Chromobacterium violaceum assay using the agar cup diffusion method. The screening revealed the anti-QS activity of six plants; namely the leaves of Adhatoda vasica Nees, Bauhinia purpurea L., Lantana camara L., Myoporum laetum G. Forst.; the fruits of Piper longum L.; and the aerial parts of Taraxacum officinale F.H. Wigg.This study investigated the effects of some plant extracts on the bacterial communication system, expressed as quorum sensing (QS) activity. Quorum sensing has a directly proportional effect on the amount of certain compounds, such as pigments, produced by the bacteria. Alcohol extracts of 23 ornamental and medicinal plants were tested for anti-QS activity by the  Adhatoda vasica Nees, Ambrosia psilostachya DC., Arctostaphylos uva-ursi (L.) Spreng., Bauhinia purpurea L., Boswellia carterii Birdw., Caesalpinia gillesii Wall. ex Hook., Chelidonium majus L., Dalbergia sisso Roxb., Datura stramonium L., Dimorphotheca ecklonis DC., Duranta erecta var. alba (Mast.) Caro, Kigelia pinnata (Jacq.) DC., Kochia indica Wight., Lantana camara L., Myoporum laetum G. Forst., Nerium oleander L., Olea europaea L., Piper longum L., Schinus molle L., Smilax aristolochiifolia Mill., Tagetes erecta L., Taraxacum officinale F.H. Wigg., and Zinnia elegans Jacq. The anti-quourm sensing activities of these plants were evaluated using the QS biosensor Chromobacterium violaceum strain ATCC 12472.Antimicrobial agents exhibit their activity through different mechanisms such as disrupting cell wall function or disrupting protein and DNA synthesis –3. As a To the best of our knowledge, studies to evaluate this activity for these selected plants have not been reported before.Chromobacterium violaceum assays. Pigment fading in the vicinity of the tested extracts indicated their QS inhibitory effect. Pigmentless zones adjacent to the clear zones of dead bacteria were observed surrounding certain samples, in comparison with the negative control, indicating the inhibition of violacein pigment secretion. Bacteria in this pimentless zones were alive but lost their QS ability.Phytochemical screening for the presence of alkaloids, saponins, carbohydrates, tannins, flavonoids, steroids, triterpenoids, and cardenolides is summurized in . The thiMyoporum laetum G. Forst. Adhatoda vasica Nees and Bauhinia purpurea L., exhibited strong anti-quorum sensing activity/AHL-mediated violacein inhibition activities , while extracts of Piper longum L., Taraxacum officinale F.H. Wigg., and Lantana camara L. showed moderate anti-quorum sensing activity of 6–9 mm radius. Literature reviews for the anti-microbial activity of the total alcohol extracts of these plants revealed moderate to strongantimicrobial activities exhibited by the leaves of Myoporum laetum G. Forst [Bauhinia purpurea L. [, Lantana camara L. [Taraxacum officinale F.H. Wigg. [, Adhatoda vasica Nees, and the fruits of Piper longum L. The activity of the last two plants, A. vasica and P. longum L., was attributed to their alkaloidal content [The assay revealed, as shown in G. Forst , Bauhinipurea L. , Lantanaamara L. , TaraxacH. Wigg. , Adhatod content , 28.Six of the screened plants may contain antimicrobial compounds that we are currently working on isolating and identifying using the same bioassay as a guide. Thus, this study serves in selecting promising plant species for discovering new antimicrobial drugs.Plants were collected either from the vicinity of Mansoura University or were purchased from commercial sources. Samples were identified by staff members in the Faculty of Agriculture. Voucher specimens were deposited in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Mansoura University.All plant samples were air-dried at room temprature and finely crushed. One hundred grams, dry weight, of each plant was extracted by soaking in 100ml 90% ethanol for two days with intermittent shaking; then the solvents were evaporated under reduced pressure on a rotary evaporator and the residues were stored frozen for testing.Phytochemical screening was performed according to a standard procedure .254,365 nm then sprayed with vanillin/sulfuric reagent and heated for 5 min. at 110 °C.The extracts were run on pre-coated silica gel plates using a mixture of ethyl acetate:hexane (3:7) as the mobile phase. Spots were visualized by UVThe strains were grown in Luria-Bertani (LB) broth , solidified with 1.5% agar when required, and supplemented with an antibiotic (Kanamycin 20 μg/ml).et al. [Chromobacterium violaceum strain ATCC 12472. In this test, bacterial growth inhibition would result in a clear halo around the cup, while a positive quorum sensing inhibition is exhibited by a turbid halo harboring pigmentless bacterial cells of the C. violaceum ATCC 12427 monitor strain. Cultures were prepared by growing bacteria in Luria Bertani broth  and incubated for 16–18 h in an orbital incubator  running at 30 °C and 150 rpm. Cultures were then adjusted to 0.5 McFarland standard (Ca.108 CFU/ml). Cups were of 10 mm diameter. Plant extracts were dissolved in sterile DMSO. A volume of 100 μl of each extract was transferred to the cups made in triplicate per extract onto C. Violaceum-inoculated (100 μl/plate) LB agar plates, which were then incubated at 30 °C for 24–48 h, and then the results were recorded (2− r1) in mm; where r2 is the total growth-inhibition zone radius and r1 is the clear zone radius [The quorum sensing inhibition activity of the plant extracts was determined by the agar cup diffusion assay, described by Zahin et al.  using threcorded . The nege radius .


Taenia multiceps, a global cestode, encysts in the central nervous system (CNS) of sheep and other livestock. This frequently leads to their death and huge socioeconomic losses, especially in developing countries. This parasite can also cause zoonotic infections in humans, but has been largely neglected due to a lack of diagnostic techniques and studies. Recent developments in next-generation sequencing provide an opportunity to explore the transcriptome of T. multiceps.The larval stage of Taenia pisiformis, Taenia solium, Echincoccus granulosus and Echincoccus multilocularis) and intestinal parasites  showed that 5,100 common genes were shared among three Taenia tapeworms, 261 conserved genes were detected among five Taeniidae cestodes, and 109 common genes were found in four zoonotic intestinal parasites. Some of the common genes were genes required for parasite survival, involved in parasite-host interactions. In addition, we amplified two full-length CDS of unigenes from the common genes using RT-PCR.We obtained a total of 31,282 unigenes (mean length 920 bp) using Illumina paired-end sequencing technology and a new Trinity de novo assembler without a referenced genome. Individual transcription molecules were determined by sequence-based annotations and/or domain-based annotations against public databases . We identified 26,110 (83.47%) unigenes and inferred 20,896 (66.8%) coding sequences (CDS). Further comparative transcripts analysis with other cestodes (T. multiceps, and demonstrates that comparative transcriptomic investigations deserve to be further studied. This transcriptome dataset forms a substantial public information platform to achieve a fundamental understanding of the biology of T. multiceps, and helps in the identification of drug targets and parasite-host interaction studies.This study provides an extensive transcriptome of the adult stage of  Taenia multiceps is a taeniid cestode, which inhabits the small intestine of dogs and other canids , making these definitive hosts a widespread infection reservoir T. multiceps) parasitizes the central nervous system (CNS) of sheep, occasionally goats, deer, antelopes, chamois, rabbits, hares and horses, and less commonly, cattle T. multiceps are discharged from infected dogs and are ingested by intermediate hosts  through contaminated food or water The gravid proglottids of Taenia species very difficult; consequently, new methods to control this parasite are required. Although traditional methods of control  help to disrupt the lifecycle of this parasite, its global distribution still includes Europe Together with this complex lifecycle, the specific immune evasion traits of parasites and even the randomness of the infection make research and drug or vaccine programs for T. multiceps remain scarce. Currently, only 101 nucleotide sequences and 103 proteins have been published on NCBI and only the mitochondrial genome of T. multiceps has been sequenced Taenia solium cysticerca, has been revealed by the ORESTES method Taenia pisiformis, T. solium, Echinococcus granulosus, Echinococcus multilocularis and Hymenolepis microstoma by NGS technology have already been studied, but only T. pisiformis transcriptome dataset has been published T. multicepsDespite its global importance, available gene sequences for T. multiceps. In addition, a comparative analysis was performed against the transcriptomes of other cestodes, including T. pisiformis, T. solium, E. granulosus and E. multilocularis, and intestinal parasites, including T. spiralis, A. caninum and A. suum, in order to help us to discover essential biological pathways and pathway-related genes for intestinal parasites or cestodes specifically. These would be essential for parasite development and survival, or for parasite-host interactions Here, we used the novel assembler Trinity and Illumina sequencing technology to gather initial insights into the transcriptome of the adult stage of T. multiceps survival and development, the RNA of T. multiceps adults was extracted. Using Illumina (paired-end) sequencing technology, we obtained a total of 28.3 million raw reads with an average length of 90 bp, a total of approximately 2.55 Gigabase pairs (Gbp). After the removal of raw reads that only had 3' adaptor fragments, ambiguous reads and low-quality reads, 27.4 million  clean reads with a Q20 percentage of 89.92% and a GC content of 49.04% remained. All clean reads were assembled de novo by Trinity, generating 53,568 contigs without gaps longer than 300 bp , with a mean contig length of 974 bp and a N50 of 1,268 bp  unigenes were annotated against animal protein of Nr, 5,925 (18.94%) against Drosophila protein of Nr, 14,350 (45.87%) obtained annotations against UniProtKB/Swiss-Prot, 16,286 (52.06%) against UniProtKB/TrEMBL, 6,653 (21.3%) received annotation against COG, and 11,645 (37.3%) against the KEGG database , in addition to the UniProtKB/Swiss-Prot, UniProtKB/TrEMBL, COG, and KEGG databases, with an e-value threshold of 1.0database . Altoget−50, while 64% of the similar sequences ranged from 1.0−5 to 1.0−50  and Danio rerio (2.09%), respectively , followed by 'Replication, recombination and repair’  and ‘Transcription’ . In contrast, the clusters for ‘Nuclear structure’ , ‘Extracellular structures’  and ‘RNA processing and modification’  were the smallest categories .T. multiceps sequences was revealed by mapping against known proteins  of the KEGG database. Out of 31,282 assembled unigenes, a total of 11,645 homologous sequences were grouped into six categories, including ‘Metabolism’, ‘Genetic Information Processing’ (GIP), ‘Environmental Information Processing’ (EIP), ‘Cellular Processes’, ‘Organismal Systems’ and ‘Human Disease’ . As a result, 25,457 (81.38%) sequences obtained the entry description against InterPro and were categorized into 4,562 domains/families. Most domains/families were found to contain more than one unigene. According to the frequency of occurrence of T. multiceps unigenes contained in each InterPro domain, InterPro domains/families were ranked and the 30 most abundant InterPro domains/families are shown in To identify domain-based annotations, unigenes were used to search the domain/families according to the InterPro and Pfam databases  unigenes showed significant similarities to know proteins in the seven public databases . After aT. multiceps and T. pisiformis is shown in T. multiceps and T. pisiformis showed the same sub-categories and a similar percentage, but still revealed their qualitative and quantitative differences Of the 17,618 Nr hits, a total of 4,706 sequences were assigned to 2,360 non-redundant GO terms according to the Blast2GO program. All GO terms were allocated into three main GO categories  and 48 sub-categories , left. BE. granulosus and E. multilocularis , on NCBI. The 9,701 ESTs of E. granulosus received 5,876 GO annotations, whereas 1,168 ESTs of E. multilocularis obtained 1,979 GO annotations against Gene Ontology. WEGO showed the similar percentages of GO terms among these three cestode species . The available transcript datasets of four species of zoonotic intestinal parasites were chosen to detect common genes that may be involved in adapting to intestinal parasitic life based on KEGG annotations. Of the 32,895 annotated nucleotide ESTs  of A. caninum, 11,645 annotated unigenes  of T. multiceps and 2,110 annotated CDSs  of T. spiralis, 145 common genes were found from the overlapping annotated part (identity ≥80%) of A. caninum, T. spiralis and T. multiceps , we obtained 5,100 common genes (E. granulosus and E. multilocularis) T. multiceps survival and parasite-host interactions, supporting the development of drug targets/vaccines and phylogenetic relationship analyses between Echinococcus and Taenia tapeworms. In addition, we validated one promising drug target (unigene 1299) using RT-PCR to support the further analysis of its protein structure and function (GenBank accession number: GU205473).In order to obtain a more detailed understanding of Taeniidae cestode biology and reveal potential drug target genes, we compared five important cestodes by making use of currently available datasets. After comparing three cestodes of the Taenia genus  and unigenes (≥500 bp) was higher than 60%, and the gap rate of all contigs and unigenes was as low as 0%. Unlike previous studies, our results showed that the mean length (920 bp) of T. multiceps unigenes was shorter than the contigs (974 bp), which might be due to the higher frequency of long sequences in contigs than in unigenes, whereas the number of contigs in each length extent was higher than that of unigenes. The reason for this is that one unigene has multiple transcripts, due to alternative splicing in eukaryotes. Of 31,282 unigenes, 26,110 (83.47%) could be annotated by seven public databases, whereas the remaining 16.53% unaligned unigenes are most likely contain Taenia- or cestode-specific genes T. pisiformisT. pisiformis). This is in accordance with a previous report that showed longer contigs are more likely to obtain BLAST matches in protein databases T. multiceps was predicted from this transcriptome dataset, with an approximate 200-fold coverage of the available proteins (for this stage/species) in the public databases. All of these results demonstrate the high quality and effectiveness of Illumina paired-end sequencing technology and the Trinity assembler.In this study, a reliable and substantial transcriptome dataset of the adult stage of T. multiceps (6.3%). In this sub-category, the ‘Wnt signaling pathway’ was one of the most abundant pathways and was particularly interesting. A comparative analysis of Wnt signaling components in parasitic and free-living flatworms  has been conducted and a hypothesis of Wnt gene loss in flatworms has been proposed H. microstoma, whereas Wnt2 was expressed only in larvae. Wnt1 was known to play a role as a segment polarity gene in adult worms T. multiceps transcriptome annotation. This may have been due to the limitation of Wnt annotations in the currently available databases, or may reveal that the presence of Wnt1 and Wnt2 differ between species. Further research is necessary to validate the role of Wnt genes in the development of T. multiceps and their loss in cestodes.Throughout the biological pathway sub-categories, ‘signal transduction’  of the environmental information processing (EIP) category was the most highly represented in the transcriptome of After the transcript comparison of four species of zoonotic intestinal helminths, we narrowed the scope of the gene group to find genes that might be used for adapting to intestinal parasitic life. The 109 genes in the common part were still imperfect due to the limited available dataset. However, they did contain conserved genes, essential genes for parasite survival or genes related to parasite-host interactions. The majority of these common genes were α-tubulin and actin  might be involved in parasite-host interaction. PEPCK is a key enzyme in malic acid disproportionation, which is the principle process for the continuous metabolism of phosphoenolpyruvate (PEP). PEP is generated from anaerobic glycolysis, in order that intestinal parasitic helminths can obtain ATP in anaerobic conditions T. multiceps, T. solium, E. granulosus, E. multilocularis and T. pisiformis are five important Taeniidae cestodes, of which the former four species are zoonotic parasites that cause huge economic losses and threaten human health T. multiceps are poor and less than 10 candidate antigens exist as drug targets, including Tm18 [EF672035], Tm16 [EF672037], 45 m [FJ461729], TPx [HQ888859] and Tm7 [FJ603044]). In this study, we used relatively large datasets that contained 31,282 unigenes of T. multiceps and 72,597 unigenes of T. pisiformis obtained by high-throughput sequencing, and 30,700 ESTs of T. solium obtained by cDNA library construction, to bring to light the scope of potential drug targets/candidate vaccines. Conserved Taenia genes and essential genes for Taenia survival were contained by this scope of common genes. There might be other new Taenia-specific genes in the remaining 2,100 un-annotated common genes.Taenia and Echinococcus tapeworms. Among 204 annotated conserved genes, we also found PEPCK (unigene 17356). As the five Taeniidae cestodes are also intestinal parasites, the PEPCK might play the same role as discussed above and could be a promising drug target if it is minimally toxic to the host. Furthermore, PEPCK has been considered as a new marker of the phylogenetic relationship within Echinococcus and Taenia tapeworms T. multiceps will help in the study of cestode phylogenetic relationships.Together with the Eg+Em common ESTs, 261 conserved genes were obtained between E. granulosus can induce the host to produce IgA, IgG1 and IgG2a T. multiceps FABP3 using RT-PCR.The 261 conserved genes were believed to contain necessary genes for the five Taeniidae cestodes that could help drug target/vaccine finding. Unigene 1299 see  of theseT. multiceps using Illumina paired-end sequencing technology and a Trinity de novo assembler without a reference genome. A total of 31,282 unigenes was produced with 26,110 sequences having annotations against seven public databases. We have demonstrated the feasibility and advantage of using a Trinity assembler. The common genes found among four zoonotic intestinal parasites  and the comparative transcript analysis with T. pisiformis, T. solium, E. granulosus and E. multilocularis established a substantial platform for the better understanding of T. multiceps survival and development, further study of parasite-host interactions, and the development of future drug targets/vaccines and phylogenetic relationship analyses within Echinococcus and Taenia tapeworms.In this in-depth study, we obtained a broad transcriptome dataset of the adult stage of T. multiceps into two parasite-free beagle dogs. Once the gravid proglottid expelled in the feces of infected dogs appeared on day 28 post-infestation, adult T. multiceps were immediately removed from the small intestine and washed thoroughly in physiological saline solution (37°C) to avoid host contamination; they were then transferred into liquid nitrogen and stored at −80°C until further use. All animals from which specimens were collected were handled in accordance with the animal protection law of the People's Republic of China . This study was approved by the National Institute of Animal Health Animal Care and Use Committee at Sichuan Agricultural University .The larvae (coenurus)  were collected from the brain of a naturally infected goat at an organic farm from Sichuan, China. The infection experiment was followed by larval morphological identification, and was performed by administering 20 larvae of T. multiceps (n = 6) using Trizol , following the manufacturer's instructions. The integrity of total RNA was verified using Agilent 2100 with the RNA integrity number (RIN). Polyadenylated (polyA) RNA was purified from 40 µl of total RNA using Sera-Mag oligo (dT) beads, fragmented into small pieces by fragmentation buffer, reverse-transcribed using random hexamers and reverse transcriptase, and then end-repaired with adapter primer attached and adaptor-ligated by the addition of a specific adapter, according to the manufacturer’s protocol (Illumina). These ligated products were purified and amplified with PCR to create the final cDNA library http://www.ncbi.nlm.nih.gov/Traces/sra_sub/sub.cgi) with the accession number: SRA048944.A paired-end transcriptome sequencing (RNA-Seq) T. multiceps. Reads that have a certain length overlap area were joined (the best method of joining was chosen by Trinity) into longer fragments, which are called contigs without gaps. TIGR Gene Indices clustering tools (TGICL) A new Trinity de novo transcriptome assembler −5. InterProScan (http://www.ebi.ac.uk/InterProScan/) http://hmmer.janelia.org/) were used to obtain domain-based annotation by InterPro (http://www.ebi.ac.uk/interpro/) http://Pfam.sanger.ac.uk) Unigene sequences were first aligned to the protein databases Non-redundant (Nr), UniProtKB/Swiss-Prot, UniProtKB/TrEMBL, Cluster of Orthologous Groups (COG) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases by Blastx, using an e-value threshold of 1.0http://www.blast2go.de/) −5) and were assigned into three ontologies  (http://www.geneontology.org/). WEGO software (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) T. multiceps, E. granulosus and E. multilocularis on a macro level.Of the Nr annotations, Gene Ontology (GO) annotations of unigenes were obtained using Blast2GO software  and T. pisiformis  were provided by our laboratory. T. pisiformis unigenes were also produced by the BGI center . For four intestinal parasites common genes finding, 80,905 nucleotide ESTs of A. caninum were downloaded from http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=29170, 16,380 CDSs in 9,267 contigs of T. spiralis  A. suum were downloaded from ‘Ascaris_cDNA_All_v1.fa.gz - Ascaris suum cDNA assembly’ (http://www.nematode.net/NN3_frontpage.cgi?navbar_selection=home&subnav_selection=asuum_ftp). All transcripts were chosen to be mapped to the known proteins in KEGG  and the further comparative analysis among the four different species of intestinal parasites were based on KEGG annotations. The overlap for common sequences was obtained with an identity threshold of ≥80%.To compare GO classification among Taeniidae cestodes, the unigenes of T. pisifomis transcriptome, we choose 31,282 and 72,597 unigenes from T. multiceps and T. pisiformisT. soliumTaenia spp. When the common Taenia genes were combined with 1,058 ESTs from Eg+Em  and conserved genes between Taenia and Echinococcus (E. granulosus and E. multilocularis) were aligned to the KEGG database using the Blastx algorithm.Based on the previous study of the T. multiceps  e-value distribution. (B) Similarity distribution of the top BLAST hits for each unigene. (C) Species distribution is shown as the percentage of the total homologous unigenes with a threshold e-value of 1.0−5.(TIF)Click here for additional data file.Figure S2COG function classification of the T. multiceps sequences.(TIF)Click here for additional data file.Table S1Detailed KEGG pathway categories of the T. multiceps unigenes.(XLS)Click here for additional data file.Table S2The 30 most abundant Pfam domains/families in T. multiceps unigenes.(XLS)Click here for additional data file.Table S3The 109 common genes among T. multiceps, T. spiralis, A. caninum and A. suum.(XLS)Click here for additional data file.Table S45,100 common genes among T. multiceps, T. pisiformis and T. solium.(RAR)Click here for additional data file.Table S5261 conserved genes among T. multiceps, T. pisiformis, T. solium, E. granulosus and E. multilocularis.(RAR)Click here for additional data file.Table S6Primers were designed for RT-PCR of two unigenes from the adult T. multiceps transcriptome.(DOC)Click here for additional data file.


Cthulhu macrofasciculumque and Cthylla microfasciculumque from Prorhinotermes simplex and Reticulitermes virginicus, respectively (both hosts confirmed by DNA barcoding). Both genera have a single anterior nucleus embeded in a robust protruding axostyle, and an anterior bundle flagella (and likely a single posterior flagellum) that emerge slightly subanteriorly and have a distinctive beat pattern. Cthulhu is relatively large and has a distinctive bundle of over 20 flagella whereas Cthylla is smaller, has only 5 anterior flagella and closely resembles several other parababsalian genera. Molecular phylogenies based on small subunit ribosomal RNA (SSU rRNA) show both genera are related to previously unidentified environmental sequences from other termites (possibly from members of the Tricercomitidae), which all branch as sisters to the Hexamastigitae. Altogether, Cthulhu likely represents another independent origin of relatively high cellular complexity within parabasalia, and points to the need for molecular characterization of other key taxa, such as Tricercomitus.The parabasalian symbionts of lower termite hindgut communities are well-known for their large size and structural complexity. The most complex forms evolved multiple times independently from smaller and simpler flagellates, but we know little of the diversity of these small flagellates or their phylogenetic relationships to more complex lineages. To understand the true diversity of Parabasalia and how their unique cellular complexity arose, more data from smaller and simpler flagellates are needed. Here, we describe two new genera of small-to-intermediate size and complexity, represented by the type species  The hindgut of lower termites has long been known for the diversity of its community of symbiotic protists The large and complex hypermastigotes are most conspicuous and have therefore attracted the most attention in studies of the diversity and taxonomy of termite symbionts. Indeed, after over 100 years of study, it is arguable that most of the major types  of large and complex hypermastigotes have probably been observed and described . The likelihood that many, if not most, of the major types of large hypermastigtes have been described is also supported by phylogenetic analysis of molecular surveys of hindgut diversity: there are many lineages in the tree of parabasalians that are made up entirely of “unidentified environmental sequences” from termite hindguts In order to fully understand the evolution of the large, complex, and conspicuous parabasalians, it is necessary to pay greater attention to the smaller forms because the phylogeny of parabasalians makes it clear that large size and structural complexity have evolved more than once Cthulhu macrofasciculumque found in Prorhinotermes simplex (Hagan), and Cthylla microfasciculumque, found in Reticulitermes virginicus (Banks), newly described genera and type species of relatively small parabasalian termite symbionts that address both these issues. These two similar but morphologically distinct genera are the first identified members of a clade of unidentified environmental sequences derived from several distantly related rhinotermitids and kalotermitids. Altogether, these new genera help provide a morphological framework to a previously uncharacterized lineage of environmental sequences, and Cthulhu in particular represents a newly identified morphological type for Parabasalia, as well as a new case of accelerating size and complexity.Here we describe R. virginicus . No comparable barcode sequence for P. simplex is available in public databases, so we also barcoded eight other isolates from across the Caribbean basin . Comparing these and our P. simplex isolate confirmed its identification with a 99.7% shared identity. , Cryptotermes cylindroceps (Scheffrahn & Krecek), and Heterotermes tenuis (Hagan), the identities of which were all confirmed by barcoding that was reported previously The misidentification of host termites has often proved to be a complicating factor in the study of parabasalian symbionts. To avoid this issue, we first acquired a positive identification of all termite species by both examining their morphology and by characterizing mitochondrial 16S rRNA (mtLSU rRNA), an established DNA barcode marker for lower termites. Barcode sequences from field identified termites confirmed the identification of rginicus , which sdentity. . We alsoP. simplex revealed a number of large and conspicuous hypermastigonts , but we also noted an intermediate sized flagellate with interesting characteristics . A single anterior nucleus (which was difficult to observe in still cells but could be seen in moving cells more easily) was cupped within the curvature of a large and robust axostyle, that extended from the nucleus to the posterior end of the cell , where it consistently terminated in an emergent point . It also bears only five anterior flagella  to a group of environmental sequences from H. tenuis (JX975351: this study) and Reticulitermes chinensis (310871926), respectively. This suggests flagellates with similar morphology may be found in these termites. We have neither species available to investigate, but we did review video footage from the same isolate of H. tenuis that our environmental sequence was derived, and identified two small flagellates from which the Cthulhu-related sequences might plausibly be derived , and more distantly to the genus Hexamastix and several more unidentified sequences from termites, including some we identified from termite whole hindgut preparations: unidentified symbionts from Cryptotermes cylindroceps (JX975352) and Cryptotermes nearcticus (JX975353)  in Zootermopsis angusticollis (Hagan) since this is the type host for the type species X975353) . AccordiAssignment. Eukaryota; Parabasalia.Cthulhu n. g. James and Keeling, 2012.urn:lsid:zoobank.org:act: 25BCAD11-B42E-4105-826C-0459AE4EC5A8Type species: Cthulhu macrofasciculumque Description: Small parabasalian flagellate with a single anterior nucleus associated with a robust axostyle. Axostyle extends the entire length of the cell, and projects at the posterior where it terminates in a point. Approximately 20 flagella emerge in a single slightly sub-anterior bundle. Anterior flagella beat coordinately, forming a single large bundle that rolls anteriorly to the fully extended position and then dissociates into loose flagella that whip posteriorly. Possibly also a single recurrent flagellum.Etymology: The name is based on the fictional many tentacled, cephalopod-headed demon found in the writings of H. P. Lovecraft, specifically The Call of Cthulhu. The tentacle-headed appearance given by the coordinated beat pattern of the anterior flagellar bundle of Cthulhu cells is reminiscent of this demon. The name is supposedly impossible to pronounce as it comes from an alien language, but currently it is most often pronounced “ke-thoo-loo”.Cthulhu macrofasciculumque n. sp. James and Keeling, 2012urn:lsid:zoobank.org:act:B5FDCF7C-386D-4793-8EB0-61F4B0063BFAType host: Prorhinotermes simplex Type locality: Ft. Lauderdale, Secret Woods County Park, Florida, USA: lat. 26.08567, long. −80.18017.Host collection: University of Florida termite collection, accession number FL1563. Collector B. Maharajh. Collected Sept. 15, 2002.Description: Parabasalian flagellate with morphological characters for the genus Cthulhu. Cells are 17 to 24 µm in length and 7 to 12 µm in width and have a minimum of 20 anterior flagella. Found in the hindgut of Prorhinotermes simplex. Distinct SSU rRNA sequence (GenBank accession number JX975349).Hapantotype: Microscope slide deposited at the Beaty Biodiversity Museum, University of British Columbia, Vancouver, Canada under accession number MI-PR200.Gene sequence: SSU rRNA accession number JX975349.Etymology: Species name refers to the large (macro) bunch (fasciculumque) of flagella.Cthylla n. g. James and Keeling, 2012.urn:lsid:zoobank.org:act 0C373970-AD9F-4A3A-930B-36D82FC8683CType species: Cthylla microfasciculumque Description: Parabasalian flagellate with a single anterior nucleus associated with a robust axostyle. Axostyle extends the entire length of the cell, and projects at the posterior where it terminates in a point. Five flagella emerge in a single slightly sub-anterior bundle. Anterior flagella beat coordinately, forming a single large bundle that rolls anteriorly to the fully extended position and then dissociates to individual flagella that whip posteriorly. Possibly also a single recurrent flagellum.Etymology: The name is based on the fictional Cthylla (often pronounced ke-thil-a), who was the secret daughter of Cthulhu in later writing inspired by H. P. Lovecraft. Though never described, Cthylla is generally portrayed as a winged cephalophod. It is here named as a smaller and simpler relative of the parabasalian genus Cthulhu.Cthylla microfasciculumque n. sp. James and Keeling, 2012urn:lsid:zoobank.org:act:C5109A93-A1EC-4362-AEBF-D2E538C55D66Type host: Reticulitermes virginicus Type locality: Ft. Lauderdale, Secrete Woods County Park, Florida, USA: lat. 26.08567, long. −80.18017.Host collection: University of Florida termite collection, accession number FL2261. Collector R. H. Scheffrahn. Collected Feb 21, 2005.Description: Parabasalian flagellate with morphological characteristics of the genus Cthylla. Cells are 10 to 15 µm in length and 6 to 10 µm in width. Found in the hindgut of Reticulitermes virginicus. Distinct SSU rRNA sequence (GenBank accession number JX975350)Hapantotype: Microscope slide deposited at the Beaty Biodiversity Museum, University of British Columbia, Vancouver, Canada under accession number MI-PR201.Gene sequence: SSU rRNA accession number JX975350.Etymology: Species name refers to the small (micro) bunch (fasciculumque) of flagella.Paraneotermes simplex was collected Sept. 15, 2002 in Secret Woods County Park, Ft. Lauderdale, Florida,  and deposited in the University of Florida termite collection, accession number FL1563. Reticulitermes virginicus was collected on Feb 21, 2005 at the same location and deposited in the University of Florida termite collection, accession number FL2261. Cryptotermes cylindroceps, Calcaritermes nearcticus, and Heterotermes tenuis were collected as reported previously P. simplex and R. virginicus were amplified using LR-N-13398 CGCCTGTTTATCAAAAACAT and LR-J-13007 TTACGCTGTTATCCCTAA under conditions previously described P. simplex barcode was available, we also barcoded P. simplex from eight other independent isolations  from Florida, Puerto Rico, Bahamas, Belize, Guatemala, and Honduras . These new barcodes were submitted to GenBank under accessions KC140183-90. These were aligned with all available Paraneotermes and Reticulitermes mt LSU termite barcodes from Genbank using MAFFT DNA barcodes at the mitochondrial 16S (LSU) rRNA marker for Whole termite guts were dissected in Trager medium U For scanning electron microscopy (SEM), whole hindgut contents were mounted on a poly-L-lysin coated coverslip, then fixed in 2.5% glutaraldehyde  diluted in Trager's medium U overnight at 4°C. Samples were dehydrated using a graded ethanol series and critical point dried in a Tousimis Sandri 795 CPD . The dried coverslips were mounted onto aluminum stubs and sputter-coated with 5-nm thick gold . The scanning electron microscopy was performed using a Hitachi S4700 .5′-TGC GCT ACC TGG TTG ATC CTG CC-3′ and 5′-TGA TCC TTC TGC AGG TTC ACC TAC-3′ as described previously Target cells were isolated by micropipette using a Zeiss Axiovert 2 microscope and each isolated cell was photographed using a QImaging MicroImager II camera (data not shown). DNA was isolated using the Epicentre Masterpure Complete DNA and RNA purification Kit . SSU rRNA gene sequences were amplified from single cells, small pools of identical cells, and whole hindgut contents using eukaryote-specific primers Parabasalian and host rRNA phylogenies were inferred using ML and Bayesian tree reconstruction methods, with PHyML v.3 Video S1Movie of live Cthulhu macrofasciculumque showing overall body plan and the flagellar beat pattern with and without cytoplasmic undulation.(MP4)Click here for additional data file.Video S2Movie of live Cthylla microfasciculumque showing overall body plan and the flagellar beat pattern.(MP4)Click here for additional data file.Figure S1Maximum likelihood tree with a broad representation of parabasalian diversity, inferred with RAxML and the GTR+gamma model of evolution. New sequences from Cthulhu and Cthylla are shown white on black.(EPS)Click here for additional data file.


Statistical methods for ranking differentially expressed genes (DEGs) from gene expression data should be evaluated with regard to high sensitivity, specificity, and reproducibility. In our previous studies, we evaluated eight gene ranking methods applied to only Affymetrix GeneChip data. A more general evaluation that also includes other microarray platforms, such as the Agilent or Illumina systems, is desirable for determining which methods are suitable for each platform and which method has better inter-platform reproducibility.t statistic (ibmT). The percentages of overlapping genes (POGs) across different test sites were mainly evaluated as a measure for both intra- and inter-platform reproducibility. The POG values for WAD were the highest overall, irrespective of the choice of microarray platform. The high intra- and inter-platform reproducibility of WAD was also observed at a higher biological function level.We compared the eight gene ranking methods using the MicroArray Quality Control (MAQC) datasets produced by five manufacturers: Affymetrix, Applied Biosystems, Agilent, GE Healthcare, and Illumina. The area under the curve (AUC) was used as a measure for both sensitivity and specificity. Although the highest AUC values can vary with the definition of "true" DEGs, the best methods were, in most cases, either the weighted average difference (WAD), rank products (RP), or intensity-based moderated These results for the five microarray platforms were consistent with our previous ones based on 36 real experimental datasets measured using the Affymetrix platform. Thus, recommendations made using the MAQC benchmark data might be universally applicable. Identification of differentially expressed genes (DEGs) under different conditions is an important goal in microarray-based gene expression analysis. For this identification, new gene ranking methods have been developed and comparative studies have been performed -8. SeverCurrently, by virtue of the MicroArray Quality Control (MAQC) project ,19, reseWe recently reported that WAD outperformed AD, which was recommended by the MAQC study with regard to inter-site reproducibility. We also reported that the use of WAD or RP, in conjunction with suitable preprocessing algorithms dedicated to the Affymetrix (AFX) GeneChip data, can increase both sensitivity and specificity of the results . HoweverIn this study, we analyzed the MAQC data ,19 measuExpression data derived from five platforms, i.e., AFX, ABI, AG1, GEH, and ILM, were obtained from the MAQC website  and anal2 transformation was performed. The GEH data, i.e., 53,517 probes, were used for analyzing sensitivity and specificity. For the other platforms , the expression data on a log2 scale were analyzed.For the GEH data, signals from multiple probes with the same ID  were averaged. This produced 53,517 unique probes from the original 54,359 probes. Then, signals under a minimum value for signals flagged as 'Good' quality were set to the minimum value . This substitution of a floor value was made for 21.75% ) of the signals. For reproducing the research, the R-code for the preprocessing of GEH data is available in Additional file Quantitative real-time polymerase chain reaction (RT-PCR) data were obtained from the MAQC website  and used2 transformed data: a t-test and an AD statistic . The false discovery rate (FDR) for the p-values from the t-test was calculated using the Benjamini and Hochberg approach [TAQ_AB_FDR = 817, denoted as "Platform_Comparison_Metric ") and the AD threshold produced 553 DEGs (TAQ_AB_AD = 553). Similarly, 767, 351, 144, 106, 114, and 60 DEGs were determined by TAQ_CD_FDR, TAQ_CD_AD, GEX_AB_FDR, GEX_AB_AD, GEX_CD_FDR, and GEX_CD_AD, respectively. The R-codes for analyzing the TAQ and GEX data are available in Additional files To determine the DEGs, we applied two metrics for the logapproach . DEGs wet statistic (modT) [t statistic (shrT) [t statistic (ibmT) [The genes were ranked according to the differential expression using R ver. 2.11.0)  and Biocc (modT) , signifi1.0  and c (shrT) , and intc (ibmT) . A higheRP is only a rank-based non-parametric method. Different from the other methods, RP independently handles up-regulated genes and down-regulated genes under one class and therefore produces two separate ranked gene lists. In other words, RP outputs two values per gene. To obtain one RP value per gene for comparison with the results of the other methods, we defined a lower value as a net value for the gene. A small net value for RP is therefore evidence of differential expression. This procedure is the same as that described in Ref. . The genFor evaluation of the methods based both on sensitivity and specificity, an AUC value was calculated for each ranked gene list, where true DEGs were mainly defined by two metrics  as mentioned above. For evaluation based on inter-site and inter-platform reproducibility, the percentages of overlapping genes (POGs) and Spearman's rank correlation coefficients between different ranked gene lists were calculated.Analyzing gene set enrichment is a common approach in functional analysis where functionally or structurally related sets of genes regarding differential expression are assessed. Methods for this purpose include Gene Set Enrichment Analysis (GSEA)  and Param associative statistics S = {1, s2s, ..., ms} composed of m genes. Given R = {1, r2r, ..., mr} as the corresponding ranks of the values of the statistics and G = {1, g g2, ..., ng} as a gene set composed of n genes, the enrichment score for the gene set, GE, is simply calculated asnd, 8th, and 14th respectively. The enrichment score is simply calculated as GE= (2+8+14)/3 = 8. Accordingly, if a gene set has a small GEvalue, it is expected to be enriched (or de-regulated) in the two classes analyzed.To compare the performances of the eight gene ranking methods as simply as possible, we calculate the average of the ranks of the genes belonging to a gene set as an "enrichment score". Consider a gene ranking method that produces Method_Comparison_Site ".The gene sets were obtained from the Molecular Signatures Databases (MSigDB) ver. 3.0 . We usedGene set information is available as gene symbols, while expression data are measured on the probe basis. Inevitably, many multiple probes are mapped to the same gene on existing microarray platforms. For example, three probes  are mapped to the ACLY gene on the AFX platform, while only one probe is mapped to that gene on the other platforms . In this case, expression signals for the three probes on the AFX platform should be collapsed to avoid inflating the enrichment scores and to facilitate biological interpretation of the analysis results . We aver2 scale were analyzed. The POGs and Spearman's rank correlation coefficients for the 186 enrichment scores between test sites (or different platforms) were calculated for estimating inter-site (or inter-platform) reproducibility of the individual gene ranking methods at the function level.The mapping information for the five platforms was obtained from the Gene Expression Omnibus  as 'GPL5Investigation of suitable gene ranking methods having high sensitivity, specificity, and (inter-site and inter-platform) reproducibility is the main purpose of this work. Similar to our previous study on the AFX platform , eight gWe and other researchers believe the human samples compared in the MAQC study, cell lines (sample A) versus brain tissues (sample B), are clearly distinct, i.e., such comparisons are unrealistic in practical biological research -29. ThisThe average AUC values for the AFX data across six test sites are shown in Table TAQ_AB_AD ) case. The ibmT method may be suitable for ABI data. RP was overall good when DEGs were determined by the AD metric for TAQ RT-PCR data. WAD was overall good when DEGs were determined by the FDR metric from a t-test, regardless of the choice of RT-PCR technology .The average AUC values for ABI, AG1, GEH, and ILM data across three test sites are shown in Table X genes are defined as DEGs . We confirmed the superiority of the WAD, RP, and ibmT methods against various thresholds.Note that these results were based on only two sets of DEGs arbitrarily defined by commonly used procedures . As the AUC values vary with the thresholds for individual metrics, the effects of changing the parameters should also be analyzed. Table For reproducing the research, the R-codes for obtaining the AUC values shown in Tables Next, we examined the inter-site reproducibility of ranked gene lists among three test sites for each platform. Our previous study using AFX data reports that WAD is the most reproducible method when POG is evaluated . The POThe POG values for WAD were clearly higher than those for the other seven methods, especially in the "Sample C versus D" comparison. The results of that comparison should take precedence over those of "Sample A versus B". This is because sample C is a 3:1 and sample D is a 1:3 mixture of samples A and B, respectively, and real biological samples consist of various kinds of tissues and cells. These results suggest that the use of WAD can be promising for users concerned about inter-site reproducibility at the DEG list level.Method_Comparison_Site "). Each list consists of (a) 54,676 gene ranks for AFX data, (b) 32,879 for ABI, (c) 41,059 for AG1, (d) 53,517 for GEH, and (e) 47,294 for ILM. When we focus on the dendrograms for the ILM data  are shown in Figure We performed the functional analysis for 186 gene sets associated with KEGG Pathway. The POG values for different settings of STEROID_BIOSYNTHESIS, VALINE_LEUCINE_AND_ISOLEUCINE_BIOSYNTHESIS, FOLATE_BIOSYNTHESIS, TERPENOID_BACKBONE_BIOSYNTHESIS, and RENIN_ANGIOTENSIN_SYSTEM ) were identical across the 5 platforms. To estimate the overall similarity among platforms, we calculated an intersection-union ratio for each gene set. For example, there are 61, 61, 60, 54, and 61 genes in the respective platforms AFX, ABI, AG1, GEH, and ILM in the GLYCOLYSIS_GLUCONEOGENESIS pathway. As the intersection and union across the 5 platforms are 53 and 61, respectively, the ratio is 53/61 = 0.87. Detailed information on the 186 gene sets is available in Additional file Note that the numbers of genes belonging to each gene set differ across platforms. Only 5 of the 186 KEGG Pathway gene sets , RP/AD, and ibmT/FDR. These combinations are logical because RP/AD are both fold-change-based statistics and ibmT/FDR are both DR Table . To our As mentioned, our previous study reports that WAD outperforms the other methods when inter-site reproducibility is evaluated for AFX microarray data . In the Li et al. recently reported high inter-platform reproducibility at the biological function level obtained by using a commercially available tool . Only thw ) in a WAD statistic; see Figure one in Ref. [w statistic is clearly inferior to that obtained from the other specialized gene ranking methods such as WAD when the AUC value (both sensitivity and specificity) is evaluated [In this study, we could perform a more detailed analysis with regard to reproducibility by using MAQC datasets. However, the original MAQC study  has ofte in Ref. ). This wvaluated ,14. Eval2 transformation . As demonstrated by our previous study [In this study, we analyzed publicly available datasets that were already pre-processed according to the MAQC philosophy . Our addus study  and anotus study , there aus study -42) and In conclusion, the superiority of WAD was confirmed with regard to sensitivity, specificity, and reproducibility by using the MAQC datasets. We thus recommend the use of WAD for more effective transcriptome analysis.t-statistic; ILM: Illumina; MAQC: MicroArray Quality Control (project); POG: percentage of overlapping genes; RP: rank products; samT: significance analysis of microarrays; shrT: shrinkage t-statistic; TAQ: TaqMan; WAD: weighted average difference (method)ABI: Applied Biosystems; AD: average difference; AFX: Affymetrix; AG1: Agilent one-color microarray; AUC: area under the curve; FDR: false discovery rate; GEH: GE Healthcare; GEX: StaRT-PCR; ibmT: intensity-based moderated KK performed the analyses and wrote the paper. KS supervised the critical discussion by KK. All authors read and approved the final manuscript.R-code for pre-processing GEH data. Input file must be obtained from http://edkb.fda.gov/MAQC/MainStudy/upload/norm_MAQC_GEH_123_Median1.zip. After execution of this R-code, expression data consisting of 53,517 unique probes can be obtained as an output file arbitrarily named "norm_MAQC_GEH_123_Median1_unique.txt". Note that the website with the normalized MAQC data was unavailable under review. The raw data may be availabl from. http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for fileR-code for analyzing TAQ data. Two input files must first be obtained from the following websites: "http://www.nature.com/nbt/journal/v24/n9/extref/nbt1239-S5.txt" and "http://edkb.fda.gov/MAQC/MainStudy/upload/norm_TAQ_1_POLR2A.zip". After execution of this R-code, FDR and AD statistics for each comparison  can be obtained in an output file arbitrarily named "data_TAQ.txt". Note that the website with the normalized MAQC data was unavailable under review. The raw data may be available from http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for fileR-code for analyzing GEX data. Two input files must first be obtained from the following websites: "http://www.nature.com/nbt/journal/v24/n9/extref/nbt1239-S5.txt" and "http://edkb.fda.gov/MAQC/MainStudy/upload/norm_MAQC_GEX_1_ACTB.zip". After execution of this R-code, FDR and AD statistics for each comparison  can be obtained in an output file arbitrarily named "data_GEX.txt". Note that the website with the normalized MAQC data was unavailable under review. The raw data may be available from http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for fileR-code for calculating average AUC values for AFX data across six test sites shown in Table 1. Two input files are required. One is the AFX gene expression data file obtained from http://edkb.fda.gov/MAQC/MainStudy/upload/norm_MAQC_AFX_123456_qPLIER16.zip. The other includes the DEG information derived from individual RT-PCR data. Specifically, the filename should be "data_TAQ.txt" (default) or "data_GEX.txt", generated by executing Additional file http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for fileR-code for calculating average AUC values for ABI data across three test sites shown in Table 2. Two input files are required. One is the ABI gene expression data file obtained from http://edkb.fda.gov/MAQC/MainStudy/upload/norm_MAQC_ABI_123_qNorm.zip. The other includes the DEG information derived from individual RT-PCR data. Specifically, the filename should be "data_TAQ.txt" (default) or "data_GEX.txt", generated by executing Additional file http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for fileR-code for calculating average AUC values for AG1 data across three test sites shown in Table 2. Two input files are required. One is the AG1 gene expression data file obtained from http://edkb.fda.gov/MAQC/MainStudy/upload/norm_MAQC_AG1_123_Median1GeneSpring.zip. The other includes the DEG information derived from individual RT-PCR data. Specifically, the filename should be "data_TAQ.txt" (default) or "data_GEX.txt", generated by executing Additional file http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for fileR-code for calculating average AUC values for GEH data across three test sites shown in Table 2. Two input files are required. One is the GEH gene expression data file obtained by executing Additional file Click here for fileR-code for calculating average AUC values for ILM data across three test sites shown in Table 2. Two input files are required. One is the ILM gene expression data file obtained from http://edkb.fda.gov/MAQC/MainStudy/upload/norm_MAQC_ILM_123_qNorm16.zip. The other includes the information about DEGs derived from individual RT-PCR data. Specifically, the filename should be "data_TAQ.txt" (default) or "data_GEX.txt", generated by executing Additional file http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for fileAverage AUC values for all methods regarding Table 3. Table 3 was obtained based on these values.Click here for fileX genes are defined as DEGsR-code for obtaining average AUC values for AFX data across six test sites when top . Two input files are required. One is the AFX gene expression data file obtained from http://edkb.fda.gov/MAQC/MainStudy/upload/norm_MAQC_AFX_123456_qPLIER16.zip. The other includes the DEG information derived from individual RT-PCR data. Specifically, the filename should be "data_TAQ.txt" (default) or "data_GEX.txt", generated by executing Additional file X genes are defined as DEGs (X = 100 by default), shown in Additional file http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/ or http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5350.Click here for filePOG values for given numbers of top-ranked genes among three test sites. (a) AFX data, (b) ABI data, (c) AG1 data, (d) GEH data, and (e) ILM data. Number of DEGs (X ) is shown on x -axis . Percentage of genes (POG) for X top-ranked genes among three test sites is shown on y -axis.Click here for fileRaw data for Additional file Click here for fileCorrelation coefficients of ranked gene lists between any two test sites.Click here for fileCorrelation coefficients of 186 ranked lists of gene sets between any two test sites.Click here for fileRaw data for Figure 2. There are a total of ten sheets (five platforms × two comparisons).Click here for fileR-code for making left side of Figure 2(a). The R-code assumes the input filename is "POG_AFX_KEGG_AB.txt," which contains data for the first sheet  in Additional file Click here for fileNumbers of genes belonging to each of 186 KEGG Pathway gene sets in individual platforms.Click here for fileRaw data for Figure 3. There are two sheets.Click here for file


Perioperative prophylactic antibiotic treatment significantly influences intestinal microflora, resulting in impaired bowel functioning in some patients, sometimes requiring further investigations. This may lead to a worse health-related quality of life (HRQoL). Probiotics administrated in the early post-operative period may help avoiding such nuisances in older people.9 daily, while Group B patients received a Placebo (cornstarch). Patients were further divided in sub-groups whether ileo-caecal valve was spared or not. Patients were followed-up every 7 days for 4 weeks. Patients daily annotated bowel frequency, stool shape and consistency - according to Bristol's scale. HRQoL was assessed every week by means of SF-36 questionnaire.We prospectively enrolled patients undergoing laparoscopic colorectal surgery aged over 70 years between 2005 and 2012. The study was approved by IRB. All patients received perioperative antibiotic treatment. Patients were randomized to one of two treatment arms: Group A patients received probiotics after surgery (VSL#3, VSL Pharmaceuticals, Inc. USA - 900 × 10Group A included 10 while Group B included 8 patients. One patient in each group experienced a postoperative complication. Group A patients had fewer bowel movements than controls, during every week. Stool consistency was higher in patients undergoing resections including ileo-caecal valve receiving VSL#3. HRQoL gradually increased in both groups; Group A patients had higher "social functioning" item scores at week 1 and 4 than controls.Elderly patients undergoing resection of ileo-caecal valve may benefit from an early probiotics administration pathway after perioperative antibiotic treatment. Intestinal microflora is profoundly influenced by antibiotic therapy needed peri-operatively in patients undergoing large bowel resections. The effects of short antibiotic therapy on gut microbiota can last more than 24 months . ElderlyAim of our study was to investigate whether the administration of probiotics in the early postoperative period after large bowel resection can facilitate the restoration of bowel function and reduce symptoms in elderly patients.This is a prospective double blind randomized trial on patients aged over 70 years undergoing laparoscopic colonic resection in our Unit between 2005 and January 2012. The study was approved by IRB and all patients were included after signing an informed consent. Patients were randomly assigned to Group A (antibiotic + probiotic) or Group B (antibiotic + placebo). In each group, patients were further divided in sub-groups 1 and 2 whether surgery preserved or not ileocaecal valve, respectively. After the discharge, patients were seen in outpatient settings by a resident every 7 days for 4 weeks. Neither patients nor residents knew which treatment (probiotic/placebo) was being administered.Dukes D colorectal cancerInflammatory bowel diseasesNeed for temporary or definitive oostomyAllergy to antibiotics/probiotics/placebo used in the studyPatients received i.v. cefotaxime 1 g and i.v. metronidazole 1 g within 30 minutes from surgery, and continued on i.v. cefotaxime 1g t.i.d. and i.v. metronidazole 500 mg t.i.d. for no more than 24 hours postoperatively.9 daily, divided in two administrations from one day after discontinuation of antibiotic through four weeks. The probiotic composition is the following: Streptococcus thermophilus DSM 24731, Bifidobacteria , Lattobacilli ; maltose; silicium dioxide. Group B patients received the Placebo composed of cornstarch (base ingredient for probiotic product), indistinguishable from VSL#3, in a similar fashion.Group A patients received probiotics  at the dose of 900 × 10During probiotic/placebo treatment patients were requested to complete a daily report annotating: bowel movement/s, body temperature, stool shape and consistency. Stool characters were defined according to Bristol scale , a 7-poiPatients completed the SF-36 questionnaire every week during probiotic/placebo treatment.t test was used.We enrolled all consecutive patients undergoing surgery in the study period fitting inclusion criteria. Data are presented as mean ± SD. Categorical variable were compared by means of Fisher's exact test; for continuous parameters Student's Group A included 10 patients ; Group B included 8 patients . Patients characteristics and surgical parameters are reported in Table No differences were observed concerning overall stool consistency; however it was higher in patients undergoing ileocolic resections including ileo-caecal valve receiving VSL#3 Figure .No febrile episodes (body temperature ≥ 37.3° C) were recorded.Both groups showed a gradual increase in HRQoL from first through fourth week; with respect to item "social functioning", group A patients showed significantly higher score at week 1 and 4 than patients receiving placebo Figure .No adverse effects to VSL#3 or placebo were observed.Perioperative antibiotic therapy, although demandable, are responsible for relevant imbalances of intestinal microbiota ,3. This It has been reported that probiotic formulations are not effective to reduce the rate of postoperative septic complications in patients undergoing elective abdominal surgery . HoweverIn our series, we observed a lower frequency of evacuations in patients receiving probiotics, although not statistically significant; stool consistency was slightly higher in patients receiving resection not preserving ileo-caecal valve treated with probiotics. HRQoL was comparable in both groups, but patients in the probiotic group achieved a better social functioning than controls at 1- and 4-week follow-up.Even if therapy with probiotics did not show a protective effect on postoperative complications, frail patients undergoing resection of ileo-caecal valve may benefit from an early probiotics administration pathway after perioperative antibiotic treatment. Bowel frequency may be lower, irrespective of the type of surgery. These observations support the usefulness of early probiotic administration in achieving a prompt restoration of function and return to everyday activities.HRQoL: health-related quality of life; IRB: internal review board; SF-36: short-form-36 questionnaire; SD: standard deviation.The authors declare that they have no competing interests.G.P.: conception and design, interpretation of data, given final approval of the version to be publishedG.S.: acquisition of data, drafting the manuscript, given final approval of the version to be publishedG.C.: acquisition of data, drafting the manuscript, given final approval of the version to be publishedA.C.: acquisition of data, drafting the manuscript, given final approval of the version to be publishedR.M.: acquisition of data, drafting the manuscript, given final approval of the version to be publishedS.D.F.: acquisition of data, drafting the manuscript, given final approval of the version to be publishedF.R.: acquisition of data, drafting the manuscript, given final approval of the version to be publishedS.C.: critical revision, interpretation of data, given final approval of the version to be publishedG.R.: conception and design, critical revision, given final approval of the version to be publishedF.S.: conception and design, critical revision, given final approval of the version to be publishedGP: Resident in General Surgery at Second University of Naples.GS: PhD, Researcher, Aggregate Professor of General Surgery at Second University of Naples.GC: Resident in General Surgery at Second University of Naples.AC: Medical Student (Research Assistant) at Second University of Naples.RM: Medical Student (Research Assistant) at Second University of Naples.SDF: M.D. (Research Assistant) at the Second University of Naples.FR: Medical Student (Research Assistant) at Second University of Naples.CS: Full Professor of General Surgery at Second University of Naples, Chief Division of General and Geriatric Surgery Second University of Naples.GR: Associate Professor of Gastroenterology, Second University of Naples.FS: Associate Professor of General Surgery at Second University of Naples.


Vitis. The phylogenetic relationships among these species are of keen interest for the conservation and use of this germplasm. We selected 309 accessions from 48 Vitis species,varieties, and outgroups, examined ~11 kb  of aligned nuclear DNA sequences from 27 unlinked genes in a phylogenetic context, and estimated divergence times based on fossil calibrations.Grapes are one of the most economically important fruit crops. There are about 60 species in the genus Vitis formed a strongly supported clade. There was substantial support for species and less for the higher-level groupings (series). As estimated from extant taxa, the crown age of Vitis was 28 Ma and the divergence of subgenera (Vitis and Muscadinia) occurred at ~18 Ma. Higher clades in subgenus Vitis diverged 16 – 5 Ma with overlapping confidence intervals, and ongoing divergence formed extant species at 12 – 1.3 Ma. Several species had species-specific SNPs. NeighborNet analysis showed extensive reticulation at the core of subgenus Vitis representing the deeper nodes, with extensive reticulation radiating outward. Fitch Parsimony identified North America as the origin of the most recent common ancestor of extant Vitis species.Phylogenetic patterns suggested origination of the genus in North America, fragmentation of an ancestral range during the Miocene, formation of extant species in the late Miocene-Pleistocene, and differentiation of species in the context of Pliocene-Quaternary tectonic and climatic change. Nuclear SNPs effectively resolved relationships at and below the species level in grapes and rectified several misclassifications of accessions in the repositories. Our results challenge current higher-level classifications, reveal the abundance of genetic diversity in the genus that is potentially available for crop improvement, and provide a valuable resource for species delineation, germplasm conservation and use. Vitis spp.) are one of the world’s most economically valuable fruit crops . Additional file The authors declare they have no competing interests.YW, CJS, AMB, HRS conceived the ideas; YW, CJS collected or arranged for the materials; AMB, YW, JAL developed the markers; YW, AMB developed the sequence matrix; HRS analyzed the data except for PCA; AMB developed and performed PCA; HRS, JAL, YW wrote the paper; CJS and GYZ managed the project. All authors read and approved the final submission.Accession Information Table_with GenBankAccNo.xlsx. Germplasm accessions information. Accession name, taxonomy, continental origin, source, data set membership, individual GenBank and EMBL accession numbers.Click here for fileFragment information.xlsx. Gene fragment information. Primer sequence, fragment length, chromosome location, original EST annotation, number of unique site patterns (an indicator of informative variation), gene.meanRate, substitution models used (from Findmodel), GenBank accession numbers.Click here for filea-d. Consensus Networks.pdf. Consensus Network of 26 single gene trees, showing all splits present in at least a. one tree , b. two trees , c. 50% of the trees (threshold = 0.5), d. 90% of the trees (threshold = 0.9).Click here for fileNode Ages.pdf. Node ages (Ma) of all nodes in maximum clade credibility tree inferred with BEAST from three combined runs.Click here for filePosterior Probabilities.pdf. Posterior probabilities of all nodes in maximum clade credibility tree inferred with BEAST from three combined runs.Click here for fileCartoon BestMLtree.pdf. Cartoon of best ML tree. For comparison with trees reconstructed with other methods. Blue = North American OTUs, Green = Asian OTUs, Red = European (mostly) OTUs.Click here for fileBest ML tree with Bootstrap supports.pdf. Best ML tree of 273 accessions with bootstrap supports from 1,000 replicates. Supports 1-100% are listed along branches. Abbreviated uncorrected taxon labels.Click here for fileBayesian Tree_48MGen.pdf. Bayesian tree, 48 million generations, not partitioned, burn in 10 million steps. Posterior probabilities (0 to 1) are listed along branches.Click here for fileBayesianTree_50MilGen.pdf. Bayesian tree, 50 million generations, not partitioned, burn in 5%. Posterior probabilities (0 to 1) are listed along branches.Click here for fileCartoon Of Bayesian Tree_48MilGen.pdf. Cartoon of Bayesian tree 48 million generations, not partitioned, burn in 10 million steps.Click here for fileNodes With Support And Character scores Genes Filled.xlsx. List of characters supporting selected nodes in strict consensus tree . Last character in each sequence was added for the ancestral area reconstruction. Can be opened using a text editor. Gene partitions are listed in Additional file Click here for fileGene partitions for the 11437bp matrix.Click here for file800mil10Klog_52taxaNoTiliWithCay_11437_LognRelYule3CalPts_NOpartK80G.xml. XML file used to date divergences in BEAST (unpartitioned).Click here for file400Mil10Klog_53taxa With Cayratia_11437_Yule_LogNrelaxedClock_partitioned3calibration Many Models.xml. XML file used to estimate evolutionary rate of change per partition (27 partitions).Click here for fileEvaluation criteria for BEAST runs.pdf. Evaluation criteria for Tracer files.Click here for file


AbstractPellamaoershanensis Song & Li, sp. n., collected from a colony of Lasius (Dendrolasius) spathepus in Maoershan Natural Reserve, Guangxi, is diagnosed, described and illustrated. The discovery represents the first record of the genus in South China. Lomechusini , Pella cooterorum Maruyama , Pella hlavaci Maruyama (Beijing), Pella jureceki Dvořák (Beijing), Pella kishimotoi Maruyama (Hunan), Pella puetzi Assing (Yunnan) and Pella zhoui Maruyama (Beijing). Members of Pella are comPageBreakmonly found in association with the ant genus Lasius Fabricius (Lasius (Dendrolasius) spathepus. A closer examination of this material revealed a new species of the genus Pella. In this paper we describe the new species, provide illustrations of its major diagnostic features, and briefly discuss the biology.According to the latest catalogue of echusini , the genabricius . RecentlSpecimens were killed with ethyl acetate and preserved in 75% ethanol before dissection;Photos of habitus were taken with a Canon EOS 50D with an MP-E 65mm Macro Photo Lens.Head length was measured from the clypeal anterior margin to the occipital constriction; elytral length at the suture from the apex of the scutellum to the elytral posterior margin.SNUC).All the types were deposited in the Insect Collection of Shanghai Normal University, Shanghai, China . Xingan County / Guilin City /  / 24.VII.2012, Song X-B & Hu J-Y // HOLOTYPE [red] / Pella maoershanensis sp. n. / Song & Li / det. 2013, SNUC’. Paratypes: 23 ♂♂, 28 ♀♀, same label data as holotype, all bearing the following label: ‘PARATYPE [yellow] / Pella maoershanensis sp. n. / Song & Li / det. 2013, SNUC’.Pella maoershanensis shares with Pella puetzi a similar form of male sexual character on the head  but can be readily separated by the sexually modified head in the male.the head . The twoBody  length: PageBreakHead  spathepus (det. by M. Maruyama) (aruyama) .Pella are usually observed walking around the host’s nest but never appear in it (Maruyama 2006). All individuals of the new species, plus three other aleocharine species were taken by sifting mixed leaf litter around the ant nest (Dendrolasiophilus Nomura species (Yin pers. comm.) were directly collected from the deep site of the nest.Species of ant nest . Three pant nest . It’s woNamed after the type locality.


Taenia multiceps larvae, and zoonotic infections occur in humans. Infections frequently lead to host death, resulting in huge socioeconomic losses. MicroRNAs (miRNAs) have important roles in the post-transcriptional regulation of a large number of animal genes by imperfectly binding target mRNAs. To date, there have been no reports of miRNAs in T. multiceps.Worldwide, but especially in developing countries, coenurosis of sheep and other livestock is caused by T. multiceps small RNA library using Illumina sequencing technology. A total of 796 conserved miRNA families  from 170,888 unique miRNAs were characterized using miRBase (Release 17.0). Here, we selected three conserved miRNA/miRNA* (antisense strand) duplexes at random and amplified their corresponding precursors using a PCR-based method. Furthermore, 20 candidate novel miRNA precursors were verified by genomic PCR. Among these, six corresponding T. multiceps miRNAs are considered specific for Taeniidae because no homologs were found in other species annotated in miRBase. In addition, 181,077 target sites within T. multiceps transcriptome were predicted for 20 candidate newly miRNAs.In this study, we obtained 12.8 million high quality raw reads from adult T. multiceps provides a substantial platform for improving our understanding of the molecular regulation of T. multiceps and other cestodes development.Our large-scale investigation of miRNAs in adult  Caenorhabditis elegans[Increasing evidence shows that control of gene expression is essential for normal development of organisms and for regulating fundamental biological processes -4. Reguls elegans,13, manys elegans. Furthers elegans,16.Taenia multiceps is a widespread cestode, which has two life cycle stages. The larval stage (coenurus) parasitizes the brain or spinal cord of domestic ruminants, such as buffalo, cattle, goats, horses, sheep, and yak, as well as wild species, causing lethal neurological symptoms [T. multiceps are found in the small intestine of dogs and other canids, and gravid proglottids in host feces are a source of infection following ingestion by an intermediate hosts [symptoms . Since hsymptoms , furthersymptoms -25. Adulte hosts .T. multiceps occurs almost all over the world [T. multiceps miRNAs and other sRNAs using high-throughput sequencing methods. In this study, we investigated T. multiceps miRNAs using an Echinococcus multilocularis reference genome and an adult T. multiceps transcriptome dataset. Our results increase the current understanding of the molecular mechanisms of cestode gene regulation [Coenurus caused by he world , especiahe world . Given tgulation , and wilgulation ,30 and dgulation .T. multiceps were obtained from the small intestine of infected dogs after gravid proglottids were observed in their feces. T. multiceps were washed thoroughly in physiological saline solution (37°C) to avoid host cell contamination, transferred into liquid nitrogen, and stored at - 80°C.Larvae (coenuri) were collected from the brain of a naturally infected goat at an organic farm in Panzhihua, Sichuan, China. Infection was performed after morphological identification of the larvae. Two parasite-free beagles were orally infected with 20 larvae each. Forty-eight days after infection, adult All animals were handled in accordance with the Animal Protection Law of the People’s Republic of China . This study was approved by the National Institute of Animal Health Animal Care and Use Committee at Sichuan Agricultural University .T. multiceps (n = 6) using TRIZOL , according to the manufacturer’s protocol. RNA integrity was checked by determining the RNA integrity number using an Agilent 2100 Bioanalyzer. RNA sequences ranging from 18–30 nt in length were isolated and purified from total RNA by Novex 15% TBE–Urea gel (Invitrogen) electrophoresis. Proprietary (Solexa) adaptors were then added to the 3 and 5 -termini of sRNAs, which were then used for cDNA synthesis. These ligation products were amplified by reverse transcription PCR (RT-PCR) using a RT-PCR kit (Invitrogen). PCR amplification products were purified for high-throughput sequencing by electrophoresis using a 6% TBE PAGE gel (Invitrogen). The produced libraries were sequenced using a Solexa sequencer at the Beijing Genomics Institute (BGI)-Shenzhen, Shenzhen, China, according to the manufacturer’s instruction.Total RNA was extracted from adult E. multilocularis genome, which was obtained from the Sanger Institute FTP site [The workflow for obtaining ‘clean’ reads involved filtering low quality tags; removing raw reads with 5’ primer contaminants; trimming 3’ adaptors; removing reads without insert tags; discarding reads with polyA tails; and removing contaminants formed by adaptor–adaptor ligation. The length distribution of clean reads was summarized, all clean reads were mapped to the FTP site , using tFTP site , and theFTP site  and GenBFTP site  to removT. multiceps, clean small RNA sequences were aligned with the miRNA precursor and mature sequences of all animals deposited in miRBase 17.0, using tag2miRNA software (provided by BGI). Sequences containing no more than two mismatches with known miRNAs were defined as conserved miRNAs, and the most abundantly expressed sequence was selected when multiple sequences were assigned as the same conserved miRNA. Sequences that could not be annotated from sequence alignments were defined as non-annotation unique reads.To identify conserved miRNAs in E. multilocularis genome [T. multiceps reads that perfectly matched the E. multilocularis genome were designated candidate miRNA precursors. Potential precursor sequences were selected from each end of the sequence matching the referenced genome, and MIREAP software [The s genome  was alsosoftware  was usedsoftware ,38. We uto select novel miRNAs with MFEIs > 0.85.T. multiceps using a Gentra Puregene Tissue Kit  following the manufacture’s protocol. Three conserved putative miRNA/miRNA* duplexes were selected randomly to check if their corresponding precursors form a characteristic pre-miRNA hairpin-like structure with low free energy and the putative miRNA in the stem. Using the candidate precursors which predicted based on the E. multilocularis genome, we designed primers for novel miRNA precursors and miRNA/miRNA* duplexes using Primer Premier 5.0. Primers successfully used for amplifying the three conserved and 20 novel miRNA precursors are shown in Additional file Conserved miRNAs and novel candidate miRNA precursors were verified using a PCR-based method. Genomic DNA was extracted from adult T. multiceps transcriptome unigenes obtained by Illumina sequencing were used to predict novel miRNA targets (Unigene: JR916739–JR948020 in the Transcriptome Shotgun Assembly Sequence Database at NCBI from our previous study [T. multiceps were predicted using RNAhybrid [.us study ). miRNA NAhybrid ,42 paramTo understand the main biological functions and identify the biochemical metabolic pathway/signal transduction pathways of novel miRNAs target gene candidates, all target gene candidates were annotated against the Gene Ontology (GO) database  and the T. multiceps to identify miRNAs, which yielded 13.5 million raw reads. The dataset was deposited in NCBI Gene Expression Omnibus [E. multilocularis genome .We employed Illumina sequencing technology on a library of small RNAs from adult  Omnibus  with theNon-coding small RNAs including 653,754 rRNAs, 1,086 snoRNAs, 12,806 snRNAs, and 728,898 tRNAs were removed after mapping all clean reads against the Rfam database (version 10.0). Subsequently, 392,874 total sRNAs  were identified as repeat associated sRNAs and then removed. A total of 11.2 million small RNA sequences were obtained. The distribution of total and unique sRNAs is shown in Figure E. multilocularis genome: tmu-miR-0004 corresponds to eight different locations on different chromosomes, while tmu-miR-0039 corresponds to two different locations  was the most abundant, with 9,463 reads, which suggests that it has an important role in adult T. multiceps. As a previous study on maize miRNAs suggested that miRNA precursors with MFEIs > 0.85 are most likely to be miRNAs [In addition to conserved miRNAs, we identified 20 candidate miRNAs. A total of 44 precursor candidates  were predicted using MIREAP software; these contained a Dicer cleavage site, and exhibited an appropriate secondary structure and MFE. Two candidate miRNAs mapped to multiple locations in the e miRNAs , we detetmu-miR-87/miR-87*; tmu-miR-124b/miR-124b*; tmu-miR-2162/miR-2162*) and three novel miRNA/miRNA* duplexes . The electrophoretic analysis of three conserved miRNA precursor PCR products is shown in Figure Echinococcus granulosus, E. multilocularis, Caenorhabditis elegans and Schistosoma japonicum from miRBase are listed in Figure T. multiceps miRNA precursors have > 90% similarity with the corresponding predicted sequences from E. multilocularis. However, one novel miRNA precursor (tmu-novel-19) has low similarity (65.1%) and a different structure to the predicted E. multilocularis precursor, suggesting that this novel miRNA may have different functions in T. multiceps and E. multilocularis. Interestingly, six of the novel miRNAs did not share homology with sequences in the miRBase [We used a PCR-based method to verify our predicted miRNA precursors, including three conserved miRNA/miRNA* duplexes , was targeted by tmu-novel-07. After mapping to the GO database, all putative target genes were classified into diverse GO functional groups. The GO functions of the predicted targets of novel miRNAs are shown in Additional file To determine the functions of the 1,006 conserved miRNAs and 20 novel miRNAs in T. multiceps miRNAs by Illumina sequencing. Since the adult worms used for RNA extraction were gravid, the identified miRNAs could also be expressed by the oncospheres. This dataset might help the further study of miRNAs of T. multiceps oncospheres. Our results showed that a low percentage (17.68%) of reads had a perfect match to sequences and only 39,765 out of a total of 1,379,196 (19.46%) unique sRNAs matched sequences in the related E. multilocularis genome, as proposed in previous study of Clonorchis sinensis sRNAs when matching which into the related genome of Schistosoma japonicum[E. multilocularis reference genome, as the T. multiceps whole-genome sequence is currently unavailable. T. multiceps and E. multilocularis are both Taeniidae cestodes, but significant differences exist between them [Animal miRNAs are important regulators of gene expression that function through imprecise complementarity to their mRNA targets . Hence, japonicum. This laeen them .Taenia saginata were expression predominated by miR-71  [T. multiceps (16.43%). Similar to T. saginata, members of the common miR-40 family were not detected in T. multiceps in this study. However, low expression of 12 members of another common miRNA family, let-7 , E. granulosus and E. multilocularis. Considering that T. saginata and T. multiceps belong to the same genus, Taenia, the absence of let-7 miRNAs in T. saginata, their presence in T. multiceps and their universal existence in some other species suggest that non-recovery of let-7 in T. saginata may be attributed to the specific stage of the parasite examined or experimental methods. In addition, we compared conserved miRNAs of T. multiceps with E. granulosus (E. multilocularis) which were obtained from miRBase. The miR-8, which was found in E. granulosus and E. multilocularis, was not identified in adult T. multiceps. While miR-96 was found both in E. multilocularis and T. multiceps but not in E. granulosus. Among these three species, two other miRNAs (miR-4990 and miR-4991) were only detected in E. granulosus. These findings could be useful for miRNAs’ function researches.Some kinds of miRNAs were expressed with high predominance. A previous data showed that known miRNAs of l reads) , which w saginata), was shT. multiceps miRNAs. Target searching identified many unigene targets of miR-1, including unigenes 754 and 757, both of which have been annotated as malate dehydrogenase. This finding is consistent with a recent report that malate dehydrogenase is regulated by miR-1 in Trichomonas vaginalis[T. multiceps are intestinal parasites that produce ATP mainly through anaerobic glycolysis (not the citric acid cycle) [T. multiceps. However, we can infer that miR-1 may interact with other miRNAs to regulate glucose metabolism in T. multiceps.The miR-1, a highly conserved muscle-specific miRNA, is represented in the top four abundant vaginalis, illustrvaginalis and T. mcel-miR-87 from miRBase, the mature miRNA tmu-miR-87-5p seemed not so highly conserved  were detected. The miR-1422 family reads therefore suggest a strong bias (99.97%) toward miR-1422* in adult T. multiceps. miR-959*, whose mature strand has only previously been reported in D. melanogaster[T. multiceps.As miRNAs* are less stable than mature miRNAs, being quickly degraded after mature miRNA enters the RISC complex ,9, we dianogaster,55. The  members . Althouganogaster, was theT. multiceps miRNAs using PCR-based methods, thus providing evidence that computational methods used for miRNA prediction were correctly executed. We also verified that miRNA precursor and flanking sequences could be folded into typical miRNA-like hairpin structures. The sequences and structures of these newly candidate miRNAs should help future studies of miRNA function in T. multiceps. All 20 novel miRNAs are regarded as candidate novel miRNAs here. Additional evidence is required to further validation their reliability. However, we failed to validate 16 other predicted novel miRNAs, including two candidate miRNAs with multiple loci in the E. multilocularis genome. We consider that this may be due to 1). the poor quality of primers and the difficulty of primer designing as we designed primers to amplify full-length predicted precursors. 2). the miRNA precursors were predicted by comparing to a related cestode species E. multilocularis instead of T. multiceps, which may highlight differences between the cestode species at the nucleotide level.We identified 20 candidate novel T. multiceps compared to E. multilocularis genome. As they both belong to Taeniidae, we speculate that these specific miRNAs might have specialized functions related to cellular processes in Taeniidae, and may therefore provide novel therapeutics for this disease [T. multiceps  determined using the MFEI formula could be validated by PCR-based method and meeting the secondary structure and the required MFE  are lacking. In previous miRNA studies of locust and pinewood nematode, researchers chose EST databases to predict miRNA targets [T. multiceps transcriptome unigenes obtained by high-throughput sequencing as an alternative method to predict miRNA targets because animal miRNAs can bind to a broad spectrum of different target mRNAs through imprecise base pairing [T. multiceps unigenes were predicted by only 20 candidate novel miRNAs. This indicates that a single novel miRNA may have a large number of unigene target sites, thus suggesting that miRNAs have diverse functions. For example, we identified 8,665 target sites in multiple unigenes for the most abundant novel miRNA, tmu-novel-03. Most unigenes were predicted to contain more than one target site for different novel miRNAs. For example, unigene 17356, annotated as phosphoenolpyruvate carboxykinase (PEPCK), was targeted by both tmu-novel-01 and tmu-novel-11  from adult  Omnibus  with theThe authors declare that they have no competing interests.XHW and GYY conceived and designed the whole experiment. DYY, RHZ, WPZ, NW and JHW performed the experiments of verification. NY, HMN and YX analyzed the data. XBG, SXW and XRP contributed reagents/materials/analysis tools. XHW and YF drafted the manuscript. All authors read and approved the final manuscript.PCR primers for three conserved and 20 novel Taenia multiceps miRNA precursors. A total of 23 pairs of primers were used successfully in validation experiments.Click here for fileDistribution of Taenia multiceps small RNAs across different chromosomes of Echinococcus multilocularis. “sense” and “anti-sense” represent “+” and “-” strands of Chromosomes, respectively.Click here for fileConserved Taenia multiceps miRNAs based on miRBase 17.0. (A) Conserved miRNA families and miRNAs (B) List of Taenia multiceps antisense miRNAs (miRNAs*).Click here for fileDetails of the novel miRNA precursors. (A) Novel miRNA precursors predicted by MIREAP software using the Echinococcus multilocularis genome. (B) Confidence level of novel miRNAs selected using the minimal folding free energy index. (C) The result of RNAfold software for predicting the secondary structure of validated miRNA precusors.Click here for fileHairpin secondary structures of 20 novel Taenia multiceps miRNA precursors. Nucleotide bases of mature miRNA-5p (miRNA-3p) are marked with red (blue) color. The stem-loop structure of all validated novel miRNA precursors was predicted by RNAfold software. “Tmu-novel candidate-17” represents the non-typical secondary structure. Three novel miRNA/miRNA* pairs  were validated. All 20 novel miRNAs noted in this Figure are the candidate names of novel miRNAs of T. multiceps.Click here for fileNovel miRNA target sites conserved in Taenia multiceps unigenes. 181,077 target unigenes were predicted for 20 novel miRNAs based on 31,282 adult T. multiceps transcriptome, which were obtained by Illumina sequencing and Trinity assembling.Click here for fileCellular component GO annotations for candidate target unigenes for novel Taenia multiceps miRNAs. 5,696 target unigenes were assigned to 324 GO-terms from “Cellular component” ontology. “Gene Ontology term” means GO terms from Component Ontology with P-value as good as or better than 1. “Cluster frequency” stands for number and frequency of target unigenes related to this term. “Genome frequency of use” represents number and frequency of coding genes related to this term.Click here for fileMolecular function GO annotations for candidate target unigenes of novel Taenia multiceps miRNAs. 7,095 target unigenes were assigned to 604 GO-terms from “Molecular function” ontology.Click here for fileBiological process GO annotations for candidate target unigenes of novel Taenia multiceps miRNAs. 6,178 target unigenes were assigned to 2,262 GO-terms from “Biological process” ontology.Click here for fileKEGG pathways for predicted target genes of Taenia multiceps novel miRNAs. 9,306 target unigenes were assigned into 240 KEGG pathways. . “Pvalue” and “Qvalue” represent P-value before correction and corrected P-value, respectively.Click here for file


Neonatal Onset Multisystem Inflammatory Disorder (NOMID) is the most severe of the Cryopyrin-Associated Periodic Syndromes (CAPS) spectrum . Given tN.D. is a 2.5 yo male with congenital hearing loss who initially presented at 2 months of age with direct hyperbilirubinemia and transaminitis. Liver biopsy showed giant cell hepatitis. He had a positive ANA (with strong cytoplasmic staining), anti-smooth muscle antibody, and mildly positive double-stranded DNA antibody although his mother had no autoimmune antibodies. At 3 months of age, he developed persistent fevers and an urticarial-like rash and at 1 year he developed hip arthritis followed by knee swelling. His physical exam was notable for poor growth, macrocephaly, and irritability. He had generalized lymphadenopathy, pronounced hepatosplenomegaly and arthralgia with effusion of the left knee. Laboratory evaluations demonstrated persistently elevated CRP up to 200 mg/L, ESR 76 mm/hr, and ferritin 114 ng/mL. Urine showed no proteinuria or mevalonic acid. MRI of the brain demonstrated severe volume loss and Xray of distal femur showed physeal irregularity. Skin biopsy showed neutrophilic urticaria particularly surrounding eccrine glands. Sequencing of NLRP3 revealed a c.926T>A mutation (Phe209Leu) which was described in a NOMID patient [in utero and suggests that in some cases, autoinflammation and autoimmunity may not be mutually exclusive.This complex patient with evidence for persistent inflammation emphasizes the variable clinical presentation of the CAPS spectrum. The severity of this case also illustrates that NLRP3-related inflammation may begin L. Broderick: None declared, J. Chang: None declared, I. Szer: None declared, H. Hoffman Consultant for: Sobi, Novartis and Regeneron


Bacillus pumilus. B. pumilus is widely used in agriculture but has recently been linked to cases of food poisoning. Here, we report the complete genome of Blastoid and discuss unique genomic characteristics.Phage Blastoid is a siphophage that infects  Bacillus pumilus is a Gram-positive, sporulating, soil-dwelling bacterium. It is used in agriculture both as a symbiont to promote plant growth and as an antifungal agent of the root microenvironment . Transmission electron microscopy was performed at the University of Mary Washington.iversity . Phage Biversity  and corrhttps://cpt.tamu.edu/cpt-software/releases/pause/) revealed an 830-bp terminal repeat.Phage Blastoid has a 49,524-bp double-stranded DNA (dsDNA) unit genome with a G+C content of 42.5%, a 92.6% coding density, and 78 coding sequences. Of those, 46 were hypothetical conserved genes, three were novel hypothetical genes, and 29 have a putative function based on BLASTp and InterProScan analysis  for peptidoglycan binding and a class II holin with two predicted transmembrane domains in an N-in C-in topology.Genomic analysis revealed a variety of genes encoding proteins whose functions include DNA replication, recombination, biosynthesis, morphogenesis, and lysis. The genes for replication and recombination proteins identified were helicase, primase, DNA polymerase III, a Holliday junction resolvase, and a variety of nucleases. Blastoid uses thymidylate synthase, deoxynucleoside monophosphate kinase, and serine/threonine kinase to aid in DNA and amino acid biosynthesis. The genes encoding morphogenesis proteins include those encoding a minor head protein, scaffold protein, tail completion protein, tape measure protein, tail fiber, and a tailspike protein with a pectin lyase domain. The tail fiber protein was identified by a fibronectin (fn 3) domain and its location in the genome . The lysA unique gene in the genome encodes a putative cell division protein, FtsK/SpoIIIE. The FtsK/SpoIIIE protein is an ATPase involved in intracellular chromosomal DNA transfer in prokaryotes. In Gram-negative cells, FtsK is a DNA translocase that mediates the segregation of sister chromosomes into the daughter cells after replication . The GraKF669648 to GenBank.The genome sequence of phage Blastoid was contributed as accession no.