Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question.MicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (pi)RNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes.The small RNAs from larval and adult stages of two parasitic species, This in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention. The processing of miRNAs from long primary transcripts to their mature functional form by Drosha and Dicer RNase III enzymes has been reviewed recently . The matmiRNAs show temporally controlled and cell-specific patterns of expression -8, with Caenorhabditis elegans led to the discovery of the first miRNA, lin-4, which controls hypodermal cell fate decisions during early larval development [C. elegans let-7 and the related miRNAs, mir-48, mir-84 and mir-241, also function to regulate the timing of developmental events [C. elegans lys-6 and mir-273, which act sequentially to control the laterality of chemosensory neurons [C. elegans miRNAs, either individually [mir-35-mir-42 [mir-51-mir-56, [mir-58, -80, -81 and -82 [C. elegans mir-1, which regulates synaptic signalling at neuromuscular junctions and influences sensitivity to levamisole [Genetic analysis in the free-living nematode elopment ,18. Simil events ,20. A ce neurons ,22. Howevidually  or by co-mir-56, , as well and -82 . AdditioC. elegans. Whether development of parasitic species is regulated by microRNAs, as occurs in C. elegans, is currently unknown. As an essential first step to address this question we have undertaken a comprehensive analysis of miRNAs in two parasitic nematodes: Brugia pahangi, a mosquito-borne parasite closely related to B. malayi and Wuchereria bancrofti, which are the causative agents of lymphatic filariasis, a condition affecting around 120 million people in 73 countries worldwide [Haemonchus contortus, a prevalent blood-feeding gastrointestinal parasite of ruminants causing substantial losses to livestock production. H. contortus is relatively closely related to C. elegans, with both belonging to nematode clade V, while Brugia spp. (clade III) are more distantly related [B. malayi [H. contortus [B. pahangi and H. contortus and compare the repertoire of miRNAs to those in well-studied species, particularly C. elegans. Surprisingly, despite the close phylogenetic relationship, particularly between C. elegans and H. contortus, we find that the majority of miRNAs from the parasitic nematodes are novel, perhaps reflecting adaptations to parasitism.Parasitic nematodes of medical and veterinary importance have body plans and developmental programs similar to orldwide ,29; and  related . Importa. malayi  and H. contortus , allowinH. contortus provides an in-depth picture of the small RNA silencing pathways operating in gene regulation and genome stability in these organisms. Identification of these molecules will facilitate detailed study of their function and may ultimately provide novel targets for parasitic nematode control.miRNAs are members of a larger group of small silencing non-coding RNAs, including endogenous small-interfering (endo-si)RNAs and Piwi-interacting (pi)RNAs, which are classified on the basis of their biogenesis and the proteins with which they associate in the RISC complex ,33. HereB. pahangi is a sister species to the human-infective filarial nematode B. malayi and shows a high level of conservation of gene sequences, including 18S rRNAs [Wolbachia endosymbionts [B. pahangi is maintained in our laboratory, allowing access to all developmental stages. To enable identification of small RNAs we exploited the almost complete and annotated genome sequence data for B. malayi [B. pahangi sequences to this genome. Sequences identified in this manner were considered common to B. pahangi and B. malayi and, as such, are referred to as Brugia small RNAs. The B. malayi genome data represents 88 Mb of assembled sequence, mostly in scaffolds , with an estimated genome size of 90-95 Mb [H. contortus genome sequencing project is ongoing with 900 Mb of sequence data estimated to represent 2-3 fold genome coverage . The majority of sequences have been assembled into contigs  and supercontigs  with annotation presently unpublished. In this study we carried out Illumina deep sequencing [B. pahangi and H. contortus which produced between 5.2 and 13.2 million raw reads . The miRNA contribution was lower in both H. contortus libraries (25-33%), which was a consequence of a higher percentage of rRNA sequences. Variable levels of rRNA sequences in small RNA libraries have been described previously [Computational approaches were then used to classify sequences as miRNA, siRNA or piRNA and identify reads derived from rRNAs and tRNAs. The proportion of reads assigned to each RNA class for the L3 and adult stage libraries of both species is shown in Figure eviously ,42.Two miRNA prediction programs, miRDeep  and MIREB. pahangi output for both programs with the 32 known sequences identified previously for B. malayi [let-7, were found only by MIREAP. Therefore, to reduce the likelihood of failing to identify genuine miRNAs, the prediction outputs from both programs  were combined. These combined output data was subsequently processed to identify identical, or near identical, sequences mapping to different genomic locations , read counts and a further hairpin classifier, CIDmiRNA [B. pahangi, this produced 125 precursor sequences representing 123 miRNA loci, encoding 99 unique mature sequences, and 81 unique star sequences. For H. contortus, the same process identified 180 precursor sequences representing 175 loci, encoding 180 unique mature sequences and 128 unique star sequences (Table HE617336-HE617661) and to miRBase, the miRNA database. The final numbers of precursors identified by each program or combinations of programs are shown in Additional File B. pahangi and 52% for H. contortus). We also find evidence for a small number of additional miRNAs in the unmapped sequence data  were used as BLAST [B. malayi and H. contortus genome sequences. The flanking genomic regions from BLAST hits were excised in two orientations and these candidate precursor sequences passed through RNAfold [Brugia and 25 H. contortus miRNAs that we had already found by deep-sequencing, this bioinformatics approach identified an additional 42 Brugia and 78 H. contortus sequences. Final high confidence sets of five Brugia and 12 H. contortus sequences , or represent more accurate annotation due to the depth of our sequencing data.A comparison of the eviously  showed t archive  with theB. pahangi and H. contortus, after normalisation of the data to the total number of reads mapping to the genome for each library. 22 B. pahangi and 19 H. contortus miRNAs were found at high abundance , either in the L3 stage, adult stage, or both  are shared with C. elegans, where all except mir-50 are also abundant (using the same read number criteria) [mir-2b and let-7 being found only in the adult stage of B. pahangi  . While mi Figure , while HB. pahangi miRNAs identified by library sequencing, 14 had reads in one stage only but below this cut-off. Of the remaining miRNAs, six were found only in the L3 stage, 10 in the adult stage only, while 69 were expressed in both stages  entries, demonstrated that the majority were not conserved in other organisms. Using a combination of BLAST and SSEARCH [Brugia miRNAs out of a total of 104 (40%) unique mature sequences, were conserved in other species based on our criteria. For H. contortus, 54 miRNAs out of a total of 192 (28%) unique mature sequences were conserved. The remainder for each species appeared novel. These numbers are comparable to findings from the necromenic nematode Pristionchus pacificus, the only non-Caenorhabditis nematode with a substantial set of miRNA sequences deposited in miRBase (release 15) where 33/124 (27%) were conserved in any other species [Importantly, comparing the full-length mature sequences (using a criteria of > 70% identity) of the  SSEARCH , 42 Brug species .Brugia and 18 H. contortus miRNAs being found in at least 20 species. Based on this analysis we find 15 miRNAs  that are found only in nematodes and not in any other species. Additionally, re-analysing these using only a seed match to indicate conservation, confirms that at least mir-36, mir-86, mir-265 and mir-63 are found only in worms. No miRNAs were found which were shared between Brugia and H. contortus only and not in any other species. Interestingly, examination of data from Ascaris suum [Ascaris but currently in no other species. These are Bpa-mir-5360, -5361, -5363, -5364, -5365a, -5365b, and -5366, and Hco-mir-5352. The greater number of miRNAs conserved between Brugia and Ascaris is consistent with their classification as clade III nematodes.The BLAST analysis was then used to identify the extent of miRNA conservation in other organisms  falling into six conserved families. For H. contortus, the seed match criterion alone was used, which grouped 12 miRNAs into five conserved families  a clear preference for positioning within introns was evident. For Brugia, 21 miRNAs were found to be located within protein coding genes , and the location of Brugia mir-100c within the intron of Bm1_29425 .miRNAs can be located intergenically or intragenically. The majority of ng genes , whereasng genes ,55. Examescribed  locationBrugia mir-50 [C. elegans mir-50 is also located intronically in the sense orientation within an essential gene, Y71G12B.11a, and that the Brugia mir-50 host gene, Bm1_56215 , is a clear homologue of this gene. Moreover, an H. contortus mir-50 homologue was identified and, although classified as intergenic by the preliminary gene predictions used here, BLASTX analysis of ~4 kb of sequence surrounding hco-mir-50 against C. elegans proteins also provided a match to Y71G12B.11a (positive strand translation). Deeper conservation of this same miRNA/protein-coding gene organisation was also found: the D. melanogaster rhea gene (NCBI gene ID: 38978) and human talin 2 (NCBI gene ID: 83660) (which show 47% identity and 67% similarity to each other), both show 38% identity and 57% similarity at the amino acid level to the C. elegans mir-50 host gene Y71G12B.11a, and both contain mir-190 homologues  which are > 70% identical to the nematode mir-50 mature sequences. Additionally, the host gene for hco-mir-7 shows homology to D. melanogaster bancal (NCBI gene ID: 43862) (39% identity and 59% similarity) and to human heterogeneous nuclear ribonucleoprotein K (NCBI gene ID: 3190) (48% identity and 68% similarity), both of which also contain mir-7, thus indicating strong selective pressure to maintain this organisation over a large evolutionary distance.The intronic-sense context of a mir-50  was likeBrugia data to include protein coding genes close to miRNAs. Although the average intergenic region for B. malayi is ~3.8 kb [C. elegans. Brugia-bantam is located 299 nucleotides 5' of the gene Bm1_08910 (sense orientation). The homologous C. elegans gene, T07D1.2, contains mir-81  and mir-82 , and the mature sequences for both show only two mismatches to bpa-bantam. Therefore, bpa-bantam appears to be homologous to Cel-mir-82 due to their mature sequence identity, conserved host gene, and similar relative positioning with respect to this gene.We extended the analysis of genomic context for the  ~3.8 kb  we foundH. contortus miRNAs were located within predicted protein coding genes, including mir-7 described above, all being within introns in the sense orientation. The lower number of miRNAs found within genes may reflect the greater genome size or the preliminary status of current gene annotations. Evolutionary conservation of mir-coding gene organisation was again demonstrated by the positioning of mir-86 within the homologous host genes in H. contortus and C. elegans (Y56A3A.7), although not in Brugia.Based on preliminary gene annotations, five C. elegans mir-50, -82 and -86 target their host gene (results not shown), the positional conservation of miRNA and protein coding genes described above suggests that, with the last shared common ancestor of C. elegans, H. contortus and Brugia being ~350 million years ago [Although our searches of the PicTar, TargetScan and mirWIP target prediction databases provide no indication that ears ago , there iBrugia and H. contortus. We find two examples in Brugia and four in H. contortus of the major mature product being generated from alternative arms of the precursor (indicated 5p and 3p) in different lifecycle stages, and one example in H. contortus of identical read numbers from both arms in the same stage. Two related novel H. contortus sequences hco-mir-5894a and hco-mir-5894b provide an example of alternative arm usage in duplicated sequences as the mature form is derived from different arms of the hairpin in each , this was not as pronounced as described previously where a 2.1-3.3-fold increase was reported [Brugia and H. contortus  does not constitute a cluster by the criteria applied here, their relative proximity in C. elegans and tight clustering in H. contortus may suggest functional linkage of these two miRNAs.Using the same criteria for s Figure . For evepoptosis  and for esponses . AlthougC. elegans [B. malayi coding sequences and H. contortus exons in both the sense and antisense orientations were identified using BLASTN, retaining only those with perfect matches over their full length. After removal of sequences corresponding to miRNAs, between 227,319 and 294,245 sense, and between 140,829 and 391,339 antisense reads were identified from the libraries  [C. elegans [P. pacificus [Brugia and H. contortus. The motif was searched for in a defined region upstream from the mapped positions of all 21 U reads which had not been already assigned to another class of RNA. Table Brugia and for the L3 stage of H. contortus. However, over 1200 reads, representing 154 loci and 151 unique sequences were identified from the adult H. contortus library . In C. elegans, piRNAs are most abundantly expressed in young adult worms [H. contortus library, and only these loci showed specific enrichment for the presence of the conserved upstream motif, we propose that these represent genuine piRNAs. Their relatively low abundance, in comparison to miRNAs and siRNAs, is in keeping with the results of others [piRNAs are critical for germline development in many species and are generated by a distinct mechanism that does not involve Dicer (the biogenesis and functions of piRNAs have been reviewed recently ). In C.  (GTTTC) ,62. Thes elegans  and P. pacificus . We therlt worms , consistf others , and conBrugia and H. contortus. Comparative analysis with C. elegans small RNA data reveals several points of interest.Using a combination of deep sequencing and bioinformatic prediction tools we have identified and characterised small RNAs from different lifecycle stages of the parasitic nematodes Caenorhabditis spp., which currently represent the most in-depth catalogue of nematode miRNAs. Only 42 of 104 Brugia and 54 of 192 H. contortus miRNAs were conserved in any other organism. Phylogenetic analysis groups H. contortus in the closest clade of parasitic nematodes to C. elegans. Despite this close relationship, the miRNA repertoire of H. contortus is no more similar to C. elegans than more distantly related parasitic species, including Brugia (this work) and Ascaris [Many parasite miRNAs identified here were classified as species-specific, based on current sequence data, and did not have homologues in any species, including  Ascaris ,64C. elegans miRNAs, with these data derived from detailed studies of miRNA function in the worm as well as from deep sequencing of multiple stages [Brugia and H. contortus genomes and these will be identified by analysis of additional lifecycle stages. Although we have classified a high number of miRNAs from H. contortus and Brugia as unique, some of these may yet be identified in closely related species. Indeed, several of the miRNAs we describe as currently being found only in Brugia and Ascaris can be found by homology in the genome databases of other filarial nematodes (unpublished data). As additional data from other parasitic nematode genomes becomes available our results will aid the annotation of miRNAs and advance the understanding of nematode miRNA evolution.The current version of miRBase (release 18) contains 223 e stages . Our curB. pahangi sequencing reads were, of necessity, mapped to the B. malayi genome. Thus any B. pahangi-specific sequences will not have been identified. While this explanation may account for the lower number of miRNAs found for B. pahangi compared to H. contortus, it is unlikely to fully explain the apparent expansion of miRNAs in H. contortus. In addition, homologous or heterologous probing of the B. malayi V2 gene array with B. malayi and B. pahangi cDNA showed no obvious bias towards B. malayi [Brugia and H. contortus. From current sequence data, the B. malayi genome is estimated to be 90-95 Mb [C. elegans (100 Mb). However, the current estimate for H. contortus is 300-500 Mb , a more precise figure awaiting the assembly and annotation of the genome. From studies on H. contortus gene structure to date, it appears that most genes are 2-3 times the size of their orthologues in C. elegans due to the greater size and number of introns [H. contortus genome remains speculative for the time being.An important proviso of this study was that the . malayi . An alte90-95 Mb , similar introns . Whetherbpa-mir-5364, and those with stage-restricted expression profiles, which will be relevant to understanding mechanisms of parasite development and responses to host signals.Understanding the functional relevance of diversity in miRNA sequences in these parasitic nematodes will require additional study; key to this will be defining their mRNA targets and demonstrating their biological relevance. As small non-coding RNAs provide a mechanism for altering levels of gene expression, their analysis may provide clues as to the evolution of parasitic life styles. The findings of this study provide the basis for further detailed analysis of miRNA expression profiles, both temporal and spatial, and of their target genes, using computational and experimental approaches. Of particular interest will be novel miRNAs that are abundantly expressed, such as mir-50 is located within a homologous gene in three different nematode species, argues strongly for the conservation of miRNA function and suggests a strong selective pressure to maintain this genomic positioning. That these partnerships have been maintained over ~350 million years of evolution adds further weight to this hypothesis. This conservation is particularly surprising as there is little conserved microsynteny between these species [In the midst of such diversity, comparison of the few evolutionary conserved miRNAs identified in this study demonstrates a remarkable degree of conservation in genomic positioning of certain miRNAs. For example, the observation that  species ,66,67 anAscaris suggested that the piRNA pathway has been lost from this parasitic nematode [H. contortus adult worms, suggests that, at least within clade V parasitic nematodes, the potential roles of piRNAs are conserved. Absolute confirmation of these sequences as piRNAs will require further study to show their association with Piwi proteins in the relevant tissues. However, sequence comparison studies have identified the Piwi related gene, prg-1, from H. contortus and related clade V parasitic species [Ascaris develops within an egg, those of Brugia within a mosquito vector, while the larval stages of H. contortus are found free within the environment), but sequence data from more species will help test such speculation.A recent study detailing small RNAs from nematode . It was nematode . Our ide species , supportBrugia and H. contortus, and compared these to C. elegans. Our findings demonstrate that many miRNAs in both parasitic species are unique and show little overlap with those of C. elegans or any other organism. Some of these novel microRNAs are both abundantly expressed and developmentally regulated. The association of certain miRNAs within host genes is highly conserved across species indicating a functional association. siRNAs and piRNAs were also identified, the latter only in H. contortus adult stage, supporting a conserved role of piRNAs in reproduction of some parasitic nematodes. Our results highlight the evolutionary diversity in microRNA repertoire between free-living and pathogenic species, and may provide clues to the evolution of parasitic lifestyles and adaptation to different environments. The availability of genome data for additional parasitic nematode species in the next few years [Using deep-sequencing and homology-based bioinformatics we have carried out the first in-depth study of small regulatory RNAs in ew years  will greB. pahangi strain used in this study was derived from the original isolate reported by Buckley and Edeson [Aedes aegypti mosquitoes (refm strain) were infected with B. pahangi by membrane feeding on rabbit blood containing first stage larvae (microfilariae). L3 were isolated from mosquitoes after 10 days at 27°C. L3 were used to infect the laboratory host, the jird (Meriones unguiculatus) and adult B. pahangi worms were isolated after 3-4 months [H. contortus worms were a gift from the Moredun Research Institute (Edinburgh) and were the strain MHco3(ISE), which is used for the on-going genome sequencing project. Ensheathed L3 stage larvae were harvested from faecal cultures, while mixed sex adult worms were collected at necropsy (day 28) from Suffolk lambs (5-9 months old) infected with 5,000 L3 larvae and maintained indoors to eliminate risk of other nematode infections. All procedures were carried out in accordance with the Animal (Scientific Procedures) Act, UK.The d Edeson . Aedes a4 months . H. contBrugia malayi genome assembly [Brugia_assembly.ghedin.fasta) was obtained from the Sanger Institute FTP site [B. malayi coding sequences were downloaded from NCBI [H. contortus genome data (combined_worms_supercontigs200808.fasta) was obtained from the Sanger Institute FTP site [H. contortus gene annotations were provided pre-publication from The Wellcome Trust Sanger Institute.The assembly   and H. contortus nuclear rRNA sequences were retrieved from the NCBI nucleotide database [Brugia rRNA" or "Haemonchus contortus rRNA". Additional Brugia sequences not already identified were retrieved from the SILVA database [Brugia 5S and 5.8S sequences. No further H. contortus sequences were identified from either SILVA or Rfam databases. The combined sequences for each organism were compared to a list of C. elegans rRNAs derived from WormBase [Brugia rRNA list contained partial sequences for B. malayi 28S , and 18S ; B. malayi and B. pahangi sequences for full length 5.8S  (EU373625 and EU373655), and B. malayi and B. pahangi sequences for full length 5S rRNA (with SL1 sequences) (D87037 and D87038), as well as a number of redundant and partial sequences for all forms. The H. contortus rRNA list contained full length sequences for 28S (AM039742), 18S (L04153), 5.8S  (EU084689) and 5S rRNA sequences (U32122) as well as a number of redundant and partial sequences.The predicted locations of tRNA encoding sequences were identified from the Ascan-SE ,75. Excedatabase  using thdatabase ,78, whildatabase  providedWormBase  which inFor both organisms, small RNA libraries were constructed from two developmental stages, infective third stage larvae (L3) and mixed-sex adult worms. Total RNA was extracted by re-suspending frozen nematodes in at least six volumes of TRIzol Reagent (Invitrogen). The material was disrupted using a 3 ml glass hand-held homogeniser (Jencons) and the RNA isolated following the manufacturer's protocol. RNA integrity was determined using an Agilent 2100 Bioanalyzer and RNA Nano kit . For eacBrugia precursors were also analysed by BLASTN against transposable element genomic sequences  and generial of ,85. Thisquencing ,87. It wquencing . From 78C. briggsae (cbr), C. elegans (cel), C. remanei (crm), P. pacificus (ppc), S. japonicum (sja), S. mansoni (sma) and Schmidtea mediterranea (sme), were downloaded from miRBase [B. malayi and H. contortus genome assemblies using the BLASTN algorithm with parameters adjusted to account for the short query length . All BLAST hits were then extended to match the length and positioning of the query sequence to give hits of ~22 bases representing candidate mature sequences. For each of these, two candidate precursors were generated by extracting genomic sequences corresponding to i) 70 nucleotides upstream and 10 nucleotides downstream and, ii) 10 nucleotides upstream and 70 nucleotides downstream, with respect to the ~22 nucleotide region. Candidates were then filtered using RNAfold, CIDmiRNA and manual scoring as detailed in Supplemental Methods in Additional File All miRNA mature and star sequences reported for the following worm species:  miRBase  release  miRBase . These smiRNAs were identified as homologous to known miRNAs present in miRBase 15 using BLASTN  to compare full-length mature sequences, allowing only sense matches and applying a 70% nucleotide identity cut-off. This process was used to assign gene names for conserved miRNA identified by deep-sequencing and to determine inter-species conservation of miRNAs. To provide additional homology assignments for gene naming, mature sequences were searched against sequences using SSEARCH  at miRBaC. elegans miRNA family members where available  identity over the first 10 bases was examined. The full-length sequences for miRNAs identified using either criteria were then aligned using ClustalX and Boxshade alongside eviously ).B. pahangi, all miRNAs were classified as exonic, intronic or intergenic (with protein coding genes within 1 kb noted) by analysing B. malayi genomic scaffold GenBank annotation files via NCBI Batch Entrez  or exons (H. contortus) using BLASTN. Only sequences with perfect identity along the full length of the read sequence were retained and those already identified as miRNAs excluded. A custom Perl script was used to map the retained sequences to coding sequences/exons. To identify putative piRNAs, library sequences of 21 nucleotides in length starting with a uracil were assessed. 21 U sequences that had been previously annotated as miRNA, coding sequence/exons, rRNA or tRNA were removed  and a custom Perl script used to extract 80 nucleotides of upstream genomic sequence. The presence of a consensus motif, GTTTC, between positions -48 and -42 of the upstream region was then determined [siRNAs were identified by matching library sequences to the coding sequences , nine supplemental tables , and supplemental references.Click here for filemiRNAs identified by deep sequencing. This file contains full details of the miRNAs identified by deep sequencing.Click here for filemiRNAs predicted by homology. This file contains full details of the miRNAs predicted by homology.Click here for fileEvolutionary conservation of miRNAs. This file contains the analysis of the evolutionary conservation of the identified miRNAs.Click here for filemiRNAs associated with protein coding genes. This file describes miRNAs associated with protein coding genes.Click here for filemiRNA clusters. This file describes miRNA found clustered in the genome.Click here for filesiRNAs. This file contains anti-sense sequences for all libraries which were identified as endo-siRNAs.Click here for filepiRNAs. This file contains sequences identified from the piRNA analysis.Click here for file


Peutz-Jeghers syndrome (PJS) is caused by mutations in the tumor suppressor gene, STK11, and is characterized by gastrointestinal hamartomas, melanin spots on the lips, and an increased risk of developing cancer.Blood samples were collected from two unrelated Chinese PJS families totaling 20 individuals , including 6 PJS patients. The entire coding region of the STK11 gene was amplified by polymerase chain reaction and analyzed by direct sequencing.A novel mutation, c.904C > T, in exon 7 was identified in both families. A C > T substitution changed codon 302 from CAG (glutamine) to TAG (stop), truncating the STK11 protein, thus leading to the partial loss of the kinase domain and complete loss of the α-helix C-terminus. Furthermore, one PJS patient from each family was diagnosed with a visceral cancer, a colon cancer and a liver cancer respectively.We predict that this novel mutation, p.Q302X, is most likely responsible for development of the PJS phenotype and may even contribute to malignancy. Peutz-Jeghers Syndrome PJS; MIM#175200) is a rare autosomal dominant disorder characterized by gastrointestinal hamartomatous polyps, mucocutaneous pigmentation, and an increased risk for the development of gastrointestinal (GI) and various extra-GI malignancies 75200 is ,4.Mutations in the serine-threonine kinase 11  gene on chromosome 19p13.3 have been considered to be the major cause of PJS ,6. The gHere, we report a novel nonsense mutation, c.904C > T (p.Q302X), in exon 7 from two unrelated Chinese PJS families that subsequently developed colon cancer and liver cancer, respectively.Family 1, from Guangdong province, China, consisted of three generations affected with PJS, including two female and two male individuals Figure . The proThe diagnostic criteria for PJS include the presence of characteristic mucocutaneous pigmentation, the presence of hamartomatous polyps in the gastrointestinal tract, and a family history of PJS. Patients need to fulfill two of these three criteria for a clinical diagnosis of the disease . This sthttp://frodo.wi.mit.edu/primer3/) to amplify the STK11 (GeneBank NM_000455) coding regions and intron-exon boundaries. Details of primer sequences and PCR conditions are available upon request. The PCR products were then sequenced in both directions on an ABI 3730 XL Automated DNA Sequencer  with the ABI BigDye Terminator v3.1 cycle sequencing kit. Standard nomenclature (http://www.hgvs.org/mutnomen/) was used to describe sequence variations, with +1 corresponding to the A of the ATG translation initiation codon of GeneBank NM_000455 for STK11.Genomic DNA was extracted from peripheral blood using the QiAamp DNA Blood Midi Kit . PCR primers were designed by Primer 3.0  and CPH models, (http://www.cbs.dtu.dk/services/CPHmodels/) were used to develop an appropriate model to mimic the effects of the mutated region.The homology modeling programs, SWISS MODEL  to the mutant sequence TAG (stop) at codon 302, and resulted in a truncation of the STK11 protein  . Codons Although the exact function of STK11 remains largely unknown, studies suggest that it plays a role in cell signaling and apoptosis . Recentlet al. [et al. [et al. [A genotype-phenotype correlation has been sought in PJS. Schumacher et al.  suggeste [et al.  suggeste [et al.  extendedIn our current study, we identified the novel mutation, p.Q302X, which was associated with cancers in both Chinese families where it was identified. Members of PJS families are at risk of developing the disease. Identification of a STK11 gene mutation in an index patient offers the possibility of a predictive diagnosis in PJS pedigrees. Furthermore, a possible genotype-phenotype correlation, especially concerning the risk of gastrointestinal and extraintestinal cancers, would be of particular clinical interest.PJS: Peutz-Jeghers syndrome; STK11: Serine threonine kinase 11; GI: gastrointestinal; CRD: C-terminal regulatory domain; Cdc37: Cell division cycle 37; Hsp90: Heat shock protein; PTEN: phosphatase and tensin homolog deleted on chromosome ten; STRAD: STE20-related adaptor; MAPK: mitogen-activated protein kinase; AMPK: AMP-activated protein kinase.The authors declare that they have no competing interests.ZQW carried out molecular genetic studies including sequencing for all the families and the controls, and drafted the manuscript. YLC did the structural predictions. BPW identified and diagnosed the patients. HXZ and JMH performed GI examinations and provided histopathological information. BJ designed, supervised the study and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/161/prepub


B-block_TFIIIC family in the Pfam database. In the NCBI and EMBL translated protein databases, there are archaeal proteins (approximately 100 amino acids in length) referred to as B-block binding subunits. Most of them contain a B-block_TFIIIC motif. DELTA-BLAST searches using these archaeal proteins as queries showed significant multiple blast hits for many eukaryotic B-block binding subunits on the same proteins. This result suggests that eukaryotic B-block binding subunits were constituted by repeating a small unit of B-block_TFIIIC over a long evolutionary period. Bacterial proteins have also been annotated as B-block binding subunits in the databases. Here, some of them were confirmed to have significant similarities to B-block_TFIIIC. These results may imply that part of the RNAP III transcription machinery existed in the common ancestry of prokaryotes and eukaryotes.Eukaryotic RNA polymerase III transcribes tRNA genes, and this requires the transcription factor TFIIIC. Promoters are within genes, with which the B-block binding subunit of TFIIIC associates to initiate transcription. The binding subunits are more than 1000 amino acids in length in various eukaryotic species. There are four regions with conserved sequence similarities in the subunits. The helix-turn-helix motif is included in one of these regions and has been characterized as the  While bacteria and Archaea have their inherent single RNA polymerases, eukaryotes have multiple types of RNA polymerase. Eukaryotic RNA polymerase III (RNAP III) is one of them and synthesizes tRNA, 5S ribosomal RNA, and other small RNAs . MamB-block_TFIIIC (PF04182) belonging to the clan HTH (CL0123) in the Pfam database, which is a large collection of protein families (http://pfam.sanger.ac.uk/): there are currently 324 sequences from 262 species in this family. This domain is considered to directly associate with the B-block sequence of the internal promoter for RNAP III.When the cDNAs for the B-block binding subunits of the yeast, rat, and human TFIIICs were cloned in previous studies, their amino acid sequences were compared for similarities  6–9]. H–9. H6–9]B-block_TFIIIC family in the Pfam database (http://pfam.sanger.ac.uk/family/PF04182#tabview=tab7). Interestingly, the promoter sequences of the A- and B-blocks are conserved in bacterial tRNA genes, and bacterial tRNA genes can serve as templates for eukaryotic RNAP III [RNAP III is generally known to be present in eukaryotes, but not in prokaryotes. However, archaeal and bacterial hypothetical proteins which have been defined or annotated as B-block binding subunits can be found in the translated protein databases of NCBI and EBI. Although some of the information in these databases has not yet been reviewed or confirmed, several proteins have been shown to be members of the RNAP III .In this study, the amino acid sequences of the eukaryotic B-block binding subunits and their possible prokaryotic relatives were investigated in silico for similarities, and their structural relationships are reported.http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=deltaBlast&PAGE_TYPE=BlastSearch&LINK_LOC=BlastHomeAd [http://www.ebi.ac.uk/Tools/msa/clustalo/; and Pfam sequence search in the website at http://pfam.sanger.ac.uk/search?tab=searchProteinBlock [http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja and NJplot [The programs used to compare the primary structures of proteins were as follows: DELTA-BLAST on the NCBI website at stHomeAd ; ClustalstHomeAd  in the EeinBlock . The seapsb.cgi) . DELTA-Bpsb.cgi) . The resd NJplot –18. The http://www.ncbi.nlm.nih.gov/protein) and UniProtKB/TrEMBL (http://www.uniprot.org/).The databases used were the NCBI protein database (B-block_TFIIIC family in the Pfam database (see http://pfam.sanger.ac.uk/family/PF04182#tabview=tab7). To date, many archaeal sequences have been defined or annotated as B-block binding subunits in the protein database of NCBI.  B-block_TFIIIC (As described in k_TFIIIC . Similark_TFIIIC . These rk_TFIIIC .B-block_TFIIIC (data not shown). In the Pfam database of B-block_TFIIIC, there are two bacterial proteins (http://pfam.sanger.ac.uk/family/PF04182#tabview=tab7). The representative proteins in which B-block_TFIIIC was detected by the CD-search or Pfam sequence search are shown in B-block_TFIIIC was not detected in any protein by both programs, and the E-values obtained by these searches were mostly higher than those of archaeal proteins . However, there were several cases in which the archaeal sequence did not hit the B-block_TFIIIC regions annotated in the NCBI database but showed significant hits for other regions in the same eukaryotic B-block binding subunits. For example, when the Metallosphaera yellowstonensis sequence (GI: 496365863 in Pediculus humanus corporis (GI: 242025343), Drosophila willistoni (GI: 195434252), Anopheles gambiae (GI: 347968303), and Oryzias latipes (GI: 432847756), the regions of aa positions 375–466, 429–516, 374–429, and 381–475 had significant E-values of 2e−6, 3e−6, 3e−5, and 5e−4, respectively , were not detected (http://dx.doi.org/10.1155/2014/609865): a combination of the Pyrolobus fumarii sequence (GI: 347523111 in Anopheles gambiae sequences (GI: 347968303) or Drosophila ananassae sequence (GI: 194765831); a combination of the Methanoplanus petrolearius sequence (GI: 307353829 in D. willistoni sequence (GI: 195434252) or Nasonia vitripennis sequence (GI: 345495267); a combination of the Methanosalsum zhilinae sequence (GI: 335930125 in Drosophila yakuba sequence (GI: 195472611) or Drosophila melanogaster sequence (GI: 20129503); a combination of the Methanofollis liminatans sequence (GI: 490137988) and A. gambiae sequence (GI: 347968303); a combination of the Methanoregula formicicum sequence (GI: 432331009 in Pediculus humanus corporis sequence (GI: 242025343) or A. gambiae sequence (GI: 347968303).As described above, archaeal B-block binding subunits are approximately 100–200 aa in length, while eukaryotic subunits are more than 1000 aa in length. Region II in the eukaryotic subunit, which is approximately 100 aa in length, contains the IC motif . DELTA-Bectively . On the detected . CD-searB-block_TFIIIC region of the A. gambiae sequence (GI: 347968303) was not detected in DELTA-BLAST searches using M. yellowstonensis, P. fumarii, M. liminatans, and M. formicicum sequences as queries. However, in the search using the Methanocella conradii sequence   in this case and the mosquito hit region mostly overlapped with regions that were detected with the M. yellowstonensis, P. fumarii, M. liminatans, and M. formicicum sequences (D. melanogaster and D. yakuba sequences (GI: 20129503 and GI: 195472611) when DELTA-BLAST searches were performed using the M. zhilinae and M. conradii sequences as queries  in four regions, and the P. fumarii sequence hit the yeast Candida tropicalis sequence (GI: 255729444) in two regions.As described above, the authentic  (7e−8) . There wB-block_TFIIIC-like regions in addition to the authentic B-block_TFIIIC regions in their entire sequences. Although the B-block_TFIIIC-like regions were repeated near the authentic B-block_TFIIIC regions in many cases, they also existed in the C-terminal regions and in the middle regions of the subunits . As described in B-block_TFIIIC  as a query: two regions were detected in each of these proteins   were conserved in this alignment. They were also conserved in regions II and III alignments of Figures 3B and 3C in [B-block_TFIIIC-like sequence commonly exists as region III in eukaryotic B-block binding subunits (The  and III . In this371–431) . When thsubunits .E-values lower than threshold in DELTA-BLAST searches using the archaeal sequences as queries (M. yellowstonensis sequence (GI: 496365863) was used as a query, in the sequences of Fibroporia radiculosa , Stereum hirsutum , Punctularia strigosozonata , and Trametes versicolor , the regions of aa positions 2013–2053, 2091–2133, 2284–2321, and 2154–2191 had significant E-values of 4e−4, 0.002, 0.002, and 0.001, respectively. When the P. fumarii sequence (GI: 347523111) was used as a query, the C-terminal region (aa positions 2285–2322) of the P. strigosozonata sequence  was hit at an E-value of 0.002  were similar to aa positions 1–33 of the Exophiala dermatitidis sequence (GI: 378726632) , and aa positions 1–27 of the M. conradii sequence (GI: 383320206) were similar to aa positions 7–33 of the Kluyveromyces lactis sequence (GI: 50302891) . All such N-terminal regions are shown in the lower part of There were no hits in the N-terminal regions of the eukaryotic B-block binding subunits at M. yellowstonensis sequence (GI: 496365863) were similar to aa positions 1539–1584 of the Selaginella moellendorffii sequence  , and aa positions 3–86 of the P. fumarii sequence (GI: 347523111) were similar to aa positions 934–1019 of the Batrachochytrium dendrobatidis sequence  . All these results suggest that the eukaryotic B-block binding subunit was mainly constructed by repeated duplication of the B-block_TFIIIC sequence  which was shown in CD-search. As shown in B-block_TFIIIC sequence. CD-search of the bacterial proteins showed that they have similarities also to other HTH motifs such as MarR, MarR_2, and HTH27, and the E-values were frequently lower than those of B-block_TFIIIC , MarR_2 (PF12802), and HTH_27 (PF13463) are members of the clan HTH (CL0123) (http://pfam.sanger.ac.uk/clan/CL0123#tabview=tab0). Proteins with the MarR and MarR_2 motifs are involved in resistance to multiple antibiotics. They repress the expression of mar operons consisting of antibiotic-resistant genes. HTH_27 is a family of the winged helix-turn-helix motif. Each of the cdd sequences of MarR, MarR_2, and HTH_27 obtained as representatives from CD-search was aligned with the six bacterial B-block binding subunits . On the other hand, comparably low amino acid preferences are found in the left central regions of the MarR, MarR_2, and HTH_27 families . Actually, the corresponding region in the alignment of the bacterial proteins and the cdd sequence of MarR, MarR_2, or HTH_27 did not show such similarities (boxed region in HTH_27 is not conserved in the motif (http://pfam.sanger.ac.uk/family/PF13463#tabview=tab4). These results confirmed that the bacterial proteins shown in As already described and shown in  queries . Therefok_TFIIIC . Like B-subunits . The amiignments . As showB-block_TFIIIC motif sequences of several archaeal short proteins were shown to be repeated in the longer B-block binding subunits of various eukaryotes. This finding suggests that the eukaryotic B-block binding subunit has been constructed by repeating duplication of the B-block_TFIIIC region in long evolutionary time. Repetition of B-block_TFIIIC sequence was common in various eukaryotic B-block binding subunits. Therefore, the B-block_TFIIIC sequence had possibly begun to be repeated in the first primitive eukaryotes. Alternatively, the sequence might have been originally repeated in the ancient B-block binding subunit. It may be imagined that the repetition was lost in the evolutionary process from the ancient organisms to prokaryotes to leave single copies on the subunits. However, this is opposite to the established view that DNA duplications have contributed to the evolution of organisms [Here, the rganisms , 20.B-block_TFIIIC motif in CD-searches (data not shown). Additionally, the M. yellowstonensis sequence (GI: 496365863) used in this study showed similarity to Rpc34: when CD-search was performed, its HTH region was aligned with the HTH region of the Rpc34 motif (PF05158) at an E-value of 0.03 (data not shown), although the value was much higher than that of the alignment with B-block_TFIIIC . In this tree, prokaryotic B-block_TFIIIC sequences are not contained. Therefore, the phylogenetic tree was constructed together with the prokaryotic B-block_TFIIIC sequences. The tree also indicated that animals are evolutionarily closer to plants than to fungi .Molecular phylogenetic studies using small subunit rRNA and the proteins like actin and o plants , 25. Howo plants . This wato fungi . Althougto fungi , this reB-block_TFIIIC motif in this study. The host species belong only to four genera although whole genome sequencing has been completed in many bacterial genera. Why is the B-block binding subunit absent in the other bacteria? Three possibilities are there. The first one is that in the other bacteria genes encoding the B-block binding subunit proteins had been lost. The protein might have been nonessential for bacterial survival. The second possibility is that the B-block binding subunit had highly diverged in the other bacteria, and those protein sequences cannot be detected with the similarity search programs that are currently used. This possibility is extensively discussed in the following paragraph. The third possibility is that horizontal gene transfers between bacteria and Archaea or eukaryotes. A few cases of horizontal gene transfer from Archaea to bacteria have been reported [Some bacterial proteins in databases were confirmed to contain a reported . HoweverE. coli [E. coli artA gene is shown to bind to the internal region of IS1 and stimulate transcription [Bacterial IS1 is a mobile DNA  and appE. coli . The procription , 30. Altcription .B-block_TFIIIC motif in archaeal and bacterial proteins and type II promoter sequences in prokaryotic genomes.The relatives of the RNAP III transcription machinery may have existed in the common ancestry of eukaryotes and prokaryotes due to the presence of the B-block_TFIIIC sites to the B-block binding subunits of the M. petrolearius, M. zhilinae, M. liminatans, M. formicicum, and M. hollandica. Details are as described in the legend to Figure 2, except that the maximum number of proteins shown in each column of the eukaryotic groups against each query is three.Supplementary Figure: Eukaryotic B-block binding subunits showing similarities in regions different from their authentic Click here for additional data file.


Retinal imaging devices made dramatic progress in these several years. These new devices reveal various anatomical and functional changes in the fundus by high resolution images, which may develop and improve diagnosis, treatment, and surgical maneuver.The special issue on multimodal imaging of the fundus published one review article and five clinical research articles. The review article of Y. Mitamura et al. showed that the three lines in the outer retina detected by spectral-domain optical coherence tomography (SD-OCT) could serve as hallmarks for evaluation of photoreceptor condition in retinitis pigmentosa and recovery process after macular hole surgery. Of the five clinical research articles, N. F. Mokwa et al. compared sensitivity and specificity of imaging techniques of color fundus photography, fluorescein angiography, and SD-OCT for detecting age-related macular degeneration (AMD) and activity of choroidal neovascularization (CNV). F. Pichi et al. evaluated multimodal visualization of retinal genetic diseases by fundus autofluorescence, FA, indocyanine green (ICG) angiography, and SD-OCT to monitor progression of the diseases. O. A. Osmanbasoglu et al. analyzed diurnal variation of central choroidal thickness by enhanced depth imaging technique of SD-OCT in healthy emmetropic subjects. M. Şahin et al. reported hyperautofluorescence after cataract surgery, and J. M. Muñoz et al. compared ranges of contrast values in autofluorescence imaging between 2 fundus cameras. All the six articles in this special issue underwent a rigorous peer-review process.


The prevalence of infections by nontuberculous mycobacteria (NTM) has steadily increased over the past decades, especially in immunocompromised patients.Mycobacterium (M.) haemophilum and M. kansasii.We present a patient with IgA-deficiency and mixed cutaneous infection by two slowly growing mycobacteria, M. haemophilum infections most often result from HIV or transplantation-associated immunosuppression. Rarely, M. haemophilum may also infect healthy patients or iatrogenically immunosuppressed patients without transplantation. M. kansasii is one of the most frequent NTM and large awareness exists about its involvement in human diseases. Mycobacterial diagnosis of cutaneous infections should be considered in long-lasting skin lesions.Cutaneous  Immunoglobulin (Ig) A-deficiency is the most common primary antibody deficiency. Although the majority of affected individuals have no apparent symptoms, selected patients suffer from recurrent mucosal infections, allergies, and autoimmune diseases . So far,Mycobacterium haemophilum is an established cause of cutaneous infections in immunocompromised hosts [M. haemophilum infections are skin lesions with a preference for cooler body sites such as extremeties, while the development of sporotrichoid-like nodular lymphangitis is exceptional.ed hosts . The mosM. kansasii is one of the most frequent NTM causing human diseases among both immunocompetent and immunocompromised patients [M. kansasii most likely causes infections resembling tuberculosis likewise with a preference for pulmonary disease [M. kansasii should be included in the differential diagnosis of skin infections with an indolent course and lack of response to standard antibiotics [M. haemophilum is still unknown, while tap water is likely the major reservoir for M. kansasii infections. Inoculation of NTM into the host usually occurs due to impairment of the physical skin barrier. Affected patients commonly show a history of fish tank cleaning, oyster shucking, swimming, or other aquatic activities [patients . M. kans disease . Cutaneoibiotics . Since Nibiotics . Unfortutivities .A 48-year old man presented with multifocal nodules of variable size arranged in a sporotrichoid-like manner on the right arm, the dorsum of the digit IV, the left arm, the right side of his back, the knees and a partly circumscribed erythematous plaque on his back Figure . The lesBorrelia-specific IgM and IgG and tuberkulin skin test were negative. Microbiological cultures showed twice growth of M. haemophilum from independent lesions on the right hand taken about 8 weeks apart. Interestingly, M. kansasii was recovered from another lesion on the right elbow indicating a mixed infection.At two occasions skin biopsies from several lesions  were initiated and subjected in parallel to histopathological and microbiological examinations. They revealed caseating epitheloid cell granuloma in the dermis with a marginal zone of lymphocytes and a subepidermal lymphohistiocytic infiltrate with perivascular accentuation Figure . By PAS-Mycobacteria-specific liquid and solid cultures were performed by using the automated BACTEC MGIT 960 system (Becton Dickinson) at 36°C and Lowenstein-Jensen Agar (LJA) at 24°C, 30°C, and 36°C. Growth of acid fast bacilli was detected from LJA at 30°C supplemented with hemin discs. First, culture isolates were identified as NTM due to the absence of an amplicon by a M. tuberculosis-specific gyrB PCR [M. tuberculosis complex, the M. avium complex, M. abscessus, M. kansasii and M. chelonae etc. The Mycobacterium AS test provides the identification of rarely found species, such as M. simiae, M. haemophilum, and M. mucogenicum. In case of M. haemophilum, Mycobacterium CM assay revealed a banding pattern specific for the M. haemophilum-M. nebraskense-M. malmoense-M. palustre group. Further species differentiation was done with the AS assay that showed positivity for M. haemophilum and M. nebraskense but not for M. malmoense or M. palustre. The growth requirement for hemin indicated the presence of M. haemophilum. Nevertheless, species identification of M. haemophilum was confirmed at the National Reference Center for Mycobacteria in Borstel (Germany). M. kansasii was recovered with the Bactec MGIT 960 and identified with the GenoType Mycobacterium CM assay.gyrB PCR  and weregyrB PCR . The GenThus, culture positive lesions were all from the right body site, while no co-infection of a single lesion was found. Interestingly, the patient was working as a scaffolder having potential exposure to diverse environmental habitats while touring around Europe. Further, he is aquarist but reports to usually wear gloves during cleaning his aquarium.®) was administered, but due to his frequent travel activities the substitution was realized irregularly. Serum IgA level was 1.8 (07-4.0) mg/ml under substitution with Beriglobin®. Biochemical laboratory parameters were within normal ranges except for serum protein electrophoresis with albumin of 57.2% (59-70.6) and beta globulin of 14.9% (7.3-12.2). ASL, AST, ACE, ANA, and ENA tests were unremarkable. Besides multiple cutaneous lesions there was no evidence for lymphadenopathy or systemic infection with M. haemophilum/M. kansasii by using abdominal ultrasound, thorax X-ray, and thoraco-abdominal computer tomography imaging. Mycobacterial sputum cultures were negative as well. Empirical triple treatment with clarithromycine 2 × 500 mg/d, rifabutine 1 × 300 mg/d and ethambutol 1 × 20 mg/kg body weight/d was initiated. This regime was given for 6 months, while a complete clinical remission of skin lesions was seen within 4 months. Finally, two years after his initial presentation the patient is still healthy.Interestingly, in the patient's history a selective IgA-deficiency was documented since childhood. His mother also suffered from IgA-deficiency. Since infancy human immunoglobulin , and optimal growth temperature, which can provide helpful information for a preliminary classification of NTM. bacteria ,2. DiagnM. haemophilum infections are most often the result of HIV- or transplantation-associated immunosuppression. It's most common clinical manifestations are cutaneous lesions with a preference for cooler body sites such as extremities. Therefore, in case of skin biopsies LJA at 30°C should routinely supplemented with hemin discs in order to fulfill special growth requirements of M. haemophilum that is known to primarily cause cutaneous infections. Rarely, M. haemophilum may infect iatrogenically immunosuppressed patients without transplantation or even healthy persons. The development of sporotrichoid nodular lymphangitis due to M. haemophilum is exceptional [Strikingly, cutaneous M. kansasii infection has been reported in up to 20 percent of nontuberculous mycobacteriosis in both immunocompetent and immunocompromised patients. Disseminated infections are uncommon but of poor prognosis [M. kansasii pulmonary infection is good if diagnosed and treated early. In one report, M. kansasii cutaneous infection occurred on the hand and forearm, with carpal tunnel syndrome complicated by concomitant pulmonary M. tuberculosis [In contrast, rognosis . The outrculosis , and in rculosis .2, the role of IgA in the protection of this surface from harmful environmental agents is extremely essential. Any changes connected with a lack or an overproduction of IgA can manifest as variable clinical disease [As the only comorbid condition our patient showed IgA-deficiency. Sustained, very low levels of IgA, IgG, or IgM, as found in primary immunodeficiency syndromes, are associated with significantly increased risk for infections, primarily respiratory tract infections of bacterial origin. IgA, a major serum Ig and the predominant antibody in external secretions that bathe mucosal surfaces, plays a key role in immune protection. IgA at concentrations of about 2-3 mg/ml is the second most prevalent serum antibody after IgG, which is normally present at about 12 mg/ml. However, IgA-deficiency is the most common primary antibody deficiency and appears to be well tolerated. Due to the large surface area of the human mucosa of up to 400 m disease . Most in disease -13. Stri disease . However disease . Rodrigu disease .M. marinum. In IgA-deficiency, beside reduced serum IgA levels often also other immune deficiencies e.g. in cytokine levels and B-cell function but also associated T cell defects have been described. Thus, we cannot exclude that IgA-deficiency may be a surrogate for another undetected less marked immune dysfunction [Most likely, in our patient NTM's are transmitted from a contaminated environmental source by a direct cutaneous route. The exact source and the infection mode remain unclear. Nevertheless, even in a healthy individual an appropriate exposition  may lead to NTM infections, as described for function .M. haemophilum and M. kansasii, both belonging to the slow growing mycobacteria, in a patient with IgA-deficiency. The patient is aquarist and was working as a scaffolder presenting a relevant risk of exposure to diverse environmental habitats while touring around Europe. Although according to current knowledge no definite pathophysiological link between mycobacterial disease and IgA-deficiency may be given, we cannot rule out that this immunological imbalance may contribute to the patient's vulnerability towards NTM. Finally, the patient responded well to triple anti-mycobacterial therapy with complete regression of entire skin lesions within 4 months.This is the first report of a mixed cutaneous infection We obtained a written informed consent from the patient for publication of this article and accompanying images. A copy of the written consent is available for the review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.VB and MH conceived the project and wrote the manuscript. AB and TR helped in the acquisition of clinical data and manuscript revision. MF carried out and interpreted the histological examinations. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-5945/11/3/prepub


In 2009, the Childhood Arthritis and Rheumatology Research Alliance (CARRA) established a longitudinal multi-center, multiple disease U.S. national registry (CARRAnet) for pediatric rheumatology with the intent of providing 60 participating clinical sites a new framework to drive observational clinical research and evidence-based care. CARRAnet seeks to enroll up to 20,000 subjects with childhood-onset rheumatic disease and twice yearly follow-up. We report baseline characteristics of the initial 6-month enrollment cohort; disease-specific results are reported separately.th birthday in subjects <=21 years . A common baseline data set and 1 disease-specific data set was completed on each participant by interview and chart review. Data cleaning and analysis employed Microsoft Excel and Access (Microsoft Corp), SAS (SAS Institute), and R .Enrollment commenced 5/29/2010 with data available through 12/28/10. Inclusion criteria comprised 1 of 8 categories of defined rheumatic disease with onset before the 161638 subjects were enrolled from 27 centers throughout the US. The analysis cohort reflected 1371 subjects, predominantly JIA; 63 variables were collected for the shared baseline form with summary statistics presented in the figures. The population reported overall good to excellent health by patient and physician report: 96% with mean HRQOL good to excellent; physician mean global assessment of disease activity (PGAS) 1.6 . PGAS correlated with subject reports . Medication use was prevalent, including 74% ever on steroids, 41% ever on biologics, and 31% currently on biologics. Growth was within normal on average, but exhibited wide deviation . A similarly wide range was seen on both objective and subjective measures, identifying probable subpopulations with high disease activities.The initial CARRAnet cohort reflects predominantly low disease activity with favorable self-reports. This is not a population study and issues of enrollment bias require further investigation. Despite the overall well-being of the population, the high use of steroids, biologics, and DMARDs, along with significant subpopulations concerning for high disease activity, are important areas of future focus.Marc D. Natter: None; Jane R. Winsor: None; Kathleen A. Fox: None; Norman T. Ilowite: None; Kenneth D. Mandl: None; Kelly L. Mieszkalski: None; Christy I. Sandborg: None; John S. Sundy: None; Carol A. Wallace: None; Laura E. Schanberg: None; CARRAnet Investigators Group: None.


Apomixis is an intriguing trait in plants that results in maternal clones through seed reproduction. Apomixis is an elusive, but potentially revolutionary, trait for plant breeding and hybrid seed production. Recent studies arguing that apomicts are not evolutionary dead ends have generated further interest in the evolution of asexual flowering plants.Pennisetum and Cenchrus. A 1 kb region from the 3' end of the ndhF gene and a 900 bp region from trnL-F were sequenced from 12 apomictic and eight sexual species in the genus Pennisetum and allied genus Cenchrus. An 800 bp region from the Apospory-Specific Genomic Region also was sequenced from the 12 apomicts. Molecular cytological analysis was conducted in sixteen Pennisetum and two Cenchrus species. Our results indicate that the Apospory-Specific Genomic Region is shared by all apomictic species while it is absent from all sexual species or cytotypes. Contrary to our previous observations in Pennisetum squamulatum and Cenchrus ciliaris, retrotransposon sequences of the Opie-2-like family were not closely associated with the Apospory-Specific Genomic Region in all apomictic species, suggesting that they may have been accumulated after the Apospory-Specific Genomic Region originated.In the present study, we investigate karyotypic variation in a single chromosome responsible for transmitting apomixis, the Apospory-Specific Genomic Region carrier chromosome, in relation to species phylogeny in the genera Cenchrus and newly investigated Pennisetum species into a single clade containing a terminal cluster of Cenchrus apomicts, the presumed monophyletic origin of Cenchrus is supported. The Apospory-Specific Genomic Region likely preceded speciation in Cenchrus and its lateral transfer through hybridization and subsequent chromosome repatterning may have contributed to further speciation in the two genera.Given that phylogenetic analysis merged  Apomixis is an intriguing trait in plants that allows multiplication of maternal clones through seed reproduction . BesidesPennisetum/Cenchrus branch of the monophyletic bristle clade of grasses [Pennisetum glaucum (L.) R. Br., and at least 17 aposporous species [ndhF (F subunit of NADH dehydrogenase) [ndhF and trnL-F [trnL-F and rpl16 [knotted) to study the phylogeny in Cenchrus, Pennisetum and related genera.The  grasses  contains species . Relatio species  and sequogenase) , ndhF and trnL-F , trnL-F nd rpl16 . Chemisqnd rpl16  also useNicotiana tabacum) ndhF gene is 2223 bp in length and has a nucleotide substitution rate [rbcL, a second extensively studied chloroplast gene [ndhF is more variable than the 5' region [ndhF and 811-872 bp region from trnL-F) and a 792-799 bp segment from the ASGR-BBM-like gene, also located within the p208 BAC used in fluorescence in situ hybridization (FISH) analysis. We furthermore report molecular cytogenetic analysis of the genomic region associated with apomixis, the apospory-specific genomic region (ASGR) that was previously identified in P. squamulatum, C. ciliaris and now in 16 Pennisetum and one additional Cenchrus species.ITS sequences provide limited resolution to estimate genetic similarities of hybrids and their parents due to concerted evolution . Though ion rate  which isast gene . More re' region . For theP. squamulatum and C. ciliaris based on high sequence similarity between putative orthologous genes within this region; syntenic relationships between chromosomal sequences identified by BAC probes; shared cytological features of hemizygosity, the heterochromatic nature of the ASGR, and a region of low copy DNA flanked by high copy sequences [Pennisetum and Cenchrus species phylogeny constructed with sequence data from chloroplast genes, ndhF and trnL-F. Variability observed in chromosomal context should enable a more precise delineation of the ASGR.The ASGR is conserved between equences -37. NevendhF sequence of 1134 bp except for P. hohenackeri (PS156) and P. polystachion (PS19). PS156 had an insertion of 21 bp while PS19 showed a 3 bp deletion. For the trnL-F region, size varied from 863 bp to 872 bp except in the case of P. polystachion, P. pedicillatum and P. subangustum which showed a length of 811 bp. ndhF and trnL-F produced an aligned matrix of 1155 and 901 nucleotide positions respectively thus giving a total aligned matrix of 2056 characters. The matrix had 1913 constant, 76 parsimony uninformative and 67 parsimony informative characters. A partition homogeneity test was done for 100 replicates, although the test was aborted during the 78th replicate due to time constraints (655:46 hr). The test gave a P-value of 0.86 supporting the combination of data sets for analysis.All species Table  generateA simple heuristic search of the aligned matrix using Phylogenetic Analysis Using Parsimony (PAUP) retained 28 trees. All trees were 166 steps in length and had a consistency index (CI) of 0.875, retention index (RI) of 0.895 and rescaled consistency index (RC) of 0.775. The log likelihood of all the trees ranged from 3974.04078 to 3971.98984. To account for homoplasy generated by gaps, the gap creating regions were ignored . After exclusion, a heuristic search generated 9 trees each showing a length of 152 steps with CI of 0.875, RI of 0.914 and RC of 0.799. The log likelihood for all the trees ranged from 3755.09184 to 3757.65531.P. ramosum, P. nervosum and P. mezianum showed low support in the Bayesian-based analysis. These species also showed variation with respect to their position in the two trees (Bayesian and Maximum Parsimony). Subgroups I, II, and V contain apomictic and obligately sexual species whereas subgroups III and IV contain apomictic species with sexual cytotypes or facultative apomixis.Phylogenetic trees with similar topologies were generated by Bayesian and maximum parsimony (MP) methods. Overall five groups emerged in the present phylogenetic study Figure , Table 2ndhF and trnL-F sequences to understand the relationship among Pennisetum and Cenchrus species. To compare their analysis with that of the present study, the sequence alignment was downloaded from TreeBase http://purl.org/phylo/treebase/phylows/study/TB2:S10252. The resultant matrix was too large to be analyzed by PAUP, hence it was only analyzed by Mr. Bayes  and amplify a region including 3 introns of 95 bp, 266 bp, and 154 bp. Based on ASGR-linked BAC clone sequencing, P. squamulatum and C. ciliaris have duplicated ASGR-BBM-like genes [P. squamulatum while polymorphism is detectable in C. ciliaris. The present analysis could differentiate two copies of the ASGR-BBM-like gene in C. setigerus, P. orientale, P. mezianum and C. ciliaris. In P. orientale, accession PS12 did not show two copies while PS15 did. Among the two types of sequences obtained in C. ciliaris and C. setigerus, one showed similarity with P. squamulatum while the other sequence grouped with the other copy from Cenchrus , a 54-chromosome accession, two ASGR-carrier chromosomes were observed  and P. massaicum (PS680) showed rDNA signals on the ASGR-carrier chromosome. The ASGR-carrier chromosome in C. setigerus was indistinguishable from C. ciliaris in the position of the rDNA locus, i.e., terminal on the short arm of the ASGR-carrier chromosome  to 7.20 μm in P. setaceum (PS25). The ASGR position was estimated based on the mid-point of the P208 signal and was always shown to be in or near a moderately to highly condensed region, again with the exception of P. massaicum.FISH experiments revealed that morphologies of the mitotic ASGR-carrier chromosomes among the species were different; therefore, image analysis was carried out to quantify the differences Table . ThreshoLengths and arm ratios of the two ASGR-carrier chromosomes in PS12 were compared to each other by a paired t-test, which showed a significant difference for chromosome length t = 3.16 P < 0.01) but not for arm ratio . The DNA distribution on the two ASGR-carrier chromosomes of PS12 showed different patterns as measured by DAPI staining intensity Figure . The ASG P < 0.01C. setigerus was similar to that of C. ciliaris  showed unique morphology among the species with a highly condensed region in the middle of the long arm  and P. massaicum (PS680) [P. mezianum with 32 chromosomes, only the ASGR-carrier chromosome showed signal from hybridization of P602  or confined to the ASGR-carrier chromosome (P. squamulatum pattern). The latter pattern occurred in only two other species, P. setaceum and P. mezianum. The Opie-2-like repeat was of low abundance flanking the ASGR in P. setaceum, P. villosum, and P. massaicum.BAC P602 contains an Opie-2-like retrotransposon abundant only in the ASGR of ciliaris . The dis (PS680) . In P. m2 Figure , whereasC Figure . This reReconstruction of ancestral states was done by Mesquite. Four characters viz mode of reproduction, basic chromosome number, distribution of Opie-2 on genomes, and distribution of Opie-2 on the ASGR Table .Pennisetum or higher taxonomic levels including Pennisetum were based on multiple approaches such as genome size variation [knotted [ndhF [rpoC2 [rpl16/trnL-F [Earlier attempts to resolve phylogenetic relationships among different species in the genus ariation , moleculariation ,41, and ariation , b) the [knotted ,26, and ed [ndhF ,26,42-44F [rpoC2 , and rpl6/trnL-F . The pretrnL-F and ndhF for conducting a phylogenetic analysis. A combined analysis of their sequences along with the sequences used in this study generated a tree presented in Figure Chemisquy  recentlyCenchrus was monophyletic and embedded in a polyphyletic Pennisetum [Cenchrus was supported by additional chloroplast sequence data and taxon sampling [Cenchrus within Pennisetum, but are incongruent with the result of Donadio [P. purpureum, P. setaceum, and Cenchrus spp. In the ITS-based phylogeny of Martel [P. setaceum and P. villosum grouped with species from section Brevivalvula in a clade that included Cenchrus ciliaris; therefore, their placement of P. setaceum, but not P. villosum, was more consistent with Donadio [P. villosum grouped away from Cenchrus species and Brevivalvula section. In contrast, results from the present study place P. setaceum as a member of a large clade that included the cultivated species P. glaucum and excluded species from section Brevivalvula while P. villosum grouped with P. hohenackeri and P. alopecuroides. Doust and Kellogg [P. setaceum to be closely related to P. glaucum. Section Brevivalvula is considered more homogeneous compared with other sections in the genus Pennisetum [ndhF is a chloroplast gene and thus likely not susceptible to the process of concerted evolution, although nuclear capture of chloroplast DNA is possible [Previous studies of nuclear and chloroplast gene sequences suggested that, within the bristle clade of grasses, the genus nnisetum . Polyphysampling . Our dat Donadio  and Chem Donadio  that shof Martel , P. seta Donadio . Accordi Donadio , P. vill Kellogg  also founnisetum  and morpnnisetum . The nonnnisetum . As withpossible  but unteP. setaceum in the P. glaucum clade is supported by the current and prior [P. purpureum, P. squamulatum, and P. schweinfurthii. P. squamulatum, recently proposed by Akiyama et al. [ndhF and cytological [P. glaucum (the primary gene pool) and P. purpureum (secondary gene pool). Since revision of the basic chromosome number of P. squamulatum from x = 9 to x = 7 [P. setaceum, have a basic chromosome number of x = 7 and all (including P. setaceum) can be crossed with P. glaucum [P. purpureum we investigated were strongly supported as sister to P. glaucum, confirming placement of this species in the secondary gene pool of Pennisetum [P. glaucum and P. purpureum, and one genome of P. purpureum has been suggested to be homologous with pearl millet [The placement of nd prior  chloroplnd prior . This cla et al.  to be a a et al. ), has stological  data forto x = 7 , all spe glaucum -55. The nnisetum . Naturall millet .ndhF data, the apomictic pentaploid P. villosum is most closely related to the sexual diploids, P. hohenackeri and P. alopecuroides. P. hohenackeri and P. alopecuroides also grouped together in an ITS-based phylogeny [P. alopecuroides grouped with P. villosum based on EST-microsatellites [knotted-1 gene sequence [P. alopecuroides was more closely related to P. glaucum than P. villosum based on the chloroplast sequence data of Donadio [P. villosum are also consistent with its derivation through hybridization [Based upon the hylogeny , while Ptellites  and the sequence , further Donadio . Signifidization , and incP. orientale and P. flaccidum. P. orientale and P. flaccidum were also closely related based upon EST- microsatellite data, even though the P. orientale cytotype examined was 2n = 2x = 18 and likely sexual [P. basedowii is uncertain. While Bayesian analysis places P. basedowii basal to subgroups IV and V, MP analysis shows it as sister to subgroups III, and IV/V. No previous phylogenetic studies have incorporated P. basedowii except for Chemisquy [P. glaucocladum, a species not included in the present analysis, and to which P. flaccidum was basal. Neither does our combined analysis , 2) Pennisetum species from outside this section, P. massaicum, and 3) the Cenchrus species. The close relationship between P. polystachion and P. pedicillatum of section Brevivalvula was also indicated by ITS and SSR data [P. massaicum where the basic chromosome number is unclear. P. massaicum, with 35 chromosomes, 8 of which do not hybridize to the Opie-2-like repeat, may be an aneuploid (4x-1) as the number of rDNA loci suggests that it is tetraploid or it may have been produced by interspecific hybridization between species with basic chromosome numbers of x = 8  and x = 9 . Another x = 8 species, P. montanum, falls outside of the Cenchrus clade [P. orientale used in the present study uniquely showed two ASGR-carrier chromosomes, each with a different morphology but able to pair at meiosis, and may have resulted from fertilization of an unreduced egg with reduced pollen, both carrying an ASGR. In the case of the derivation of the apomictic P. massaicum, such hybridization would have necessarily involved at least one parental apomictic lineage that contributed the ASGR-carrier chromosome. Pollen fertility in Pennisetum apomictic lineages is required for pseudogamous apomixis, in which the endosperm develops only after fertilization of the central cell. Normal male meiosis, giving rise to reduced pollen is typical, but irregular segregation of genomes through aberrant meioses also occurs. Either interspecific or intergeneric hybridization may have precipitated speciation of P. massaicum. Genomic in situ hybridization (GISH) studies could provide valuable insights regarding the putative hybrid origin of this species.Clade IV included 1) all three SSR data ,41. FurtSSR data . All speus clade , but wasus clade . One accP. ramosum, P. nervosum and apomictic P. mezianum with divergent basic chromosome numbers . Apomictic P. mezianum did show deviation in its grouping among MP and Bayesian trees. The two sexual species in this group differ in their distribution of Opie-2-like repeats, abundant throughout the genome of P. nervosum, but barely detectable by FISH in P. ramosum. Even though P. ramosum has the smallest number of chromosomes of any Pennisetum species (2n = 2x = 10), it has one of the highest DNA contents estimated per haploid genome  and the as derived states which originated independently of each other , we speculate that the association of this repeat with the ASGR was derived by either 1) translocation of the repeat-poor portion of the ASGR into a repeat-rich region of the genome or 2) transposition and accumulation of retrotransposons in proximity to the ASGR. Ancestral stage analysis  involving apomicts in diploid-polyploid-dihaploid cycles . HybridiBoechera . One expPennisetum species that either have diploid or higher ploidy sexual cytotypes [The ASGR is present only in apomictic cytotypes of ytotypes  or are cPennisetum and Cenchrus, but also on the origin and evolution of the ASGR. FISH results reveal structural similarity within the ASGR across Pennisetum and Cenchrus apomictic species. This similarity is further supported by an ~800 base pair sequence generated from the ASGR. The fact that apomictic species cluster with sexual species in the chloroplast sequence-based phylogeny supports the view that apomixis probably originated once and then spread through repeated hybridization between the species. The presence of different morphologies for the ASGR-carrier chromosome in different species and variation in linkage with rDNA infers that the ASGR can be translocated within the genome and those genomes can support gross chromosomal aberrations as a consequence. Polyploidy associated with apomixis likely increases tolerance of the genome to mutation and chromosomal aberrations. It has often been speculated that apomicts cannot be sustained in nature for a long period of time due to their propensity for accumulating mutations in the absence of sexual reproduction. Low rates of sequence variation at the ASGR, therefore, suggest that the ASGR might be of recent origin.The present investigation provides interesting insights not only on the phylogeny of genus P. squamulatum and C. ciliaris have shown association of high-copy Opie-2-like sequences with the ASGR. Various species within the present investigation have an ASGR containing fewer copies of Opie-2-like sequences, from which we infer that association of the Opie-2 sequence with the ASGR is only a genome specific feature rather than a unique feature associated with apomixis. Ancestral state analysis suggests that the ASGR might have originated in a genome with low abundance of Opie-2 and was transferred to a high Opie-2-copy genome while independent duplication of low-copy regions within the ASGR also occurred. P. massaicum, which groups with Cenchrus spp. and section Brevivalvula species, is a species where genomes with low Opie-2 at the ASGR and high abundance of Opie-2 across the chromosome complement coexist, and most likely were merged by hybridization. Section Brevivalvula shows variation in distribution of Opie-2 on the ASGR and throughout the genome with P. polystachion displaying lower Opie-2 signal than the other two species examined. These observations warrant investigation into other species from the section. Additional sequence data from the ASGR also could resolve the extent of gene duplication in this region.Earlier studies from In conclusion, the present investigations have provided new insights into structure of the ASGR and its evolution based on experimental evidence. Further studies in this direction can lead to information which can help in deciphering the intrigues of apomixis.Plant materials and their origins are listed in Table http://simon.bio.uva.nl/object-image.html) and modified CHIAS3 [Fluorescence in situ hybridization was carried out according to Akiyama . Enzymatd CHIAS3 . The sta® PCR system 9700 thermal cycler . The 3' end of the ndhF gene was amplified using the primers 972 and 2110R [2, 0.2 mM dNTPs, 1.5 U Taq polymerase, and 0.2 μM of each primer. Cycling conditions were denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and a final extension for 7 min at 72°C. The amplified products were purified using the Qiaquick PCR purification kit . The purified products were sequenced using primers 972, 1603, 1603R and 2110R. In a few species, primers 1318, 1318R, 1955 and 1955R were used to complete the sequencing of amplified fragments. Sequencing was carried out on a Beckman CEQ8000  according to the manufacturer's instructions. The trnL-F region was amplified using the primers trnL-F_c and trnL-F_f as per [ASGR-BBM-like region was amplified using the primers p779/p780. These primers amplify only apomicts from segregating F1 populations of both Pennisetum and Cenchrus (unpublished results) and showed amplification only in apomictic and not the sexual species in the present study. Amplification was carried out in 50 μl reactions containing 50-75 ng template DNA, 1X iProof GC buffer, 200 μm each dNTP, 0.5 μM each of trnL-F or ASGR-BBM-like primers and 0.02 U/μl iProof DNA polymerase . The cycling conditions were one cycle of 98°C for 30 s followed by 30 cycles of 98°C for 8 s, 60°C (trnL-F)/52°C (ASGR-BBM like) for 20 s, 72°C for 25 s with a final 72°C extension for 10 min. The amplified products were ligated with the pCR®-BluntII-TOPO® vector (Invitrogen) and transformed into NEB E. coli DH5α following the manufacturer's instructions . For each trnL-F DNA template 3 clones were fully sequenced. Sequencing was performed by the Georgia Genomics Facility,  using M13 Forward and Reverse primers. For each ASGR-BBM-like DNA template 5 clones were sequenced using M13 Forward primer.Total DNA was extracted from fresh leaves using the procedure of Tai and Tanksley  or follond 2110R . Amplifif as per . The ASGndhF available from the whole chloroplast genome sequence in Nicotiana tabacum (Accn. No. Z00044 S54304). Sequence chromatograms were cross-checked at positions where mutations were predicted. The corrected alignment was used for phylogenetic analysis. The analysis was done by PAUP 4.0 beta 10 for windows [The nucleotide sequences generated were aligned by Clustal X ver 1.81 . The ali windows  and Mr.  windows . Mr. Mod windows  was used windows , analysi windows . Mesquit windows  was usedndhF chloroplast gene, produced and analyzed all the evolutionary data and wrote those corresponding parts of the paper. YA produced and did image analysis of the chromosomal and FISH data and wrote those corresponding parts of the paper. JC produced the sequence for the ASGR-BBM-like and trnL-F chloroplast genes and participated in their analysis and writing of results. PO-A conceived of the project, provided guidance, secured funding for the study, and coordinated manuscript preparation. WWH provided plant research material and contributed to cytological analysis. HY-A contributed with image analysis and reconfirmed the PS680 rDNA results. All authors have read and approved the manuscript.SG produced sequence for the Current addresses:YA: Livestock and Forage Research Division, Tohoku Agricultural Research Center (TARC), National Agriculture and Food Research Organization (NARO), Akahira 4, Shimokuriyagawa, Morioka, Iwate 020-0198, JapanSG: Department of Botany, University of Delhi, Delhi 110007, IndiaName and sequence of ASGR primers tested on the species.Click here for file