There is a need for more objective outcome measures for chronic cough. In this pilot study we sought to investigate the utility of the mannitol challenge as a cough-provocation test in non-asthmatic chronic cough. We studied 16 healthy controls and 13 subjects with chronic cough. We assessed cough severity using a visual analogue score, capsaicin cough sensitivity, health status using the Leicester Cough Questionnaire and the dose of mannitol to cause 2 (C2) or 5 (C5) coughs. In all of the subjects with chronic cough and 6 of the controls we assessed the 1-week repeatability of the mannitol challenge. We found that in those subjects with chronic cough the geometric mean (logSEM) mannitol C2 and C5 was heightened compared to controls (C2: 4 (0.2) versus 16 (0.1); p = 0.04 and C5: 63 (0.1) versus 251 (0.1); p = 0.04). Cough visual analogue score, capsacin-induced cough sensitivity and health status were also altered in chronic cough compared to healthy controls, but in those subjects with chronic cough none of these outcomes was correlated with the mannitol C2 or C5. The repeatability of the mannitol challenge assessed by intraclass correlation was C2 = 0.53 and C5 = 0.59. A cut-off in the dose of mannitol of 62 mg/ml for C2 and 550 mg/ml for C5 had a sensitivity of 69 and 62% and specificity of 69 and 81% respectively to distinguish chronic coughers from healthy controls. In conclusion, the mannitol challenge my have potential as a novel cough challenge test and further work is required to extend our findings and to assess whether it has utility in different causes of chronic cough. Chronic cough is the most common presenting symptom in primary care; is a significant cause of morbidity and a considerable health economic burden . The nee1 was measured between each dose of mannitol to measure a drop in lung function. An empty capsule was administered prior to the 5 mg dose, as a placebo. The numbers of coughs within the first 30 seconds following mannitol administration were counted. The challenge was terminated if the subject's FEV1 dropped by 15% (PD15) or more or when the highest dose of mannitol had been attained.Subjects were recruited from hospital staff or from the respiratory clinics at Glenfield Hospital, Leicester, UK. Chronic cough was defined as a cough > 8 weeks as per American College of Chest Physician Guideline (ACCP) . HealthyAll statistical tests were performed using Prism version 4. The concentration of capsaicin or mannitol that caused two coughs (C2) and five coughs (C5) were calculated by the log-dose-response curves. Comparisons between groups were made using t-tests or Mann-Whitney-U test for parametric and non-parametric data respectively. Repeatability was assessed by intra-class correlation and presented as Bland-Altman plots. Receiver-operator curves were generated for mannitol C2 and C5. Correlations were made between the mannitol challenge and other cough outcome measures using spearman rank correlations. A p-value of < 0.05 was considered to be statistically significant.Clinical characteristics for subjects are as shown in table In this pilot study, we report for the first time that mannitol-provoked cough is a repeatable test and is increased in non-asthmatic chronic cough. The advantages of the mannitol challenge compared to more established cough challenge tests is that administration is simple, whereas other tests often require preparation of solutions needing laboratory support, and the mannitol challenge provides additional information on AHR. Our findings support our hypothesis that mannitol-provoked cough is increased in non-asthmatic chronic cough as has been reported in asthma . HoweverThe cause of mannitol-provoked cough is unknown. It is likely that it mediates its effect indirectly via activation of mast cells in the superficial airway to release mediators, which in turn activate local cough receptors. An earlier study in asthma demonstrated that the degree of bronchoconstriction in response to mannitol and the cough response are independent . This suThere a number of potential shortcomings of our study. This was a pilot study of a small number of subjects with non-asthmatic chronic cough. We are therefore unable to determine whether mannitol-provoked cough has a strong association with different causes of cough. Its utility as a cough outcome may therefore differ dependent upon the aetiology of the cough. We have only assessed the short-term repeatability. To fully address the clinical utility of mannitol in the assessment of cough future work will need to determine repeatability over a longer period and investigate responsiveness to therapy in more subjects.In conclusion, the mannitol challenge may have potential as a novel cough challenge test. However, its repeatability and ability to discriminate between subjects with non-asthmatic chronic cough in this pilot study was not yet sufficient to recommend its routine use. Our findings need to be replicated in a larger series of chronic coughers and in particular it may be informative to examine response in chronic coughers with different aetiologies.The authors declare that they have no competing interests.AS and SM undertook the clinical assessments. AS and CB prepared the manuscript. All authors have read and approved the manuscript. CB supervised the study.


We read the recent article by  with keen interest and would like to congratulate them for a job well done. However, we would like to raise a few points relating to the limitations of the study. Antimalarial use in pregnancy is an important determinant of the level of malaria parasitaemia at delivery but Mokuolu et al. , like thThe authors claimed that they took specific note of those patients who used preventive antimalarial drugs, yet they did not provide the proportion of such patients. These patients are likely to procure the drug from the community pharmacies and are at risk of using counterfeit and adulterated antimalarials which are very abundant in Nigeria . In the The inclusion criteria used in the study appear not to be clearly defined by the authors. We are not sure if pregnant women with HIV infection and unbooked patients were part of the cohorts they studied. Perhaps, it would have been proper to exclude these groups of patients because of the possible bias their inclusion may introduce into the study and this may be better avoided by recruiting patients from antenatal clinics and followed to the time of delivery rather than recruiting at the point of delivery. HIV and malaria coinfection has more adverse effects on pregnancy than malaria alone . The preMokuolu et al. found a significant association between malaria parasitaemia at delivery and maternal age less than twenty years. It may be true that pregnancy associated with acquired immunity is lower in younger women than older ones who have obtained adequate immunity from repeated exposures to malaria infections. Teenage pregnancies are likely to be associated with poverty, lack of good education, and poor nutrition. The necessary proteins needed in the synthesis of antibodies against malaria parasites may therefore be reduced in young pregnant women and may indirectly contribute to the high prevalence of malaria parasitaemia observed by the authors in young parturients.Given that the authors are from various disciplines, a future study that will involve a multidisciplinary approach is therefore suggested. The ability of the contributing authors, who are clinical pharmacologists, to assay blood levels of antimalarial drugs in pregnancy will add novelty to the future study.


Acropora palmata, a foundation-species of reefs in the Caribbean and western Atlantic, will advance our understanding of the genetic basis of ecologically important traits in corals and comes at a time when sequencing efforts in other cnidarians allow for multi-species comparisons.Cnidarians, including corals and anemones, offer unique insights into metazoan evolution because they harbor genetic similarities with vertebrates beyond that found in model invertebrates and retain genes known only from non-metazoans. Cataloging genes expressed in A. palmata transcripts were tagged, and provided a rough estimate of the total number genes expressed in our samples . The coral annotation data contained many of the same molecular components as in the Bilateria, particularly in pathways associated with oxidative stress and DNA damage repair, and provided evidence that homologs of p53, a key player in DNA repair pathways, has experienced selection along the branch separating Cnidaria and Bilateria. Transcriptome wide screens of paralog groups and transition/transversion ratios highlighted genes including: green fluorescent proteins, carbonic anhydrase, and oxidative stress proteins; and functional groups involved in protein and nucleic acid metabolism, and the formation of structural molecules. These results provide a starting point for study of adaptive evolution in corals.A cDNA library from a sample enriched for symbiont free larval tissue was sequenced on the 454 GS-FLX platform. Over 960,000 reads were obtained and assembled into 42,630 contigs. Annotation data was acquired for 57% of the assembled sequences. Analysis of the assembled sequences indicated that 83–100% of all Currently available transcriptome data now make comparative studies of the mechanisms underlying coral's evolutionary success possible. Here we identified candidate genes that enable corals to maintain genomic integrity despite considerable exposure to genotoxic stress over long life spans, and showed conservation of important physiological pathways between corals and bilaterians. Hydra magnipapillata and Nematostella vectensis) have revealed that basal metazoans retain genes known only from non-metazoans Cnidarians are valuable for understanding the evolution of metazoan genomes because they comprise a sister group to the Bilateria. From a developmental perspective, comparisons among these groups have yielded much information regarding the evolution of body plans in complex multicellular animals A. palmata are short by comparison  that are responsible for the formation of reefs worldwide de novo assembly of whole transcriptomes from non-model organisms Progress in sequencing technology, chemistry, and bioinformatics now enable the sequencing and Acropora palmata) remains an ecologically important community member A. palmata is one of only a few Caribbean species for which population genetic data is available A. palmata's range Despite a population reduction of >80% throughout its range in recent decades Acropora millepora, Acropora hyacinthus, Montastraea faveolata, Pocillopora damicornis, and Porites astreoidesNematostella vectensis and Hydra magnipapillata were recently completed Transcriptome and EST data are now available from several coral species including A. palmata we present here its transcriptome sequenced with 454 GS-FLX technology, and survey the assembled and annotated sequences for genetic markers, and functional enrichment of important biological processes (with a particular focus on stress response and DNA repair pathways). The genetic resources presented here are a major advance for studies of this species and will promote the study of adaptive trait variation in wild populations of A. palmata as well as comparative genomics among basal metazoans.To develop genome scale tools for 2 treatments, and sampled over the course of development from fertilization to the planula stage. A small amount of adult tissue was also included to represent adult transcripts. We thus expected to find developmental and stress related genes derived from the larval temperature and CO2 treatments, as well as genes related to symbiosis and calcification from the adult tissue.RNA was acquired from genetically diverse larvae collected over a broad geographic range previously identified as comprising two divergent populations A. palmata sample following the library preparation methods of Meyer et al.One plate of a normalized cDNA library generated from a pooled A. palmata ESTs . Assembly yielded 42,630 contigs averaging 1,030 bp long (σ = 623). Contig sizes ranged from 132 to 9,066 bp. The mean depth of coverage was 5.6 sequences  to all 119 genes. Comparison of the distributions of coverage depth between all A. palmata transcripts and those with hits to the 119 single copy orthologs showed that the mean depth of coverage for contigs with hits to these 119 genes (9.7) is greater than that for contigs in general (5.6). This analysis indicated that depth of coverage was high even for single copy genes and that the set of 119 orthologs can serve as a representative sample of the A. palmata transcriptome.Estimation of transcriptome completeness based on two different methods suggest that our single sequencing run tagged between 83–100% of the genes expressed in N. vectensis transcriptome we found hits to 83% of all N. vectensis transcripts. The lengths of the A. palmata sequences were generally greater than those of N. vectensis, suggesting that full length transcripts were obtained for the majority of the A. palmata genes  reflected the longer average lengths of the A. palmata transcripts, and taken together with the high proportion of hits to the N. vectensis transcriptome data indicated very high coverage of the A. palmata transcriptome.Secondly, by comparing our assembled data to the published ta genes . The disAll contigs and singletons  were used to search against the UniProt protein database. Searches of both Swiss-Prot and TrEMBL resulted in hits for 50,118 (57%) of the queried sequences, of which 32,114 (36%) represented unique subject names. 31,888 of these hits corresponded to Gene Ontology (GO) A. palmata dataset (derived from UniProt). A clear match was found for 88% of the 119 annotation descriptions. An additional 9% of matching A. palmata transcripts were described with uninformative species specific codes from UniProt, but functional matches were easily found by looking up the codes in the NCBI UniGene database. Only three of the 119 genes did not match with the A. palmata annotation data, and in all three cases the A. palmata annotation was derived from blast hits to the Florida lancelet or the green puffer, species with limited annotation data. Overall this suggests high (97%) annotation efficiency.To estimate annotation efficiency, the description terms associated with each of the 119 single copy genes were compared to those in the blast results from the 119 orthologs were used to examine the average number of hits to each putatively singe copy gene. Results of this test showed that 63% of the 119 single copy genes had multiple sequence matches , the number of assembled sequences matching to known single copy genes can give an estimate of the level of residual gene fragmentation (either from sequencing gaps or splice variants), and/or assembly difficulty. A. palmata sample. The mean value of 2.2 contigs per transcript was used as an estimate of the redundancy in our dataset. This yielded an estimate of ∼19,377 genes  expressed in our A. palmata sample. Finally, based on the blast results with N. vectensis we found that 43,036 of the A. palmtata sequences matched to a non-redundant set of 17,988 proteins from N. vectensis. Thus we predict that the number of transcripts expressed in A. palmata is in the range of ∼18,000–20,000 genes.The degree of fragmentation in our assembly was also used to generate a rough estimate of the total number of transcripts expressed in the The majority of taxonomic associations of the annotated transcripts matched to metazoan invertebrates and vertebrates (44% and 48% respectively). A small proportion of sequences matched to bacteria (3%), protists (2%), plants and algae (2%). All other groups including fungi, archaea and viruses accounted for 1% or less of matches. Stony coral (Scleractinian) sequences accounted for only 1% of the annotated sequences primarily due to a paucity of annotated coral sequences present in current protein databases.blast annotation associates a given sequence with its closest match in the database, results are limited to previously identified genes. Thus, the vast majority of annotated transcripts in this dataset showed matches to the Starlet Sea Anemone, Nematostella vectensis , the most closely related organism with a sequenced genome blast hits reflected the sequencing efforts on the Florida Lancet, Branchiostoma floridaeblast hits in our annotation database when the best sequence match lacked any functionally relevant data. Viewed this way the number of A. palmata sequences with best matches to N. vectensis was reduced from 36% to 12%. Thus, despite slightly higher sequence similarity of hits to N. vectensis, the search for useful functional annotations was greatly improved by exploring more distantly related hits.Because A. palmata transcripts, we placed A. palmata homologs in the context of known biological pathways using ipa software . Information in the ipa database reflects the level of interest certain pathways have received. Thus, comparison of A. palmata transcripts to the IPA database identified well annotated metazoan pathways for which a high proportion of pathway components were present in A. palmata. These pathways were then used as guides for further analysis. Note that this approach did not make inferences about expression levels of transcripts. Among the most highly represented pathways were many that are expected to be important to heat stressed embryos and who are thus of interest to the current study including protein ubiquitination, cell cycle control, NRF2 mediated oxidative stress response, p53 signaling and DNA double stranded break repair , were chosen to test for positive selection with the program pamlNext, we placed genes involved in the response to DNA damage and oxidative (and irradiant) stress in a phylogenetic context to test for signatures of natural selection. 498 bp of the conserved DNA binding domain from homologs of two A. palmata transcriptome. These included 68 transmembrane ion transporters, including 10 sodium driven bicarbonate exchangers which may play an important role in supplying bicarbonate ions to the reaction site of calcification. Another enzyme, carbonic anhydrase found in the coral calicodermis  Stylophora pistillataCalcification is crucial for coral growth and survival, and numerous genes involved in coral calcification were found in the N. vectensis and A. palmata, 7,754 ortholog pairs were detected, with 9,604 in-paralogs in N. vectensis and 9,365 in-paralogs in A. palmata. The size distribution of paralog groups showed that the number of groups detected declines with increasing group size, and groups of 5 or more were rare  were found, with a large majority consisting of trinucleotide repeats n = 170; . Within A total of 72,605 candidate SNPs were identified from 13,803 contigs spanning 19.8 Mb of sequence. The overall SNP frequency was 1 per 272 bp, with 71% transitions (Ts) and 29% transversions (Tv). Frequencies of different transitions types were similar, as were frequencies of different transversion types . The traTo date, 18 SNPs from 6 contigs have been targeted for amplification using PCR, and the products were sequenced on an ABI3700 fragment analyzer. Of these 18 SNPs, 8 were observed in the sample population (success rate 44%), which consisted of 11 individuals from Florida, 8 from Curacao, and 7 from Puerto Rico. The SNPs detected were in genes for NFkB, melanopsin, Cyano Fluorescent Protein, NRF2, galaxin, and LITAF . OngoingAcropora palmata transcriptome presented here joins transcriptomes and EST data available for several other scleractinian corals, including that of the Pacific congener A. millepora (Matz and Traylor-Knowles pers. comm.) A. palmata  was comparable to that of N. vectensis , and the estimated number of genes expressed in this species  was within the range estimated for A. millepora  and N. vectensis .Coral species are the foundation of reefs because of their association with intra-cellular photosymbionts and ability to calcify. Comparative functional genomic analyses are now possible to elucidate the mechanisms underlying coral's evolutionary success. The A. palmata possesses many of the molecular components present in the more complex Bilateria (particularly in pathways associated with oxidative stress and DNA damage repair) and homologs of p53, a key player in several DNA repair pathways, showed evidence of natural selection along the lineage separating Cnidaria and Bilateria.The high quality and comprehensiveness of the assembled data enabled functional analysis of pathways shared among metazoans as well as those of particular importance to the Cnidaria. A. palmata. Indeed, approximately half of the sequences with blast hits in the ‘protists’ category (555) were associated with the superphylum which includes symbiotic zooxanthellae.Xenobiotic sequences from intracellular parasites are commonly found in transcriptome data of wild species blast with reasonable cutoff parameters.Hits to sequences from scleractinians were rare because of the small number of coral genes in current databases . As such, the genes identified are likely to be highly conserved among the scleractinians, with remaining unannotated transcripts  representing a combination of coral specific genes A. palmata transcriptome  A. palmata.p53 proteins play a central role in the response to DNA damage in vertebrates by initiating pathways leading to apoptosis, or cell cycle arrest and DNA repair N. vectensis and A. palmata are potentially very long lived, as genets can reproduce asexually more or less indefinitely. Thus, minimizing the impact of DNA damage to somatic and germ line cells is critical to survival and fitness. Indeed, our analysis suggests that p53 family proteins have experienced positive selection along the lineage separating Cnidaria from Bilateria  and the Scleractinia  is approximately 225 mya and divergence between the Cnidaria and Bilateria is estimated at 650–1000 mya ilateria . Additio palmata .A. palmata transcriptome. We found similar patterns in the group size distributions of paralogs in both the N. vectensis genome and the A. palmata transcriptome. While it is possible that additional paralogous copies of some genes were not sequenced, such missing transcripts add additional conservatism to this analysis.Paralog group analyses have proven informative in identifying gene and genome duplication events and how they relate to speciation in a variety of organisms A. palmata consisted of immunoglobulin superfamily proteins in the IgLON family involved in the organization of neuronal connections in the developing nervous system in other metazoans N. vectensis and are expressed in developing larvae and planulae Given the rarity of large paralog groups, members of such groups must serve important functions to justify the maintenance of multiple copies. The largest paralog group in A. palmata, including TRAF 2, 3, 6 and a lipopolysaccharide induced TNF alpha factor. In mammals TRAF proteins are important signal transducers mediating innate immune response, apoptosis, bone metabolism and response to stress (including DNA damage) The second most abundant paralog group consisted of homologs of the TRAF proteins. Several TRAF homologs showed multiple paralogs in Drosophila and Caenorhabditis (n = 3 & 1 respectively) These annotations were determined solely by bioinformatic means, thus further verification is required, but it is of interest to note that the six TRAF proteins found in vertebrates are thought to be the result of an evolutionarily recent diversification because few TRAF homologs have been detected in A. palmata transcriptome numerous microsatellite repeats were observed (n = 333), with tri and hexanucleotide motifs representing the most common types (A. millepora (pers. obs.) Microsatellite repeats commonly occur in portions of transcribed mRNA including 5′ and 3′ untranslated regions on types . This isAmong trinucleotide repeats, motifs with high AT content were most frequent in both acroporid transcriptomes . SpecifiA. palmata was 1 per 272 bp . The frequency in A. millepora was slightly higher (1 per 207 bp) A. palmata samples. Only a small set of SNPs were chosen for preliminary validation here, but their wide distribution and the presence of frequency differences in even a small sample size is encouraging  were obtained from the local authorities  for all samples used in this study.Acropora palmata adults contain intracellular symbionts known as zooxanthellae. Like many other corals, symbionts are taken up after settlement and metamorphosis of the planula larvae et al.2 levels.To obtain larval tissue, gametes were collected from adult colonies as they were released during annual spawning events in 2008. Larvae were acquired from colonies at multiple locations in the upper Florida Keys, Puerto Rico, and Curacao to minimize ascertainment bias Low, mean, and elevated temperatures  were used to stimulate the expression of thermal stress response genes. Larval batches were housed in 1 L plastic containers with mesh sides, suspended in four separate 45 L plastic bins filled with filtered sea water at each treatment temperature. Water was circulated with an aquarium pump and changed daily with filtered sea water preheated to the target temperature. Temperatures were maintained within ±1°C with aquarium heaters and chillers, and were monitored with HOBO data loggers .Larval samples were collected throughout development, from immediately following fertilization until day five, to include a full range of developmental genes in the sequencing results. Approximately 100 larvae from each container at each time-point were incubated in RNA later  then frozen in liquid nitrogen following manufacturer's recommendations. Samples were stored at −80°C prior to extraction. Additional samples from adult colonies were also collected to include a component of post metamorphosis transcripts.2 treatments, larvae from Florida were raised in the Climate Change Laboratory at the Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL. Water in the treatment aquaria was held at 28°C and CO2 concentration was manipulated by bubbling with CO2-enriched air to maintain control and high CO2 treatment conditions (400 and 800 µatm respectively) as described in Albright et al. For the COPurification of RNA from all samples was performed using a modified Trizol extraction protocol. Samples were removed from the preservative and immediately submerged in 1 ml of Trizol for 5 minutes. Following the addition of 0.2 ml of Chloroform and incubation for 3 minutes, the samples were centrifuged at 12,000 rpm for 15 minutes at 4°C to separate the phases. The upper aqueous phase was collected and mixed with an equal volume of 70% ethanol, then applied to a Qiagen RNeasy mini spin column  and purified following manufacturer's instructions. Sample concentration was determined using a Nanodrop ND-1000 spectrophotometer  and RNA quality was assessed with an Agilent Bioanalyzer .2 treatment samples, and 4 adult samples from Puerto Rico and Curacao were pooled into a single batch sample such that the contribution of genetic material from each geographic location was equivalent (∼17 ug each). An aliquot of the resulting pool was submitted to the Indiana University Center for Genomics and Bioinformatics  for cDNA preparation, normalization and sequencing on the 454-GS FLX using Titanium chemistry following previously published methods A total of 71 samples, including 1 to 5 replicates each of 13 larval developmental time-points from all 3 locations, 10 COperl pipeline (pipemeta) following the methods of Vera et al.smartscreener script in the pipemeta package (SMART “CAP” primer 5′-AAGCAGTGGTATCAACGCAGAGT-3′), then entered into seqmanprov8 (DNAstar) for additional quality filtering and assembly. Default quality filtering parameters for 454 data were used, and assembly was performed in seqman using parameters suggested by the manufacturer for short read data. Prior to assembly an additional 36,239 high quality EST Sanger reads from a pool of A. palmata samples from different life stages and stress conditions were included to promote the assembly of long contigs. Sanger sequences are available from the SymBioSys database (sequoia.ucmerced.edu/SymBioSys/). 454 sequences and quality scores generated in this study are available from the NCBI short read archive , and the assembled annotated sequences can be downloaded from http://main.g2.bx.psu.edu/u/nickpolato/h/apalmataassembly.The resulting sequencing reads were preprocessed and annotated using a custom blastx to the complete Uniprot database . Acceptable hits were determined by a bitscore of >45 and a corresponding e-value of <1−5. Because two of the most closely related genomes in publicly available databases (Nematostella vectensis and Branchiostoma floridae) are largely unannotated, a large proportion of the highest ranking blast hits were uninformative. To maximize the information content, results were augmented with lower ranking (but more informative) annotations using a perl script and a list of keywords to avoid (i.e. ‘uncharacterized protein’ and ‘predicted protein’). Contig and singleton sequences were also submitted to the KEGG automated annotation server (www.genome.ad.jp/tools/kaas/) for further functional annotation.The resulting contigs and remaining singletons were aligned with blast search was performed comparing all A. palmata transcriptome data against a subset of transcripts from the N. vectensis genome project (“transcripts.Nemve1FilteredModels1” downloaded from genome.jgi-psf.org). The N. vectensis sequences were first compared to themselves to eliminate multiple copies of sequences with >90% similarity (i.e. multi copy genes). Only blast hits between A. palmata and N. vectensis sequences with a bitscore ≥45 were considered. The length of the A. palmata sequence was then divided by the length of the N. vectensis transcript to obtain a percent coverage estimate for each unigene  was downloaded from OrthoDB (cegg.unige.ch/orthodb4) blast was used to identify homologs of these genes in the A. palmata data.A  unigene , using oA. palmata dataset to compare with functional pathway relationships known in other model organisms. IPA was then used to test whether some pathways were more highly represented then others considering the number of sequences from A. palmata that mapped to a given pathway and the pathways' size. This application of IPA did not depend on expression level (i.e. the number of sequencing reads observed per gene). This is appropriate here because the A. palmata transcriptome was generated from a normalized library. IPA bases its statistical analyses on a Fisher's Exact Test (corrected for multiple testing) which is comparable to other well-known enrichment analysis methods Pathway analysis was performed in the Ingenuity Pathway Analysis (IPA) software. IPA is a web-based application that performs functional enrichment analyses to determine the probability that a given gene set is associated with pre-defined reference pathways beyond what would be expected by random chance. Pathway data in IPA is based on information in the Ingenuity Knowledge Base which is a manually reviewed database of pathways and relationships taken directly from the primary literature and public sources including GO, KEGG and EntrezGene. While this software is capable of other functions that consider gene expression levels based on microarray or RNAseq data, our analyses used only presence or absence of pathway components in the A. palmata dataset. Our analyses compared successfully mapped A. palmata transcripts to the IPA reference set consisting of all molecules present in the Ingenuity Knowledge Base. Significance of the association between the A. palmata data set and a reference pathway was measured in two ways: 1) A ratio was calculated by dividing the number of A. palmata transcripts that map to a given reference pathway by the total number of molecules associated with that pathway in the Ingenuity Knowledge Base, and 2) A Fisher's exact test determined the probability that there was a significant enrichment of A. palmata transcripts mapping to a given pathway beyond what was expected by chance alone given the total number of genes involved in that reference pathway. Because some pathways are better characterized than others these results should be interpreted as a guide for selecting conserved and well annotated functional metazoan pathways present in the A. palmata transcriptome.A canonical pathway analysis identified those pathways in the Ingenuity Knowledge Base that were the most well represented in the codeml program in pamlA. palmata homologs of N. vectensis genes (pVS53a and p63) were aligned with 17 other sequences representing a variety of taxa from anemones to humans . jmodeltestgarli (www.molecularevolution.org/software/phylogenetics/garli) with the TIM2+G model. One hundred bootstrap replicates were performed and bootstrap values were matched to the maximum likelihood tree using sumtreespamlh0−lnLh1)) of the difference between the log likelihood of the null model  relative to that of the alternative model , that incorporated positive selection along the branch leading to the Cnidaria by allowing the dN/dS ratio to be >1 along that branch, was performed and the result compared to a χ2 distribution to assess statistical significance.Tests of natural selection in p53 family genes were performed with the A. palmata and N. vectensis was assessed using inparanoidv4.1 N. vectensis (downloaded from genome.jgi-psf.org), and 43,081 ORFs from the A. palmata transcriptome were used in the analysis. Acceptable hits were determined by a bitscore of >40 using the BLOSUM62 scoring matrix.The identification of in-paralog groups in snphunter script in pipemetamysql database and will be made available to the public via the Dryad data archive (http://datadryad.org/) along with sequences containing potential microsatellite markers.Potential SNPs were detected in contigs with sufficient depth of coverage using the snphunter SNPs were validated using PCR and the amplified products were sequenced on an ABI3700. Primers were designed using primer3 , 2 mM of MgCl2, 0.2 mM of dNTPs, and 2 U of Biolase polymerase . PCR was carried out in an Eppendorf Mastercyler Gradient with an initial denaturation step of 95°C for 5 min followed by 35 cycles of 95°C for 20 s; annealing at 56°C for 20 s; and 72°C for 30 s, and a final extension of 30 min at 72°C.To confirm the results obtained by snphunter for each contig. Enrichment analysis of GO terms associated with transcripts showing the top and bottom 5% of all Ts/Tv ratios (<1 or >5) was carried out using wego (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) Transition and transversion ratios were determined based on the number of SNPs in each class identified by http://tandem.bu.edu/cgi-bin/trdb/trdb.exe) tandem repeats finder v4.04 A. millepora transcriptome (www.bio.utexas.edu/research/matz_lab/matzlab/Data.html) To detect repeat elements, the full set of sequences was uploaded to the Tandem Repeats Database Click here for additional data file.Information S2codeml.Results of tests for natural selection using the program (XLSX)Click here for additional data file.Information S3Top and bottom 5% of Ts/Tv ratios.(XLS)Click here for additional data file.Information S4SNP validation summary and primer sequences.(XLSX)Click here for additional data file.


We expand the anecdotic report by Johansson that back-and-forth linear harmonic motions appear uniform. Six experiments explore the role of shape and spatial orientation of the trajectory of a point-light target in the perceptual judgment of uniform motion. In Experiment 1, the target oscillated back-and-forth along a circular arc around an invisible pivot. The imaginary segment from the pivot to the midpoint of the trajectory could be oriented vertically downward (consistent with an upright pendulum), horizontally leftward, or vertically upward (upside-down). In Experiments 2 to 5, the target moved uni-directionally. The effect of suppressing the alternation of movement directions was tested with curvilinear (Experiment 2 and 3) or rectilinear (Experiment 4 and 5) paths.  Humans make striking perceptual mistakes in judging even the simplest kinematics of visual stimuli. Thus, a spot moving at constant velocity along a rectilinear path is perceived as moving fast upon entering the visual field and then decelerating to a constant velocity Runeson The idea that the expected dynamics of natural stimuli biases visual motion perception has been extensively tested in the case of biological movements The motions of masses under force fields afford another rich array of natural events that can bias perception. Gibson A more stringent test of Runeson's hypothesis is possible by considering stimuli compatible with more than one ecological model. The present study focuses on the perception of a very common visual template, the oscillation of an object around a pivot. Many real-world events, including biological movements, are described approximately by such a template that arises whenever a mass is subjected to elastic and/or gravitational forces. In particular, the perceived motion of the pendulum has received considerable attention. When friction is negligible, the instantaneous velocity of a suspended mass under Earth gravity depends only on its initial position and on the length of the pendulum rod. Moreover, in the small-angle approximation, the oscillation is harmonic with a period that depends only on the length of the rod.The earliest evidence that the motion of the simple pendulum is perceptually salient was provided by measuring the accuracy with which one can point to the bob Under specific circumstances, gravity and elastic forces induce similar harmonic oscillations. In general, however, the two forces are very different. Unlike elastic tension, gravity is ubiquitous, almost constant at Earth surface, and acts invariably in the downward direction. In fact, the expected constraints imposed by gravity on object motion can affect visual perception more generally than in the simple oscillatory case We designed six experiments to test these predictions and to investigate the perceptual consequences of modifying some of the features that are normally associated with the motion of a gravitational pendulum or a mass-spring system. We presented a spot moving along curvilinear or linear trajectories with different velocity profiles including both harmonic and constant velocity motion, and asked the observers to choose the profile that appeared most uniform. By varying the law of motion, the shape of the trajectory, and its orientation relative to gravity, we explored a significant range of possible departures from the canonical motion of a simple pendulum. In Experiment 1, the target oscillated back-and-forth along a circular arc. The effects of target kinematics and of the orientation of the trajectory relative to gravity were manipulated independently. In Experiments 2 to 5, the target moved uni-directionally. The effect of suppressing the alternation of movement directions that characterizes the motion of a pendulum was tested with curvilinear (Experiment 2 and 3) or rectilinear (Experiment 4 and 5) paths. In Experiments 1 to 5, no instruction was given concerning eye movements and observers were free to eye-track the target. The anecdotal report by Johansson First, we presented stimuli moving along curvilinear trajectories with constant radius, which are always compatible with the motion of a pendulum. Second, rather than asking a qualitative judgment on just one velocity profile, we asked observers to select the most uniform motion from a full range of alternative profiles, which also included the uniform one. The range of velocity modulations was wide enough to exclude that perceptual misjudgments of the kind reported by Johansson If perception relies heavily on statistical prior models of physics, and if “only natural motions look constant”, the harmonic motion should be chosen as prototype of uniform motion for the three stimulus orientations, which are all consistent with the assumption that oscillations are driven by elastic forces. However, if a prior related to the direction of gravity also affected perception, one would expect that the harmonic motion should be chosen with greater accuracy, precision and inter-individual consistency in the canonical upright orientation of a pendulum than in the other two orientations incongruent with gravity.Note that, although the stimuli could be construed also as a schematic description of a voluntary biological movement (such as the back and forth swing of a limb), they were point-to-point motions with a constant curvature throughout and a reversal of velocity direction at the endpoints of the trajectory. One cannot expect the responses to comply with the 2/3 Power Law, which does not apply to such movements.Twelve participants  volunteered for the experiment. They were all right-handed , had normal or corrected-to-normal vision, and no past history of psychiatric or neurological diseases. All participants in this and the following experiments gave written informed consent to procedures approved by the Institutional Review Board of Santa Lucia Foundation, in conformity with the Declaration of Helsinki on the use of human subjects in research, but were otherwise unaware of the purpose of the experiments.1g) of an ideal simple pendulum under Earth gravity (g = 9.81 m s−2) obeys the differential equation:θ(t) is the angular displacement from the equilibrium position (θ = 0), and L is the rod length. With the initial conditions:0 = π/4. Thus, T = 1.68 s. In the following, A = 2θ0 = π/2 denotes the amplitude of the oscillation. Angular velocity (V1g = dθ(t)/dt) was computed by solving 1g and the law of motion computed in the small-angle approximation (harmonic function) was 0.7% and 1.3% for angular position and velocity, respectively. We use the term Velocity to indicate the unsigned time derivative of the angular displacement.The motion  with the same parameters , but different velocity profiles −V1g (T/4) = V1g (T/4)−V0g (T/4), and V3g (T/4)−V2g (T/4) = V2g (T/4)−V1g (T/4), respectively. The labels 2 g and 3 g denote conditions with a maximum velocity twice and three times as large as 1 g, but do not describe a pendulum motion under a gravity level twice or three times as large as Earth's gravity. In fact the corresponding velocity profiles depart significantly from harmonic functions , and then under-sampling the sequence to match the vertical refresh rate of the display.ions see . FinallyTask and procedures). Stimuli were programmed in C++ and rendered using Autodesk Maya 2009 (Autodesk Inc) with a PNY NVIDIA Quadro FX5600 graphics card.Participants sat 0.58 m in front of a display  in a dimly illuminated room. The height of the chair was adjusted so that participant's eyes were ∼10° above the display midpoint. Responses were entered by pressing one of 4 keys (button-box Empirisoft Corp.) labeled  “Start”, “Forward”, “Backward”, and “OK” (see 2), moving against a uniformly bluish-purple background . Luminance and chromaticity were measured with a Tektronix J17 LumaColor photometer. In each trial, the target oscillated back-and-forth along the arc of a circle  around an invisible pivot  and 275% (K20). For all kinematic profiles except K5 (constant velocity), the ratio exceeded the detection threshold of 25% reported for stimuli with comparable duration and modulation frequency Human sensitivity to visual accelerations is based on the comparison of velocity estimates averaged over short, successive time windows 2, K6, K10, K14, K18,], with the constraint that successive trials could not have the same initial kinematics. There were 5  × 10 (repetitions)  = 50 trials in each session.Participants were tested in a counterbalanced order in three experimental sessions about 15 days apart. In each session, we presented one of the three orientations defined above (block design). In all trials, the target initial position was at π/4 clockwise relative to the trajectory midpoint. The target oscillated continuously with the same law of motion until the participant intervened either to modify the target kinematics (see below), or to end the trial with a response. The initial kinematics for a trial was selected pseudo-randomly from the set . If Ki+1 or Ki−1 fell outside the preset range of conditions, the computer replaced it randomly by one of the three conditions . Participants were informed about neither the number of steps in the sequence nor the condition they were currently exploring. No feedback about response accuracy was provided. No instruction was given concerning eye movements so that participants were free to eye-track the target. Responses were stored together with the preceding sequence of changes. On average, reaching a final decision required 12 adjustments. The typical duration of a session was 40 min. None of the participants reported any sensation of motion-in-depth.The effect of pressing the buttons was to move one step backward  Wnij, respectively. Moreover, in order to treat the stimuli as an ordinal random variable, we define the 3-vector I =  indexing the corresponding kinematics. The results were analyzed at both population and individual level. At population level , the effect of orientation was estimated in several different ways. First, by computing the median (M) and the interquartile range (IQR) of the index of the kinematics judged as uniform. Second, global estimates of the central tendency index and of the variability were derived from a Probit analysis of W: −1 is the probit link function, 0β the intercept and 1β the slope of the linear regression. Goodness of fit was assessed by testing that the deviance was not significantly different from 0 In the following, we use a vector notation (boldface) to denote the performance for the three orientations of the trajectory. Let the 3-vectors I of experimental kinematics) and random-effects predictors (to account for the variability among participants). For participant j the complete model, which also includes the interactions between fixed and random factors, is:Wj is the CDF of participant j, 90D and 180D are dummy variables coding for the orientation (with respect to the 0° orientation reference), 90I·D and I·180D are the interaction terms, 0β, …, 5β are the fixed-effects coefficients (independent of participants), and j0u, …, j5u are the random-effects coefficients for participant j. In particular, 1β estimates the precision of the response in the reference (0°) orientation , 2β (3β) tests whether the intercept is significantly different between the reference and the 90° (180°) orientation, and 4β (5β) tests the same difference for the slope. The model was applied twice, by taking as reference first the 0° orientation (as detailed above), and then the 90° orientation (with the appropriate dummy variables D). The model was fitted to the CDF's using the R package ‘lme4’ As a further analysis at population level, we applied to the data the Generalized Linear Mixed Model GLMM, . The GLMwww.mixedpsychophysics.wordpress.com).Median and JND for the population were again estimated from GLMM using the R package “MERpsychophysics” . There was a significant (P<.001) attractive effect of the initial kinematics on the median. However, the size of the effect was very small, the difference between maximum and minimum median values, computed separately for each starting condition, being equal to 1. Therefore, in the subsequent analyses the results were pooled across repetitions and starting conditions.10 which simulated the effects of a virtual gravity (1 g) acting downwards (0° orientation), leftwards (90° orientation), or upwards (180° orientation). However, the specific distribution of the responses differed as a function of orientation. In particular, the variability was smaller for the 0° orientation (IQR = 1) than for both the 90° (IQR = 3) and the 180° orientation (IQR = 3). Moreover, the proportion of trials in which velocity profiles close to K5 (constant absolute velocity) were judged as uniform was lower for the 0° orientation than for the other two orientations.These results were confirmed by a probit analysis of the population CDFs of the response variable. There was a highly significant difference  between the CDF for orientation 0° and those for either 90° or 180° orientation. Both the median and the slope (response precision) were higher for 0° orientation than for the other two orientations. Differences remained significant  even when the CDFs were computed after equalizing the individual medians. In contrast, the CDFs for 90° and 180° orientation appeared to have the same shape , the same variance , but different median value .Data analysis and modeling). With the only exception of 5β, all parameters of the model were significantly different from zero . Clearly, the responses were much more consistent among participants for 0° orientation than for the other two orientations. The difference across orientations is dramatized in 10 (1 g), but was significantly higher than the median for the other two orientations (90° and 180°). The medians for all stimulus orientations were significantly closer to K10 than to K5 (constant velocity). The slope of the CDF for orientation 0° was significantly higher than the slope for 90° orientation , whereas the slope was not significantly different between 0° and 180° orientations  and between 90° and 180° orientations .The effects of orientation were investigated further by modeling the individual CDFs of the response variable. 0–K9], which includes the uniform motion case (K5), the kinematic profiles presented a discontinuity, because velocity does not go to zero at the endpoints of the trajectory  for the 0° orientation , and the maximum angular velocity of K10 was 32.3° s−1  and were less variable across trials and participants than for the other two orientations (90° and 180°). In other words, the bias was stronger for a trajectory orientation consistent with the interpretation of the stimulus as a canonical upright pendulum accelerated by physical gravity. In the general discussion we elaborate the implications of these findings vis à vis the general notion that perception is influenced by the interpretation of the stimuli in terms of physical events. Two issues must be addressed. 1) Both gravitational and elastic oscillations are characterized by the inversion of movement direction at both ends of the trajectory and by the curvilinear trajectory of the target. The role of these features on velocity perception is investigated in the next 4 experiments. 2) The possible role of eye movements. The last experiment tests whether eliminating the possibility to track the target with eye movements affects velocity judgments.In sum, we found that a large misperception of target kinematics exists for all three orientations of the trajectory. Observers perceived as uniform a quasi-harmonic velocity profile that was strongly non-uniform, velocity changes being greater than 150% within each oscillation. The perceptual bias was significantly modulated by trajectory orientation. Responses for the 0° orientation clustered closer to the 1g-condition  volunteered for the experiment. None of them had served in Experiment 1.Apparatus, task, and general experimental procedures were the same as in Experiment 1. The target moved along the same trajectory and with the same 21 velocity profiles as in the 0° orientation of that experiment. However, motion was unidirectional, from left to right. Its duration was the same (840 ms) as that of a single sweep of the pendulum-like motion in Experiment 1. Upon reaching the rightmost position along the path, the target disappeared for 140 ms; then it reappeared in the leftmost position and immediately started a new sweep. The inter-sweep interval was chosen to permit the eyes to re-foveate the target with a saccade . However, the JND was considerably higher than in the previous experiment. Taken together, the results of Experiment 1 and 2 show that a target moving with quasi-harmonic kinematics is perceived as uniform irrespective of whether full back and forth oscillation cycles or just repeated unidirectional half cycles are displayed. However, the responses tend to be considerably more variable in the latter than in the former case.There was no significant effect of repetition on the response median , and a significant (P<.0001) effect of the starting condition in the sequence . In this experiment we asked whether the perceptual bias toward a quasi-harmonic velocity profile disappears when the trajectory is incompatible with any simple physical model capable of sustaining an oscillatory behavior. The velocity of the targets was modulated as in the previous experiments, but with a different trajectory. Here, the target moved along a circle without ever changing direction and was visible only in the bottom and top quadrants. These two segments of trajectory shown sequentially in time can be perceived as a partially occluded circular motion. Crucially, no physical system is compatible with a harmonic motion along a circular trajectory.Eleven participants  volunteered for the experiment. None of them had served in the previous experiments.Apparatus, task, and general experimental procedures were the same as in Experiment 1. The target moved uni-directionally, anticlockwise, along a circular trajectory centered on the display midpoint . The rad6 (IQR = 3) , and a small but significant effect of the initial kinematics . = 3) see . ParticiIn summary, the change in target configuration abolished almost completely the perceptual bias in favor of the harmonically modulated velocity profiles. Although the trajectory was partly occluded, the stimulus may have evoked the motion of a rotating system. As argued above, no simple such system exhibits harmonic oscillations. By contrast, a mass rotating under the effect of a central force - arguably the simplest physical model for such motion - does indeed move at constant velocity. Therefore, the results are still compatible with the general hypothesis that perception is permeable to pre-conceptions about the possible physical interpretation of the stimuli.This and the next experiment investigate the role of trajectory curvature. In Experiment 2 we showed that even repeated unidirectional sweeps along a circular arc were sufficient to make participants perceive the quasi-harmonic motion of the target as uniform. Here we tested whether the same bias is present also when the target follows a horizontal rectilinear path.Twelve participants  volunteered for the experiment. Ten of them had participated also in Experiment 3. There were about 20 days between Experiment 3 and 4.The target moved on a linear, horizontal path from left to right, encompassing a visual angle of 17.85°. The path length was the same as that of the circular segments in Experiments 1 to 3. The midpoint coincided with the center of the display. Motion duration was the same (840 ms) as that of a single sweep of the pendulum-like motion in Experiment 1. As in Experiment 2, upon reaching the rightmost position along the path, the target disappeared for 140 ms and reappeared in the leftmost position to start a new sweep. Target velocity at any time along the linear trajectory was equal to the tangential velocity at the same time along the curvilinear path of Experiment 1. No instruction was given concerning eye movements. 6 (IQR = 2). 6 or the median of the previous experiment. Finally, the response in this and in the previous experiment had indistinguishable distributions . Therefore, the single difference with respect to Experiment 2 (rectilinear versus curvilinear trajectory) was responsible for eliminating almost completely the perceptual bias toward quasi-harmonic motion. As in the case of Experiment 3, this suggests that the bias disappeared because the stimuli no longer evoked an oscillating physical system.There was no significant effect of repetition on the median of the responses , and a small but significant (P<.005) effect of the initial kinematics (difference between maximum and minimum median  = 1). We tested whether the results of the previous experiment are confirmed when the rectilinear unidirectional motion is vertical instead of horizontal.Twelve participants  volunteered for the experiment. Five of them had participated also in Experiment 1. One of them had participated one year before also in Experiments 2 and 3.The target moved downward along a linear, vertical path of the same length as in Experiment 4 (17.85°). The midpoint coincided with the center of the display. Motion duration was the same (840 ms) as that of a single sweep of the pendulum-like motion in Experiment 1. Upon reaching the bottom position, the target disappeared for 140 ms and reappeared in the top position to start a new sweep. Target kinematics was the same as in Experiment 4. No instruction was given concerning eye movements.5 as the constant velocity profile. 6 (IQR = 5). 6. Taken together, the results of the last 3 experiments show that the perceptual bias towards quasi-harmonic motion is not inevitable, but requires that some cues in the stimuli evoke a context of an oscillatory physical system.There was no significant effect of repetition on the median of the responses , and a small but significant (P<.005) effect of the initial kinematics . Observers were often able to correctly identify KIn all previous experiments, no instructions were given concerning eye movements to participants, who were free to track the target. It has been reported that motions that can be construed as natural events  are easier to track with eye movements than motions deviating from such natural models Six participants  volunteered for the experiment. Two of them had participated also in Experiment 1 about 30 days before.−1), the retinal eccentricity of the target (10.74°) relative to the fovea does not alter velocity estimates substantially The apparatus and general experimental procedures were the same as in Experiment 1. The target oscillated back and forth along the same trajectory and with the same 21 velocity profiles as in the 0° orientation condition. However, throughout each trial, participants were asked to fixate a white cross-hair (0.37°) located at the pivot of rotation of the target, 10° above the display midpoint. The head of the participants was stabilized with the help of a forehead-chin rest. The height of the chair and of the forehead-chin rest was adjusted individually so that the eyes were level with the fixation point. It is known that, in the range of values tested here (average angular velocity: 19.96° s10 (IQR = 3). 10 or from the corresponding median in the 0°orientation condition of Experiment 1. The probit fit to the population CDF and the corresponding CDF in Experiment 1 had the same shape , variance , and median . In conclusion, this control confirmed fully the results of Experiment 1. Preventing the possibility to track the target with eye movements did not suppress the perceptual bias present when target tracking was possible.All participants correctly reported the words presented at the fixation point. Although the target moved in the periphery of the visual field, performance in the main task was very similar to that in Experiment 1. The median response depended on neither the initial kinematics , nor repetition . 10, here denoted as 1g-kinematics), that is, the kind of motion naturally associated with these two physical systems. The bias towards quasi-harmonic motion was most consistent in the upright 0° orientation of Experiment 1, where both the gravitational pendulum and the rotational mass-spring system afford a plausible physical model with almost identical kinematics. When the predictions of the two dynamic models were mutually incompatible (90° and 180° orientation), the bias tended to diminish and to be less consistent across participants. Finally, in the experiments in which the trajectory was incompatible with both a simple pendulum and a rotational mass-spring system  was still compatible with a mass-spring system, but became incompatible with the action of physical gravity. In fact, for trajectories at 180° orientation, gravity would decelerate the target toward the trajectory midpoint and accelerate it away from the midpoint, whereas at 90° orientation gravity would accelerate the target on the way down and decelerate it on the way up. We suggest that, in these conditions, the mass-spring model is the dominant one and is a sufficiently strong prior to attract the perceptual responses much closer to 1g-kinematics than to 0g-kinematics even at these orientations. However, we also argue that the powerful pendulum model is still somehow evoked. The shift of the perceptual bias slightly away from the 1g-kinematics and towards the 0g-kinematics, and the larger variability across trials and participants with respect to the 0° orientation see  is then 6), they were often able to correctly identify K5 as the constant velocity profile (The comparison of the results of Experiment 1 and Experiment 2 (0° and 0°ss  profile  and 7. A6), but they were often able to correctly identify K5 as the constant velocity profile  with a prior related to the statistical distribution of velocities in the natural world File S1Contains: Movies S1–S21. Down-sampled (50 Hz) version of the stimuli presented in the 0° orientation condition of Experiment 1, , Movie S6 to K5 (0 g), Movie S11 to K10 (1 g), Movie S16 to K15 (2 g), and Movie S21 to K20 (3 g). Because each movie shows only one stimulus period, to see the stimuli repeatedly the media player must be set in reply (continuous) mode.(ZIP)Click here for additional data file.File S2Contains: Movies S22–S42. Stimuli presented in the 180° orientation condition of Experiment 1 (0 (−1 g), and Movie S42 to K20 (3 g).riment 1 . Movie S(ZIP)Click here for additional data file.File S3Contains: Movies S43–S63. Stimuli presented in Experiment 4. Movie S43 corresponds to K0 (−1 g), and Movie S63 to K20 (3 g).(ZIP)Click here for additional data file.


Background. Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma. Extensive studies on Iranian MF patients are absent. The present study aimed to produce updated clinical information on Iranian MF patients. Methods. This was a retrospective, descriptive, single-center study, including all cases of MF seen in the Department of Dermatology, University Hospital of Isfahan, Iran, between 2003 and 2013. Data systematically recorded for each patient included clinical, biological, histological, and molecular findings. Results. Eighty-six patients with clinical and histologic diagnosis of MF were included in the study. Thirty-nine patients (45.3%) were male. Female predominance was observed in patients . Patients were between 7 and 84 years of age (median: 41). The interval from disease onset to diagnosis ranged from 0 to 55 years (median: 1 year). Eighteen cases (20.9%) had unusual variants of MF. The most common types included hypopigmented and poikilodermatous MF. Childhood cases of MF constituted 5.8% (5/86) of all patients. The early stages were seen in 82 cases (95.34%). Conclusion. The major differences in epidemiologic characteristics of MF in Iran are the lack of male predominance and the lower age of patients at the time of diagnosis. Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). MF differs from other primary CTCLs by virtue of unique clinical features and histopathology , 2. PatiThe natural history of MF is characterized by an indolent progression through four stages: patch, plaque, tumor, and visceral involvement, but this progression is not necessarily seen in all patients . Many vaThe peak age at presentation is in excess of 55 to 60 years, with a 2 : 1 male : female ratio , 8.Extensive studies on Iranian MF patients are absent. The present study thus aimed to produce updated clinical information on Iranian MF patients, including epidemiology, patient characteristics, clinicopathologic features, and treatment.Between 2003 and 2013, patients with MF from single-center resident training hospitals had been retrospectively enrolled into the study.A diagnosis of MF was confirmed according to International Society for Cutaneous Lymphoma (ISCL)/EORTC criteria , 9 and, The analyzed clinical data included the age of the patients at the time of diagnosis, the age of the patients at the time of initiation, the cutaneous lesion, sex, and the status of disease at the last follow-up examination. Hematoxylin and eosin-stained slides, immunohistochemical stains for basic B- and T-cell markers (CD20 and CD3 and/or CD45RO), and additional immunostaining, including CD4, CD8, CD30, and CD5, were histologically evaluated by an expert dermatopathologist. Polymerase chain reaction analyses of T-cell receptors (TCR) b or c and immunoglobulin heavy chain gene rearrangement were reviewed.In all the patients, the presence of extracutaneous disease at the time of diagnosis had been excluded by standard staging procedures.Stage workup  and overall survival were obtained by reviewing the clinical records.To determine the stage of MF, the tumor-node-metastasis (TNM) system was used . The cliAfter data collection, statistical analysis was performed by the SPSS software version 19.Eighty-six patients with clinical and histologic diagnosis of MF were included in the study.The interval from disease onset to diagnosis ranged from 0 to 55 years (median: 1 year).From 68 patients with classic MF, 42 patients presented with the patch, 21 with plaque, 4 with a tumor, and only one with erythroderma . From 86Only one patient had folliculotropic MF . One patChildhood cases of MF (younger than 18 years) constituted 5.8% (5/86) of all the MF patients. There were two male and three female patients .There was no familial clustering. None of the patients had HIV or other forms of immunosuppression.According to the TNM classification, among 86 cases of MF, the early stage (stage I + IIA) included 82 cases (95.34%) and the advanced stage (stage IIB + III + IV) included four cases (4.66%). One female patient had a large cell transformation during the course of disease. Progression to the late stages of MF is not seen in childhood/juvenile-onset MF patients.Immunophenotypic analysis demonstrated that 77.6% of the patients with MF were CD4 positive. CD20 was negative in all patients. In our patients, the conventional T-helper phenotype (CD4+/CD8−) was the most common one (63% of patients) .A clonal T-cell receptor rearrangement was detected in the skin biopsy in 9 patients (36%) from a total of 25 patients in whom T-cell receptor analysis was possible.Antibodies to HTLV-1 were not detected in the sera of any patients with MF by ELISA. Seven patients (8.1%) had abnormal lactate dehydrogenase (LDH). None of the patients died during the study period in the early stages.The major differences in the epidemiologic characteristics of MF in Isfahan, Iran, are lack of male predominance and the lower age of patients at the time of diagnosis in those reported from the West. It was reported that the incidence rate of MF in Isfahan is the same as that in other parts of Iran, which is largely similar to the data of other countries .MF is generally rare among Asians and it has a higher incidence rate in blacks , 14; it There is a male predominance in almost all studies on CTCLs and MF, with a male : female ratio of 1.3 : 1 to 2 : 1 –17.The proportion of our patients complaining on the onset of their disease before 21 years was much higher, similar to the epidemiological study in Kuwait . The occSome studies have found an increased incidence of CTCL among workers in chemical science, transportation, and manufacturing industries, whereas other studies have not .Hydrocarbons and petrochemical exposure have been reported to carry an increased MF risk , 21. WheIn the Iran-Iraq War (1980–1988), it is not known whether chemical weapons by Iraq contained Sulfur mustard (SM), a DNA alkylation, and a well-known carcinogenic agent . This hyThe prevalence of childhood MF among different studies has varied between 2.7% and 16.6% , 24. In Among the atypical forms of MF, hypopigmentation is one of the most prevalent forms in Asians . HypopigOur data is compatible with the study of Quaglino et al.  in the fThe median time from symptom onset to diagnosis in retrospective studies is 3-4 years, but it may exceed four decades , 32, 33.The current study differs from that of Quaglino et al. in the fact that the median time from the onset of cutaneous lesions to diagnosis was 2 years .In the present study, the mean time from symptom onset to definite diagnosis was about 5–10 years, which is significantly different in male and female patients.Several studies have shown that nearly all patients with MF are HTLV-1 seronegative . Our finUsually, MF is characterized by an infiltrate of T-helper memory lymphocytes  . HoweverAlthough the group of this study is really not very large and also the number of the patients with more advanced disease is small, it may be the starting point for a larger multicenter study in Iran. Whether the occurrence of MF in younger patients in Iran is related to some ethnic or genetic variations or to environmental factors needs to be determined. A prospective multicenter study on a larger population of patients with a longer follow-up might be useful to confirm the possible causes of these epidemiological differences.


Objectives: Schizophrenia patients often experience relapses once and even more with no limit on number of relapses. The time among relapses are rarely considered in studies. The aim of this study was to identify some risk factors of time to elapses in schizophrenic patients with recurrent events model in survival analysis.Methods: In a retrospective longitudinal study, the medical records of 159 schizophrenic patients who referred to Razi hospital in Tehran from 2003 to 2005 were conveniently sampled, investigated and followed until the end of 2009. The time to recurrent relapses were considered in weeks. The patients with at least one relapse in this duration were included in the study. Event-dependent frailty model, using Bayesian approach, was applied to fit the data and identify the risk factors of time to relapses.Results: In this recurrent failure time model, the effects of age of onset ), gender ), marital status ) and family history ) were significant on the hazard time to relapses. According to the credible interval of frailty variance, elapsed time to relapse is dependent on patients’ characteristics ). Subsequent relapses are likely to be influenced by the occurrence of the first relapse, too ), with decreasing hazard of time to relapse.Conclusions: Subsequent relapses are likely dependent on the first and previous relapses. Age of onset, gender, marital status and family history are important risk factors influencing hazard of time to relapses. More studies are required to clear out the effect of other covariates with this model. Mental disorders have profound social, cultural and economic effects on communities worldwide . Burden The illness tends to develop between the ages of 16 and 30, and mostly persists in the course of patient’s lifetime. Although the causes of schizophrenia are unknown, evidences suggest that genetic factors, early environmental influences , and social factors  play an important role .The inciParticipants and ProcedureA retrospective longitudinal study was carried out on 162 conveniently sampled schizophrenic patients with at least one relapse, who were hospitalized at least once at Razi psychiatric hospital in Tehran between 2003 and 2005, and who were followed up until the end of 2009; data were collected from their medical records. Three patients who had missing values on all covariates in their records were excluded from the total sample. For the rest of the patients, the onset of schizophrenia was considered as the time of their follow up, and they were followed in terms of their relapses. Relapse was defined as observing schizophrenia symptoms in the first re-hospitalization after discharge and following re-hospitalizations. We assessed time to relapses and the number of weeks patients remained to be re-hospitalized. Some patients right censored at their last time period to re-hospitalize. Statistical Analysis Recurrent time data are commonly encountered in longitudinal studies when failure events can occur repeatedly over time for each study subject . Time toThe participants for the studied sample were 134 (84.28%) males and 25 (15.72%) females. The mean age of the patients at the onset of the disease was 21.52 ± 6.84 years (range 10-43). The relative frequency of married patients was 19.59% (31), and 85 (53.46%) patients experienced sudden onset of disease while 76 (47.8% ) patients had a history of head injury. Only 10 (6.29%) patients had schizophrenic patients in their family (grades one to three kinship). In common survival models, positive signs of regression coefficients increase the risk of event occurrence. Distribution of the number of relapses in schizophrenic patients shows that 45.3% of males who did not have a family history of schizophrenia had experienced 3 relapses during the study period . With reChronic diseases such as schizophrenia are, roughly speaking, lifelong transitions between the states of relapse and recovery. The long-term pattern of recurrent times-to-relapse can be investigated with routine register data on hospital admissions. The relapses indicate the time for readmissions to hospital and the time spent at the hospital are considered gaps between subsequent times-at-risk . In thisSubsequent relapses are likely dependent on the first and previous relapses. Age of onset, gender, marital status and family history are important risk factors influencing hazard of time to relapses. More studies are required to clear out the effect of other covariates such as place of residence , education level, economic status and season of birth of patients on time to relapses with this model.


Transformed mycosis fungoides (T-MF) is a rare variant of MF with an aggressive course. In this study, we aimed to describe characteristics of MF/Sezary syndrome (SS) patients with transformation.Patients diagnosed with T-MF among MF/SS patients between 2000 and 2014 in a tertiary single center were evaluated retrospectively. Demographic data, clinical data, laboratory data, immunophenotype features, response to treatment, survival, and histopathologic features were analyzed.Among 254 MF patients, 25 patients with T-MF were identified (10.2%) and included in the study. The male-to-female ratio was 2.6/1. The median time between MF diagnosis and transformation was 32 months (range: 0-192). Nine (36%) patients were diagnosed initially with T-MF. Advanced disease stage and high serum lactate dehydrogenase (LDH) levels were indicators of poor prognosis and treatment response. Five of the 18 patients with progressive disease had undergone allogeneic hematopoietic stem cell transplantation . Allo-HSCT resulted in complete remission in three (60%) patients. Ten (40%) patients died as a result of disease progression. Mean survival time was 25.2±14.9 (2-56) months after transformation.Advanced stage, high serum LDH levels, and loss of CD26 and CD7 expression in the peripheral blood are poor rognostic factors in T-MF. Treatment-resistant tumors and nodules should be cautionary for T-MF. Patients with T-MF have a shortened survival. Some patients may respond to first-line treatments. However, the majority of patients who do not respond to first-line therapies also are unresponsive to second or third-line therapies. Allo-HSCT may be an alternative option in patients with T-MF. Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). Generally, MF has an indolent course with slow progression from patch/plaque-stage disease to cutaneous tumors . HoweverDiagnosis of transformed MF (T-MF) is based on the presence of large cells CD30 +/-) exceeding 25% of the infiltrate throughout the lesion or forming microscopic nodules of large cells0 +/- exc.Advanced stage of MF at the time of transformation and folliculotropism are suggested as the most important factors affecting survival. AdditioRisk factors associated with an aggressive course of T-MF are not well described in the literature due to the low incidence of MF/SS and thus T-MF. In different series, the incidence of T-MF has been reported to range between 8% and 55%among MF patients ,10,11. TWe retrospectively evaluated all MF/SS patient records in a single reference center in Ankara, Turkey, from 2000 to 2014. Among all MF/SS patients, T-MF patients with at least one histopathologically confirmed biopsy were included in the study.For each case, clinical features were evaluated by three dermatologists and histopathological findings were reviewed by one pathologist who was an expert in this area.All patients were classified according to the International Society for Cutaneous Lymphomas and European Organisation of Research and Treatment of Cancer revised criteria of 2007 . StagingThe accompanying prognostic factors were also analyzed: age, sex, age at diagnosis of T-MF, presence of folliculotropism in skin lesions, CD30 expression in more than 75% of cutaneous neoplastic T cells, serum lactate dehydrogenase (LDH) levels, serum b2-microglobulin levels, and eosinophilia. The time interval between MF and T-MF, clinical stage at the time of T-MF, and survival were analyzed.Therapies were classified as first-, second-, and third-line treatments according to the 2014 National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology ,14. AlloResponse to treatment was evaluated as follows: complete response (CR), complete resolution of the disease; partial response (PR), at least 50% improvement compared with baseline; stable disease (SD), some improvement (25% to 50% improvement in lesions) plus reduction in the size of axillary and inguinal lymph nodes in the absence of significant evidence of disease; or progressive disease (PD), more than 25% increase in the number or size of clinically abnormal lymph nodes, or development of novel tumors or pathologically positive nodes or visceral disease .The data obtained from patients were analyzed with SPSS 16.0. The Mann-Whitney U test, chi-square test, Spearman’s test, and Mantel-Cox analysis were used to compare variables. The Kaplan-Meier method was used to determine overall survival.The disease stage and exact TNMB stages of patients, initial treatments, and follow-up data of each patient are summarized in 1 , stage IVA2 , and stage IVB ]. Most patients had transformation only at a skin site (96%); in one patient skin and lymph, node transformations were detected simultaneously (4%). Furthermore, 32% of T-MF patients presented with a new or enlarging tumor accompanied by long-standing plaque lesions. Dermatological examination at the time of T-MF diagnosis for the rest of the patients revealed the following: two (8%) patients had long-standing enlarging tumors, four (16%) patients had a new tumor accompanied with erythroderma (one bullous), one (4%) patient demonstrated ichthyosiform erythroderma, three (12%) patients had an enlarging plaque with erythroderma, three (12%) patients had long-standing plaques, two (8%) patients had newly scattered papules distinct from MF plaques, one (4%) patient showed an abrupt onset of multiple pink scattered nodules, and another patient (4%) had follicular papules associated with hair loss within the involved area (Two of 25 patients with T-MF (8%) had early patch and plaque MF . Twenty-three patients had advanced-stage disease [stage IIB , stage III , stage IVAved area .Histopathological examinations of the transformation site showed tumoral lesions in 18 (72%) cases and plaque lesions in seven (28%) cases. Lesion subtype (plaque or tumor) was not significantly correlated with survival (p=0.678). Less prominent or focal epidermotropism was present in 15 (60%) of the 25 patients in our study, and only 2 (8%) patients had Pautrier microabscesses. Folliculotropism was observed in ten cases (40%) with LCT. In eight (80%) of them, there was progression under treatment, while in one (10%) patient PR to treatment and in 1 (10%) patient CR was observed. Folliculotropism was not correlated statistically with survival (p=0.568).+CD4+CD8− T-cell phenotype and one (4%) patient had a double CD4+CD8+ T-cell aberrant phenotype. In most cases (88%), there was partial loss of one or more pan-T-cell antigens. Loss of CD7 expression was seen in 22 (88%) patients. Immunophenotype analysis of the skin biopsies showed that 24 (96%) patients had a CD3CD30 positivity in more than 75% of all the large T cells was present in skin biopsies of five patients (20%). In the remaining 20 (80%) cases, CD30 staining was either completely negative or expressed by only a very few (<5%) large T cells. There was no statistically significant difference either in disease stage or treatment response among CD30-positive and CD30-negative patients . Twelve patients with early  and concurrent  transformation were also evaluated separately for survival (p=0.582).Advanced disease stage at the time of transformation correlated with poor survival (p=0.003). In our study, among the deceased patients, 80% had stage IV disease, whereas only 20% of patients had stage IV disease among the surviving patients (p=0.002). During follow-up, 10 patients died of disease-related events (32%). Three (30%) of 10 patients who died in our study had SS, and the other patients’ stages were as follows: stage IIB , stage III , and stage IVA . The survival curve of the patients is presented in Flow cytometry of peripheral blood showed an increased ratio of CD4/CD8 (>2) in 13 (52%) patients. The ratio was between 2 and 10 in ten (40%) patients and higher than 10 in three (12%) patients. The patients’ disease stages and CD4/CD8 levels showed a statistically significant positive correlation (p=0.038). Increased CD4+/CD26 cell ratio was significantly correlated with poor survival (p=0.017). Loss of CD7 expression (more than 40%) was significantly related to poor survival (p=0.001).Laboratory findings are summarized in All patients received first-line therapy as a combination treatment of two or more of the following: psoralen plus ultraviolet, interferon-alpha, extracorporeal photopheresis, vorinostat, bexarotene, retinoid, low-dose methotrexate, local radiotherapy, or total skin electron beam radiotherapy. Of the 18 (72%) patients showing PD with first-line treatment modalities, 12 (48%) patients received either second- or third-line treatments. Of these 12 patients, six (48%) received second-line treatments either for the induction of remission or in an attempt to decrease tumor burden before allo-HSCT. Second-line therapy included single-agent chemotherapy of either gemcitabine or pralatrexate in 5 (20%) patients to decrease the tumor burden before allo-HSCT. One patient had a lymph node biopsy consistent with concomitant natural killer cell lymphoma and received an Aurora A kinase inhibitor as second-line therapy following five cycles of multiagent chemotherapy. All of the patients’ treatment responses with second-line treatment were evaluated as PD.Seven (28%) patients received third-line therapy due to PD. Additionally, three (12%) patients had received multiagent chemotherapy before first- and second-line treatments before admission to our center. In all patients, the treatment responses of the third-line treatments were evaluated as PD.Five (20%) patients with PD underwent allo-HSCT and CR was achieved in 3 (60%) of them after the procedure. Two patients’ disease recurred 2 and three months after allo-HSCT, and these two patients died 9 and 11 months following transplantation, respectively. One patient in follow-up with complete remission died 24 months after allo-HSCT due to sepsis. Autologous stem cell transplantation was performed in one patient in 2000, and the patient died four months after the procedure due to disease progression. The outcome of patients with HSCT is given in LCT of MF can occur at any stage of MF, and it has been associated with disease progression and poor outcome. Unfavorable prognostic factors for T-MF have been reported previously as advanced stage, presentation of MF with transformation, generalized skin tumors, increased LDH level, and use of combination chemotherapy ,15. CD30LCT at initial diagnosis of MF or within two years has been associated with worse prognosis in several studies ,16,17,18In previous studies, LCT of MF has been reported mainly in advanced disease. In a series with 22 T-MF patients, Arulogun et al.  reportedTransformed folliculotropic MF patients were previously found to have shorter survival time ,21,22. IAccording to a recent study, several clinical characteristics such as a new solitary nodule on MF plaques or rapidly presenting scattered papules may be indicators of the development of LCT for dermatologists . We woulThe treatment strategy is challenging in T-MF. It is important to note that, among our patients with advanced stages of T-MF, none had a CR to treatment under first-line therapies. Among patients receiving first-line therapy, 20% had either SD or PR with these therapies. Notably, all the patients who received second- and third-line therapies had PD. This finding highlights the refractory nature of T-MF. In fact, aggressive treatment strategies and multiple chemotherapies for MF result in a short period of CR, followed by an aggressive relapse . Allo-HSA limitation of the present study was the small number of patients with T-MF, highlighting the rarity of MF/SS. A second limitation was the retrospective design of the study, which may have restricted retrieval of the data from patient archives.Unfavorable prognostic factors in T-MF include advanced stage, high serum LDH levels, and loss of CD7 and CD26 expression in T helper cells. In patients with treatment-refractory tumors and unusual lesions, a biopsy is warranted to exclude T-MF. Patients with T-MF have a short life expectancy. Patients may have CR, PR, or SD with first-line treatments, which underlines the value of less aggressive therapies. However, nonresponders usually do not respond to second- or third-line therapies. Allo-HSCT may be an alternative option for patients with T-MF.