only in the patient groups, but also in the physicians that have applied this therapy.The 3:1 proportion between case and controls certainly represent a bias of this study, mainly in terms of statistics. However, this has been simply due to the fact that our Institute is well-known with regard the treatment of psycho-physical stress-related dysfunctions and, paradoxically, it is been easier to enlist "stressed" than normal subjects. According to us, the physician's rotation in the different rooms and the fact that each one of them didn't known if were administering the "active" or "placebo" REAC, represent a attempt to achieve more "scientific" strictness.We propose that NPPO treatment with REAC will help to improve the physiological capability of the organism's recovery, optimizing the adaptive response to environmental stressors.Moreover, NPPO treatment with REAC is a non-pharmacological treatment and can represent an efficient support in many medical fields, because it does not interfere with the simultaneous use of other therapeutic approaches.Obviously, though these observations on the effectiveness of NPPO - REAC treatment in the psychometric evaluation of stress related disorders are certainly important and suggestive, they are only preliminary and require further confirmation.SR and VF are the inventors of the Radio electric Asymmetric Conveyer.SR and VF conceived of the study, participated in its design and coordination and in drafting of the manuscript. LA has critically revised the manuscript and PM has done the psychiatric evaluation. All authors read and approved the final manuscript.


MRI reveals restricted diffusion on diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) in the region of cytotoxic edema, while no such restriction is observed in vasogenic or perilesional edema. We present a case where the patient manifested with status epilepticus (SE) due to a granuloma, possibly neurocysticercosis, with reversible restricted diffusion and cytotoxic edema on DWI and ADC in the perilesional zone of altered signal intensity.th May 2006 within 24 h of cessation of SE and showed a right frontal ring-enhancing lesion (8 mm in diameter), with perilesional hyperintensity on T2W, FLAIR, and PDW sequences [−3 mm2 per second on the side of the lesion as compared to 83.6 × 10−3 mm2 per second in the corresponding region on the healthy side, suggesting a percentage decrease of 71.8% in the ADC coefficient. The area measured contained white matter. A repeat DWI study after 96 h revealed no restriction and the ADC value in the perilesional zone was 71 × 10−3 mm2 per second on the side of the lesion as compared to 76 × 10−3 mm2 per second in the corresponding region on the healthy side. However, there was no further serial MRI study. EEG immediately after control of seizures showed diffuse theta slowing of background activity with intermittent, frontally dominant, delta waves, which was more from the right side.A 10-year-old boy presented with recurrent left partial motor seizures (20-24/day) for 6 days with secondary generalization and unconsciousness. He had only received 50 mg of oral phenytoin twice a day prior to admission. There was no other significant history. The vital signs were stable and he had crackles in the chest. The Glasgow coma score was 6/15 . There was no lateralizing sign. The seizures were brought under control with intravenous lorazepam, phenytoin, and valproic acid in recommended dosages 45 min after admission. Hemogram and serum biochemical parameters, including glucose, liver and renal function tests, and electrolytes, were normal. MRI was performed on 15equences , findingequences , the zonet al.[Conventionally, the edema in the vicinity of brain tumors, cerebral abscesses, and granulomas is vasogenic, because there is disruption of the blood-brain barrier. It is characterized by variable signal intensity on DWI and high ADC values. Symptomatic SE secondary to granulomas is characterized by surrounding vasogenic edema and this is often noted following a seizure. By contrast, cytotoxic edema is characterized by high signal intensity on DWI with low ADC values . In this patient, MRI findings were suggestive of neurocysticercosis. The DWI revealed hyperintense signal change in the perilesional zone, which was hypointense on ADC, suggesting restricted diffusion (cytotoxic edema). A repeat study with DWI and ADC 4 days later revealed resolution of the cytotoxic edema. To the best of our knowledge, this is a novel observation. Evidence of cytotoxic edema secondary to epileptic activity itself has been reported by several authors,2 along wet al.[+-K+-ATPase pump in the acute stage of cerebral ischemia and subsequently leads to cytotoxic edema, thereby creating an environment that hinders water diffusion. In ischemic brain tissue, DWI intensity is high and the ADC, a measure of freedom of water diffusion, is low.[Lansberg et al. reported, is low.5+-K+-ATPase pump due to depletion of metabolic substrates, may lead to a disturbance of membrane ion homeostasis, with influx of Na+ into the cell,[In experimental animals with kainate-induced SE, decreased ADC values similar to that seen in acute cerebral ischemia have been documented. These chthe cell, which hathe cell, The incrthe cell, In the pthe cell, and revethe cell, have beeThe finding of restricted diffusion surrounding a lesion is an interesting observation and suggests cytotoxic edema. Resolution of cytotoxicity is of great interest because it offers scope for research in epilepsy and ischemia.


These intriguing data of a deuterostome-specific gene that might be implicated in FGF signalling prompted us to search for putative FGFRL1 orthologues in the completely sequenced genomes of metazoans.Fibroblast Growth Factors (FGF) and their receptors are well known for having major implications in cell signalling controlling embryonic development. Recently, a gene coding for a protein closely related to FGFRs (Fibroblast Growth Factor Receptors) called FGFR5 or FGFR-like 1 (FGFRL1), has been described in vertebrates. An orthologous gene was also found in the cephalochordate amphioxus, but no orthologous genes were found by the authors in other non-vertebrate species, even if a FGFRL1 gene was identified in the sea urchin genome, as well as a closely related gene, named Nematostella vectensis as well as in many bilaterian species. Our analysis also shows that FGFRL1 orthologous genes are linked in the genome with other members of the FGF signalling pathway from cnidarians to bilaterians (distance < 10 Mb). To better understand the implication of FGFRL1 genes in chordate embryonic development, we have analyzed expression patterns of the amphioxus and the mouse genes by whole mount in situ hybridization. We show that some homologous expression territories can be defined, and we propose that FGFRL1 and FGF8/17/18 were already co-expressed in the pharyngeal endoderm in the ancestor of chordates.We found FGFRL1 genes in the cnidarian Our work sheds light on the existence of a putative FGF signalling pathway actor present in the ancestor of probably all metazoans, the function of which has received little attention until now. Nematostella vectensis [Interaction between many different signalling pathways is necessary to form a metazoan starting from a single egg cell. Fibroblast Growth Factor (FGF) signalling represents one such developmental pathway. FGFs are small proteins that act by binding to their transmembrane receptors, FGFRs. The latter are characterised by three Immunoglobulin-like (Ig-like) extracellular domains, which are implicated in ligand and heparan sulphate binding, as well as an intracellular tyrosine kinase domain responsible for signal transduction. In mammals, the analysis of several completely sequenced genomes shows the presence of 22 FGF and 4 FGFR genes . InteresBesides the 4 classical FGFRs known in mammals, a fifth evolutionarily related protein, called FGFR5 or FGFR-like 1 (FGFRL1), has recently been described in vertebrates -8. FGFRLin vivo function of FGFRL1 during development [Danio rerio, two copies of FGFRL1 have been identified, fgfrl1a and fgfrl1b, which probably arose after the genome duplication that took place early in the evolution of actinopterygian fish lineage [fgfrl1a and fgfrl1b mRNAs are detected in other organs such as pectoral fin buds and lens, as well as in the midbrain-hindbrain boundary. However, fgfrl1a-morpholino injected embryos have a phenotype resembling that of FGF3 mutants that is not consistent with a putative dominant negative activity of FGFRL1 [Gene inactivation experiments performed in mouse and zebrafish have shed light on the possible elopment ,10-12. I lineage . Injectif FGFRL1 . Indeed,Branchiostoma floridae [nou-darake gene in the planarian Dugesia japonica, which clearly shows a structure related to FGFRL1 [Caenorhabditis elegans, the sea squirt Ciona intestinalis, and the crustacean Daphnia pulex. We also identified a clear FGFRL1 orthologue in the genome of Nematostella vectensis. To further analyse the role of this gene, which has been conserved from diploblastic animals to mammals, and to better understand its ancestral function in chordates, we characterised mRNA expression during amphioxus embryonic development, as well as in early embryonic stages in mouse. Our data highlight two embryonic regions that might correspond to FGFRL1 expression territories in the ancestor of chordates, namely the pharyngeal endoderm and the anterior neural tissues. Finally, our work sheds light on a possible new actor of the FGF signalling pathway that has been poorly studied until now, particularly outside vertebrates.An orthologue of FGFRL1 was isolated in the cephalochordate amphioxus floridae , but seao FGFRL1 . It was o FGFRL1 . In orde to analyse the domain organization of the nou-darake protein from the planarian Dugesia japonica and found that contrary to previous descriptions, the protein possesses not two but three Ig loops [nou-darake with the non-redundant CDS database from Genbank yielded vertebrate and amphioxus FGFRL1 sequences as best reciprocal hits. We also performed a phylogenetic reconstruction for FGFR and FGFRL1 families. The topology of the tree we obtained clearly shows that nou-darake is more closely related to the FGFRL1 family than to FGFRs  and tunicates . Some of the sequences we obtained are complete CDS, whereas others are incomplete, probably as a result of prediction/assembly mistakes or incomplete sequencing. We also looked at the genome sequences of vertebrate species for which FGFRL1 was described to ensure there were no additional paralogues. As previously described, we only found one gene in human, mouse, chicken and Xenopus, and two genes in zebrafish as well as in Tetraodon and Takifugu . Interestingly, in Nematostella vectensis, as in human, the gene is encoded by 6 exons. The first exon includes the 5'UTR as well as the signal peptide sequence. Each Ig loop domain is coded by a unique exon, namely exons 2, 4 and 5 for IgI, II and III respectively. Exon 3 codes for a box between IgI and IgII, while the sixth exon codes for the transmembrane as well as the cytoplasmic and 3'UTR sequences. In Nematostella, similarly to human, the exon/intron limit ends at phase 1 for exons 1 to 5. Thus, exon/intron structure of FGFRL1 genes is highly conserved between human and Nematostella. In the other vertebrate species analysed, the gene organisation is conserved overall, with few differences. Indeed, for Xenopus, chick, and the fgfrl1a zebrafish duplicate, there is an additional exon coding for part of the 5'UTR. Furthermore, in Xenopus, the box between IgI and IgII is encoded not by one, but by two exons. As in human and Nematostella, all the exon/intron limits end at phase 1, except for Xenopus exon 3. This conservation of genomic structure between sea anemone and vertebrate genes further confirms their putative orthology.We have analyzed exon/intron structure for the species for which genomic data are available, the results of which are shown in Figure Ciona species, the exon/intron data available are incomplete; however, we observed that the gene organization is clearly not conserved with that of the other deuterostomes. Indeed, the three Ig loop coding domains are separated into two exons.In the deuterostome group, although not all gene predictions are complete, the exon/intron structure is also quite similar to that of vertebrates, except in the urochordates. Indeed, in the sea urchin the only obvious difference is that the box between IgI and IgII is coded by four exons instead of one. In amphioxus, it is the IgII domain that is cut into two exons compared to one in vertebrates and sea urchin . For theLottia gigantea and an annelid Capitella species I), the gene structure is close to that observed for both vertebrates and the sea anemone. In effect, only what corresponds to the first exon is separated into two sections, as well as the IgIII domain coding part, which is split into two exons.In lophotrochozoans , which are orthologous to the vertebrate FGF8/17/18 group. In Caenorhabditis elegans and Ciona intestinalis , FGFRL1 orthologue genes are also placed in close proximity to the FGF8/17/18 orthologues (named egl-17 for the nematode). In contrast, the FGFRL1 orthologue is close to the FGFR orthologues (htl and btl) in Drosophila, to the FGFR1 gene in the sea urchin genome, and to the FGFR3 gene in human. However, it has been proposed by others, using evidence from the conserved syntenic position of several genes, that FGFR and FGF8/17/18 orthologues were linked in the ancestor of bilaterian animals [Nematostella we did not find any orthologous genes of the bilaterian FGFR/FGFRL1/FGF8/17/18 syntenic block in the same scaffold as FGFRL1 with the exception of FGF8a and FGF8b. Nevertheless, the fact that these three genes are positioned close together in this species may suggest a functional link between them. It is noteworthy that the position of the Drosophila FGFRL1 putative gene confirms its orthology with the other species' FGFRL1, in spite of a poorly supported position in our phylogenetic tree reconstructions.We have analyzed, when data were available, the genomic position of the FGFRL1 genes and the presence of known FGF signalling pathway genes in their vicinity. Figure  animals . Given tin situ hybridization in two species: amphioxus, a cephalochordate placed at the base of the chordate clade; and mouse, a vertebrate for which only late embryonic expression has been described [To better understand the putative implication of FGFRL1 during embryonic development in chordates, we chose to analyze its expression pattern by whole mount escribed .In amphioxus, expression starts very early during embryogenesis since we can detect FGFRL1 mRNA from the 4 cell stage. Before gastrulation, expression is ubiquitous. In late gastrula a higher expression level is visible in the anterior dorsal part of the embryo Figure . In earlin situ hybridization at embryonic days E7.5, E8.5, E9.5, E10.5 and E11.5. At the earliest stage, expression is restricted to the most anterior and posterior embryonic regions with a gradient from the axial to the lateral parts , but it is still unpublished. The expression pattern is very restricted throughout embryogenesis, which might indicate a specific function that still needs to be studied. With respect to Caenorhabditis elegans, we looked at the data available in WormBase [Dugesia japonica strongly suggest interaction between FGFRL1 and FGF signalling during regeneration. Our work reveals the existence of a widely conserved gene, FGFRL1, whose characteristics may shed light on the most ancestral FGF functions. Detailed developmental studies are needed to decipher its full evolutionary history and the diversification of FGF signalling pathways in metazoans.Data on the embryonic role of FGFRL1 orthologues outside the chordate group remain sparse. In the FlyBase database , we haveWormBase  for the WormBase . In plannou-darake sequence [We searched for FGFRL1 orthologous sequences using Blast with the mouse FGFRL1 peptide sequence and the planarian sequence . We firsPredicted amino acid sequences were aligned automatically using ClustalW  with manB. lanceolatum and mouse FGFRL1 were cloned by RT-PCR on adult RNA in pGEM-T Easy vector (Promega). An insert was then subcloned in pBluescript II SK (Stratagene) for subsequent RNA probe synthesis. Accession number for the B. lanceolatum sequence is FJ694966.Partial cDNAs from B. lanceolatum) were collected in Argelès-sur-Mer (France), and gametes were obtained by heat stimulation [in situ hybridization were performed as described in [in situ hybridization was performed according to [Ripe animals of the Mediterranean amphioxus . All the FGFRL1 sequences described in the manuscript were included. VEGFR sequences from vertebrates were used as the outgroup. FGFR sequences are indicated in blue and FGFRL1 sequences in red. Sequences not included in Figure AEDAE: Aedes aegypti, ANOGA: Anopheles gambiae, BRABE: Branchiostoma belcheri, BRAFL: Branchiostoma floridae, BRUMA: Brugia malayi, CAEBR: Caenorhabditis briggsae, CAEEL: Caenorhabditis elegans, CAPSP: Capitella sp.I, CIOIN: Ciona intestinalis, CIOSA: Ciona savignyi, DANRE: Danio rerio, DAPPU: Daphnia pulex, DROME: Drosophila melanogaster, DROPS: Drosophila pseudoobscura, DUGJA: Dugesia japonica, GALGA: Gallus gallus, HOMSA: Homo sapiens, LOTGI: Lottia gigantea, MUSMU: Mus musculus, NEMVE: Nematostella vectensis, RATNO: Rattus norvegicus, STRPU: Strongylocentrotus purpuratus, TAKRU: Takifugu rubripes, TETNI: Tetraodon nigroviridis, TRICA: Tribolium castaneum, XENLA: Xenopus laevis, XENTR: Xenopus tropicalis.Click here for filePhylogenetic analysis of the putative FGFRL1 orthologues. Phylogenetic tree estimated under the JTT+I+G model . All the FGFRL1 sequences described in the manuscript were included, with the exception of FGFR orthologue sequences from protostomes. VEGFR sequences from vertebrates were used as the outgroup. FGFR sequences are indicated in blue and FGFRL1 sequences in red. Sequences not included in the Figure AEDAE: Aedes aegypti, ANOGA: Anopheles gambiae, BRABE: Branchiostoma belcheri, BRAFL: Branchiostoma floridae, BRUMA: Brugia malayi, CAEBR: Caenorhabditis briggsae, CAEEL: Caenorhabditis elegans, CAPSP: Capitella sp.I, CIOIN: Ciona intestinalis, CIOSA: Ciona savignyi, DANRE: Danio rerio, DAPPU: Daphnia pulex, DROME: Drosophila melanogaster, DROPS: Drosophila pseudoobscura, DUGJA: Dugesia japonica, GALGA: Gallus gallus, HOMSA: Homo sapiens, LOTGI: Lottia gigantea, MUSMU: Mus musculus, NEMVE: Nematostella vectensis, RATNO: Rattus norvegicus, STRPU: Strongylocentrotus purpuratus, TAKRU: Takifugu rubripes, TETNI: Tetraodon nigroviridis, TRICA: Tribolium castaneum, XENLA: Xenopus laevis, XENTR: Xenopus tropicalis.Click here for file