We demonstrate that the present-day spatial distribution and orientation of the principal stresses within Larsen C ice shelf are akin to those within pre-collapse Larsen B. When Larsen B’s stabilizing frontal portion was lost in 1995, the unstable remaining shelf accelerated, crumbled and ultimately collapsed. We hypothesize that Larsen C ice shelf may suffer a similar fate if it were not stabilized by warm and mechanically soft marine ice, entrained within narrow suture zones.The collapses of the Larsen A and B ice shelves on the Antarctic Peninsula in 1995 and 2002 confirm the impact of southward-propagating climate warming in this region. Recent mass and dynamic changes of Larsen B’s southern neighbour Larsen C, the fourth largest ice shelf in Antarctica, may herald a similar instability. Here, using a validated ice-shelf model run in diagnostic mode, constrained by satellite and  Signs of instability in the Antarctic Larsen C ice shelf have raised concerns that it might soon collapse like its northern neighbour Larsen B. Kulessa et al. combine an ice-shelf model with satellite and geophysical data to show that despite dynamic similarities, Larsen C is presently stabilized by marine ice. The stability of an Antarctic ice shelf depends on the balance between the constructive stresses that ensure its integrity and the destructive stresses that compromise it. The spatial distribution and temporal evolution of the first and second principal stresses on Antarctic ice shelves depend on the geometry of the embayment in which the shelf is situated, the spatial and temporal attributes of the ice recharge it receives from feeding glaciers, environmental factors such as the evolution of atmospheric surface and oceanographic basal conditions2Fractures, such as horizontally extending rifts and vertically and horizontally extending surface and basal crevasses can be inherited from feeding glaciers. Fractures may also originate near the grounding line as the ice begins to float, or open up on the shelf in response to bending or shearing forces or thermal processes4We adopt our previous approachOur first stage of argument considers that both the first and the second principal stresses have highest absolute magnitudes near Larsen C’s grounding line and decrease non-uniformly towards the calving front . Stress-56Our second stage of argument considers that the distribution of stress-flow angles on Larsen B ice shelf in 1986 before its collapse -derived flow velocities of Larsen C ice shelf2021Our model sensitivity experiments with simulated softening of marine ice-bearing suture zones and major rifts on Larsen C ice shelf  reveal tSuture zones on Larsen C ice shelf tend to become thicker and narrower as they are advected downstream, owing to lateral compaction and the balance between continued accretion and basal melting2241328The presence of variably sized bodies of marine ice has already been inferred for several other Antarctic ice shelves, including the three largest, Filchner-Ronne18Our continuum-mechanical ice-shelf modelOur perturbation experiments implemented the most prominent marine ice-bearing suture zones and rifts on Larsen C ice shelf for the purpose of sensitivity testing . Adoptin−1 based on common-midpoint surveys conducted in both field seasons4The GPR data presented in Our seismic reflection data were acquired in walk-away mode in 2008–09 ref.  using a B.K. and A.J.L. respectively led NERC projects NE/E012914/1 and NE/I016678/1 that made this work possible; and D.J. was the post-doctoral scientist for both. E.C.K. and P.R.S. were co-investigators on NE/E012914/1. D.J. implemented and conducted the ice-shelf model runs presented here. Building on E.C.K.’s expertise, B.K. and A.J.L. collected the field geophysical data used as model constraints; and B.K., E.C.K. and D.J. processed them. A.J.L. and D.J. acquired and processed the satellite data used as model constraints. P.R.S. contributed on the mechanical properties of and fracture processes operating on ice shelves.How to cite this article: Kulessa, B. et al. Marine ice regulates the future stability of a large Antarctic ice shelf. Nat. Commun. 5:3707 doi: 10.1038/ncomms4707 (2014).Supplementary Figures 1-3


These included 26 first-borns and 24 second-borns. Insulin sensitivity was assessed by the Matsuda method from an oral glucose tolerance test. Other assessments included DXA-derived body composition, lipid profiles, 24-hour ambulatory blood pressure, and carotid intima-media thickness. First-born men were 6.9 kg heavier (p = 0.013) and had greater BMI  than second-borns. Insulin sensitivity in first-born men was 33% lower than in second-borns , despite adjustment for fat mass. There were no significant differences in ambulatory blood pressure, lipid profile or carotid intima-media thickness between first- and second-borns. Thus, first-born adults may be at a greater risk of metabolic and cardiovascular diseases.We aimed to assess whether birth order affects metabolism and body composition in overweight middle-aged men. We studied 50 men aged 45.6 ± 5.5 years, who were overweight (BMI 27.5 ± 1.7 kg/m There has been a steady reduction in birth rates throughout the worldThere is some evidence that birth order influences growth and metabolism, from infancy to early adulthood424While there are limited data on the effects of birth order on growth and metabolism in the first two decades of life, there are no data on such outcomes in mid-adulthood. It is important to assess whether the metabolic effects observed in early years are sustained or magnified throughout adulthood, particularly since middle age is a time where identification of risk factors and early intervention may be most appropriate. Therefore, we aimed to assess whether birth order affects metabolism and body composition in overweight middle-aged men.2, including 26 first-borns and 24 second-borns. The vast majority of participants (92%) were of European descent, and men in both groups were of similar age and ethnicity  than second-borns  , despiteThis study has shown that within a cohort of overweight middle-aged men, first-borns had greater BMI and lower insulin sensitivity than second-borns. Importantly, the difference in insulin sensitivity was independent of fat mass. Reduced insulin sensitivity is an independent predictor of type 2 diabetes mellitus, hypertension, coronary heart disease, stroke and cancer in non-obese middle aged menRecently, there has been increasing interest in two atypical groups. Firstly, the metabolically healthy obese (MHO)The mechanisms by which birth order influences long-term metabolism are unknown, but differences in placental blood flow may play a role. During pregnancy, structural changes occur to uterine spiral arteries to facilitate placentation2It is worth noting that the difference in insulin sensitivity was also independent of the age of participants and of their parents. Recent studies indicate that there are age-related changes in the β-adrenergic system that lead to altered cardiovascular functionpost hoc analysis and a relatively small sample size. In addition, we studied a relatively narrow range of individuals , which could also limit the applicability to the general population, particularly females. Lastly, as we did not study sibling pairs, our findings could have underestimated the magnitude of birth order effects on insulin sensitivity and other metabolic outcomes.The major strength of this study is the comprehensive metabolic assessment. Although the study of a narrow BMI range enabled characterization of overweight middle-aged men, this may limit wider application of our findings. Given first-borns had greater BMI than second-borns, using BMI as an inclusion criterion for the parent clinical trials might have introduced a subtle selection bias. Other limitations include the In conclusion, first-born overweight middle-aged men have greater BMI and lower insulin sensitivity than second-borns. Larger studies are required to better evaluate the long-term health effects of birth order across the BMI range. Ideally, future research should focus on sibling pairs.Ethics approval was provided by the Central and Northern Y Regional Ethics Committees , and written informed consent was obtained from all participants.2) and middle-aged (35–55 years) were eligible to participate. Note that only males were recruited to the clinical trials so that the effects of the menstrual cycle and/or oral contraception on insulin sensitivity (the primary outcome) could be avoided. Exclusion criteria were: diabetes mellitus, hypertension (systolic blood pressure >145 mmHg or diastolic blood pressure >95 mmHg), known dyslipidaemia, or the use of tobacco, or prescription medications likely to affect blood pressure, lipid profile or insulin sensitivity. From this group, all participants born at term (37–41 weeks) from singleton pregnancies were included. Note that when assessing birth order, miscarriages of less than 20 weeks were not counted. Where subjects participated in more than one clinical trial, the data from the most recent trial was used.Participants were recruited for two clinical trials investigating the metabolic effects of supplementation with olive leaf extractAll clinical assessments were carried out at the Maurice & Agnes Paykel Clinical Research Unit . Insulin sensitivity was assessed via a 75 g oral glucose tolerance test using the Matsuda method, with glucose and insulin samples collected at 0, 30, 60, 90, and 120 minutesFasting blood samples were used to assess lipid profile, namely triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Auxological assessment included height measurement using a Harpenden stadiometer. Weight and body composition were assessed using whole-body dual-energy X-ray absorptiometry . Apart from BMI, the fat mass index was also calculated24-hour ambulatory blood pressure monitoring was carried out prior to the clinical assessment. Participants were fitted with a Spacelabs 90207 or 90217 monitor , with each subject being assigned the same device model for all assessments. Measurements were performed every 20 minutes between 07:00 and 22:00, and every 30 minutes from 22:00 to 07:00. Only profiles with >14 daytime and >7 night time recordings over a 24-hour period were analysed.tm v.4.1, Sonosite). The maximal cIMT measurement from both views, approximately 10 mm proximal to the carotid bulb was used for comparative analysis. To assess reproducibility, triplicate measures were taken of seven healthy volunteers over a 7-day interval, and resulted in an intra-observer CV of 3.7% (unpublished data).Carotid intima-media thickness (cIMT) was also measured, as it is a validated and reproducible measure that is predictive of cardiovascular and cerebrovascular risks. cIMT was measured using an M-Turbo ultrasound system  by trained investigators , with longitudinal images attained using a standard protocolPhysical activity levels were assessed using the International Physical Activity Questionnaire (IPAQ)Insulin concentrations were measured using an Abbott AxSYM system  by microparticle enzyme immunoassay with an inter-assay coefficient of variation (CV) of 5.4%. Glucose, triglyceride, total cholesterol, HDL-C, and LDL-C concentrations were measured on a Hitachi 902 autoanalyser  by enzymatic colorimetric assay  with a CV lower than 2.5%. Highly sensitive CRP was measured using commercially available ELISA kits  or a Hitachi 902 autoanalyser .Demographic characteristics between first- and second-born men were compared using one-way ANOVA and Fisher's exact tests in Minitab v.16 . Multivariate linear regression models were carried out in SAS v.9.3 . All models accounted for important confounding factors, namely age, ethnicity, socio-economic status (NZDep2006), physical activity levels (IPAQ), and mean parental age at childbirth. Age and total body fat percentage were also controlled for, when assessing potential differences in lipids and outcomes associated with glucose homeostasis. All statistical tests were two-tailed and significance level maintained at 5%. Demographic data are presented as means ± standard deviations, while other data are means and 95% confidence intervals adjusted for the confounders in multivariate models.B.B.A., W.S.C., M.d.B., P.L.H. and J.G.B.D. conceived and designed the study. B.B.A., M.d.B., C.B. and J.B. recruited and performed the tests. B.B.A. and M.d.B. collected and compiled the data. J.G.B.D. carried out the statistical analyses. B.B.A., J.G.B.D. and W.S.C. wrote the manuscript with input from other authors. All authors have approved the submission of the final version of this manuscript.


Helicobacter (H.) suis has been associated with chronic gastritis and ulcers of the pars oesophagea in pigs, and with gastritis, peptic ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma in humans. In order to obtain better insight into the genes involved in pathogenicity and in the specific adaptation to the gastric environment of H. suis, a genome analysis was performed of two H. suis strains isolated from the gastric mucosa of swine. Homologs of the vast majority of genes shown to be important for gastric colonization of the human pathogen H. pylori were detected in the H. suis genome. H. suis encodes several putative outer membrane proteins, of which two similar to the H. pylori adhesins HpaA and HorB. H. suis harbours an almost complete comB type IV secretion system and members of the type IV secretion system 3, but lacks most of the genes present in the cag pathogenicity island of H. pylori. Homologs of genes encoding the H. pylori neutrophil-activating protein and γ-glutamyl transpeptidase were identified in H. suis. H. suis also possesses several other presumptive virulence-associated genes, including homologs for mviN, the H. pylori flavodoxin gene, and a homolog of the H. pylori vacuolating cytotoxin A gene. It was concluded that although genes coding for some important virulence factors in H. pylori, such as the cytotoxin-associated protein (CagA), are not detected in the H. suis genome, homologs of other genes associated with colonization and virulence of H. pylori and other bacteria are present. Helicobacter (H.) suis is a very fastidious, spiral-shaped, Gram-negative bacterium requiring a biphasic culture medium at pH 5 enriched with fetal calf serum, and a microaerobic atmosphere for in vitro growth [H. suis colonizes the stomach of more than 60% of slaughter pigs [H. suis in gastric disease in pigs is still unclear, it has been associated with chronic gastritis [H. suis, compared to the non-infected control animals [o growth . H. suister pigs ,2. Althoastritis ,4 and ulastritis . This maastritis . A reduc animals .Helicobacter pylori [Helicobacter pylori helicobacters (NHPH) have also been associated with human gastric disease with a prevalence ranging between 0.2 and 6% [H. suis is the most frequent NHPH species found in humans, where it was originally named "H. heilmannii" type 1 [H. suis has been associated with peptic ulcer disease [Bacterial gastric disorders in humans are mainly caused by r pylori . However2 and 6% . H. suis" type 1 . There a" type 1 ,12. In t disease , gastric disease  and chro disease . In rode disease .H. suis infections. To improve understanding in the genes playing a role in pathogenicity, gastric colonization and persistence of H. suis, a genome-wide comparison with the well-investigated H. pylori genome was performed. Some virulence factors may indeed be similar for both bacteria. As there may also be differences, ab initio annotations of the H. suis genome were performed as well.Up to now, little is known about the pathogenesis of H. suis (HS1T = LMG 23995T = DSM 19735T) and H. suis strain 5 (HS5), isolated from the gastric mucosa of two different swine, according to the method described by Baele et al. [A pyrosequencing  assay was applied to the genome of the type strain of Helicobacter pylori strains , and ab initio annotation.In order to maximize the number of quality gene annotations, two different annotating approaches were followed: cross-mapping with three T and HS5 genomic contigs. The H. pylori proteome and non-coding RNAs were aligned  to the H. suis database. For each BLAST hit the following additional information was analysed: 1) (secretion) signal peptide cleavage site if present, as assessed by the SignalP 3.0 program [H. suis, the Shine-Dalgarno sequence was determined to be a subsequence of AGGAGGU (which is the reverse complement of the 3' end of the 16S rRNA), and the minimum AU-richness  of the preceding region was arbitrarily set to 10/16. For each theoretical ORF a range of possible start codons was scored; the higher the similarity to the ideal Shine-Dalgarno sequence, or the AU-richer the preceding region, or the better a combination of both, the more likely the potential start codon is to be the actual start codon.A custom BLAST  database program ,19; 2) s program ; 3) an e program ,22; ii)  program ,24. For -15 against the Uniprot-KB universal protein database. The generalist algorithm of getorf yielded roughly a tenfold of the expected natural ORFs, reducing the risk of false negatives. In order to keep the false positive rate low, extra parameters were considered: 1) percentage alignment between query and hit ORFs; 2) percentage similarity or conservation between aligned portions of query and hit ORFs; 3) ribosome binding strength (for more details see above). To determine the presence of one or more conserved domains a rpsblast search  was carried out for every single theoretical ORF against the compiled Conserved Domain Database which holds protein domain alignments from several other database sources [For ab initio annotation, theoretical open reading frames (ORFs) were first determined using the EMBOSS getorf tool  . All ORF sources .T genome a total of 1 635 292 base pairs and in the HS5 genome 1 669 960 bp were sequenced, both with an average GC content of 40%. In contrast to H. pylori, only one copy of both the 16S and 23S rRNA genes was detected, but like H. pylori, H. suis has three copies of the 5S rRNA gene. Thirty-eight transfer RNAs were identified. On the whole, 1266 ORFs from HS1T and 1257 from HS5 were detected, of which 194 and 191 encoded hypothetical proteins respectively. In 98 and 92 ORFs a signal peptide cleavage site was detected, demonstrating predicted secreted proteins of HS1T and HS5 respectively. The TMHMM program predicted 210 and 206 proteins with at least one transmembrane helix for HS1T and HS5 respectively. The sequence fraction identical for HS1T and HS5 is henceforward described together as the "H. suis genome".In the HS1H. pylori genes involved in acid acclimation, chemotaxis, adhesion to gastric epithelial cells, oxidative stress resistance  , sialic acid synthase (NeuB) , and UDP-N-acetylglucosamine-2-epimerase (WecB)  were observed as well.Homologs of ce Table , and mot. pylori . MoreoveH. pylori OMPs are presented in Additional file H. pylori OMP families  could be aligned with the H. pylori genome. Both H. suis strains contain the hof genes hofA, C, E, F, the hop genes hopE, G-2 and H, and the hor genes horB, C, D, and J. Additionally, HS1T contains homologs of the hopW protein precursor and horE, whereas HS5 possesses additional homologs of horA, horF, and horL. No members of the Helicobacter outer membrane (hom) family were detected in H. suis. Besides the major H. pylori OMP family proteins, the H. suis genome contains some predicted OMPs based on their N-terminal pattern of alternating hydrophobic amino acids similar to porins, encompassing omp29 for HS1T and omp11 and omp29 for HS5. A 491 amino acids membrane-associated homolog of the virulence factor MviN, aligned for 92% with the MviN homolog of H. acinonychis (Hac_1250), was also present in H. suis.Genes encoding putative outer membrane proteins (OMPs) in relation to H. pylori type IV secretion systems (T4SS), only two members of the cag pathogenicity island (cagPAI) were identified in the H. suis genome (cag23/E and cagX). Most members of the comB transport apparatus were present. These include comB2, B3, B6, B8 and a number of additional genes not classified as comB: recA, comE, comL and dprA. H. suis possesses genes encoding VirB- and VirD-type ATPases , all designated members of the H. pylori type IV secretion system 3 (tfs3). The HS1T and HS5 T4SS are presented in Table Of the H. pylori genes involved in induction of gastric lesions in the H. suis genome are summarized in Table H. pylori vacuolating cytotoxin A gene (vacA) identified HSUHS1_0989 in HS1T. The corresponding protein, which is exceptional in that it is one of the longest in the world of prokaryotes, possesses three small conserved VacA regions , followed by an autotransporter region (residues 2730-2983). The amino acid sequence of the HS5 homolog (HSUHS5_0761) could be aligned for 22% with the H. pylori strain HPAG1 sequence, and possesses only one conserved VacA region (residues 242-298), followed by an autotransporter region (1258-1510). In both vacA homologs, no signal sequence was determined. Additionally, an ulcer-associated adenine-specific DNA methyltransferase  coding sequence was identified, whereas a molecular homolog of the ulcer-associated restriction endonuclease (iceA) could not be discovered in H. suis. H. suis contains homologs of pgbA and pgbB encoding plasminogen-binding proteins, though both lacking a transmembrane helix or signal peptide cleavage site according to the bioinformatics tools mentioned earlier. H. suis harbours homologs of genes coding for the H. pylori neutrophil-activating protein (HP-NapA) and γ-glutamyl transpeptidase (HP-GGT). Homologs encoding the H. pylori flavodoxin fldA and the pyruvate-oxidoreductase complex (POR) members porA, porB, porC, and porD were also identified in H. suis.Homologs of H. pylori genes involved in acid acclimation, chemotaxis and motility, have counterparts in the H. suis genome. These genes are known to be essential for colonization of the human gastric mucosa [The results of the present study demonstrate that several c mucosa -32.H. pylori and other bacterial species. H. suis contains some similar members of the major OMP families described in H. pylori [H. pylori to the gastric mucosa, which is widely assumed to play an important role in the initial colonization and long-term persistence in the human stomach. These include the gastric epithelial cell adhesin HorB [H. pylori adhesin A (HpaA). HpaA, also annotated as neuraminyllactose-binding hemagglutinin, is found exclusively in Helicobacter and binds to sialic acid-rich macromolecules present on the gastric epithelium [H. suis lacks homologs of several other H. pylori adhesion factors, including genes coding for the blood group antigen binding adhesins babA (hopS) and babB (hopT), the sialic acid binding adhesins sabA (hopP) and sabB (hopO), and the adherence-associated lipoproteins alpA (hopC) and alpB (hopB) [Several OMP coding sequences were identified by comparative analyses with . pylori . Some ofsin HorB  and the ithelium . On the B (hopB) .H. suis contains a fibrinonectin/fibrinogen-binding protein coding gene, which may enhance its adherence to injured gastric tissue. Damage to host epithelial cells may indeed expose fibronectin and other extracellular matrix components. Strong homology was found with fibronectin-binding proteins of H. felis (YP_004072974), H. canadensis (ZP_048703091) and Wolinella succinogenes (NP_907753). To our knowledge, no exact function has been given to these proteins in these species. In Campylobacter jejuni, however, fibronectin-binding proteins CadF and FlpA have been shown to be involved in adherence to and/or invasion of host's intestinal epithelial cells [H. suis fibronectin-binding protein lacks a transmembrane helix or signal peptidase cleavage site, indicating that it is not surface exposed or secreted. Its real role in colonization therefore remains to be elucidated.al cells ,38. AcconeuA, neuB, and wecB) were annotated in the H. suis genome, indicating that this bacterium may decorate its surface with sialic acid. The presence of surface sialylation has been studied extensively in pathogenic bacteria, where it contributes to evasion of the host complement defense system [Three genes involved in sialic acid biosynthesis . This indicates that H. suis may harbour a defense mechanism against the host inflammatory response, contributing to the ability of chronic gastric colonization by this bacterium [Additionally, acterium .H. suis genome, namely the comB cluster and the tfs3 system. The H. suis comB system probably plays a role in genetic transformation [H. suis strains as has been recently demonstrated by multilocus sequence typing of available H. suis strains [H. pylori tfs3 secretion system in pathogenesis is not exactly known. Seven genes of the tfs3 cluster are homologs of genes involved in type IV secretion: virB4, virB11, and virD4 code for ATPases which move substrates to and through the pore. The latter is coded by transmembrane pore genes virB7, virB8, virB9, and virB10 [virB7 were identified in H. suis, indicating that the H. suis tfs3 can be important in transmembrane transport of substrates in H. suis.Two partial T4SS were predicted in the ormation ,42. Tran strains . The rold virB10 . All theH. pylori cag pathogenicity island (cagPAI) region encodes a T4SS allowing H. pylori to insert the cytotoxin-associated antigen A (CagA) into the host cell. This process results in altered host cell structure, an increased inflammatory response, and a higher risk for gastric adenocarcinoma [H. suis possesses two members of the H. pylori cagPAI (cag23/E and cagX), the majority of genes, including the gene coding for pathology-causing protein (CagA), were not identified. This indicates that HS1T and HS5 lack a functional cag protein transporter secretion system.The arcinoma . AlthougH. suis with H. pylori resulted in the identification of additional genes possibly associated with virulence in H. suis. A H. suis homolog of the H. pylori vacA was detected. VacA is both a cytotoxin of the gastric epithelial cell layer, and an immunomodulatory toxin of H. pylori [H. pylori contains either a functional or non-functional vacA. The H. suis vacA homolog exhibits no vacA signal sequence, indicating that it might encode a non-functional cytotoxin [H. suis vacA could clarify the functionality of the vacA homolog in this Helicobacter species.Genomic comparison of . pylori . H. pyloytotoxin . In vitrH. pylori virulence-associated genes namely napA, encoding the HP-NapA and ggt, encoding HP-GGT. The H. pylori GGT has been identified as an apoptosis-inducing protein [H. pylori from oxidative stress by binding free iron [H. suis contains homologs of two H. pylori genes coding for plasminogen-binding proteins, pgbA and pgbB. The corresponding proteins, PgbA and PgbB bind host plasminogen, which subsequently can be activated to plasmin and may contribute to obstructing the natural healing process of gastric ulcers [H. suis pgbA and B homologs in chronicity of gastric ulceration is uncertain, as no exact membrane association was found in the corresponding proteins.Strong homology was found with two  protein ,49. The  protein ,51. Moreree iron . H. suisc ulcers ,54. The H. suis infected human patients is higher than after infection with H. pylori [H. pylori flavodoxin (fldA) and its electron donor, the POR enzyme complex (porA to D) were found in H. suis. The H. pylori flavodoxin protein (FldA) has been proposed to play a role in the pathogenesis of H. pylori-associated MALT lymphoma, as antibodies against the H. pylori FldA protein were more prevalent in patients with MALT lymphomas compared to patients with other H. pylori-related diseases [fldA and the por complex has shown that these genes are essential for the survival of H. pylori [fldA and its por complex may play a role in gastric colonization of H. suis and MALT lymphoma development in H. suis infected people.The risk to develop MALT lymphomas in . pylori ,14. Homodiseases . Besides. pylori . These oH. pylori strain B38 and the carcinogenic and ulcerogenic Helicobacter mustelae have been sequenced [H. pylori virulence genes , which are also absent in the H. suis genome. Additionally, H. mustelae lacks a vacA homolog. Despite this absence, infection with H. pylori strain B38 and H. mustelae has been associated with gastric MALT lymphomas and other gastric disorders. Whole genome sequencing data are also available from H. acinonychis strain Sheeba, a gastric pathogen of large felines. Similar to H. suis, H. acinonychis lacks a cagPAI as well as genes encoding BabA/B and SabA/B. Both species contain a vacA homolog, which for H. acinonychis has been described to be fragmented [Recently, the genomes of the carcinogenic equenced ,58. Bothagmented ,60.H. suis contains a mviN homolog. This gene has been described to be a virulence factor of several bacterial species, such as Burkholderia pseudomallei and Vibrio alginolyticus [mviN in the Helicobacter genus, however, remains to be elucidated.olyticus ,62. In aolyticus -63. The H. suis lacks homologs of some major H. pylori virulence genes, other candidate virulence factors, such as napA, ggt, mviN, and fldA were detected. H. suis also possesses genes known to be essential for gastric colonization. Future in vitro and in vivo research of the currently presented genes of this porcine and human gastric pathogen should elucidate their precise role in colonization and virulence.Although T and ADHO00000000 for HS5. The versions described in this paper are the first versions, ADGY1000000 and ADHO1000000.The genome sequences have been deposited at GenBank/EMBL/DDBJ under the accession ADGY00000000 for HS1The authors declare that they have no competing interests.MV designed the study, analysed the data and drafted the manuscript. TTMV carried out the sequence alignment and participated in the design of the study. BF, AS and DDG participated in the design of the study. FP, WVC, RD, and FH coordinated and participated in the design of the study. All authors read and approved the final manuscript.H. suis strain 1 (HS1Table S1 Classification of TH. pylori OMPs) and strain 5 (HS5) outer membrane proteins (OMPs) in relation to . Additional file Table S1 presents the classification of H. suis outer membrane proteins in relation to H. pylori outer membrane proteins. Although this table is not essential, we believe that it is both a relevant and interesting addition to the content of the article.Click here for file


Many edible plants are used in Cameroon since ancient time to control microbial infections. This study was designed at evaluating the antibacterial activities of the methanol extracts of ten Cameroonian vegetables against a panel of twenty nine Gram negative bacteria including multi-drug resistant (MDR) strains.β-Naphtylamide (PAβN), an efflux pumps inhibitor (EPI). The phytochemical screening of the extracts was performed using standard methods.The broth microdilution method was used to determine the Minimal Inhibitory Concentrations (MIC) and the Minimal Bactericidal Concentrations (MBC) of the studied extracts. When chloramphenicol was used as a reference antibiotic, the MICs were also determined in the presence of Phenylalanine-Arginine L. sativa, S. edule, C. pepo and S. nigrum being active on all the 29 bacterial strains tested meanwhile those from Amaranthus hybridus, Vernonia hymenolepsis, Lactuca.carpensis and Manihot esculenta were active on 96.55% of the strains used. The plant extracts were assessed for the presence of large classes of secondary metabolites: alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, steroids, tannins and triterpenes. Each studied plant extract was found to contain compounds belonging to at least two of the above mentioned classes.All tested extracts exhibited antibacterial activities, with the MIC values varying from 128 to 1024 mg/L. The studied extracts showed large spectra of action, those from These results confirm the traditional claims and provide promising baseline information for the potential use of the tested vegetables in the fight against bacterial infections involving MDR phenotypes. Amarantus hybridus Linn (Amarantaceae), Vernonia hymenolepis (H.F.) Hook., Lactuca sativa Linn. and Lactuca capensis Thumb. (Asteraceae), Manihot esculenta Crantz (Euphorbiaceae), Phaseolus vulgaris Linn (Fabaceae), Cucurbita pepo Linn and Sechium edule (Jacq) Sw. (Cucurbitaceae), Solanum nigrum Linn. and Capsicum frutescens L. (Solanaceae) against MDR bacteria expressing active efflux pumpsInfectious diseases are still a major health concern, accounting for 41% of the global disease burden measured in terms of Disability-Adjusted Life Years (DALYS), close to all noninfectious diseases 43%) and far more than injuries 16%)  in the f% and far%  in theAmarathus hybridus, Vernonia hymenolepis, Lactuca sativa, Lactuca capensis, Sechium edule, Manihot esculenta, Curcubiata pepo, Solanum nigrum, the cloves of the Green bean (Phaseolus vulgaris), and the fruits of Capsicum frutescens. These plants were identified by Mr Victor Nana of the National Herbarium (Yaoundé-Cameroon) where all the voucher specimens were deposited with the corresponding reference number  of each plant was extracted with methanol (MeOH) for 48 h at room temperature (25°C), using Whatman Grade No.1 filter paper and concentrated under reduced pressure, then dried to give the crude extracts. All extracts were stored at 4°C until further use.The major secondary metabolites classes such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, tannins, sterols and triterpenes were screened according to the common phytochemical methods previously described by Harbone, 1973 .Providencia stuartii, Pseudomonas aeruginosa, K. pneumoniae, Escherichia coli, Enterobacter aerogenes and Enterobacter cloacae  was used for all antibacterial assays.The studied bacteria included both reference (from the American Type Culture Collection) and clinical strains of 6 CFU/ml) prepared in MHB was then added. The plate was covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37°C for 18 h. The final concentration of DMSO was 2.5% and did not affected the microbial growth. Wells containing MHB, 100 μl of inoculum and DMSO at a final concentration of 2.5% served as negative control. The MICs of samples were detected after 18 h incubation at 37°C, following addition of 40 μl of a 0.2 mg/ml INT solution and incubation at 37°C for 30 minutes. Viable bacteria reduce this yellow dye to pink. MIC was defined as the lowest sample concentration that exhibited complete inhibition of microbial growth and then prevented this change [The MICs were determined using the rapid INT colorimetric assay ,72. Bries change . The MBCs change .Chloramphenicol, used as reference antibiotic, was tested also in the presence of the PAβN, at 30 mg/L final concentration to confirm the resistance of bacterial strains.A. hybridus contains anthocyanins, while triterpenes were found both in this extract as well as that of C. frutescens. The extract from C. frutescens as well as those from S. edule and M. esculenta contained the highest number of classes of the studied secondary metabolites (five). Alkaloids and phenols were present in all vegetable extracts except that of A. hybridus.The results of the qualitative analysis showed that each of the studied plant extract contains at least two classes of secondary metabolites such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, steroids, tannins and triterpenes Table . Only thC. frutescens showed inhibitory activities against 16 (55.17%) of the 29 tested bacteria whilst that of P. vulgaris inhibited the growth of 12/29 (41.38%) pathogens (narrowest spectrum). None of these two extracts showed any antibacterial activity against Pseudomonas species, but were active against at least one bacterial strain of other studied genus. Extracts from L. sativa, S. edule, C. pepo and S. nigrum displayed the largest spectra of activity, their inhibitory effects being observed on all the 29 Gam-negative bacteria (100% of activity). The extracts from A. hybridus, V. hymenolepis, L. sativa, L. carpensis and M. esculenta also exhibited large spectrum of activity as they were active on 28/29 tested bacteria. The top eight active extracts, with large spectra of activity, showed MIC values generally ranging from 128 to 512 μg/ml. These MIC values were in some of the cases better than those of choramplenicol (Table V. hymenolepis (MIC of 128 μg/ml) against E. aerogenes EA27. The extracts from A. hybridus, S. edule and C. pepo as well as those from L. capensis and M. esculenta were more active than chloramphenicol on at least one of the tested MDR bacteria. The activity of chloramphenicol increased in the presence of PAβN in the majority of the tested bacteria  tested bacteria. The extracts from P. vulgaris and C. frutescens did not show any MBC value at up to 1024 μg/ml. Concering the eight other vegetable extracts, the MBC results showed values equal to or below 1024 μg/ml in many cases. The extract from C. pepo leaves showed the best MBC spectrum with the values below to 1024 μg/ml recorded on 58,62% (17/29) of the studied microorganisms, followed by those from M. esculenta leaves on 51,72% (15/29), A. hybridus, V. hymenolepis and L. capensis extracts on 44.83% (13/29) and L. sativa on 31.03% (9/29) ,is its he plant .C. pepo, but these reports are based on the components of the fruits and the seed’s oil [Many reports have also been published about the biological properties of ed’s oil ,55,57,78P. vulgaris are in accordance with some other reports [Phaseolus vulgaris was found to inhibit also the growth of Gram-positive bacteria B.subtilis[P. vulgaris contains tannins with good antimicrobial properties against Listeria monocytogenes. Therefore, the low antibacterial effects of this plant as obtained herein   Table  could beC. frutescens against Staphylococcus aureus as well as K. pneumoniae and P. aeruginosa have been reported [S. aureus[The antibacterial effects of the extract from reported . The ethS. aureus. The preS. edule have already been proved against bacteria of clinical relevance by Ordonez et al. [” antimicrobial activities against MDR staphylococci and enterococci [The antibacterial properties of z et al.  which sherococci . Herein,erococci } observeM. esculenta possess antibacterial activities against Listeria monocytogenes, Vibrio cholerae, Shigella flexneri and Salmonella typhi whilst ethanol extract was found active against P. aeruginosa, Corynebacterium diphtheriae and V. cholera[M. esculenta against MDR strains of P. aeruginosa, E. coli, E. cloacae, K. pneumoniae, P stuartii and E. aeorogenes. The activity of Amaranthus hybridus was reported against E. coli, S. typhi, K. pneumoniae and P. aeruginosa with MICs ranged between 200 and 755 mg/ml [S. aureus and B. subtilis whilst the ethanol extract was found effective against E.coli[The chloroform extract of . cholera. This rest E.coli.P. vulgaris and C. frutescens which did not show any MBC below 1024 μg/ml, other values further confirmed the bactericidal effect of the 8 remaining extracts as they were generally less than 4-fold greater than corresponding MIC values [The high MIC values observed with chloramphenicol can be explained only if we take into account the non-specific resistance mechanism: active efflux of the toxic compound by pumps belonging to the small multidrug resistance (SMR) proteins family . The facC values ,83.The overall results of the present investigation confirmed the traditional uses of the studied vegetables in the treatment of bacterial infections. This study also provide baseline information for the possible use of the methanol extracts of the tested plant samples in the control of infectious diseases involving Gram-negative MDR bacteria. The arising question is of course which are the active compounds responsible for these effects. Our research group is currently focusing on the characterization of these plants extracts in terms of chemical composition and synergistic effects.The authors declare that they have no competing interest., MM, STL and MS carried out the study; VK designed the experiments. JAKN, MM and VK wrote the manuscript; VK and JRK supervised the work; VK provided the bacterial strains; all authors read and approved the final manuscript.JAKNThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/26/prepubBacterial strains and features. Click here for file


Vespadelus vulturnus Thomas, V. pumilus Gray, Miniopterus australis Tomes, Nyctophilus gouldi Tomes and Chalinolobus gouldii Gray) ranging in size from 4-14 g in coastal forest, using molecular analysis of fecal DNA. Abundances of potential mosquito and non-mosquito prey were concurrently measured to provide data on relative prey abundance. Aedes vigilax was locally the most abundant mosquito species, while Lepidoptera the most abundant insect order. A diverse range of prey was detected in bat feces, although members of Lepidoptera dominated, reflecting relative abundance at trap sites. Consumption of mosquitoes was restricted to V. vulturnus and V. pumilus, two smaller sized bats (4 and 4.5 g). Although mosquitoes were not commonly detected in feces of V. pumilus, they were present in feces of 55 % of V. vulturnus individuals. To meet nightly FMR requirements, Vespadelus spp. would need to consume ~600-660 mosquitoes on a mosquito-only diet, or ~160-180 similar sized moths on a moth-only diet. Lower relative profitability of mosquitoes may provide an explanation for the low level of mosquito consumption among these bats and the absence of mosquitoes in feces of larger bats. Smaller sized bats, especially V. vulturnus, are likely to be those most sensitive to reductions in mosquito abundance and should be monitored during mosquito control activities.Insectivorous bats have often been touted as biological control for mosquito populations. However, mosquitoes generally represent only a small proportion of bat diet. Given the small size of mosquitoes, restrictions imposed on prey detectability by low frequency echolocation, and variable field metabolic rates (FMR), mosquitoes may not be available to or profitable for all bats. This study investigated whether consumption of mosquitoes was influenced by bat size, which is negatively correlated with echolocation frequency but positively correlated with bat FMR. To assess this, we investigated diets of five eastern Australian bat species ( Mosquitoes may cause serious nuisance biting and serve as vectors of mosquito-borne pathogens such as Ross River virus (RRV) and Barmah Forest virus (BFV) . In respCulex spp. associated with bat predation. However, the suggestion by Tuttle =0.491, p=0.487; F[62]=0.462, p=0.500; F[62]=3.463, p=0.070; Total nightly insect abundance during spring tides (267.29 ± 9.69) was not significantly different to total nightly insect abundance during neap tides (286.00 ± 8.90) (F(1) = 1.982, p = 0.166; Ae. aegypti produced readable sequences that provided species-level matches to either T. molitor (0 % mosquito) or Ae. aegypti (100 % mosquito) , the proportion of sequences corresponding to mosquito DNA in clone libraries do not correspond to ratios of mosquito-mealworm in the original samples .All mosquito-mealworm mixtures produced visible PCR products after amplification. Mosquito-mealworm mixtures containing 0 % and 100 % osquito) . For eacosquito) . Sequencmixtures . These rC. gouldii, V. pumilus and V. vulturnus, but were not commonly present in fecal samples  (Aedes vigilax was detected only in the feces of two bat species (V. pumilus and V. vulturnus). Mosquitoes were present in the feces of 2 of 10 V. pumilus individuals, both of which were trapped during neap tides (Jan and Mar). Mosquitoes were detected in the feces of 11 of 20 V. vulturnus individuals, trapped during spring and neap tides (Dec-Feb). A total of 40 prey taxa were identified from the feces of 52 insectivorous bats representing five species. Lepidopterans were the most frequently detected prey, present in the feces of 49 individuals . Coleopt1 of 20) . Diptera1 of 20) . Aedes vChalinolobus gouldii, N. gouldi, V. pumilus and V. vulturnus all consumed prey in proportion to the abundance of arthropods in light trap collections .Ae. vigilax consumption and abundance of lepidopterans, coleopterans, other insects or all insects combined (r(7)=0.018, P=0.969; r(7)=0.274, P=0.552; r(7)=-0.226, P=0.627; r(7)=0.178, P=0.703). A significant positive relationship between Ae. vigilax consumption and dipteran (culicid and non-culicid) abundance was observed (r(7)=0.889, P=0.007). Correlation analyses revealed no significant relationships between V. pumilus and V. vulturnus). However, the proportion of individuals of these species that consumed mosquitoes was low. Although mosquitoes were not a common prey item consumed by smaller sized bats in coastal forest, it is not possible to fully assess their importance as a prey item for bats in the study area as bats were not trapped in saltmarsh where small prey might be most efficiently preyed upon.This study used prey DNA within bat feces to investigate whether consumption of mosquitoes was restricted to small bats with high frequency echolocation calls in the study area, whose activity was correlated with mosquito abundance . AlthougSince sampling of bats was undertaken in the course of one field season, the sample size for most species investigated was relatively small. Additionally, some DNA sequences had low (<98 %) similarity to reference sequences in BOLD and these identifications should be treated conservatively. Consequently, interpretation of dietary results must be taken with caution. Although it was possible to identify prey in bat feces using molecular techniques, it remains impossible to quantify the amount of prey consumed. While at least two studies have discussed the potential use of clone library proportions to infer quantitative information about consumed prey ,38, give2-baited EVS traps during the study, Ae. vigilax was consistently the most abundant, irrespective of tidal cycle. This trend has been observed during long-term mosquito surveillance in the study area, in which Ae. vigilax represented 41.2 % of all mosquitoes trapped over nine consecutive trapping seasons . The consistent presence of highly abundant populations of this species in the study area provides bats, particularly small sized species, with a consistent prey resource during summer. Nightly abundance of Aedes vigilax populations recorded in the forest habitat during this study (364.67 ± 86.96 and 229.10 ± 60.00 during neap and spring tides) was comparable to nightly abundance of Ae. vigilax populations recorded in saltmarsh habitats over the same period in 2008-09 (528.2 ± 347.9 and 261.1 ± 127.5 during neap and spring tides) [While fourteen mosquito species were represented in COg tides) .The most abundant taxa in light trap collections were lepidopterans. While it is acknowledged that certain insect taxa may be more attracted to particular attractant traps  and therAe. vigilax (229±60 during spring tides and 365±87 during neap tides) suggests that mosquitoes, like lepidopterans, represent a highly abundant prey resource in the study area. Since two different trapping techniques were used to survey mosquito populations and aerial insect fauna, it is not possible to directly compare the abundance of mosquitoes to the abundance of insects in light trap collections. However, the abundance of The diets of the five insectivorous bat species we investigated consisted of a diverse range of prey. Previous dietary studies of the five bat species also report a diverse range of prey ,45,46. WAe. vigilax was only detected in the feces of V. pumilus and V. vulturnus. Mosquitoes were detected in < 60 % of individuals of these species , though the numbers consumed are unknown given the molecular techniques used can only provide presence/absence data. These two congeneric species are morphologically similar with echolocation calls that overlap to such a degree that it is not possible to differentiate between the two species in the study area [Ae. vigilax. However, similar to findings from studies of diets of many medium-large sized European bats [N. gouldi and M. australis) on occasion consumed prey that were smaller than Ae. vigilax .The prey detected in the feces of all bats reflected the locally abundant prey taxa. However, udy area . Both baean bats ,25,47, tC. gouldii; 25-34 kHz) supports the suggestions of Barclay and Brigham [Generally, smaller predators acquire small prey, while larger predators are capable of consuming both, small and large prey . However Brigham . N. gouldi and M. australis both consumed prey smaller than mosquitoes, the absence of mosquitoes from their feces, given mosquito DNA was still detectable when mosquitoes were present as 5 % of insect material in artificial bat feces , suggests that it is unlikely that larger (> 6 g) bats actively seek mosquitoes as prey in forest. Additionally, N. gouldi employs a gleaning foraging strategy, with microscopy revealing a diet predominantly consisting of moths in other areas [While er areas . Gleaniner areas . Additio-1 (V. vulturnus) and 19.51 kJd-1 (V. pumilus). If it is assumed that the two bat species were specialist foragers and consumed only mosquitoes, V. vulturnus and V. pumilus would be required to consume ~604 and ~659 mosquitoes, respectively, each night just to maintain day-to-day activity . Converenergy - ). While Ae. vigilax emerges and proportional feeding activity by bats is greatest [Ae. vigilax in association with fluctuations in the abundance of the mosquito in the two habitats [Despite the low energetic profitability of mosquitoes relative to other prey, bats may still pursue them if they are extremely abundant , particugreatest . Bats argreatest . Similarhabitats . V. vulturnus. This species is likely to be most sensitive to reductions in mosquito abundance and should be monitored during mosquito control activities. While it is beyond the scope of this study to infer potential impacts of broadscale mosquito control and the associated reductions in larval mosquito populations on insectivorous bat diet and health, declines in bat populations have previously been attributed to deteriorating feeding conditions . In AustMethods S1(DOCX)Click here for additional data file.Results S1(DOCX)Click here for additional data file.


Epidermophyton floccosum revealed susceptibility to more tested agents than those of Trichophyton mentagrophytes. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common pharmacological group that has three primary therapeutic effects including anti-inflammatory, anti-pyrexia, and analgesia. In this study, seven of NSAIDs were tested against two species of skin pathogenic fungi (dermatophytes). Percentage inhibition was determined for effective agents. Diclofenac, Aspirin and Naproxen showed much more ability to inhibit dermatophytes growth. In conclusion, many of NSAIDs may have the ability to inhibit pathogenic fungi. Others may also have potential activity toward fungal growth. Candida albicans  or Diclofenac revealed strong and dose-dependent inhibition of colonies growth  are the centerpiece of pharmacotherapy for most rheumatological disorders and are used in large numbers as analgesic and antipyretics . In addialbicans . Furthers growth . Sulindais agent . Herman  Aspirin . Trichophyton, Microsporum, and Epidermophyton . Naproxen was supplied from Medical Bahri Company, (Damascus (AI-Gassoulaeh), Syria). Piroxicam was supplied from Shaghai Pharmaceutical Co. LTD, . Celecoxib was supplied from Micro-Labs, Limited, Sipcot Industrial Complex . Mefenamic acid was supplied from Brown and Burk Pharmaceutical Limited, Spicot, . Aspirin was supplied from SDI, .Antifungal assay°C for 1 week. Perpendicular colony diameters (mm) of grown strains were measured and percentage inhibition calculated according the formula: The colony diameter method recorded by Kücüc and Kivan  was usedPercentage inhibition=(C-T)×100CWhere: C = colony diameter (mm) of the control; T = colony diameter (mm) of the test plate.Minimal inhibitory concentrations determination (MIC)4 cfu/mL. MIC was performed according to Santos and Hamdan . Serial °C and were read visually after 7 days of incubation.Microdilution plates (96 wells) were set up in accordance with the NCCLS reference method . Each miT. mentagrophytes and at 280 μg/mL for E. floccosum. Aspirin and Naproxen also ceased fungal development through decreased colonies diameter of growing fungi, but less than Diclofenac  as the first NSAID in 1897 , NSAIDs Candida albicans did not influence by Mefenamic acid, Meloxicam or Naproxen , a hemeprotein that exists in two isoforms (COX-1 and COX-2), and converts arachidonic acid to prostanoids such as prostaglandin . NSAIDs COX-1 is important in the protection of human gastrointestinal mucosa. Inhibition of this enzyme can result in formation of ulcers. On the other hand, stimulation of COX-2 results in pain and inflammation . TherefoDipodascopsis uninucleata fungus can use its COX to incorporate arachidonic acid within lipids synthesis  In addition to mammalian, COX can be found in several species of fungi. ynthesis . Cells oCandida albicans and Cryptococcus neoformans can secrete prostaglandins de nova or via conversion of exogenous arachidonic acid   , otherwioformans .Aspergillus nidulans (Dermatophytes, the main subject of this study, also have the ability to produce prostaglandin that responsible for chronic fungal colonization . Therefonidulans  or functnidulans .The usage of NSAIDs at therapeutic doses could cause many adverse effects. Some of NSAIDs cause less damage in human than others, especially Celecoxib or Rofecoxib  and coatIn conclusion, NSAIDs contain many promising agents within their group which can be used against dermatophytes. Diclofenac, Aspirin and Naproxen are more effective on two main species of dermatophytes. Other NSAID agents have variable actions on dermatophytes, based on fungal species.


Chorispora bungeana is a subnival alpine plant possessing strong cold tolerance mechanisms. To get a deeper insight into its cold tolerance mechanisms, the transcriptome profiles of chilling-treated C. bungeana seedlings were analyzed by Illumina deep-sequencing and compared with Arabidopsis.The plant tolerance mechanisms to low temperature have been studied extensively in the model plant Arabidopsis at the transcriptional level. However, few studies were carried out in plants with strong inherited cold tolerance. de novo assembly, and 3,484 chilling up-regulated and 4,571 down-regulated unigenes were identified. The expressions of 18 out of top 20 up-regulated unigenes were confirmed by qPCR analysis. Functional network analysis of the up-regulated genes revealed some common biological processes, including cold responses, and molecular functions in C. bungeana and Arabidopsis responding to chilling. Karrikins were found as new plant growth regulators involved in chilling responses of C. bungeana and Arabidopsis. However, genes involved in cold acclimation were enriched in chilling up-regulated genes in Arabidopsis but not in C. bungeana. In addition, although transcription activations were stimulated in both C. bungeana and Arabidopsis, no CBF putative ortholog was up-regulated in C. bungeana while CBF2 and CBF3 were chilling up-regulated in Arabidopsis. On the other hand, up-regulated genes related to protein phosphorylation and auto-ubiquitination processes were over-represented in C. bungeana but not in Arabidopsis.Two cDNA libraries constructed from mRNAs of control and chilling-treated seedlings were sequenced by Illumina technology. A total of 54,870 unigenes were obtained by C. bungeana, a subnival alpine plant with inherited cold tolerance. Comparative transcriptome analysis suggests that cold acclimation is not a major chilling tolerance mechanism of C. bungeana. Activation of protein phosphorylation and ubiquitination may confer chilling tolerance to C. bungeana in a more rapid and flexible way than cold acclimation. Such differences may have contributed to the differences in cold tolerance between C. bungeana and Arabidopsis. The results presented in this paper will be informative for gene discovery and the molecular mechanisms related to plant cold tolerance.We conducted the first deep-sequencing transcriptome profiling and chilling stress regulatory network analysis of  Chorispora bungeana Fisch. & C.A. Mey (C. bungeana) is a perennial subnival alpine plant that can survive freezing temperature.C. bungeana unigene was normalized by the number of RPKM (reads per kilobase exon region per million mapped reads)], and in the NCBI SRA repository, [http://www.ncbi.nlm.nih.gov/sra?term=SRA054354].The data sets supporting the results of this article are available in the NCBI GenBank repository, [COR: Cold-responsive; CBF: CRT binding transcription factor; RNA-seq: RNA sequencing; DEG: Differentially expressed gene; GO: Gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; POG: Putative orthologous gene; qPCR: Quantitative real-time PCR; TF: Transcription factor.The authors declare that they have no competing interests.ZZ designed the experiments and drafted the manuscript (zgzhao@lzu.edu.cn). LT contributed to data analysis and interpretation (tanll@lzu.edu.cn). CD prepared plant materials and carried out qPCR analysis (tsdcy2006@126.com). HZ participated in plant preparations (zhanghua@lzu.edu.cn). QW provided part of the financial support (qbwu@lzb.ac.cn). LA conceived of the study and provided financial support for the project (lizhean@lzu.edu.cn). All authors read and approved the final manuscript.Complete list of unigenes with BLASTX hits.Click here for fileComplete list of chilling regulated DEGs identified with fold change > =2 and q < 0.001.Click here for fileComplete list of chilling regulated DEGs identified with fold change > =2, q < 0.001 and RPKM > =1.Click here for fileChilling up-regulated TFs. 1. List of chilling up-regulated TFs in both ATH-SR and ATH-MA. 2. List all chilling up-regulated TFs in Arabidopsis (ATH-SR or ATH-MA). 3. All chilling up-regulated TFs (orthologs) in C. bungeana.Click here for fileList of chilling up-regulated protein serine/threonine kinase in C. bungeana.Click here for fileList of chilling regulated DEGs and all expressed genes of ATH-SR. 1. List of chilling up-regulated DEGs (SR). 2. List of chilling down-regulated DEGs (SR). 3. List all genes RPKM > =1 (SR).Click here for fileList of chilling regulated DEGs and all expressed genes of ATH-MA. 1. List of chilling up-regulated DEGs (MA). 2. List of chilling down-regulated DEGs (MA). 3. List all expressed genes (MA).Click here for filePrimers and reference gene selections. 1. Primers for reference gene selection. 2. Primers for qPCR verification. 3. Unigenes for qPCR reference gene selection. 4. geNorm results of reference gene selection.Click here for file


Panax notoginseng (Burk) F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown.P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST) similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS), which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158) and UDP-glycosyltransferase (Pn00082) gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH), and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR) were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif.Using the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root acquired by next-generation sequencing (NGS) technology. The candidate genes involved in triterpene saponin biosynthesis, including the putative CYP450s and UGTs, were obtained in this study. Additionally, the identification of SSRs provided plenty of genetic makers for molecular breeding and genetics applications in this species. These data will provide information on gene discovery, transcriptional regulation and marker-assisted selection for P. notoginseng. The dataset establishes an important foundation for the study with the purpose of ensuring adequate drug resources for this species.This study is the first to present a large-scale EST dataset for  Panax notoginseng (Burk) F.H. Chen is highly valued medicinal plant of the Araliaceae family [P. notoginseng is distributed in the southwestern region of China, Burma and Nepal [P. notoginseng is cultiviated commercially in Wenshan County, Yunnan province [P. notoginseng include triterpene saponins, non-protein amino acids, polyacetylenes, phytosterols, flavonoids, and polysaccharides, many of which have pharmacological activities and are useful in the treatment of some diseases [P. notoginseng including ginsenosides, notoginsenosides, and gypenosides [P. notoginseng root [Panax ginseng) and American ginseng (Panax quinquefolius), have not been found in P. notoginseng based on the evidence from phytochemical studies [e family . P. notond Nepal . Presentnd Nepal . In Chinprovince . The rooprovince . The ingdiseases . Among tdiseases -7. Approenosides . The majeng root . All theeng root . The ole studies .dammarenediol synthase (DS), β-amyrin (β-AS), lupeol synthase (LS) and cycloartenol synthase (CAS) have been isolated in plants [P. ginseng [Panax plants, are proposed to catalyze the conversion of dammarenediol-II or β-amyrin to various ginsenosides and the modification on ginsenosides. In our previous study, one candidate CYP450 and four candidate UGTs most likely to be involved in ginsenoside biosynthesis have been selected from P. quinquefolius [P. quinquefolius root based on the analysis of tissue-specific expression pattern and methyl jasmonate (MeJA) induction [Triterpene saponins are synthesized via the mevalonic acid (MVA) pathway , which in plants -13. The  ginseng ,14. Howeuefolius . These cnduction .P. notoginseng are very limited and only 95 ESTs are available in the National Center for Biotechnology Information (NCBI) database. The limited transcriptomic data hinder the study of triterpene saponin biosynthetic mechanisms in P. notoginseng. Expressed sequence tag (EST) analysis is a useful tool for the purposes of gene discovery especially in non-model plants for which no reference genome sequences are available [Saponaria vaccaria [Glycyrrhiza uralensis [Despite its pharmacological importance, the transcriptomic and genomic data of vailable . ESTs revailable ,18 and cvailable . The disvailable ,20,21. Tvaccaria . The licralensis . In addiralensis . The nexralensis -27.P. notoginseng root using NGS technology based on 454 GS FLX Titanium platform. Our ultimate goal is to discover the candidate genes that encode enzymes in the triterpene saponin biosynthetic pathway and provide an overview of transcriptome, as well as produce molecular markers of EST-SSRs for facilitation the marker-assisted breeding of this species.Herein we present the results of the study designed to characterize the transcriptome of P. notoginseng root produced 188,185 reads with an average length of 410 bp , NCBI non-redundant protein (Nr), and NCBI non-redundant nucleotide (Nt) database. The number and percentage of the annotated unique sequences were summarized in Additional file P. notoginseng unique sequences were annotated and the remaining (29.8%) unique sequences had no match to any sequences in the public databases  were performed. The result of the comparison between P. notoginseng and P. quinquefolius [P. notoginseng unique sequences had sequence similarity to P. quinquefolius unique sequences, and the remained 11,626 unique sequences were the P. notoginseng special transcripts. Similarly, a total of 19,479 P. notoginseng unique sequences had sequence similarity to P. ginseng unique sequences [P. notoginseng special transcripts.The annotation for es Table . The annius root  and P. gemaining .8% uniquuefolius  and P. gemaining .8% uniquuefolius  unique semaining .8% uniquP. notoginseng root were shown in Table P. notoginseng transcript  had no hit to any sequences of the public databases. The transcripts encoding enzymes associated with sugar and energy metabolism were highly expressed in this cDNA library, including 1,4-alpha-glucan-branching enzyme protein, sucrose synthase isoform, and formate dehydrogenase (Table P. notoginseng root. It is noteworthy that three transcripts encoding the key enzymes of Acetyl-CoA acetyltransferase (AACT), squalene epoxidase (SE) and dammarenediol synthase (DS) were also abundant in P. notoginseng root  numbers . 16,300 P. notoginseng unique sequences with 8.98%  frequency  are the most feasible genetic markers for plant breeding and genetic applications . A total species . These mP. notoginseng. In particular, several SSR motifs were linked with the unique sequences encoding enzymes  involved in triterpene saponin biosynthesis  families  was found in the cDNA library. The alignment of amino acid sequences of DS from P. ginseng, P. notoginseng, and P. quinquefolius was shown in Figure P. quinquefolium and P. notoginseng were deposited in NCBI GenBank with Accession No. GU997679 and GU997680, respectively. All three DSs have 769 amino acid residues and display high similarity. At the protein level, DSs exhibit 99.5% identity between P. ginseng and P. quinquefolius, 99.0% identity between P. quinquefolius and P. notoginseng, and 98.7% identity between P. ginseng and P. notoginseng, indicating that P. ginseng and P. quinquefolius are more closely related. This conclusion agrees with that drawn from the alignment of TrnK and 18S rRNA sequences [Most of all the known enzymes involved in MVA pathway for triterpene saponin biosynthesis were discovered in et Table . As showet Table . After tet Table . Dammara ginseng ,14. It iequences .P. notoginseng based on the phytochemical studies [P. notoginseng singleton sequences (FW1NBNE02D50IR and FW1NBNE02D30JP) matched to β-AS of Panax ginseng. The presence of the β-ASs in P. notoginseng was further confirmed by RT-PCR and the PCR products were sequenced (Data not shown). The existence of the transcripts for β-ASs in P. notoginseng was seemingly conflicted with previous reports claiming that oleanane-type ginsenosides do not exist in P. notoginseng [P. notoginseng at levels too low to be detected phytochemically, or oleanane-type ginsenosides in fact did not exist in P. notoginseng, despite the presence of β-AS, might due to the lack of biosynthetic genes downstream of β-AS.The oleanane-type saponin (Ro) has not been detected in  studies . Surprisoginseng . TherefoPanax genus will facilitate to elucidation of the triterpene saponin biosynthetic pathway. CYP450s are generally involved in the biosynthesis of terpenoids, sterols, lignins, hormones, fatty acids, pigments, and phytoalexins in plants [Glycyrrhiza uralensis (CYP85 clan) [Glycine max (CYP71 clan) [Panax genus. Glycosylation, catalyzed by glycosyltransferases (GTs), transfers the activated saccharides to an aglycone substrate in the modification on ginsenoside biosynthesis. This enzymatic conjugation based on glycosylation can stabilize the product and alter its physiological activity [Medicago truncatula [Saponaria vaccaria [ found as candidate genes involved in ginsenoside biosynthesis in P. quinquefolius and P. ginseng in our previous studies [P. quinquefolius [Characterization of specific CYP450s or UGTs involved in triterpene saponin biosynthesis in n plants . Some CYn plants ,45. Prev85 clan)  and CYP971 clan) , both ofactivity . The damuncatula  and UGT7vaccaria  have bee studies ,29. Partuefolius .P. notoginseng cDNA library. As reported for P. ginseng and other plants, enzymes in the same biosynthetic pathway are usually co-expressed [DS was expressed abundantly  in the root of P. notoginseng, indicating that other genes in the triterpene saponin biosynthetic pathway might also expressed at higher levels in the P. notoginseng root. Based on this knowledge, CYP450- and UGT-unique sequences that contain more than 10 reads from the P. notoginseng root cDNA library were found as candidate enzymes involved in triterpene saponin biosynthesis. Thus, 25 CYP450s and 16 UGTs were selected, among which 15 CYP450s and 8 UGTs had full-length sequences after assembly and using RACE (rapid amplification of cDNA end) method. The primers used for RACE were listed in Additional file CYP450s and GU997656-GU997663 for UGTs.In this study, a total of 174 CYP450  [The phylogenetic relationship between the15 full-length CYP450s of yrrhizin   . TherefoP. notoginseng UGT sequences to other functionally characterized members of plant UGT families . The total RNA was treated with DNase I  at a concentration of 1.5 units/μg of total RNA. The RNA quality was tested using 1% ethidium bromide-stained (EtBr-stained) agarose gels and the concentration was assessed using a GeneQuant100 spectrophotometer  prior to cDNA synthesis.P. notoginseng according to the instructions provided with Clontech's SMART cDNA synthesis kit  with slight modifications as our previous study [GT GCA GT(20)VN-3') containing a BsgI digestion site upstream of the poly (T) segment was used in combination with the Clontech SMART IV primer to synthesize the first-strand cDNA. The cDNA was amplified using PCR Advantage II polymerase  to synthesize the double-strand cDNA (ds cDNA) with the following thermal profile: 1 min at 95°C followed by 19 cycles of 95°C for 15 sec, 65°C for 30 sec, and 68°C for 6 min. And then, 5 μl of PCR product were electrophoresed in a 1% agarose gel to determine the amplification efficiency and quality. Approximately 13 μg of amplified ds cDNA was purified using the PureLink ™ PCR purification kit . Then the purified cDNA was treated with BsgI  overnight at 37°C and recovered by QIAquick PCR Purification Kit . Finally, a total of 10 μg of ds cDNA was used for pyrosequencing with the GS FLX Titanium Kit.The first-strand cDNA was produced using 2.1 μg of total RNA extracted from the root of us study : in ordeDe Novo Assembler software v2.0.01.14  with a quality score threshold set at 40.The 454 raw read sequences were screened and trimmed for weak signals by GS FLX pyrosequencing software to yield high-quality (HQ) (> 99.5% accuracy of single-base reads) sequences. The resulting HQ reads were then submitted to the Short Read Archive at NCBI and assigned the accession number SRX017444. The primer and adapter sequences were trimmed from the HQ sequences, followed by removing the sequences shorter than 50 bp from the clean ESTs before assembly. Then, the data from the 454 read sequences were assembled into unique sequences (including contigs and singletons) using 454 GS E-value cut-off of 10-5 to find and remove ribosomal RNA sequences [Arabidopsis protein database at The Arabidopsis Information Resource  (version Tair9), SwissProt protein database , and the NCBI non-redundant protein (Nr) database  using the BLASTX algorithm with an E-value cut-off of 10-5. The functional categories of these unique sequences were further analyzed using the Gene Ontology (GO) database. The unique sequences were categorized according to GO terms based on AGI codes and TAIR GO slim provided by TAIR.The assembled unique sequences were first searched for sequence similarities against the NCBI non-redundant nucleotide (Nt) database using the BLASTN algorithm with equences . And thehttp://www.genome.ad.jp/kegg/kegg2.html) (version KEGG 50) [E-value cut-off of 10-5 upon against the KEGG database. The unique sequences were assigned to special biochemical pathways according to the corresponding EC distribution in the KEGG database.Pathway assignments were carried out according to the Kyoto Encyclopedia of Genes and Genome (KEGG) mapping (KEGG 50) . Enzyme http://www.gramene.org/db/markers/ssrtool) [The total unique sequences were searched to determine the composition, frequency, and distribution of simple sequence repeats (SSRs) using an online SSR identification tool - SSRIT (Simple Sequence Repeat Identification Tool) (ssrtool) . The seaDS transcript was much more abundant in P. notoginseng , suggesting the other genes encoding enzymes in the same biosynthetic pathway were also expressed at much higher levels in the former species. For CYP450s and UGTs, each isozyme with more than ten reads in P. notoginseng is arbitrarily considered a candidate involved in the biosynthesis of triterpene saponins. The screening of CYP450s and UGTs was performed according to phylogenetic analysis.The P. notoginseng root and cloned into T easy Vector  for Sanger sequencing. Then the full-length cDNA of each gene was generated by assembly of the corresponding EST sequence and 5' and/or 3'end sequences.Primers listed in Additional file Distances between each clone were calculated with the CLUSTAL W program . The indMeJA: methyl jasmonate; NCBI: National Center for Biotechnology Information; NGS: next-generation sequencing; RACE: rapid amplification of cDNA end; SSR: simple sequence repeat; TAIR: the Arabidopsis Information Resource; UGT: UDP- glucosyltransferase.β-AS: β-amyrin synthase; BLAST: basic local alignment search tool; bp: base pair; cDNA: complementary DNA; CYP450: cytochrome P450; DS: dammarenediol-II synthase; EST: expressed sequence tag; GGDP: geranylgeranyl diphosphate; GO: gene ontology; GT: glucosyltransferase; KEGG: Kyoto encyclopedia of genes and genomes; The authors declare that they have no competing interests.HML contributed to the cDNA sample preparation, data analysis and manuscript writing. CS and YZS participated in the design of the study, the tissue sample collection, and the data analysis. QW helped with RNA extraction, gene cloning and the generation of the phylogenetic tree for CYP450s and UGTs. YL contributed to the bioinformatic analysis and helped with the RNA extraction. YYN helped with RNA extraction and cDNA amplification. JYS, XLC, HXX, CYL, JYL and AS participated in the study design and discussed the results. This work was conducted in the laboratory of SLC, who initiated the 454-sequencing projects and contributed to the evaluation and discussion of the results as well as to the revision of the manuscript. All authors have read and approved the final manuscript.P. notoginseng 454-ESTs as compared to public databasesSummary of the annotation percentage of . The annotation for P. notoginseng unique sequences was based on sequence similarity searches against public databases including SwissProt, KEGG, TAIR, NCBI non-redundant protein (Nr), and NCBI non-redundant nucleotide (Nt) database. A total of 70.2% of P. notoginseng unique sequences were annotated by BLAST searches against the above public databases.Click here for fileP. notoginseng unique sequences to KEGG biochemical pathwaysMapping of . List of the number of P. notoginseng unique sequences involved in metabolism, genetic information processing, environmental information processing, cellular processes, human diseases, unclassified and unassigned in the 454-EST dataset.Click here for fileP. notoginseng unique sequences involved in the biosynthesis of secondary metabolitesThe . The number of unique sequences involved in the biosynthesis of alkaloid, brassinosteroid, caffeine, carotenoid, diterpenoid, flavone and flavonol, flavonoid, limonene and pinene, monoterpenoid, novobiocin, phenylpropanoid, streptomycin, terpenoid, tetracycline and zeatin.Click here for fileP. notoginsengThe detection of SSR motifs in the unique sequences of . The list of unique sequences (including contigs and singletons) containing potential microsatellite loci including the name of unique sequence, motif (SSR-repeat type), number of repeat, SSR start, SSR end and sequence length from P. notoginseng 454-EST dataset.Click here for fileThe discovery of SSR motifs in the putative triterpene saponin-biosynthetic genes. The SSR motifs were detected in the putative triterpene saponin-biosynthetic genes including AACT (acetyl-CoA acetyltransferase), HMGR (HMG-CoA reductase), SS , SE  and DS (dammarenediol-II synthase).Click here for fileP. notoginseng unique sequences encoding putative transcription factors based on Inter-Pro searchesThe . The 906 unique sequences of P. notoginseng containing transcription factor domains using Inter-Pro searches.Click here for fileP. notoginseng using Inter-ProMajor transcription factor families identified from . The unique sequences from P. notoginseng with similarities to genes encoding transcription factors.Click here for fileCytochrome P450 discovery. The unique sequences from P. notoginseng with sequence similarities to cytochrome P450s.Click here for fileGlycosyltransferase discovery. The unique sequences from P. notoginseng with sequence similarities to glycosyltransferase.Click here for fileThe primers used for RACE in this study. The primers used in 5'-RACE for the amplification of unique sequences including Pn01024, Pn02132, Pn03717, and Pn00788.Click here for file


Chiroptera) are one of the most diverse groups of mammals which carry out important ecological and agricultural functions that are beneficial to humans. However, they are increasingly recognized as natural vectors for a number of zoonotic pathogens and favourable hosts for zoonotic infections. Large populations of the Straw-Coloured Fruit Bat (Eidolon helvum) colonize the main campus of the Obafemi Awolowo University (OAU), Ile-Ife, Nigeria, but the public health implications of faecal contamination and pollution by these flying mammals is unknown. This study characterized S. aureus obtained from faecal samples of these migratory mammals with a view to determining the clonal types of the isolates, and to investigate the possibility of these flying animals as potential reservoir for zoonotic S. aureus infections.Bats  were assigned in coagulase type VI, while accessory gene typing classified 69 isolates into the following: type I , type II , type III  and type IV . On the whole, the isolates were grouped in five (A-E) main genotypes. Of the ten representative isolates selected for multilocus sequence typing (MLST), nine isolates were assigned with new sequence types: ST1725, ST1726, ST1727, ST2463-ST2467 and ST2470. Phylogenetic analysis provided evidence that S. aureus isolates in group C were closely related with ST1822 and associated clones identified in African monkeys, and group D isolates with ST75, ST883 and ST1223. The two groups exhibited remarkable genetic diversity compared to the major S. aureus clade.One hundred and seven (107) S. aureus isolates of E. helvum is low and multiple unique S. aureus lineages co-existed with E. helvum. The Straw-Coloured Fruit Bat in Ile-Ife, Nigeria is colonized predominantly by ST1725, ST1726, ST2463 and ST2470 with distinct genotypic characteristics that are rarely found in humans. This study has demonstrated on the possible existence of a reservoir of indigenous and anciently-divergent S. aureus clones among mammals in Africa.Antibiotic resistance in faecal  Chiroptera) are the only mammals capable of true sustainable flight and one of the most diverse and species rich mammals on earthagr[39] ae typing. The MAFer. 5.05. Furtherer. 5.05 was carr CCM7213. A phyloE. helvum: Eidolon helvum; S. aureus: Staphylococcus aureus; MSA: Mannitol salt agar; DNase: Deoxyribonuclease; CLSI: Clinical Laboratory Standards Institute; MRSA: Methicillin resistant Staphylococcus aureus.OAU: Obafemi Awolowo University; PVL: Panton Valentine Leukocidin; Agr: Accessory gene regulator; Coag: Coagulase; MLST: Multilocus sequence typing; ST: Sequence type; The authors declare that they have no competing interest.AS, OA, TS conceived the study, BA conducted the sample collection, preliminary identification and susceptibility testing of the isolates; TS carried out the molecular characterization. All authors read and approved the final version of the manuscript.


Podospora anserina and Sordaria macrospora with closely related non-coprophilous members of the order Sordariales. An increase of MCO genes in coprophilic members of the Sordariales most probably occurred by gene duplication and horizontal gene transfer events.Multicopper oxidases (MCO) catalyze the biological oxidation of various aromatic substrates and have been identified in plants, insects, bacteria, and wood rotting fungi. In nature, they are involved in biodegradation of biopolymers such as lignin and humic compounds, but have also been tested for various industrial applications. In fungi, MCOs have been shown to play important roles during their life cycles, such as in fruiting body formation, pigment formation and pathogenicity. Coprophilous fungi, which grow on the dung of herbivores, appear to encode an unexpectedly high number of enzymes capable of at least partly degrading lignin. This study compared the MCO-coding capacity of the coprophilous filamentous ascomycetes  A largeMagnaporthe grisea [Ophiostoma novo-ulmi [Gaeumannomyces graminis [Aspergillus nidulans [Penicillium chrysogenum [Stachybotrys chartarum [Trichoderma reesei [Xylaria species [Melanocarpus albomyces has been solved as one of the first complete laccase structures [While plant laccases mainly participate in lignin polymer formation, fungal laccases are involved in the degradation of lignin and humic acids, but also have important roles in developmental processes such as fruiting body development and pigmentation. Because they oxidize, polymerize, or transform phenolic or anthropogenic compounds into less toxic derivatives, fungal laccases have been used for various biotechnological applications such as food processing, bioremediation of waste water, and removal of lignin from wood fibers -7. To dae grisea , Ophiostovo-ulmi  and Gaeugraminis , 12 as wnidulans  and Peniysogenum  and fresysogenum . In addihartarum  and Trica reesei  and in w species . The thrructures , 20.Podospora anserina  revealed an unexpectedly large number of putative ligin-degradating enzymes, among them several laccases [P. anserina lives exclusively on the dung of herbivores and usually fructifies at the late stage of dung decomposition when simple carbohydrates are depleted. The genome of P. anserina evolved a more comprehensive coding capacity for enzymes that degrade complex biopolymers than the close relative Neurospora crassa, which in nature is often found on scorched vegetation after wildfires or agricultural burns [P. anserina, Chaetomium globosum is also publicly available (http://www.broadinstitute.org/annotation/genome/chaetomium_globosum). Recently, the genome sequence of another coprophilous member of the Sordariales, Sordaria macrospora, was published [P. anserina, S. macrospora in nature lives exclusively on the dung of herbivores and also fructifies at the late stages of dung decomposition [C.  globosum is a wood-destroying fungus causing the so-called mildew-rot [N. crassa and P. anserina were purified to apparent homogeneity by classical purification techniques [P. anserina produce multiple laccases isoenzymes [N. crassa and P. anserina were cloned [Analysis of the genome of the coprophilous fungus laccases , 22. In al burns . The genublished . Similarposition . C.  gloldew-rot . It is fldew-rot . In the chniques , 29 Furte cloned , 31. TheThe data presented here revealed gene duplication, acquisition and gene loss events in the evolutionary history of MCO genes in the genomes of Sordariales members. An increase in MCO genes in coprophilic members most probably occurred by gene duplication and horizontal gene transfer events and might contribute to their ability to grow in an extremely competitive habitat.2.2.1.http://www.broad.mit.edu/annotation/fgi/), (http://podospora. igmors.u-psud.fr) and the Institut für Allgemeine und Molekulare Botanik Ruhr-Universität Bochum, 44780 Bochum, Germany [Fungal genomic sequences used for this study are available at: Fungal Genome Initiative  as query, and key word searches were performed. Annotations of several laccases were found to be incorrect, because conserved domains were not detectable under the given annotated genes or accession numbers. Because often domains could be found within the accordant open reading frames or in the sequence of the annotated gene, the gene locus is given in Results and Discussion instead of the accession numbers.To identify laccases and other multi-copper oxidases of  tblastn  searches2.2.http://evolution.genetics.washington.edu/phylip.html). Distance matrices were calculated using program PROTDIST and were then used for constructing trees with the neighbor-joining program NEIGHBOR. Statistical significance was evaluated by bootstrap analysis with 1000 iterations of bootstrap samplings generated with SEQBOOT. A majority rule consensus tree was subsequently generated using the program CONSENSE. The consensus trees were graphically displayed using the program TreeView (Win 32) 1.6.6 [125 and ends with Pro209 of the Laccase precursor NCU04528.4 from N. crassa (XP_956939.1) [S1).Multiple protein sequence alignments were performed using the clustalX program . Phyloge2) 1.6.6  and save56939.1) . This re56939.1) . For the2.3.http://www.cbs.dtu.dk/services/TMHMM-2.0/) and HMMTOP [The online programs SignalP was used to determine cleavage sites of putative signal peptides . Programd HMMTOP , 39 were3.3.1.N. crassa, S. macrospora, P. anserina and C. globosum were mined using blastp and tblastn searches with the protein sequence of the MaL laccase of the thermophilic ascomycete M. albomyces, and with keyword searches. A total of 49 MCO amino acid sequences were obtained from the genomic databases of N. crassa, S. macrospora, P. anserina and C. globosum, including putative laccases, ascorbate oxidases, bilirubin oxidases and ferroxidases (Table 1). The highest number of MCOs was identified in the coprophilous fungi S. macrospora and P. anserina (15 MCO genes), whereas N. crassa and C. globosum encoded only 11 and 8 MCOs, respectively. Based on the analyses of more than 100 laccases, four ungapped sequence regions, L1-L4, have been identified as the overall signature sequences for distinguishing laccases within the broader class of MCOs [S. macrospora (SMAC09721 and SMAC03318) and one of C. globosum (CHGG_08215) lack the L3 and L4 signature in the predicted sequence. This could be due to annotation and sequence errors or the identified genes could encode non-functional pseudogenes. Consistent with an extracellular function established for other well-characterized fungal laccases, including laccases of the basidiomycete Coprinopsis cinerea [N. crassa [M. albomyces [1) Signal peptides length were 17- 32 amino acids (Table 1). However, at least one isoform was present in each fungus that was predicted to contain no signal peptide. This again may be due to annotation errors or members of the order Sordariales may contain intracellular laccase isoforms. Intercellular laccase activity has been identified in basidiomycetes and ascomycetes, and is proposed to be involved in the transformation of low molecular weight phenolic compounds [Pleurotus ostreatus when co-cultivated with the ascomycete Trichoderma longibrachiatum, suggesting expression of these enzymes can be induced by interspecies interactions [To compare the MCO coding capacity of coprophilous and non-coprophilous members of the fungal order Sordariales, the genomes of  of MCOs . In the  cinerea  and N. clbomyces , signal es Table . The higractions . Thus, tet al. [et al. [N. crassa. In contrast to Espagne et al. [P. anserina. The putative laccase Pa_1_16470 was not identified in this analysis. However, Pa_6_2550 was predicted as an MCO since it contains all of the conserved laccase signature sequences. As in Hoegger et al. [C. globosum and one C-terminally truncated (CHGG11082.1) laccase lacking L3 and L4. S. macrospora encodes at least nine putative laccases and two C-terminally truncated laccase-like proteins without the L3 and L4 domains.Consistent with previous studies by Espagne et al.  and Hoeg [et al. , eight pe et al. , only niSaccharomyces cerevisiae plasma membrane ferroxidase Fet3p. In S. cerevisiae, Fet3p receives iron(II) ions from cell-surface iron reductases and passes iron(III) ions to the iron permease Ftr1p [fet3 homologs of the Sordariales is located directly downstream of a ftr1 homolog (Table 1). With the exception of C. globosum (CHGG08215.1), only Fet3p homologs clustered with ftr1  are predicted to have one transmembrane domain (Table 1). Thus, these homologs may have a similar function as in S. cerevisiae, while the other seems to be an extracellular enzyme and may fulfill another role.Each of the four fungi analyzed here encodes two homologs of the se Ftr1p , 44. Simse Ftr1p , one of S. macrospora, P. anserina and C. globosum encode a putative ascorbate oxidase. This enzyme is highly specific for the reducing substrate, ascorbate, and other compounds with a lactone ring with an enediol group adjacent to a carbonyl group [In addition to laccases and ferroxidases, yl group . In planyl group . The funC. globosum, the fungi analyzed here encode at least one bilirubin oxidase. This class of MCO oxidizes bilirubin to biliverdin. Putative bilirubin oxidases from N. crassa (NCU05042.4), S. macrospora (SMAC_07604) and P. anserina (Pa_6_11170 and Pa_5_1710) are highly similar to the bilirubin oxidase from the ascomycete Myrothecium verrucaria (BAA03166) [M. verrucaria bilirubin oxidase was identified in a screen for microorganisms decolorizing urine and feces in raw sewage [M. verrucaria was also identified on the dung of horses [S. macrospora and P. anserina seem to have the ability to oxidize bilirubin, the degradation product of hemoglobin, which is mainly excreted via feces.Except for AA03166) , which iAA03166) . Interesw sewage . M. verrassa NCU042.4, S. 3.2.S. macrospora is a close relative of N. crassa. Both fungi exhibit a 90% nucleic acid identity within coding regions of orthologous genes, as well as a high degree of synteny over large genomic regions [S. macrospora and N. crassa share ∼95% amino acid identity (similar to mice and humans) [P. anserina was shown to be more distantly related to N. crassa and S. macrospora, but more closely related to C. globosum [S. macrospora and N. crassa as well as of P. anserina and C. globosum .Previous studies identified that  regions , 51. Wit humans) . P. anseglobosum , 53. To N. crassa and S. macrospora and of P. anserina and C. globosum. The neighbor-joining trees shown in Fig. (2) are based on a ClustalX alignment of a protein region spanning the laccase signature sequences L1-L2 [2a) shows that two putative gene duplication events and the gain of one MCO gene by a putative horizontal gene transfer event may have occurred in S. macrospora. These events may have led to the large laccase gene family in S. macrospora. However, note that the annotated ORFs SMAC_03318 and SMAC09721 encode C-terminally truncated proteins without laccase domains L3 and L4. Thus, these paralogs may be pseudogenes that resulted from a gene duplication event and subsequent loss of function or these ORFs may be wrongly annotated. No N. crassa ortholog could be identified for SMAC_09572. A BLAST analysis of SMAC_09572 revealed a putative MCO of the necrotrophic fungal phytopathogen Sclerotinia sclerotiorum order Helotiales  as the closest ortholog. Thus, SMAC_09572 may have been acquired by horizontal gene transfer. Also previous studies revealed that S. macrospora contains more polyketide biosynthesis genes than N. crassa and phylogenetic analyses suggest that some of these genes may have also been acquired by horizontal gene transfer from a distantly related ascomycete group [N. crassa and S. macrospora MCOs revealed that N. crassa may have lost a gene coding for an ascorbate oxidase, which is not only present in S. macrospora but also in C. globosum and P. anserina .To analyze whether gains, duplications and losses of MCO genes occurred when two closely related fungi inhabited different ecological niches, a phylogenetic analysis was performed using the identified MCO proteins of es L1-L2 . Fig. . C. globosum may have lost the bilirubin oxidase gene and perhaps one laccase gene. No C. globosum ortholog could be identified for Pa_6_2550. A BLAST analysis of the encoded protein identified a putative MCO of the plant pathogen Glomerella graminicola order Hypocreales  as the closest ortholog. Thus, similar S. macrospora P. anserina seems to have acquired one additional laccase gene by horizontal gene transfer. Previously horizontal transfer of a mitochondrial plasmid from the discomycete Ascobolus immersus to P. anserina was reported [3) revealed a strict separation of bilirubin oxidases, ascorbate oxidases, ferroxidases and laccases. According to the phylogenetic tree seven different groups of laccases may be distinguished. Group I, II, IV and VII comprises laccases orthologs of all four species. Not surprisingly, laccases SMAC0572 of S. macrospora and Pa_6_2550 of P. anserina, presumed to have been acquired by horizontal gene transfer, do not cluster within these groups. Laccase group IV contains Pa_5_1200 and NCU4529.4, which have been demonstrated to be active laccase enzymes [3 and Table 1). Laccase group III contains mainly laccases from the coprophilous fungi S. macrospora and P. anserina and may be subdivided into two subgroups. Group VII consist of putative intracellular isoforms of laccases enzymes laccases without N-terminal signal sequence (Table 1). Fet3-like ferroxidases comprise two subgroups, with each containing homologs of all four species. One subgroup consists of fet3 homologs located adjacent to a putative iron permease ftr1 homolog, while the other subgroup is not clustered with a permease gene . As already seen in Fig. (2), N. crassa lacks an ascorbate oxidase and C. globosum does not encode a bilirubin oxidase.Similar to reported . The com enzymes . Laccase4.S. macrospora and P. anserina can be attributed to horizontal gene transfer and gene duplication events. While the complete set of MCO genes was maintained in coprophilous fungi some MCOs disappeared in their closely related non-coprophilous relatives, N. crassa and C. globosum.Closely related members of the order Sordariales which inhabit different ecological niches exhibit different coding capacities for MCOs. The analysis of the phylogeny of MCO gene families in coprophilous and non-coprophilous members of the order Sordariales revealed that the laccase gene family is particularly large in coprophilous fungi. The increase of laccase genes in the coprophilous fungi Supplementary material is available on the publishers Web site along with the published article.