We used a comparative approach spanning three species and 90 million years to study the evolutionary history of the avian sex chromosomes. Using whole transcriptomes, we assembled the largest cross-species dataset of W-linked coding content to date. Our results show that recombination suppression in large portions of the avian sex chromosomes has evolved independently, and that long-term sex chromosome divergence is consistent with repeated and independent inversions spreading progressively to restrict recombination. In contrast, over short-term periods we observe heterogeneous and locus-specific divergence. We also uncover four instances of gene conversion between both highly diverged and recently evolved gametologs, suggesting a complex mosaic of recombination suppression across the sex chromosomes. Lastly, evidence from 16 gametologs reveal that the W chromosome is evolving with a significant contribution of purifying selection, consistent with previous findings that W-linked genes play an important role in encoding sex-specific fitness. Drosophila), or incipient sex chromosome systems, such as those observed in plants and fish  and Y  chromosome coding content . In spitand fish .Older sex chromosomes support the classic model of sex chromosome evolution, where intra-chromosomal rearrangements are proposed to initiate the suppression of recombination across large regions . TheoretGasterosteus aculeatus) and white campion (Silene latifolia) X and Y chromosomes originated around 30 million years ago (Mya) and less than 10 Mya, respectively. Work on these systems indicates that recombination suppression evolves heterogeneously along the length of the chromosome  and wild turkey  W chromosomes, we used a combined approach based on RNA-seq data and sequence similarity to known red jungle fowl  W-linked genes.Previous efforts to identify W-linked sequences in birds  have resA. platyrhynchos and M. gallopavo, taken at the start of their first breeding season . All samples were collected in accordance with national guidelines and with permission from institutional ethical review committees. The left gonad was collected separately from five male and five female A. platyrhynchos, and seven male and five female M. gallopavo. The spleen was collected from five male and five female A. platyrhynchos and four male and two female M. gallopavo. The samples were homogenized and stored in RNAlater until preparation. We extracted RNA using the Animal Tissue RNA Kit (Qiagen) and The Wellcome Trust Centre for Human Genetics, University of Oxford, prepared and barcoded samples using standard methods. RNA was sequenced on an Illumina HiSeq 2000 resulting in on average 26 million 100 bp paired-end reads per sample.RNA-seq data were obtained from captive populations of www.bioinformatics.babraham.ac.uk/projects/fastqc) and filtered using Trimmomatic version 0.22 , M. gallopavo, and A. platyrhynchos cDNA sequences were obtained from Ensembl version 74 . The dS of three newly identified gametologs were also consistent with the previously identified strata, with overlapping 95% confidence intervals  coding sequences were obtained from Ensembl version 74  we identified the orthologous G. gallus W-linked gene using BLAST and the G. gallus Z-linked ortholog from A. platyrhynchos and M. gallopavo Z-linked orthologs were defined as the reciprocal best hit of the G. gallus Z-linked ortholog; (3) in a few instances there was no reciprocal best hit, often because of high sequence similarity between Z and W gametologs. For these genes, the Z-linked reciprocal ortholog was identified as the next best BLAST hit of the G. gallus Z-linked ortholog.For A. platyrhynchos, M. gallopavo, and G. gallus. We verified Z-linkage of reciprocal orthologs using positional information in the M. gallopavo genome assembly. The A. platyrhynchos genome assembly lacks annotated chromosomes and as recently diverged Z- and W-linked genes share a high degree of sequence similarity, to avoid misidentifying the Z-linked reciprocal ortholog as a W-linked paralog, we verified the reciprocal ortholog was not located on the W chromosome using PCR. However, this approach does not distinguish Z chromosome to autosomal gene duplication, but given the pronounced stability of avian genomes   and nonsynonymous (dN) divergence in pairwise comparisons and generate standard errors. We verified dS ≤ 1 for all gametologs, thereby avoiding inaccurate divergence estimates due to mutational saturation and double hits (To identify the number and boundaries of evolutionary strata in nce (dS) , as dS ph MUSCLE ,2004b inh MUSCLE . Alignmeh MUSCLE  was usedble hits .A. platyrhynchos gametologs had dS < 0.02. We verified that the annotated Z and W genes were distinct coding sequences by identifying female-limited SNPs using RNA-seq data and W-linked SNPs from Ensembl annotated sequences.Three dS estimate to avoid potential problems of relaxed or diversifying selection acting on gene copies  years  years .M. gallopavo gametologs to independently confirm the four strata previously identified in G. gallus  obtained coding sequences for gametologs in the G. gallus, A. platyrhynchos, M. gallopavo, and T. guttata from Biomart, and aligned the translated protein sequences with MUSCLE  we constructed maximum-likelihood gene trees with 1000 bootstrap replicates in MEGA5  and specified the –group option to look only for conversion between gametologs in G. gallus, A. platyrhynchos, and M. gallopavo. We used the –Seqtype = SILENT option to specify coding sequence and that gene conversion fragments should only be detected using silent site amino acid polymorphisms. High false-positive rates have been documented when this option is not implemented, particularly if genes are subject to contrasting selective regimes  where no mismatches are permitted between aligned putative conversion fragments and repeated the analysis with a relaxed mismatch penalty  to ensure the number of mismatches did not bias our analysis. GENECONV detects inner fragments, which arise from gene conversion events between the ancestors of two aligned sequences and outer fragments, which represent a conversion events originating outside of the alignment or events whose signature has been weakened by subsequent mutation. We identified significant inner gene conversion fragments between gametologs as evidence of ongoing recombination between the sex chromosomes.To measure the completeness of recombination suppression between gametologs, we tested for evidence of gene conversion using GENECONV . GENECON regimes , 2010. WOn closer inspection, gene conversion events identified in this study span several exons that in turn are separated by extremely dissimilar intronic sequences. As GENECONV identifies potential gene conversion sites as long fragments of sequence bounded by variable sites, it estimates the maximum sequence length over which recombination acts. To avoid falsely identifying gene conversion sites, we excluded exons where GENECONV fragments span less than 50 bp. We used informative polymorphic sites and maximum-likelihood exon trees to establish the direction of gene conversion.To assess the selective regime acting on Z and W coding sequences, we used the branch models and branch-site test in the CODEML package in PAML version 4.7 .dN/dS) to vary across branches. By comparing a model where ω is estimated to one where it is fixed to equal 1  or 0 (strict purifying selection) it is possible to assess the contribution of neutral and purifying selection to coding sequence evolution. To do this, we used the multiple sequence alignments described above (used to build gene trees and test for gene conversion). If A. platyrhynchos and M. gallopavo gametologs ceased recombining before the last common ancestor of the Galloanserae the following gene tree was specified , A. platyrhynchos W), , A. platyrhynchos Z))) and if A. platyrhynchos gametologs diverged independently the following gene tree was used , ), )).Branch models  allow ω  allows ω to vary across branches and among sites and permits tests to detect positive selection acting on a subset of sites along specific lineages. Under this model, we estimated ω across W and Z branches separately and then tested whether ω was significantly different to 1 using the settings fix_omega = 1, omega = 1.M. gallopavo) and mallard duck (A. platyrhynchos). Our combined dataset comprised 14 W-linked genes in M. gallopavo and 21 in A. platyrhynchos, together with 27 previously identified W-linked genes in the red jungle fowl   Table and thesdS) divergence estimates  Z-linked ortholog included as an outgroup, indicate that all four strata predate the G. gallus–M. gallopavo divergence estimated at roughly 30 MYA , arose between 41 and 71 Mya and Stratum IV, corresponding to 0–11 Mb (dS = 0.137–0.257), between 36 and 68 Mya , 54–61 Mb , and arose between 75 and 106 Mya, and 45 and 71 Mya, respectively.For the three nes Figs.B, S3. FoA. platyrhynchos gametologs located in the youngest Galliform strata, dS estimates for five gametologs are significantly lower than the corresponding G. gallus gametolog dS estimates, where 95% confidence intervals are nonoverlapping  and found no significant difference between the results. These gametologs are uniformly distributed across the Z chromosome, located in Conserved Strata I, II, and Anseriform-specific Stratum III, indicating ongoing recombination along the whole length of the avian sex chromosomes. In total, we detected four Z- and four W-linked exons subject to gene conversion .We recalculated Studies examining the evolutionary forces acting after recombination suppression are mainly limited to male heterogametic systems . As a redN/dS) was significantly lower than 1 in 14 gametolog families, indicating these genes are evolving primarily due to purifying selection .For W-linked branches, ω   defined a priori . We foun0) Tables, S4.dS is constant across a gene and the GC bias in the mismatch repair process can inflate ω (dN/dS) . ATP5A1W encodes a mitochondrial ATP synthase subunit. As the W chromosome and mitochondrial genome are both maternally inherited and effectively linked (Gene conversion violates the assumption in PAML that  (dN/dS) . Consequ (dN/dS) . Althougs genome  and may y linked , one plaThe process of recombination suppression is a crucial component of sex chromosome evolution, facilitating the divergence and differentiation of gametologs . StudiesWe used a comparative approach to study the dynamics of sex chromosome recombination suppression on the largest collection of W-linked coding content yet assembled across 90 million years of avian evolutionary history. Our results reveal that recombination between the Z and W chromosomes ceased independently multiple times across different avian lineages. The pattern we uncover is consistent with both the inversion model  where evG. gallus (G. gallus and M. gallopavo (Using both maximum-likelihood gene trees and synonymous divergence estimates, we confirm that four strata are conserved across the Galliformes Fig.. These f. gallus , and fol. gallus . Using nallopavo .A. platyrhynchos sex chromosomes, and the topology of gene trees revealed that gametologs diverged independently in this region (Fig.dS across strata.Similarly, maximum-likelihood gene trees reveal that the two oldest Galliform strata are conserved on the Anseriform Z chromosome. In contrast, recombination suppression is heterogeneous across the remainder of the gion Fig.. DivergeA. platyrhynchos Z chromosome means we cannot rule out the possibility that Anseriform-specific Stratum III is actually made up of two distinct strata formed by a lineage specific inversion. Under this scenario, the three gametologs with dS estimates <0.02 would comprise the youngest stratum with ongoing recombination between the W and Z orthologs. Positional information may support this scenario, as two of the three gametologs are located between 0 and 10 Mb on the Z chromosome.The lack of positional information for the A. platyrhynchos gametologs showed evidence of significant gene conversion, and were distributed across the length of the Z chromosome. Therefore, even for highly differentiated gametologs, certain exons are prevented from diverging. This is consistent with previous studies documenting gene conversion between X- and Y-linked orthologs in mammals (A. platyrhynchos W chromosome is highly divergent in comparison to closely related avian W chromosomes (Some gametologs are subject to gene conversion, lending further support to the notion that recombination suppression is not complete. Three  mammals . Gene co mammals  in addit mammals . Interesomosomes . We findG. aculeatus and S. latifolia sex chromosomes. The S. latifolia XY system originated 10 Mya, and a neo-sex chromosome formed 2 Mya in G. aculeatus. In both these system, recombination suppression evolves heterogeneously (This heterogeneous pattern of sex chromosome divergence and ongoing recombination is consistent with eneously . The undeneously , or locaeneously , may be G. gallus, A. platyrhynchos, and M. gallopavo with the exception of Conserved Stratum II. Using a molecular clock that accounts for sex-specific mutation rates in Z- and W-linked genes, the divergence of Conserved Stratum II was estimated to have occurred between 45 and 71 Mya. Galliformes and Anseriformes diverged 90 Mya (van Our divergence estimates for recombination suppression are consistent with divergence dates for  Mya van . Althoug Mya van .Drosophila (Studies examining the evolutionary forces acting after recombination has ceased have mainly been limited to male heterogametic systems, such as the Y chromosomes of mammals and osophila . These sosophila  togetherosophila . Howeverosophila .Drosophila and primate Y chromosomes (Theoretical models predict key differences between W and Y chromosome evolution . When seomosomes .Using the largest catalog of W-linked genes to date, we find that avian W-linked genes are evolving with a significant contribution of purifying selection. This is consistent with a previous study showing that sex-specific selection drives evolutionary changes in the expression of W-linked genes , and indWe found evidence of positive selection in one W-linked gene that encodes a mitochondrial ATP synthase subunit. As the W chromosome and mitochondria are both maternally inherited and are effectively linked , we mighOur findings indicate that adaptive evolution together with conservation of gene sequence is not restricted to male heterogametic systems and may be a fundamental feature of heterogametic sex chromosome evolution.Recombination suppression is a key force driving sex chromosome evolution, and sex chromosomes are therefore important for the study of genome evolution, particularly the interplay between selective forces and recombination. Here, we use a comparative approach to examine short- and long-term dynamics of sex chromosome evolution. Our results reveal that recombination has ceased multiple and independent times over 90 million years of avian evolution. We show that sex chromosome divergence over the long term is characterized by regions of similar divergence among gametologs, consistent with the inversion model of sex chromosome evolution . In cont


Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus virulence factors. As part of the innate immune system in the skin, cationic antimicrobial peptides (CAMPs) such as the β-defensins and cathelicidin contribute to host cutaneous defense, which prevents harmful microorganisms, like S. aureus, from crossing epithelial barriers. Conversely, S. aureus utilizes evasive mechanisms against host defenses to promote its colonization and infection of the skin. In this review, we focus on host-pathogen interactions during colonization and infection of the skin by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). We will discuss the peptides  and other mediators  that comprise the host defense against S. aureus skin infection, as well as the various mechanisms by which S. aureus evades host defenses. It is anticipated that greater understanding of these mechanisms will enable development of more sustainable antimicrobial compounds and new therapeutic approaches to the treatment of S. aureus skin infection and colonization. Approximately 30% of healthy people are asymptomatically colonized by S. aureus, which permanently colonizes the anterior nares in 10%–20% of the population and intermittently colonizes 30%–50%; the rest of the population never becomes colonized . I. IS. aur7.S. aureus is evidenced by the mechanisms that have evolved in S. aureus to resist and evade these peptides. As shown in S. aureus counteracts CAMPs and antimicrobial fatty acids through at least four mechanisms: (i) production of CAMP-binding molecules, like the fibrinolytic enzyme staphylokinase (SAK), which binds to and inhibits α-defensins; (ii) proteolytic degradation of CAMPs by secreted proteases such as aureolysin, which cleaves and inactivates LL-37; (iii) reduction of the bacterial cell surface net negative charge by modification of teichoic acids using d-alanine or phospholipids with l-lysine; and (iv) alteration of bacterial cell surface hydrophobicity [The importance of CAMPs in host cutaneous defense against hobicity ,108. In 7.1.S. aureus resists α-defensins through the production of SAK, which binds human α-defensins with high affinity, thereby mediating significant α-defensin resistance. In vitro, SAK levels correlate inversely with the susceptibility of S. aureus isolates to α-defensins  as well as clinical isolates expressing lower levels of dltA showed greater susceptibility to CAMPs, including dermcidin, RNase 7, hBD2 and hBD3 [N-terminal part of the peptide appear crucial for interaction with the negative bacterial cell surface, which likely explains why d-alanylation influences its efficacy [S. aureus [Products of the nd LL-37 ,73. Teic-alanine . These pand hBD3 . With deefficacy . Also, s. aureus –117.7.4.S. aureus produces IsdA, which alters its surface hydrophobicity, thereby reducing the efficiency with which fatty acids gain access to the cells [S. aureus resistant to hBD2 and LL-37 on human skin . The fact that the efficacy of hBD3 appears unaffected may be due to its more positive net charge, as compared to LL-37 (+11 vs. +6), which would favor stronger interaction with the bacterial cell surface [dltABCD could be an effective treatment strategy. Moreover, in addition to its essential role in mediating resistance to CAMPs [S. aureus to the novel lipopeptide antibiotic daptomycin [S. aureus, it would render it more susceptible to CAMPs, thus assisting host cutaneous defenses to successfully combat skin infections.Ouhara the hBD3 . The gre surface . This suto CAMPs ,111, it ptomycin ,121. ThuS. aureus prompt further investigation into the mechanisms that enable this pathogen to cause infection and overcome the broad spectrum of human cutaneous antimicrobial defenses. We anticipate that future studies will provide further information about the host and bacterial determinants involved in skin colonization and infection by S. aureus. Targeted drug development around highly conserved bacterial resistance mechanisms against host CAMPs is a promising pharmacologic approach in this era of highly virulent and drug-resistant strains of S. aureus.The increasing numbers of reports of virulent and drug-resistant strains of


Helicobacter pylori and the etiology of gastric cancer was substantially certain. Cholesterol-rich microdomains , which provide platforms for signaling, are associated with H. pylori-induced pathogenesis leading to gastric cancer. Patients who have been prescribed statins, inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, have exhibited a reduced risk of several types of cancer. However, no studies have addressed the effect of statins on H. pylori-associated gastric cancer from the antineoplastic perspective. In this study, we showed that treatment of gastric epithelial cells with simvastatin reduced the level of cellular cholesterol and led to attenuation of translocation and phosphorylation of H. pylori cytotoxin-associated gene A (CagA), which is recognized as a major determinant of gastric cancer development. Additionally, a nationwide case-control study based on data from the Taiwanese National Health Insurance Research Database (NHIRD) was conducted. A population-based case-control study revealed that patients who used simvastatin exhibited a significantly reduced risk of gastric cancer (adjusted odds ratio (OR) = 0.76, 95% confidence interval (CI) = 0.70–0.83). In patients exhibiting H. pylori infection who were prescribed simvastatin, the adjusted OR for gastric cancer was 0.25 (95% CI = 0.12–0.50). Our results combined an in vitro study with a nationwide population analysis reveal that statin use might be a feasible approach to prevent H. pylori-associated gastric cancer.Gastric cancer is the second leading cause of cancer-related death worldwide. The correlation of  Helicobacter pylori, a Gram-negative microaerophilic spiral bacterium, colonizes the human stomach and infects over 50% of the worldwide population [H. pylori infection is associated with several gastroenterological illnesses including gastritis, peptic ulcer, and gastric adenocarcinoma [H. pylori can penetrate the mucosal layer and survive intracellularly in the gastric epithelial cells, thereby escaping host immune response or antimicrobial therapy [pulation ,2. Persiarcinoma . H. pylo therapy ,5.H. pylori-induced pathogenesis [H. pylori [cag-pathogenicity island (cag-PAI)-encoded type IV secretion system (TFSS) results in phosphorylation of the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs and induction of host cell pathogenesis, such as cell elongation (hummingbird phenotype) [Several virulence factors characterize ogenesis . Cytotox. pylori ,8. Transenotype) , inductienotype) , and carenotype) .H. pylori exploits cholesterol-rich microdomains  for internalization of cells [H. pylori can delicately manipulate membrane cholesterol which contributes to CagA functions and pathogenesis.It has been reported that of cells ,13, as mof cells –16. The of cells . Severalof cells . Treatinof cells . Depletiof cells ,21. AddiH. pylori-induced pathogenesis and its progression to gastric cancer [H. pylori-associated gastric cancer has not been elucidated. In this study, we examined the effect of statins on gastric cancer risk in this nationwide population-based case-control study and investigated the interaction of cholesterol-lowering statins and H. pylori CagA-induced pathogenesis.According to the World Cancer Report in 2014, gastric cancer is the fifth most common cancer, and the third leading cause of cancer-related deaths, worldwide . Cholestc cancer ,25. A rec cancer . HoweverCagA antibody and phosphotyrosine (4G10) antibody were purchased from Santa Cruz Biotechnology . Lipofectamine 2000 was purchased from Invitrogen . Luciferase substrate and β-galactosidase expression vector were purchased from Promega . Simvastatin, lovastatin, RhoA inhibitor (Y27632) and all other chemicals were of the highest grade commercially available and purchased from Sigma-Aldrich .H. pylori-infected assay. H. pylori 26695 (ATCC 700392) were routinely cultured on Brucella blood agar plates  containing 10% sheep blood under 5% CO2 and 10% O2 conditions at 37°C for 2–3 days.Human gastric epithelial cells  were cultured in F12 . MKN45 cells  were cultured in Dulbecco’s minimum essential medium . TSGH9201 cells were cultured in RPMI1640 medium . Ten percent de-complemented fetal bovine serum (Hyclone) was added to all cultures. Penicillin and streptomycin (GibcoBRL) were used if necessary. Antibiotics were not added to the cell culture medium in the Gastric epithelial cells were treated with or without various concentrations of simvastatin  for 1 h. Trypan blue staining was used to measure the effects of statin on cell viability .H. pylori CagA translocation and phophorylation were prepared using the relevant techniques [Immunoprecipitates for analysis of chniques . The immH. pylori at an multiplicities of infection (MOI) of 100 for 6 h. The cells were scraped from dishes and an equal volume of luciferase substrate (Promega) was added to the samples. The luminescence was measured using a microplate luminometer . Luciferase activity was normalized to transfection efficiency, which was determined using the β-galactosidase activity of a cotransfected β-galactosidase expression vector (Promega).AGS cells were grown in 12-well plates for 20 h and transfected with a NF-κB-luc reporter plasmid using Lipofectamine 2000  . After aH. pylori at an MOI of 100 and incubated for 16 h. The supernatants from cell cultures were collected and the levels of IL-8 were determined using a sandwich enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions [AGS cells were treated with 25 μM simvastatin and thereafter infected with ructions .6 cells) were cultured in 12-well plates at 37°C for 20 h. After one wash with PBS, cells were treated with simvastatin (25 μM), then infected with H. pylori 26695 at an MOI of 100 for 6 h. Elongated cells were defined as cells that were typically elongated and had thin needle-like protrusions > 20 μm long. All samples were cultured in triplicate in 3 independent experiments. The proportion of elongated cells was determined and cells with the hummingbird phenotype were counted [AGS cells  program was implemented in 1995 and covers approximately 99% of the Taiwanese population, and has contracts with 97% of all medical providers . The detIn this population-based case-control study, patients aged at least 20 years, with recently diagnosed gastric cancer (ICD-9-CM code 151), were selected from the Catastrophic Illnesses Patient Database (CIPD) as the case group during the period 2005–2010. The NHI offers a catastrophic illness program that exempts patients from co-paying for the corresponding medical services, and the CIPD includes cancer patients. The index date for each case was the date of diagnosis of gastric cancer. Control subjects were identified from the Longitudinal Health Insurance Database 2000 (LHID 2000), a database containing the claims data from 1996 to 2011 for 1 million people randomly sampled from 2000 Registry of Beneficiaries of the Taiwan NHI program. For each case, one control patient, frequency-matched for sex, age (in 5-y bands), and year of gastric cancer diagnosis, was selected as the control group. Patients with any other cancer (ICD-9-CM codes 140–208) diagnosed before the index date were excluded. To explore the association between individual statins and gastric-cancer risk, the medication history of two statins commercially available before the index date, simvastatin and lovastatin, was analyzed.H. pylori-infection (ICD-9-CM code 041.86), gastritis , gastric diseases (ICD-9-CM codes 531–533), gastric polyp (ICD-9-CM code 211.1), gastroesophageal reflux disease (ICD-9-CM codes 530.81 and 530.11), and cirrhosis (ICD-9-CM code 571). These variables were potential confounders associated with gastric cancer.Co-morbidities were identified at least three times from principal/secondary diagnoses in the outpatient visits and/or hospitalizations. For each patient, records of co-morbidities present since the index date were obtained. Covariates included gender, age, and co-morbidities including t test for continuous variables. Univariate and multivariate unconditional logistic regression model was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs). The ORs and 95% CIs were measured to explore the risk of gastric-cancer associated with medication use and co-morbidities. The multivariate analysis was performed to adjust for possible confounders . The defined daily dose (DDD), recommended by the World Health Organization (WHO), was assumed to be the average maintenance dose per day of a drug. The annual mean DDD was calculated by diving the total cumulative DDD by the number of days in a year. Patients were classified into 3 groups according to the first quartile and second quartile of dose distribution. All analyses were conducted using SAS statistical software . All statistical tests were performed at the 2-tailed significance level of 0.05.The differences in sex, age , medication history of simvastatin and lovastatin, and co-morbidities were compared between the gastric-cancer cases and the controls by using a chi-square test. We used a H. pylori 26695; the levels of cellular cholesterol were then determined. As indicated in H. pylori and the cells was barely affected by treatment with simvastatin  and infected with wild-type vastatin .H. pylori-infected gastric epithelial cells was examined. As indicated in H. pylori-infected cells.The influence of a lower level of cellular cholesterol on translocation and phosphorylation of CagA in H. pylori CagA functions, NF-κB luciferase activity and IL-8 production were analyzed. The results revealed that treatment of cells with 25 μM simvastatin and subsequent infection with H. pylori reduced the levels of NF-κB promoter activity and secretion of IL-8 , gastric diseases (75.3% vs 40.4%), gastroesophageal reflux disease (14.6% vs 5.00%), gastric polyp (2.65% vs 0.37%), cirrhosis (31.9% vs 26.6%), and gastritis (54.1% vs 40.6%).A total of 19728 patients with recently diagnosed gastric cancer were identified from 2005 to 2010 and 19727 patients comprised the control group. Among the enrolled patients, 63.0% were men and 61.0% were aged 65 years or older . The meaH. pylori infection , gastric diseases , gastroesophageal reflux disease , gastric polyp , and gastritis  were associated with increased odds of gastric cancer. We performed an analysis of the dose-related response, using nonusers of statins as the reference group , compared with those not prescribed simvastatin. The same trend was also observed in patients treated with lovastatin, but the reduction in risk was nonsignificant. We further analyzed whether the use of statin was associated with the occurrence of different types of gastric cancers. As shown in Patients who used simvastatin exhibited a significantly reduced risk of gastric cancer  . A reducce group . Comparenfection . The resH. pylori infection, gastric diseases, gastroesophageal reflux disease, and gastritis. Therefore, the results of the present study were highly reliable and consistent with the findings of Chiu et al, who reported the association of statin use with reduced gastric cancer risk by conducting a population-based case–control study [The inhibitors of HMG-CoA reductase, commonly known as statins, are widely prescribed for lowering serum cholesterol. Statins are associated with multiple protective functions including reducing the risk of hepatocellular carcinoma , enhanciol study .Staphylococcus aureus with high concentrations of statins resulted in considerable antimicrobial activity [H. pylori with 0–50 μM statins (H. pylori-induced gastric inflammation [H. pylori–induced-inflammation action of statins was mediated by a depletion of cholesterol which might inhibit CagA-functions in host cells rather than H. pylori growth.The mechanisms underlying the immunoregulatory effects of statins have been studied by other researchers. Exposure of murine macrophages to statins attenuated lipopolysaccharide (LPS)-induced proinflammatory cytokine production . Moreoveactivity . However statins . CagA isammation . Previouammation ,21. TherChlamydia pneumonia [Clostridium difficile [Streptococcus pneumonia [Staphylococcus aureus [H. pylori-induced gastritis in mice [H. pylori infection [H. pylori eradication rates of standard antibiotics [H. pylori and reduced H. pylori-associated diseases required further investigation.Statins are known to reduce the risk of severe bacterial infections, including neumonia , Clostriifficile , Streptoneumonia , and Stas aureus . The imms aureus . Additio in mice . Long-tenfection . Statinsibiotics . AlthougH. pylori etiology and gastric cancer is known, the exact mechanism of statins’ influence on H. pylori-associated gastric cancer remains unclear. H. pylori CagA is a bacterial oncoprotein that can contribute to the development of H. pylori-associated neoplasms in mammals [H. pylori-induced carcinogenesis. The results of this study revealed that treatment of cells with statins reduces cellular cholesterol and inhibits H. pylori CagA actions, which might exert a protective effect against gastric cancer.The incidence and mortality rate of gastric cancer remain high worldwide. The association of statins with a reduced risk of gastric cancer has been reported by epidemiological meta-analysis studies ,41,53. T mammals . Further mammals ,20. TherH. pylori-infected rodent models, pitavastatin was ineffective in inhibiting gastric carcinogenesis [Studies on the various effects of statins are contradictory. Some studies have indicated that statins do not reduce the risk of gastric cancer ,56. In aogenesis . This miogenesis .H. pylori-associated distal gastric cancer, but might also affect H. pylori-independent proximal gastric cancer. We further showed simvastatin use resulted in much lower odds ratios for distal gastric cancer than for proximal cancer (OR = 0.65 vs. 0.82). Our results demonstrated that the inhibition of cancer development by simvastatin was more pronounced in distal than in proximal gastric cancer, suggesting that simvastatin may affect H. pylori-induced pathogenesis. Although H. pylori infection is associated with distal gastric cancer, it is not the only risk factor for development of gastric cancer. The main risk factors for distal gastric cancer include H. pylori infection and diet, while gastroesophageal reflux disease and obesity play important roles in proximal gastric cancer [H. pylori infection. Long-term follow-up studies or randomized controlled trials are necessary to elucidate the role of statins in the prevention of different types of gastric cancer.In this study, we found that simvastatin reduced the risks of both distal and proximal gastric cancer . The datc cancer . Previouc cancer  and assoc cancer . Moreovec cancer , indicatc cancer . These lH. pylori infection  are not covered by the insurance system. The data regarding H. pylori infection was under-estimated using the NHIRD. Therefore, large and long-term clinical studies on statin usage are required to further clarify statins’ mechanism of inhibiting H. pylori-associated gastric cancer.Some limitations might have been present in those studies, including potential confounders, concomitant use of other chemopreventive drugs, short enrolment periods, and small sample sizes. In particular, individuals of higher socioeconomic classes are more likely to be prescribed statins. A social gradient in statin use may reflect social inequalities in health care use or health care quality, as well as systematic differences in factors such as smoking, nutrition, physical activity, and obesity . In addiH. pylori infection is associated with a high risk of gastric cancer, which is attenuated by the prescription of statins. It was also demonstrated that statins inhibit H. pylori-induced pathogenesis by preventing CagA actions in host cells. Therefore, statins, which are cholesterol-lowering agents and proven to be extremely safe, can be used as chemopreventive drugs for protecting against H. pylori-gastric cancer.In conclusion, we determined that S1 FigH. pylori at an MOI of 100 for 6 h. The cells were then prepared for luciferase activity assays. Results are expressed as means ± standard deviations. *, P < 0.05 was considered statistically significant.AGS cells were transfected with κB-Luc vector and incubated for 24 h. The cells were then treated with 25 μM simvastatin or 10μM RhoA inhibitor (Y27632), followed by infection with (TIF)Click here for additional data file.S2 FigH. pylori at an MOI of 100 for 6 h. (B) The proportion of cells with the elongated (hummingbird) phenotype was evaluated as described in the materials and methods section. The quantitative results represent the means and standard deviations for three independent experiments. *, P < 0.05, compared with H. pylori-infected cells without simvastatin pretreatment. Scale bar, 10 μm.(A) AGS cells were pretreated with simvastatin (25 μM) or RhoA inhibitor Click here for additional data file.


Lignosus rhinocerotis (Cooke) Ryvarden or Tiger milk mushroom  is a valuable folk medicine for indigenous peoples in Southeast Asia. Despite the increasing interest in this ethnobotanical mushroom, very little is known about the molecular and genetic basis of its medicinal and nutraceutical properties.The sclerotium of de novo assembled 34.3 Mb L. rhinocerotis genome encodes 10,742 putative genes with 84.30% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed a close evolutionary relationship of L. rhinocerotis to Ganoderma lucidum, Dichomitus squalens, and Trametes versicolor in the core polyporoid clade. The L. rhinocerotis genome encodes a repertoire of enzymes engaged in carbohydrate and glycoconjugate metabolism, along with cytochrome P450s, putative bioactive proteins  and laccases. Other genes annotated include those encoding key enzymes for secondary metabolite biosynthesis, including those from polyketide, nonribosomal peptide, and triterpenoid pathways. Among them, the L. rhinocerotis genome is particularly enriched with sesquiterpenoid biosynthesis genes.The L. rhinocerotis provides insights into the genetic basis of its reported medicinal properties as well as serving as a platform to further characterize putative bioactive proteins and secondary metabolite pathway enzymes and as a reference for comparative genomics of polyporoid fungi.The genome content of The online version of this article (doi:10.1186/1471-2164-15-635) contains supplementary material, which is available to authorized users. Lignosus rhinocerotis (Cooke) Ryvarden, which belongs to the family of Polyporaceae, is characterized by a centrally stipitate pilei arising from its distinct tuber-like sclerotium. This mushroom is widely used by natives of Southeast Asia as a general health tonic for immune enhancement, or as a treatment regime for numerous ailments including cancer, asthma, and bronchitis. It is also used to treat discomfort caused by fright, fever, coughing, vomiting, and cuts [L. rhinocerotis with medicinal value. It is a compact mass of hardened fungal mycelium and represents one of the stages in the fungal life cycle. This structure is a morphologically variable, nutrient-rich, multihyphal aggregate that serves as a food reserve and can remain dormant until favourable growth conditions arise [L. rhinocerotis are shown in Figure L. rhinocerotis have been found in Penang Island, Cameron Highlands, Hulu Langat, and Gerik. All of these isolates showed high nucleotide sequence identity (approximately 98%) in their internal transcribed spacer (ITS) gene regions [and cuts . The sclns arise . Differe regions , 4.FigurL. rhinocerotis against hypertension, cancer cell cytotoxicity along with enhancement of immunomodulatory activity and antioxidant properties [Polyporus rhinocerus, a synonym for L. rhinocerotis, was also reported as a potential novel prebiotic for gastrointestinal health [L. rhinocerotis signalled for an immediate need to decipher its biochemical functions, at the genetic level and the identification of its bioactive components.This mushroom is rich in carbohydrates and dietary fiber with moderate amounts of protein while being low in fat . Previouoperties –8. The nl health . The recGanoderma lucidum (lingzhi) by Chen et al. and Liu et al. where the genes in the triterpene biosynthesis and wood degradation pathways were described [Volvariella volvacea (straw mushroom) by Bao et al. [Agaricus bisporus (button mushroom) by Foulongne-Oriol et al. [Rapid advancements in technology has led to the sequencing of numerous fungal genomes with the fungal kingdom becoming one of the most sequenced amongst the eukaryotes . This isescribed , 12. Otho et al.  and Agarl et al. .de novo draft genome sequence of L. rhinocerotis TM02 sclerotium. The recent availability of several genome sequences of polyporaceous fungi, especially from the JGI CSP Saprotrophic Agaricomycotina Project [L. rhinocerotis genome through comparative analyses. We have also surveyed its secondary metabolite production capabilities and identified putative genes that may be involved in the biosynthesis of bioactive proteins and polysaccharides. To our knowledge, this is the first detailed description of the genomic features of L. rhinocerotis, an ethnobotanical mushroom of Southeast Asia.In this paper, we present the  Project , has allL. rhinocerotis sclerotium with more than 100× coverage produced a total of 6,187 Mb clean data which was further assembled into a 34.3 Mb draft genome . The tandem repeats (<4 kbp), not clearly associated with transposons, vary in copy number from 1.8 to 362.5. While among the retrotransposons, long terminal repeats (LTRs) and non-LTR retrotransposons (long and short interspersed nuclear elements) make up 1.26% and 0.55% of the genome, respectively. DNA transposons (Class II) comprised 0.24% of the genome. The DNA transposons elements were mainly categorized into three classes: Enhancer (En/spm), Tigger (TcMar), and Activator (hAT).Repeat elements of 74 diverse families make up about 4.01% or 1,374,638 bp of the assembled genome of L. rhinocerotis, its average gene length, the proportion of repeat sequences, and the average number of exons and introns were comparable to the recently sequenced polyporaceous G. lucidum genome [The total 10,742 predicted genes, 216 tRNAs, 17 snRNAs and a single rRNA together comprise 44.33% of the assembled genome. Gene density and the average size of protein coding genes are 5.42 genes/10 kb and 1,414.33 bp, respectively. Among the tRNAs, 13 are possible pseudogenes, four with undetermined anticodons and the remaining 199 anti-codon tRNAs correspond to the 20 common amino acid codons. About half of the tRNAs (109) are predicted to not contain an intron. The genome size of m genome .L. rhinocerotis genome revealed a total of 1,686 predicted genes that encode for hypothetical proteins with no apparent homologs to currently available sequences. This is indicative of the uniqueness of L. rhinocerotis. On the other hand, up to 8986, 5883, 6669 and 8997 genes are homologous to known proteins in the NCBI nr, SwissProt, InterPro and TrEMBL databases, respectively. These homologous proteins represent 84.30% of the assembled genome.The L. rhinocerotis because such a lifestyle results in exposure to more diverse carbohydrate forms and sources as opposed to the wood saprophytes.We further mapped the genome to the Eukaryotic Clusters of Orthologs (KOG), Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to further characterize the predicted proteins. Based on the phylogenetic classification of the KOGnitor, 4,948 proteins (46.06%) were assigned to KOG Figure . The R cL. rhinocerotis, we successfully assigned 4,980 (46.36%) putative proteins to their orthologs in the KEGG database , a basidiomycete (S. commune) and tremellomycete (Tremella mesenterica), were selected for comprehensive comparison. L. rhinocerotis is relatively enriched with genes for catabolism and biosynthesis of secondary metabolites  number classification was used to link the respective enzyme genes to their repertoire of metabolic pathways . In the L. rhinocerotis and not present in any other Basidiomycota fungi compared  is centrally stipitate with an isodiametric cap. On the other hand, the cap of G. lucidum and T. versicolor is offset and sometimes indistinct with either a bare stipe for the former or lacking one for both fungi. On the contrary, D. squalens has a basidiocarp with poroid hymenophore and lacks a stipe. Although L. rhinocerotis shows similar growth habit to W. cocos with the presence of a sclerotium, the latter has resupinate fruiting body and spherical sclerotium. Therefore, L. rhinocerotis is relatively unique among the sequenced Basidiomycota mushrooms.It should be noted that, although lerotium . The sclL. rhinocerotis is known to thrive on cellulosic substrates, its genome was mapped to the CAZy database to identify the presence of carbohydrate-active enzymes (CAZymes) and auxiliary proteins [As proteins . A totalproteins , 13. TheL. rhinocerotis has 33 genes engaged in “Metabolism of xenobiotics by cytochrome P450” and 36 in “Drug metabolism - cytochrome P450” KEGG sub-pathways (Table The cytochrome P450 (CYP) superfamily is a diverse group of enzymes involved in various physiological processes, including detoxification, degradation of xenobiotics and the biosynthesis of secondary metabolites . Althougys Table , respectL. rhinocerotis and seven other basidiomycetes were retrieved using BLAST searches against the P450 database  and W. cocos . On the other hand, T. mesenterica, a tremellomycete, formed the smallest group among the eight fungi compared with 32 functional genes and a known pseudogene. L. rhinocerotis had a total of 136 CYP sequences , which can be classified into 37 families according to Nelson’s nomenclature  and domain architecture with the orsellinic acid synthase from Coprinopsis cinerea (CC1G_05377), the only basidiomycete PKS gene that has been characterized so far [L. rhinocerotis genome also harbours a single multidomain NRPS gene. The NRPS has a single adenylation domain along with three thiolation and condensation domains, and are conserved among several basidiomycetes, including D. squalens DICSQDRAFT_132068 (61% identity) and T. versicolor TRAVEDRAFT_27949 (59% identity), but none are characterized.Secondary metabolite biosynthetic genes are often clustered . The L. d so far . Like CCG. lucidum. The triterpenoid ganoderic acids, for example, have been reported to have anti-tumor, immuno-regulation, and antioxidative functions [Clitopilus mushrooms [L. rhinocerotis, we identified 15 key enzymes involved in the mevalonate (MVA) pathway but not the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway  is one group of secondary metabolites that are well recognized for their pharmaceutical uses and are known to be one of the major groups of therapeutic compounds in unctions . Other nushrooms , as wellushrooms . In the u et al. , we founL. rhinocerotis genome for potential triterpenoid biosynthesis genes and found an open reading frame (GME631_g) that encodes a single copy gene for lanosterol synthase . LSS is a squalene/oxidosqualene cyclase family enzyme that catalyzes the cyclization of the triterpenes squalene or 2-3-oxidosqualene to lanosterol, the precursor of all steroids [G. lucidum[L. rhinocerotis can be involved in biosynthesis of bioactive triterpenes. The CYP5144 and CYP512 families of P450 genes have been previously implicated in triterpenoid biosynthesis in G. lucidum due to their co-expression with LSS [L. rhinocerotis with 35 genes, while six CYP512 family genes are present in its genome as well. This suggests that L. rhinocerotis may be a potential triterpenoid source.We next searched the steroids . This en lucidum. Similarwith LSS . As mentL. rhinocerotis genome is enriched with sesquiterpenoid biosynthesis genes compared to some of the other seven Basidiomycota genomes , and four putative laccases  are homologous to LZ-8 (64% identities), a member of the FIP family from G. lucidum that has been shown to possess immunomodulation and anti-cancer activity [Flammulina velutipes with immunomodulatory activity [Mushrooms have also been an important source of bioactive proteins, which include lectins, fungal immunomodulatory proteins (FIP), ribosome inactivating proteins (RIP), antimicrobial proteins, ribonucleases, and laccases . The genactivity , 36. Botactivity .L. rhinocerotis genome sequencing has allowed us to perform comparative genomics and phylogenetic analysis. This has provided valuable insights into the biology of this medicinal mushroom from Southeast Asia. A survey of the secondary metabolite biosynthesis genes in the L. rhinocerotis genome shows that it is particularly enriched with sesquiterpenoid biosynthesis genes. Thus, future bioactive molecule discovery efforts should focus on this class of metabolites. Furthermore, L. rhinocerotis appears to encode the capabilities to produce 1,3-β- and 1,6-β-glucans as well as bioactive proteins, such as lectins and FIPs. Our genomic analysis of L. rhinocerotis will provide the foundation for future research and exploitation of L. rhinocerotis in pharmacological and functional food applications.L. rhinocerotis was collected from tropical forest at Lata Iskandar, Cameron Highland, Pahang, Malaysia  in 2010. The fungus was deposited at Royal Botanic Gardens, Kew  with the accession number K(M) 177812. Genomic DNA was extracted using a modified cetrimonium bromide (CTAB) procedure [L. rhinocerotis TM02 strain cultivated by Ligno Biotech Sdn. Bhd. . Paired-end reads were generated by sequencing of four cloned insert libraries of 200, 700, 2,000, and 5,000 bp using Hiseq2000 system  at BGI-Shenzhen, China. Reads of low complexity and low quality with adapter and duplication contamination were removed from the raw data to strengthen the accuracy of follow-up analysis. To avoid issues associated with heterozygosity and/or low sequence quality, reads with significant poly-A structure and kmer frequency of 1 were removed. The clean short reads were then assembled using SOAPdenovo based on de Bruijn graph theory [Sclerotium of rocedure  from theh theory . The gapab initio gene predictors Augustus [http://www.repeatmasker.org and RepeatProteinMasker software (transposon protein library). Tandem repeat sequences were predicted using Tandem Repeat Finder (TRF) [de novo prediction software [Protein coding genes were predicted using the Augustus , GeneMarAugustus , and SNAAugustus . The resAugustus  using Reer (TRF) . rRNA sesoftware . tRNA gesoftware  while otPredicted genes were functionally annotated based on homology to annotated genes in various databases via BLAST. Protein models were aligned to SwissProt, TrEMBL, InterPro and NCBI nr ; and further classified according to GO , KOG 4848, and KL. rhinocerotis, 14 Basidiomycota of agaricomycetes , Laccaria amethystina, P. ostreatus), basidiomycete (S. commune), tremellomycete (T. mesenterica), pucciniomycete (P. graminis), and ustilaginomycete (U. maydis) were selected for analysis. Three Ascomycota of eurotiomycetes  and saccharomycete (Saccharomyces cerevisiae) were added to root the phylogenetic trees. All the selected genomic gene models were downloaded from the US Department of Energy Joint Genome Institute website at http://genome.jgi.doe.gov/  at Cytochrome P450 homepage (http://drnelson.uthsc.edu/CytochromeP450.html) [L. rhinocerotis genome was carried out by BLASTP analysis against CAZy database at http://www.cazy.org/[50.html) . Annotatazy.org/.http://jcvi.org/smurf/index.php) [http://antismash.secondarymetabolites.org/) [Secondary metabolite gene clusters were determined using Secondary Metabolite Unique Regions Finder   based ones.org/) ; a web-bThis Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AXZM00000000. The version described in this paper is version AXZM01000000.Heterogeneous sequence (k-mer) and repeat rate analyses ofL. rhinocerotisgenome.(PDF 207 KB)Additional file 1:L. rhinocerotis and the other seven compared Basidiomycota fungi in the third layer of KEGG pathway. Table S2. Enzyme distribution of L. rhinocerotis with not less than 2-fold genes more than the other seven compared Basidiomycota fungi in the fourth layer of KEGG pathway. Table S3. The classes of 26 enzymes of L. rhinocerotis with not less than 2-fold genes more than the other seven compared Basidiomycota fungi in the fourth layer of KEGG pathway. Table S4. The 535 enzymes which are exclusive to only L. rhinocerotis. Table S5. Cytochrome P450 genes in L. rhinocerotis. Table S6. The putative backbone genes and clusters involved in L. rhinocerotis secondary metabolism. Table S7. Putative genes involved in the biosynthesis of bioactive polysaccharides and proteins in L. rhinocerotis. Table S8. Download portal of additional fungi used in this study. (XLS 298 KB)Additional file 2: Table S1: Gene distribution of Assignment ofL. rhinocerotisgenes to CAZy families and their respective EC activities.(XLSX 31 KB)Additional file 3:Potential terpenoids biosynthesis pathway inL. rhinocerotis.(PDF 120 KB)Additional file 4:


Aedes aegypti, the principal vector of yellow fever and dengue fever, is responsible for more than 30,000 deaths annually. Compounds such as carbon dioxide, amino acids, fatty acids and other volatile organic compounds (VOCs) have been widely studied for their role in attracting Ae. aegypti to hosts. Many VOCs from humans are produced by associated skin microbiota. Staphyloccocus epidermidis, although not the most abundant bacteria according to surveys of relative 16S ribosomal RNA abundance, commonly occurs on human skin. Bacteria demonstrate population level decision-making through quorum sensing. Many quorum sensing molecules, such as indole, volatilize and become part of the host odor plum. To date, no one has directly demonstrated the link between quorum sensing  by bacteria associated with a host as a factor regulating arthropod vector attraction. This study examined this specific question with regards to S. epidermidis and Ae. aegypti. Pairwise tests were conducted to examine the response of female Ae. aegypti to combinations of tryptic soy broth (TSB) and S. epidermidis wildtype and agr- strains. The agr gene expresses an accessory gene regulator for quorum sensing; therefore, removing this gene inhibits quorum sensing of the bacteria. Differential attractiveness of mosquitoes to the wildtype and agr- strains was observed. Both wildtype and the agr- strain of S. epidermidis with TSB were marginally more attractive to Ae. aegypti than the TSB alone. Most interestingly, the blood-feeder treated with wildtype S. epidermidis/TSB attracted 74% of Ae. aegypti compared to the agr- strain of S. epidermidis/TSB (P ≤ 0.0001). This study is the first to suggest a role for interkingdom communication between host symbiotic bacteria and mosquitoes. This may have implications for mosquito decision-making with regards to host detection, location and acceptance. We speculate that mosquitoes “eavesdrop” on the chemical discussions occurring between host-associated microbes to determine suitability for blood feeding. We believe these data suggest that manipulating quorum sensing by bacteria could serve as a novel approach for reducing mosquito attraction to hosts, or possibly enhancing the trapping of adults at favored oviposition sites. Mosquitoes are important vectors responsible for the transmission of viruses, bacteria, parasitic protozoans, and filarial, which cause diseases in humans; such as dengue fever, yellow fever, and malaria . Among aIn addition to heat and carbon dioxide cues, mosquitoes locate their hosts through olfaction systems sensing chemical cues emanating from humans . As deteStaphylococcus epidermidis is a commensal bacteria associated with human skin that can attract mosquitoes [Anopheles gambiae (Diptera: Culicidae), a species that vectors the malaria causative agent, were attracted more by blood agar plates incubated with S. epidermidis than by sterile blood agar plates [squitoes . Anophelr plates . Volatilr plates . For exar plates  and indor plates , 16 are r plates –20.S. epidermidis contributes to the formation of volatile fatty acids [agr) and luxS systems [As a predominant bacterium associated with human skin , S. epidty acids , a VOC cty acids . Current systems , which r systems . In QS, Enteromorpha, have been shown to detect the presence of bacteria (Vibrio anguillarum) through recognition of their auto-inducer produced via their QS system which subsequently attaches to the bacterial biofilms and begins growth [Pseudomonas aeruginosa which activates its QS dependent virulence determinant so that it enhances its virulence phenotype [rfaL strain of Proteus mirabilis, which is unable to swarm, a QS-controlled phenotype, is less attractive to the blow fly, Lucilia sericata  [P. mirabilis, the rfaL strain had fewer eggs deposited on it by the flies. Such interactions are speculated to regulate arthropod attraction and colonization of ephemeral resources, such as vertebrate carrion, which is an important aspect of nutrient recycling within larger ecosystems [Interspecies interactions regulated by QS compounds produced by microbes have been identified for a number of systems . Such sys growth . Convershenotype . A previhoridae) . Furtherosystems –30.agr operon, which encodes a S. epidermidis QS system, was deleted to construct the mutant S. epidermidis strain (Tü3298) that cannot regularly perform QS [S. epidermidis contributes to attracting Ae. aegypti to a blood meal.The rform QS . The goaThe average percent response of adult female mosquitoes to the blood-feeders treated with TSB for each trial is presented in H = 9.309, df = 3, P = 0.025)  . HoweverS. epidermidis/TSB or solely TSB for each trial is presented in S. epidermidis/TSB attracted, in general, 57% of the mosquitoes to respond   . One of idis/TSB . The remagr- strains of S. epidermidis/TSB or solely TSB for each trial is presented in S. epidermidis, the blood-feeder treated with agr- strain S. epidermidis/TSB attracted, in general, 57% of the mosquitoes   . Howeverlely TSB . The remlely TSB . The aveS. epidermidis/TSB or agr- S. epidermidis/TSB for each trial is presented in S. epidermidis/TSB attracted 72% of the Ae. aegypti over the blood-feeder treated with the agr- strain of S. epidermidis/TSB   . Three oidis/TSB . The remAe. aegypti.Bacteria living inside, as well as on a human body outnumber its cells by an estimated 10-fold . The intVibrio fischeri, are able to respond to bacterial QS and conversely produce molecules that are sensed directly by bacteria [Euprymna scolopes) and the bacterial symbiont (Vibrio fischeri). Salmonella typhimurium, a bacterial pathogen, uses the QS regulator QseC and QseE to detect norepinephrine–a hormone from vertebrate hosts such as mouse [Delisea pulchra, can block bacterial QS by producing halogenated furanones that act as signal mimics [Acetobacter pomorum, of Drosophila (Diptera: Drosophilidae) is able to promote host growth rates and reduce sugar and lipid levels according to the activity of its pyrroloquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) [This “interkingdom cross-talk” between bacteria and eukaryotic organisms has gained increased recognition in recent years . Some hobacteria , resultias mouse . This abas mouse . The marl mimics . A recenPQQ-ADH) . This inPQQ-ADH) . For exaPQQ-ADH) , when thPQQ-ADH) , 42. Des2) is a strong volatile cue for mosquito attraction over long distances [In the case of mosquitoes, VOCs produced by humans play a role in influencing their host-seeking behavior. For example, carbon dioxide , compared to TSB alone.Quorum sensing by bacteria results in the production of VOCs, which then serve as a mechanism allowing for interkingdom interactions , 35. S. man skin . It is k of skin , 47, parthe feet . In this armpits . We reco armpits , which we strain . FurtherP < 0.0001) among trials was determined for each set of experiments conducted , as they were more attracted by a warmer with more humidified human emanations air stream [Ae. aegypti host-seeking behavior [Results were relatively consistent towards a given treatment; however, heterogeneity P < 0.000 among trd Tables  Although 26–28°C . Humiditr stream . Furtherbehavior . Future The statistical approach implemented for analyzing response data in these experiments indicate care should be taken by researchers when determining the methods they employ to analyze their behavior data. Some commonly employed analyses, such as chi-square, would overlook such variation as we observed in our experiments, which could be crucial for deciphering the factors  influencing host-seeking behavior by mosquitoes. Less rigorous methods could also potentially mask  true treatment effects leading to misleading interpretations of the behaviors observed.An. gambiae is highly associated with microorganisms. Ponnusamy et. al [Ae. aegypti. Based on these previous studies, QS systems of the bacteria associated with mosquito oviposition sites should also be explored for the potential development of new and effective methods for mosquito control.Results from these experiments could be used to develop novel methods for shifting VOC profiles by manipulating the QS system of host associated bacteria, resulting in reduced mosquito attraction. In addition to host-seeking behavior, previous research also demonstrated that mosquitoes preferred to oviposit on substrates containing live microorganisms, rather than sterilized substrates , indicaty et. al  reportedAedes aegypti colony originated from the Liverpool strain. Aedes aegypti eggs were hatched in a container with 1 L distilled water at 25°C, 70% RH and 12:12 L:D photoperiod. Larvae at approximately 2-d-old were separated into containers at a density of 100–200 larvae/L, for optimal larval growth [The l growth . Larvae l growth  were useagr deletion mutant S. epidermidis strain (S. epidermidis TüF38) was used in this experiment because of the deletion of QS agr gene. Both the mutant (agr-) and wildtype S. epidermidis strains were grown in mannitol salt agar (MSA), as MSA contains a high concentration of NaCl (approximately 7.5%-10%), it is inhibitory to most other bacteria, and selective for gram-positive bacterium Staphylococci . The bacteria were diluted to a concentration of 107 cfu in tryptic soy broth (TSB), for use in the experiments.The 2 cell culture flask (Corning Incorporated NY) . The blood-feeders were made by 25 cmated NY) . Parafilated NY) , so that7 colony forming units (cfu)/mL of the S. epidermidis wildtype strain, mutant (agr- S. epidermidis TüF38) strain or 0.5 ml TSB solution respectively. An aliquot of 1 ml of rabbit blood (HemoStat Laboratories) was added in the gap space between the blood-feeder and the parafilm. Initial experiments indicated there was no bias towards either side of the cage (see below); however, treatments were still rotated after each trial. Furthermore, the cage and feeders were cleaned using a vacuum and ethanol between trials.A clear Plexiglas cage  containiAe. aegypti females actively seeking hosts several hours before sunset [The experiments were performed 3–4 h before dark (chamber at 12:12 L:D), based on e sunset , 58. A ce sunset –61, the P ≤ 0.05) as it allowed us to determine if variation from the expected proportion (50:50 in our case) and significant variation across trials [In order to determine if the mosquito response was significantly different between treatments and trials, the data were analyzed using the repeated G test of goodness-of-fit Click here for additional data file.


To the editor,We read with great interest in the article “Efficacy of botulinum toxin type A 100 Units versus 200 units for treatment of refractory idiopathic overactive bladder”. Osama Abdelwahab, et al. (Lack of voiding diary is one of the weak points of the article. Number of incontinence episode or functional bladder capacity is important in the evaluation of OAB. In addition, the mean pretreatment cystometric capacities in group A and B were 277.7±75.29 and 289.2±70.83ml, respectively. The high mean values mean that some of the patients actually had large cystometric bladder capacity before BoNT-A treatment and that they might not gain benefits from this intervention. So, it would be great to see the results in the subgroups of various functional or cystometric bladder capacity. Shag-Jen Chang, MD and Stephen Shei-Dei Yang, MDDepartment of SurgeryTaipei Tzu Chi General HospitalNew Taipei, Taiwan No.289Jianguo Rd., Xindian Dist.New Taipei City 23142, Taiwan E-mail: krissygnet@yahoo.com.twMeng-Jen Huang, MD


Ketogulonicigenium vulgare has been widely used in vitamin C two steps fermentation and requires companion strain for optimal growth. However, the understanding of K. vulgare as well as its companion strain is still preliminary. Here, the complete genome of K. vulgare Hbe602 was deciphered to provide insight into the symbiosis mechanism and the versatile metabolism. K. vulgare contains the LuxR family proteins, chemokine proteins, flagellar structure proteins, peptides and transporters for symbiosis consortium. Besides, the growth state and metabolite variation of K. vulgare were observed when five carbohydrates  were used as carbon source. The growth increased by 40.72% and 62.97% respectively when K. vulgare was cultured on D-mannitol/D-sorbitol than on L-sorbose. The insufficient metabolism of carbohydrates, amino acids and vitamins is the main reason for the slow growth of K. vulgare. The combined analysis of genomics and metabolomics indicated that TCA cycle, amino acid and nucleotide metabolism were significantly up-regulated when K. vulgare was cultured on the D-mannitol/D-sorbitol, which facilitated the better growth. The present study would be helpful to further understand its metabolic structure and guide the engineering transformation. K. vulgare was identified as a member of the ProteobacteriaK. vulgare have been sequenced completely3K. vulgare is still preliminary partly because of its unique nature. It was found that the K. vulgare mono-culture system grew poorly, even on rich natural media such as yeast extracts, peptones and corn steep liquor. The Bacillus species are usually co-cultivated with K. vulgare to achieve a better growth rate of K. vulgare and an adequate 2-KGA yieldK. vulgare, implying the defects of sulfur and oxidation metabolism, vitamin and amino acid synthesis. However, further research is needed to know the reason for the weak growth of K. vulgare and how to adapt its growth state in symbiosis system.K. vulgare and Bacillus strainLactococcus lactis in K. vulgare could enhance the growth and 2-KGA productionUsing GC-TOF/MS technology, metabolomics was suitable for analyzing the variations in primary metabolites, such as intermediates in central carbon metabolism and amino acid biosynthesis. The metabolomics approach has been demonstrated in our previous studies on the interactions between K. vulgare Hbe602 consists of one circular chromosome and two plasmids, which encodes 3,178 predicted proteins, 58 tRNAs and 15 rRNAs in total (maf gene (282-887 nt) encoding septum formation protein Maf. The analysis of replication origins for bacteria in DoriC, a database of oriC regions in bacterial and archaeal genomes10oriC region is adjacent to a maf gene is conserved in the family Rhodobacteraceae. For the plasmids, the putative origins of replication are also at 0 kb, and both contain repeat sequences. Phylogenetic analysis of K. vulgare Hbe602 with other species could provide putative evolutionary histories and phenotypic diversity , which was related closely to Rhodobacter and Paracoccus. Furthermore, the genome-scale sequence comparison by LAST software available at http://last.cbrc.jp shows the genome similarity between K. vulgare Hbe602 with WSH-001 is higher than that with Y25  is similar to those in E. coli and R. sphaeroides and higher than that in G. oxydans. Those functional genes encode many transporters, which absorb the nutrients to compensate its metabolic defect. Besides, the number of transcriptional regulation related genes (K) is even more than that in E. coli. The strong transcriptional regulation ability may be activated to form a symbiotic relationship with other microorganisms. With the aid of KEGG analysis, metabolic network is obtained, including the carbohydrate-active enzymes, nitrogen and central carbon metabolism, amino acid and cofactor metabolism.The genome of in total . For theiversity . To datewith Y25 12. Throuwith Y25 . Previoue Hbe602 . ConsequProteobacteria species shows that at least 68 species contain LuxI (AHL synthase) and LuxR , while 45 species only contain LuxRK. vulgare only contains LuxR and lacks LuxI, so it may produce AHL by other means, or obtains the AHL signaling molecules from other microorganismsK. vulgare also contains phosphorylated protein CtrA taking part in flagella mobile and cell division, which is widely present in the alpha-ProteobacteriaK. vulgare contains two HK97 family proteases, which are generally encoded by phage. They were also found in R. capsulatus as gene transfer agent, which can mediate gene transferK. vulgare and R. capsulatus are the neighbor strains, the HK97 family protease in K. vulgare may not be used for the degradation of protein, but the gene transfer agent. Furthermore, K. vulgare Hbe602 contains several chemotaxis proteins  for observing the growth phenotype and analyzing the key genes. The growth conditions of K. vulgare varied dramatically on five carbon sources and the highest growth value with D-mannitol was about 1.63 times than that with L-sorbose  was analyzed by metabolomics studies. The genomics analysis found that K. vulgare lacks the endoenzyme, which transforms D-mannitol, L-sorbose, D-sorbitol and D-glucose to D-frucose  biosynthesis pathway was isolated and over-expressed in K. vulgare Hbe602K. vulgare mutant harboring a plasmid with the complete pqqABCDE cluster achieved 3-folds higher level of PQQ biosynthesis than the wild-type. PqqN was annotated as a channel protein based on the best blast hit with the bestrophin in Shinella strainpqqN and pqqABCDE decreased the PQQ levelK. vulgare, and the addition of purine improved growth and 2-KGA production of K. vulgareK. vulgare in the co-cultureIn the cofactor and nucleotide metabolism of K. vulgare, three reactions during the conversion of succinate to glutamic acid are defective. In the methionine cycle, K. vulgare converses methionine to S-Adenosylmethionine (SAM), and then transforms to S-adenosyl homocysteine cysteine and homocysteine, again into methionine. The last step of the methionine cycle reaction need N5-methyl-tetrahydrofolate as the methyl transfer agent, while tetrahydrofolic acid synthetic pathway is defective in K. vulgare, which may stop the normal circulationK. vulgare only contains some isolated reactions and the conversion steps for tryptophan to acetyl CoA/FAD/FADH2 are absent. Besides, K. vulgare is absent of the fatty acid metabolism pathway so that it can’t obtain acetyl CoA directly. The concentration of acetyl CoA significantly contributed to the formation of citric acid in TCA cycle, which can be used not only as an electron acceptor, but also as a precursor for amino acid biosynthesis and ATP production. Hence, the lack of tryptophan metabolism pathway severely affected the growth of K. vulgare. Furthermore, K. vulgare harbores the complete valine, leucine and isoleucine biosynthesis pathways, but only contains several isolated reactions in the related degradation pathway. The lack of degradation ability may lead to the amino acid accumulation in K. vulgare, providing extracellular amino acids to companion strain in consortiumIn the amino acid metabolism of K. vulgare Hbe602 strain was cultured in 250 mL flasks with 50 mL of seed medium  supplied with 2% carbon source for 35 h. The seed medium contains 3 g/L beef extract, 3 g/L yeast powder, 3 g/L corn steep liquor, 0.2 g/L MgSO4, 1 g/L KH2PO4, 1 g/L urea and 10 g/L peptone. The genome sample was carried out by CTAB/NaCl extractionThe 2SO4 was used as the mobile phase at a speed of 0.6 mL/min and the RI temperature was kept at 65 °C.2-KGA and different substrates were detected by HPLC using an Aminex HPX-87H column  coupled with a refractive index (RI) detector. 5 mM HUsing the 454 GS FLX system, single-end libraries with 15-fold coverage and mate paired-end libraries with 10-fold coverage were constructed. The genome was sequenced by the Sanger shotgun approach. The reads were assembled into 6 contigs by using the 454 Newbler assembler and the gaps between the contigs were closed by PCR amplification.oriC) and putative DnaA boxes were identified by Ori-FinderThe genome analysis was carried out by the Rapid Annotation using Subsystems Technology (RAST) analysis platformK. vulgare Hbe602 genome has been deposited at DDBJ/EMBL/GenBank under the accession numbers CP012908, CP012909 and CP012910.The sequence of the d4 acid (0.1 mg/mL) was used as an internal standard to correct for minor variations occurring during sample preparation and analysis. The extracts of intracellular were lyophilized and four independent experiments were performed for each sample. Two-stage chemical derivatization was performed as described previouslyCells cultured in different carbon source of logarithmic growth phase were quenched and extracted as intracellular metabolites according to our previous methodMetabolites were analyzed by GC-TOF/MS  as described previouslyHow to cite this article: Jia, N. et al. Insights into mutualism mechanism and versatile metabolism of Ketogulonicigenium vulgare Hbe602 based on comparative genomics and metabolomics studies. Sci. Rep.6, 23068; doi: 10.1038/srep23068 (2016).


Copy number variation (CNV), a complex genomic rearrangement, has been extensively studied in humans and other organisms. In plants, CNVs of several genes were found to be responsible for various important traits; however, the cause and consequence of CNVs remains largely unknown. Recently released next-generation sequencing (NGS) data provide an opportunity for a genome-wide study of CNVs in rice.Oryza glaberrima as the outgroup, 80 % of the CNV events turned out to be insertions in Nipponbare. There were 2,806 annotated genes affected by these CNV events. We experimentally validated 28 functional CNV genes including OsMADS56, BPH14, OsDCL2b and OsMADS30, implying that CNVs might have contributed to phenotypic variations in rice. Most CNV genes were found to be located in non-co-linear positions by comparison to O. glaberrima. One of the origins of these non-co-linear genes was genomic duplications caused by transposon activity or double-strand break repair. Comprehensive analysis of mutation mechanisms suggested an abundance of CNVs formed by non-homologous end-joining and mobile element insertion.Here, by an NGS-based approach, we generated a CNV map comprising 9,196 deletions compared to the reference genome ‘Nipponbare’. Using This study showed the impact and origin of copy number variations in rice on a genomic scale.The online version of this article (doi:10.1186/s12864-016-2589-2) contains supplementary material, which is available to authorized users. Drosophila . Th. Thde nojaponica and indica subspecies [.PCR validation was performed in five randomly selected rice accessions, along with Nipponbare. The primers were designed by Primer5 , and thebspecies , and BACbspecies , were ushttp://rice.plantbiology.msu.edu/). CNV genes were annotated by InterProScan to assign Gene Ontology annotations [S. bicolor [O. brachyantha using Blast software. For validation of the functional genes affected by CNVs, the orthologous regions of CNVs in O. nivara, O. barthii, O. glaberrima, O. glumaepatula, and O. meridionalis were used for alignments. These genome sequences were provided by I-OMAP ; PCR validation was performed in 18 selected rice accessions. The visualization of alignments used the ACT v11.0.0 software.Gene positions were obtained from the TIGR database (otations . The Genotations . The ric bicolor , and O. O. glaberrima was performed according to the previous described method [The co-linearity analysis of CNV genes compared to d method . CNV ford method .Statistical analyses were performed using the R v2.15.1 software . FiguresThe CNV data have been deposited into NCBI dbVar, with the submission number of nstd96.


Nelumbo nucifera is an evolutionary relic from the Late Cretaceous period. Sequencing the N. nucifera mitochondrial genome is important for elucidating the evolutionary characteristics of basal eudicots. Here, the N. nucifera mitochondrial genome was sequenced using single molecule real-time sequencing technology (SMRT), and the mitochondrial genome map was constructed after de novo assembly and annotation. The results showed that the 524,797-bp N. nucifera mitochondrial genome has a total of 63 genes, including 40 protein-coding genes, three rRNA genes and 20 tRNA genes. Fifteen collinear gene clusters were conserved across different plant species. Approximately 700 RNA editing sites in the protein-coding genes were identified. Positively selected genes were identified with selection pressure analysis. Nineteen chloroplast-derived fragments were identified, and seven tRNAs were derived from the chloroplast. These results suggest that the N. nucifera mitochondrial genome retains evolutionarily conserved characteristics, including ancient gene content and gene clusters, high levels of RNA editing, and low levels of chloroplast-derived fragment insertions. As the first publicly available basal eudicot mitochondrial genome, the N. nucifera mitochondrial genome facilitates further analysis of the characteristics of basal eudicots and provides clues of the evolutionary trajectory from basal angiosperms to advanced eudicots. Brassica napus)Silene conica)4The size of the mitochondrial genome differs among angiosperm species, ranging from approximately 220 kb (Nelumbo nucifera Gaertn. (Sacred lotus) is considered an evolutionary relic, which like Ginkgo, Sequoia, Metasequoia and Liriodendron, has survived since the Late Cretaceous periodN. nucifera was a perennial aquatic plant that flourished during the middle Albian8N. nucifera has been classified in the monotypic family Nelumbonaceae, which contains a single genus Nelumbo. This genus includes two species, N. nucifera and N. lutea. As a eudicot whose lineage emerged prior to the divergence of core eudicotsN. nucifera provides new insights into the origin of eudicots. The nuclear11N. nucifera have recently been released. However, no information on the N. nucifera mitochondrial genome has been reported. Thus, it is important to sequence the N. nucifera mitochondrial genome to reveal the evolutionary characteristics of this plant and provide clues concerning the evolutionary trajectory from basal angiosperms to advanced eudicots.de novo assembly through the PacBio RS II platform17181920Third-generation sequencing through single molecule real-time sequencing technology (SMRT)14N. nucifera mitochondrial DNA and sequenced the genome using SMRT technology. The mitochondrial genome map was constructed after de novo assembly and annotation of the sequence data. Our analyses provide insights into the evolution of gene content and order, RNA editing patterns, positively selected sites and chloroplast DNA insertions in core eudicots.In the present study, using an optimized method for mitochondrial DNA isolation, we prepared N. nucifera etiolated seedlings after discontinuous sucrose gradient centrifugation and DNase I digestion. Janus Green B staining showed that most isolated mitochondria were intact , with a mean read quality of 0.83. After trimming off adapters and low quality regions and correcting by mapping short reads to long seeds, we have obtained 9,165 reads  with an accuracy of 99%. After filtering chloroplast reads, a total of 7,151 reads  were used for the assembly process, reaching a coverage depth of 59 over the l genome , making N. nucifera mitochondrial genome is assembled into a single circular-mappingN. nucifera has the second highest GC content of all plant mitochondrial genomes, while the Butomus umbellatus mitochondrial genome has the highest GC content of 49.1%N. nucifera mitochondrial genome also contained many small repeats (20- to 500-bp), comprising 3.2%  of the total length. Two hundred and one simple sequence repeats (SSRs) were identified , 13 complete native mitochondrial tRNA genes and seven chloroplast-derived tRNA genes  were also predicted in the present study, comprising 7.3%  of the total length N. nucifera. The results showed that A or U were more frequently used compared with G or C at the third position of N. nucifera mitochondrial codons -nad7, nad5_exon1,2-atp9, nad3-rps12, trnN(GTT)(cp)-trnY(GTA), ccmB-trnK(TTT)-trnQ(TTG), trnS(GCT)-trnF(GAA)-trnP(TGG)-sdh3-a, cob-rps14-rpl5, trnP(TGG)(cp)- trnW(CCA)(cp) and petGψ-petLψ. Among these, six N. nucifera mitochondrial gene clusters were found conserved in other angiosperm species. Atp8-cox3-sdh4 occurred in both the ‘early-diverging’ angiosperm L. tulipifera and some eudicot species. Cox1-rps10, nad5_exon1,2-atp9, trnN(GTT)(cp)-trnY(GTA) and petGψ-petLψ were represented only in eudicots. Nad5_exon4,5-trnE(TTC)-nad7 was found only in L. tulipifera and N. nucifera. The nine remaining gene clusters were variously distributed among all of these taxa.Based on the endosymbiont hypothesis that mitochondrial genomes are the remnants of a free-living prokaryote (most likely an extant α-proteobacterium), some small DNA fractions of the original genes have been identified in the mitochondrial DNA of higher order plants29lipifera : rrn18-rrpl10, all of the mitochondrial protein-coding genes of N. nucifera were successfully amplified, and a total of 33,719-bp cDNA sequences were sequenced; the amplicon for ccmFC was derived from pre-mRNA, with unspliced introns rather than mature mRNA. After aligning the DNA and cDNA sequences, 700 RNA editing sites were identified, including 612 nonsynonymous, 58 synonymous and 30 unpartitioned edits of the pseudogene mttB  to GCG (A), UGC (C) to CGC (R) and UCU (S) to CCU (P), respectively. The most frequently edited genes were atp9 and ccmB, with editing frequencies of 7.111 edits/100 nt and 6.924 edits/100 nt, respectively, while the frequencies of other genes were less than five edits/100 nt. The genes with no RNA editing sites included ccmC, rps12, rps2 and sdh4. N. nucifera showed a pattern of relative editing levels across genes that was similar to other angiospermsIn the present study, with the exception of ene mttB . As the Amborella trichopoda, L. tulipifera, Vitis vinifera and Oryza sativa with that of N. nucifera to identify similarities in the RNA editing sites  were higher compared with V. vinifera and O. sativa . To examine similarities in RNA editing, we summarized the number of unique and shared edits in A. trichopoda, the maximum number of shared edits was 444 in L. tulipifera, followed by 361 in N. nucifera, 209 in O. sativa and 176 in V. vinifera. Compared with L. tulipifera, the maximum number of shared edits was 482 in N. nucifera, followed by 261 in O. sativa and 205 in V. vinifera. Moreover, N. nucifera contained the fewest number of unique edits  tree of 79 plant mitochondrial genomes, including N. nucifera, was built using the sequences of 41 mitochondrial protein-coding genes and rooted with the green algae Mesostigma virideHuperzia squarrosa was a sister of the gymnosperm Cycas taitungensis and Angiospermae. Within the Angiospermae clade, Amborella trichopoda was sister of all other angiosperms. The monocots and the eudicots were monophyletic; Spirodela polyrhiza was sister of all other monocots; and N. nucifera formed an independent basal branch as a sister of all other eudicots. Each of these relationships has 100% bootstrap value support.To examine the phylogenetic evolution of N. nucifera mitochondria, Maximum Likelihood (ML) trees were reconstructed from 41 individual gene alignments using optimal substitution models  of highly divergent sequences, because the alignment demonstrated that the rps7 sequence of E. fimbriata was highly divergent from that of S. miltiorrhiza and B. hygrometrica. NCBI-BLASTN searches of these rps7 genes against the Nucleotide Collection Database showed that rps7 in S. miltiorrhiza and B. hygrometrica mitochondria have shown 100% identity with rps7 of their own chloroplast genomes, while rps7 in E. fimbriata showed low similarity with other nucleotides. The abnormalities in rps11 tree and rpl2 tree could also be caused by genes of chloroplast origin, because BLASTN showed a high similarly of V. vinifera mitochondrial rps11 with its chloroplast rps11, the rpl2 of S. miltiorrhiza, A. reptans and D. carota also showed 100% similarities with their chloroplast ones. The sdh3 of M. pinnata showed extremely low similarity with records in both nucleotide collection database and non-redundant protein database of NCBI, which leads to the embedding of M. pinnata into alga cluster with a extremely long branch. The abnormalities in nad7 tree may be caused by lacking of informative sites due to the limited sequence variations, since the deletion of S. polyrhiza and Phoenix dactylifera leads to the relocation of V. macrocarpon as sister to D. carota in nad7 tree with higher branch support values. And the alignments showed that V. macrocarpon shows higher similarity with D. carota than with S. polyrhiza. The abnormalities in atp9 tree may be also caused by lacking of informative sites considering its short length  and the high similarity between eudicots and monocots.To explore potential horizontal gene transfer (HGT) events that occurred in n models . The supria tree . In cox1ial tree  or the pN. nucifera using the alignments and gene trees of the 79 mitochondria genomes. After likelihood ratio tests (LRT), alternative models were significantly favoured in 27 genes. However, in other genes, alternative models were not preferable to the null model  and the two shortest fragments (positions 91514-91569 and 342137-342190) were unique to the N. nucifera mitochondrial genome. The third shortest fragment (positions 413440-413496) was only identified in A. trichopoda. The longest fragment (positions 469237-471234) was only identified in L. tulipifera. The 84-, 81- and 78-bp fragments containing the trnN(GTT), trnH(GTG) and trnM(CAT) genes, respectively, had homologues in more than 40 species. The 84-bp fragment was commonly identified in angiosperms, but was absent in the gymnosperm C. taitungensis, while the 81- and 78-bp fragments were commonly observed in both angiosperms and C. taitungensis.Nineteen fragments of chloroplast DNA ranging from 54 to 1,998-bp were identified in ra mtDNA , Table 3nt mtDNA . None ofN. nucifera mitochondrial genome using third-generation SMRT sequencing technology and constructed a genomic map. In our previous workN. nucifera chloroplast assemblies obtained using Sanger, Illumina MiSeq, and PacBio RS II platforms indicated that SMRT sequencing technology showed great promise for the production of highly accurate genome sequences because it produces long reads and lacks bias. In this study, the assembly validation experiment showed a great accuracy of N. nucifera mitochondrial genome assembly.We sequenced the N. nucifera mitochondrial genome provides valuable insights into the pattern of the evolution from basal angiosperms to advanced eudicots, and shows ancient evolutionary features in many aspects:N. nucifera mitochondrial genome retains 40 of all the 41 protein-coding genes present in ancestral flowering plant mitochondrial genomesNelumbo mitochondrial genome contains the full set of protein-coding genes present in the ancestral angiosperm. To our knowledge, of all available angiosperm mitochondrial genomes, only A. trichopodaL. tulipiferasdh) genes, has occurred in other angiosperm mitochondrial genomes and results in variable gene content among angiospermscis-spliced introns of the N. nucifera mitochondrial genome is longer than any other sequenced angiosperm mitochondrial genomecis-spliced introns has only been identified in the mitochondria of three angiosperm species, including N. nucifera (in the present study), the ‘early-diverging’ angiosperm L. tulipifera and the ‘early-diverging’ core eudicot Vitis vinifera. In contrast, the five trans-spliced introns in N. nucifera were conserved in all of the sequenced angiosperm mitochondrial genomes.Conservation of genomic content. The L. tulipifera occurred prior to the divergence of monocots and eudicots, showing primitive features in gene content and at RNA editing sitesL. tulipifera mitochondrial genome is a scale to explore the evolution of N. nucifera and other angiosperms. The N. nucifera mitochondrial genome has gene clusters that can date back to the original bacterial ancestor of mitochondria , gene clusters unique to angiosperms , a gene cluster unique to eudicots (petGψ-petLψ) and a gene cluster -nad7) that is only shared with L. tulipifera. This further indicates that the N. nucifera mitochondrial genome was more primitive than other advanced angiosperms in angiosperm evolution.Conservation of gene clusters. As a sister group, the ‘early-diverging’ angiosperm N. nucifera, approximately 87% of the total editing sites changed the translated amino acid sequence. Comparing RNA editing sites of A. trichopoda, L. tulipifera, V. vinifera and O. sativa with that of N. nucifera clearly shows that the degree of editing loss in N. nucifera is much lower than that in V. vinifera and O. sativa, suggesting that the N. nucifera mitochondrial genome was more ancient.Retention of ancient RNA editing sites. During the evolution of advanced angiosperms, RNA editing sites showed various degrees of subsequent losses in different lineages3738As the first publicly available basal eudicot mitochondrial genome, the N. nucifera was originally considered to be the closest relative to Nymphaea alba because of morphological similarities, reflecting convergent evolution with N. alba in the same aquatic environmentN. nucifera is a member of the Proteales within the basal eudicots and phylogenetic analyses using chloroplast sequences clustered N. nucifera into the basal eudicot branches as a sister to Meliosma and PlatanusN. nucifera as a sister to all other eudicots. Finally, the phylogenetic analysis in the present study, along with previous studies based on chloroplast and nuclear genome data, provided a comprehensive view of the basal location of N. nucifera in eudicots. Thus, we concluded that N. nucifera is an ‘early-diverging’ basal eudicot. In the most of the 41 gene trees, the N. nucifera sequences were clustered in the basal location in eudicots, suggesting that N. nucifera mitochondrial genes maintained low evolutionary rates. Topological incongruence was observed in different species of many single gene trees in the present study. The embedding of H. squarrosa into the bryophytes clusters confirmed Liu’s study on H. squarrosa mitochondriaC. vulgaris is considered to be the most closely related alga to land plantsC. vulgaris mitochondriaC. vulgaris mtDNA strikingly resembles Marchantia polymorpha mtDNA, supported by both genome comparisons and phylogenetic analyses. The sister-group relationship between C. vulgaris and N. hyalina in our study suggested that C. vulgaris mtDNA may have more similarities with N. hyalina mtDNA. The embedding of species into totally unrelated plant phyla identified in six gene trees was expected to be possible HGT candidates. But our further analysis showed that this topological incongruence was caused either by long branch attraction of highly divergent genes, or by lacking of informative sites due to the limited sequence variations. Of the eight highly divergent genes, six genes were found similar to their own chloroplast genes, and three of these six genes were reported as chloroplast-derived by former studies -contained chloroplast-derived fragment was absent in the gymnosperm C. taitungensis while the trnH(GTG)- and trnM(CAT)-containing fragments were commonly observed in both angiosperms and C. taitungensis. This finding is consistent with the analysis of chloroplast-derived tRNA gene evolutiontrnH(GTG) and trnM(CAT), previously present in C. taitungensis, were obtained from an earlier transfer, while trnN(GTT) might have occurred after the separation of gymnosperms and angiosperms. The short stretches of these three fragments in N. nucifera suggested that the selection of the flanking sequences might have occurred prior to the separation of early-diverging angiosperms and basal eudicots. Plant mitochondrial tRNAs were derived from three origins: (i) ‘native’ mitochondrial tRNAs, orthologous to those in moss mitochondria and derived from a mitochondrial ancestor; (ii) chloroplast-like mitochondrial tRNAs, highly homologous to chloroplast DNA; and (iii) unknown-origin mitochondrial tRNAs, such as the bacterial-like trnC(GCA), obtained through horizontal gene transfer events45N. nucifera mitochondrial genome had a complete set of 13 intact ‘native’ mitochondrial tRNAs, which were also identified in the ‘early-diverging’ core eudicot V. vinifera, but variously presented in other angiosperms. The ‘native’ mitochondrial tRNAs retained in gymnosperms, trnL(CAA) and trnD(GTC), have become pseudogenes in N. nucifera during the evolution of angiospermstrnL(CAA) and trnD(GTC) was compensated with chloroplast-derived trnL(CAA) or cytosolic tRNAsN. nucifera mitochondrial genome contained seven chloroplast-derived tRNA genes present in the ‘early-diverging’ core eudicot V. vinifera. Notably, the chloroplast-derived trnT(GGT) gene included in the 141-bp unique fragment in N. nucifera was also identified within a 3,571-bp chloroplast-derived fragment in V. vinifera mitochondriatrnT(GGT) gene in N. nucifera and V. vinifera mitochondria were separate events.However, intracellular gene transfer (IGT) of DNA from the plastid to the mitochondrial genomes of higher plants is a common phenomenon on an evolutionary timescale. Sometimes, these IGT events may initiate a gain of functional tRNAs. Due to the slow rate of mitochondrial sequence change, some of the chloroplast-derived fragments identified in Though dozens of flowering plant mitochondrial genomes are published, early-derived angiosperms or basal eudicot mitochondrial genomes are still limited. The difficulty of isolating mitochondrial DNA is a bottleneck in acquiring complete mitochondrial genome sequence data. The optimized method for the isolation of mitochondrial DNA from plants rich in secondary metabolites used in the present study would promote the sequencing of more plant mitochondria. The genomic data of other basal eudicots, such as Platanus, will provide more detailed insights into mitochondrial genome evolution in basal eudicots.N. nucifera were harvested from the Wuhan Moshan Botanical Garden  of Hubei Province, China and germinated in the dark at room temperature. Discontinuous sucrose gradient centrifugation combined with DNase I digestion in vitro was used to separate mitochondrial DNA from etiolated seedlings. Three pairs of primers specific to genes in the nuclear, mitochondria and chloroplast were designed to evaluate the purity of the mtDNA through semi-quantitative PCR. A full description of the mtDNA isolation method has been provided in Mature seeds of de novo assembly were performed at Yale University, Connecticut, America and Nextomics, Wuhan, China, respectively.A total of 20 μg of standard mtDNA was required to construct a size-selected ~20 kb SMRT-bell library. Subsequently, the SMRT-bell library was sequenced using three SMRT cells (Pacific Biosciences) with C2 chemistry on a PacBio RS II sequencing platformNelumbo nucifera chloroplast genome [GenBank:KM655836] and the NCBI chloroplast genome database using BlatThe primitive reads from the Pacbio RS II were filtered and corrected by mapping short reads to long “seed” reads  using SMRT Analysis 2.1 . Then, the reads were mapped to both the N. nucifera mitochondrial genome was manually annotated based on the output of NCBI-BLASTN and NCBI-BLASTX searches to a custom local database. The online tool tRNAscan-SEN. nucifera was drawn using Genome VxN. nucifera chloroplast genome with 70% identity and an e-value of 1e–5. To identify the nuclear-derived sequences, the protein-coding genes, cis-spliced introns, rDNAs, tRNAs and chloroplast-derived sequences were masked; subsequently, the masked sequences were searched in the N. nucifera nuclear genome. The RSCU (relative synonymous codon usage) of the N. nucifera mitochondrial genome was calculated from the coding sequences of 40 different protein-coding genes. MISAThe N. nucifera mitochondria genome were compared to 37 representative species of angiosperm mitochondria genomes available in NCBI using BLASTP/BLASTXTo evaluate the collinear relationships between genes, all protein-coding genes, tRNA genes and rRNA genes from the E. coli and subsequently sequenced. The sequenced cDNAs were compared with the genomic sequence and aligned using ClustalWA. trichopodaL. tulipiferaO. sativaV. viniferaTotal RNA was isolated from tender leaves using the modified CTAB methodN. nucifera, the protein-coding sequences from 78 published complete mitochondrial sequences of angiosperms were downloaded from the NCBI database  were performed, and the P value was calculated using the chi2 program in PAML packages.The program Codeml in the PAML packageN. nucifera mitochondrial genome were masked to eliminate BLAST hits of the conserved household genes in chloroplast and mitochondrial genomes. The masked genome sequence was BLASTed against the chloroplast genome of N. nucifera. The chloroplast-derived fragments were subsequently BLASTed against a local database containing all Viridiplantae mitochondrial genomes available in NCBI to identify homologues with an identity greater than 80% and full-length or nearly full-length (more than 90%) alignment. The maps of N. nucifera mitochondrial and chloroplast genomes and the chloroplast fragment transferences were drawn using Circos v.0.67After sequence annotation, the protein-coding genes, tRNA genes and rRNA genes of the N. nucifera has been submitted to GenBank [GenBank: KR610474]. The raw sequence data have been deposited in the Short Read Archieve (SRA) database of NCBI [SRA: SRP066826], note that only data from SMRT cell 3 (named “mt_s3.fastq”) was used for the assembly in this study. Other data sets supporting the results of the present study are included within the article and The precise mitochondrial genome of How to cite this article: Gui, S. et al. The mitochondrial genome map of Nelumbo nucifera reveals ancient evolutionary features. Sci. Rep.6, 30158; doi: 10.1038/srep30158 (2016).