Candidatus Caldiarchaeum subterraneum” (Ca. C. subterraneum). A phylogenetic analysis based on the concatenated sequences of proteins common among 142 archaea and 2 bacteria, and among 137 archaea and 13 unicellular eukaryotes suggested that Ca. C. subterraneum is closely related to thaumarchaeotic species. Consistent with the results of the phylogenetic analysis, clustering and principal component analyses based on the completion ratio patterns for all KEGG modules in 79 archaeal species suggested that the overall metabolic and physiological potential of Ca. C. subterraneum is similar to that of thaumarchaeotic species. However, Ca. C. subterraneum possessed almost no genes in the modules required for nitrification and the hydroxypropionate–hydroxybutyrate cycle for carbon fixation, unlike thaumarchaeotic species. However, it possessed all genes in the modules required for central carbohydrate metabolism, such as glycolysis, pyruvate oxidation, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle, as well as multiple sets of sugar and branched chain amino acid ABC transporters. These metabolic and physiological features appear to support the predominantly aerobic character of Ca. C. subterraneum, which lives in a subsurface thermophilic microbial mat community with a heterotrophic lifestyle.In this study, the metabolic and physiological potential evaluator system based on Kyoto Encyclopedia of Genes and Genomes (KEGG) functional modules was employed to establish a functional classification of archaeal species and to determine the comprehensive functions (functionome) of the previously uncultivated thermophile “ Since the existence of undescribed physiological types of archaea in addition to extremophiles and methanogens in marine plankton was demonstrated in 1992 . In addiNitrosopumilus maritimus and Cenarchaeum symbiosum. However, we proposed that this crenarchaeotic group should be considered a novel phylum, Aigarchaeota, owing to its unique genomic traits that were distinct from those of known phyla and candidate divisions such as Crenarchaeota, Euryarchaeota, “Korarchaeota”, Nanoarchaeota, and Thaumarchaeota; accordingly, we named the microbe Candidatus Caldiarchaeum subterraneum (Ca. C. subterraneum) [We obtained the complete genome of an uncultivated archaeal species in the candidate phylum, Hot Water Crenarchaeotic Group I, from the metagenomes of a microbial mat community that flourishes along a subsurface geothermal stream –15. The rraneum) .Ca. C. subterraneum is to deduce its lifestyle on the basis of potential comprehensive functions (functionome) of genes harbored in its genome. We performed a detailed genomic analysis of its metabolic and physiological features, but it was difficult to capture its overall functional potential and to compare it with that of other archaea. The functional categories defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) [http://www.genome.jp/) to the functional classification of archaea and characterized the potential functionome of the Ca. C. subterraneum genome.A primary objective of genomic analyses of uncultivated s (KEGG)  and SEEDs (KEGG)  databases (KEGG) . These ms (KEGG) . We alsos (KEGG) . In the Ca. C. subterraneum. We also determined that the metabolic potential of Ca. C. sunterraneum based on overall MCR patters was similar to that of thaumarchaeotic species.In this study, we performed clustering and statistical analyses to determine functional classifications based on the MCR patterns of 79 archaeal species, including the uncultivated Ca. C. subterraneum gene and the genes of other prokaryotic species were identified using the DomClust program [Ca. C. subterraneum and 143 prokaryotic species . To determine the phylogenetic position of Ca. C. subterraneum among archaeal species, phylogenetic analyses were performed using a concatenated alignment of single-copy protein-coding genes without domain splitting, which were selected using the MBGD database. Twenty-two orthologous families conserved in 142 archaea and two bacteria, or 12 orthologous families conserved in 137 archaea and 13 unicellular eukaryotes were used for each phylogenetic analysis. The sequences of genes in these families were aligned using MUSCLE [Ca. C. subterraneum was inferred by MEGA6 using the maximum-likelihood method [The orthologous relationships between each  program  based ong MUSCLE  with theg MUSCLE  with thed method . The subd method . AccordiCa. C. subterraneum) listed in MCRs of all KEGG functional modules  in each type of archaea were calculated based on a Boolean algebra-like equation  using thCa. C. subterraneum based on comparisons with those of the other 78 archaea, the pairwise Euclidean distances between the overall MCR patterns for each archaea were used and the complete-linkage clustering method was employed for the functional classification of 79 archaea using an R statistical package [Ca. C. subterraneum, were excluded from this analysis. Similar to the clustering analysis, the overall MCR patterns of Ca. C. subterraneum and the other 78 archaea, except for the modules with no differences in the MCR values among all archaea, were subjected to a principal components analysis (PCA). PCA was performed using the R statistical environment.To characterize the overall MCR pattern of  package . KEGG moCa. C. subterraneum among archaea, we conducted a genome-wide phylogenetic analysis using the maximum-likelihood method with the LG+G substitution model, which was not used in the previous study [Ca. C. subterraneum and two representative bacterial species (Escherichia coli and Bacillus subtilis) as an outgroup (Ca. C. subterraneum formed a cluster with five species within Thaumarchaeota (Candidatus Nitrososphaera gargensis” (Ca. N. gargensis) [Nitrosopumilaceae and Cenoarchaeaceae comprised a sub-cluster. Thus, Ca. C. subterraneum was closely related to the thermophile Ca. N. gargensis based on this tree  , 30 was his tree . We cons species . “KorarcCa. C. subterraneum, was calculated to evaluate their metabolic and physiological potential using the MAPLE system [Ca. C. subterraneum, suggesting similar overall metabolic and physiological potential -thermophilic acidophiles or neutrophiles. The members that formed this cluster were sulfur compound reducers, except Fervidicoccus fontis [Thermosphaera aggregans [The MCR of all KEGG modules in 79 archaea, including E system  . In this case, modules, such as the archaeal exosome (M00390), which was identified in archaea, except for the halophiles and half of the methanogens, and DNA polymerase II complex (M00261), were not identified in Crenarchaeota and contributed to the variance in PC2, as shown in Ca. C. subterraneum is probably an aerobe that is similar to thaumarchaeotic species in overall metabolic and physiological potential; however, more careful examinations of these properties are required owing to the low percentages of variance explained -thermophilic heterotrophs. These exceptional euryarchaeotic species were mixed within the crenarchaeotic zone, together with equitans  and “Ca.tofilum” . Consequined see .Ca. C. subterraneum is similar to thaumarchaeotic species, both phylogenetically and in terms of its overall metabolic and physiological potential. Thus, we focused on the MCR patterns of six types of carbon fixation modules in Ca. C. subterraneum and thaumarchaeotic species, i.e., Nitrosopumilus maritimus [Cenarchaeum symbiosum [Ca. Nitrososphaera gargensis” [Ca. C. subterraneum could not complete any of the carbon fixation modules and the MCR values were very low except for the reductive tricarboxylic acid (TCA) cycle (M00173)  catalyzed by ATP-citrate lyase [Ca. C. subterraneum possesses enzymes related to all reaction steps shared between both cycles. If Ca. C. subterraneum harbors different types of key enzymes for the reductive TCA cycle, it could perform carbon fixation, but it is parsimonious to infer that it has an extremely poor carbon fixation potential. The thaumarchaeotic species were also unable to complete any of the carbon fixation modules, but are known to possess the HP-HB cycle for carbon fixation  . Apparente lyase  or citryte lyase , 47, wasfixation . The HP- species .Ca. C. subterraneum was extremely poor despite previous suggestions that it has an autotrophic lifestyle [Ca. C. subterraneum did not complete the KEGG module for the Embden–Meyerhof (EM) pathway comprising 10 reaction steps because two enzymes responsible for step 3, ATP-dependent 6-phosphofructokinase (ATP-PFK) (K00850 or K16370) and ADP-dependent phosphofructokinase/glucokinase (K00918), were not identified in the genome (Aeropyrum pernix and Desulfurococcus amylolyticus [A. pernix (APE_0012) belonging to the PFK-B family is defined as ribokinase (EC: 2.7.1.15) and the different K number of K00852 is assigned to this orthologous group. At present, K00852 has not yet been assigned to the module for the EM pathway, but it should be assigned to reaction step 3 in this module together with currently assigned KOs. In the Ca. C. subterraneum genome, there are two K00852-assigned genes, CSUB_C0883 and CSUB_C1035, and APE_0012 and CSUB_C1035 share 74.4% similarity (ca. 30% identity). Thus, the gene product of CSUB_C1035 presumably acts as ATP-PFK, responsible for reaction step 3.The carbon fixation potential of ifestyle , 13. Thue genome . Howeverolyticus –51. BothCa. C. subterraneum completed the gluconeogenesis module with a high MCR of 85.7%, although phosphoenolpyruvate carboxykinase, which is responsible for reaction step 1, was not identified. Ca. C. subterraneum possesses genes encoding malate dehydrogenase: oxaloacetate-decarboxylating (CSUB_C1205) and pyruvate orthophosphate dikinase (CSUB_C1231), which enable the conversion of malate to phosphoenolpyruvate via pyruvate. A series of reactions have been defined as the KEGG module for crassulacean acid metabolism (CAM)-light (M00169), comprising 2 reaction steps, and K00029 and K01006 were assigned to each step as shown in Sulfolobus solfataricus [Ca. C. subterraneum cannot complete the current M00169 module, to which K00027 is not assigned for unclear reasons. One of two specific enzymes in the module for gluconeogenesis, fructose 1,6-bisphosphate aldolase/phosphatase, was identified in both Ca. C. subterraneum and thaumarchaeotic species [Ca. C. subterraneum also completed the pyruvate oxidation module connected to the TCA cycle.Similar to the enzymes involve in the reductive TCA cycle and TCA cycle, the enzymes used for 4 to 9 reaction steps in the EM pathway modules are shared with those involved in reaction steps 2 to 7 of the gluconeogenesis module . Actuallataricus , but a d species , 54. Ca.N. maritimus and C. symbiosum, completed the EM pathway with an MCR of 70%, but lacked glucokinase (step 1) and pyruvate kinase (step 10) as well as the enzyme (6-phosphofructokinase or ADP-dependent phosphofructokinase/glucokinase) responsible for step 3 (Ca. N. gargensis” possessed an ATP-PFK within the PFK-B family showing 75% similarity (ca. 32% identity) to that of Ca. C. subterraneum, unlike the two other thaumarchaeotic species. Ca. C. subterraneum possesses at least three sets of multiple sugar and simple sugar ABC transporters (M00207 and M00221), while the thaumarchaeotic species possess no sugar transporters as shown in All of the thaumarchaeotic species showed a high MCR value for the EM pathway because they completed the gluconeogenesis modules. Two of three thaumarchaeotic species, r step 3 . HoweverCa. C. subterraneum did not complete the current KEGG module for the TCA cycle, which comprises eight reaction steps; the genes encoding the γ (K00177) and δ (K00176) subunits of 2-oxoglutarate ferredoxin oxidoreductase (step 3) were not identified in the genome, but those for the α (K00174) and β (K00175) subunits were identified. Another enzyme type with a different subunit composition, such as an αβ (heterodimer), α2β2 (dimer of heterodimer), and α2 homodimer, has been identified [Ca. C. subterraneum genome show sequence similarity of more than 84% to each subunit of the αβ-type enzyme from Sulfolobus tokodaii strain 7 [Ca. C. subterraneum is thought to function in reaction step 4 of the TCA cycle module, similar to in S. tokodaii. In addition, Ca. C. subterraneum possesses the glyoxylate cycle to bypass the decarboxylation steps in the TCA cycle. As mentioned above, because this organism also possesses malate dehydrogenase (CSUB_C1205) and pyruvate orthophosphate dikinase (CSUB_C1231), which enable the conversion of malate to phosphoenolpyruvate, this cycle connected to the gluconeogenesis pathway is thought to be involved in the production of cell components. In contrast to Ca. C. subterraneum, thaumarchaeotic species possess almost none of the key enzymes responsible for the glyoxylate cycle , such as leucine, isoleucine, and valine ABC transporters, whereas among the thaumarchaeotic species, C. symbiosum possesses only one set , which is one of the metabolites in the TCA cycle that possibly stimulates growth, was identified in the Ca. C. subterraneum genome. one set . Except Ferroplasma type II [Ca. C. subterraneum in a comparative analysis with other archaea based on MCR patterns.A primary objective of genomic analyses is to deduce the potential functionome of individual organisms. Since the genome of uncultured  type II  from a n type II , 14, 60. type II . Recentl type II . In the Euryarchaeota, Crenarchaeota, and Thaumarchaeota, according to PC2. These interpretations according to the loadings in PC1 and PC2 are biologically plausible , the genomic sequence of which has almost been completed [Ca. A. autotrophicum demonstrated its chemolithoautotrophic potential based on homo-acetogenesis via H2 and CO2, although the detailed mechanisms for energy generation still remain unclear. In contrast to Ca. A. autotrophicum, Ca. C. subterraneum has an extremely poor carbon fixation potential, but completed the modules responsible for major central carbohydrate metabolism. Ca. C. subterraneum also possesses all enzymes responsible for the glyoxylate cycle, which has been well studied in Escherichia coli [E. coli can grow using the glyoxylate cycle, which is an anaplerotic pathway of the TCA cycle when acetate serves as the sole carbon source for growth. Therefore, Ca. C. subterraneum also may use acetate produced by acetogenic Ca. A. autotrophicum as a carbon source for growth in the microbial mat community. Moreover, Ca. C. subterraneum possesses multiple sets of ABC transporters required for the uptake of sugars and BCAAs, thereby supporting its heterotrophic lifestyle in the microbial mat community. In addition to Ca. A. autotrophicum, another potentially chemolithoautotrophic phylotype that is closely related to “Ca. Nitrosocaldus yellowstonii” (Thaumarchaeota), Hydrogenobacter thermophilus (Aquificae), Hydrogenophilus thermoluteolus (Betaproteobacteria), and a methanotrophic phylotype similar to Methylohalobius crimensis (Gammaproteobacteria), which probably use CO2 and CH4 supplied by the geothermal aquifer, coexist with Ca. C. subterraneum. However, the abundance of these phylotypes was low in the microbial mat community [Ca. C. subterraneum in the anaerobic organic-depleted state.The genome of mal mine . Because aquifer –65. The ompleted . The genhia coli . E. coliommunity . Given tCa. C. subterraneum using the MAPLE system and subsequent metabolic analyses based on the MCR patterns of individual KEGG modules, supported the predominantly aerobic nature of Ca. C. subterraneum, which lives in a subsurface thermophilic microbial mat community with a heterotrophic lifestyle. However, it is still challenging for the archaeal science community to elucidate metabolic features using bioinformatics methods because many of the unusual features of archaeal metabolism revealed to date are not reflected in the KEGG modules. Thus, the MAPLE system will be more helpful for characterizing archaeal metabolism, even in uncultured archaea, when the KEGG modules incorporate more archaeal metabolic features. Also, manual data curation and literature mining are still important strategies when implement MAPLE.In conclusion, using the MAPLE system, the archaeal species registered in the KEGG GENOME database were classified into various groups that possess similar phenotypic properties, such as halophiles, methanogens, ammonia oxidizers, heterotrophic (hyper)-thermophilic acidophiles or neutrophiles, and hyperthermophilic facultative autotrophic sulfur reducers. Moreover, methanogens were classified into two major groups with different ecological niches, the human gut and other earth environments. The functional classification of previously uncultivated S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Introduction. Helicobacter pylori (H. pylori) infection could lead to chronic local and systemic immune response. The resulting increase in proinflammatory cytokines could affect bone resorption and might increase the risk of osteoporosis. This study aimed to investigate the association between H. pylori infection and osteoporosis in elderly female patients with upper gastrointestinal diseases. Methods. A retrospective patient record review study was conducted in a regional teaching hospital in south Taiwan. Relevant information on female patients aged 65 and over who were diagnosed with diseases of esophagus, gastric ulcer, or duodenal ulcer during January 2008 to December 2010 were abstracted. Association between H. pylori infection and osteoporosis was evaluated using multivariate logistic regression analysis. Results. Of the 365 patients with a mean age of 77.3 years, 77 (21.1%) had H. pylori infection and 101 (27.7%) had been diagnosed with osteoporosis. Multivariate logistic regression analysis revealed that osteoporosis was significantly associated with H. pylori infection  after adjusting for age group, body mass index group, and use of proton pump inhibitor. Conclusion. Osteoporosis was found to be associated with H. pylori infection in Taiwanese female patients with upper gastrointestinal diseases. Further studies with information on potential confounders are needed to confirm the association. Osteoporosis is a major public health problem affecting up to one in three women . It has Helicobacter pylori (H. pylori) is a spiral-shaped, Gram-negative bacterium that colonizes the gastric mucosa of more than 50% of human population. It can elicit lifelong inflammatory and immune responses in infected individuals. It is strongly associated with chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer [ H. pylori infection has previously been suggested to be involved in the pathogenesis of a number of extradigestive disorders including cardiovascular, skin, rheumatic, liver cirrhosis, diabetes mellitus, and respiratory diseases [c cancer . In addiH. pylori infection can increase the systemic levels of inflammatory cytokines and bone turnover regulating cytokines such as tumor necrosis factor-alpha, interleukin-1, and interleukin-6 [ H. pylori infection are at an increased risk of osteoporosis. However, few studies have investigated this association except in a study of 80 osteoporotic male patients and 160 controls where the results showed that individuals infected by CagA-positive H. pylori strains was more prevalent in men with osteoporosis [ H. pylori infection was not accompanied by significant changes in biochemical markers of bone metabolism in a study of 41 H. pylori positive children [ H. pylori infection and osteoporosis among elderly Taiwanese women with upper gastrointestinal diseases.oporosis . On the children . TherefoA retrospective patient medical record analysis was performed using the data on the computerized patients' record database in a regional hospital in south Taiwan. The study protocol was approved by the institutional review board of the Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (no. B10003001). Informed consent was not required because of the nature of the study. Female patients were included in the study if they were 65 years or older and were diagnosed with diseases of esophagus, gastric ulcer, or duodenal ulcer  between January 1, 2008, and December 31, 2010. H. pylori testing, diagnosis of osteoporosis, the use of proton pump inhibitor (PPI), and antiosteoporotic medications. The presence of H. pylori infection was determined by histological examination [2), normal weight (BMI = 18.5–23.9 kg/m2), overweight (BMI = 24.0–26.9 kg/m2), and obese (BMI ≥ 27 kg/m2). Osteoporosis was defined by either dual energy X-ray absorptiometry (DXA) scan [A medical record abstraction form was used to collect information on age, body weight, height, smoking, alcohol use,mination , rapid umination , or bothXA) scan  or use o H. pylori infection, adjusting for age, BMI, and the use of proton pump inhibitor. Hosmer-Lemeshow statistics were used to assess model fit. A two-tailed P value <0.05 was considered statistically significant.Categorical variables were expressed as frequencies and percentages and Fisher's exact test was used for their comparisons. Multiple logistic regression analyses were used to evaluate the association between osteoporosis and H. pylori and a further 129 patients were excluded from the final analysis due to missing data in other biochemical variables. The mean age of these 365 patients was 77.3 years . Of them, 77 (21.1%) were tested positive for H. pylori and 101 (27.7%) had been diagnosed with osteoporosis.A total of 671 female patients with upper gastrointestinal diseases were identified from the medical records. Of them, 494 (73.6%) had undergone testing for H. pylori. The group tested negative for H. pylori was younger (P = 0.061) and had a higher proportion with gastric ulcer (P = 0.051). The group tested positive for H. pylori had significantly higher proportion of duodenal ulcer (P = 0.033). No significant differences were observed in the distributions of tertiles of PPI use between the two groups. Results from multivariate logistic regression analyses revealed that osteoporosis was significantly associated with H. pylori infection  after adjusting for age group, body mass index group, and use of proton pump inhibitor . Nevertheless, with only 50 subjects, the study suffered from a low statistical power to detect the association when it truly exists (power = 33%). Studies with adequate sample sizes are needed to ensure that a reasonable statistical power can be achieved.To date, there was only one cross-sectional study designed to specifically evaluate the association betweenal women . The aut H. pylori infection and osteoporosis are not clear, several possible explanations have previously been postulated. First, chronic infection of H. pylori can increase the release of cytokines, such as tumor necrosis factor- (TNT-) α, interleukin-1, and interleukin-6. It has been demonstrated that increased levels of interleukin-1 and TNF-α can lead to bone resorption [Although the exact mechanisms underlying the observed association betweensorption  and, the H. pylori infection has shown to decrease the levels of vitamin B12 [12 are considered as a risk factor for osteoporosis [ H. pylori positives and negatives for the same reason of lack of long-term insurance coverage.In addition,amin B12 , 14. Lowoporosis , 16. Furoporosis . A revieoporosis . In our  H. pylori. Therefore, we could not rule out the presence of selection bias. Fourth, measurement of systemic inflammatory markers was not available to confirm whether they were elevated in patients with H. pylori infection.Findings from this study should be weighed within the confines of the limitations of our data source. First, the study subjects came from a single regional hospital, which might not be representative of other settings. Second, retrospective studies based on abstraction of medical records are constrained by the accuracy and completeness of the records. Third, not all patients with upper gastrointestinal diseases were tested for presence of H. pylori infection was associated with osteoporosis in elderly female Taiwanese. Further studies using prospective design with information on systemic inflammatory markers and potential confounding factors are needed to confirm the association.Results from our retrospective patient medical record analysis indicated that


Pseudomonas aeruginosa is the most common gram-negative pathogen causing pneumonia in immunocompromised patients. Acute lung injury induced by bacterial exoproducts is associated with a poor outcome in P. aeruginosa pneumonia. The major pathogenic toxins among the exoproducts of P. aeruginosa and the mechanism by which they cause acute lung injury have been investigated: exoenzyme S and co-regulated toxins were found to contribute to acute lung injury. P. aeruginosa secretes these toxins through the recently defined type III secretion system (TTSS), by which gram-negative bacteria directly translocate toxins into the cytosol of target eukaryotic cells. TTSS comprises the secretion apparatus (termed the injectisome), translocators, secreted toxins, and regulatory components. In the P. aeruginosa genome, a pathogenic gene cluster, the exoenzyme S regulon, encodes genes underlying the regulation, secretion, and translocation of TTSS. Four type III secretory toxins, namely ExoS, ExoT, ExoU, and ExoY, have been identified in P. aeruginosa. ExoS is a 49-kDa form of exoenzyme S, a bifunctional toxin that exerts ADP-ribosyltransferase and GTPase-activating protein (GAP) activity to disrupt endocytosis, the actin cytoskeleton, and cell proliferation. ExoT, a 53-kDa form of exoenzyme S with 75% sequence homology to ExoS, also exerts GAP activity to interfere with cell morphology and motility. ExoY is a nucleotidal cyclase that increases the intracellular levels of cyclic adenosine and guanosine monophosphates, resulting in edema formation. ExoU, which exhibits phospholipase A2 activity activated by host cell ubiquitination after translocation, is a major pathogenic cytotoxin that causes alveolar epithelial injury and macrophage necrosis. Approximately 20% of clinical isolates also secrete ExoU, a gene encoded within an insertional pathogenic gene cluster named P. aeruginosa pathogenicity island-2. The ExoU secretory phenotype is associated with a poor clinical outcome in P. aeruginosa pneumonia. Blockade of translocation by TTSS or inhibition of the enzymatic activity of translocated toxins has the potential to decrease acute lung injury and improve clinical outcome.The online version of this article (doi:10.1186/2052-0492-2-10) contains supplementary material, which is available to authorized users. Pseudomonas aeruginosa is one of the most common gram-negative pathogens causing pneumonia in immunocompromised patients . TT. TTYersivia TTSS .Figure 1P. aeruginosa is highly homologous to the prototypical Yersinia TTSS , 62transic cells , 64. His strains , 66. Pcrectively , 64.TablP. aeruginosa is known to secrete at least four effector molecules (type III secretory toxins) via TTSS: ExoS, ExoT, ExoU, and ExoY  for Rho GTPases , suggestting Rho .Two immunologically undistinguishable proteins, with apparent molecular sizes of 53- and 49-kDa, co-fractionated with exoenzyme S activity . Later, exoU was cloned from the cytotoxic PA103 strain. A region downstream of exoU was found to encode a specific Pseudomonas chaperone for ExoU (SpcU) [P. aeruginosa, ExoU and SpcU are coordinately expressed as an operon controlled at the transcriptional level by ExsA [P. aeruginosa ExoU caused increased bacterial virulence and systemic spread in a mouse model of acute pneumonia [in vitro assays indicated that ExoU most closely linked to mortality in animal models was secreted in detectable amounts in vitro by 10 (29%) of the 35 isolates examined [In 1997, a novel cytotoxin, ExoU , was foU (SpcU) . In P. a by ExsA . Acquisineumonia . Hauser examined .in vitro models, it was shown that ExoU exhibits Ser-142- and Asp-344-dependent catalytic PLA2 activity, which requires eukaryotic cell factors for its activation [P. aeruginosa causes acute lung injury, thereby causing sepsis and mortality, through cytotoxic activity derived from the patatin-like phospholipase domain of ExoU [ExoU has a potato patatin-like phospholipase (PLA) domain ; Figure tivation , 83. The of ExoU . The cel of ExoU . Through of ExoU , 87.FiguBortedella pertussis (CyaA), Bacillus anthracis (EF), and Yersinia pestis  [P. aeruginosa producing catalytically active ExoY resulted in the elevation of intracellular cAMP levels and changes in cell morphology [ExoY is the fourth type III secretion effector protein controlled by ExoS regulon. ExoY is homologous to the extracellular adenylate cyclases of l toxin) . In assarphology , 89. It rphology .P. aeruginosa infections [P. aeruginosa isolates expressing at least one of the type III secretory proteins was 21% compared with the rate of 3% in patients with isolates expressing no type III secretory protein. In another study, infection with isolates secreting TTSS proteins, particularly isolates with an ExoU-positive phenotype, correlated with severe disease [P. aeruginosa pneumonia. exoU-positive isolates were more likely to be fluoroquinolone resistant and exhibit both a gyrA mutation and efflux pump overexpression [exoU gene were more likely to be resistant to cefepime, ceftazidime, piperacillin tazobactam, carbapenems, and gentamicin [P. aeruginosa in pneumonia [P. aeruginosa isolates from bacteremic patients confers a poor clinical outcome, independent of antibiotic susceptibility [P. aeruginosa bacteremia [Analysis of type III secretory protein phenotypes was performed in 108 isolates derived from patients with fections . The mor disease . Recentlpression . Clinicantamicin . A fluorneumonia . Howevertibility . Severitcteremia .P. aeruginosa expresses a variety of factors that confer resistance to a broad array of antibacterial agents. Multidrug-resistant P. aeruginosa (MDRP) is defined as the resistance to carbapenems, aminoglycosides, and fluoroquinolones. The current increase in the incidence of lethal outbreaks of MDRP is especially a serious concern. Multiple genetic rearrangements, such as chromosomal mutations or horizontal gene transfers , are associated with the acquisition of multidrug resistance in these bacteria. The various mechanisms, such as β-lactamases, carbapenemases or aminoglycoside-modifying enzymes, and mutations in antibiotic targets, efflux pumps, impermeability, are associated in these multidrug resistances. In the management of P. aeruginosa pneumonia, the increasing resistance level of these bacteria to most classes of antibacterial agents frequently leads to failure of effective treatment, which is associated with high mortality of the infected patients. Therefore, choosing adequate antibiotics is crucial to increase the survival rate, especially in patients infected with MDRP. Therefore, surveillance in antibiotic resistance must be important to reduce the risk of inadequate antibacterial therapy. In addition, surveillance in TTSSgenotype- and phenotype-associated acute lung injury and sepsis may help to predict the higher risk of lethal outbreaks.P. aeruginosa has been already reported as a caution of the emergence of pan-resistant strains in the near future [P. aeruginosa is highly depending on its TTSS-associated virulence as described above. PcrV has a critical role in the TTS-associated virulence of P. aeruginosa as follows [P. aeruginosa-induced lung injury greatly increased survival [P. aeruginosa strains expressing PcrV disabled macrophage phagocytosis. However, antibodies against PcrV blocked this critical antiphagocytic effect [2 fragments of polyclonal anti-PcrV were also effective [P. aeruginosa pneumonia in artificially ventilated patients and cystic fibrosis patients [Polymyxin E (colistin) remains the most consistently effective agent against MDPR, while colistin-resistant r future . Differe follows . In a sesurvival . Virulenc effect . Passivec effect . The proffective . A murinffective . The admffective . Based opatients –103.P. aeruginosa possesses a sophisticated toxin secretion system to directly inject toxins into the cytosol of target eukaryotic cells. This system, called TTSS, is regulated by the exoenzyme S regulon of P. aeruginosa. Through TTSS, P. aeruginosa translocates the type III secretory toxins ExoS, ExoT, ExoU, and ExoY. By injecting these toxins into the cytosol of eukaryotic cells, P. aeruginosa exploits mammalian enzyme functions to modulate eukaryotic cell signaling.P. aeruginosa pneumonia. Virulent strains of P. aeruginosa possess the exoU gene, whereas nonvirulent strains lack the same. The major pathogenesis of P. aeruginosa-induced acute epithelial lung injury and subsequent bacteremia and sepsis is highly dependent on the ExoU phenotype of the strain, while the type III secretory toxins ExoS, ExoT, and ExoY modulate host immunity and cause lung edema (Figure P. aeruginosa pneumonia. What we have learned from our attempts to elucidate the molecular mechanisms underlying acute lung injury over the last 30 years is how well pathogenic bacteria utilize our cell signaling to cause diseases: bacteria know our cell signaling better than we do.Of these four toxins, ExoU is the major virulence factor responsible for alveolar epithelial injury in a Figure . ProgresTS is a professor in Anesthesiology at Kyoto Prefectural University of Medicine, Japan.


Prunus tree species exhibit a homomorphic gametophytic self-incompatibility (GSI) system, in which the pollen phenotype is encoded by its own haploid genome. To date, no identification of S-alleles had been done in Prunus africana, the only member of the genus in Africa. To identify S-RNase alleles and hence determine S-genotypes in African cherry (Prunus africana) from Mabira Forest Reserve, Uganda, primers flanking the first and second intron were designed and these amplified two bands in most individuals. PCR bands on agarose indicated 26 and 8 different S-alleles for second and first intron respectively. Partial or full sequences were obtained for all these fragments. Comparison with published S-RNase data indicated that the amplified products were S-RNase alleles with very high interspecies homology despite the high intraspecific variation. Against expectations for a locus under balancing selection, frequency and spatial distribution of the alleles in a study plot was not random. Implications of the results to breeding efforts in the species are discussed, and mating experiments are strongly suggested to finally prove the functionality of SI in P. africana.In flowering plants, self-incompatibility is an effective genetic mechanism that prevents self-fertilization. Most  Prunus africana (Hook. f) Kalkman is a medicinal tree indigenous to the montane regions of West, Central, East and Southern Africa, including Madagascar. It is the only member of the genus Prunus which comprises of more than 200 species that are indigenous to Africa 12] [12] S-al [12] S-aThe second intron was initially amplified using a pair of documented primers . AmplifiEscherichia coli competent cells using a heat shock protocol . Po. PoPyrusquencies . Based oelow 90% . This neafricana  has alsoP. africana, although most species with gametophytic SI support the equal allelic frequencies (isoplethy) hypothesis  e. e.P. afrus avium . Howeverus avium . The devquencies .Prunus lannesiana var. speciosa . Se. SePrunu 3400 bp  relative 2217 bp  respectiP. africana S-RNases was 1.18 which is substantially higher than has been found in both the cultivated P. avium and P. dulcis as well as other wild Prunus species like P. cerasifera, P. lannesiana (0.793) . Dr. DrP. afafricana . It has P. africana trees that were monitored in this study showed a prolific fruit and seed set but seedlings exhibited limited survival and were commonly found under the mother trees. This indicated that either limited seed dispersal or the microenvironment after dispersal doesn’t favour recruitment, with strong implication on the SGS. Reduced reproductive success has also been associated with local genetic effects in Prunus virginiana  wit witP. afafricana .P. africana may also be attributed to the inefficiency of the SI system at preventing selfing or crossing between relatives. Observations indicate that P. africana may exhibit mixed mating system especially when clumped [S-alleles into the system may disrupt functionality of the SI system of the plant [Besides constrained dispersal and regeneration, the observed SGS for  clumped  as it wahe plant .P. africana. The observed similarity of P. africana S-alleles to other Prunus species strengthens the taxonomic placement of the species within this genus. Although empirical evidence is missing that the observed alleles operate a functional SI system, the high number of alleles detected largely indicate an active SI in P. africana. Crossing experiments involving individuals with known S-genotype are strongly encouraged to finally solve this functionality issue.This study has provided important insights into the SI system of S- alleles. As indicated above, further empirical studies are needed to finally prove SI in P. africana.The results also have important implications for future breeding purposes. To ensure reproductive success and hence sustainability of the species, genebank establishment and clonal development has to maximally diversify the S1 Data(XLS)Click here for additional data file.


Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids . This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity. Bacillus cereus infections are typically associated with foodborne illnesses, periodontal diseases, and other opportunistic diseases [B. cereus can be associated with more severe and even fatal infections. Within the last decade, B. cereus isolates associated with severe infections have been described harboring B. anthracis toxin genes and/or capsule biosynthesis genes. Hoffmaster et al. described the first B. cereus with B. anthracis toxin genes, B. cereus G9241, to cause a severe pneumonia in a metal worker from Louisiana [B. cereus resembling G9241 isolated from severe pneumonia cases in metal workers in Texas [Bacillus isolates harboring B. anthracis virulence genes, now termed B. cereus biovar anthracis, cultured from deceased great apes in Cote d’Ivoire and Cameroon. [diseases . Howeverin Texas , 4, 5. Wameroon. .B. cereus isolate, BcFL2013, cultured from a swab of a facial lesion resembling an anthrax eschar from a 70-year-old Florida resident. (B. anthracis-specific polymerase chain reaction assay (LRN PCR) on the isolate from the skin lesion and detected one of the B. anthracis plasmids, pXO1. The isolate was then forwarded to the Centers for Disease Control and Prevention (CDC) for further characterization. In addition to isolate characterization, plasma and serum samples from the patient were collected and forwarded to the CDC for serological testing.In this report, we characterize a esident. . The patB. anthracis virulence genes  were performed as previously described [The BcFL2013 isolate was inoculated on trypticase soy agar with 5% sheep blood  and tested to determine susceptibility to gamma phage as previously described . Capsuleescribed , 9. The pagA) sequencing prior to performing whole genome sequencing (WGS). MLST and pagA sequence typing was performed as previously described [glpF, gmk, ilvD, pta, pur, pycA, and tpi) and is described online at http://pubmlst.org/bcereus. For WGS, a 101 X 101 paired-end run was performed in an Illumina GAIIx using TruSeq chemistry and yielded 10,182,400 reads. A de novo assembly was performed using CLC Genomics Workbench 6.0.4  which yielded 84 contigs consisting of 5.46 Mb, with an N50 of 139,961 bp. [http://enve-omics.ce.gatech.edu/ani/index [The isolate was also molecularly characterized by multilocus sequence typing (MLST) and protective antigen gene  and two subsequent convalescent serum samples (collected on 03/14/2013 and 04/04/2013) taken from the patient was performed to detect lethal factor (LF), anti-protective antigen IgG, and anthrax lethal toxin neutralizing activity (TNA) immune response. All serological assays were performed as previously described , 16.Because the samples were collected in the course of clinical care and management, and sent to CDC for routine diagnostic purposes, informed consent was not obtained, and IRB approval was not sought. However, we received express written permission from the patient to publish the photograph presented in this report.B. cereus: large, umbonate (raised center), dark tan and granular. This isolate also produced a capsule  and negative for the pXO2-encoded capsule (capA) gene. However, unlike G9241, this isolate was negative by the pBC210 plasmid assay. Molecular subtyping showed the isolate had an identical MLST and pagA sequence type as G9241 .BcFL2013 was beta-hemolytic on sheep blood agar and resistant to gamma phage lysis . The col capsule , Fig 2. pagA, cya, and lef) and hyaluronan capsule synthase genes (hasACB) which were determined to be 100% identical to those found in pBCXO1. Mapping the Illumina reads of BcFL2013 to plasmid pBC210 of G9241 indicated a homolog of the plasmid was present but had only approximately half the sequence of pBC210 and did not include the bpsXABCDEFGH operon. The 108,352 kb of homologous pBC210 sequence had an ANI of 99.84% when compared to G9241.We performed WGS to further characterize and determine the presence of virulence genes and plasmids in this isolate. Whole genome sequence Illumina reads for BcFL2013 were mapped to that of plasmid pBCXO1 of G9241, which indicated that a homolog of this plasmid is present, although with a ~ 2.5 kb deletion. Comparison of this homolog with pBCXO1 yielded an ANI of >99.98%. This included the anthrax toxin genes , the putative BcFL2013 chromosomal sequence yielded an ANI of 99.98%. The shotgun sequence for BcFL2013 was deposited into DDBJ/EMBL/GenBank under accession no. JHQN01000000.Serological testing of acute-stage plasma and two subsequent convalescent serum samples taken from the patient was performed to detect anthrax toxin lethal factor (LF), anti-protective antigen (PA) IgG, and anthrax lethal toxin neutralizing activity (TNA) immune response . LF was B. cereus isolates, associated with human infections, have been described containing B. anthracis toxin genes: four isolates  from metal workers in Texas and Louisiana with respiratory infections and two isolates (laboratory-acquired G9241 infection from Illinois and BcFL2013) resulting in cutaneous infections [B. cereus isolate harboring B. anthracis toxin genes (LA4726) from Louisiana in 2007 from a pneumonia patient who was also a metal worker (data not shown). Four of these isolates  had identical subtypes (ST 78) by MLST. The two remaining metal worker isolates (03BB102 and Elc2) were MLST subtypes ST 11 and ST 108, respectively. All seven metal worker isolates had sequence homology to B. anthracis toxin genes  on pXO1. Only one isolate, 03BB102, had homology to the B. anthracis capsule genes (cap operon) on the pXO2 plasmid; however, it was not observed to produce a polyglutamate capsule like B. anthracis [B. cereus isolates from the metal workers produced a capsule  although they differed in composition from the B. anthracis capsule. The BcFL2013 isolate described here has one of the capsule operons associated with G9241, hasACB. This would suggest that BcFL2013 produced, at very least, a hyaluronic acid capsule, but further studies would be required to determine the exact capsule composition.To date, six fections , 19. In B. cereus isolates have been isolated from non-human primates in Cote d’Ivoire and Cameroon (CI and CA) which harbor B. anthracis virulence genes and have been described as B. cereus biovar anthracis [B. cereus biovar anthracis isolates harbor both B. anthracis virulence plasmids (99–100% identity) [plcR gene, a transcriptional regulator, due to an insertion at the 3’ end of the gene [B. anthracis also has a non-functional plcR gene resulting from of a nonsense mutation in the gene [In addition to these isolates, two nthracis . Unlike dentity) . Additiothe gene . B. anththe gene . ConversB. anthracis as they are motile, gamma-phage resistant, and, in some cases, penicillin-resistant and, unlike most B. cereus, are non-hemolytic on blood agar. [B. cereus isolates in that they express both a hyaluronic acid and polyglutamate capsules [B. anthracis [B. cereus biovar anthracis isolates have not been cultured from humans. Whether this is the result of virulence differences, decreased human exposure, or an artifact of limited diagnostic capacity in regions where these strains have been detected remains to be determined.Phenotypically, the CA and CI isolates differ from od agar. . The CA capsules . By MLSTnthracis , 20. It B. cereus isolate harboring B. anthracis toxin genes associated with a naturally occurring cutaneous infection. However, as previously mentioned, there was a laboratory-acquired cutaneous infection associated with B. cereus G9241 in 2011 [B. cereus isolates, the Florida patient did not have any previous history of metal work. The patient had recent contact with apparently healthy horses but this is not a known risk factor for infection. Previous cases caused by these types of isolates occurred in Texas and Louisiana, neither of which were visited by this patient recently. It remains unknown how the Florida patient became infected.This is the first report of a  in 2011 . Unlike B. cereus infection resembling cutaneous anthrax, this is the first report of demonstrated LF toxemia and a detectable humoral response to the toxin with in vitro toxin neutralization activity from an infection caused by a B. cereus. However, Brezillon et al. showed that the B. cereus biovar anthracis isolates expressed both B. anthracis toxin and capsule genes in animal models [B. cereus infections did not include serologic testing of patients to determine if the toxin genes were expressed in vivo resulting in toxemia. These methods have been used successfully to detect toxin and antibody response in systemic and cutaneous anthrax cases [B. anthracis toxin genes are not only present but are also expressed in vivo and, in this case, associated with the clinical presentation resembling an anthrax eschar.In addition to being the first report of a naturally acquired l models . Previouax cases , 23. Thi


The development of organized, informative, robust, user-friendly, and freely accessible molecular markers is imperative to the Musa marker assisted breeding program. Although several hundred SSR markers have already been developed, the number of informative, robust, and freely accessible Musa markers remains inadequate for some breeding applications. In view of this issue, we surveyed SSRs in four different data sets, developed large-scale non-redundant highly informative therapeutic SSR markers, and classified them according to their attributes, as well as analyzed their cross-taxon transferability and utility for the genetic study of Musa and its relatives. A high SSR frequency (177 per Mbp) was found in the Musa genome. AT-rich dinucleotide repeats are predominant, and trinucleotide repeats are the most abundant in transcribed regions. A significant number of Musa SSRs are associated with pre-miRNAs, and 83% of these SSRs are promising candidates for the development of therapeutic SSR markers. Overall, 74% of the SSR markers were polymorphic, and 94% were transferable to at least one Musa spp. Two hundred forty-three markers generated a total of 1047 alleles, with 2-8 alleles each and an average of 4.38 alleles per locus. The PIC values ranged from 0.31 to 0.89 and averaged 0.71. We report the largest set of non-redundant, polymorphic, new SSR markers to be developed in Musa. These additional markers could be a valuable resource for marker-assisted breeding, genetic diversity and genomic studies of Musa and related species. Musa spp.) is an edible fruit crop that is widespread in tropical and subtropical regions around the world. It is a large, herbaceous, monocotyledonous flowering plant belonging to the order Zingiberales and the family Musaceae [in silico EST sequence-mining of several Musa spp. [Banana  analyze and compare SSR frequencies, densities, and distributions among Musa genomic regions, EST and GSS sequences, (ii) develop large scale non-redundant SSR markers, (iii) assign putative functions to developed SSR markers, (iv) estimate SSR marker transposon and miRNA association, (v) analyze the cross-taxon transferability of markers in silico, (vi) classify and characterize developed Musa SSR markers according to their attributes, (vii) map markers to the Agenome (M. acuminata) to determine their physical positions within the genome, and (viii) evaluate a subset of markers for cross-species transferability and potential for use in Musa diversity studies.The whole genome sequences of two banana varieties, more than 0.1 million EST and several thousand GSS sequences, are publicly available, facilitating the M. acuminata  and M. balbisiana  were downloaded from the Banana Genome Hub (http://banana-genome.cirad.fr/). GSS sequences were retrieved from NCBI. EST sequences were generated by cDNA library sequencing [http://esttik.cirad.fr/). All of the EST sequences were assembled into a single fasta file using an in-house custom Perl script. The est_trimmer.pl (http://pgrc.ipk-gatersleben.de/misa/download/est_trimmer.pl) was then used to remove poly-A repeats, low-quality sequences and low-complexity regions at the 5' and 3' ends. Cleaned and high-quality EST sequences were then assembled by the CAP3 assembler  using the default parameters. GSS sequences were also assembled by CAP3.The whole genome sequences of quencing  of CavenMIcro SAtellite, http://pgrc.ipk-gatersleben.de/misa/). The search was restricted to perfect di-, tri-, tetra-, penta- and hexanucleotide motifs with a minimum of six, five, five, four and four repeat units, respectively. For compound SSRs, no interrupting nucleotide sequences were permitted between the two SSRs. A Perl module was used to generate a unique id for each SSR locus and extract the SSR length, nucleotide composition  and approximately 400bp flanking the repeat region. Forward and reverse primers were designed for the identified SSR using primer3 software with the default parameters. Redundant primer sets were removed by an in-house Perl script called non-redudantSSR.pl. SSR loci and primer information were stored in text files for further use.Genomic, GSS and EST sequences were searched using the MISA program  libraries. TE libraries were generated by combining the results of different signature-, homology- and Xu et al.. The resTo identify miRNAs associated with Musa SSR markers, sequences flanking non-redundant Musa markers were subjected to a BLASTN search against known mature miRNA sequences in the miRNA Registry Database V20 (released June 2013) [M. acuminata chromosomes using the ePCR program. To further validate the ePCR results of the in silico physical mapping, we re-analyzed the markers using BLAST-searches against M. acuminata whole-genome sequences. The forward and reverse primers of the physically mapped markers were then mapped to the whole-genome sequences of 23 plant species  using the following conditions: 94°C for 3 min, 35 cycles of 94°C for 30 sec, 55–60°C  for 30 sec, and 72°C for 45 sec, followed by a final step at 72°C for 7 min. The PCR products were then run on 3% agarose gels in 1× Tris–borate–EDTA buffer for 45 min at 80V to determine the amplicon size and assess PCR specificity. DNA bands were visualized by ethidium bromide staining, and a 100bp molecular ladder was used to estimate the amplicon size. In addition, the PCR products of selected primer pairs were resolved on a denaturing 6% polyacrylamide gel and visualized by silver staining.A subset of 330 primer pairs was selected from eleven chromosomes (1 marker from every 1.5 Mbp of the genome) and synthesized by Sangon Company, Shanghai, China. These primers were tested for their utility by PCR amplification of 8 Musa accessions representing diverse genomic groups within the Musa germplasm core-collection maintained at FTRI, Guangzhou, PR China. Genomic DNA was extracted from the young leaves of the accessions as previously described by Gawel and Jarnet  with minM. acuminata genome (data set AA), 403Mbp M. balbisiana genome (data set BB), 41 Mbp EST (Expressed Sequences data) and 19 Mbp GSS(Genome survey sequences) sequences, and the results are presented in O. sativa, A. chinensis, C. papaya, C. sativus, C. melo, P. persica, F. ananassa and V. vinifera  and 15274(15%) SSR loci were monomorphic and polymorphic, respectively. Furthermore, we analyzed the correlation of extent of polymorphism with repeat length for each SSR type; the results of this analysis are presented in the supplementary data. As shown in SSR primers were categorized as either monomorphic or polymorphic based on their BLASTX was used to assign putative functions to SSR-loci. This approach revealed that 5% of all SSR loci were annotated in functional protein-coding sequences in the public non-redundant protein database, whereas 95% had no significant homology to known protein-coding sequences. SSR-loci were grouped in to five major categories based on their functions. The largest category comprised hypothetical/putative/uncharacterized proteins (50%), followed by house-keeping-related proteins, stress-related proteins and transcription factors .A possible association between Musa non-redundant SSR markers and plant transposable elements (TEs) was estimated by BLASTX analysis, which showed that less than 1% of SSR loci are associated with known plant TEs. Among the TE-associated SSR loci, 71% were associated with LTR/Copia-like TEs and 15% were associated with LTR/Gypsy-like TEs .Musa SSR loci were analyzed for possible associations with miRNA candidates. In total, 30% of the SSR loci were predicted to be miRNA-associated , and amoA total of 330 SSR primer pairs from eleven chromosomes were selected for wet lab PCR validation. This analysis revealed that 312 (94%) primer pairs can be used for amplification in at least one Musa spp. accession, with the prominent PCR products having the expected size. Polymorphisms were identified for 243 markers (74%), with allele numbers ranging between 2 and 8 per locus  for a toPinus taeda L. [M. domestica, F. ananassa, and O. sativa; lower than those of A. chinensis and C. papaya; and higher than those of S. italica, B. distachyon, and P. virgatum. Sonah [et al. [et al.[et al.[et al.[Knowledge of the SSR frequency and distribution in the genome provides insight into the possible roles of SSRs in genome organization, evolution and function. We determined the SSR frequency and distribution among four different Musa data sets, finding that the SSR density was nearly two-fold higher in the GSS data set compared with the EST data set. Similar results were reported for taeda L. . Moreovetaeda L. –17, and taeda L. , it is dm. Sonah  demonstrm. Sonah . Our com [et al.  found th. [et al.. The oppl.[et al. reportedl.[et al. examinedE. guineensis [et al. reported that [et al. as we found that dinucleotide repeats were the most frequent repeat class in several higher plants. The high trinucleotide repeat frequency in the transcribed regions of many plant and animal genomes can be explained by the fact that changes to trinucleotide repeat length do not alter the reading frame[We found that dinucleotide repeats were the most common repeat class in the Musa genome. Similar observations were reported for many plant species including sweet orange and ineensis , 23, whiineensis , the genineensis  and cereineensis . Victorited that  dinucleoing frame, 26–28.S. bicolor (monocots), AT-rich SSRs were predominant, while for B. distachyon, O. sativa, P. virgatum and S. italica, GC-rich SSRs were more frequent; however, the differences in AT-rich SSRs were minor  non-redundant Musa SSR primer pairs were identified, meaning that17% of the primer pairs designed from 936 Mbp of sequence data were identified as being redundant.Overall, 15% of the SSRs detected were not adequate for primer design due to insufficient flanking regions. Similar findings were reported in other genome-wide SSR mining studies in plants , 17–18. in silico estimate SSR marker cross-taxon transferability in a wide set of taxonomic groups. The virtual PCR shows a relatively low transferability of Musa SSR markers compared with other plant species tested to date. Transferability of Musa SSRs ranges from 0.29 to 2.14% was related to functional loci, and similar results were reported for citrus. In contL. bicolor[Copiaand 15% LTR/gypsy) were predominantly associated with SSRs compared with other TE classes. Indeed, LTR retrotransposon repeats constitute the largest TE class found in the Musa genome [Considerable proportions of SSRs associated with transposable elements (TE) have been reported in . bicolor, barely . bicolor and Lepi. bicolor. In conta genome . Consequa genome . The TE in silico analysis revealed that some of the TE-associated SSR markers amplified multiple M. acuminita loci (data not shown), and Tay et al.[H. armigera, H. zea, Y. padellus, A. epimuta, and B. betularia. TE-associated SSR markers often generate multiple bands as TEs generally occur in the genome with higher copy numbers; consequently, SSRs within the TEs also occur with higher copy numbers. As a result, primers designed for such loci may produce multiple bands or even fail to generate distinct, detectable PCR products because they amplify too many different sites, limitingthe general use of TE-associated SSRs as informative molecular markers. Additionally, TE-associated SSRs facilitate the development of other types of co-dominant markers such as S-SAP, which are also useful for genetic studies including plants and animals. The combination of our findings and published reports suggest that TE-associated SSRs are ineffective for the development of reliable microsatellite markers that target single-copy regions within the genome.Our ay et al. reportedSSR miRNA-association is not well studied in plants, and until now, nothing was known with regards to the association of Musa SSRs with miRNAs. Our results demonstrated that a considerable number (30%) of Musa SSRs are miRNA-associated. This finding is in agreement with a similar study in black pepper . Dinuclein silico results. These criteria were: (i) a single hit on the A genome (ii) transferability to other species including Musa (iii) in silico polymorphism, (iv) elimination of TE-associated SSR markers and (v) priority for SSR markers with functions. Our results suggest that the primer selection criteria based on our in silico results helped to increase the robustness and quality of SSR markers. Furthermore, our pre-selection criteria significantly reduced the cost and time of SSR marker development.Several features are crucial to successful marker development and primer design: (i) a high proportion of PCR products with the expected size, (ii) a strong banding pattern and (iii) distinct allelic peaks for the tested markers. High-quality and strong PCR amplification of SSRs, in addition to their polymorphism and cross-taxon transferability enhances their value and utility especially for germplasm characterization, marker assisted breeding programs and population genetics studies. We found that 312(94%) of the 330 primer pairs that we selected for wet the lab validation amplified scorable distinct strong PCR product, bands of the expected sizes from the genomic DNA of the tested Musa spp. The PCR success rate was higher than previously reported for Musa 65%-88%)%-88%5, –39 and m and m39 et al., Passos et al. and Passos et al.,[We found that 74% of the tested markers were polymorphic and 94% were transferable to Musa relatives. These results are higher than those previously reported for Musa SSR markers where 43%, 14.7% and 24% were reported as polymorphic by Backiyarani s et al.,, 38, resin silico and experimentally, and developed a freely available Musa SSR marker database.In the present study, we introduced a concise procedure for SSR marker development that is more efficient and effective than published SSR marker development procedures for obtaining highly informative, therapeutic and robust SSR markers. The present method has significantly increased the proportion of polymorphic markers, thereby reducing marker development costs, time and labor. We developed a large number of SSR markers, characterized them both S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOC)Click here for additional data file.S7 Fig(DOC)Click here for additional data file.S1 File(TXT)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOC)Click here for additional data file.


Periodontitis and Alzheimer disease (AD) are associated with systemic inflammation. This research studied serum IgG to periodontal microbiota as possible predictors of incident AD.Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans Y4, Treponema denticola, Campylobacter rectus, Eubacterium nodatum, and Actinomyces naeslundii genospecies-2). In all analyses, we used antibody threshold levels shown to correlate with presence of moderate-severe periodontitis.Using a case-cohort study design, 219 subjects (110 incident AD cases and 109 controls without incident cognitive impairment at last follow-up), matched on race-ethnicity, were drawn from the Washington Heights-Inwood Columbia Aging Project (WHICAP), a cohort of longitudinally followed northern Manhattan residents aged >65 years. Mean follow-up was five years (SD 2.6). In baseline sera, serum IgG levels were determined for bacteria known to be positively or negatively associated with periodontitis  was associated with increased risk of AD . This association was stronger after adjusting for other significant titers . In this model, high anti-E. nodatum IgG  was associated with lower risk of AD .Mean age was 72 years (SD 6.9) for controls, and 79 years (SD 4.6) for cases (p<0.001). Non-Hispanic Whites comprised 26%, non-Hispanic Blacks 27%, and Hispanics 48% of the sample. In a model adjusting for baseline age, sex, education, diabetes mellitus, hypertension, smoking, prior history of stroke, and apolipoprotein E genotype, high anti-Serum IgG levels to common periodontal microbiota are associated with risk for developing incident AD. Poor oral health, including caries, periodontal disease, and edentulism are highly prevalent globally, particularly in the elderly. 6 is ubiquitous in older adults, Periodontitis is a chronic inflammatory disease, initiated by a bacterial biofilm adhering to the tooth surfaces adjacent to the gingiva. Epidemiological evidence supports an association between the level of serum antibodies to periodontal pathogens and stroke, Porphyromonas gingivalis, and poor cognitive test performance among people aged >60 years in the Third National Health and Nutrition Examination Survey (NHANES-III). Poor dental status, a late-life marker of the cumulative effects of oral inflammatory pathologic conditions including periodontitis, is associated with prevalent cognitive impairment and incident dementia. In this study, pre-morbid levels of serum IgG antibodies to selected periodontal microbiota were explored for possible association with risk for incident AD. We hypothesized that serologic markers of periodontitis can serve as predictors of incident cognitive impairment among older adults, and tested this hypothesis in a case-cohort study of the Washington Heights Inwood Columbia Aging Project (WHICAP).WHICAP has longitudinally followed a multiethnic elderly community population in northern Manhattan with serial neuropsychological assessments repeated every 18–24 months. Study design, criteria for diagnosis of AD, and vascular risk factor definitions have been described elsewhere. The present study has a case-cohort design, comparing individuals with incident probable Alzheimer disease with controls without incident dementia through the last available follow-up, matched by race-ethnicity. This design is advantageous for the purposes of an exploratory study because of its cost-efficiency, Individuals to be included in this study were selected in October 2010. Cases were identified as persons without prevalent AD at the time of phlebotomy who were subsequently determined clinically to have AD during follow-up. Thus, the sample included 135 incident AD cases and 134 controls, of whom 110 cases and 109 controls had available serum. Controls comprised approximately 10% of the overall control sample; for these analyses, the sampling fraction was equal to 1 for cases and 1/10 for controls.Porphyromonas gingivalis (strain FDC381), Tannerella forsythia (ATCC43037), Actinobacillus actinomycetemcomitans (FDCY4), Treponema denticola (OMGS3271), Campylobacter rectus (ATCC33238), Eubacterium nodatum (OMGS3356), and Actinomyces naeslundii genospecies 2 (ATCC15987).Serum IgG antibody levels to periodontal bacteria were determined by means of checkerboard immunoblotting a priori approaches: a) those exceeding single antibody thresholds distinguishing clinical periodontitis from healthy periodontal conditions th percentile titer of individuals free of periodontitis according to the definitions by the Centers for Disease Control and Prevention/American Academy of Periodontology (CDC/AAP). Antibody threshold levels were used as a proxy for exposure to common periodontal pathogens, based on the strong associations with clinical periodontitis identified by our group in NHANES-III. 2 tests for comparing categorical variables. Multivariable Cox proportional-hazards regression models were used to examine individual associations between each of the seven antibody thresholds and incident AD. Time-to-event was defined as duration from phlebotomy to time of AD diagnosis. To minimize small-sample bias, conservative methods suited for case-cohort analyses were used, including derivation of an “exact” pseudolikelihood estimator for the point estimate, and robust variance estimation for derivation of the 95% confidence interval for each association. Descriptive analyses used t-tests for comparing means of continuous variables, and χC. rectus were found in 69% of the participants, followed by 63% to T. forsythia, 53% to T. denticola, 23% to P. gingivalis, 19% to E. nodatum, 11% to A. actinomycetemcomitans and 10% to A. naeslundii. The proportions did not differ between cases and controls , were less educated, and were more likely to have a history of prior stroke Table 1.A. naeslundii serum IgG (>640 ng/ml) had a consistently higher risk for incident AD (E. nodatum (>1755 ng/ml) approached statistical significance with a decreased risk of incident AD . Fully adjusted survival curves are provided for each of these analyses in S1 and S2 Figures. Next, when high titers to both A. naeslundii and E. nodatum were included into similar models , but a lesser risk in the presence of high E. nodatum levels  in the fully adjusted model. Neither antibody demonstrated evidence of being an effect modifier of the other in this relationship.Participants with elevated ident AD Table 3,A. naeslundii (>380.9 ng/ml) and E. nodatum (>1609.6 ng/ml) individually, no significant association with risk of incident AD was identified in these models , as when using the individual thresholds .When considering the highest quintile levels for e models Table 5.A. naeslundii IgG antibody, and this finding was further strengthened when high E. nodatum levels were also included in the model. To our knowledge, this is the first longitudinal study to identify an association between these serological markers of periodontal infection and incident AD.In this community-based, multiethnic cohort of elders, an increased risk of incident AD was observed among participants with high serum There are several plausible mechanisms relating periodontal infections to impaired cognition: A. naeslundii IgG is associated with increased risk of incident AD. A. naeslundii is a gram positive rod associated with early dental plaque formation, A. naeslundii host response and general health outcomes has not been explored to the same extent as with established periodontal pathogens such as P. gingivalis that has been implicated in atherogenesis. A. naeslundii antibodies are associated with periodontal health A. naeslundii. A. naeslundii, and specifically the peptidoglycan from its cell wall, likely has a strongly pathogenic role in osteoclast activation and alveolar bone loss similar to gram negative periodontal organisms such as P. gingivalis, including elevation of IL-1β, IL-6, and TNF-α mRNA. A. naeslundii can elicit strong expression of several cytokines and chemokines in oral epithelial cells, including IL-1α, IL-6, TGFα, IL-8, fractalkine, and interferon gamma-induced protein (IP-10). The primary finding of the study is that high serum E. nodatum IgG levels were associated with lower AD risk in the most conservative regression models. E. nodatum is a gram positive, anaerobic rod that has been implicated in periodontitis. E. nodatum oral colonization are associated with generalized aggressive periodontitis among younger individuals E. nodatum IgG was associated with lower risk for clinical periodontitis in NHANES-III. Additionally, high serum P. gingivalis were cross-sectionally associated with poor cognitive test performance among patients aged >60 years in NHANES-III. Our group previously reported that high antibody levels to The clinical significance of a high antibody response to specific periodontal microbiota is uncertain. As previously demonstrated, A. naeslundii and E. nodatum and incident AD were strengthened when these antibody responses were explored jointly in regression models. Taken together, these findings suggest the host response to these two periodontal species, although primarily targeted towards control of the localized periodontal infection, may nevertheless stimulate a systemic inflammatory cascade, and support a hypothesis of mediation of incident AD risk through systemic host inflammatory responses. Since data on clinical periodontal status and inflammatory markers such as CRP were not available in this study, a potential interaction between clinical periodontitis and systemic antibody responses cannot be determined.Interestingly, the observed relationships between high levels of IgG to th percentile, which as shown in A. naeslundii.Several additional limitations and potential strengths of the present study are acknowledged. Measurable socioeconomic covariates provide a restricted scope of lifelong socioeconomic status, and could lead, at least in part, to residual confounding. However, the influence of socioeconomic differences was likely diminished by matching cases and controls on race-ethnicity. Additionally, adjusting for chronological age alone might not capture residual confounding due to age-related illnesses that are variably distributed between people of similar ages. Although analyses were limited to the study of antibodies to seven periodontal bacteria, these were preselected on the basis of their documented association with clinical periodontal status A. naeslundii and E. nodatum in the model resulted in nearly identical findings using both statistical methods. Moreover, the prevalence of high antibody levels was similar to the prevalence of other risk factors for dementia including DM Table S1. Finally, persons with baseline mild cognitive impairment were not excluded and thus the possibility of reverse causation, i.e., that cognitive impairment may have affected the antibody levels to periodontal microbiota, cannot be ruled out.The possibility of a small-sample bias is also recognized. However, we used previously described methodology The levels of serum antibodies to periodontal bacteria alone do not capture the complex interplay between bacterial infection and host response in the context of periodontitis, and as suggested here, possibly AD. The present findings are based on a limited sample, and their external validity needs to be confirmed in independent cohorts. Notably, since the development of this study, serum antibodies to other periodontal organisms not included in the panel investigated here have also been identified as potential risk markers for AD and mild cognitive impairment. S1 FigureA. naeslundii antibody levels above a clinically defined threshold A. naeslundii antibody levels (dotted line) in a fully adjusted model .Cox-proportional hazards regression of dementia-free survival of the association with high (DOCX)Click here for additional data file.S2 FigureE. nodatum antibody levels above a clinically defined threshold E. nodatum antibody levels (dotted line) in a fully adjusted model .Cox-proportional hazards regression of dementia-free survival of the association with high (DOCX)Click here for additional data file.S1 TableA. actinomycetemcomitans, Pg: P. gingivalis, Tf: T. forsythia, Td: T. denticola, Cr: C. rectus, En: E. nodatum, and An: A. naeslundii.Aa: (DOCX)Click here for additional data file.S1 DataThis dataset provides the periodontal antibody levels (ng/ml) for all 219 participants included in this study.(XLSX)Click here for additional data file.


Iatrogenic skin and soft tissue infections by rapidly growing mycobacteria are described with increasing frequency, especially among immunocompromised patients.Mycobacterium fortuitum skin and soft tissue infections after subcutaneous injections to relieve joint pains by a Vietnamese traditional medicine practitioner. Moreover, we present dilemmas faced in less resourceful settings, influencing patient management.Here, we present an immunocompetent patient with extensive This case illustrates the pathogenic potential of rapid growing mycobacteria in medical or non-medical skin penetrating procedures, their world-wide distribution and demonstrates the dilemmas faced in settings with fewer resources.The online version of this article (doi:10.1186/s12879-014-0550-z) contains supplementary material, which is available to authorized users. Mycobacterium fortuitum is a member of the group of non-pigmented Rapidly Growing Mycobacteria (RGM), and is found ubiquitously in nature , over a wide geographical area. Infections with non-tuberculous mycobacteria have been described increasingly, especially in immunocompromised patients and as iatrogenic infections in immunocompetent patients, causing a variety of local and disseminated disease. RGM in particular can cause local skin and soft tissue infections (SSTI) and have also been described in outbreak and pseudo-outbreak settings involving infections after surgery or other invasive procedures [M. fortuitum infection of the skin and soft tissues covering several joints after injection of traditional Vietnamese medicine to relieve joint pain. We discuss the diagnostic process and treatment for this patient in a setting with fewer resources.ocedures -3]. Her. HerMycoAn immunocompetent, 61-year old female patient from Ho Chi Minh City, Vietnam, was admitted to the Hospital for Tropical Diseases with multiple painful fluctuating and non-fluctuating masses on both hands and feet , neutrophiles 81.2% (reference range: 49-65.5%). Blood cultures  were taken and empiric antimicrobial therapy was started with intravenous (i.v.) oxacilline to cover Staphylococcus aureus and group A beta-haemolytic streptococci.Laboratory findings showed: procalcitonin 0.083 ng/ml (reference range <0.05 ng/ml), leucocytes 14.8 • 10A pulmonary X-ray, and X-ray imaging of hands and feet were unremarkable, and incision and drainage of the abscesses followed. Gram stains of the collected fluids showed slender beaded and fragmentally stained Gram positive rods, suggestive of mycobacteria. This was subsequently confirmed by Ziehl-Neelsen staining  with rapidly growing mycobacteria was made, and treatment was initiated with oral clarithromycine (500 mg/b.i.d.) and intramuscular (i.m.) amikacin (500 mg/t.i.d.).Mycobacterium spp. This was confirmed by Gram and ZN staining and the isolate was later genotyped as Mycobacterium fortuitum by 16S DNA PCR and sequencing analysis.Purulent fluid was cultured on standard culture media (blood agar and McConkey agar) for three days and yielded grey wrinkled colonies on blood agar (no growth on McConkey agar)  by disk diffusion on Müller-Hinton agar showed the strain to be resistant to fluoroquinolones , azithromycin and tobramycin, but susceptible to amikacin, imipenem and amoxicillin-clavulanic acid. Clinical improvement during the 120-day admission was variable and slow, with subfebrile temperatures (38°C) last monitored on day 14, and the appearance of new fluctuating lesions on days 23 (hands/feet), 29 (hands/feet), 32 (right shoulder and hip), and 72 (shoulder), which were incised and drained. Antimicrobial therapy was adjusted several times during admission based on repeated DST results and financial incentives. Empiric treatment was started with amikacin (i.m. 500 mg/t.i.d.) and oral clarithromycin (500 mg/b.i.d.). This was later changed to amikacin (i.m. 500 mg/t.i.d.), doxycycline (100 mg/b.i.d.) and trimethoprim-sulfamethoxazole , after culture results and pending additional DST. Imipenem, to which the isolate was susceptible but which the patient could not afford, was not given at this time. Once resistance to tetracyclines and co-trimoxazole were reported and amikacin had been administered for 14 days, therapy was adjusted to imipenem monotherapy i.v. 500 mg/t.i.d, and later (day 50) to imipenem 1 gram/q.i.d, based on international guidelines. Imipenem therapy was funded by the hospital. The patient was discharged on day 120, at which point imipenem was changed to oral amoxicillin-clavulanic acid (500/125 mg/t.i.d.), and follow-up visits transpired for 8 months. Antimicrobial treatment was stopped 4 months after discharge, while clinical improvement was observed 1 month after discharge (Table et al. describe M. fortuitum infection with multiple skin lesions after acupuncture. This was treated with doxycycline and ciprofloxacin for three months which led to full recovery . The. Theet aOur patient’s treatment regimen was altered on many occasions, but overall, 105 days of imipenem monotherapy was prescribed, in addition to the 14 days of amikacin therapy and 4 months of amoxicillin-clavulanic acid, for which the isolate was sensitive according to disk diffusion. After discharge and an 8 months follow-up period, she remained free of symptoms.M. fortuitum and other RGM are not very pathogenic, but can cause infections after direct inoculation into sterile sites, e.g. trauma with skin damage, use of contaminated surgical instruments or fluid for injection or after use of contaminated tap water during invasive procedures [M. fortuitum appear to occur in younger patients who are generally not immunocompromised, and manifest more often after surgical procedures [M. fortuitum is more sensitive to antibiotics than the related M. abscessus or M. chelonae and infections have a better recovery rate [In conclusion, ocedures . Infectiocedures . M. fortery rate . In our Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.NVVC and DHL were responsible for patient treatment, NPHL, NTT, JC and HRVD participated in the described diagnostic process, NVD and MEK collected data and information and drafted the report, HRVD finalized the manuscript, all authors have seen and approved the manuscript.


Ailanthus altissima and Robina pseudoacacia are two successful invasive species of floodplains in central Spain. We aim to explain their success as invaders in this habitat by exploring their phenological pattern, vegetative and sexual reproductive growth, and allometric relations, comparing them with those of the dominant native tree Populus alba. During a full annual cycle we follow the timing of vegetative growth, flowering, fruit set, leaf abscission and fruit dispersal. Growth was assessed by harvesting two-year old branches at the peaks of vegetative, flower and fruit production and expressing the mass of current-year leaves, stems, inflorescences and infrutescences per unit of previous-year stem mass. Secondary growth was assessed as the increment of trunk basal area per previous-year basal area. A. altissima and R. pseudoacacia showed reproductive traits  different from P. alba and other native trees, which may help them to occupy an empty reproductive niche and benefit from a reduced competition for the resources required by reproductive growth. The larger seeds of the invaders may make them less dependent on gaps for seedling establishment. If so, these invaders may benefit from the reduced gap formation rate of flood-regulated rivers of the study region. The two invasive species showed higher gross production than the native, due to the higher size of pre-existing stems rather than to a faster relative growth rate. The latter was only higher in A. altissima for stems, and in R. pseudoacacia for reproductive organs. A. altissima and R. pseudoacacia showed the lowest and highest reproductive/vegetative mass ratio, respectively. Therefore, A. altissima may outcompete native P. alba trees thanks to a high potential to overtop coexisting plants whereas R. pseudoacacia may do so by means of a higher investment in sexual reproduction. In the last decades, the scientific interest in explaining causes and consequences of biological invasions has drastically increased Recent studies have emphasized the relevance of phenological differences between exotic and native plants as a mechanism favouring invasions In the Mediterranean part of Europe, floodplains are one of the habitats more susceptible to exotic plant invasions Populus alba L. dominates clay-rich basic soils of middle or low river stretches, where it coexists with other less abundant native trees, such as Salix alba L., Populus nigra L., Fraxinus angustifolia L. and Ulmus minor Mill. th century these species also co-occur with naturalized exotic trees, such as Ailanthus altissima (Mill.) Swingle and Robinia pseudoacacia L., which are native to E-China and the Appalachian Mountains (USA), respectively R. pseudoacacia, profuse seed production and fast growth in A. altissima and vegetative reproduction by root suckering in both species A. altissima may increase soil fertility R. pseudoacacia may increase the input of atmospheric N into the soil R. pseudoacacia leaves by aquatic invertebrates P. alba-dominated riparian forests of Central Spain.In Mediterranean riparian forests of central Spain, the native A. altissima and R. pseudoacacia in the above described community by comparing their life-history strategies with that of the dominating native tree in the recipient community, P. alba. We hypothesize that (1) the exotic species differ in phenology from the dominating natives We aim to explain the invasive success of Populus alba, which is accompanied by S. alba and to a minor extent, P. nigra, F. angustifolia and U. minorA. altissima and R. pseudoacacia are frequent, mainly in human-disturbed areas P. alba has been found to be an exotic invasive species in other regions, such as Australia and South Africa This study was conducted in the floodplains of a medium-low stretch of the Henares River in central Spain (Province of Madrid), which is a public non protected area where no permit access is required. The study area spans over 22 km, from Alcalá de Henares to Mejorada del Campo. Environmental conditions and vegetation structure along this stretch are considered to be homogeneous Populus alba, A. altissima and R. pseudoacacia, are considered as fast-growing pioneer trees which can reach up to 25 m of height P. alba and A. altissima are dioecious, while R. pseudoacacia is monoecious but self-pollination is prevented by a hairy collar below the stigma P. alba is wind-pollinated P. alba and A. altissima have specific structures assisting wind-dispersal P. alba, the two exotics are able to form seed banks, both in the soil and in the plant canopy The three studied species, A. altissima and P. alba, respectively, to have representation of both sexes. Mean ± SE trunk diameter at breast height (DBH) of selected trees were 34.7±6.2, 28.0±3.3 and 33.5±4.0 cm for A. altissima, R. pseudoacacia and P. alba, respectively, which was representative of the average tree size in the studied populations. Monitoring of phenology spanned from 7th February 2011 until 12th April 2012, when fruit dispersal of A. altissima was completed. Every 1–3 weeks, depending on the activity of the plant, we noted whether the following phenophases were active or not: leaf development, flowering, fruit set, fruit dispersal, leaf abscission. Phenophases were considered active in a tree when they were easily observed at naked eye in at least 5% of the crown. These data are available at In January 2011, before any sign of budbreak, we selected 10–11 healthy adult trees per species to performed phenological observations. After flowering, we selected 5 and 8 additional trees in R. pseudoacacia, 6 female and 6 male adult trees of A. altissima. This year we could not sample P. alba due to technical reasons. To minimize the risk of branch collection interfering with tree growth, in January 2011 we selected 6 new trees per species and sex plus 6 female adult trees of P. alba  and previous-year (t-1) cohorts, and then each cohort was separated into different organs, oven-dried (>48 h at 60°C) and weighted . To be representative of the full tree, current-year FL and FR values were multiplied by the proportion of branches in the canopy holding flowers or fruits. This proportion was estimated at the time of flower and fruit sampling as follows: we divided the canopy of each tree in four quarters and randomly selected 10 branches in each quarter. The proportion of the selected branches holding flowers/fruits was considered to be representative for the full tree. To correct for the allometric effects of previous-year shoot mass on current-year production, the oven-dried weight of every current-year fraction  was divided by that of the previous-year stem (t−1S) bearing the fraction. In this way we obtained the relative production of stems (t/St−1S), leaves (t/St−1L), inflorescences (t/St−1FL) and infrutescences (t/St−1FR). In the case of P. alba, the six initially selected trees failed to produce fruits, so they were replaced by 6 additional trees from the same population for the last sampling of fruit production. These data are available at In January 2010 we selected 6 adult trees of 10RW) and 2011 (11RW), was measured with an accuracy of 1/100 mm using a LINTAB measuring table and the software TSAP P) to derive the trunk radius (R = P/2π) and assessed the average cork thickness (thC) with a cork calliper  in four equidistant points around the trunk. Using these data we estimated the trunk basal area in 2011 excluding the cork (2011 = π(R-CthBA)2), the 2010 basal area (2010 = π(R-Cth-RW11BA)2 and the 2009 basal area (2009 = π(R-Cth-RW11-RW10BA)2). The basal area increment of each tree in 2010 and 2011 t(ΔBA) was the difference between current- and previous-year basal areas. These data are available at In February 2012, before the onset of the new growing season, 3–4 radial wood samples were extracted at 1.30 m of trunk height using an increment core borer  from the same trees used in 2011 for branch production. Wood samples were glued on wooden holders and sanded until tree-rings were clearly visible under a binocular microscope. The width of the last two xylem rings, corresponding to 2010 (st and transformed into angles of a 52-week circle) were pair-wise compared across species using the multisample Watson-Williams F test Phenological data were analysed using circular statistics with Oriana 2.0 package. Dates of beginning and end of each phenophase . Therefore, we decided to perform all analyses separately for 2010 and 2011. Although in 2010 we lack of native control, we decided to compare the two invasive species to check the extent to which the patterns found for them were consistent across years.In previous analyses we found that some growth variables differed between years in some species. For instance, in t−1S with tS, tL, tFL and tFR were fitted using standard major axis estimation (SMA) after log-transforming both variables A. altissima and across species using the likelihood ratio statistic (LRS). When cross-species differences were detected, we performed pairwise comparisons, lowering the significance level to 0.017, following a Bonferroni correction for multiple comparisons The allometric relations of t, Lt, FLt, FRt,St/St−1, Lt/St−1, FLt/St−1S and t/St−1FR were compared among species, using linear mixed models, with species as fix factor, DBH as covariate and tree as random factor. When species was significant, pairwise comparisons were performed using a Tukey post-hoc test. In the case of A. altissima, variables were also compared among sexes (fix factor), using DBH as additional fix factor and tree as random factor. When necessary, variables were log-, 0.25- or 0.01-power-transformed to achieve homoscedasticity and normal distribution of residuals. Analyses were performed using R 3.0.2 and packages “nmle” and “multcomp”.A. altissima) and each year. Species above the 1∶1 line had a relative higher investment in sexual reproduction while species below this line invested more in vegetative growth.To assess the relative investment in vegetative growth vs. sexual reproduction, we represented in a plane defined by the mean vegetative (leaves+stems) and reproductive (inflorescences+infrutescences) mass produced per previous-year stem, each species, sex (in the case of t(ΔBA) and previous-year basal area t−1(BA) was fitted using standard major axis estimation (SMA). SMA lines were tested for different slope, elevation and shift across species as explained above.The allometric relation between basal area increment A. altissima arrested growth significantly earlier than R. pseudoacacia and P. alba , intermediate in R. pseudoacacia (late April–mid May), and the latest in A. altissima (June) , followed by R. pseudoacacia (May–September) and by A. altissima (June–October) , P. alba ended later (late December), and R. pseudoacacia was intermediate  . AccordiOctober) . Leaf abrmediate . Finally and May .A. altissima the slope of the allometric lines did not differ between sexes , but the tL − t−1S line showed higher elevation in females, whereas the t- St−1FL line showed higher elevation in males . By contrast, t/St−1FL and t/St−1FR followed the ranking R. pseudoacacia ≥ P. alba ≥ A. altissima . In 2010 comparisons between A. altissima and R. pseudoacacia showed similar trends, but less often significant . In 2011, the random factor (tree) explained between 37 and 57% of the variance not explained by the fixed factors, while in 2010 this proportion varied from 6% to 83% , i.e. this species produced less infrutescence mass for a given value of previous-year stem mass , as a mechanism to exploit temporal empty niches, to reduce competition and to promote coexistence Populus nigra, Salix alba and Ulmus minor) show a reproductive phenology similar to P. alba (early flower and fruit set), only F. angustifolia showing a pattern in between P. alba and the invaders  and vegetative (V) buds, which allows any combination of vegetative and reproductive phenology A. altissima and R. pseudoacacia enclose vegetative and reproductive primordia within the same buds (VR buds), which forces these species to start shoot elongation and leaf expansion before bloom P. alba, and the other native trees  A. altissima and R. pseudoacacia, like the above mentioned native shrubs/vines, are insect-pollinated and present late flowering The period when a plant flowers is determined by the interaction between genetic factors and environmental variables  A. altissima and R. pseudoacacia. The invaders may benefit from the reproductive asynchrony with native trees due to a reduced competition for the resources demanded for flowering and fruit set, i.e. nutrients, water and pollinators, which in A. altissima and R. pseudoacacia are mainly honeybees, beetles and other nectar- and pollen-feeding generalist insects R. pseudoacaciaA. altissima and R. pseudoacacia only have to face competition for pollinators by the sub-dominant undercanopy entomophilous shrubs and vines. Moreover, the latter species may facilitate exotic tree invasion by providing an established pollinator community, as found before A. altissima and R. pseudoacacia also contrasted with P. alba in the year-round seed dispersal period. Seed viability is very short in P. alba and the remaining native trees of the studied community P. alba was not significantly different from any of the invaders  and only F. angustifolia possesses seeds larger than those of the invaders Acer negundo L. and Elaeagnus angustifolia L, which are potentially invasive in Spanish riparian forests The niche for insect-pollinated late-flowering trees seems to be empty in the studied native community and successfully colonized by invaders . AlternaA. altissima and P. alba, respectively, and intermediate in R. pseudoacacia, either for stems, leaves, inflorescences or infrutescences  over the native at the adult stage. However, when plant production was relativized per unit of previous-year stem mass , differences between R. pseudoacacia and P. alba tended to disappear  and A. altissima only retained a superior relative production for stems but showed the lowest reproductive relative production  found in A. altissima agrees with the above reports of great potential for height growth A. altissima to have a less steep allometric relation between basal area increment and trunk area than the other species  different to those of the tree species dominating the studied riparian forest community of central Spain. These differences may help them to occupy an empty reproductive niche, and then benefit from a reduced competition for the resources required by flower/fruit development and for pollinators. Moreover, the long fruit set period of invaders and the associated larger seed mass may make them less dependent on gaps for seedling establishment than the tiny-seeded native tree P. alba. Such ability may help the invaders to expand their reproductive niche in flood-regulated rivers of the study region. The two invasive species showed a superior gross production than the native which was due to a higher size of pre-existing stems rather than to a faster relative growth rate. At the studied adult stage A. altissima and R. pseudoacacia showed the lowest and highest reproductive/vegetative mass ratio, respectively. Therefore, A. altissima might outcompete the native trees thanks to a higher vegetative growth whereas R. pseudoacacia may do so by means of a higher investment in sexual reproduction. Altogether, our findings suggest that preserving the natural flood regime of the river is the best strategy to favour the native trees over the two invaders in this case study. Given the great vegetative production of the invaders at adult stage, especially in the case of A. altissima, an efficient management should focus on early detection and prevention of establishment. Further information on the growth potential of all native and invasive tree species at different ontogenetic stages would help to predict the dynamics of this invaded riparian forest community.Figure S1A. altissima (data of 2011). Different symbols and lines represent different sexes. The Wald statistic indicates a significant difference in line elevation. No significant difference was found between slopes.Allometric relationships between previous-year stem mass and current year leaf (left) and inflorescence (right) mass in (TIF)Click here for additional data file.Figure S2Left- Allometric relationships between previous-year stem mass and current year stem (A), leaf (C), inflorescence (E) and infrutescence (G) mass . The Likelihood Ratio Statistic (LRS) comparing slopes across species and its significance is shown in each chart. When slopes were equal, we also show results for the Wald satistics (WS) comparing elevation and shift along a common slope. Right- current-year stem (B), leaf (D), inflorescence (F) and infrutescence (H) mass per unit of previous-year stem mass. Different letters across species indicate significant differences . Data from 2010 collection.(TIF)Click here for additional data file.Figure S3Ailanthus altissima, showing that the same current-year stem bears leaves and inflorescences, which were derived from the same VR bud (see text) (April 2009). Centre- Picture of two recently opened buds of Robinia pseudoacia. It can be observed that both leaves and inflorescence derived from the same VR buds (March 2009). Right- Picture of a female inflorescence of Populus alba. It can be seen that the inflorescence grew on the previous-year stem from a R bud, while the apical vegetative (V) bud, enclosing the current-year vegetative organs, is still close (March 2009). Pictures by the authors.Left- Picture of a current-year shoot of (TIF)Click here for additional data file.Table S1Phenological data of the studied species.(XLSX)Click here for additional data file.Table S2Branch production data of the studied species.(XLSX)Click here for additional data file.Table S3Secondary growth of the studied species.(XLSX)Click here for additional data file.Table S4Ailanthus altissima.Comparison of shoot production between sexes in (DOC)Click here for additional data file.