Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77  Caenorhabditis elegans. This pathway can be triggered experimentally using double-stranded (ds)RNA to selected gene targets, thereby allowing researchers to ‘silence’ individual genes and so investigate their function. It is hoped that this technology will facilitate gene silencing in important parasitic nematodes that impose a considerable health and economic burden on mankind. Unfortunately, differences in RNAi susceptibility have been observed between species. Here we investigated the possibility that differences in the complement of effector proteins involved in the RNAi pathway are responsible for these differences in susceptibility. Our data revealed that most facets of the RNAi pathway are well represented across parasitic nematodes, although there were fewer pathway proteins in other nematodes compared to C. elegans. In contrast, the proteins responsible for uptake and spread of dsRNA are not well represented in parasitic nematodes. However, the importance of these differences is undermined by our observation that the protein complements in all the parasites were qualitatively similar, regardless of RNAi-susceptibility. Clearly, differences in the RNAi pathway of parasitic nematodes do not explain the variations in susceptibility to experimental RNAi.Many organisms regulate gene expression through an RNA interference (RNAi) pathway, first characterized in the nematode  RNA interference (RNAi) is a reverse genetics technique which permits the ablation of mRNA by introduction of complementary double-stranded RNA (dsRNA), through cellular mechanisms common to most eukaryotes  in which RNAi is an established experimental technique Ostertagia ostertagi genes Haemonchus contortus genes H. contortus, one feature of successful RNAi appears to be the location of target gene expression, since genes predicted to be expressed in environmentally-exposed tissues are more readily silenced Heligmosomoides polygyrusPristionchus pacificus and Oscheius sp1 CEW1 Caenorhabditis, where C. briggsae (unlike C. elegans) is unable to take up dsRNA from the environment, due to a SID-2 which displays aberrant RNAi functionality Several groups have attempted to apply the RNAi protocols pioneered in in vitro culture systems for parasitic nematodes C. elegansPlasmodium spp. , which are refractory to RNAi due to deficiencies in key pathway components C. elegans RNAi pathway proteins as query sequences, we performed BLAST trawls of nematode-derived genomic and transcriptomic resources. Our searches focused on high-quality sequence datasets, including the draft genomes of Trichinella spiralis , Brugia malayi (Clade III/clade 8), Meloidogyne incognita (Clade IV/clade 12), Meloidogyne hapla (Clade IV/clade 12), Caenorhabditis brenneri, Caenorhabditis briggsae, Caenorhabditis japonica, Caenorhabditis remanei (Clade V/clade 9), Haemonchus contortus (Clade V/clade 9), and Pristionchus pacificus (Clade V/clade 9) as well as the transcriptomes of Oesophagostomum dentatum (Clade V/clade 9) and Ancylostoma caninum (Clade V/clade 9). We find that the RNAi effector complements of these species, whilst quantitatively different are qualitatively similar with regard to the presence of functional groupings, yielding no major inter-species differences except that all were notably less diverse than in Caenorhabditis spp. These data suggest that variable susceptibilities to RNAi amongst parasitic nematodes cannot be adequately explained by differences in RNAi effector complement between such species.Hypotheses to explain RNAi difficulties in parasitic nematodes have been reported, and include: (i) the lack of appropriate C. elegans proteins known to be involved in core aspects of RNAi were identified from literature  and used as search strings in a series of primary translated nucleotide (tBLASTn) and protein BLASTs (BLASTp) www.expasy.ch/tools/dna.html), and analysed for identity and domain structure by BLASTp (through NCBI's Conserved Domain Database service) and InterProScan (www.ebi.ac.uk/Tools/InterProScan). The appropriate reading frame in each case  was then subjected to reciprocal tBLASTn and BLASTp against the C. elegans non-redundant nucleotide and protein databases on the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST), using default settings. The identity of the top-scoring reciprocal BLAST hit was accepted as identity of the relevant primary hit, as long as that identity was also supported by domain structure analysis  and used as reciprocal tBLASTn and BLASTp searches against C. elegans, as before.Seventy-seven terature . These pysis see , S4, S5.M. incognita (http://www.inra.fr/meloidogyne_incognita/genomic_resources) and B. malayi (http://blast.jcvi.org/er-blast/index.cgi?project=bma1) genomes were searched using BLASTp to predicted protein sets, in addition to tBLASTn against available contig assembly, unplaced reads and associated ESTs M. hapla genome was searched using BLASTp against public release 4 (HapPep4: www.hapla.org) of the hand annotated and experimentally-validated M. hapla protein set H. contortus genome was searched using tBLASTn against the supercontig 26/08/09 database (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/h_contortus). A. suum, A. caninum, T. spiralis and O. dentatum primary BLASTp and tBLASTn searches were performed using the datasets generated at Washington University, St Louis  available through GenBank (www.ncbi.nlm.nih.gov), using methods as described above.The C. elegans RNAi pathway (H. contortus (which at the time of searching lacked adequate gene predictions), due to the central importance of this species to the question of RNAi's applicability to animal-parasitic nematodes. While we recognise that we have omitted several other publically-available nematode genome datasets from our analyses, we considered that they did not meet our criteria for inclusion, as described above. Whilst several authors refer to the apparent presence/absence of a few RNAi effector proteins in single nematode species, one small scale study examined the occurrence of 18 such proteins across H. contortus, B. malayi and the flatworm parasite Schistosoma mansoniIn the absence of tractable methods for transgenesis or genetic manipulation, RNAi represents one of the few molecular genetics tools that can currently be applied to parasitic nematodes. However, reports documenting difficulties in the application of RNAi to some nematodes  pathway  proteinsC. elegans RNAi proteins  orthologs of the C. elegans proteins are absent from the species in question; (ii) they have diverged to a degree that is unrecognisable on a primary sequence level, or (iii) our datasets possess significant areas of inadequate coverage such that additional RNAi effector genes await discovery in these species) was observed across all of the functional groupings in our dataset, but was most pronounced within the proteins responsible for uptake/spread of dsRNA. In contrast, the other Caenorhabditid species possessed an RNAi effector complement much closer to that of C. elegans; C. briggsae 65, C. remanei 65, C. brenneri 63, and C. japonica 60 . Much interest has centered on SIDs as core determinants of dsRNA uptake/spreading mechanisms. These transmembrane proteins were first described in C. elegans as mediators of systemic and environmental RNAi due to their role in transmembrane transport of dsRNA Drosophila melanogaster does not possess known SID orthologs, heterologous expression of C. elegans SID-1 sensitizes Drosophila cells to RNAi by soaking C. elegans neurons reverses the neuronal intractability of this species C. elegans SID-2 in C. briggsae, a transformation which confers susceptibility to environmental RNAi in this species C. elegans, it is surprising that these proteins are so poorly conserved in other nematodes, where putative SID-1 orthologs were identified in H. contortus and O. dentatum only (sid-2 was not identified outside the Caenorhabditis genus. Similarly poor conservation was observed with RSD-2 (not identified) and RSD-6 (seen only in P. pacificus). RSD-3 is the sole perfectly conserved spreading protein in our dataset, occurring in all 13 species  and endogenous (miRNA) small RNA pathways, which could indicate that uptake is not limiting for these nematodes , which appears in seven species  In  species . RRF-3 ( species -interactC. elegans possesses at least 27 distinct AGOs (including pseudogenes C06A1.4 and C14B1.7) C. elegans BLAST match) included the miRNA-interacting AGO, ALG-1 (Argonaute [Plant]-Like Gene), as well as several endo-siRNA-interacting AGOs including the 26G-RNA-interacting ALG-4 C. elegans, was only identified in the animal parasitic nematodes A. suum, H. contortus and A. caninum. Thus the AGOs known in C. elegans to be responsible for endogenous regulation of gene expression are well conserved, while the AGOs responsible for executing RNAi triggered by exogenous dsRNA are not. However, as previously stated, our identification strategy does not account for the possibility that other uncharacterized AGOs exist in each nematode species, performing roles comparable to those AGOs which we could not identify. A further four C. elegans AGOs  did not appear to be present within our parasite dataset. The AGO NRDE-3, is responsible for nuclear translocation of RNAi triggers in C. elegans, and is involved in processes which lead to heritability of gene silencing events. As NRDE-3 is completely absent from the parasite datasets considered here, this may indicate that silencing events cannot be passed between generations of parasitic nematodes. Our data suggest that most nematodes have smaller AGO complements than C. elegans, although the impact this has on functional diversity is unknown. The contracted complement of AGOs identified in the parasite species relative to C. elegans is consistent with their propensity for gene loss C. elegans, or conversely a reduced functionality within the parasites considered here. Interestingly, ERGO-1 is involved in the function of endogenous siRNA populations within C. elegansC. elegans. Our analysis reveals that TSN-1 , which is a common component of RISC in C. elegans, Drosophila and mammalian cells C. elegans ortholog of Drosophila VASA intronic gene which regulates transition between larval and adult cellular fates though interaction with the let-7 miRNA In addition to the catalytic AGO protein, RISCs also comprise several protein co-factors, including multiple dsRNA-binding proteins and exonucleases which are thought to pass from elements of the biosynthetic machinery , althougC. elegans, leading to the identification of RNAi-hypersensitive null mutant strains of RRF-3 Caenorhabditis spp.Proteins with RNAi-inhibiting function were first characterized in Caenorhabditis spp.The RNAi pathway affects a number of poorly understood nuclear silencing mechanisms. We found that an uncharacterized nuclear effector, EKL-1 (DOC)Click here for additional data file.Dataset S2Nematode proteins putatively responsible for secondary amplification, uptake and intercellular spread of siRNA; domains and sequence data. (DOC)Click here for additional data file.Dataset S3Components of nematode RNA-induced silencing complex (RISC); domains and sequence data. (DOC)Click here for additional data file.Dataset S4Nematode RNA interference (RNAi) inhibitor proteins; domains and sequence data. (DOC)Click here for additional data file.Dataset S5Nematode chromatin modifiers, histone methylation factors, and other nuclear effectors; domains and sequence data. (DOC)Click here for additional data file.Table S1Distribution of RNAi pathway components in nematode expressed-sequence tag, genome and transcriptome databases. Select tab at page bottom to view RISC-associated proteins, or all other proteins. Key to colours described below. Species were assigned to Clades according to nematode molecular phylogeny as described by (XLS)Click here for additional data file.


O. tsutsugamushi were obtained and established in culture. Phylogenetic analysis based on the 56-kDa type-specific antigen gene demonstrated that the isolates fell into 4 genetic clusters, 3 of which had been previously reported and 1 that represents a new genotype.During a scrub typhus outbreak investigation in Thailand, 4 isolates of  Orientia tsutsugamushi  is transmitted to humans, we genetically typed O. tsutsugamushi obtained from these 26 children and small mammals. The Royal Thai Army Medical Department Ethical Committee approved all procedures (protocol S014q/45). Small mammals were handled according to guidelines in the Guide for the Care and Use of Laboratory Animals .To better characterize the specific strain(s) of O. tsutsugamushi.We obtained clinical information and blood samples from 26 scrub typhus–infected children from Ban Pongyeang after their parents gave informed consent. Blood specimens were stored in liquid nitrogen and shipped on dry ice to the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, for serologic testing, genetic characterization, and isolation of O. tsutsugamushi by using an indirect fluorescence antibody assay . Using PAUP 4.0b10 software and maximum parsimony methods, we generated phylogenetic relationships  were successfully established from EDTA whole blood samples of 7 patients . The specimens were kept in liquid nitrogen and delivered to the Armed Forces Research Institute of Medical Sciences for laboratory testing. Chiggers were removed from captured mammals and stored in 70% ethanol. The chiggers were slide-mounted and identified to species by using a microscope.To investigate Bandicota indica), Savile's bandicoot rats (B. savilei), black rats (Rattus rattus), small white-tooth rats (R. berdmorei), Polynesian rats (R. exulans), Berdmore's ground squirrels (Menetes berdmorei), a common tree shrew (Tupaia glis), and a small Asian mongoose (Herpestes javanicus)  . B. indica and a B. savilei rat had the highest chigger densities. Collected chiggers were classified to 4 species: Leptotrombidium deliense , Gahrliepia  rustica (35.1%), G. (Schoengastiella) ligula (14.6%), and Ascoschoengastia spp. (2.7%) (Forty-five (81.8%) mammals were infested with a total of 2,277 chiggers . A B. in. (2.7%) . O. tsutsugamushi , M. berdmorei ground squirrels (2), and H. javanicus mongoose (1). Two O. tsutsugamushi isolates (PYA5 and PYA6) were established from livers and spleens of 2 B. indica rats  of 51 animals tested were seroreactive to ugamushi . Compareacterium . Becauseica rats . TogetheO. tsutsugamushi obtained from the infected children and small mammals was characterized on the basis of Orientia spp.–specific 56-kDa gene fragments. Multiple alignment and phylogenetic analysis demonstrated that the 4 O. tsutsugamushi isolates from Ban Pongyeang fell into 4 clusters. Sequences for 3 of the isolates clustered with Gilliam, LA, and TA, 3 genotypes that are commonly found in Southeast Asia (B. indica bandicoot rats (isolate PYA5), the most commonly found rats in the village and the small mammals with the highest densities of L. deliense chiggers. These findings indicate possible transmission between animals and humans. Many studies have demonstrated that chiggers can acquire O. tsutsugamushi during the feeding process (O. tsutsugamushi and for feeding vector mites, causing widespread distribution of O. tsutsugamushi in Ban Pongyeang.ast Asia . Most ofast Asia . MoreoveO. tsutsugamushi infection in children and rodent hosts, and it demonstrated the potential for transmission between small mammal reservoirs and humans. Campaigns concerning protection from scrub typhus should be established in areas where O. tsutsugamushi is endemic, and local medical clinics should be made aware of the campaigns. Specific plans for protecting against/preventing O. tsutsugamushi transmission are crucially needed to prevent scrub typhus infection in humans.Investigation of scrub typhus in Ban Pongyeang, northern Thailand, demonstrated Clinical manifestations and laboratory test results for 26 scrub typhus–infected children from Ban Pongyeang, Thailand, who were hospitalized during June 2006–May 2007.


Alternative splicing (AS) of mRNA is a vital mechanism for enhancing genomic complexity in eukaryotes. Spliced isoforms of the same gene can have diverse molecular and biological functions and are often differentially expressed across various tissues, times, and conditions. Thus, AS has important implications in the study of parasitic nematodes with complex life cycles. Transcriptomic datasets are available from many species, but data must be revisited with splice-aware assembly protocols to facilitate the study of AS in helminthes.Caenorhabditis elegans using 454/Roche technology for use as an experimental dataset. Reads were assembled with Newbler software, invoking the cDNA option. Several combinations of parameters were tested and assembled transcripts were verified by comparison with previously reported C. elegans genes and transcript isoforms and with Illumina RNAseq data.We sequenced cDNA from the model worm C. elegans sequences, decreased mis-assembly rates , and improved the coverage of the geneset. The optimized protocol was used to update de novo transcriptome assemblies from nine parasitic nematode species, including important pathogens of humans and domestic animals. Our assemblies indicated AS rates in the range of 20-30%, typically with 2-3 transcripts per AS locus, depending on the species. Transcript isoforms from the nine species were translated and searched for similarity to known proteins and functional domains. Some 21 InterPro domains, including several involved in nucleotide and chromatin binding, were statistically correlated with AS genetic loci. In most cases, the Roche/454 data explored in this study are the only sequences available from the species in question; however, the recently published genome of the human hookworm Necator americanus provided an additional opportunity to validate our results.Thoughtful adjustment of program parameters increased the percentage of assembled transcripts that matched known Our optimized assembly parameters facilitated the first survey of AS among parasitic nematodes. The nine transcriptome assemblies, their protein translations, and basic annotations are available from Nematode.net as a resource for the research community. These should be useful for studies of specific genes and gene families of interest as well as for curating draft genome assemblies as they become available. Caenorhabditis elegans[Alternative splicing (AS) is a post-transcriptional, mRNA modification process that allows a single gene to give rise to multiple protein isoforms ,2. Theses elegans-12, but s elegans-16.de novo into contiguous sequences (contigs) that faithfully represent the full-length transcripts from which they were derived. Graph-based assembly algorithms have been developed to maintain associations between transcripts with shared contigs, making it possible to identify different isoforms of the same gene [de novo cDNA assemblers typically overestimate the number of isoforms associated with a given locus and that many of the predicted isoforms are illegitimate [High throughput cDNA sequencing is the preferred method for detecting and quantifying AS. Today’s most prevalent sequencing protocols  involve fragmentation of nucleic acid molecules, construction of sequencing libraries, and generation of many thousands to millions or even billions of short reads. In the absence of a well-curated genome for comparison, these reads must be re-assembled ame gene . Howevergitimate -21. Carede novo transcriptome assemblies have been reported [Cooperia oncophora and Ostertagia ostertagi[cDNA sequencing is a cost-effective means of gene discovery in non-model organisms, so it often serves as the first line of investigation into an organism’s genetic complement. Thus, the transcriptomes of many parasitic nematodes (often including multiple sexes and life cycle stages) have been sequenced and relevant datasets are readily available . Severalreported -30, but reported ,32. For ostertagi,33.Caenorhabditis elegans to define a set of optimized parameters for high-confidence splice isoform prediction using the Newbler assembler. The optimized protocol was then applied to existing and novel cDNA sequences from a diverse array of parasitic nematodes, including Ancylostoma caninum, C. oncophora, Dictyocaulus viviparus, Necator americanus, Oesophagostomum dentatum, Onchocerca flexusoa, O. ostertagi, Teladorsagia circumcincta, and Trichostrongylis colubriformis[In this study, we used cDNA data from the well-characterized model organism briformis-30 in thC. elegans (Bristol N2) RNA according to previously described methods [One splice-leader SL1) and four oligo(dT) cDNA libraries were constructed from DNase treated  methods . Librari methods , and raw and four methods -30).http://bioinf.comav.upv.es/seq_crumbs/). The remaining reads were aligned to rRNA [Canis lupus famliaris (CanFam3.1) for A. caninum; Bos taurus (Btau4.6.1) for C. oncophora, D. viviparus, and O. ostertagi; Sus scrofa (Sscrofa10.2) for O. dentatum; Ovis aries (Oar3.1) for T. colubriformis and T. circumcincta. O. flexuosa, a parasite of European red deer (Cervus elaphus), and N. americanus, maintained in golden hamsters (Mesocricetus auratus), were screened against Bos taurus (Btau4.6.1) and the GenBank rodent database , respectively, as close substitutes for the unavailable host genomes.Raw reads were edited and filtered prior to assembly. Relevant adapter sequences were removed with Cutadapt , and rea to rRNA ,37 and b to rRNA  sequence to rRNA ) and to  to rRNA ) for conC. elegans Roche/454 reads were mapped to C. elegans coding sequences (WormBase [http://gmt.genome.wustl.edu/gmt-refcov/) and coverage was reported in Additional file Cleaned WormBase  release WormBase ) in ordeC. elegans Roche/454 reads were assembled into isotigs (transcript isoforms) and clustered into distinct isogroups (putative genetic loci) using the Newbler assembler , invoking the cDNA option. Various combinations of parameters were tested (see Table C. elegans coding sequences (CDSs) and coding transcripts (CDS + UTRs) included in WormBase [C. elegans RNA as previously described [http://gmt.genome.wustl.edu/gmt-refcov/), is reported in Additional file Cleaned, decontaminated WormBase  release escribed ). The coC. elegans data . Large or complex datasets were reduced to a manageable size by digital read normalization prior to assembly using khmer with a word size of 31 bp (http://ged.msu.edu/papers/2012-diginorm/). N. americanus isotigs were compared with transcript isoforms reported along with the genome of N. americanus[Parasitic nematode transcriptomes were assembled with parameters that showed the optimal performance on mericanus by BLASTParasite isotigs were searched against the GenBank non-redundant protein database , and non-overlapping top hits with e-value ≤1e-5 were recorded  correction [P values was used to determine which InterPro domains were significantly more often associated with AS isogroups than non-AS isogroups.The number of alternatively spliced and non-alternatively spliced isogroups associated with each InterPro domain was counted  showed ≥50% breadth of coverage. This level of coverage is comparable to the level seen in previous transcriptomic surveys of non-model organisms [C. elegans; studies of this nature have been reported elsewhere [cDNA libraries were generated from mixed stage rganisms ,28,29 anlsewhere .C. elegans sequences  of these isogroups are associated with more than one isotig, with an average of 2.2 isotigs per AS isogroup . In the best version of our transcriptome assembly, 38.07% of the isotigs were matched to 7,027 distinct CDS isoforms from 5,727C. elegans have steadily increased over time . The identification of novel AS isoforms and genes is to be expected given that the reported rates of AS in me Table . TherefoAltogether, 12,265 of the 16,772 isotigs included in our best transcript assembly (73.1%) were verified either by a match to previously reported transcript isoforms included in WormBase or by our orthologous sequencing chemistries. Given the limitations presented by today’s sequencing technologies and assembly software, no combination of parameters will provide a perfect assembly. Some rate of error is to be expected given the challenges presented by complex, dynamic eukaryotic transcriptomes . However, the error rates we detect are lower than those reported in other studies . CleThe Newbler assembler relies on overlap-layout-consensus (OLC) algorithms for read assembly. These OLC algorithms may be less likely to overestimate the number of isoforms associated with a given gene compared to de Bruijn graph assemblers -20,52, bC. oncophora[O. flexuosa[O. ostertagi[T. circumcincta[T. colubriformis[A. caninum[D. viviparus[N. americanus[O. dentatum[Necator americanus, one of the two human hookworms, is thought to infect hundreds of millions of people across the Americas, Sub-Saharan Africa and Asia, and is a leading cause of morbidity in children. A. caninum, the canine hookworm, is an important pathogen in domestic dogs and a model for the study of human hookworm infections. O. flexuosa is a filarial nematode and a close relative of Onchocerca volvulus, the causative agent of African river blindness. O. flexuosa is unique among the Onchocercids in that it is devoid of the bacterial endosymbiont required for development and reproduction in its sister taxa. D. viviparus, the bovine lungworm, is the only Trichostrongylid nematode that resides in the lung during its adulthood. C. oncophora, O. dentatum, O. ostertagi, T. circumcincta, and T. colubriformus are all intestinal worms of livestock animals and are responsible for significant financial losses in the beef, dairy, sheep, goat and pork industries. Projects have been initiated to sequence the genomes of these species , but we expect that this will be possible in future studies aimed at exploring AS profiles of particular species in greater detail.The number of isogroups obtained from each assembly ranged from 15,828 from paration ,54. It iT. colubriformis. The AS rate reported for T. colubriformis (11.68%) was much lower than AS rates reported for other species represented by a single cDNA library derived from mixed-sex adults (24.44% AS in O. flexuosa and 21.14% AS in T. circumcincta). This disparity may be due to decreased transcriptomic complexity in T. colubriformis, but there may be other explanations. In the case of T. colubriformis, material was obtained from an inbred laboratory strain [O. flexuosa and T. circumcinta material were collected in the field [O. flexuosa nodules tend to be dominated by large, adult females [T. circumcinta is a polymorphic species with sex ratios biased towards females [T. colubriformis or specifically studying adults of other species would provide additional data needed to better understand the obtained results.It stands to reason that sampling and sequencing more life cycle stages would lead to increased resolution of AS events. Indeed, including more stages tended to increase the number of isogroups  identified, but overall AS rates and the average number of isotigs associated with each isogroup remained relatively consistent with the notable exception of y strain , while Ohe field ,29. O. f females . Likewis females . This isB. malayi was published in 2007, but the report made no mention of AS [B. malayi WS236) includes multiple isoforms for 16% of the reported genes, but no comprehensive studies on the subject of AS in B. malayi have been reported despite an abundance of representative RNAseq data [N. americanus genome paper was unique in that it included an estimate of AS based on Illumina RNAseq data generated from L3 and adult worms. Multiple isoforms were identified from approximately 25% of the 19,151 predicted protein coding genes. Some 1,209 of the 3,354 AS isogroups from N. americanus match 1,114 genes reported as AS in the genome study (≥90% nucleotide sequence identity over ≥75% of the length of the isotigs in a single high-scoring segment pair) [The assembled sequences from these nine species, as well as their functional annotations and predicted translations are available from Nematode.net  for use on of AS . The mosseq data . The recnt pair) , while aO. ostertagi to 2,212 in O. flexuosa  associated and parasitic nematode species associated with a given InterPro protein domain. In order to reduce false positives resulting from poorly represented domains, InterPro domains represented by fewer than ten isogroups and/or fewer than four species were excluded from enrichment analyses. Red lines indicate cutoff values.Click here for file


Rosa damascena mill L., known as Gole Mohammadi in is one of the most important species of Rosaceae family flowers. R. damascena is an ornamental plant and beside perfuming effect, several pharmacological properties including anti-HIV, antibacterial, antioxidant, antitussive, hypnotic, antidiabetic, and relaxant effect on tracheal chains have been reported for this plant. This article is a comprehensive review on pharmacological effects of R. damascena.R. damascena. Searches also were done by going through the author's files and the bibliographies of all located papers. Online literature searches were performed using Medline, medex, Scopus, and Google Scholar websites backed to 1972 to identify researches about  Rosa damascena mill L, commonly known as Damask rose , because they believe its nice aroma reminds them of prophet Mohammad .R. damascena and the lack of natural and synthetic substitutes, essential rose oil of this plant is one of the most expensive ones in the world markets  for preparing rose water and essential oil , 10. Bec markets . R. damascena has also been used for medicinal purposes  in , is washed yearly by unique and special rose water of Kashan. Rose water is also of high value in the food industry and some special foods are prepared using this product   , 26. Bec markets .Dried flowers Two kinds of dried flowers are produced in . A) Dried bud which is mostly for export. B) Dried petals for different purposes; its major use is for eating, as it can solve problems with digestive system. Some Iranians eat it with yogurt. Another reason for drying petals is to store them when distilleries cannot accept the whole produced flower anymore. They use them later for distillation ,16.HipsR. damascena processed or not processed, are used in Iran  of this plant. In comparison with rose oil, hydrosol and absolute are less expensive. The ethanolic, aqueous, and chloroform extracts are also prepared for research purposes .Chemical compositionR. damascena including terpenes, glycosides, flavonoids, and anthocyanins  found more than 95 macro- and micro-components in the essential oil of R. damascena from the Kashan regions of . Among them, eighteen compounds represented more than 95% of the total oil. The identified compounds were; β-citronellol (14.5-47.5%), nonadecane (10.5-40.5%), geraniol (5.5-18%), and nerol and kaempferol were the major components of the oil (2). Analyses of rose absolute showed that phenyl ethylalcohol (78.38%), citrenellol (9.91%), nonadecane (4.35%) and geraniol (3.71%) ethanol (0.00-13.43%), and heneicosane were the major compounds (35). In another study, the composition of rose was phenyl ethylalcohol (72.73–73.80%), citrenellol (10.62–11.26%), nerol (2.42–2.47%), and geranial (5.58–5.65%) (36). Hydrosol was also found to contain four constituents; geraniol was the major compound (30.74%) followed by citrenellol (29.44%), phenyl ethylalcohol (23.74%), and nerol (16.12%) .Several components were isolated from flowers, petals and hips (seed-pot) of ocyanins -30. Thisocyanins , myrceneocyanins , vitaminocyanins , kaempfeocyanins . Flowersocyanins . LoghmanThe medicinal functions of Rosaceae are partly attributed to their abundance of phenolics compound. Phenolics possess a wide range of pharmacological activities, such as antioxidants, free-radical scavengers, anticancer, anti-inflammatory, antimutagenic, and antidepressant , 38-42.R. damascena are as follows . The effects of this plant on CNS are extensive. Several Pharmacological studies have been performed on R. damascena has been shown to possess a potent depressant activity on CNS in mice  of hypnotic effect of this plant was (were) not clarified. R. damascena contains several components such as flavonoids and terpenes  of this plant were evaluated. It has been shown pentobarbital induced sleeping time increased by these fractions. Among these fractions, the ethyl acetate fraction has the best hypnotic effect. The ethanol crude extract of diazepam . Althougterpenes , 46. Theterpenes ,47. Ther studies , 43,44. eceptors . TherefoThe analgesic effectR. damascena is also reported. In a study, the effect of aqueous, ethanolic and chlorphormic extracts in mice on hot plate and tail flick was evaluated and only ethanolic extract showed analgesic effect  showed neurite outgrowth activity of rose extract (R. damascena significantly induced the neurite outgrowth activity and inhibited the amyloid β (Aβ) ( extract . They fod β (Aβ) . Aβ is td β (Aβ) -35 is mad β (Aβ) -35 caused β (Aβ) -61.R. damascena was isolated which is a very long polyunsaturated fatty acid (VLFA) having molecular formula C37H64O2. This isolated compound protected atrophy induced by Aβ -induced seizure in rats, delays the start of epileptic seizures and decrease the duration of tonic-clonic seizures (stage 4)  ,63. In cstage 4) . R. damascena retarded the development of behavioral seizures in amygdale electrical kindling and possesses the ability to counteract kindling acquisition  of these effects of eceptors . Other cc effect . HoweverR. damascena as an adjunct in treatment of children with refractory seizures were also studied and showed a significant reduction in the mean frequency of seizures in patients using essential oil of the plant. Therefore, the essential oil of R. damascena has beneficial antiepileptic effect in children with refractory seizures  not clarified. However, this effect of R. damascena might be due to its possible tachykinin inhibitory substance(s) content mediating both bronchodilatory and antitussive effects  unknown. This effect may be produced by several different mechanisms. Because the relaxant effect of adrenoceptors on guinea pig airway and bronchodilatory effect of H1 blocking drugs have been shown previously (1) receptors. In fact, the extract and essential oil from R. damascene did not show any significant relaxant effect on incubated tracheal chains with β-adrenergic and H1 receptors antagonists. These results indicated a stimulator effect for this plant on β-adrenoceptors and/or histamine (H1) receptors blocking effect. Based on bronchodilatory effect of calcium channel blockers, an inhibitory effect of this plant on calcium channels of guinea pig tracheal chain also suggested (The mechanism(s) of relaxant effect of eviously -69, we suggested ,66.R. damascena also showed relaxant effect on tracheal smooth muscle of guinea pigs  activity. Because ACE is a key enzyme in production of angiotensin II, R. damascena may be effective to improve the cardiovascular function  showed that essential oil and absolute have strong antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, B. subtilis, Staph. aureus, Chromobacterium violaceum and Erwinia carotovora strains. The C. violaceum was the most sensitive microorganism against rose essential oil and absolute. E. coli was also sensitive against rose essential. However, hydrosol had no antimicrobial activity against any of the microorganisms  and four Methicillin-sensitive Staph. aureus (MSSA) was studied. The results showed that synergism effect between antimicrobial agents and plant extracts was occurred in both sensitive and resistant strains but the magnitude of minimum fold inhibition in resistant strains especially MRSA strain was higher than the sensitive strains , gram-negative E. coli (ATCC 25922), gram-negative Ps. aeruginosa (ATCC 27853), and yeast Candida albicans (ATCC 14053). The tested essential oils exhibited inhibitory and bactericidal activities against all tested microorganisms at low concentrations  was reported ,80. TherAntioxidant effectsR. damascena similar to many aromatic and medicinal plants exhibits antioxidant properties. Sources of natural antioxidant are primarily phenolics compound that are found in all parts of plants such as the fruits, vegetables, seeds, leaves, roots and barks . They determined antioxidant activity of this extract compare to standard antioxidant L-ascorbic acid by 1, 1-diphenyl-2-picryl hydrazyl (DPPH) free-radical method. This study showed that R. damascena has high antioxidant activities  which has antioxidant and anti-inflammatory effects . Because intraperitoneal (i.p.) injection of extract showed symptoms of constipation (no feces in 24 hr), it seems the laxative effects is partly due to osmotic infiltration of fluids into intestinal lumen  consisting of seven medicinal plants namely activity .Anti-aging effectsDrosophila melanogaster was evaluated in a recent study. Supplementing Drosophila with the plant extract resulted in a statistically significant decrease in mortality rate in male and female flies. Moreover, the observed anti-aging effects were not associated with common confounds of anti-aging properties, such as a decrease in fecundity or metabolic rate. Therefore, R. damascena can extend Drosophila life span without affecting physiological mechanisms. This study postulated that the plant’s antioxidant properties could have contributed to prolongation of life span in Drosophila  containing different herbs including R. damascena in patients suffering from various ophthalmic disorders namely, conjunctivitis, conjunctival xerosis (dry eye), acute dacryocystitis, degenerative conditions (pterygium or pinguecula), and postoperative cataract patients was studied. These herbs have been conventionally used in the Ayurvedic system of medicine since time immemorial and reportedly possess anti-infective and anti-inflammatory properties. An improvement was observed after receiving the herbal eye drop treatment in most of the cases. These results showed that herbal eye drop, Ophthacare®, has a useful role in a variety of infective, inflammatory and degenerative ophthalmic disorders  constituents of this plant are mainly responsible for most of the above-mentioned effects.This plant contains several components such as terpenes, glycosides, flavonoids, and anthocyanins that have beneficial effects on human health. The pharmacological effects of


Oligotropha carboxidovorans OM5 T.  is a chemolithoautotrophic bacterium capable of utilizing CO (carbon monoxide) and fixing CO2 (carbon dioxide). We previously published the draft genome of this organism and recently submitted the complete genome sequence to GenBank.Oligotropha carboxidovorans OM5 consists of a 3.74-Mb chromosome and a 133-kb megaplasmid that contains the genes responsible for utilization of carbon monoxide, carbon dioxide, and hydrogen. To our knowledge, this strain is the first one to be sequenced in the genus Oligotropha, the closest fully sequenced relatives being Bradyrhizobium sp. BTAi and USDA110 and Nitrobacter hamburgiensis X14. Analysis of the O. carboxidovorans genome reveals potential links between plasmid-encoded chemolithoautotrophy and chromosomally-encoded lipid metabolism. Comparative analysis of O. carboxidovorans with closely related species revealed differences in metabolic pathways, particularly in carbohydrate and lipid metabolism, as well as transport pathways.The genome sequence of the chemolithoautotrophic bacterium Oligotropha, Bradyrhizobium sp and Nitrobacter hamburgiensis X14 are phylogenetically proximal. Although there is significant conservation of genome organization between the species, there are major differences in many metabolic pathways that reflect the adaptive strategies unique to each species. Oligotropha carboxidovorans  ligase), which is involved in degradation of long chain fatty acids. This gene is present in B. japonicum. Fatty acid methyl ester analysis on strain OM5 indicated an accumulation of some long chain fatty acids when it is grown under different environmental conditions (unpublished results). We believe the absence of these genes in O. carboxidovorans may contribute to accumulation of long chain fatty acids under certain conditions.Compared with O. carboxidovorans is an autotrophic organism that can grow at the expense of inorganic compounds and use CO2 as a carbon source. Its nitrogen comes from inorganic compounds such as NH3, NO3-, or N2. During chemolithoautotrophic growth, it derives energy from oxidation of CO to CO2. This function is encoded on a megaplasmid encoding the enzyme CO dehydrogenase (cox gene cluster). Plasmid-cured mutants (or mutants in which deletions have been introduced into the plasmid) either lose all chemolithoautotrophic growth capabilities or cannot utilize H2 plus CO2 but retain the ability of using CO plus CO2 [plus CO2 ,25,28.N. hamburgensis genome contains genes that are found in carboxidotrophic bacteria. For example, it has several genes encoding cytochrome b561, an important electron transfer component in aerobic carboxidotrophic bacteria [N. hamburgensis genome also has four gene clusters and a lone CDS encoding multiple homologs of molybdopterin-containing carbon monoxide dehydrogenase (Mo-CODH). The largest of these clusters (Nham_2601 to Nham_2608) has high similarity to B. japonicum USDA110 and few other species [N. hamburgensis genome has more copies of Mo-CODH-like genes than it does of nitrite oxidoreductase (membrane bound Mo-dependent enzyme required for converting nitrite to nitrate) [N. hamburgensis. B. japonicum USDA110 also encodes a Mo-CODH, and it can oxidize CO at the expense of nitrate reduction under anaerobic conditions. However, it does not grow under these conditions [O. carboxidovorans in their chemolithoautotrophic lifestyle, they have similar enzyme(s) for this purpose, particularly CODH.The bacteria . The N.  species . In factnitrate) . This sunditions . In stranditions ,31. ThusO. carboxidovorans, the cox genes are specifically and coordinately transcribed under chemolithoautotrophic conditions in the presence of CO [O. carboxidovorans genes encode proteins (CoxC and CoxH) that are putatively associated with CO sensing based on sequence motifs similar to two component regulatory systems [O. carboxidovorans sequence upstream of the cox cluster does not have genes with unambiguous functions in the sensing of CO or the regulation of cox genes. Other species, such as Azotobacter vinelandii and Bradyrhizobium, contain a gene encoding CooA transcriptional receptor that may be involved in CO sensing and regulating the function of CO dehydrogenase [O. carboxidovorans has chromosomally encoded acyl-CoA dehydrogenase genes; some of them are directly involved in fatty acid metabolism . Therefore, it is possible that chromosomally encoded lipid metabolism in O. carboxidovorans and plasmid-encoded CO fixation are linked, but further studies are needed to confirm this.In ce of CO ; therefoce of CO . Two O.  systems . Howeverrogenase . BLASTP O. carboxidovorans genes are responsible for cell motility, binding, and membrane transport. By contrast, 6% of N. hamburgensis genes are in this category. This difference likely reflects their contrasting lifestyles. As an environmental species, O. carboxidovorans likely has increased need for a diversity of these functions, while the symbiotic species N. hamburgensis is in a relatively constant environment.One tenth of O. carboxidovorans codes for 3 Fhu gene(s), OCAR_4564, OCAR_4560, and OCAR_4561, that are periplasmic proteins and may function as an ABC-type transport mechanism driven by ATP hydrolysis. The E. coli fhu system consists of four genes, designated fhuA, fhuC, fhuD and fhuB, that are arranged in one operon [3+-hydroxamate compounds  across the cytoplasmic membrane from the periplasm into the cytoplasm [O. carboxidovorans.Iron transport and related proteins are responsible for specific uptake of ferric-siderophore complexes, which are high affinity iron chelators in gram-negative bacteria. e operon . Periplaytoplasm -36. The ytoplasm ,38 proviRhizobium leguminosarum (a symbiotic nitrogen fixing species) contains a fhuDCB operon for siderophore uptake [Bradyrhizobium also has iron transport systems, but unlike O. carboxidovorans, it does not utilize TonB iron transport. The presence of Fe siderophores and transporter genes in non pathogenic, non-nitrogen fixing O. carboxidovorans reflects its similarity to nitrogen fixing symbiotic bacteria. The presence of these genes in O. carboxidovorans also indicates that its natural environment, like most natural environments, is restricted in iron [Iron acquisition is important in bacteria, both as a requirement for growth and as an environmental signal that regulates gene expression . Iron tre uptake . Bradyrh in iron .N. hamburgensis, O. carboxidovorans and B. japonicum have nickel/peptide transporting genes. Other O. carboxidovorans transport related proteins  are also present in N. hamburgensis and B. japonicum.Unlike O. carboxidovorans megaplasmid revealed mechanisms for its chemolithoautotrophic growth. The complete O. carboxidovorans genome reveals metabolic pathways used during heterotrophic growth and how chemolithoautotrophic growth is linked with central metabolism. Further research is needed to identify genes involved in the regulation of chemolithoautotrophic growth versus heterotrophic growth.The previously reported sequence of the O. carboxidovorans appears to be adapted to a relatively stable microenvironment because its genome contains remarkably few phage- or transposon-related genes in comparison to N. hamburgensis and other soil bacteria. The lack of any typical Gram-negative communication system based on acylated homoserine lactones (AHLs) also suggests an exclusive microhabitat, similar to Azoarcus BH72 [O. carboxidovorans genome is the presence of TonB-dependent receptors for iron transport across the cell membrane, unlike N. hamburgensis and B. japonicum. The role of iron acquisition in O. carboxidovorans metabolism is another very interesting area for future research.cus BH72 . Productcus BH72 . AnotherO. carboxidovorans, N. hamburgensis, and B. japonicum each have unique genetic pathways that enable them to have specialized functions such as CO oxidation, nitrite oxidation, and N2 fixation. These genetic pathways have historically been the most well studied from these species. However, comparison of their genomes reveals that there are many metabolic pathways that are conserved between these closely related species. Comparison of these three genomes also revealed previously unknown differences in the "conserved" metabolic pathways. The cumulative effects of these metabolic differences, along with the unique pathways, allow these phylogenetically related species to adapt to diverse environments [O. carboxidovorans proteins will yield valuable information on how this species survives in its unique environment and how it adapts to both heterotrophic and chemolithoautotrophic lifestyles.ronments . TherefoO. carboxidovorans OM5 (ATCC no. 49405) was grown on carboxydobacterium medium (ATCC no 1789) using acetate as a substrate at 30°C with continuous shaking for 72 h under aerobic conditions. DNA isolation, sequencing, and assembly were conducted as described previously [x Enhancer Solution A (Invitrogen), which enables longer reads on templates containing regions with secondary structure caused by high G+C content, small nucleotide repeats, hairpins, direct repeats, and inverted repeats. Other techniques were used by MWG Biotech (Eurofins) to resolve hard sequencing stops to complete the reads. The complete genome was submitted to NCBI.http://aclame.ulb.ac.be/Tools/Prophinder/, and CGVIEW were used to determine the origin of the circular genome, identify prophages, and to obtain a circular graphical representation of the genome.Once the complete genome was submitted to NCBI, Comprehensive Microbial Resource (CMR) and Integrated Microbial Genomes (IMG) were used to compare OM5 against all of the publicly available, complete prokaryotic genomes. Orifinder, Prophinder http://acO. carboxidovorans genome sequence was submitted to Magnifying Genomes Microbial Genome Annotation System (MaGe) [The m (MaGe) , which bm (MaGe) . Unique O. carboxidovorans-specific genes (genes encoding proteins where no orthologs were detected in the compared species and not in any synteny group) using the two most closely related species with fully sequenced genomes for comparison, Bradyrhizobium sp. (strain USDA110 and BTAi1) and N. hamburgensis X14. Cartographic representations in the MaGe web interface allowed us to confirm the manual annotations and detect specific gene(s).We determined For tree construction, 16 S rRNA sequences were aligned in ClustalX , followehttp://www.genome.jp/kegg-bin/show_organism?org=oca. The KEGG database was also used to trace the enzymes/pathways in Bradyrhizobium USDA110 and N. hamburgensis X14 and compare with strain OM5.For pathway analysis, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY Database, which includes the completed sequence of strain OM5 DP conducted sequencing and closure of the genome as well as annotation and analysis. SMB, SCB, YSD, and MLL conceived and directed the project. SMB and YSD oversaw computational analyses, SCB and MLL oversaw sequencing aspects, and MLL oversaw microbiological aspects. DP wrote the manuscript draft, and all authors edited and approved the final manuscript.O. carboxidovorans OM5, N. hamburgensis X14, and Bradyrhizobium spp. USDA110Orthologous proteins in .Click here for fileO. carboxidovorans OM5 as compared to N. hamburgensis X14, and Bradyrhizobium spp. USDA110Proteins unique to .Click here for fileO. carboxidovorans enzyme complexes involved in some metabolic pathways.Click here for file


Zea mays ssp. mays L.) is an important model for plant basic and applied research. In 2009, the B73 maize genome sequencing made a great step forward, using clone by clone strategy; however, functional annotation and gene classification of the maize genome are still limited. Thus, a well-annotated datasets and informative database will be important for further research discoveries. Signal transduction is a fundamental biological process in living cells, and many protein families participate in this process in sensing, amplifying and responding to various extracellular or internal stimuli. Therefore, it is a good starting point to integrate information on the maize functional genes involved in signal transduction.Maize , which endeavours to identify and classify protein kinases/phosphatases, transcription factors and ubiquitin-proteasome-system related genes in the B73 maize genome. Users can explore gene models, corresponding transcripts and FLcDNAs using the three abovementioned protein hierarchical categories, and visualize them using an AJAX-based genome browser (JBrowse) or Generic Genome Browser (GBrowse). Functional annotations such as GO annotation, protein signatures, protein best-hits in the http://bioinfo.cau.edu.cn/ProFITS.ProFITS, which utilizes both the B73 maize genome and full length cDNA (FLcDNA) datasets, provides users a comprehensive platform of maize annotation with specific focus on the categorization of families involved in the signal transduction process. ProFITS is designed as a user-friendly web interface and it is valuable for experimental researchers. It is freely available now to all users at  Zea mays ssp. mays L.) is an important economic crop, and has served as a model organism for plant genetic research for several decades. The B73 maize genome was sequenced in 2009 [http://www.maizesequence.org[http://www.maizegdb.org/[Maize  of maize, a database which categorizes TFs, protein kinases/phosphatases (PKs/PPs) and ubiquitin-proteasome-system (UPS)-related genes in maize.http://www.maizesequence.org/index.html[cis-element databases, PLACE [The B73 maize genome dataset version 4a.53) which includes gene, transcript and protein sequences were downloaded from MaizeSequence ndex.html. Four mandex.html-8 were ondex.html by the sndex.html was appls, PLACE  and Atcis, PLACE . These ta.53 whicArabidopsis and rice genomes were identified using BLAST .First of all, InterProScan was perfAfter series analyses above of the maize genome and FLcDNA data, we integrated the comprehensive annotation into ProFITS  was done on candidate sequences against PPC classified Arabidopsis PKs/PPs sequences. The candidates were assigned to different PPC groups according to their best hit in the reference. The required InterPro accessions and a modified PPC criterion which intend to gather all the protein phosphorylation related genes in one category can be explored in Additional File As for PKs/PPs, a modified PlantsP kinase Classification/PlantsP Phosphatase Classification (PPC)  is used E-value ≤ 1e-10 and Coverage ≥ 0.5) against protein sequences of these family members in Arabidopsis were implemented for identification.Lastly, we identified UPS-related genes employing same method as in plantsUPS . A grouphttp://www.mysql.com, and the web interface was built by PHP scripts http://www.php.net on Red Hat Linux, powered by an Apache server http://www.apache.org. Server-side scripts were developed using Python http://www.python.org.We constructed and configured ProFITS upon a typical LAMP (Linux + Apache + MySQL + PHP) platform. The dataset was stored in MySQL 5.0 Arabidopsis. The genome dataset is displayed on two levels (gene and transcript levels) as one gene may have one or more corresponding transcripts. In the page for gene level, comprehensive annotations  are provided. In the page for transcript level, more information generated by protein signatures analysis and gene function prediction is displayed because protein sequence is available for each transcript  [Arabidopsis, rice and maize for phylogenetic tree analysis using similar parameters as Hamel et. al. [Completed profiles of  PlantsP  and RKD  PlantsP ; howeverctively) ,23, the  et. al.   repressors are targeted for proteasome degradation, releasing MYC2 and de-repressing transcriptional activation [Jasmonate (JA) is a plant hormone (phytohormone) which participates in multiple developmental processes. The core of the JA-signalling module in  defined . SCFCOI1tivation . We checArabidopsis genes classified in the signal transduction process (GO:0007165), and then explored their annotation of molecular function. Interestingly, among 1,169 genes annotated to have catalytic activities, > 60% have protein kinase activity (725) and about 10% have phosphatase activity (133). Only 0.8% of genes have protein ligase activity; however, this is threefold that of the 0.28% of all annotated with protein ligase activity genes in the Arabidopsis genome, which indicates their important roles in signal transduction processes. Other genes such as receptors, TFs, two-component response regulators and protein phosphatase type 2A regulators are under molecular transducer activity, transcription regulator activity and enzyme regulator activity terms, respectively (see Additional File We collected all 1,230 As ProFITS provides a platform of maize information, its expansibility will be useful when new data is available or a new gene family needs to be categorized.http://bioinfo.cau.edu.cn/ProFITS.ProFITS provides users with a comprehensive profile of genes involved in signal transduction. Sequences of the maize genome and four maize FLcDNA projects are available, making it valuable for experimental researchers. It is freely available now to all users at YL performed protein kinases/phosphatases classification, and compiled the Background and Discussion parts of the manuscript. ZD performed data collection and annotation, the database and web server construction, and compiled the Results part of the manuscript. ZZ provided system support. ZS supervised the project. All authors read and approved the final manuscript.Classification rules in ProFITS. Detailed classification rules including a modified PPC criterion which is intended to gather all the protein-phosphorylation-related genes into one category.Click here for fileSnapshot of JBrowse in ProFITS. In ProFITS, the text annotation and graphical exploration are interrelated to each other.Click here for fileArabidopsis genesGO enrichment analysis on . The hierarchal GO graph of 1230 Arabidopsis genes involved in signal transduction are subjected to GO enrichment analysis using agriGO http://bioinfo.cau.edu.cn/agriGO/. The aspect of molecular function is presented here.Click here for file


Halomonas anticariensis strain FP35T is a moderately halophilic bacterium isolated from a soil sample taken from Fuente de Piedra, a saline wetland in the province of Málaga (Spain), which produces an exopolysaccharide and quorum-sensing signaling molecules of the type N-acylhomoserine lactone. We report here the draft genome sequence of this gammaproteobacterium. Halomonas anticariensis FP35T was isolated from saline soil at Fuente de Piedra, Málaga, in southern Spain  systems  sequencing technology. The raw data obtained were subjected to treatment, including several steps of data filtering. Then, we performed de novo assembly using SOAPdenovo version 1.05 (www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html) was employed for gene annotation. The tRNA and rRNA genes were predicted with tRNAscan-SE (T contains a total of 61 tRNA genes and one rRNA operon.Little genome data are currently available from this group of bacteria. In this study, we report the draft genome sequence of ion 1.05 , generatAscan-SE  and RNAmAscan-SE , respectThe draft genome information reported in the present study might be valuable for future studies to investigate the role of the quorum-sensing regulatory system in the adaptation to high salinities in hypersaline environments.H. anticariensis strain FP35T whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. ASTJ00000000. The version described in this paper is the first version, accession no. ASTJ01000000.The


The Greek coastal waters are subjected to harmful algal bloom (HAB) phenomena due to the occurrence of species characterized as toxic (TX), potentially toxic (PT), and non-toxic, high biomass (HB) producers causing harm at multiple levels. The total number of (TX), (PT) and (HB) algae reported in this work are 61, but only 16 species have been associated with the occurrence of important HABs causing damage in the marine biota and the water quality. These phenomena are sporadic in time, space and recurrence of the causative species, and are related to the anthropogenically-induced eutrophication conditions prevailing in the investigated areas. The coastline 18,000 km) of the Greek mainland is located in the Eastern Mediterranean Sea, it is surrounded by the Aegean, Ionian and Cretan Seas and its morphological regime shows a variety of gulfs and semi-enclosed gulfs. All these basins are eutrophic [8,000 km Eutrophication triggers various physical and chemical changes in the marine environment and exerts a pressure on algal populations, allowing the intensive growth of certain harmful-toxin producing species or nuisance blooms that may create problems in the structure of the ecosystem and public health. These blooms are collectively called Harmful Algal Blooms (HABs). The greatest number of toxic species is found among dinoflagellates, but evidence has been provided for several species of other taxa  suggesting that they belong in this category ,3,4. Concern about harmful algae in Greek coastal waters has been growing since the late 1970s, when the first symptoms of “fish kills” due to the increased anthropogenic effects led to the fact that HABs-often quoted as the phenomenon of red tides-acquired the attention of scientists and the public. Since then, routine records of phytoplankton samples from almost all major gulfs along the Greek coastline during the last 30 years have revealed the presence of toxic and potentially toxic algae  and non-toxic, high biomass producing species , although their destructive effects were occasional.The European Commission has funded a number of projects such as EUROHAB  to generate the required research to better manage the effects of toxic/harmful marine microalgae that have caused problems in European marine waters . This paThe investigated area  is locatThe collection of data covers the period 1977-2008. The methodology of sampling, preservation of samples, quantitative-qualitative analysis and the toxicity detection/evaluation of each one of the phytoplanktonic species under investigation are given in the literature cited in A traditional system has been adopted for the eukaryotic species taxonomy . CyanobaAmphora coffeaeformis (from Canada) was found to produce Domoic acid. Other strains examined so far were non-toxic. However, the precise identification of the Canadian strain has been questioned [Family Catenulaceae. A strain of the species estioned ,9.It is estioned .Family Bacillariaceae. The five species of this family are Domoic acid producers: Pseudo-nitzschia delicatissima , Pseudo-There are five important orders of Dinophyceae identified and presented in this work: Peridiniales, Prorocentrales, Dinophysiales, Gymnodiniales, and Noctilucales.Alexandrium  and one of the genus Gambierdiscus that are among the well known harmful algae. A. catenella is a producer of c1-c4 toxins, Saxitoxins and Gonyautoxins [A. tamarense, A. minutum and A. taylori produce Gonyautoxins [A. balechii and A. insuetum have been characterized in the literature as species of unknown toxicity, but they have been associated with harmful algal blooms [Gambierdiscus sp. is known to be toxic producing Ciguatoxin and Maitotoxine [Family Goniodomataceae. This family comprises six species of the genus autoxins ,17. A. tautoxins ,19. A. bl blooms ,20 and Gtotoxine .Coolia monotis produces Cooliatoxin, an analog of Yessotoxin [Family Ostreophidaceae. ssotoxin . Heterocapsa circularisquama produces the photosensitizing hemolytic toxins H2-a, H3-a [Family Heterocapsaceae. -a, H3-a . Ostreopsis are O. ovata producing putative Palytoxin and Ovatoxin compounds and O. siamensis, putative Palytoxin [Family Ostreopsidaceae. The two toxic species of the genus alytoxin ,25,26.Protoceratium reticulatum is a species known as a Yessotoxin toxin producer [Scrippsiella trochoidea is a bloom forming species of unknown toxicity [Family Gonyaulacaceae. producer . Scrippstoxicity .Diplopsalis lenticula, a bloom forming species [Protoperidinium crassipes, producingAzaspiracid toxins [Family Protoperidiniaceae. Two species of this family have been recorded,  species  of unknod toxins .Peridinium quinquecorne is a bloom forming species [Family Peridiniaceae.  species .Ceratium, C. furca, C. fusus, C. lineatum, and C. tripos occasionally form non-toxic blooms [Family Ceratiaceae. The four species of the genus c blooms  that mayc blooms ,34,35,36Prorocentrum. The four toxic species are: P. borbonicum, producing Borbotoxins [P. levis and P. lima, producing Okadaic acid and Dinophysistoxins [P. rhathymum [P. arcuatum [P. obtusidens [P. redfeldii [P. micans [P. minimum [P. dentatum [P. emarginatum [Family Prorocentraceae. All species of this family are in the genus botoxins , P. leviistoxins ,39, and hathymum . Speciesarcuatum , P. obtutusidens , P. redfedfeldii , P. mica. micans , P. mini minimum , P. dentdentatum  and P. erginatum . Dinophysis are toxic. D. sacculus produces Okadaic acid [D. tripos and D. rotundata, Dynophysistoxin [D. acuminata and D. acuta, Okadaic acid/Dynophysistoxin [D. fortii, Okadaic acid/Dynophysistoxin/Palytoxin [D. caudata, Okadaic acid/Palytoxin [Family Dinophysiaceae. All species of this family representing the genus aic acid ; D. tripsistoxin ,49; D. asistoxin ; D. fortalytoxin ; D. caudalytoxin . Gyrodinium impudicum, a non-toxic, bloom forming species [Amphidinium carterae, is a producer of the Maitotoxin [G. aureolum produces 1-acyl-3-digalactosylglycerol and octadecapentaenoic acid [Karenia, K.brevis produces Polyether Neurotoxins called Brevetoxins [K. mikimotoi Gymnocin-A [Gymnodinium catenatum produces Gonyautoxins and Saxitoxin [Karlodinium veneficum, Karlotoxins [Family Gymnodiniaceae. All species of this family are toxic except  species . Amphidiitotoxin  and G. aoic acid . One of vetoxins  and K. mmnocin-A . The speaxitoxin , whereaslotoxins . Noctiluca scintillans is the single species of this family. It is a non-toxic bloom forming species [Family Noctilucaceae.  species  responsiPhaeocystis puchetii: toxic species producing polyunsaturated aldehyde [Family Phaeocystaceae. aldehyde .Prymnesium parvum: toxic species producing Prymnesins [Family Prymnesiaceae. ymnesins .Order ChattonellallesChattonella globosa and C.verucolosa are unknown toxicity high biomass forming species [Family Chattonellaceae. Both species of this family  species . Microcystis aeruginosa produces the toxin Microcystin-LR [Chroococcus gelatinosus and Synechocystis sallensis are bloom forming species [Family Chroococaceae. The species ystin-LR , and the species .Lyngbya agardhii is a high biomass forming species [ Trichodesmium erythraeum produces Saxitoxin [Family Oscillatoriaceae.  species  and Tricaxitoxin .Pseudonitzschia seriata, a domoic acid producer, caused [Dinophysis sacculus, an okadaic acid producer, caused [Microcystis aeruginosa,a microcystin-LR producer, caused [In the present article  we nomin, caused  amnesic , caused  diarrhet, caused  extensivAlexandrium minutum, whose presence did not produce toxic symptoms in the Greek coastal waters since their nutritional status did not favor blooms of this species [Potentially toxic (PT) are characterized as species carrying the toxigenic potential according to toxicological analyses, but their presence in an area has not been accompanied by toxic blooms and the relevant symptoms. A noticeable example is the toxic dinoflagellate (GTX1-4)  species .Noctiluca scintillans (late winter-early spring), Chatonella globosa (spring)and several species of the genus Prorocentrum in autumn  caused severe water discoloration in Thermakos Gulf during the years 2000-2004 [Certain non-toxic species create high biomass (HB) blooms that have been characterized as harmful, because their occurrence produces discoloration of the water, undesirable aesthetic symptoms and anoxic harmful conditions to the ecosystem. They also cause severe economic problems due to loss to fisheries and tourism operations . Massive000-2004 .Dinophysis acuminata, Gyrodinium aureolum, Karenia brevis), seven species are forming high biomass (HB) harmful blooms and the rest (36) are potentially toxic species. Dinoflagellates are referred [The total numbers of (TX), (PT) and (HB) algae reported in this work are 61 species. Dinoflagellates included 46 species contributing the 75% of total number . Among treferred  as the gDiatoms were represented by only six species-all potentially toxic-and this might be attributed to their nutrition requirements for a well balanced ratio (N:P:Si) of all nutrients. This necessity makes them poorer competitors than the non-siliceous dinoflagellates that seem to have a competitive advantage over diatoms if the stoichiometry of nutrients is deviated from its normal status in seawater . Another advantage of dinoflagellates over diatoms is their nutritional mode, since several dinoflagellates are not exclusively phototrophic but heterotrophic/mixotrophic because they can shift to osmotrophy (uptake of dissolved organic substances) and/or phagotrophy (feeding on particulate organic carbon) under changes in nutrient supply ratios  and light-depleted conditions .Ceratium furca, Dinophysis acuminata, Gymnodinium catenatum, Gyrodinium impudicum, Noctiluca scintillans, Prorocentrummicans, Protoperidinium crassipes, Scrippsiella trochoidea, Prymnesium parvum) have been reported as phagotrophic, having the ability to feed on prokaryote prey  and/or eukaryote algae . However, the prey of phagotrophic Gambierdiscus sp., Ostreopsis ovata, O. siamensis, is unknown. For species supplementing their nutrition with osmotrophy (Alexandriumcatenella) or osmotrophy and phagotrophy , urea proved to be an important nitrogen source, with the exception of the cyanobacterium Microcystis aeruginosa, which may utilize leukine.The 19 identified mixotrophic species in this investigation  includedAmong the 61 species presented in 7 cells.L−1) of Karenia brevis (Gymnodinium breve) were recorded only in the Saronikos Gulf, three times  with massive fish kill. Outbreaks of Dinophysis acuminata (up to 8.5 × 104 cells.L−1) were recorded only in the Thermaikos Gulf in January 2000, April 2001, February 2002, March 2003 and May 2004, and they were associated with extensive shellfish deaths. However, this species was also observed in the Amvrakikos and Malliakos Gulfs at several times in low abundances and without toxic symptoms. The huge growth (5.4 × 106cells.L−1) of Noctiluca scintillans caused water discoloration in late winter-early spring occasionally during 2000-2004 in Thermaikos and in Kavala Gulfs. The outbursts of four species of the genus Prorocentrum were also associated with water discoloration. P. obtusidens, P. redfeldii and P. micans occurred in the Thermaikos Gulf during the winter 2000-2001 at abundances up to 6.0 × 106 cells.L−1 and P. minimum was recorded (up to 1.2 × 105 cells.L−1) in April 2003 along the N. Aegean coastal line and in the Saronikos Gulf, and in autumn 2003 in the Amvrakikos Gulf. However, the presence of P. minimum in the Kalloni Gulf did not cause any undesirable incidents [5-2.5 × 106 cells.L−1) of Alexandrium insuetum caused water discoloration in the Amvrakikos Gulf in the spring of 2003 and 2004, but in Kalloni Gulf did not create harmful effects [The present data demonstrate that HAB episodes in Greek coastal waters are sporadic in time, space and recurrence of the causative species. Blooms (up to 5.0 × 10ncidents . Mass oc effects .Trichodesmium erythraeum (7.1 × 104 trichomes.L−1) produced a serious harmful bloom in the Evoikos Gulf. The sea surface was covered by mucus-forming “blankets” and “marine snow” transported horizontally and vertically and causing problems to recreation, public health and fish harvesting. The two Rhaphidophyte species of Chattonella were also involved in severe HAB phenomena. The species C. globosa grew massively (>104 cells.L−1), causing water discoloration during spring 2001-2003 in the Thermaikos Gulf, whereas considerable growth of C. veruculosa caused finfish mortality in the Amvrakikos Gulf in December 1998. The Prymnesiophyte Phaeocystis pouchetii, growing at concentrations up to 3.5 × 107 cells.L−1, caused water discoloration in the Saronikos Gulf  and “mucilage” problems in the Evoikos Gulf (September 1999). In September 1999, the co-occurrence of five species of the cyanophyceae, Microcystis aeruginosa (9.9 × 105 cells.L−1), Lyngbya agardhii (4.8 × 103 filaments.L−1), Chroococcus gelatinosus (8.2 × 105 cells.L−1), Synechocystis sallensis (8.9 × 104 cells.L−1) and −3) in relation to the values (<<0.5 mg chlα. m−3) prevailing in the oligotrophic open oceanic waters [From the ecological point of view, most (TX), (PT) and (HB) algae  are “norc waters . The infc waters .Alexandrium insuetum, A. tamarense, Gymnodinium catenatum, Gyrodinium aureolum, Coolia monotis, Ostreopsis ovata and O. siamensis are not indigenous, but alien species of the Mediterranean Sea. They have been introduced via ship traffic for the Atlantic, Pacific and Indian Oceans [It is interesting to notice that the species n Oceans  and it in Oceans .The available data indicate that 61 identified HAB species  have spread across the Greek coastline during the last 30 years. Among these, certain algae (16) were associated with the occurrence of important HAB incidents causing damage in the marine biota and the water quality. There is a strong indication that these incidents were eutrophication-induced phenomena, but sporadic in time, space and recurrence of the causative species.


Anaplasma spp., Babesia spp., Theileria spp., and Ehrlichia ruminantium, was for the first time studied in Bié Province, central Angola. We examined 76 blood samples of cattle originated from seven farms, and 13 blood samples of goats from two farms employing molecular genetic tools (PCR). Most prevalent was A. ovis-infection in goats (100%) and A. marginale-infection in cattle . B. bigemina-infection was detected in only one specimen at Andulo, whereas B. bovis was not detected in Bié. We did not detected T. parva, the causative agent of serious diseases in cattle; nevertheless, infection by T. velifera was quite frequent . Causative agent of heartwater disease – E. ruminantium, was not detected. Taking into account short-term perspective of PCR methods in monitoring of epidemiological status in herds, the number of infected animals and distribution of detected pathogens should not be ignored.Distribution of  Altogether 76 cattle blood samples were obtained by puncture of ventral coccygeal vein at seven selected farms operating by mixed farming system – namely in Andulo (n = 6), Cunhinga (n = 4), Kuito (n = 4), Kukema (n = 11), Kunje (n = 11), Lioema (n = 21), and Vila Graça (n = 19) . All indBabesia/Theileria spp., Ehrlichia ruminantium, and Anaplasma marginale/ovis, following modified protocols published by (Babesia/Theileria spp.), 1 μl of PCR product from the first round of amplification was used instead of DNA template.DNA was isolated from ethanol preserved blood using NucleoSpin Tissue kit  following the manufacturer’s protocol, and then stored at - 20 °C until further analysis. Polymerase chain reactions were conducted to amplify specific DNA fragments of Babesia/Theileria spp., E. ruminantium, and A. marginale/ ovis, respectively. Final extension step lasted eight minutes at 72° C for Babesia/Theileria spp., ten minutes at 72 °C for E. ruminantium, and seven minutes at 68 °C in case of A. marginale/ovis. All PCR products were subjected to 1.2 % agarose gel with ethidium bromide and visualized under UV light.The sequences of the primers and PCR conditions used in the study are presented in Representative part of positive PCR products were selected, purified after gel electrophoresis using Gel/ PCR DNA Fragments Extraction Kit  and sequenced . Similarity of the obtained sequences was compared with sequences available at GenBank database using BLAST  algorithm (http://blast.ncbi.nlm. nih.gov/) and MEGA program .All 13 goats’ samples were positive only for the presence of partial sequence of  protein . AltogetA. marginale, and Babesia/Theileria spp. in bovine blood samples , Italy (AY702922) and Zimbabwe (AY666011). A. marginale was detected at six out of seven farms, with prevalence ranging between 16 % and 55 %. Sequencing of 12 amplified 18S rRNA fragments of Babesia/Theileria spp. and their following analysis employing MEGA software showed that one sample from Andulo displayed the highest similarity (99 %) with B. bigemina isolate originated from Kenya (accession number EF458200). All 11 remaining sequences revealed the highest similarity (99 %) with Theileria velifera strain from Tanzania (AF097993). The prevalence of Theileria sp. within particular herds ranged between 9 % and 33 %, whereas samples of cattle from Kunhinga (n = 4) and Kuito (n = 4) was PCR negative. In total, we detected six animals being infected by both A. marginale and T. velifera. We did not find any E. ruminantium positive sample. The results are summarized in We detected gene segments of  samples . The mspA. ovis (100 %) in goats, and A. marginale (38 %) in cattle. Such a high prevalence could be explained by the easy transmission routes, when species of the genus Anaplasma can be transmitted by ticks, but mechanical transmission mediated by biting flies and vertical transmission can play a role as well  decoloratus were recorded also from Angola , January disease, and Corridor disease. Whereas Rhipicephalus appendiculatus, the vector of ECF, is distributed in eastern Africa, R. duttoni and R. zambeziensis, both important vectors of Corridor disease, occur naturally also in Angola . Moreover, earlier reports on clinical cases of Corridor disease in Angola are available  decoloratus and R. evertsi  microplus (the main vector of B. bovis) in Angola, as published by previous authors (e.g.B. bigemina, 87 % prevalence of Anaplasma sp., and absence of B. bovis among studied animals. Despite the fact that we found only 1 % prevalence of B. bigemina, due to the time-limited nature of parasitaemia leading to relatively smaller chance to detect Babesia by PCR method, this result should not be overlooked.Ehrlichia ruminantium, the causative agent of heartwater disease, was not detected among our samples, despite the fact that Amblyomma pomposum and A. variegatum, both considered as competent heartwater vectors, were reported from Angola as a frequent tick species infesting cattle (E. ruminantium in Angola.g cattle . RegardiA. marginale and Theileria sp. in six animals. As confirmed by many previous authors, multi-infections in both wild and domestic ruminants of sub-Saharan Africa, are frequent (e.g.Notably, despite the presence of four above mentioned blood parasites in Central Angola, all cattle and goats were without symptoms of disease. Asymptomatic development of infections could testify their endemic stability in the area. Such a state is defined as relationships between host, vector, and pathogen in environment, when clinical cases or symptoms of disease absent, or occur rarely (PCR methods give us short-term perspective in monitoring of epidemiological situation calling for employing serological methods. To clarify recent epidemiological threats and to reduce the economic losses in cattle farming in Angola, we suppose as basic the following measures - extensive epidemiological survey employing serology together with molecular genetic methods, monitoring of distribution of tick vectors, availability of vaccination programs, and tracking of animal transports.


Turnera aphrodisiaca aerial and its segregated parts  so that plant can be standardized on the basis of its bioactive marker. The apigenin content in methanol extract of T. aphrodisiaca aerial parts was found to be about fourteen times less than acid hydrolyzed methanol extract of the plant indicating the presence of most of apigenin in glycosidic form. Amongst different plant parts, flowers possessed maximum content of apigenin followed by leaves. The apigenin content was also determined in three marketed formulations of T. aphrodisiaca viz., NLK, DWSG and SBL. DWSG contained higher content of apigenin. Aerial parts of the plant were collected at bimonthly intervals over a period of one year in the months of January, March, May, July, September and November. The plant material collected in September showed maximum content of apigenin.An HPTLC densitometric method has been developed to estimate apigenin in  Turnera aphrodisiaca Ward  is commonly known as Damiana. The leaves of T. aphrodisiaca have been used traditionally as a stimulant, aphrodisiac, tonic, diuretic, nerve tonic, laxative, and in kidney, menstrual and pregnancy disordersT. aphrodisiaca indicate that the plant contains tetraphyllin B (cyanoglycoside)T. aphrodisiaca revealed sporadic pharmacological reports on the plant. Aguilara et al.T. aphrodisiaca leaves possesses significant hypoglycaemic activity in rabbits upon oral administration. Aqueous extract of the plant was reported to exhibit aphrodisiac activity in sexually sluggish male rats at a dose of 1 ml/kgT. aphrodisiaca aerial parts, only methanol extract  exhibited significant anti-anxiety activity on elevated plus maze apparatusT. aphrodisiaca aerial parts using bioactivity-guided fractionationT. aphrodisiaca aerial and its segregated parts viz., leaves, stems, flowers and fruits so that plant can be standardized on the basis of its bioactive marker.A survey of literature on T. aphrodisiaca whole plant was procured from a cultivated source, Rati Ram Nursery, Village Khurrampur, district Saharanpur (UP) in the month of August 2002, and dried in shade. Identity of the plant was confirmed through Botanical Survey of India, Howrah. A voucher specimen (No. 314) of the plant has been deposited in the Herbarium-cum-Museum of the University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh. The mother tinctures from three different manufacturers viz., National Laboratory, Kolkata, India , DHU-Arzneimittel, Germany  and SBL Private Limited, Sahibabad, Ghaziabad, India  were procured from the local market.The HPTLC system consists of Linomat-IV applicator and Camag TLC Scanner – III with CATS 4 software. TLC plates were developed in TLC chamber (Merck) saturated (10 min) with toluene:ethyl acetate (1:4) as the mobile phase. The development length was 50 mm. The plates were dried and scanned at 336 nm. Stock solution of apigenin (1.25 mg/10 ml) was prepared in methanol. From this stock solution, four working standard solutions were obtained by appropriate dilution with methanol. The concentrations of working standard solutions were 1, 0.75, 0.5 or 0.25 mg/10 ml.T. aphrodisiaca (1 g), packed in a filter paper sachet, were defatted by refluxing in 250 ml round bottom flask on boiling water bath with 3×50 ml quantity of petroleum ether (1 h each). The marc obtained was air dried and refluxed under similar conditions with 3×50 ml quantity of methanol. The methanol extracts were pooled, filtered and concentrated under reduced pressure. Dried methanol extract was reconstituted in methanol, in a volumetric flask, and its volume was made up to 10 ml.Dried powdered aerial parts of T. aphrodisiaca aerial parts was prepared. Dried methanol extract, thus obtained, was heated in a 50 ml round bottom flask with 6% aqueous hydrochloric acid  for 45 min on water bath in order to hydrolyze flavonoid O-glycosidesAcid hydrolysis was performed following the procedure mentioned above, methanol extract of T. aphrodisiaca (30 ml each) from NLK, SBL and DWSG were concentrated under reduced pressure, and dried extracts were acid hydrolyzed as per the procedure described above. The final volume was made up to 10 ml (NLK) and 50 ml (SBL and DWSG) with methanol. Aerial parts of T. aphrodisiaca (1 g each), collected in different seasons , were also extracted separately as per the procedure optimized, followed by acid hydrolysis described above. The final volume for each sample was made up to 25 ml with methanol.Segregated aerial parts viz., leaves, stems, flowers and fruits (1 g each) were extracted as per the procedure optimized, followed by acid hydrolysis described above. The final volume was made up to 25 ml (leaves), 10 ml (stems), 25 ml (flowers) and 10 ml (fruits) with methanol. Mother tinctures of T. aphrodisiaca. A standard graph was plotted against mean area under the peak and apigenin amount (μg). Aliquots (2 μl) of test samples were applied, in triplicate, to the TLC plates. T. aphrodisiaca. The area under the curve of every sample was recorded, and apigenin content was determined from the regression equation of the standard graph.Two microlitres each of the stock solution and the four working standard solutions of apigenin were applied, in triplicate, on 20×20 cm TLC plates. The developed plates were scanned at 336 nm using HPTLC densitometer. T. aphrodisiaca aerial parts in three separate flasks. The extraction was carried out as per the procedure mentioned earlier. The resulting solutions were concentrated under reduced pressure, finally making up their volume to 50 ml. A 2 μl sample of every solution was subjected to HPTLC for the estimation of apigenin. The estimated quantity of apigenin was expressed as % w/w. The recovery graph was obtained by plotting % w/w-added versus % w/w-determined  were added to 1 g of termined . RecoverT. aphrodisiaca. The content of apigenin in the marketed formulations of the plant is shown in T. aphrodisiaca aerial parts, collected in different seasons, is shown in T. aphrodisiaca has long history of use in CNS disorders. Authors have isolated anxiolytic flavone apigenin from the plant using bioactivity-guided fractionation. In this work, apigenin content was determined in T. aphrodisiaca aerial and its segregated parts by HPTLC densitometry. Linearity of the calibration curve was achieved between 0.05 and 0.25 μg for apigenin (r2 = 0.998). Apigenin content in the hydrolyzed methanol extract of T. aphrodisiaca aerial parts was found to be about 14 times more than direct methanol extract (O-glycosidic form in T. aphrodisiaca aerial parts. Amongst different plant parts, flowers possessed maximum content of apigenin followed by leaves. DWSG possessed maximum content followed by SBL and NLK (T. aphrodisiaca, i.e., apigenin, NLK showed maximum anxiolytic activity at lower dose extract . From th and NLK . ContentT. aphrodisiaca was also observed (T. aphrodisiaca was validated by adding known quantities of reference apigenin to the test samples, and determining its content (The effect of season on content of apigenin in observed . Further content ;Table 4.