Tourist-like MITE, Monkey King, was identified from the promoter region of a flowering time gene, BnFLC.A10, in Brassica napus. Based on this MITE, the characteristics and potential roles on gene regulation of the MITE family were analyzed in Brassicaceae.Miniature inverted repeat transposable elements (MITEs) are important components of eukaryotic genomes, with hundreds of families and many copies, which may play important roles in gene regulation and genome evolution. However, few studies have investigated the molecular mechanisms involved. In our previous study, a Tourist-like MITE family Monkey King in Brassicaceae, including its distribution, copies and insertion sites in the genomes of major Brassicaceae species were analyzed in this study. Monkey King was actively amplified in Brassica after divergence from Arabidopsis, which was indicated by the prompt increase in copy number and by phylogenetic analysis. The genomic variations caused by Monkey King insertions, both intra- and inter-species in Brassica, were traced by PCR amplification. Genomic sequence analysis showed that most complete Monkey King elements are located in gene-rich regions, less than 3kb from genes, in both the B. rapa and A. thaliana genomes. Sixty-seven Brassica expressed sequence tags carrying Monkey King fragments were also identified from the NCBI database. Bisulfite sequencing identified specific DNA methylation of cytosine residues in the Monkey King sequence. A fragment containing putative TATA-box motifs in the MITE sequence could bind with nuclear protein(s) extracted from leaves of B. napus plants. A Monkey King-related microRNA, bna-miR6031, was identified in the microRNA database. In transgenic A. thaliana, when the Monkey King element was inserted upstream of 35S promoter, the promoter activity was weakened.The characteristics of the Monkey King, a Brassicaceae Tourist-like MITE family, has amplified relatively recently and has induced intra- and inter-species genomic variations in Brassica. Monkey King elements are most abundant in the vicinity of genes and may have a substantial effect on genome-wide gene regulation in Brassicaceae. Monkey King insertions potentially regulate gene expression and genome evolution through epigenetic modification and new regulatory motif production.The online version of this article (doi:10.1186/s12870-015-0490-9) contains supplementary material, which is available to authorized users. They weke MITEs -bp, TAA -bp, TA) . Studiesectively –7. Laterectively , 9 and Mectively . In addiectively .mPing, a Tourist-like MITE, which originated from an internal deletion of a transposase-encoding element Ping, is activated by tissue culture and γ-ray irradiation in rice[mPing insertions presented different profiles  among four rice strains under selection during domestication [mPing insertions might benefit the host by creating potentially useful allelic variants and novel, stress-inducible regulatory networks [There are hundreds of families of MITEs and they are present in high copy numbers, making them important genome constituents. These elements are widely, but not randomly, distributed in the genome, and their distribution density in each chromosome varies . Thousann in rice–16. mPintication . It was networks , 18.kiddo, a MITE located in the rice ubiquitin2 promoter, has a dual function in gene regulation: its presence not only increases transcription rates but induces epigenetic modifications [DICER-LIKE 3, RNA-dependent RNA polymerase 2, and possibly DICER-LIKE 4 [Although selection pressure tends to eliminate most insertions that reside in gene exons and introns in the early stage of MITE amplification in the genome , studiesications . Small Rications . In SolaR-LIKE 4 .Brassica, a close relative of Arabidopsis, is an agriculturally important genus that includes a wide range of diploid and allotetraploid species, including oil crops, vegetables, and forages. B. napus, an allotetraploid species , originated from natural hybridization between the ancestral forms of the diploid species B. rapa  and B. oleracea  ~ 7500 years ago [Brassica A and C genomes were estimated to have diverged ~ 4.6 million years ago [B. napus and its progenitors were defined as subgenomes of each other. The A genomes in B. rapa and B. napus were assigned the Ar and An subgenomes, respectively; Co and Cn represent the C genome in B. oleracea and B. napus, respectively [r and An subgenomes is 97.5 ± 3.1 %, and is 93.1 ± 4.9 % between the Ar and Cn subgenomes [ears ago , 24. Theears ago . The samectively ,27. Sequbgenomes . It has bgenomes , 28, 29.Stowaway-like MITE, BraSto, first reported in B. rapa, was found in the gene space and is still active both in diploid and allotetraploid Brassica species [B. napus, a Tourist-like MITE, Monkey King, was identified in the promoter region of BnFLC.A10, a homologue of Arabidopsis FLOWERING LOCUS C (FLC) [Monkey King elements are not restricted to Brassica species, but are specific to the Brassicaceae family. We further investigated its sequence features, distribution, and phylogenetic relationships, and inferred its potential role in the evolution of Brassicaceae genomes. Monkey King-related intra- and inter-species polymorphisms were confirmed experimentally. DNA methylation analysis, electrophoretic mobility shift assay (EMSA) analysis, identification of a Monkey King-related microRNA (miRNA), and transgenic analysis revealed its effects on gene expression and genome evolution in Brassicaceae.A  species . In B. n C (FLC) . In thisMonkey King sequence in the promoter of BnFLC.A10 included 14 bp TIRs and was flanked with a trinucleotide TAA TSD, which are typical features of Tourist MITEs , respectively, were screened in the published plant MITE database (P-MITE) [A. thaliana, 52 Monkey King homologous sequences were identified in the A. thaliana genome sequence by BLAST analysis , while the smallest genome  has the lowest density (0.43 MITEs/Mb). In the same species, no significant differences were found in Monkey King density among different chromosomes, except for chromosome 3 from A. thaliana and A. lyrata. In silico mapping of 504 complete elements on the B. rapa chromosomes also showed that they were approximately evenly distributed in their respective chromosomes . Correlation analysis between the AT-content and the length of complete Monkey King sequences showed that longer Monkey King sequences have relatively higher AT-contents in the B. rapa genome   at the 9th nucleotide in the 3′ terminal regions between the Brassica and Arabidopsis genomes.mes Fig. . In geneMonkey King sequences mined from the four Brassicaceae genomes were used for phylogenetic analysis. In addition, the Monkey King sequence in the promoter of BnFLC.A10 from B. napus was included. From the phylogenetic tree  were located within introns of genes  were more than 3kb from a gene. Most of the members (26/38) were within less than 1kb from a gene, and two (5.1 %) were within introns (Table Monkey King elements and untranslated regions (UTR) of genes in A. thaliana  database at NCBI. Sixty-seven ESTs carrying Monkey King fragments were mined from B. rapa, B. oleracea and B. napus. Thirty ESTs matched with annotated B. rapa and (or) A. thaliana genes  analysis to check for interacting proteins. The methylation level of cytosine residues inside and flanking the Monkey King sequences was investigated using bisulfite sequencing. In B. napus cultivar Tapidor, cytosine methylation occurred in the Monkey King sequence, while no apparent cytosine methylation was observed in the flanking sequences; in cultivar Ningyou7, no DNA methylation occurred in the corresponding flanking regions  were also recognized by nuclear proteins from Tapidor leaves; however, the binding was much weaker than to the ES7 fragment. Some fragments (e.g. ES4 and ES5) were non-specifically bound by nuclear proteins, because retarded bands were observed in both the noncompetitive (without plus unlabeled probes) and competitive (plus 50-fold unlabeled probes) binding assays. This result indicated that the Monkey King insertion produced new binding motifs for some nuclear proteins , which may regulate BnFLC.A10 expression in winter varieties of B. napus.The EMSA results clearly revealed that nuclear protein(s) extracted from Tapidor leaves specifically bound to a fragment (ES7) from the middle of the ses Fig. . Some MoB. napus Monkey King sequence was used to scan the microRNA database (miRBase) [Monkey King sequence  could form a stem-loop structure by itself   and a mince Fig. . The miRo et al. . Furtherelf Fig. , which sSTs Fig. . This miMonkey King element on gene expression, we studied the influence of the Monkey King element on the activity of the 35S promoter that drives a GUS reporter gene. Two expression vectors, pBI121 (without Monkey King) and pBI121m (with Monkey King) . Seedlings hosting the pBI121m construct had lower levels of the GUS transcript compared with the pBI121 seedlings  Fig.  were usengs Fig. . Chemicangs Fig. . These rTourist-like MITE, termed Monkey King. Monkey King possesses all of the typical features of Tourist MITEs and has the consensus TIR GGGC(orT)CTGACTGGTT. Interestingly, a similar TIR, GGGGNTGTTTGGTT, is present in kiddo and Tourist-D in rice and Hbr in maize [Monkey King from the Brassicaceae family and kiddo, Tourist-D, and Hbr from the grass family.In this study, we conducted molecular and genomic characterization of a Brassicaceae in maize , 34, 35.in maize . TIRs ofin maize , 37. TIRBrassica species with A. thaliana revealed that, besides genome triplication and allopolyploidization, TEs contribute to the increased genome size of the Brassica species [Monkey King density in the B. rapa genome was three and 10 times more than that in the A. lyrata and A. thaliana genomes, respectively, demonstrating that the elements were actively amplified in the Brassica after divergence from Arabidopsis. On average, the B. rapa genome has the shortest complete Monkey King sequence, followed by the A. lyrata genome, while the A. thaliana genome has the longest sequence. Given the MITE density differences among these Brassicaceae genomes, we inferred that shorter Monkey King sequences may be more easily amplified than longer Monkey King sequences.Comparative analysis of  species , 39. In Monkey King sequences also suggested that they have amplified relatively recently in the Brassicaceae family. The high conservation of TIRs was also considered a sign of recent proliferation in the maize Tourist MITE ZmV1 [B. rapa Stowaway MITE BraSto [Monkey King members have exactly the same TIR sequence, which supports the recent activity and ongoing mobilization of Monkey King. This hypothesis is also supported by the phylogenetic relationships among complete Monkey King sequences, because some species-specific clusters were found in the phylogenetic tree. In addition, the PCR amplification results from different Brassica DNA samples also suggested that some Monkey King insertions have arisen from independent activation in each species after speciation. In fact, our previous study suggested that the Monkey King insertion in the BnFLC.A10 promoter occurred in winter rapeseed after B. napus speciation [Monkey King members inserted into their respective genomes independently after allopolyploidization ~7500 years ago.The high conservation of complete ITE ZmV1  and the E BraSto . Some Moeciation . These rMonkey King is a high copy number Tourist MITE found in B. rapa, which parallels BRAMI-1, a recently identified high copy Stowaway MITE [A. thaliana, we identified only 52 Monkey King members. The physical association between MITEs and genes showed that, in the B. rapa and A. thaliana genomes, most complete Monkey King elements were located in gene-rich regions, less than 3 kb from genes. Many members were within less than 1 kb from a gene, while a few members were within introns. Although the MITE density and copy number showed obvious differences between B. rapa and A. thaliana genomes, a similar distribution trend was observed in the two genomes: the Monkey King sequences were most abundant in the vicinity of genes. The insertion preference of this Brassicaceae Tourist MITE is similar to that of the two Brassica Stowaway MITEs, BroSto and BRAMI-1 [A. thaliana, nearly half of the members were within less than 0.5kb from a UTR and two members fell within UTR regions. Additionally, 30 of 67 Brassica ESTs carrying Monkey King fragments matched the annotated B. rapa and (or) A. thaliana genes. Thus, Monkey King insertions could play a role in gene regulation and evolution in Brassicaceae.way MITE . By cont BRAMI-1 , 40. MIT BRAMI-1 , 41. In mPing were considered as enhancers that render adjacent genes stress inducible [kiddo was responsible for up to 20 % of neighboring gene expression in both transient and stably transformed rice calli [kiddo DNA methylation was blocked with 5-azaC, ubiquitin2 transcript accumulation increased threefold [kiddo has a dual function in regulating gene expression. In our previous study, the Monkey King insertion upstream of BnFLC.A10 was positively associated with induced BnFLC.A10 expression during vernalization [Monkey King DNA methylation in the BnFLC.A10 promoter was observed. This suggested that epigenetic modification may regulate BnFLC.A10 expression by changing the Monkey King DNA methylation status. Monkey King contains important motifs, such as TATA-box and CAAT-box [Monkey King bound nuclear protein(s) extracted from Tapidor leaves. These results suggest that Monkey King insertions can produce new regulatory sites, which probably recruit new transcription factors. New alleles caused by MITE insertions may benefit the host by producing useful allelic variants and novel regulatory mechanisms. In fact, BnFLC.A10 allelic diversity caused by the Monkey King insertion is one of the major causes of differentiation of winter and spring B. napus genotypes [Monkey King-related miRNA, bna-miR6031, and its potential targets, indicated that the Monkey King silencing pathway may be involved in expression regulation of genes bearing Monkey King insertions. In Arabidopsis, a transposable element was inserted in an intron of FLC, resulting in low FLC RNA levels. The intronic TE renders FLC subject to repressive chromatin modifications mediated by TE-derived siRNAs [VRN-A1a and influenced the flowering [BnFLC.A10 expression through chromatin modifications of the Monkey King sequence in the BnFLC.A10 promoter remains to be investigated. bna-miR6301 is a new class of miRNA found in B. napus [Brassica species because it may be generated by a certain Monkey King sequence. Moreover, no similar miRNAs from other species are present in miRbase. To date, many MITEs have only been identified in a given species, which suggests that MITEs can generate species-specific small RNAs. The Monkey King insertion was positively associated with BnFLC.A10 expression [A. thaliana experiment revealed that the Monkey King element inhibited promoter activity when inserted upstream region of a promoter. These results indicate that a new Monkey King insertion may enhance or repress the expression of a gene with which it is associated, by causing DNA epigenetic modification, nuclear protein binding sites and(or) possible miRNA-mediated regulation. Given the wide distribution of Monkey King elements in some Brassicaceae genomes, this Tourist MITE family may contribute considerable phenotypic diversity to Brassicaceae plants.Although many MITEs have been found in plant genomes and are associated with genes, few studies have examined the effects of MITE insertions on neighboring gene expression , 20, 22.nducible . Promotelization . In thisCAAT-box . Certainenotypes . In addid siRNAs . In whealowering , 44. Reclowering . WhetherB. napus  and may Tourist-like MITE family Monkey King. Comparative analysis of Brassica species with Arabidopsis species revealed its putative role in the evolution of Brassicaceae genomes. Phylogenetic analysis and investigation of intra- and inter-species polymorphisms supported recent proliferation of Monkey King in Brassica species. Monkey King elements are closely associated with genes and influence gene regulation and evolution. DNA methylation detection, EMSA analysis, identification of a Monkey King-related miRNA, and transgenic experiments suggested that Monkey King insertions could regulate gene expression and genome evolution in the Brassicaceae family in a variety of ways, such as epigenetic modification and new regulatory motif production.In this study, we characterized a Brassicaceae Monkey King sequence inserted in the BnFLC.A10 promoter was first used as a query to search for similar sequences in the NCBI database and was employed to find its coding capacity. When similar sequences were only found in Brassicaceae genomes, the BLAST program was used to screen the homologous sequences in the published plant MITE databases (P-MITE) [Monkey King sequences in B. rapa and A. lyrata genomes were downloaded from the P-MITE database for further analysis. Monkey King homologous sequences were also identified in A. thaliana genome in the NCBI database by BLAST screening  and manual inspection. Additionally, complete Monkey King sequences (with TSDs at the two ends of one Monkey King sequence) were identified in the preliminary assembled B. oleracea genome sequence (http://brassicadb.org/). Nucleotide composition of the complete Monkey King sequences was computed in MEGA 5 [Monkey King sequences among four Brassicaceae plant genomes, B. rapa, B. oleracea, A. thaliana and A. lyrata, was visualized using the program Pictograms (http://genes.mit.edu/pictogram.html). Then, the Monkey King sequence was used against the microRNA database (miRBase) (http://www.mirbase.org/search.shtml) [The (P-MITE) . All Monn MEGA 5 . Conservh.shtml)  to find Monkey King sequences were used for multiple sequence alignment using ClustalW in MEGA 5 [All of the complete n MEGA 5 . Phylogen MEGA 5  followinMonkey King elements and their flanking regions were annotated using the B. rapa genome database (http://brassicadb.org/) and the A. thaliana genome database (http://www.arabidopsis.org/), respectively. The distances between Monkey King elements and their respective nearest genes were calculated for analyzing the relationship between the elements and genes. Additionally, Potential transcription activity of Monkey King was examined by searching Brassica EST database at NCBI. The Brassica ESTs carrying Monkey King fragments were used for localizing the Monkey King fragments in their respective genes, by comparison with the corresponding annotated B. rapa genes (http://brassicadb.org/brad/blastPage.php) and A. thaliana genes (http://www.arabidopsis.org/wublast/index2.jsp).The insertion sites of 504 and 38 complete Monkey King insertions were checked by PCR amplification. The flanking sequences of 5 Monkey King members were extracted for primer design , three B. napus  and two B. oleracea (CA25 and A12DHd) accessions. Sequencing and sequence comparisons were performed for further identification of the Monkey King insertions.Intra- and inter-species genomic variations caused by Monkey King member inserted in the BnA10.FLC promoter in winter-type B. napus cultivar Tapidor, compared with that in a semi-winter-type B. napus cultivar Ningyou7 [Monkey King to regulate gene expression.Previous studies have identified the Ningyou7 . TherefoMonkey King member in the BnFLC.A10 promoter was selected to detect the DNA methylation level of its inside and flanking sequences. The primers  [Genomic DNA was extracted from young Tapidor and Ningyou7 leaves, and 2 μg DNA were subjected to bisulfite treatment using the EpiTect Bisulfite Kit (Qiagen) following the manufacturer’s instructions. The kismeth) . Ten clokismeth) .2, 5 mM EDTA, 1 mM DTT, 200 mM PMSF, 250 mM sucrose, and 0.5 % Triton X-100) and stirred for over 20 min. The mixture was filtered with a Miracloth and the supernatant was centrifuged at 3000 × g for 20 min at 4 °C. The pellet was re-suspended in a low salt buffer  and centrifuged at 3000 × g for 10 min at 4°C (this step was repeated for 2 times). The pellet was suspended in a high salt buffer  in a volume of the estimated PNV (pellet nuclear volume). The nuclear lysate was incubated for 30 min at 4 °C with shaking and then centrifuged at 12 000 × g for 20 min at 4 °C. The supernatant was dialyzed in the low salt buffer for over 1 h. Finally, the protein content was measured by the Coomassie Brilliant Blue method. The EMSA was conducted as described by Hellman [Monkey King sequence were synthesized  were harvested from Tapidor seedlings. Protein extraction was performed as described by Mazabal et al.  with somMonkey King sequence was amplified from a B. napus BAC (JBnB75D10) carrying the Monkey King insertion in the BnA10.FLC promoter [HindIII site of the binary vector pBI121 . The correct orientation was confirmed by DNA sequencing. The original pBI121 vector was used as a control. The two vectors were introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into A. thaliana using the floral dip method [2 transgenic plants on half-strength MS medium containing 50 mg L−1 of kanamycin. T3 lines carrying a single transgene copy were assumed to be homozygous lines when they did not segregate for kanamycin resistance.The promoter  using spBAC JBnB7D10 carryp method . A segre3 homozygous lines were used to study the effect of the Monkey King element on gene expression. Ten-day-old seedlings grown on MS medium were harvested for RNA collection and GUS staining. Total RNA was extracted from about 25 seedlings using a Universal Plant Total RNA Extraction Kit . RNA samples were treated with DNaseI and used for reverse transcription using M-MLV reverse transcriptase (Promega). SYBR-Green qRT-PCR was used to quantify the transcription level of the GUS gene in transgenic seedlings. The 25 μL reaction contained 12.5 μL SYBR Green PCR Master Mix (Toyobo), 600 nM primers, and 2 μL of 5 × diluted cDNA sample. The PCR profile was as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 5 sec and 60 °C for 1 min. Fluorescence data were collected after the 60 °C step. Three replicate reactions were performed with each cDNA sample and individual primer pairs 6, 0.5 mM K4Fe(CN)6, 10 mM Na2EDTA, 0.1 % (v/v) Triton X-100, 20 % (v/v) methanol, and 100 mM sodium phosphate (pH 7.0). After staining, the samples were rinsed with ethanol and photographed.Thttp://purl.org/phylo/treebase/phylows/study/TB2:S17406 ).Phylogenetic data is available in the TreeBASE as accession number S17406 (


The concept of united airway diseases (UAD) has received increasing attention in recent years. Sustained and increased inflammation is a common feature of UAD, which is inevitably accompanied with marked gene modification and tight gene regulation. However, gene regulation in the common inflammatory processes in UAD remains unclear. MicroRNA (miRNA), a novel regulator of gene expression, has been considered to be involved in many inflammatory diseases. Although there are an increasing number of studies of miRNAs in inflammatory upper and lower airway diseases, few miRNAs have been identified that directly link the upper and lower airways. In this article, therefore, we reviewed the relevant studies available in order to improve the understanding of the roles of miRNAs in the interaction and pathogenesis of UAD. The close link between the upper and lower airways has been carefully investigated in recent years, leading to recognition of united airway diseases (UAD) ,2. The eThe link between upper and lower airways also stems from similarities in anatomical structures and functions. The respiratory tract is lined by ciliary epithelium, lamina propria, and glands. The upper airway, especially the nose, functions as a physical filter for the lower airways. Any impairment of this physical filter may result in an alteration of the homeostasis of the lower respiratory airways. For instance, postnasal drip is a possible mechanism causing pulmonary inflammation in patients with rhinitis .Immunological studies have shown that the inflammatory processes in allergic upper and lower airway diseases share overlapping immunopathologic features. In allergic inflammation, the following occur in both upper and lower airways: activation of epithelial immune response, infiltration of eosinophils, IgE production, and activation of mast cells, all of which exaggerate Th2 response . During While regulation on the transcriptional level has been a focus of research for a long time, the discovery of gene regulation by small non-coding RNAs has revolutionized the picture of gene expression recently ,11. Micret al. found that 10 miRNAs were differentially expressed in asthmatic patients and controls, but independent of concomitant AR [Few studies have investigated the miRNAs that may potentially participate in the pathogenesis of both AR and asthma. Two reports from one laboratory studied miRNA expression profiles in nasal biopsy from patients with a coexistence of AR and asthma ,22. In titant AR . Asthmatitant AR  compareditant AR . This isAlthough those two studies did not demonstrate an obvious interplay between AR and asthma on nasal mucosal miRNA expressions, it may be due to the improper time point of sampling and the mild severity of asthma and AR. When study was conducted out of seasonal allergy exposure or out of an episode of asthma exacerbation, the alterations in miRNA expression might not be easily detectable. Therefore, further studies with careful patient and time point selection would be helpful to clarify the interaction of asthma and AR on the miRNA expression. Moreover, despite total RNA samples being obtained from all frozen nasal biopsies in both studies ,22, onlyet al. measured the expression of 420 miRNAs by using qRT-PCR technology in plasma obtained from healthy, AR and asthmatic subjects. They found that 30 miRNAs were differentially expressed among healthy, AR and asthmatic subjects, with those miRNAs being classified into five expression pattern groups based on differences in expression between the asthma and AR, AR and healthy, and asthma and healthy groups. One of those groups showed 10 miRNAs  that were concordantly dysregulated by a similar magnitude in patients with AR and asthma. These findings showing a common expression pattern of circulating miRNAs might be reflective of the common pathways involved in AR and asthma. Further studies are therefore needed to validate these promising findings, which suggest that circulating miRNAs have potential for use as non-invasive biomarkers for the diagnosis of UAD, as well as to characterize AR, asthma and other allergic diseases.That notwithstanding, a very recent article  has estaAlthough upper and lower airway diseases share a lot of common pathways, few studies have investigated the same particular miRNA in the pathophysiology of both AR and asthma. Therefore, we summarized the miRNAs involved in the pathways common to AR and asthma, such as IL-13, GATA binding protein 3 (GATA-3), and mucin secretion, which have been discovered either in asthma or in the study of AR. We did not include the miRNAs involved only in the unique processes of AR or asthma. Those miRNAs involved particularly in AR or in asthma have been well described in recently published reviews ,23.et al. screened for the expression of a panel of 157 miRNAs by using RT-PCR in mononuclear leucocytes from human umbilical cord blood (CB) samples and analyzed the association of miRNAs expression and development of AR [et al. showed that TGFBR2 expression on monocytes was significantly up-regulated in CB with elevated IgE levels and in AR patients. These results indicated the regulatory association between miR-21 and TGFBR2 in AR [et al. observed that miR-21 was increased in lung biopsy of IL-13 transgenic mice, with allergic inflammation, and demonstrated that miR-21 is responsible for decreased IL-12 expression, which would partially account for the exaggerated Th2 response seen in asthma , a typical Th2 cytokine, is a central mediator of IgE-mediated allergic diseases. Given its well-established role in modulating Th2 immune responses in AR and asthma, great efforts have been taken to investigate the modulation of IL-13. Using miRNA microarray technology, Shaoqing controls . Furthercontrols . Importacontrols . Likewiscontrols . Inducedcontrols . Given tGATA binding protein 3 (GATA-3) is identified as an important transcription factor of mast cells and Th2 cells, which are key effector cells in the pathogenesis of AR and asthma through releasing inflammatory mediators and cytokines upon exposure to allergens. miRNAs have been shown to regulate the development of mast cells. The forced expression of miR-221 in mast cells led to increased degranulation, cytokine production, and adherence . miR-146in vitro data. Further investigations on the therapeutic potential of manipulating miR-146a in the management of mucus overproduction are needed in the context of airway diseases.Goblet cell hyperplasia and mucus hypersecretion are common characteristics of UAD. Muc5AC is recognized as one of the major components of mucus, which is linked to goblet cell hyperplasia. Recent work demonstrated that ectopic expression of miR-146a reduced Muc5AC expression, while depletion of intrinsic miR-146a increased the level of Muc5AC in airway epithelial cells . The favBesides the regulation of mucus production, miR-146a was also associated with the clinical efficacy of immunotherapy. In a recently published report, miR-146a expression level in peripheral blood mononuclear cells of 24 AR patients treated with immunotherapy was examined . The resTechnological advances have spawned a multitude of platforms for miRNA profiling , and an A number of individual miRNAs have been associated with upper or lower airway diseases , and eme


The perception of a visual stimulus can be markedly altered by spatial interactions between the stimulus and its surround. For example, a grating stimulus appears lower in contrast when surrounded by a similar pattern of higher contrast: a phenomenon known as surround suppression of perceived contrast. Such center–surround interactions in visual perception are numerous and arise from both cortical and pre-cortical neural circuitry. For example, perceptual surround suppression of luminance and flicker are predominantly mediated pre-cortically, whereas contrast and orientation suppression have strong cortical contributions. Here, we compare the perception of older and younger observers on a battery of tasks designed to assess such visual contextual effects. For all visual dimensions tested , on average the older adults showed greater suppression of central targets than the younger adult group. The increase in suppression was consistent in magnitude across all tasks, suggesting that normal aging produces a generalized, non-specific alteration to contextual processing in vision. The perceived properties of a visual stimulus can be markedly affected by the spatial context in which it is presented. For example, the perceived contrast of a textured patch or grating appears to be of lower contrast when surrounded by a high-contrast pattern than when it is presented on a uniform field . Visual Of specific interest to our study is the fact that some perceptual effects of background context are considered to arise from pre-cortical neural circuits, whereas others are more consistent with cortical neuronal properties. Two effects that have been linked to pre-cortical processing are suppressive effects on the perception of (a) luminance and (b) flicker. For example, a gray patch appears darker when placed on a bright background, and vice versa . Models Pre-cortical and cortical mechanisms are also implicated in the contextual processing of contrast. Perceptually, the contrast of a pattern appears lower when embedded in a high-contrast background . FunctioIn recent years, perceptual contextual effects in vision have been widely used to study a range of human conditions as an assay of the cortical balance between excitatory and inhibitory processes. An example of this is the study of healthy normal aging, where such tasks have been applied to indirectly study the presumed effects of altered cortical inhibition on visual processing. Older adults show stronger suppression of contrast for foveal viewing under a wide range of stimulus conditions, including variations of center–surround contrast , 2015, sd = 1.75) in older observers relative to younger observers.Two groups of observers were recruited to the study: 18 younger adults  and 18 older adults . A power analysis was performed using data from previous work that reported significantly increased perceptual surround suppression of contrast in older observers, relative to a younger cohort . The anaEthics approval was granted by the Human Research Ethics Committee of the University of Melbourne. All participants provided written consent prior to participation in compliance with the tenets of the Declaration of Helsinki. An eye examination was conducted  to ensure the following eligibility criteria were met: best corrected visual acuity at least 6/7.5 or better with a refractive error between ± 5.00 D sphere and less than –2 D astigmatism, normal ocular health, no significant lens opacities defined as Grade 1.5 or less on the Lens Opacities Classification System III scale , and no 2 mean luminance.The experimental software was written in Matlab V7.6 , with experimental stimuli presented on a gamma-corrected Sony G500 CRT monitor  using a ViSaGe graphics system . Participants were refractively corrected for the 80 cm working distance. Stimuli were viewed binocularly. The background was a homogenous gray screen of 50 cd/mFigure 1A). All stimuli shown were supra-threshold. Fixation was assisted by four white nonius lines, which appeared after each trial and disappeared during stimulus presentations.Participants typically required 2 h to complete all of the tasks including regular rest breaks. There were eight tasks in total: four stimuli  and two conditions (‘surround’ and ‘no surround’). The ‘no surround’ condition (0.67° radius center stimulus only) was tested first, followed by the ‘surround’ condition (center + 4° radius surround). The inclusion of a ‘no surround’ condition was important to establish that participants were able to accurately judge the stimuli (the specific judgments required are described below) and to account for any baseline biases. The order of the four stimuli was randomized for every participant and counterbalanced between older and younger groups to avoid order-dependent effects of learning or fatigue. Each task was tested twice using a Method of Constant Stimuli (MOCS) consisting of seven stimulus levels of 10 repeats . For training purposes and to decide which stimulus levels to formally test for the ‘surround’ conditions, an initial abbreviated MOCS was performed . On each trial, participants viewed two stimuli (500 ms duration each) that were presented one after the other  separated by a 500 ms interstimulus interval . Participants were required to indicate the central stimulus  that appeared to be brighter by pressing one of two response buttons.For the luminance task, the monitor was configured to run at a frame rate of 100 Hz and have a resolution of 1024 × 768 pixels. Stimuli were homogenous circular patches, with the reference target of 55 cd/mThe monitor configuration was altered to allow for greater temporal resolution . Similar to the luminance task, the flicker task involved homogenous circular patches except that the luminance contrast was temporally modulated. Both center and surround regions had a temporal contrast of 50% and were flickering at a rate of 15 Hz. To perceptually segment the center from the surround region, the two regions were presented with 180° phase difference given there was no gap between the center and the surround. The task was to choose which central stimulus appeared to be flickering with a greater depth of modulation.Figure 1B). The luminance noise images were constructed as per The monitor configuration used for the contrast task was the same as for the luminance task . To avoid the potential confound of orientation information, the stimulus was a textured patch consisting of filtered noise . The reference center was oriented at 55° and the surround was oriented at 75°, given that the repulsive tilt illusion is greatest when the center and surround orientation differs by 20°  is the cumulative Gaussian distribution with mean μ and standard deviation σ for value t. FP and FN represent the false positive and false negative error rates, respectively, assuming that false responses are made independently of the underlying Gaussian distribution of responses. Two parameters of interest were extracted for statistical analysis: (a) the mean of the fitted psychometric function (μ) or the ‘point of subjective equality’ (PSE), when both the reference and target stimulus appeared subjectively the same, and (b) the spread of the Gaussian (σ), which provides an estimate of precision, or the slope of the psychometric function. To quantify the strength of surround suppression for each task, a suppression index was calculated (1 – PSE ‘surround’/PSE ’no surround’). A positive suppression index indicates suppression (maximum suppression = 1), a negative index indicates facilitation, and an index of 0 indicates no effect of the surround.where Figure 2). To avoid removing all data from a single observer if only one data point was missing, a linear mixed effect model was employed to compare older and younger group performance across all tasks. The fixed factors of the linear mixed effect model were ‘group’  and ‘stimulus’  and the random factor was ‘participant’ to control for non-independence among repeated observations for an individual.Statistical comparisons were performed using SPSS Version 22.0 . Data were tested to determine the probability that the sample was derived from a Gaussian distribution (Kolmogorov-Smirnov test). Three participants (one younger and two older observers) could not satisfactorily match one of the four ‘no surround’ percepts, whereas data were incomplete for the flicker suppression task for five older adults, who perceived zero flicker in the ‘surround’ condition, despite being able to reliably judge the depth of modulation in the ‘no surround’ condition  = 0.79, p = 0.38; group × stimulus interaction: F = 2.42, p = 0.07]. This shows that both older and younger participants approximately matched to the same percept when there was no surround. Moreover, the spread of the psychometric functions  was not different between groups  confirming no between-group difference in the precision with which stimulus comparisons were made.A single mixed model analysis was used to first compare the performance of the groups when there was no surround . The PSE for older and younger observers did not differ for the baseline ‘no surround’ conditions . The difference in group performance was consistent across stimuli . Effect sizes  were calculated to compare the magnitude of increased suppression in the older group, relative to the control group:Next, we used a separate mixed model analysis to compare the effect of introducing a surround by calculating suppression indices, as illustrated in where,1 and μ2 are the mean suppression indices for the younger and older groups, respectively, and σ1 and σ2 are the standard deviations. Effect sizes were medium-large (Cohen’s d > 0.5) for all stimuli tested . To take into account differences in task variability, suppression indices were normalized against the performance of the control group . The relative increase in surround suppression in older adults (increased positive z-score) was not stimulus-dependent . Thus, differences in contextual effects between older and younger observers appear to be widespread and of similar magnitude (medium–large effect size) across different visual dimensions.and μFigure 5 depicts the inter-task Pearson correlation analyses across the entire dataset based on the suppression indices, which takes into consideration baseline (no surround) performance. Statistical significance was defined at an alpha level starting at 0.008 , given there were six multiple comparisons. Under this criterion, we found a statistically significant positive correlation between contrast and orientation suppression . Similarly, although these did not reach statistical significance based on sequential multiple comparisons , trends for a positive relationship between luminance and contrast suppression , and luminance and flicker suppression , were also observed. Table 1 shows the correlation analyses for each group (younger and older) separately. None of these reached statistical significance once the 12 multiple comparisons were considered.Does stronger suppression on one task predict stronger suppression on other tasks? Given that visual contextual performance is relatively uniform within each age group (younger versus older), we pooled the data from the two groups to obtain a range of suppressive strengths in the presence of a surround. Figure 2), as a group older participants showed a consistent increase in surround suppression for all targets tested . Our findings are congruous with previous observations that foveal suppression of perceived contrast is increased in older adults . Hence, age-related changes to contextual processing are widespread in the visual system. Our results are in agreement with two previous reports of greater age-related surround suppressive effects for luminance stimuli , providing indirect support for a common process underlying age-related differences in center–surround perception. Perhaps not surprisingly, the two center–surround stimuli presumed to rely predominantly on pre-cortical processing (luminance and flicker) were correlated, as were the tasks with principal V1 involvement (contrast and orientation). In addition, the significant correlation between the strength of perceptual suppression of contrast  and luminance  is consistent with surround suppression at V1 being, at least partly, inherited from pre-cortical surround suppression at the retina and LGN. On the other hand, previous reports find no such correlations between different tests of surround suppression , and conclude that these tasks reflect distinct neural mechanisms , possibly owing to button response bias and/or differences in masking/adaptation due to the 2IFC design. Given that the magnitude of contextual effects depends on the relative difference in percept once a surround is introduced, group differences in the strength of suppression may be less evident if the ‘no surround’ PSE is assumed to be a single constant that is common to all observers.By testing the same observers across all tasks, we could examine whether performance was correlated between tasks and hence, infer whether the neuronal mechanisms involved in the contextual processing of fundamental visual attributes are independent. Performance was significantly, albeit modestly, correlated between certain stimuli across our range of ages , as evidenced by the medium–large effect sizes reported in this study, but that our correlational analysis  might only have reached statistical significance had more participants with complete data been tested. It should also be noted that our older participants may not be representative of all older adults – rather, they are healthy, lead active lives and live independently within the community.In interpreting our results, it is important to note that our small sample sizes were sufficient  to detect differences in suppression strength between older and younger observers (We demonstrate that surround suppression of luminance, flicker, contrast, and orientation is increased in older adults, implying a generalized contextual processing change with normal aging. Further work is needed to disentangle the specific neural mechanisms involved; however, this study provides a basis for targeted behavioral and neurophysiological approaches to be applied to the study of the aging visual neural system.Study design: BN and AM. Subjects and data collection: BN. Data analysis and interpretation: BN and AM. Paper drafting and revision: BN and AM. Final approval of the version to be published: BN and AM. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: BN and AM.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.


Factors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth.We characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy.In LuCaP35 tumors  dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone . In LuCaP96 tumors (T:DHT 10∶1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels , reconstitution of intra-tumoral DHT (to ∼30% of untreated tumors), and ∼2 fold increased expression of full length AR. In contrast, LuCaP96 demonstrated higher levels of steroid catabolizing enzymes , persistent suppression of intra-tumoral DHT, and 6–8 fold induction of full length AR and the ligand independent V7 AR splice variant. Human metastases demonstrated bio-active androgen levels and AR full length and AR splice-variant expression consistent with the range observed in xenografts.Intrinsic differences in basal steroidogenesis, as well as variable expression of full length and splice-variant AR, associate with response and resistance to pre-receptor AR ligand suppression. Expression of steroidogenic enzymes and AR isoforms may serve as potential biomarkers of sensitivity to potent AR-axis inhibition and should be validated in additional models. The clinical responses to new potent AR antagonists and novel steroid synthesis inhibitors, coupled with evidence of intracrine androgen biosynthesis in prostate cancer suggest that concepts of maximal androgen blockade should be revisited and refined. A recent study designed to target multiple steps in androgen biosynthesis in men failing ADT  dutasteride) produced substantial PSA declines in most patients and a median response duration of 20 months. In this study, we sought to determine tumor-specific characteristics that influence response of prostate cancers to inhibition of pre-receptor AR signaling, using androgen deprivation combined with the dual SRD5A inhibitor dutasteride. T and DHT are biologically active high-affinity AR ligands. However, between 3–10 fold higher concentrations of T are required to exert the same AR-mediated transcriptional effects of DHT. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments involving animals were performed in accordance with protocols approved by the Fred Hutchinson Center Institutional Animal Care Use Committee (file 1775). All surgery was performed under isofluorane anesthesia, and all efforts were made to minimize suffering. The LuCaP35 and LuCaP96 lines are patient derived xenografts that were established as part of the University of Washington tissue bank as previously described 3 tumor pieces. When tumors reached an average of 300 mm3, mice  were castrated (Cx) and randomized to treatment with vehicle or dutasteride (Dut) therapy over 8 weeks. Dutasteride was administered (PEG monolaurate (Sigma 460133)/1% Tween 80 (Sigma P4780)) by oral gavage 5 days per week in a volume of 200µl. Tumor volume was determined by the following formula (long and short axis lengths in mm): long×(Short∧2)/2. Tumors from a subset of mice in each cohort were harvested at early time points of treatment . When tumors reached approximately 750–1000 mg in size, the animals were euthanized according to institutional protocol and the xenografts harvested and flash frozen for determination of tissue androgens and extraction of total RNA. The average number of days mice in the Cx+Dut groups had been off dutasteride treatment at the time tumors were resected was 211 days for LuCaP35, and 96 days for LuCaP96. Dutasteride was generously provided by GlaxoSmithKline.Male C.B-17 SCID mice  were implanted subcutaneously with 30 mmAll procedures involving human subjects were approved by the Institutional Review Board of the University of Washington Medical Center, and all subjects signed written informed consent. Patients with metastatic prostate cancer underwent rapid autopsies under the aegis of the University of Washington Prostate Cancer Donor Autopsy Program as previously described Androgen levels in xenograft tissues were determined by mass spectrometry (MS) using methods we have described. Samples were used for RNA isolation, cDNA synthesis and quantitative RT-PCR using methods and primers we have previously described. 3) was determined via Kaplan Meier analysis with comparison of curves using the Mantel-Haenszel logrank test. To predict the approximate sample size in each treatment group that might be necessary to detect a statistically significant effect of castration plus dutasteride versus castration alone, we utilized complete case resampling for a range of sample sizes n = 10, 20, …, 200 over 500 bootstrap replicates. For each sample size and bootstrap replicate, we fit a Cox proportional hazards model and recorded the estimated hazard ratio and p-value.To evaluate differences in tumor volume trajectory, we first determined change points in the tumor growth curves using t-tests to sequentially compare tumor volumes every two days. After segmenting the curves, we applied a linear mixed model to quantify the treatment effect on tumor growth for each linear period by regressing log tumor volume on day of measurement, on treatment group, and on an interaction between day and treatment while accounting for variability in initial tumor volume across mice. Progression free survival in castration vs. castration + dutasteride treated mice (defined as tumor size <750 mmΔΔCt). The raw dCT’s, ddCT’s and fold changes are presented in the Supplemental data.For analysis of qRT-PCR data, the mean cycle threshold (Ct) for each gene was normalized to expression of the housekeeping gene RPL13A in the same sample (delta Ct). The mean expression of RPL13A was 15.6+/−0.5 (mean +/− SD) and 14.1+/−0.8 in LuCaP35 and LuCap96 tumors respectively. Welch’s two sample t-tests were used to compare differences in androgen levels and differences in mean delta Ct’s for each gene between treatment groups without correction for multiple testing. P values<0.05 were considered significant. The fold change was calculated by the delta-delta Ct method (fold = 2Table S1).To evaluate tumor characteristics associated with response and resistance to AR pathway-directed therapy, we first determined intratumoral androgens and steroidogenic gene expression in two prostate cancer patient derived xenografts, LuCaP35 and LuCaP96. These lines express AR and PSA, . While many steroidogenic genes were expressed in LuCaP35 within one order of magnitude of their expression in LuCaP96 (Table S2). Since these tumors were propagated in genetically-identical murine hosts, these data identify intrinsic tumor-specific variation in androgen metabolic programs culminating in marked intratumoral differences in AR ligand concentrations.To identify mechanisms contributing to basal differences in intra-tumoral T and DHT concentrations, we evaluated tumor-specific expression of transcripts encoding the primary enzymes involved in steroid synthesis and degradation  at the time of enrollment ; however, this may reflect decreased power associated with the post-hoc sub-classification of the groups.To further examine tumor characteristics associated with response to pre-receptor AR pathway inhibition, we determined whether response to treatment was related to tumor size at initiation of therapy. Although the study was designed to begin treatment at a tumor volume of ∼300 mm3 at enrollment (3 at enrollment (Inspection of tumor volume curves suggested a suppressive effect of dutasteride was maintained even after discontinuation of therapy for tumors <250 mmTable S1).To determine whether the differential impact of SRD5A inhibition on tumor growth reflected a difference in suppression of intratumoral androgens, we measured T and DHT levels in tumors resected after treatment. In both LuCaP35 and LuCaP96 tumor types, mean tumor DHT levels measured at 3–21 days (indicated by two-headed arrows in Table S1). Of note, the half-life of dutasteride in mice is less than 2 days, Interestingly, while T levels in LuCaP35 tumors remained suppressed in either treatment group . Collectively, these data show that dutasteride effectively inhibited DHT production in both tumor types, and that LuCaP35 tumor growth is susceptible to the enhanced pre-receptor suppression of DHT achieved with the combination of castration plus SRD5A inhibition, whereas LuCaP96 is not.The tendency toward reconstitution of DHT levels in LuCaP35 and of T in LuCaP96 is consistent with the intrinsic variation in androgen metabolic programs favoring DHT vs. T identified in the untreated tumors (FL), and as recently reported, by increased expression of ARsv that lack the LBD and consequently confer constitutive ligand-independent AR activity. Differences in ARFL and ARsv levels may contribute to the variation in tumor growth responses following pre-receptor ligand suppression. Therefore, we evaluated the expression of ARFL and the prevalent ARsv identified in human CRPC samples, ARV7 (which encodes the same protein as ARV3) in LuCaP35 and LuCaP96 tumors recurring after ADT and dutasteride Castration resistant prostate tumors are frequently characterized by increased expression of full-length AR . In contrast, while LuCaP96 tumors recurring after castration alone demonstrated a similar 1.9-fold increased expression of ARFL, they also showed a substantial 10-fold increase in the expression of ARV7 . However, significant differences were generally not observed in recurrent tumors resected at late time points, consistent with the increase in AR expression (Table S3) and reconstitution of tumor androgen levels observed in these samples. Interestingly, expression of FKBP5 remained quite low in recurrent LuCap96 tumors despite the marked increase in ARV7, suggesting ARV7 does not necessarily completely recapitulate the transcriptional activity of full length AR.Compared to tumors resected from intact mice, LuCaP35 tumors recurring after castration or after castration plus dutasteride demonstrated a 2.0-fold increase in expression of ARV7 in LuCaP96, we did not observe a parallel increase in the absolute expression of genes previously suggested to constitute the variant transcriptome . (13–16) However the induction of ARV7 in LuCap96 was associated with a significant shift in the correlation of its expression with AKT. Whereas the expression of ARV7 and AKT was correlated in LuCaP35 tumors under all treatment conditions , expression of these genes only became correlated in LuCaP96 tumors when ARV7 was induced after treatment .Despite the increased expression of ARsv may account for the differential impact of pre-receptor androgen suppression on tumor-growth inhibition, with the marked rise in ARV7 observed in LuCaP96 rendering it correspondingly less sensitive to ligand suppression by dutasteride. Of note, consistent expression of the ARv567 variant also reported in human CRPC tissues was not detected in these xenografts (data not shown) These data suggest that tumor-specific differences in the induction of full-length AR and ARFL (as represented by LuCaP35), whereas another is characterized by robust induction of ARsv, rather than metabolic adaptations directed at increased DHT synthesis (as represented by LuCaP96). To determine whether these observations are relevant in context of in vivo human prostate cancer, we compared relative levels of T, DHT and AR expression in tumor metastases directly obtained from men with CRPC. Given the 5–10 fold higher potency of DHT for engaging and activating AR relative to T, sv are not available, AR immunostaining was performed using antibodies recognizing the N-terminus (ARN+) or C-terminus (ARC+) of the AR protein to identify tumors expressing predominantly ARFL , and those with C-terminal truncated ARsv  The studies of LuCaP35 and LuCaP96 xenografts support the existence of at least two subtypes of CRPC. One type is reliant on androgen biosynthesis and intratumoral maintenance of the potent AR ligand DHT, with modest upregulation of ligand-activated AR). Moreover, patients 1 and 3 whose metastases were characterized by a higher androgenicity index with a relatively larger contribution of DHT (similar to castration recurrent LuCaP35) demonstrated primarily ARN+ and ARC+ staining, consistent with detection of ARFL, and were consistently PSA positive  compared to LuCaP35 (0.40±0.56 ng/gm), as emerging data suggest that induction of AR variants reflects a dynamic response to ligand inhibition. V7 is not markedly induced by suppression of DHT to 0.02±0.04 ng/gm in LuCaP35 tumors treated with castration plus dutasteride, suggesting tumors may also possess intrinsic differences in the propensity for generation of ARSV. Our data demonstrating significant levels of intratumoral T in LuCaP96 are in contrast to the study of Chang et al, which suggested that the route to intratumoral DHT synthesis can bypass T. Our findings indicate that this is not the case in every instance, and emphasize the range of tumor resistance mechanisms likely to be encountered in CRPC tumors Factors responsible for the preferential induction of steroidogenesis versus induction of ARSV, as has been observed in a subset of hormone naïve primary prostate cancers, The impact of DHT suppression on tumor growth inhibition in LuCaP35 was influenced by tumor size at initiation of therapy, with smaller tumors demonstrating a more profound suppression of tumor growth which also appeared to be durable after discontinuation of therapy, but larger tumors demonstrating suppression of tumor growth only while actively receiving treatment. These data suggest that even in tumors that are sensitive to androgen suppression, the potential efficacy associated with ligand synthesis inhibitors may be more pronounced and/or more durable if initiated earlier at a lower volume of disease. These data also provide a hypothesis for results of the PCPT and REDUCE clinical trials using SRD5A inhibitors for prostate cancer chemoprevention. in situ metastases demonstrate a patient-specific range of tumor androgen levels and associated differences in AR expression similar to that observed in LuCaP35 and LuCaP96 xenografts. Specifically, human metastases characterized by higher tissue androgen index demonstrated evidence of full length AR expression (similar to LuCaP35), whereas metastases with a relatively lower tissue androgen index demonstrated loss of C terminal AR staining, consistent with expression of ligand independent AR variants (as observed in LuCaP96). These data suggest that our findings regarding response and resistance to treatment strategies targeting pre-receptor androgen pathway suppression in the LuCaP35 and LuCaP96 xenograft models are likely to be relevant to the clinical spectrum of disease encountered in patients with CRPC.Notably, direct examination of human CRPC tumors showed that FL or ARSV as potential mechanisms of resistance in tumors recurring after treatment with abiraterone. SV, at a magnitude that may be sufficient to impede sensitivity to additional androgen ablation, can occur following castration alone, as observed in LuCaP96 tumors. Clearly, these represent prostate tumors for which agents acting through mechanisms independent of ligand binding have a higher likelihood of efficacy than agents targeting ligand synthesis or the LBD. Consistent with this hypothesis, recent data demonstrate that patients with detectable ARSV in circulating tumor cells have a poor response to the LBD antagonist enzalutamide. Importantly, the observations reported in the present study may be relevant to optimizing first-line treatment strategies with novel ligand-synthesis and AR targeted agents such as abiraterone and enzalutamide. While the clinical responses to abiraterone and enzalutamide have been impressive, not all patients respond, the duration of response has been variable, and mechanisms of resistance are not well understood. SV and steroidogenic enzyme expression as biomarkers for treatment success. Collectively, however, these studies suggest that intrinsic differences in basal steroidogenesis and expression of ligand-independent ARSV may associate with response and resistance to pre-receptor suppression of AR ligands, and may directly underlie the variable clinical efficacy observed in clinical studies of abiraterone and enzalutamide in men with CRPC. Recent data demonstrating that enzalutamide, which targets the AR LBD, had decreased efficacy against tumors expressing LBD-deleted ARSV, suggests these tumors may require agents targeting the AR N terminus, and is a critical subject of ongoing research. FL and ARSV as potential biomarkers of response and resistance requires assessment in ongoing clinical studies employing molecular evaluation of tumor tissue from men with CRPC.An important limitation of this study is that only two xenograft models were characterized, and evaluation of further models will be required to establish the validity of ARFigure S1The Androgen Synthetic Pathway. The schema depicts the enzymatic steps in the conversion of cholesterol to androgens. Steroid intermediates are given in the boxes. Steroidogenic enzymes mediating each step are given next to the arrow denoting the direction of the conversion. After being generated from cholesterol by CYP11A C21 steroids are converted to the C19 adrenal androgens DHEA by the sequential hydroxylase and lyase activity of CYP17A. DHEA is acted on by HSD3B to form androstenedione (AED), which in the classical pathway is then acted on by HSD17B3 (or AKR1C3) to form testosterone, which is converted to DHT via SRD5A. In the 5-alpha-Androstanedione pathway AED is converted first by SRD5A to 5α-Androstanedione and then by HSD17B3 (or AKR1C3) to DHT. In the backdoor pathway the progestin intermediates are acted on first by the activity of SRD5A and the reductive activity of AKR1C2 prior to the lyase activity of CYP17A to form androsterone (not shown). Androsterone is then acted on by HSD17B3 (or AKR1C3) and must undergo an oxidative step mediated by RL-HSD (or others) to generate DHT.(TIF)Click here for additional data file.Figure S2Expression of steroidogenic genes in LuCap35 and LuCaP96 prostate cancer xenografts. Relative expression of the indicated steroidogenic genes in LuCaP35 and LuCaP96 in tumors grown in intact mice  and in tumors re-growing after castration (B) or castration+8 weeks dutasteride (C). Transcript levels were measured by qRT-PCR and normalized to the housekeeping gene RPL13A within each sample to yield the delta cycle threshold (dCT). For each gene the relative difference in mean expression between LuCaP35 and LuCaP96 was calculated using the delta dCt method (fold change = 2∧ddCtT). Genes differentially expressed in LuCaP35 vs. LuCaP96 within one order of magnitude are indicated within the gray lines. Significant differences  are indicated by black circles and colored triangles; white circles indicate genes that were not significantly different between LuCaP35 and LuCaP96. Upward red triangles indicate highly differentially expressed genes leading to increased T (AKR1C3) and increased DHT levels . Downward green triangles indicate highly differentially expressed genes mediating DHT catabolism .(TIF)Click here for additional data file.Table S1Androgen levels in LuCaP35 and LuCaP96 tumors after pre-receptor androgen suppression with castration or castration + dutasteride.(TIF)Click here for additional data file.Table S2Fold difference in mean transcript expression of steroidogenic genes between LuCaP35 and LuCaP96 tumors grown in intact mice.(TIF)Click here for additional data file.Table S3Expression of AR and AR-regulated genes in LuCaP35 and LuCaP96 tumors after pre-receptor androgen suppression with castration or castration + dutasteride.(TIF)Click here for additional data file.


In the present study, the removal of phenol from synthetic wastewater was evaluated in a fixed bed reactor with up flow sludge blanket filtration (FUSBF) in comparison with a typical USBF system. At hydraulic retention time (HRT) of 24 hours and solid retention time (SRT) of 20 day, the effect of initial concentration of phenol (260-1020 mg/L) on phenol and chemical oxygen demand (COD) removal efficiency (%) was investigated in both systems. The effect of the presence of fixed bed was determined throughout the operational period.-3.h-1, the amount of phenol removal was 82.1%. At OLR of 30 g/m-3.h-1, role of fixed bed in phenol and COD removal was 25.01% and 29.3%, respectively, overall, the FUSBF system has a higher efficiency and ability than that of typical USBF and can be used for the purification of industrial wastewater containing refractory organic compounds such as phenol.The results showed that the FUSBF system had a better ability than the typical USBF system in terms of phenol and COD removal. The average phenol and COD removal at phenol concentration of 312 mg/L was 97.52% and 92.82% for the FUSBF system and 92.80% and 82.18% for the typical USBF system, respectively. At HRT of 24 h and organic loading rate (OLR) of 30 g/m The amount of RAS to the anoxic area was 3 times of the average inflow. The reactors were operated at room temperature (25 °C). The required air for aeration was mixed in the aerobic zone by an air compressor with a maximum output of 90 L/min along with three air stone diffusers. Air flow was adjusted by hand valves so that the concentration of dissolved oxygen in the aerobic zone liquid was 4.0±0.5 and oxidation-reduction potential (ORP) in the anoxic area was about –30±40 mV. The pH of solution was maintained neutral throughout the experiments.For FUSBF hybrid bioreactor, an aerobic fixed film was installed as an appropriate unit for further biological removal in the aerobic zone. Twenty percent of the aerobic reactor volume was filled by a media made of high density polyethylene (HDPE). Support material surface area, porosity, mean diameter and density were 400 m3NH4 as the nitrogen (N) source, and KH2PO4 and K2HPO4 as the source of phosphate (P), entered into the system throughout the operation and minor elements for biomass were added into the synthetic wastewater as follows: FeCl3.6H2O: 1.5 g/L, H3BO3: 0.15 g/L, CuSO4.5H2O: 0.03 g/L, KI: 0.03 g/L, MnCl2.4H2O: 0.12 g/L, NaMnO4.2H2O: 0.06 g/L, ZnSO4.H2O: 0.12 g/L and CoCl2.6H2O: 0.15 g/L (31). The composition of wastewater was determined based on the ratio of COD/N/P: 100/5/1. The required sludge was obtained as seed from a municipal sewage treatment plant in Arak. The operational flow rate for both FUSBF and USBF bioreactors were 10-60 L/dThe operational parameters for both FUSBF and USBF bioreactors are presented in Synthetic wastewater, containing phenol as the carbon source, NOPhenol was added increasingly and stepwise (from 0 to 260 mg/L) to the FUSBF and USBF bioreactors during the startup. Phenol was the sole carbon source for growth of microorganisms and formation of biofilm. Synthetic wastewater was continuously prepared and supplied into both FUSBF and USBF reactors and was pumped with a flow of 20 L/day under the mentioned operational conditions. During the startup period, this flow level was used for 3.7 h and 10.8 h in anoxic and aerobic zones, respectively, at a HRT of 24 hours and a SRT of 20 days. Microorganisms and the biofilm were well grown under control in different concentrations of pollutants. Treatment of inflow began when the biofilm was formed and the pollutants concentration in the bioreactors outflow became constant. Under these conditions, it takes 165 and 172 days to startup the developed FUSBF and typical USBF bioreactors, respectively. Then the efficiency of the systems was evaluated with different initial concentrations of phenol and different HRT from 12 to 48 hours. The conditions for implementation of the stages were determined using a combination of parameters given in Using synthetic wastewater, the systems reached to stable conditions and then were utilized. After seeding, the biofilm formed in aerobic reactor of the developed FUSBF bioreactor was visualized (in terms of morphology) using scanning electron microscopy (SEM). For this, the media was randomly sampled from various parts of the aerobic reactor at the end of the experiment in the stable stage. The withdrawn media were dried in darkness at room temperature for 12 hours before observation . The bioDuring acclimatization of the liquid mixture, the initial concentration of phenol was gradually increased from 60 to 260 mg/L. During this period, increase in phenol degradation at each stage of phenol initial input was considered as the fulfilling of acclimatization. Then the next stage of phenol increase started. In other words, increased rate of phenol biodegradation in a range of increasing phenol concentrations confirmed that acclimatization was fulfilled, the microbial population boosted and propagated, and biomass gained the capacity and ability to degrade phenol under the conditions existing in both bioreactors of USBF and developed FUSBF. So biomass was prepared as a suitable seed for utilization and operation of bioreactors. The total time required for the acclimatization stage for both USBF and developed FUSBF bioreactors was 172 and 165 days, respectively. The total amount of inoculated biomass in the anoxic and aerobic reactors at startup in the USBF reactor was almost 2100 and 2480 mg/l, respectively.SEM image of the initial clean media and the biofilm containing media was used to view and characterize the surface of fixed film medium in the aerobic FUSBF reactor. As shown in The results showed that the media used was suitable for biofilm formation or formation of biofilm and no pre-seeding was required. After a successful startup and achieving a stable stage (less than 5% change in phenol output in a week at phenol concentration of 260 mg/L) and confirmation of biofilm formation via SEM, the reactors were operated under conditions of HRT=24 h and SRT=20 d for evaluation of phenol removal at different input concentrations of 312, 416, 520, 780, and 1020 mg/L. Experiments were performed when the conditions were stable; these stable conditions were the same for all experiments and had no statistically significant difference (less than 5%) for about 5-7 days between the results of experiments. The behavior of typical USBF and FUSBF bioreactors at HRT=24 h and SRT=20 d was evaluated at different initial concentrations of phenol. The removal of phenol and COD was displayed as a function of initial concentration of phenol . The remThis increase in the efficiency of daily removal of 312 mg/L phenol during the operation shows that the seed inoculated or injected into the USBF and FUSBF bioreactors was able to degrade high concentration of phenol. In a similar approach, phenol concentration was increased in a stepwise manner to 416, 572, 728, and 1020 mg/L and both bioreactors were operated and evaluated until achieving steady-state condition. As seen, after a sharp increase in the concentration, for example from 416 mg/L to 572 mg/L and at any other concentration increments, the removal rate decreased at both reactors immediately the next day, although the rate of removal decline in the FUSBF reactor was less than the USBF reactor after each step of increase in concentration. For example, immediately after an increase in the concentration of phenol to 572 mg/L, the removal rate of the FUSBF reactor reached to 72.3% the next day, while declined to 63.5% in the USBF reactor. However, the removal rate increased again in both bioreactors in the next few days of operation. In general, by increasing the concentration of phenol, the removal process had a descending trend in both bioreactors, although the highest removal rate of decline was observed at 1020 mg/L in both reactors. At this concentration, the decrease in removal efficiency was higher in the typical USBF reactor than the FUSBF reactor. In fact, although the removal rate increased again after its sudden decline at the day next to sudden increase in phenol concentration to 1020 mg/L, and although both bioreactors were restored again and achieved steady state conditions, the time required to reach the steady state conditions was 20 days for FUSBF and 35 days for typical USBF bioreactor.To confirm the capabilities of both FUSBF and typical USBF systems under the minimum inhibitory concentration of phenol and to test the revival behavior of the system caused by excessive load entered, the input concentration of phenol was reduced in a stepwise manner from 1020 mg/L, to 728, 572, 468, and 312 mg/L. At each concentration, both bioreactors achieved the stable conditions of removal. Changes in effluent COD concentration and COD removal along with initial phenol concentration are shown in Biological wastewater treatment processes are cost effective only when they act highly efficient in a short time, otherwise the facilities size may not meet the requirements. Removal of phenol was evaluated at HRTs of 48, 36, 24, 12, and 8 hours in FUSBF under steady-state conditions. The phenol and COD removal under different HRTs are shown in 3.h-1) were used and compared in both FUSBF and typical USBF. The average phenol and COD removal efficiencies are shown in To better understand and explain the role of fixed bed media in operation of FUSBF system, two different loads of phenol  may be due to consumption and attraction of more phenol load by the fixed film biofilm that result in lowering of the inhibitory effect of phenol on biomass in the reactor and delaying of the inhibitory effect by the fixed film biofilm. The role of fixed film media was greater in COD removal than in phenol removal. The role of fixed film media for COD removal of phenol loads of 13 and 30 g phenol/mIn the present work, an FUSBF process was developed and studied removal. The fix film USBF intergraded bioreactor at the optimum condition can be an effective technology for phenol removal from synthetic wastewater.In this type of system microbial fixation and biofilm formation the support surface are two of the most important factors. Since they affect the levels of elimination of every pollution concentration in a synthetic wastewater. The results reveal that the FUSBF provides inproved phenol and COD removal efficiencies better than the conventional USBF, when it is operated as a single reactor with 20%media volume. The inhibition concentration of phenol in FUSBF and USBF are 1020 mg/l. The optimum HRT for the FUSBF is 24 h, at which the removal efficiencies of phenol and COD are greater than90% The critical phenol leading rate for the FUSBF is 30 g phenol/m-3.h. Which gives a phenol removal efficiency of 82.54(%) The reactor FUSBF demonstrates a high capacity for phenol removal compared to the USBF reactor. Accordingly the FUSBF is an efficient reactor phenol compound removal.


Congracilaria babae was first reported as a red alga parasitic on the thallus of Gracilaria salicornia based on Japanese materials. It was circumscribed to have deep spermatangial cavities, coloration similar to its host and the absence of rhizoids. We observed a parasitic red alga with morphological and anatomical features suggestive of C. babae on a Hydropuntia species collected from Sabah, East Malaysia. We addressed the taxonomic affinities of the parasite growing on Hydropuntia sp. based on the DNA sequence of molecular markers from the nuclear, mitochondrial and plastid genomes . Phylogenetic analyses based on all genetic markers also implied the monophyly of the parasite from Hydropuntia sp. and C. babae, suggesting their conspecificity. The parasite from Hydropuntia sp. has a DNA signature characteristic to C. babae in having plastid rbcL gene sequence identical to G. salicornia. C. babae is likely to have evolved directly from G. salicornia and subsequently radiated onto a secondary host Hydropuntia sp. We also recommend the transfer of C. babae to the genus Gracilaria and propose a new combination, G. babae, based on the anatomical observations and molecular data. Red algal parasites have been described from at least eight orders, including Ceramiales, Corallinales, Gigartinales, Gracilariales, Halymeniales, Palmariales, Plocamiales and Rhodymeniales A previous study Traditionally, the evolutionary relationships between red algal parasites and their host species were assessed by morphological similarity. However, determination of taxonomic positions of red algal parasites based solely on morphological inference was hindered by the complicated evolutionary history of the parasites, which may result in the morphological dissimilarity between the parasites and their hosts, a broad host range, and possible host-switching events. Molecular phylogenetic techniques have been successfully used to resolve the evolutionary relationships between red algal parasites and their host species Gracilariophila Setchell and Wilson Holmsella Sturch Gracilariocolax Weber van Bosse Congracilaria Yamamoto Gracilariocolax and Congracilaria are documented as pigmented pustules devoid of rhizoids penetrating into the host tissues, differing only in their sporangial division pattern and host species Gracilariocolax and Congracilaria may essentially be congeneric considering the similar morphological and reproductive features exemplified, as well as the de-emphasized diagnostic value of sporangial division pattern for Congracilaria, Ng et al. Gracilariocolax obtained from the type host species is available.Gracilariaceae, known for several economically important seaweeds, hosts several genera of red algal parasites, including Congracilaria babae Yamamoto on the host Hydropuntia species attached to the monolines of Kappaphycus in aquaculture farms. In addition to morphological and anatomical study, phylogenetic analyses based on the DNA sequences of the nuclear ITS region, mitochondrial cox1 gene and plastid rbcL gene were conducted to confirm the identity of the parasite from Hydropuntia sp. We sequenced DNA of the parasite from Hydropuntia sp. and compared the DNA sequences with those of Malaysian and Japanese C. babae found on G. salicornia generated from an earlier study Hydropuntia sp. as well as the relationship between the host-parasite association using molecular tools.In an algal collection from Sabah, East Malaysia, we found a red algal parasite suggestive of C. babae is found on Hydropuntia, a seaweed species that grows in close association with Kappaphycus on the monolines in the aquaculture sites. Hydropuntia is largely regarded as nuisance to Kappaphycus and thus does not require specific permission for sampling. The specimens are not endangered or protected species. For collection of specimens from farms, consents were granted from respective owners.No specific permits were required for the described field studies as the specimens were not collected from any national parks or protected areas. The red algal parasite A small part of each host individual bearing red algal parasites was fixed in 5% formalin/seawater, and an additional part of the specimen was desiccated in silica gel for molecular analyses. The remainder of each parasitized sample was pressed into a voucher herbarium specimen and deposited in the herbarium of the University of Malaya. Sections for anatomical study were prepared using paraffin method as outlined in rbcL, F7/RrbcS start, or F7/R753 and F577/RrbcS start cox1, COXI43F/COXI1549R Hydropuntia sp. was amplified for all the markers and the amplicons were sent for cloning  to check if the host DNA was co-extracted. Three to five clones of the representative parasite individual were sequenced for each marker.Molecular analyses were conducted on at least two parasite individuals and the actual individual host plant from which each parasite was isolated, for each site. The host and parasite tissues were carefully sampled for DNA extraction under a stereomicroscope. Only the top half of the parasite pustule farthest from the host thallus was sampled to avoid host tissue contamination. The host tissues were sampled preferably at the tip or another part without discernible swelling. Extraction of genomic DNA was performed using the i-genomic Plant DNA Extraction Mini Kit  according to the manufacturer’s recommendations. Parameters for PCR amplification and sequencing followed Hydropuntia sp.-C. babae and G. salicornia-C. babae obtained by direct sequencing  and with two model-based approaches, maximum likelihood (ML) and Bayesian analysis. MP phylogenies were constructed using PAUP* 4.0b10 rbcL datasets, and GTR+I+G for the cox1 dataset. The default priors of MrBayes were used: (1) tratiopr = Beta  for the ITS and rbcL datasets, and Revmatpr = Dirichlet  for the cox1 dataset, (2) statefreqpr = Dirichlet , (3) shapepr = uniform , (4) topologypr = uniform, and (5) brlenspr = unconstrained: exp (10.0). Bayesian analyses were initiated with a random starting tree and two parallel runs, each of which consisted of running one cold chain and three hot chains of Markov chain Monte Carlo (MCMC) iterations for 2×106 generations. The trees in each chain were sampled every 200th generation. The convergence of the two MCMC runs to the stationary distribution was determined by looking at the standard deviation of split frequencies  and by the convergence of the parameter values in the two independent runs. The first 200 trees were discarded as burn-in, and the remaining trees were used to calculate a 50% majority rule tree and to determine the posterior probabilities for all datasets.Bayesian inference was conducted with MrBayes v.3.1.2 Gracilariopsis lemaneiformis, Gp. tenuifrons and Gracilariophila oryzoides were designated as the outgroup taxa for the ITS dataset; Gp. lemaneiformis, Gp. andersonii, Gp. longissima, Gp. chorda and Gl. oryzoides for the cox1 dataset; as well as Curdiea crassa, C. racovitzia, Melanthalia abscissa and M. intermedia for the rbcL dataset.For comparison purposes, nodal support was considered strong for those with BP≥85% and PP>0.95, moderate for 70%≤BP<85% and 0.90≤PP≤0.95 and weak for BP<70% and PP<0.90. The outgroup taxa for each dataset were selected based on the phylogenetic relationships inferred from global searches for the Gracilariaceae The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix Hydropuntia sp. found attached on the monolines of Kappaphycus around Kampung Lok Butun in Pulau Bum Bum, Sabah, Malaysia. Infestations can be heavy but no apparent deleterious effects on the host were evident. All sexual stages were found in samples collected in every sampling trip.Habit: The species was parasitic on a Specimens examined: The voucher specimens included in this study were collected from Sabah, Malaysia : Pulau Bum Bum .Vegetative structure: The parasites can be recognized as swellings on various places of the host plant, and becoming spherical upon maturation. The parasite pustules assumed a lobed appearance with the presence of cystocarps. They formed protuberances up to 1.5 mm high and 2.1 mm in diameter. The color was almost the same as that of the host, usually dark olive upon collection from the field . When obThe pigmented parasite pustule was enveloped in a layer of gelatinous mucilage. The parasite was pseudoparenchymatous, being composed of large-celled axial filaments forming a medulla, from which small-celled branched filaments arise forming a peripheral cortex. Cortical cells measured up to 12 µm long by 5 µm wide and stained densely; whereas the medullary cells were lightly staining, reaching up 175–290 µm in diameter . Refractverrucosa type measuring up to 70 µm deep at the periphery of thallus . Two out of five clones of a parasite individual yielded rbcL sequence characteristic of Hydropuntia sp.; the remaining clones provided rbcL sequences attributed to the parasite with genetic divergence less than 0.8%. The occurrence of host plastid DNA in the clones of parasite was not considered as an experimental artifact (see Discussion). It is important to note that the genetic variation within individual was not the focus of this study, as the clones were sequenced to verify if host DNA was co-amplified with the parasite DNA for molecular analyses.Cloning and sequencing of the ITS region and C. babae and their host species Hydropuntia sp. and G. salicornia based on the ITS region, cox1 and rbcL gene sequences are summarized in Hydropuntia sp. did not have mitochondrial cox1 and plastid rbcL gene sequences identical to their current host, unlike the parasites from G. salicornia. The sequence divergences for C. babae regardless of their host species were 0.1–0.9%, 0.1–1.5% and 0.0–0.2% each for the ITS region, cox1 gene and rbcL gene. The red algal parasite C. babae differed from G salicornia and Hydropuntia sp. by 1.1–2.7% and 51.8–52.8% of the aligned ITS region. C. babae growing on Hydropuntia sp. had rbcL gene sequence identical to C. babae in the Peninsular Malaysia.The sequences included in the phylogenetic analyses were those determined by direct sequencing. For each marker, there was no sequence variation between all parasite individuals examined. The corrected distances between samples of red algal parasite Presented here are separate phylograms inferred from different genetic markers with bootstrap values from the MP analyses, SH-like aLRT bootstrap values from the ML analyses, as well as the Bayesian posterior probabilities appended. Phylogenies inferred from the ITS region using different reconstruction methods resulted in identical topology. The ITS phylogeny recovered a fully supported Gracilaria sensu lato ingroup consisting of three clades: (1) Gracilaria sensu stricto clade with no nodal support, (2) Hydropuntia clade , and (3) fully supported clade consisting of G. chilensis and G. tenuistipitata . The parcox1 and rbcL genes. The parasites from G. salicornia possess cox1 and rbcL gene sequences identical to those of the host from which they originated, and this was indicated in the inset box in cox1 gene recovered a monophyletic Gracilaria sensu lato clade , the Gracilaria sensu stricto clade  and the Hydropuntia clade . Similarly, the parasites from Hydropuntia sp. formed a well-supported monophyletic cluster along with C. babae from G. salicornia within the Gracilaria sensu stricto clade in the phylogeny.All phylogenetic analysis methods recovered largely congruent topology in the reconstructions based on the to clade . HydropubcL gene  identifiCongracilaria to accommodate C. babae, a red algal parasite that grows on G. salicornia, taking the form of pustules with bisporangia, coloration similar to that of its host, without any rhizoids, and the presence of spermatangia in deep conceptacles. Yamamoto C. babae in the Philippines, in which the specimens were no different from the type specimens in terms of external morphology, cellular structures and reproductive organs, apart from being slightly larger in pustule size. Despite growing on specific host species and some qualitative and quantitative differences  and a continuous zone of similar cells between the parasite and the host , tetrasporangia instead of bisporangia, and the occurrence on a different host species. The morphological and anatomical features of the parasite from Hydropuntia sp. are in common with those circumscribed for C. babae, prominently the pigmented pustule, absence of rhizoids penetrating into the host tissues, projecting cystocarps with tubular filaments extending to the pericarp and spermatangia borne in deep conceptacles of verrucosa type The parasite from G. salicornia into C. babae despite some discernible anatomical variations the Malaysian parasite exhibits from the Japanese counterpart. Molecular analyses in the present study demonstrated that the parasites from Hydropuntia sp. have DNA signatures similar to that of C. babae in having mitochondrial and plastid DNA highly similar or identical to G. salicornia. The parasites from Hydropuntia sp. were recovered in a monophyletic cluster along with the Malaysian and Japanese C. babae from G. salicornia in the phylogenies inferred from the genetic markers belonging to three different genomes  C. babae from Hydropuntia sp. had nuclear, mitochondrial and plastid DNA dissimilar to its current host. These parasites, regardless of their host species and geographical origin, formed a well-supported monophyletic clade sister to G. salicornia in the nuclear phylogeny inferred from the complete ITS region. The evolutionary relationships between C. babae and its hosts were also well-reflected in the differences in the staining reaction, which may indicate the differences in the chemical and physical constitution of cell walls between the parasite and its different host species. The uniform staining reaction across C. babae and G. salicorniaG. salicornia, in contrast to the consistently differential staining reaction across C. babae and Hydropuntia sp. which may indicate the distant relationship between the parasite and its current host.ts hosts  revealedC. babae which is parasitic on Hydropuntia sp. instead of G. salicornia provided a model to look into the evolutionary pattern of a red algal parasite. It is likely that C. babae had developed using organelles derived from G. salicornia via host cellular transformation Hydropuntia sp., C. babae may have developed in a manner which necessitates the maintenance of its own organelles. The parasite had retained its mitochondria copy rather than using those of its host, as the cox1 gene sequences characteristic of its Hydropuntia host were not obtained from three separate clones of a parasite individual. The parasite was shown to have maintained its copy of plastid, while co-opting the host-derived plastid. Two out of the five clones of a parasite individual yielded rbcL sequence which featured DNA characteristic of Hydropuntia. This observation was not surprising as red algal parasites had been shown to maintain the host-derived proplastids which were considered instrumental in the parasitic establishment C. babae would be identical to that of its original host, G. salicornia, while retaining its distinct nuclear identity even after radiation onto a secondary host species. The radiation of C. babae from one host to another is possible as G. salicornia and the Hydropuntia sp. are sympatric in Southeast Asia. C. babae corresponded to the concept of promiscuous alloparasites The observation of C. babae remained elusive, but the parasite most likely had acquired the organelles from the G. salicornia host species it originated from for development via host cellular transformation. The recovery of identical plastid rbcL and mitochondrial cox1 gene sequences for both C. babae and its G. salicornia host echoed the fate of parasite organelle DNA during host cellular transformation elucidated from the RFLP patterns obtained for Gardneriella and PlocamiocolaxC. babae isolated from G. salicornia, it will be detected in the sequence of nuclear marker; we did not encounter this. Instead, the occasional observation of C. babae DNA in the DNA of G. salicornia host indirectly supported the occurrence of host cellular transformation event where the host tissues sampled for DNA extraction were actually cellular syncytia with a proliferating parasite nuclear genome cox1 sequences for C. babae from Hydropuntia sp. indicated that the parasite was the only copy amplified despite a low level of genetic variation within an individual. With all the precautionary steps taken in this study, as well as the concurrence of our data with previous findings by other independent researchers where a parasite can have DNA sequence identical to its host G. salicornia obtained for the parasite C. babae were indeed attributed to the nature of the parasite, rather than an experimental artifact or an inability to differentiate between the parasitic entity and G. salicornia.The actual evolutionary mechanism for C. babae from each of G. salicornia and Hydropuntia sp. be delineated by the use of host race , despite forming a monophyletic cluster in molecular analyses  P.-K. Ng, P.-E. Lim et S.-M. Phang comb. nov.Congracilaria babae Yamamoto in Bull. Fac. Fish. Hokkaido Univ. 37(4): p. 281–290, 1986.Basionym: C. babae encompasses pigmented pustules which lack rhizoids that penetrate into the host tissues; it has deep spermatangial conceptacles and projecting cystocarps characteristic of Gracilaria. C. babae is genetically very closely related to G. salicornia, and thus should be transferred to the genus Gracilaria. G. babae most likely have evolved directly from G. salicornia and radiated onto a distantly related host species Hydropuntia sp. Further comparative developmental study and functional genomics analysis of G. babae from G. salicornia and Hydropuntia sp. may shed light on the factors involved in red algal parasitism.Molecular phylogenies based on genetic markers belonging to different genomic compartments are useful in resolving the evolutionary relationships between a red algal parasite and its host species, as well as revealing the possible original host species of a red algal parasite which may be obscured by the reduced morphological complexity and the biology of the interaction between the parasite and its host. Irrespective of the host species, Figure S1Agarose gel electrophoresis of PCR products obtained from DNA extracts of representatives of the host-parasite associations for the rbcL gene, cox1 gene and ITS region. Samples 1, 2, 3 and 4 represent Gracilaria salicornia, Congracilaria babae parasitic on G. salicornia, Hydropuntia sp., and C. babae parasitic on Hydropuntia sp. respectively. Lanes M and N are 1 kb ladder and negative controls.(TIF)Click here for additional data file.