Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging viral pathogen that primarily causes respiratory illness. We conducted a seroprevalence study of banked human serum samples collected in 2012 from Southern Saudi Arabia. Sera from 300 animal workers (17% with daily camel exposure) and 50 non-animal-exposed controls were examined for serological evidence of MERS-CoV infection by a pseudoparticle MERS-CoV spike protein neutralization assay. None of the sera reproducibly neutralized the MERS-CoV-pseudotyped lentiviral vector. These data suggest that serological evidence of zoonotic transmission of MERS-CoV was not common among animal workers in Southern Saudi Arabia during July 2012. First reported in the Kingdom of Saudi Arabia (KSA) in September 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) is now frequently detected and causing high mortality among persons living or traveling in the Arabian Peninsula.3The majority of the initially reported cases of MERS-CoV in KSA were sporadic, associated with severe illness, and diagnosed in tertiary care hospitals.This study was approved by two institutional review boards . Banked serum samples were collected using convenience sampling in July 2012 from 300 animal-exposed and 50 non-matched human controls residing in Jazan region, KSA, as part of a study investigating human Rift Valley fever exposure. The cross-sectional, convenience sample of human sera was chosen for its self-reported exposure to domestic animals, such as camels, as studies have documented increasing evidence that camels are a reservoir for MERS-CoVAfter informed consent was obtained, demographic and domestic animal exposure data were collected using a structured questionnaire. Blood samples were then collected and transported on wet ice to the KSA Ministry of Health (MoH) laboratory in the Jazan region, KSA. Aliquots of sera were shipped to the University of Florida for serological assessment.Questionnaire data were used to analyze demographic characteristics and generate animal exposure data. In addition, an epidemic curve was developed using published reports to identify the dates of onset for the MERS-CoV cases in KSA.44 cells/well) with HIVMERS-S-pseudotyped lentiviral vector having 5 ng p24. The MERS-S pseudoparticles were pre-incubated with serially diluted sera for 60 min at 4°C and then added to the cells in triplicate. After 2 days of incubation, 100 μl of luciferase substrate, ONE-Glo luciferase Assay System , was added. The relative level of MERS-S pseudoparticle infection of the cells as luciferase activity was measured in a BioTek Synergy MX multimode plate reader  control. Polyclonal rabbit antisera to MERS-CoV obtained from the NIH/NIAID Rocky Mountain Research Laboratory was used as a positive control. Sensitivity of the assay in our laboratory was compared to that of the Peiris Laboratory at University of Hong Kong, where it was developed, by shipping an aliquot of this positive control which was independently titrated in the ppNT assay. Both our and Peiris' laboratory found the neutralizing titer of the polyclonal rabbit sera to be comparable to that measured by the Rocky Mountain Laboratories live virus neutralization assay (1:2560).A MERS-CoV spike protein pseudotyped lentiviral vector was constructed and provided by the laboratory of Malik Peiris of Hong Kong University. This assay is considered to be a more sensitive test compare to virus neutralization assay.The study population was predominately male between 15 and 84 years of age, of them, approximately 84% of participants were between the ages of 20 and 59 years. The participants were enrolled from the governorates of Abu Arish, Al Aridah, Al Darb, Al Aydabi, Baish, Dhamad, Fifa, Jazan, Sabya, and Samtah. A small percentage (16.9%) of participants reported themselves to be semi-nomadic, with possible movement outside of the Jazan region. Nearly half (44%) of participants were from the Jazan Governorate. Among participants, 25% reported some form of contact with domestic animals including camels, cattle, horse, goats, or sheep and 17% reported daily contact with domestic camels during the 12 months to enrollment see .All serum samples were screened in triplicate at a dilution of 1:20. A specimen was considered as screening positive if the average of the three wells containing the sera exhibited a >50% reduction in luminescence in comparison to the luminescence negative (BSA) or no serum control. Thirteen sera  screened positive at 1:20, but upon titration of these sera in subsequent assays, none of them were reproducibly able to neutralize the MERS-CoV pseudotyped lentiviral vector at a serum dilution of 1:20.The epidemiological curve of human MERS-CoV cases in KSA Figure reflectsThis study evaluated 350 banked serum samples collected in July 2012 from individuals with self-reported domestic animal exposure and controls in Jazan Region, KSA, for the presence of MERS-CoV neutralizing antibodies. None of the specimens tested positive using the MERS-S ppNT assay. Our negative results suggest population naivety to MERS-CoV infection in this region of KSA 1 month after the first earliest case was reported in another region of the country.As the first cases of MERS-CoV were identified, studies have postulated that camels are likely a reservoir for the virus in the Middle East.Similar to MERS-CoV, severe acute respiratory syndrome (SARS) virus began causing sporadic self-limited outbreaks in late 2002, after which it seemingly developed sustained human-to-human transmission, and spread internationally.There still remain many questions regarding designing appropriate interventions to prevent or reduce MERS-CoV transmission. Of them, identification of risk factors for MERS-CoV infection, a comprehensive understanding of natural animal reservoirs and vectors, and better determining transmission pathways seem to be paramount in developing appropriate interventions. Priority should be given to addressing these questions if we hope to improve public health response to future index cases in new geographical areas. It seems prudent to allocate resources to further globally enhance surveillance to quickly identify MERS-CoV and to monitor for viral genetic changes as well as to conduct the necessary epidemiological studies to fill the aforementioned gaps in our knowledge regarding this new and dangerous threat.


Thus, estrogen exposure induced feminization of the male liver transcriptome in a sex-role-reversed pipefish. These results suggest that the ancestral state of estrogen-regulated female reproductive physiology has been retained in all sex-role-reversed vertebrates thus far studied, despite substantial evolution of the hormonal regulation of ornamentation and mating behavior in these interesting taxa.Species exhibiting sex-role reversal provide an unusual perspective on the evolution of sex roles and sex differences. However, the proximate effects of sex-role reversal are largely unknown. Endocrine disruptors provide an experimental mechanism to address hormonal regulation of sexually dimorphic gene expression in sex-role-reversed taxa. Here, we investigate gene expression patterns in the liver of the sex-role-reversed Gulf pipefish, because the liver is known to be sexually dimorphic and estrogen-regulated in species with conventional sex roles. Using next-generation RNA-sequencing technology (RNA-seq), we detected sexually dimorphic hepatic gene expression patterns, with a total of 482 differentially expressed genes between the sexes in Gulf pipefish. Two-thirds of these genes were over-expressed in females, and the sex-specific transcriptomes of this sex-role-reversed pipefish’s liver were superficially similar to those of fishes with conventional sex-roles. We exposed females, pregnant males, and non-pregnant males to 17α-ethinylestradiol (EE2) at ecologically relevant concentrations of 5ng/L and compared gene expression patterns in the livers of exposed fish to control fish. Several genes that were up-regulated in EE2-exposed males relative to control males were also found to be female-biased in control animals. These genes included several of the classic estrogen biomarkers, such as  Sex-role-reversed species, in which females tend to evolve elaborate secondary sexual traits and males tend to be choosy, have enjoyed a rich history in the study of the evolution of sex differences and sex roles. In the sexual selection literature, sex-role reversal is usually defined as the situation in which sexual selection acts more strongly on females than on males , and it The phenotypic manifestations of sex-role reversal include traits such as showy ornaments and increased aggression in females, as well as marked mating preferences in males . In addiThe endocrinology of sex-role-reversed species has perhaps generated more questions than answers. One general pattern is that female maturation and cycles of gamete production are controlled by estrogens, whereas male primary sexual traits are controlled by androgens, in much the same way as reproduction is controlled in species with conventional sex roles ,10. HoweSyngnathus scovelli) liver exposed to the potent endocrine disruptor 17α-ethinylestradiol (EE2). We chose to study the Gulf pipefish because it has become an excellent model for the study of many aspects sex-role reversal, and it is an emerging model in the realm of sex-role-reversed endocrinology [vitellogenin and zona pellucida isoforms, are known to be estrogen dependent [One noticeable void in the study of proximate mechanisms of sex-role reversal exists in the realm of transcriptome-level responses of sexually dimorphic tissues to hormonal manipulation. Here we begin to fill this gap by studying the transcriptome of the Gulf pipefish  and Nerophis lumbriciformis [Studies regarding the effects of environmental estrogens in Gulf pipefish and its close relatives provide context for the present study. In particular, we know that external female morphology is estrogen-dependent, because exposure of male Gulf pipefish to 17α-ethinylestradiol (EE2) results in the development of female-like secondary sexual traits ,26. In aciformis ,27,28, iciformis . This obciformis ). HoweveDanio rerio), a model organism with conventional sex roles.Our second goal was to quantify gene expression changes in the liver in response to estrogen exposure in females, pregnant males, and non-pregnant males. We hypothesized that the general pattern of expression changes would be toward feminization of male livers. However, if multiple endocrine signaling pathways have been altered to different degrees by the evolution of sex-role reversal, then we might expect an intermediate pattern, in which some genes are very strongly affected by EE2 while others are relatively resistant even though they are nevertheless sexually dimorphic in their expression patterns. We further assessed whether our observed gene expression patterns are sex-role reversed by comparing our results to those from the zebrafish  and the remaining 33% showed higher expression levels in males (157 contigs)  . The gencontigs) .vitellogenin b and c, choriogenin h, and zona pellucida sperm-binding protein 4-like, which have all been shown to be involved in egg development and maturation [estrogen receptor alpha (esr1) was also highly up-regulated in control females. Several of the genes with the largest differences in expression levels, such as extracellular serine threonine kinase fam 20c-like (Fam20c), were labeled as “cellular response to estrogen stimulus” genes in the Blast2GO analysis. Many of these strongly sexually dimorphic genes have also been shown to have female-biased hepatic expression profiles, particularly in response to estrogen exposure [3-hydroxy-3-methylglutaryl-coenzyme a reductase (hmgcra), methylsterol monooxygenase 1(msmo1), and lanosterol 14-alpha demethylase-like (CYP51A1), in addition to the aforementioned female-specific genes. Of the 157 genes that were up-regulated in males, only one transcript, histidine triad nucleotide-binding protein 3-like  were upregulated by greater than 20 fold in pregnant males, and one gene (cartilage acidic acid 2) was upregulated greater than 20-fold in non-pregnant males , extracellular serine threonine protein kinase fam20c-like, and choriogenin h, among others , the majority were not. In fact, most EE2 responsive, down-regulated genes in zebrafish were male-biased (38 of 57). However, in pipefish, only 11 of 51 down-regulated genes were male-biased.We found that the majority of the sex-biased genes in zebrafish and pipefish did not overlap, with only nine female-biased genes shared between non-exposed females of the two species . In the This first study of the effects of a potent endocrine disruptor on gene expression patterns in a sex-role-reversed organism shows that the liver of the Gulf pipefish is markedly sexually dimorphic and responds very strongly to an estrogenic compound. For instance, several female-biased genes respond to EE2 exposure in males in a direction indicative of feminization of the male liver transcriptome. Control fish follow a pattern predicted by the hypothesis that both pregnant and non-pregnant males should differ from females in terms of expression patterns as a consequence of the major role of the liver in producing egg-related proteins. Our results also show that fish exposed to synthetic estrogen show expression patterns consistent with the idea that, even in sex-role-reversed species, female-biased liver genes should be regulated by estrogen. In particular, we see a shift toward more feminine patterns of gene expression in males exposed to EE2. Some aspects of our analysis raise the intriguing possibility that the liver plays a role in not only female reproduction but also male reproduction, as sperm-related transcripts occur in the liver, an observation that leads to the hypothesis that males may also be capable of transporting proteins from the liver to the brood pouch. In addition, as we will explain below, we believe our results leave the door open for sexual selection to act on genes expressed in the liver that ultimately play a role in postcopulatory sexual selection or sexual conflict.Sex-role-reversed organisms represent an evolutionary challenge from an endocrinology perspective ,10, becavitellogenin b and vitellogenin c, showed greater than 1000-fold higher levels of expression in females compared to males. Our EE2 results establish that these expression patterns are largely estrogen-regulated, because males exposed to EE2 expressed genes that were normally only found at high levels in females. Estrogen profiles have been studied in other sex-role-reversed taxa, notably birds, and in general results show that estradiol profiles are similar to non-sex-role-reversed birds [Our study is most relevant to female reproductive physiology and indeed represents the first dataset addressing transcriptome-wide effects of hormone manipulation in any reproduction-related tissue from a sex-role-reversed species. Our results provide the first evidence that the pipefish liver shows genome-wide patterns of dimorphism, with hundreds of genes differentially expressed between males and females. Some transcripts, such as ed birds ,38,39. Ted birds .The situation becomes more confusing when we turn attention to ornamentation in sex-role-reversed taxa. In pipefish, female secondary sexual traits occur in males exposed to EE2, suggesting that female ornamentation is controlled by estrogens ,26,28. HPerhaps the most interesting and demanding area of research in the realm of sex-role-reversed endocrinology concerns the modulation of female behaviors involved in mating competition, and researchers have thus far only scratched the surface of this difficult topic. Almost no work has addressed this topic in the family Syngnathidae, but one study  and our In many ways, the Gulf pipefish liver transcriptome behaves in a similar fashion to those of the few other fish species studied in this regard. In control fish, pregnant and non-pregnant males shared more similar gene expression patterns than either group shared with females, and both groups of males responded in a female-like manner to synthetic estrogen. The most telling result is that genes involved in egg production are highly upregulated in females relative to males, a typical pattern in the livers of fish species with conventional sex roles. In addition, some of the most highly expressed genes in the livers of females also encode egg-related proteins and EE2 exposure induces expression of these genes in males.However, at face value, our patterns of gene expression look quite different from those observed in zebrafish , with onAnother intriguing result from our analysis is that the low doses of EE2 used in our study resulted in masculinization of the female liver. This pattern is evident from our observation that 90 percent of sexually dimorphic genes showed expression changes in opposition to the pattern of sexual dimorphism in exposed females . At face value, this observation might be interpreted as evidence that the female liver transcriptome is sex-role-reversed with respect to its response to estrogens. However, this sort of pattern has been observed in other taxa without sex-role reversal ,46, and One interesting possibility, which is hinted at by our data but not definitively supported by any of our analyses, is that the liver somehow plays a role in sexual selection in sex-role-reversed pipefish. The female liver produces egg-related proteins, which are transported to the ovaries, processed and packaged in the eggs. In sex-role-reversed species, females typically have greater potential reproductive rates than males, which can result in pre- and postcopulatory competition among females for mating and reproductive success. Hence, female-specific genes in the liver controlling egg production could be under stronger sexual selection in females of sex-role-reversed species compared to related species with less mating competition among females, such as monogamous pipefishes and seahorses. Another plausible hypothesis is that other proteins produced by the liver are also transported to the female’s reproductive tract and eventually transferred to the male during mating. Even though most studies of reproductive protein evolution focus on the gonads, the liver may be a neglected organ with an underappreciated role in postcopulatory processes. Interestingly, our data also show that some sperm-related proteins in males are produced in the liver, so males also could experience selection on genes expressed in the liver whose products are ultimately transported to the gonads or brood pouch. This idea remains speculative at the moment, but we feel that the role of the liver in postcopulatory processes, especially in sex-role-reversed organisms, is a topic that deserves further exploration.vitellogenin is upregulated in EE2-exposed pipefish males [Syngnathid fishes are potentially useful for the study of endocrine disruptors in marine environments, because many pipefishes and seahorses occur in shallow coastal waters throughout the world . Furthersh males ,28. Our sh males ,49,50. HOur results provide an important advance in the endocrinology of sex-role-reversed vertebrates. In particular we found that the pipefish liver is sexually dimorphic and estrogen regulated, suggesting that female reproductive physiology is regulated by estrogens in pipefish in much the same way as in fish without sex-role reversal. In light of other work on sex-role-reversed taxa, primarily birds, this result suggests that the ancestral mechanism of hormonal control of female reproductive physiology has been retained across sex-role-reversed vertebrates. However, a survey of the literature shows that secondary sexual traits are controlled by a variety of mechanisms, and pipefish are somewhat unusual in that estrogens induce female ornaments in males. Finally, sex-role-reversed mating behaviors remain a mystery, as their proximate modulation has not yet been resolved in any sex-role-reversed taxon. Our results also provide novel pipefish transcripts that respond to endocrine disruptors, which could be extremely useful for screening coastal marine habitats for environmental estrogens. In short, this study provides novel insights into sexual dimorphism and hormonal regulation of gene expression patterns in the pipefish liver, setting the stage for future studies regarding the endocrinology of sex-role-reversed mating behavior in this interesting marine fish.th, 2013 under the Texas Parks and Wildlife permit number SPR-0808-307. This study was approved by the Institutional Animal Care and Use Committee at Texas A&M University  and fish were killed using an overdose of MS-222. We collected only pregnant males and females with well-developed iridescent bands, ensuring that all individuals were sexually mature. Males were allowed to give birth before being used in the experiment. Fish were dipped in freshwater for ten minutes to remove any external parasites and then acclimated to 26-ppt salinity tanks at Texas A&M University. The 17α-ethinylestradiol powder, of 98% purity, was acquired from Sigma (#SLBF2546V) and dissolved in 200-proof ethanol from Sigma (#SHBD6226V). All tanks held 7 liters of water and were initially dosed with 50 μl of either a 7ng/10μl stock EE2 ethanol solution to have tank concentrations of 5ng/L EE2 or 50 μl of ethanol without EE2 for the controls. The chosen concentration of 5 ng/L EE2 was selected because it is within the range of EE2 found in contaminated bodies of water in nature and also because long-term exposure to this concentration has been shown to cause entire breeding populations of some fish species to collapse [Gulf pipefish were collected from Redfish Bay near Aransas Pass, Texas  on July 8collapse ,51,52. TTo generate each pregnant male, we placed one non-pregnant male and one female together in a clean, EE2-free saltwater tank until the male became pregnant. Each pair was also assigned a non-pregnant male, size matched with the pregnant male but not paired with a female. On the second day of the male’s pregnancy, all three fish were photographed, measured, and randomly assigned to treatments of either experimental tanks of 5ng/L EE2 concentrations or the EE2-free control tanks on the second day of pregnancy. The three fish were held in 7L tanks with glass dividers between the fish that allowed for water exchange but otherwise kept the fish isolated from one another. After seven days of exposure, that is, on the ninth day of the male’s pregnancy, fish were sacrificed using MS-222 and the liver was dissected. Dissections were done with the fish soaked in RNAlater under a microscope with tissue immediately frozen on dry ice and stored in the -80°C freezer. The control and 5ng/L EE2 treatments were replicated 5 times resulting in a total of 30 fish, including 5 females, 5 pregnant males, and 5 non-pregnant males from each treatment .RNA was isolated from the dissected livers using a TRIzol® Reagent  extraction method modified from . Total RA total of 805 million 150 bp paired end reads were obtained from the MSU Genomics core and were then trimmed with Trimmomatic  using thAfter assessing differential expression in all pairwise treatment comparisons, we identified genes that had at least a 2-fold expression difference between control females and control males. These genes were considered to be sexually dimorphic, and their expression patterns were analyzed in all groups, including EE2-exposed fish, using heat maps. Heat maps were generated in R  using thTo address whether the expression patterns we found showed signs of sex-role reversal, we compared our results to those from a similar study in zebrafish , a speciS1 FigAlthough the treatments of the same sex group together, EE2 exposed pregnant males cluster closer to the female treatments than other male groups.(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


This miRNA has a restricted phylogenetic distribution, providing an opportunity to examine the origin and evolution of a new miRNA within the intron of a developmentally-important homeobox gene.Introns represent a potentially rich source of existing transcription for the evolution of novel microRNAs (miRNAs). Within the Hox gene clusters, a miRNA gene, HOXC5 in humans is accompanied by expression of miR-615 in all cases, but that the miRNA can also be transcribed independently of its host gene through the use of an intragenic promoter. We present scenarios for the evolution of miR-615 through intronic exaptation, and speculate on the acquisition of independent transcriptional regulation. Target prediction and transcriptomic analyses suggest that the dominant product of miR-615 is involved in the regulation of growth and a range of developmental processes.Alignment and structural analyses show that the sequence is highly conserved across eutherian mammals and absent in non-mammalian tetrapods. Marsupials possess a similar sequence which we predict will not be efficiently processed as a miRNA. Our analyses suggest that transcription of miR-615 gene evolved within the intron of Hoxc5 in the ancestor of placental mammals. Using miR-615 as a case study, we propose a model by which a functional miRNA can emerge within an intron gradually, by selection on secondary structure followed by evolution of an independent miRNA promoter. The location within a Hox gene intron is of particular interest as the miRNA is specific to placental mammals, is co-expressed with its host gene and may share complementary functions.The The online version of this article (doi:10.1186/s13227-015-0027-1) contains supplementary material, which is available to authorized users. We collate, analyse and integrate data on the regulation, expression pattern and likely biological roles of miR-615, in concert with comparative analysis, and propose how the origin of this miRNA contributed to eutherian evolution and development.Intronic miRNAs provide an opportunity to gain insight into de novo miRNA innovation and its contribution to the evolution of development. Pre-existing transcribed RNAs, particularly in the form of introns, are thought to be a major source from which new miRNAs evolve , 2. HeremiR-10 gene is conserved between arthropods and mammals and occurs in the same position in insect and vertebrate Hox clusters [miR-196 and the bi-directionally transcribed miR-iab-4 genes are chordate-specific [miR-615, is less well-characterised and has been reported to be restricted to either mammals or amniotes [miR-615 is currently lacking. Unlike miR-10, miR-196 and miR-iab-4, which are intergenic, miR-615 is nested within an intron of the Hoxc5 gene in mouse and human. This positioning facilitates the evolutionary analysis of miR-615 because the highly conserved nature of Hox gene exons allows alignment and comparison of intronic regions.Homeobox genes form a large, diverse superclass with a wide range of roles in metazoan developmental biology. Most encode transcription factors which exert regulation over the expression of other genes during embryogenesis, thereby having a direct influence on the resultant adult morphology . Perhapsclusters . The miRamniotes . A detaimiR-615 occurs in the same orientation as Hoxc5, it is likely to be co-transcribed with its host gene. Work on other miRNAs supports a general concept of co-expression of intronic miRNAs with their host transcripts [As nscripts , 13, as nscripts , althougnscripts , 15, 16.mir-615 across tetrapods. We perform structural predictions and evaluate minimum folding energies to evaluate whether candidate pre-miR-615 transcripts could be efficiently processed into mature miRNAs, and argue that this most likely occurs only in eutherian mammals. We also integrate RNA-seq data with chromatin modification and expressed sequence tag (EST) studies to argue for the existence of a promoter specific to mir-615 located within the transcription unit of Hoxc5. Finally, we carry out target predictions for the dominant 3p product of miR-615 and compare these to expression domains to suggest likely in vivo functions of this miRNA, and how these contributed to the evolution and development of mammals.Using DNA sequence data, derived from the public domain and obtained through our own experiments, we investigate the phylogenetic distribution of Hoxc5 intronic sequences, along with those from Xenopus tropicalis and Anolis carolinensis were obtained from the UCSC Genome Browser [Sarcophilus harrisii sequence was obtained from Ensembl while Macropus eugenii sequence data were obtained from GenBank (accession: JN378720.1). Monotreme DNA was obtained from Stephen Donnellan in the South Australian Museum via the Oxford University Museum of Natural History (CITES registration number: GB026). Three species were sampled: Platypus (Ornithorhynchus anatinus), Short-beaked echidna (Tachyglossus aculeatus) and Long-beaked echidna (Zaglossus bruijni). Xenarthran samples were provided by Mads Bertelsen at the Copenhagen Zoo; xenarthran genomic DNA was obtained from sloth (Choeloepus didactylus) heart and anteater (Myrmecophaga tridactyla) liver by phenol–chloroform extraction. Data for American pika (Ochotona princeps), lesser Egyptian jerboa (Jaculus jaculus) and common shrew (Sorex araneus) were obtained by running BLASTn using the hsa-miR-615 sequence against draft genome assemblies for these species. Alignment was carried out using MAFFT [Eutherian  Browser  and the  Browser . The Sarng MAFFT .Hoxc5 intronic sequences are: 5′-TTGGACTTAAGCATCACTTTCCCACCG-3′ and 5′-CCAGAGTCTGGTAGCGCGTGTAACTGG-3′. These were designed to bind a region in the flanking coding exons conserved between most tetrapods .Data used to determine expression of mir-615 and mRNA expression is provided in Additional file p value of <0.05 was used to determine expression. Raw data for the transcriptome assemblies were quality checked using FastQC, followed by trimming of the first 15 bases using the fastqtrim.py script. Trimmed reads were assembled using Trinity and resultant contig FPKMs determined with RSEM. BLASTn with standard parameters was used to determine Hox gene expression in each transcriptome, with appropriate sequences from the corresponding species obtained from GenBank. Small RNAseq data were analysed through the miRDeep2 pipeline using the quantifier.pl script to map reads onto known human miRNAs. Handling of raw reads prior to mapping was carried out as described in Quah et al. [A list of publicly released datasets used in bioinformatic analysis of h et al. . Read coh et al.  were obth et al. .http://www.uniprot.org/ [Target prediction for hsa-miR-615 was run using the downloadable executable for the PITA prediction algorithm  on all hrot.org/ ). RPKM vrot.org/ , except mir-615 promoter were obtained using ENCODE data tracks available through the UCSC Genome Browser (http://genome.ucsc.edu/ENCODE/). The EST data track used (‘Human ESTs Including Unspliced’) was generated at UCSC from publicly available EST sequencing data.Histone methylation, DNase I and CAGE data used to find the position of the miR-615 is reported as being restricted to either mammals or amniotes  but currmir-615 on miRBase [Monodelphis domestica), a monotreme (Ornithorhynchus anatinus) and three non-mammalian vertebrates . The non-eutherian species did not produce definitive matches, while a positive control BLASTn run using the same parameters successfully recovered mir-615 in mouse (Mus musculus). Within eutherian mammals, we were able to identify the mir-615 gene by BLASTn in the elephant , but not in the sloth .Available entries for  miRBase  are limimir-615 within a highly conserved Hox gene allows for direct comparisons of the corresponding region to be made between vertebrate species. In human and mouse, mir-615 is located within the intron of Hoxc5. We performed alignments of this intron and its flanking coding exons across the four main eutherian lineages , non-eutherian mammals and other tetrapods to examine sequence conservation across the syntenic position of mir-615  and anteater (Myrmecophaga tridactyla) using primers designed against highly conserved regions within vertebrate Hoxc5 coding regions  and the tammar wallaby (Macropus eugenii) possess a sequence resembling eutherian mir-615 within Hoxc5. However, the regions corresponding to the mature miR-615 products differ slightly from the corresponding eutherian versions. We were unable to amplify the Hoxc5 gene from any monotremes with the same primers. This genomic region is also missing in the platypus (Ornithorhynchus anatinus) genome assembly.The syntenic position of oci Fig. . In marsmir-615 region is strongly conserved at the nucleotide sequence level within the Eutheria, and to a lesser extent in Metatheria, and confirm the lack of an orthologous sequence in monotremes and non-mammalian tetrapods  are higher than those observed for all eutherian miR-615 precursors . Both marsupial sequences also have large internal loops, bulges and mismatches which are likely to preclude efficient entry into the miRNA pathway  studied possess a sequence similar to the annotated se Figs. , we analway Fig. . These pUsing small RNA deep sequencing data from the ENCODE project  obtainedmir-615 is increased during pregnancy  revealsmir-615 and those of Hoxc5 would help test whether expression of mir-615 is solely driven by its host transcription unit, or whether there is independent regulation of this intronic miRNA. Out of the 36 cell lines and tissue types used to survey miR-615 expression, 34 also had available RNA-seq data  data, which identify potential initiation sites within the HOXC5 locus through the detection of 5′ capped RNA transcripts. These sites map to both the known HOXC5 TSS and a region about ~200 bp upstream of the exon–intron boundary, corresponding well with the 5′ ends of several expressed sequence tags (ESTs). This position also aligns with the DNase I hypersensitivity sites. Collectively, these analyses of chromatin accessibility and transcription at the HOXC5 locus, coupled with evidence for expression of mir-615 in the absence of HOXC5, strongly support of the existence of an internal miRNA-specific promoter, in addition to the HOXC5 gene promoter.The strongest indication of an independent promoter for mir-615, hence we undertook target prediction analysis for miR-615-3p against all known human cDNAs  genome assembly, or amplify its intron through PCR in three monotreme species . It is plausible that monotremes have undergone lineage-specific loss of part of the Hoxc cluster; posterior Hoxc genes (Hoxc10–Hoxc13) are present in the platypus draft genome while Hoxc4, Hoxc5, Hoxc6, Hoxc8 and Hoxc9 are absent [miR-615 could therefore either have been lost with the Hoxc5 gene, and we did not detect in any other genomic region. Given that that none of the 17 eutherian species studied across the four major eutherian lineages have lost this miRNA or significantly altered either of its mature products, we argue that mir-615 is more likely to be a eutherian novelty rather than the alternative scenario where it arose on the common mammalian lineage and was lost in monotremes and marsupials.The presence of functional miR-615 in eutherians and its absence from marsupials could be explained by either the acquisition of e absent . A hypotIntronic exaptation has been proposed as a major contributor to miRNA innovation , 2. UndeHoxc5 intronic regions in marsupials and eutherians, with a precise stem-loop structure present in eutherians but not marsupials, presents a strong case for the evolution of miR-615 through intronic exaptation.A possible model for the origin of miR-615, involving eutherian-specific origin via intronic exaptation, is shown in Fig. Hoxc5 intron of modern marsupials resembles an approximate hairpin more than a completely random fold , the corresponding region is not annotated as a CpG island, and the GC content of the Hoxc5 intron is lower in marsupials than in eutherians . It is more likely that the CpG island was gained in eutherians, rather than present in a common ancestor and lost in marsupials [We hypothesise that the acquisition of the CpG island in eutherian en noted , and is en noted . Interna rapidly , which wrsupials .insulin-like growth factor 2 (IGF2) transcript. However, the involvement of miR-615 in non-pathologic biological processes is currently unknown.Several studies have explored the roles of miR-615 in the context of human pathologies , 42–45. mir-615 across the Eutheria is indicative of biological function. Our analysis of data from Chiang et al. indicates that whole-body expression of miR-615 increases throughout mouse embryonic development and persists in the newborn [The conservation of  newborn . Althoug newborn . Known t newborn  is strikHoxc5 during axial patterning is likely to result in production of miR-615-3p and may account for some of the observed expression, axial Hox gene expression is initiated prior to E9.5 [Ovis aries) lends some weight to the hypothesis that miR-615 may be involved in gonadal differentiation [We also find that GO terms associated with regulation of growth and development are overrepresented by in a high stringency target prediction dataset for miR-615-3p. This observation, coupled with the high expression of miR-615-3p in the mouse embryo, suggests a potential role for miR-615-3p in the regulation of growth and development during embryogenesis. Although the expression of  to E9.5  and is u to E9.5  suggests to E9.5 , 50, sex to E9.5  and outg to E9.5 . A studyntiation , since tntiation , 43.mir-615 represents an interesting case of acquisition of a novel miRNA by intronic exaptation and an unusual example of a new functional gene arising within a conserved Hox gene cluster. We demonstrate using sequence analysis and structural prediction that miR-615 is a eutherian-specific miRNA, although a similar sequence exists in extant marsupials that may have a low probability of miRNA processing. Using a data mining approach combining epigenetic studies with RNAseq and CAGE tag data, we show that miR-615 can be expressed under the control of a Hox gene promoter, as well as independently from its host gene transcript. Based on these observations, we propose a model for intronic miRNA evolution through selection on intronic secondary structure followed by evolution of an independent miRNA promoter. Target prediction data support the involvement of miR-615 in growth and developmental pathways.The Hox cluster miRNA


The link between mental representation (MREP) structures and motor performance has been evidenced for a great variety of movement skills, but not for the human gait. Therefore the present study sought to investigate the cognitive memory structures underlying the human gait in young and older adults. In a first experiment, gait parameters at comfortable gait speed (OptoGait) were compared with gait-specific MREPs  in 36 young adults. Participants were divided into a slow- and fast-walking group. The proven relationship between gait speed and executive functions such as working memory led to the hypothesis that gait pattern and MREP differ between slow- and fast-walking adults. In a second experiment, gait performance and MREPs were compared between 24 young (27.9 years) and 24 elderly (60.1 years) participants. As age-related declines in gait performance occur from the seventh decade of life onward, we hypothesized that gait parameters would not be affected until the age of 60 years accompanied by unchanged MREP. Data of experiment one revealed that gait parameters and MREPs differed significantly between slow and fast walkers. Notably, eleven previously incurred musculoskeletal injuries were documented for the slow walkers but only two injuries and one disorder for fast walkers. Experiment two revealed no age-related differences in gait parameters or MREPs between healthy young and older adults. In conclusion, the differences in gait parameters associated with lower comfortable gait speeds are reflected by differences in MREPs, whereby SDA-M data indicate that the single limb support phase may serve as a critical functional period. These differences probably resulted from previously incurred musculoskeletal injuries. Our data further indicate that the human gait and its MREP are stable until the age of 60. SDA-M may be considered as a valuable clinical tool for diagnosis of gait abnormalities and monitoring of therapeutic effectiveness. It is widely accepted that skilled motor performance draws on general and task-specific mental representations (MREPs) that are linked to perceptual effects and correspond to functional structures of movement kinematics cf. . MREPs aAging is commonly associated with changes in the biomechanics of gait  from theAlthough gait is a largely automated motor task, cognitive resources are required for normal walking . The relGait patterns can drastically change in the course of normal aging  and in cA well-documented method that provides psychometric data on the structure and dimensions of the MREP of complex movements in LTM is the structural dimensional analysis of MREP (SDA-M) introduced by That said, while nearly all aspects of the human gait have been examined in the past, to date there are no studies which have investigated the cognitive memory structures underlying the human gait either in young or in older adults. The present study sought to investigate these issues within two experiments.In a first experiment, it was investigated whether gait-specific LTM structures reflect kinematics and kinematic differences in the human gait. Therefore, spatio-temporal  and temporophasic  gait parameters as well as gait variability (coefficient of variation of gait parameters) at comfortable gait speed (assessed with OptoGait) were compared with gait-specific LTM structures (assessed with SDA-M) in 36 young adults. As gait speed has been established as an important performance measure , subjectn = 24) from experiment one. As age-related declines in gait performance occur from the seventh decade of life onward .n = 18) and fast- (n = 18) walking adults by splitting height-adjusted gait speed at the median. Demographic and clinical subject characteristics are provided in Table 1.Thirty-six young adults volunteered to participate in this study. Participants who suffered from severe, acute gait-affecting injuries or reported to be restricted in their gait were excluded prior to testing. The remaining subjects were divided into slow-  in a quiet room, with no auditory or visual interference for the subjects. Six transmitting and six receiving bars were placed parallel to each other (distance between bars: 1 m). The subjects were instructed to wear closed shoes with heel height not exceeding 3 cm . They waFigure 1).With regard to generally recognized models of the human gait , a gait Figure 1), were used as basic action concepts  of the human gait in the splitting procedure of the SDA-M. The last period, terminal swing, was not used for the splitting procedure as pilot data demonstrated that terminal swing and initial contact are not clearly distinguishable from each other and, thus, would distort the results. During the splitting procedure, the pictures were presented on a 19 inch computer screen with always one picture in the anchoring position  to which participants classified the remaining N-1 pictures  as similar or dissimilar to the anchoring picture . Participants were asked to decide via pressing keys for ‘yes’ and ‘no’ whether the presented pictures are closely related in the gait cycle or not. After all judgments were made for an anchoring picture, another BAC randomly occupied the anchor position, and all other BACs were compared to this anchoring picture until each of the BACs was in the anchoring position. This splitting procedure delivered an Euclidean distance scaling between all BACs of the gait cycle. A following hierarchical cluster analysis then outlined the individual structure of the given set of BACs with a factor analysis revealing the feature dimensions of these individual cluster solutions  adjusted for physical activity. The level of significance was set at P ≤ 0.025 . All data were analyzed using the SPSS statistical package 20.0 . The effect size was calculated with G∗Power  in the tables. If appropriate, data are reported as mean difference (MD) and 95% confidence interval (95% CI).Data were checked for normal distribution using the Kolmogorov–Smirnov test. Differences between the groups  were tested for significance with the chi-squared tests, unpaired Student’s dcrit = 3.53, reflecting an alpha level of α = 0.050, was used to determine the statistical relevance of links between BACs with only links below this critical value being considered as statistically relevant . Between-group comparisons  of the cluster solutions derived from SDA-M were performed by determining the structural invariance (λ) between the grouped cluster solutions . According to crit = 0.68 was used to determine significance at an alpha level of α = 0.050. Moreover, we captured the number of sequencing errors  from the individual cluster solutions and compared the average values between the groups using the unpaired Student’s t-test.For all cluster analyses a critical value of Slow- and fast-walking young adults did not differ significantly in anthropometric data and physical activity.Table 1). The mean values of self-selected walking speeds were as follows: 1.32 m/s (±0.14 m/s) for the slow-walking group; 1.53 m/s (±1.52 m/s) for the fast-walking group.A total number of 32 and 28 steps was analyzed in slow- and fast-walking young adults, respectively . The two spatio-temporal gait parameters  were reduced in the slow-walking group by 11.5%  and 11.6% , respectively. Slow-walking adults further demonstrated a significantly longer stance time  and shorter swing time . Within the stance phase, single support time was shorter  and load response time  as well as pre-swing time  were longer in the slow-walking group. The only difference in CV could be documented for single support time with a higher value  in slow-walking adults. Spatio-temporal and temporophasic gait parameters and corresponding CV are reported in Figures 3A,B, cluster analysis of mean group dendograms revealed distinct clustering  in both slow- and fast-walking young adults. In both groups BACs have been clustered in two functional units, but of different structures. In slow-walking adults there were distinct clusters for BACs 1–2  and BACs 3–7  while in fast-walking adults there were two distinct clusters for BACs 1-3 and BACs 4–7 . Statistical analysis of invariance revealed the structural differences between slow- and fast-walking adults to be significant . The number of sequencing errors did not differ between slow-  and fast-walking adults .As can be seen in In the first experiment, we evaluated whether gait-specific LTM structures reflect kinematic differences in the human gait. The established relationship between gait speed and executive function such as working memory  led to tTable 1). In the slow-walking group, 64% of the participants suffered from knee injuries.Cluster analysis revealed two functional clusters for each group with BAC 3 (mid stance) clustered together with BACs 1–2  in the fast-walking group or with BACs 4–7  in the slow-walking group, suggesting that the mid stance  is a critical functional period for gait speed. The separation of the two functional periods within the single limb support phase (BACs 3–4) in the fast-walking adults suggests that for a well-coordinated gait cycle the single limb support phase serves as a  transition between the two double support phases (and the changing responsibilities of the two feet). In slow-walking adults this transition seems to be impaired  resulting in prolonged double support phases and a reduced gait speed which are probably due to a history of musculoskeletal injuries. We have documented a total of 11 previously incurred musculoskeletal injuries for the slow walkers (13–144 months before) but only two injuries and one disorder for fast walkers . Consequently, all spatio-temporal gait parameters were normalized to height and physical activity was entered as a covariate into an ANCOVA to account for these differences between groups.With regard to the differences between slow- and fast-walking adults found in experiment one, 12 young adults from the first experiment with a history of musculoskeletal injuries/disorders were excluded from the following analyses. Healthy young and elderly adults differed in height and physical activity. Elderly subjects were significantly smaller and less physically active than young adults (Table 2). The mean values of self-selected walking speeds did not differ significantly between groups , 22.3%  and 28.2%  in young subjects, respectively. Further between-group differences in CV were not observed. Figures 3C,D. Cluster analysis of mean group dendograms revealed that in both healthy young  and healthy older adults  BACs have been clustered in two functional units forming distinct clusters for BACs 1-3  and BACs 4-7 . Statistical analysis of invariance revealed the cluster solutions to be identical . The number of sequencing errors was higher in older  than in young adults .Gait-specific representation structures of healthy young and older adults are displayed in In the second experiment, healthy young and elderly subjects were compared with respect to gait performance and MREPs. Although the higher number of sequencing errors is not reflected by the average representation structure, it is probably associated with the higher CV in older adults.Spatio-temporal and temporophasic parameters of gait at a comfortable gait speed differed between slow- and fast-walking young adults. These differences in human gait patterns associated with lower comfortable gait speeds are reflected by structural differences in gait-related memory structures in LTM, probably due to previously incurred musculoskeletal injuries. The results further indicate that the human gait and its MREP are stable until the age of 60 in healthy subjects.Figure 1). Thereby, the single limb support phase (BACs 3–4) seems to be an important transition phase for a well-coordinated gait performance which is not strongly reflected in LTM structure and clustered to the preceding and subsequent phases. The results indicate that MREP of the human gait rather draws on an optimal interaction between both feet and the changing responsibilities than on functional phases within one foot.Notably, LTM representations of the gait were found to be functionally structured in tree-like hierarchies across all participants in the present study. As well-structured, hierarchically organized mental representations are typically associated with expert performance see  is not cWith regard to a potential clinical use of the SDA-M, future research should examine how gait-related memory structures are affected by different disorders of the lower extremities, how long structural abnormalities persist in LTM and which therapeutic measures are most efficient for regaining normal gait patterns. Moreover, normative cluster solutions of human gait patterns for various age groups, including smaller age spans in children and older adults , should be developed to inform and support clinicians regarding the diagnosis and monitoring of gait abnormalities based on cognitive structures.Notwithstanding the necessity of extended research on this topic, SDA-M may be considered as a valuable clinical tool for the diagnosis of (current and previous) gait abnormalities and for monitoring the effectiveness of therapeutic measures.TS, AM-M, and MB developed the study concept, and all of these authors contributed to the design in collaboration with RS and RB. RJ, MB, and AM-M collected the data and analyzed it in collaboration with TS. AM-M and TS wrote the first draft of the manuscript, and MB, RJ, RS, and RB helped to edit and revise it. All authors approved the final, submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DearEditor,Apoptosis is a fundamental physiological process in mammals in which cells die by activating a suicide mechanism. The mitochondria are one of the major checkpoints in apoptotic regulation because they serve as sensors and amplifiers of cellular damage . These data suggested the involvement of chymotrypsin in mitochondrial fission during apoptosis.We found that mitochondrial fission was an event that occurs downstream of LMP in early stage apoptotic cells. Confocal microscopic images indicated that the mitochondria tended to fuse when the lysosomes are intact, whereas 6-h LeuLeuOMe treatment, which induced LMP, ultimately led to mitochondrial fission Fig. F. PretreMitochondrial fission depends on the translocation of cytoplasmic Drp1 to mitochondria, where it binds to Fis1, oligomerizes, and constricts the organelle, ultimately leading to mitochondrial fission. After LeuLeuOMe exposure, which triggered LMP, the confocal microscopic images showed that cytosolic Drp1 was translocated to the mitochondria  and a calmodulin (CaM)-binding domain  in the C-terminus segment , and a ~13-kDa fragment could be detected with a C-terminus-specific antibody (BD Pharmingen 65061A). Thus, we reasoned that the cleavage site of calcineurin by chymotrypsin should be located at its regulatory CaMBD, which results in the removal of the AID domain. However, the catalytic domain and the calcineurin B-binding domain (CNBBD) remained intact in the ~47-kDa fragment of truncated calcineurin. As expected, the ~47-kDa fragment of truncated calcineurin showed high phosphatase activity independent of CaTo confirm that the proteolytic activation of calcineurin affects the translocation of Drp1 and mitochondrial fission, we transfected SH-SY5Y cells with GFP-tagged full-length (CN-FL) or truncated calcineurin  and observed the morphology of the mitochondria. The results shown in Fig. 2+-evoked activation of calcineurin mediates the dephosphorylation of Drp1 at its conserved serine 637 site, triggers the translocation of Drp1, and results in the fragmentation of depolarized mitochondria Below is the link to the electronic supplementary material.


Insulin-like growth factor binding protein-2 (IGFBP-2) is widely abundant in fetal life and remains highly expressed in the brain throughout life. It plays a key role in targeting IGFs to their receptors in developing organs . It is aWe have been investigating the role and mechanisms of action of IGFBP-2, and have developed unique insights into its structure-function relationships. Our work has mostly focused on neuroblastoma cell lines of varying aggressiveness and motility, and our manipulations of the IGFBP-2 molecule have allowed functional characterisation.We have demonstrated that the IGFBP-2 molecule contains a critical central basic region, the heparin-binding domain (HBD), which accounts for its binding to cell surface, critical in targeting IGFs to their receptors. Ablation of this domain dramatically reduces both proliferation and motility of these cancer cells, a process also involving integrin receptors [This same region of the molecule also contains a Nuclear Localisation Sequence (NLS) accounting for the ability of IGFBP-2 to enter the cell nucleus. This process leads to induction of transcription of a range of cancer-promoting genes including VEGF, promoting angiogenesis in a chick embryo model, intrinsic to carcinogenesis. Ablation of the NLS blocks these processes .Circulating IGFBP-2 is reduced in obesity and other insulin-resistant states and in animal models can enhance insulin sensitivity. It is regulated by the insulin sensitiser, leptin, and in a sheep model we have demonstrated that centrally administered leptin induces IGFBP-2 expression in skeletal muscle, via the sympathetic nervous system. Similarly, leptin applied directly to cultured skeletal muscle cells induces IGFBP-2 expression, leading to enhanced insulin sensitivity. This suggests that IGFBP-2 may mediate leptin’s insulin-sensitising effects .IGFBP-2 also acts on visceral, but not subcutaneous adipose tissue, inhibiting fat accumulation, an effect not seen when the HBD region is mutated or integrin blockade is applied. Thus IGFBP-2 appears to play a beneficial role in regulating the function of visceral fat, which is critical in the pathogenesis of metabolic complications of obesity.IGFBP-2 is thus a critical player in both cancer biology and in metabolic syndrome. There is potential for targeted pharmacologic manipulation of IGFBP-2 activity in order to reduce the morbidity and mortality of these major diseases in our society.


Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development.Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by  Recently, we and others have reported that miRNAs may control functional pathways in cells by targeting, in a coordinated manner, sets of functionally correlated genes thus making co-expression analysis a valid tool to gain insight into miRNA function (MicroRNAs (miRNAs), a class of short non-coding RNAs (∼22 nucleotides in length), have emerged as important regulators of gene expression in development . At the function ,21.Oryzias latipes)  ,25,26. Mntiation ,25. In t biology ,28, thusO. latipes) and Ath5:GFP Medaka transgenic line  and the donor splice site of olEfnb3 exon2 , respectively. The specificity and inhibitory efficiencies of both morpholinos were determined as previously described (In vitro synthesis of the EphB2a full-length coding mRNAs was performed as described (EphB2 mRNAs were injected at 10–50 ng/μl. The selected working concentration was 50 ng/μl. Control embryos were injected with 50 ng/μl of eGFP mRNA.The Cab-strain of wild-type (WT) Medaka fish  corresponding to E.12.5dpc in mouse] WT and injected embryos was extracted using TRIZOL (Invitrogen) according to the manufacturer's instructions and used for subsequent RNA-Seq-based profiling . The RNAAt least 250 embryos were pooled in each assay. Complementary DNA (cDNA) synthesis was performed using the SuperScript III First-Strand Synthesis System for RT-PCR using random hexamers (Invitrogen). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed as previously described . Each asrp function from RankProd Bioconductor package. The matrix of 156 putative targets and the matrix with all expressed transcripts were constructed using the normalized reads count from LIMMA 'voom' function. After the result of rp function, we selected those transcripts with a positive correlation, an average FC greater than 0 and a percentage of false prediction (pfp) value less than 0.01.The RNA-Seq paired-end sequence reads were mapped to the Medaka genome (UCSC oryLat2) using SOAPSplice with default parameters. The SOAPSplice  results http://www.microrna.org), TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/). All three of these computational programs allow the researcher to enter a specific miRNA symbol and the algorithm will determine all putative mRNA targets. This approach yielded 3720 putative Medaka targets which were obtained by taking human gene targets predicted by any of these programs  and searching for their orthologs in Medaka using Biomart database. Since Medaka annotation provides poor data on 3′UTRs, we performed our own 3′UTR reconstruction from the RNA-Seq data generated, allowing us to verify the conservation of the miR-204 seed sequences in the newly annotated UTRs .Several computational algorithms designed to predict target genes of miRNA sequences have been used. The basis for these programs is the degree of sequence complementarity between a miRNA and a target 3′UTR, including the presence of a consecutive string of base pairs at the 5′ end of the miRNA known as a 'seed' or 'nucleus'. Due to sensitivity and specificity in target prediction, we combined three bioinformatics algorithms to predict miRNA target sites: miRanda (http://david.abcc.ncifcrf.gov/home.jsp using default parameters (http://amp.pharm.mssm.edu/Enrichr/).Enrichment analyses were performed with the web tool DAVID at rameters . The KyoEFNB3, DPYSL3, KIF5 and DDX3Y genes, and psiUx plasmid constructs containing the hsa-pre-miR-204 sequence were used in Luciferase assays, as described previously  and cloned into the Topo-TA-vector (Invitrogen). Ath5:GFP Medaka transgenic lines were used for cryosections, confocal image stacks were acquired to detect native GFP fluorescence using Zeiss (LSM 710).Whole-mount RNA escribed . DigoxigWe previously demonstrated that miR-204 is required for lens differentiation and dorso-ventral patterning of the vertebrate optic cup through the modulation of several target genes ,25,26. HWe generated and analysed a profile of the Medaka early embryonic transcriptome using RNA-Seq. RNA-Seq libraries were prepared for each RNA sample in biological triplicates, i.e. three WT, three miR-204 KD and three miR-204 OE whole (St24) embryo RNAs. Each library was sequenced using 100 bp paired-end sequencing on the Illumina HiSeq 2000 system. The sequences were aligned to the Medaka genome using SoapSplice, a splice-aware mapping tool . The mapP-value for the statistical model applied lower than 0.05. This analysis yielded a total of 5843 differentially expressed transcripts  lower than 0.05. The complete results of the enrichment analysis are presented in Supplementary Table S2. Overall, the analysis indicated a strong enrichment for genes involved in biological processes such as nervous system development , neurogenesis  and in particular in the KEGG pathway ‘axon guidance’   or expressed at higher levels in the KD model compared to the WT (KD/WT > 0). This procedure yielded a dataset of 5105 putative target transcripts of miR-204 based on RNA-Seq results , neurogenesis  and axon guidance  (Supplementary Table S3).Furthermore, we retrieved a dataset of predicted miR-204 direct targets in the human genome by using three bioinformatics microRNA target prediction tools  and then we used the Ensembl BioMart data-mining tool to identify their Medaka orthologs. Using this approach we found 3720 Medaka transcripts as potential miR-204 direct targets. The overlap between transcripts predicted to be direct targets of miR-204 and differentially expressed Medaka transcripts with the expected behavior was of 785 genes corresponding to 595 human orthologs. The functional enrichment pointed, once again, to categories related to nervous system development . We tested them in triplicate by qRT-PCR on total RNA derived, respectively, from WT, miR-204 over-expressing and miR-204 morphant Medaka embryos. We found that 76% (16/21) of the analysed genes confirmed their behavior as putative miR-204 targets with expression changes concordant with those detected by RNA-Seq Figure . To furts Figure , indicatIn order to verify the conservation of the miRNA target sequences in transcripts, we reconstructed the 3′UTR starting from RNA-Seq data . We integrated Ensembl annotated data with our own sequencing information to identify UTRs as they were truly transcribed in our own dataset. As reported in Supplementary Figure S1, the alignment data show an extension downstream of the last exon compared to the standard notation, demonstrating that using standard annotation is not sufficient to verify the conservation of the miRNA target sequences in the UTRs of these transcripts.P-value and their fold change in KD or OE condition. For this analysis, we decided to seed the correlation from a stringent dataset of putative targets by applying more stringent conditions. Specifically, we took into consideration only transcripts which have the expected behavior in both conditions (KD and OE) and which are also predicted as targets by bioinformatics tools. This approach led to a set of 156 transcripts corresponding to 116 human orthologous genes  value less than 0.01 , neurogenesis  and axon guidance  (Supplementary Table S4). Overall, the correlation approach allowed us to identify more functional enrichments than the LIMMA approach. The total number of significant Gene Ontology Biological Process (GO BP) Terms and KEGG pathways identified by LIMMA approach is 721 while in the correlation approach it is 985. Moreover, on the specific enrichments that were common to both approaches (580 pathways), the correlation approach yielded stronger statistical significance , as shown in Figure We thus obtained a total of 7350 Medaka transcripts, , with those in common (intersection), with predicted targets (predicted) and the overlapping of the first three with predicted targets Figure . Since tIn addition, when looking not only at the number of pathways but also at the fold enrichment, the correlation approach and its combination with LIMMA, tended to perform better also in terms of the mean fold enrichment of pathways, as seen by the distribution of fold enrichments using LIMMA, correlation and the intersection Figure .To better describe the potentials of using the correlation analysis, we took a look at the top pathways that were produced when using genes coming only from the correlation analysis (Supplementary Table S5). Interestingly, ‘Axon guidance’ and ‘Axonogenesis’, as well as other neuron-related pathways appeared as the most significantly enriched pathways. Indeed, when the genes in the ‘axon guidance’ KEGG pathway were inspected in terms of the approach used , we could observe that while LIMMA pointed to 11 predicted genes, the Correlation pointed to 22, only 8 of them in common. Hence, by using the correlation approach in combination with LIMMA, we could expand to 14 new putative target genes within the highly enriched ‘Axon guidance’ pathway.EphB2 and Efnb3, are bona fide miR-204 targets, as recently reported by Ying et al.  and with a significant score in the LIMMA or Correlation analysis, corresponded to either Ephrin ligands or EPH receptors (see Supplementary Table S6). Two members of these axon guidance molecules , EphB2 ag et al.  or showng et al. . Both Ep and withEphB2 and Efnb3 function during the outgrowth of retinal ganglion cell (RGC) axons in Medaka fish. First, we compared the distribution of the Efnb3 and EphB2 transcripts in WT, miR-204 KD and miR-204 OE Medaka embryos by RNA in situ hybridization. We found a significant expansion of the retinal expression domains of both Efnb3 and EphB2 in KD compared to control injected embryos  . In these embryos, RGC axons approached the midline but subsets of axons misprojected to the telencephalon or failed to reach the appropriate topographic target at the tectum . Axon misprojections were not a consequence of defects in RGC specification because (i) Ath5 was normally expressed at St26 (2 days and 6 h pf), i.e. when the retina begins to differentiate Efnb3 and EphB2, we performed a series of rescue experiments. Indeed, the concomitant injection of Mo-Efnb3 and Mo-miR-204 , indicating that EphB2 counteracted the effect of miR-204.To define if these defects were a consequence of alterations in the expression of the miR-204 targets, All in all, these data demonstrate that miR-204-mediated control of the Ephrin-EPH signaling system regulates retinal axon outgrowth, revealing a previously unidentified role for miR-204 in this developmental event.in vivo gene manipulation in a fish model is an efficient approach to investigate the function of a specific miRNA, both by allowing an efficient recognition of their relevant targets and the identification of their role in the control of specific functional pathways. In particular, the RNA-Seq procedure and analysis approach we described in this study was instrumental to uncover a role of miR-204 in the control of retinal axon guidance, which was further confirmed by in vivo miR-204 KD and OE in appropriate transgenic lines. A great deal of information on miRNA biology was recently obtained by exploiting microarray-based transcriptome analyses, which were carried out using different strategies (in silico analysis of this kind of data is not easy and there are no standard pipelines that can be used to reliably identify microRNA targets, so far.We demonstrated that the integration of RNA-Seq transcriptome analysis, bioinformatics approaches and rategies . Our stuMeis2, which we previously validated as a key miR-204 target (P-value in the LIMMA analysis was higher than 0.05 for all the transcripts associated with the gene, however fold change values for KD and OE were as expected, up-regulated in KD and down-regulated in OE and highly correlated to other targets.In this study, we used two different approaches, a conventional differential expression approach based on the LIMMA tool, and a novel method developed by us based on correlation to validated targets. These methods were used to identify putative miRNA-204 target pathways as well as direct target genes. A direct comparison of the transcripts identified by two methods highlights that the two strategies reported qualitatively similar results in terms of pathways enrichment. On the other hand, the correlation method was able to recover gene lists with stronger statistical significance for common enriched terms, as shown in Figure 4 target , was fouIt is widely accepted that direct miRNA targets have their levels regulated in a fine-tuning fashion with fold changes that usually do not exceed 1.1–1.3. In that scenario, many potential targets could be penalized when analysed through classical statistical tests as LIMMA whose significance strongly depends on the extent of variation. On the other hand, simply reducing stringency in the statistical analysis based on LIMMA would not necessarily yield useful results. Using the correlation analysis, we are being less stringent in looking at fold changes because it scores that fine-tuning behavior that takes into account not only perturbations of miRNA levels in the experiment but also minimal baseline variance within biological replicates due to effect strength of the KD or OE models, fine developmental staging differences or cell numbers for specific target tissues. However, false discovery correlations are filtered out by using a consistent group of inter-correlated targets as input and by applying a false discovery correction based on non-parametrical Rank Product statistics. By using the combination of the two approaches, we take advantage of correlation to uncover a higher number of putative targets, specially those with very small changes that may not pass the classical significance filters of state-of-the-art RNA-seq differential expression tools as LIMMA while focusing our attention on those with the expected biological behavior.Such an approach is still affected by some caveats and could be improved in further settings. Our study has been conducted in whole Medaka embryos. It is of course clear that the expression of miR-204 could be restricted to some tissues and areas and hence the KD experiment would work as expected only as far as the miRNA is highly expressed in those tissues with respect to the whole embryo. Moreover, it can be argued that transcriptome analysis on well-defined tissues is advisable to identify targets more clearly; however, utilizing whole embryo has allowed us to identify targets expressed across both ocular and non-ocular tissues of great importance for visual function and axon guidance. Finally, our study is only able to address microRNA targets, which are regulated at the transcript level. Recent studies have demonstrated the importance of translation inhibition as a mode of microRNA targeting ,48, whicDespite the availability of high-throughput gene expression data, we still lack a standard method that could use this data to aid in the identification of true miRNA targets amongst the in-silico predicted based on seed sequences. Yet, some attempts have been successfully applied assuming the fact that targets of the same miRNA tend to be co-expressed and have their expression levels correlated, either to find targets of a given miRNA  or to fiin vivo through, at least in part, the targeting of some Eph receptors, such as EphB2 (Efnb3.Recently, emerging evidences highlight a direct and prominent role for the miRNAs in the control of neurite outgrowth, spinal morphology and function during nervous system development . In mostas EphB2  and of sin vitro and in vivo validations, we were able to explore and further define a key player in axon guidance, mir-204, and reveal novel target genes and pathways of this microRNA.In conclusion, by integrating transcriptome, existing and novel bioinformatics approaches, Supplementary Data are available at NAR online.


Objectives: This study attempted to find a simple adhesive restorative technique for class I and II cavities on posterior teeth.Study Design: The tested materials were a self-etching adhesive  and a dual-cure composite  used in bulk to restore the cavities. Class II MO cavities were performed and assigned to 4 groups depending on the orthophosphoric acid (H3PO4) conditioning of enamel and polymerization method used . Specimens were subjected to quantitative marginal analysis before and after thermo-mechanical loading.Results: Higher percentages of marginal adaptation at the total margin length, both before and after thermo-mechanical loading, were found in groups in which enamel was etched with phosphoric acid, without significant differences between the chemically and dual-cured modes. The restorations performance was similar on enamel and dentin, obtaining low results of adaptation on occlusal enamel in the groups without enamel etching, the lowest scores were on cervical dentin in the group with no ortophosphoric acid and self-cured.Conclusions: A dual-cure composite applied in bulk on acid etched enamel obtained acceptable marginal adaptation results, and may be an alternative technique for the restoration of class II cavities. Key words:Dual-cure composite, bulk technique, class II restoration, selective enamel etching, marginal adaptation. Amalgam has been the material of choice worldwide for class I and class II restorations for more than a century due to its high strength, good wear resistance, low technique sensitivity and low cost . HoweverAdvances in adhesive technology made during the last 40 years have simplified dental procedures and given a more esthetic result to patients and a conservative alternative treatment to clinicians . While tPrevious studies of composite class II resin restorations have shown that their clinical longevity is adversely affected by several factors such as achieving a good proximal contact , lack ofPolymerization contraction of dimethacrylate-based composites ranges from 2-6% of volumetric shrinkage . VariousDual-cured resin composites have been mainly used as a core material for the reconstruction of non-vital teeth , and as A previous study reported that the tooth-restoration complex is more prone to fail at the interface rather than in the composite or tooth material . TherefoIn an attempt to find a restorative material that could potentially be used as an amalgam substitute, i.e. an adhesive material that can be applied in bulk with a simple restorative technique, we evaluated the marginal integrity of cavities entirely restored with a dual-cured resin composite. The null hypotheses tested were that [i] The differences in the etching procedure would not affect the marginal adaptation before and after thermo-mechanical loading and [ii] Marginal adaptation would not be affected by the polymerization mode [chemical or dual-cure].The materials used for the study consisted of a chemically-cure self-etch adhesive system  composed by a non-rinse conditioner and 2 liquids [A and B] that need to be mixed before application, and a dual-cure radiopaque core material  . Thirty-Standardized box-shaped mesio-occlusal class II cavities with cervical margins located on dentin were prepared on each tooth using diamond burs with a grain size of 80 microns  and finished with diamond burs with a grain size of 40 microns [Diatech Dental] under 12x magnification. The dimensions of the cavities were 4 mm height, 4 mm of bucco-lingual width, 2.5 mm mesio-distal depth and approximately 1.5 mm below the cemento-enamel junction.In groups 1 and 2 the adhesive system was applied following manufacturer recommendations  . In grouOne day after polymerization, finishing and polishing of the restorations was carried out using flexible discs  for proximal and fine-grain diamond burs for the occlusal surface. Then, impressions with a polyvinylsiloxane material  were made of each restoration and poured with epoxy resin  and 24 h after resin replicas were gold sputtered. They were subjected to a computer assisted quantitative margin analysis in a scanning electron microscope  by using a custom made module programmed with an image processing software . For the quantitative evaluation, a blinded and trained lab technician examined the specimens Fig. . The marAnalysis of data was performed by using an analysis of variance test (ANOVA) of variance [ANOVA] for each variable separately for two time points [before and after loading] in SPSS 19 for Windows. Significant differences were observed between groups with Tukey HSD post-hoc pairwise comparasion. In all analyses, the level of significance was set at 0.05.The scores of marginal adaptation expressed as percentages of continuous margins [% CM] attained by the different groups at the total margin length, occlusal, proximal and cervical margins are shown in - Quantitative margin analysisp=0.001] and group 4 [p=0.004]. However, no significant differences between groups were seen at the proximal margins or cervical portion.Before loading , higher p=0.002] and with group 4 [p=0.013]. At the cavity segments, we obtained again lower % of CM at the occlusal side in groups in which phosphoric acid on enamel was not used. No differences between groups were observed at the proximal enamel margins. However, at the cervical portion, the lowest % of CM were obtained in group 1 being statistically different from group 2 [p=0.008] and group 3 [p=0.024].After loading, the highest scores of marginal adaptation at TML were attained, once again, by the groups that used H3PO4 . StatistRepresentative SEM images Fig.  with marThe rationale for including the evaluation of marginal adaptation before and after thermo-mechanical loading relies on the fact that both methodologies constitute and adequate model to assess the performance of adhesive interfaces -21. In rIt is known from previous studies that when etch and rinse adhesives are used on enamel, a reliable and favorable bonding interface is produced . HoweverMarginal adaptation at the occlusal enamel margins were lower for the groups 1 and 2 where the self-etching primer was applied on enamel without previous conditioning with phosphoric acid. In addition, unsupported enamel prisms [margins were not beveled] may have resulted in enamel fractures at the marginal level especially after loading .Gap formation or microleakage at the tooth-composite interface is related with polymerization shrinkage and shrinkage stresses. This issue may induce postoperative sensitivity and secondary caries, leading to restoration failure . MinimizShrinkage strain rates of both self and light-curing modes would become almost identical after about 60 min for dual-cured core materials . In the Despite the similar marginal adaptation scores, when dual-cure composites are used, higher dentin bond strength are reported when light-cured as compared to self-curing . This coIn addition, final light polymerization would enhance significant mechanical properties, making the selection of a dual-cured composite an improvement over a self-cured or a light-cured at the gingival margin .To conclude, materials insertion in one single layer, with the additional benefit of chemical cure in deep areas where light is attenuated has several clinical advantages in terms of handling, curing efficiency and time saving. Possibly, the restorative technique used in the present study could be a cost-effective alternative to traditional sophisticated adhesive restorative techniques, and a potential substitute to amalgam . NeverthWithin the limitations of the present study, the use of a dual-cure composite for the restoration of class II cavities, applied in bulk on phosphoric acid etched enamel, has provided the most reliable marginal adaptation.