Vibrio vulnificus to rapidly propagate in the host. This problematic pathological scenario can be circumvented by employing an antivirulence strategy, treating Vibrio infections without hindering the bacterial growth. We developed a genome-integrated orthogonal inhibitor screening platform in E. coli to identify antivirulence agents targeting a master virulence regulator of V. vulnificus. We identified 2′,4′-dihydroxychalcone (DHC) from the natural compound library and verified that it decreases the expression of the major toxin network which is equivalent to the ∆hlyU deletion mutant. 2′,4′-DHC also reduced the hemolytic activity of V. vulnificus which was tested as an example of virulence phenotype. The electrophoretic mobility shift assay confirmed that 2′,4′-DHC specifically targeted HlyU and inhibited its binding to PrtxA1 promoter. Under in vivo conditions, a single dose of 2′,4′-DHC protected ~50% wax-worm larvae from V. vulnificus infection at a non-toxic concentration to both V. vulnificus and wax-worm larvae. In the current study, we demonstrated that an orthogonal reporter system is suitable for the identification of antivirulence compounds with accuracy, and identified 2′,4′-DHC as a potent antivirulence agent that specifically targets the HlyU virulence transcriptional regulator and significantly reduces the virulence and infection potential of V. vulnificus.The emergence of antimicrobial resistance and rapid acclimation allows  Vibrio vulnificus is Gram-negative, halophilic, highly adaptable, opportunistic, and one of the most lethal human pathogens among foodborne infectious diseases [V. vulnificus is predominantly present in seafood such as oysters, shrimps, fish, and shellfish [V. vulnificus and then consumed by humans causes systemic infections such as lethal septicemia with an exceptionally high mortality rate surpassing 50% [V. vulnificus can easily acclimatize to its human host and cause wound infections upon exposure to seawater, which may progress rapidly to cellulitis, ecchymosis, and finally result in necrotizing fasciitis [V. vulnificus infections are one of the most life-threatening foodborne infectious diseases and are treated with quinolone, tetracycline, and cephalosporin antibiotics [V. vulnificus strains have recently been reported as developing antimicrobial resistance (AMR) in many countries, against the antibiotics currently used for the treatment of V. vulnificus infections [V. vulnificus infections. diseases . V. vulnhellfish . Raw or sing 50% . Owing tasciitis ,5. V. vuibiotics ,7,8. Howfections ,10. TherVibrio strains. As an alternative approach to bactericidal antibiotics that cause AMR development, the antivirulence strategy is rapidly progressing to treat bacterial infections by disarming their virulence factors and subjecting them to be cleared by the host’s immune system ,12. The V. vulnificus is tightly controlled by a master virulence transcriptional regulator/factor (VTF), HlyU, which is responsible for the induction of various virulence proteins including critical offensive toxins such as repeat-in-toxin (RtxA1) and hemolysin (VvhA) to establish successful infection via wound and intestinal routes [The pathogenesis of l routes ,14,15,16Escherichia coli inhibitor assay reporter platform for the identification of antivirulence compounds by screening the natural product library. The novel heterologous and isolated reporter platform identified 2′,4′-dihydroxychalcone  as an antivirulence agent that specifically targets HlyU and inhibits virulence factors under its control, similar to the hlyU deletion mutant (∆hlyU). In the present study, 2′,4′-DHC targeted HlyU and inhibited the virulence factors under its control to fight V. vulnificus pathogenesis under both in vitro and in vivo conditions using the Galleria mellonella (wax-worm) infection model.We developed an orthogonal genome-integrated, stable rtxA1, and keeps the rtxA1 gene repressed under normal conditions [V. vulnificus makes contact with the host cells, the transcription of rtxA1 is induced several-fold upon host–pathogen interaction, as reported earlier [V. vulnificus  promoter, Pnditions  , which was required for identifying the antivirulence compound against V. vulnificus, a 951 bp intergenic region of wild-type (WT) E. coli genome was chosen between two convergent genes, iap and ygbF, encoding an aminopeptidase and CRISPR  adaptations RNA endonuclease, respectively. A DNA cassette PrtxA1::luxCDABE_rcm of 7997 bp from the pBBRMCS2_PrtxA1::luxCDABE plasmid (rtxA1 promoter (754 bp) tagged with luxCDABE scorable reporter genes along with the chloramphenicol acetyltransferase (cat/rcm) gene was integrated by deleting a 0.4 kb region of the innocuous intergenic region of the E. coli genome, and the obtained recombinant clone was designated as E. coli-PrtxA1::lux_rcm  inducible Ptaclac promoter in the pMAL-c2X plasmid to mimic the higher level of expression of HlyU under pathophysiological condition. The ampicillin cassette was deactivated by inserting the kanamycin cassette at the ScaI site, and the resultant hlyU expression plasmid was designated as pMAL-c2X_rkm_hlyU  showed leaky expression of HlyU that increased after IPTG induction  carrier control was analyzed. Twenty-one hits were selected, of which 14 fell under the primary antivirulence hits category and seven belonged to known antimicrobial compounds   than thed 128 µM . The gro to 8 µM . TherefoV. vulnificus strain MO6-24/O, as demonstrated by the growth curve (V. vulnificus growth because it is 16- fold lower than the MIC concentration against V. vulnificus (128 µM).The half maximal inhibitory concentration (IC50) of 2′,4′-DHC against HeLa and HEK293 cells was calculated to be 100.3 µM and 60.0 µM, respectively . The IC5th curve . The worrtxA1 (repeat-in-toxin) and vvhA (hemolysin)  A,B withopression D. The anU strain E,E”. The32-labelled PrtxA1 (261 bp) showed two shifts at higher concentrations of HlyU protein in Electrophoretic Mobility Shift Assay (EMSA)  was administered. As a result, 2′,4′-DHC averted the pathophysiology of V. vulnificus and showed ~50% larvae protection with a relatively better health-index based on their motility, cocoon formation, melanization, and survival, as compared to 10% larvae survival in the no treatment infection group  was achieved by mutating leucine 91 and 17 residues with alanine (L91A/L17A), as reported previously . Freshlyy (EMSA) A. The hiy (EMSA) B. This sy (EMSA) C, indicaon group D,D’.Vibrio vulnificus contaminates nutrient-rich seafood and is extremely efficient in evading the host immune system and antibiotic treatments and thus causes a high rate of fatality. Most of the antivirulence approaches that are applied for bacterial pathogens target solitary virulence genes. As expected, such approaches show only marginal protection effects under in vivo infection conditions. To overcome this limitation, we focused on the master virulence regulator, HlyU, which is known to induce multiple virulence factors during infection. The antivirulence strategy targeting HlyU effectively disarmed the most primary and potent virulence factors (toxin network) and accessory multiple virulence factors. 2′,4′-DHC was identified using a newly developed orthogonal heterologous and integrated stable inhibitor/drug assay platform targeting HlyU . Three antivirulence natural products, namely resveratrol 4′-methyl ether, curcumin, and 2′,4′-DHC, were identified in the primary screening . Resveraucidated . Curcumilocation . HoweverV. vulnificus at higher concentrations (hlyU strain (>30-fold inhibition) at a non-growth inhibitory concentration (5 µM), whereas isoliquiritigenin at 10 µM inhibited transcription of rtxA1 only up to 2.5-fold. These results clearly indicate that the mild bacteriostatic nature of chalcones at higher concentrations against V. vulnificus is not the reason for the reduced transcription of major toxin genes (vvhA and rtxA1); rather the antivirulence activity of 2′,4′-DHC is specific and presumably occurs via targeting the master VTF, HlyU  a chalcone backbone and (b) the mild growth inhibition of trations . NeverthTF, HlyU . The natTF, HlyU B.V. vulnificus pathogenesis [kB-dependent mitochondrial cell death via the production of reactive oxygen species mediated by lipid rafts in intestinal cells [rtxA1 and vvhA transcripts as well as haemolysis phenotype  infection model for studying V. vulnificus pathogenesis in an earlier study [V. vulnificus infection  and ∆hlyU deletion mutant [V. vulnificus was assessed by measuring turbidity at 600 nm (OD600) and counting the colony forming units (CFU). E. coli strains were grown at 37 °C in LB medium (broth/agar) with the appropriate concentrations of antibiotics  to select for recombinant strains. All strains and plasmids used in the present study are listed in MO6-24/O   [rtxA1 promoter tagged with luxCDABE genes and chloramphenicol acetyltransferase gene using specific primers  to achieve pMAL-c2Xr_hlyU._kmAll recombinant DNA techniques were performed using various molecular biology kits for plasmid and genomic DNA isolation , and gel purification of DNA  as per the manufacturers’ protocols. An expand long PCR amplification kit  was used to amplify the long DNA cassette (P.997 kb)  harborin primers . The ~8 5 strain  harborincI sites  of the m plasmid   in 48-well plate format at 20 µM. Overnight grown cultures were diluted by 1:500 in fresh LB medium supplemented with appropriate antibiotics and 1 mM IPTG. Each plate was complemented with the E. coli PrtxA1::rlux_cm strain with an empty pMAL-c2X_kmr plasmid as the negative control for background signal assessment. The natural product library (800 compounds) was screened with the pMAL-c2X_V. vulnificus cultures were adjusted to obtain an initial 2 × 106 CFU/mL bacterial count. The WT strain was supplemented with or without secondary screening hit compounds or 2′,4′-DHC or DMSO in 3 mL fresh LBS medium at concentrations that were assessed as being non-growth inhibitory. The cultures were allowed to grow up to OD600 ~1.8–2.0. The ∆hlyU V. vulnificus strain was included as the control. RNA was isolated using a QIAGEN RNeasy Mini kit and cDNA was synthesized using the Ecodry Premix (random hexamer) kit  as described earlier [rtxA1, vvhA, hlyU, and hns genes wherein the gyrB gene served as an endogenous control. Relative gene expression was analyzed using the 2−ΔΔCT method [Overnight  earlier . The qRTT method .2. The minimum inhibitory concentration (MIC) of 2’,4’-DHC was determined according to the Clinical and Laboratory Standards Institute  guidelines in Mueller Hinton (MH) broth [The toxicity of lead antivirulence small molecule 2′,4′-DHC was evaluated against HeLa and HEK293 human cell lines using an EZ-Cytox cell viability assay kit  as described earlier . HeLa anH) broth . V. vulnificus strains were diluted in fresh LBS medium, treated with DMSO or 2′,4′-DHC, and incubated for 3 h. The culture supernatant was diluted half in phosphate-buffered saline  before mixing with 1% hRBC suspension in PBS and incubated at 37 °C under shaking conditions for 2.5 h. After incubation, the reaction mixture was centrifuged and the hemolysis was determined by measuring the absorbance of the supernatant at 540 nm (A540). Triton X-100 (1%) treated hRBC suspension was used as a 100% hemolysis control and the percent hemolysis was calculated as described earlier [The hemolysis assay was performed using hRBCs as described previously . Briefly earlier .6 tag (Invitrogen). The dimerization mutant protein of HlyU (L91A/L17A) [6-HlyU WT and the point mutant were expressed in E. coli BL21 (DE3) and grown until OD600 0.4–0.5 for induction with 1 mM IPTG and incubated at 16 °C for 8 h. Cells were harvested and purified by Ni-NTA and size exclusion chromatography  in a final buffer containing 25 mM Tris-Cl (pH 7.5) and 100 mM NaCl. The protein purity was verified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).The 297 bp HlyU coding region was directionally cloned at BamHI and XhoI site of pProEx-HTb vector to express the recombinant HlyU with N-terminus His1A/L17A)  was gene1A/L17A)  (see Tab1A/L17A) . BrieflyrtxA1 promoter region (PrtxA1) was amplified from the genomic DNA of V. vulnificus MO6 and labelled with γ-ATP32. DNA and protein were mixed in 20 µL by adding 3 nM radio labelled probe with varying concentrations of protein in 1× binding buffer 2SO4, 5 mM dithiothreitol, 1% (v/v) Tween 20, and 150 mM KCl) [For EMSA, 261 bp DNA from the  mM KCl) . For theG. mellonella (130 ± 30 mg) were purchased from a local vendor in Korea. V. vulnificus strains were sub-cultured for 6 h and the bacterial cells were washed and resuspended in phosphate buffer saline . A total of 200–300 CFU of V. vulnificus was injected into the hemocoel of larvae through the left posterior proleg. The antivirulence 2′,4′-DHC  was administered via the right posterior proleg after 1 h of infection [Larvae of nfection ,38, and nfection .V. vulnificus pathogenesis by inhibiting the HlyU mediated toxin-network necessary for establishing V. vulnificus infection.The development of an elegant, isolated, and noise-free orthogonal inhibitor screening platform identified 2′,4′-DHC as a novel, high potential antivirulence natural product that protected the host from


Impulse control disorders (ICD) are common neuropsychiatric disorders that can arise in Parkinson’s disease (PD) patients after commencing dopamine replacement therapy. Approximately 15% of all patients develop these disorders and many more exhibit subclinical symptoms of impulsivity. ICD is thought to develop due to an interaction between the use of dopaminergic medication and an as yet unknown neurobiological vulnerability that either pre-existed before PD onset (possibly genetic) or is associated with neural alterations due to the PD pathology. This review discusses genes, neurotransmitters and neural networks that have been implicated in the pathophysiology of ICD in PD. Although dopamine and the related reward system have been the main focus of research, recently, studies have started to look beyond those systems to find new clues to the neurobiological underpinnings of ICD and come up with possible new targets for treatment. Studies on the whole-brain connectome to investigate the global alterations due to ICD development are currently lacking. In addition, there is a dire need for longitudinal studies that are able to disentangle the contributions of individual (genetic) traits and secondary effects of the PD pathology and chronic dopamine replacement therapy to the development of ICD in PD. It is nowadays generally appreciated that Parkinson’s disease (PD) should not be regarded as merely a movement disorder. PD patients also commonly suffer from a broad range of non-motor symptoms. Of these, neuropsychiatric disorders are the most salient. Examples include anxiety, depression, apathy, psychosis and impulse control disorders (ICD)  the dopamine system. Before we delve into studies that implicate the dopamine system in the pathophysiology of ICD, it must be noted that dopaminergic medication—specifically dopamine D2-family receptor agonists—may also increase impulsivity by preventing pauses in D2-signaling through phasic endogenous dopamine release in the reward-related ventral striatum  exhibit several alterations in the dopamine system that may make them more vulnerable to the impulsivity inducing effects of dopamine replacement therapy. On the genetic level, multiple studies have associated gene polymorphisms of components of the dopamine signaling cascade with ICD but results have been mixed and generally based on small samples , was able to increase inhibition-related activity in the inferior frontal gyrus   provides a measure for the integrity of white matter tracts. An often used diffusion parameter as a marker for white matter integrity is fractional anisotropy (FA), which represents diffusion along the axon relative to two orthogonal radial directions , cortical thinning was observed in PD + ICD versus PD − ICD patients in the superior orbitofrontal gyrus, rostral middle frontal gyrus and bilateral caudal middle frontal region, concomitant with lower volume of the accumbens but increased volume of the amygdala  challenging. Furthermore, except for two (Smith et al.


Acute exacerbation of chronic fibrosing idiopathic interstitial pneumonias (AE-IIPs) is associated with a high mortality rate. In 2016, an international working group proposed a revised diagnostic criteria for AE-IIPs, suggesting that it be classified as idiopathic or triggered. Many factors are known to trigger AE-IIPs, including surgery, infection, and drugs. However, it is unknown which AE-IIPs triggers have a worse prognosis. We aimed to investigate the prognosis of patients with various clinical types of AE-IIPs, particularly infection-triggered, non-infection triggered, and idiopathic AE-IIPs.We retrospectively collected data from 128 chronic fibrosing IIPs (CF-IIPs) patients who were hospitalized by respiratory failure between April 2009 and March 2019 at Juntendo University Hospital. Among these patients, we evaluated 79 patients who developed AE-IIPs and 21 who developed pneumonia superimposed on CF-IIPs. Patients with AE-IIPs were classified into three types: idiopathic, infection-triggered, and non-infection-triggered AE-IIPs. We analyzed differences in patient characteristics, examination findings; level of serum markers, results of pulmonary function, and radiological findings, prior treatment for baseline CF-IIPs, and prognosis. We then evaluated the risk factor for early death (death within 30 days from the onset of AE-IIPs) associated with AE-IIPs.Among the patients who developed AE-IIPs, 34 were characterized as having idiopathic, 25 were characterized as having infection-triggered, and 20 were categorized as having non-infection-triggered AE-IIPs. Survival time for pneumonia superimposed on IIPs was significantly longer than that for any AE-IIPs. Survival time for bacterial pneumonia superimposed on CF-IIPs was significantly longer than that for AE-IIPs . Thereafter, survival time for infection-triggered was significantly longer than for idiopathic or non-infection-triggered AE-IIPs. The mortality rate was significantly lower in infection-triggered AE-IIPs than in other types of AE-IIPs. Furthermore, the incidence of infection-triggered AE-IIPs in winter was significantly higher than that in other seasons. Moreover, the clinical AE-IIPs types and radiological findings at AE-IIP onset were significant risk factors for AE-IIPs-induced early death.Our findings suggest that patients with infection-triggered AE-IIPs can expect a better prognosis than can patients with other clinical types of AE-IIPs. Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) was first reported in 1993 . FurtherDiagnostic criteria for AE-IPF were first published in 2007 . These iIn 2016, Collard et al. published an International Working Group Report on AE-IPF –17. Desp2/FiO2 ratio, laboratory data , C-related protein [CRP], Krebs von den Lungen-6 [KL-6], and surfactant protein-D [SP-D]), HRCT patterns at time of diagnosis of AE-IIPs, survival time, mortality, and seasonal variations. We evaluated HRCT findings at the time of diagnosis of AE-IIPs according to Akira’s previous publication, defining them as diffuse, multifocal, and peripheral patterns  aPost-operative AE-IIPs were defined as having been induced by any operation performed in an operating room that developed within a month after surgery. Drug-induced AE-IIPs were defined as having occurred after administration of the suspected drug and presence of previous reports of a drug-induced lung injury that developed within 3 months after drug initiation. Post-lung biopsy AE-IIPs were defined as those induced by bronchoscopy or percutaneous lung biopsy that developed within a month after the examinations. Infection-triggered AE-IIPs were defined as those induced by infection of the upper respiratory tract, contracted within 2 weeks prior to development of AE-IIPs, presenting with symptoms associated with upper respiratory tract inflammation, including high fever .We used the chi-square test, Fisher’s exact test, or Wilcoxon two-sample test to evaluate the frequency of any types of acute exacerbation and compare patient characteristics among these groups. Parametric and non-parametric data were compared with Student’s t-test and the Mann-Whitney U test, respectively. Differences in median survival time (MST) were analyzed with the log-rank test. Cox proportional hazard analysis was used to calculate hazard ratios (HRs), and univariate and multivariate logistic regression analyses were used to determine the risk factors for the AE-IIPs. A n = 16), CHF (n = 2), or terminal stage of any cancer (n = 10). Among them, 21 patients developed bacterial pneumonia superimposed on CF-IIPs; thus, we included 79 patients with CF-IIPs and with clinically diagnosed AE-IIPs , infection-triggered (n = 25), and non-infection-triggered (n = 20). Among the patients with infection-triggered AE-IIPs, 5 patients had a possible influenza infection, and all patients had symptoms related to upper respiratory tract infection, high fever or sore throat. Among patients who developed non-infection-triggered AE-IIPs, 12 had a history of new drug initiation, 5 had recently undergone operations, and 3 had received recent bronchoscopy or percutaneous lung biopsy. Patients with drug-induced AE-IIPs had received anti-cancer drugs (n = 6), including paclitaxel (n = 2), gemcitabine (n = 1), doxolbicine (n = 1), and axitinib (n = 1), antihypertensives (amlodipine) (n = 1), antibiotics (levofloxacin) (n = 1), anti-inflammatory drugs (n = 2), including loxoprofen (n = 1) and ibuprofen (n = 1), and herbal medications (n = 2), including Bofu-tsusho-san (n = 1) and Sho-saiko-to (n = 1).We identified 128 CF-IIPs patients who were hospitalized by respiratory failure. From these patients, we excluded patients with pneumothorax or mediastinal emphysema . For other symptoms related to AE-IIPs or CF-IIPs, including worsening cough and dyspnea, there were no significant differences in these symptoms among these groups. In contrast, there were no significant differences in other symptoms, including worsening cough and dyspnea.HRCT indicated a diffuse pattern in 34 patients, a multifocal pattern in 14 patients, and a peripheral pattern in 31 patients at the time of diagnosis of AE-IIPs. There were no significant differences in HRCT patterns associated with AE-IIPs among the three groups. For AE-IIPs-associated clinical symptoms, including high fever (> 37.5 degree), worsening cough, and worsening dyspnea, the number of patients with high fever tended to be higher in infection-triggered AE-IIPs than in other clinical AE-IIPs types (p = 0.037). Moreover, serum LDH levels tended to be higher in non-infection triggered patients than those in other types of AE-IIPs. In contrast, there were no significant differences in other serum markers, including KL-6 and SP-D, as well as the PaO2/FiO2 ratio among the three groups. Among pulmonary function measures, although forced vital capacity (FVC) and vital capacity (VC) were significantly higher in non-infection-triggered AE-IIPs, there was no significant difference in diffusion capacity (DLco) among the three groups .Although there was no significant difference in the serum WBC counts, serum KL-6, SP-D, and D-dimer levels in patients with AE-IIPs were significantly higher than those with pneumonia superimposed on CF-IIPs. In contrast, serum CRP and procalcitonin level was significantly lower in patients with AE-IIPs than in those with pneumonia superimposed on CF-IIPs. In addition, the serum procalcitonin was significantly higher in bacterial pneumonia superimposed on CF-IIPs than in infection-triggered AE-IIPs (p = 0.001).In addition, symptoms, serum marker levels, and pulmonary function were compared between patients with pneumonia superimposed on CF-IIPs and those with AE-IIPs. The number of patients with high fever and sore throat in the bacterial pneumonia group were significantly higher than those in the AE-IIPs group . However, there were no significant differences in the incidence of idiopathic and non-infection-triggered AE-IIPs between any seasons . Multivariate analysis performed using three variables  indicated that HRCT patterns at AE-IIPs onset and clinical AE-IIPs types were significant independent risk factors for early death .Tables p = 0.0001, Fig. p = 0.012, Fig. We evaluated differences in survival among the different types of AE-IIPs and pneumonia superimposed on CF-IIPs. Although there was no significant difference in MST between idiopathic and all triggered AE-IIPs , MST was significantly longer in patients with pneumonia superimposed on CF-IIPs  than that in patients with all triggered or idiopathic AE-IIPs  and to evaluate differences in prognosis among the three groups. Our main findings are as follows: (i) survival time was significantly longer in patients with infection-triggered AE-IIPs than in patients with idiopathic or non-infection-triggered AE-IIPs, and moreover, the survival time was significantly longer in patients with pneumonia superimposed on CF-IIPs than in patients with AE-IIPs; (ii) mortality rate was significantly lower in infection-triggered AE-IIPs than in either other type; (iii) the incidence of infection-triggered AE-IIPs in winter was significantly higher than in any other season; (iv) variation in AE-IIPs was not significantly associated with HRCT patterns at AE-IIPs onset and previous treatment; and (v) clinical AE-IIPs types and HRCT patterns at AE-IIPs onset were significant independent risk factors for early death induced by AE-IIPs.Recently, several Japanese publications have reported differences in prognosis between idiopathic and triggered AE-IIPs , 22. HowIn terms of prognosis, MST for infection-triggered AE-IIPs was the longest, followed by idiopathic, and then non-infection-triggered AE-IIPs. Moreover, there was a significant difference in the most common cause of death between infection-triggered and non-infection-triggered/idiopathic AE-IIPs. All patients received antibiotics. Therefore, control of infection may be associated with attenuation of infection-related inflammation and injury, thereby improving the survival time in patients with infection-triggered AE-IIPs. In contrast, we suggest that for any triggers other than infection it is extremely difficult to inhibit or attenuate the lung injury induced by drugs, biopsy, surgery, or anesthesia because these triggers cause chemical or physical injury. Survival in patients with idiopathic AE-IIPs was intermediate between patients with infection-triggered and non-infection-triggered AE-IIPs.Moreover, it may be also difficult to accurately classify idiopathic and infection-triggered AE-IIPs. In this study, we classified patients with symptoms related to upper respiratory tract inflammation, as having infection-triggered AE-IIPs. Corticosteroid treatment is known to interfere with the development of a fever. When a patient receives corticosteroids, they may not develop fever and other symptom associated with influenza virus infection. This patient would be classified as having the idiopathic type. Thus, treatment of baseline CF-IIPs may increase the selection bias, and idiopathic AE-IIPs may include unknown triggers that are both infection and non-infection related. Although the frequency of high fever was significantly higher in bacterial pneumonia superimposed on CF-IIPs patients than that in AE-IIPs, there were several patients who received corticosteroids in all categories and almost all of these patients did not have high fever in all clinical AE-IIPs types. Therefore, the data may be indeterminate for evaluating whether a high fever is associated with AE-IIPs in patients who received corticosteroids.The evaluation of procalcitonin levels may be useful for the distinguishing among clinical AE-IIPs types, and between AE-IIPs and bacterial pneumonia. In this study, the levels of procalcitonin was significantly higher in patients with infection-triggered than in patients with other clinical AE-IIPs. Then, the levels of procalcitonin was significantly higher in patients with bacterial pneumonia superimposed on CF-IIPs than in patients with AE-IIPs, especially in infection-triggered AE-IIPs. Therefore, evaluation of procalcitonin levels may be reasonable to distinguish bacterial pneumonia and AE-IIPs and to classify among clinical AE-IIPs types. However, the patients could easily succumb to other bacterial after viral infection compared with healthy individuals; thus, only evaluating procalcitonin and/or CRP levels may not be useful for distinguishing among the clinical AE-IIPs types, and between AE-IIPs and bacterial pneumonia superimposed on CF-IIPs.Further, whereas there was significant difference in survival time among three groups, the three survival curves almost overlapped immediately after the development of AE-IIPs. Therefore, we were unable to evaluate the prognosis and severity during from the early phase to the development of AE-IIPs, for distinguishing the clinical AE-IIPs types. In contrast, no patients with bacterial pneumonia superimposed on CF-IIPs were admitted to only the intensive care unit (ICU). In addition, the survival curve evidently differed between AE-IIPs and bacterial pneumonia superimposed on CF-IIPs from the early phase of AE-IIPs. Thus, we believe that the difference in ICU admission, which reflects on the severity, may be associated with the difference in clinical course and pathogenesis between AE-IIPs and bacterial pneumonia superimposed on CF-IIPs.2 and a > 45 Torr in Japan. Following the development of chronic respiratory failure in CF-IIPs patients, the patient’s quality of life decreases, and the risk of comorbidities associated with CF-IIPs increases. These patients die due to respiratory failure. In this study, there were no patients who died of lung cancer after the development of AE-IIPs. The difference in the cause of death between this study and the recent study [The major causes of death in IPF have been shown to be AE-IIPs, development of lung cancer, and chronic respiratory failure, based on previous reports , 24. Resnt study , 24.The incidence of AE-IIPs in winter was only significantly higher in infection-triggered AE-IIPs compared with other types of AE-IIPs. In the INPULSIS trial, a phase 3 clinical trial of nintedanib for IPF, the incidence of AE-IIPs was also higher in winter than in other seasons . In thisThis study is subject to several limitations. First, this was a small retrospective study. Although there was a moderate number of patients with CF-IIPs at our hospital, the incidence of AE-IIPs was approximately 10–20% per year , 24. TheOur findings suggest that patients with infection-triggered AE-IIPs have a better prognosis than do patients with other types of AE-IIPs.


Antimicrobial resistance (AMR) represents a major global health threat. Without urgent action to reverse its course, AMR could reverse a century of medical progress, cause irreparable damage to the environment, push more people into extreme poverty, imperil global health security and jeopardize progress towards the Sustainable Development Goals and the attainment of universal health coverage.More alarmingly, new challenges are on the horizon. Recent evidence shows that AMR is emerging in conflict zones, where chronic and recurrent infections, coupled with scarce resources, can drive the emergence and spread of AMR, leading to a reservoir of resistance that will be difficult to eradicate. Studies also indicate similar trends in the emergence and spread of AMR linked to natural and man-made disasters, as well as large movements of populations and animals where organized, cross-boundary coordinated activities may be very difficult to plan.It is very clear that AMR cannot be controlled by the health sector alone. Drug resistance recognizes no borders, as it is transmitted to humans through the food chain and through environmental degradation. All government sectors, from health to trade to agriculture, must be engaged urgently.But managing and coordinating action across the health sector and beyond to include animal health, the environment and food production is complex and time-consuming. Unfortunately, many countries lack the human and financial resources to do this. Addressing the root causes of AMR requires an integrated ‘One Health’ approach.Economically, the impact of AMR is immense. A recent OECD report  estimatePreventing the extraordinary human, social, economic and environmental costs of AMR requires significant investments in research, as well as sustained technical, societal and political action at the highest levels of global leadership.I therefore welcome this special issue devoted to the prevention of AMR. Sharing best practices and evidence-informed approaches to AMR control is critical to turn back the tide of this global public health crisis.


IDH-wild-type GBM cohorts: an original Japanese cohort and a dataset from The Cancer Genome Atlas (TCGA). The molecular disproportions between these cohorts were dissected in light of cohort differences in GBM. The Japanese cohort was collected from cases registered in Kansai Molecular Diagnosis Network for CNS tumors (KNBTG). The somatic landscape around CNAs was analyzed for 212 KNBTG cases and 359 TCGA cases. Next, the clinical impacts of CNA profiles were investigated for 140 KNBTG cases and 152 TCGA cases treated by standard adjuvant therapy using temozolomide-based chemoradiation. The comparative profiling indicated unequal distribution of specific CNAs such as EGFR, CDKN2A, and PTEN among the two cohorts. Especially, the triple overlap CNAs in these loci (triple CNA) were much higher in frequency in TCGA (70.5%) than KNBTG (24.3%), and its prognostic impact was independently validated in both cohorts. The KNBTG cohort significantly showed better prognosis than the TCGA cohort . This survival difference between the two cohorts completely resolved after subclassifying all cases according to the triple CNA status. The prognostic significance of triple CNA was identified in IDH-wild-type GBM. Distribution difference in prognostic CNA profiles potentially could cause survival differences across cohorts in clinical studies.The diagnosis and prognostication of glioblastoma (GBM) remain to be solely dependent on histopathological findings and few molecular markers, despite the clinical heterogeneity in this entity. To address this issue, we investigated the prognostic impact of copy number alterations (CNAs) using two population-based The online version of this article (10.1186/s40478-019-0749-8) contains supplementary material, which is available to authorized users. The recent comprehensive molecular analysis of glioblastoma (GBM), including The Cancer Genome Atlas (TCGA) projects, has revealed the tumor genetic landscape and various functional relations between genes and pathways in tumorigenesis . SeveralIDH1/2 mutations [IDH-wild-type GBM came to be regarded as a more delineated entity: the most common and malignant astrocytic glioma [6-methylguanine-DNA-methyltransferase (MGMT) methylation status is also regarded as a prognostic/predictive marker [The most important molecular change in glioma is utations , 29, whiutations . In the c glioma . O6-methe marker , 26, whie marker .IDH1/2 mutation, a chromosomal aberration in GBM was initially detected by gain of EGFR [IDH-wild-type GBM [NFKBIA deletion [The CNA in GBM is another significant somatic alteration which often shows a distinctive landscape with synchronous genomic gains and/or losses. Long before discovering  of EGFR , which wtype GBM , and havtype GBM . Some CNtype GBM  and NFKBIDH1/2 and MGMT [IDH-wild-type GBM and even in the subsets with the same status of MGMT methylation. For achieving accurate prognostic stratification in GBM, we investigated molecular distribution of CNAs and its relationship to clinical outcome using two independent population-based cohorts of GBM including an original Japanese cohort and a large dataset from TCGA. When targeting two different population from east Asia and TCGA, another concern is the geographical diversity in clinical and molecular profiles of GBMs. For instance, the lower EGFR amplification rates of GBM patients from Asia were recently reported during a screening for two randomized GBM trials with depatux-m: INTELLANCE1 and INTELLANCE2 (10.1093/annonc/mdx366.002). Therefore, a comparative study between two cohorts should deserve special consideration to help in identifying the cohort disproportion in GBM practice.It remains a problem that the diagnosis and prognostication of GBM largely depend on histopathological findings and few molecular features in and MGMT , despiteEGFR, CDKN2A, and PTEN CNA status had a prognostic impact in GBM patients, and that the differences in the frequencies of these molecular profiles resulted in the different survival between the two cohorts.Here, we examined the survival impact of CNAs in Japanese and TCGA GBM cohorts. The differences of molecular profiles and clinical outcome were carefully investigated between these two cohorts. This investigation conclusively showed that a combination of IDH wild-type GBM and compared the somatic landscapes of the two cohorts with each other. In Step2, we investigated the clinical impact of CNA profiles in primary IDH wild-type GBM cases treated with chemoradiotherapy with TMZ after initial surgery using the Japanese and TCGA cohorts [The analyses of this retrospective study included two steps. The detail of this study design and flowchart of patient selection are provided below and are summarized in Fig.  cohorts . The JapJapanese cohort was collected from the cases registered in Kansai Molecular Diagnosis Network for CNS tumors (KNBTG): a consortium where neuro-oncologists, neurosurgeons, pediatricians, pathologists, and basic scientists are conducting cooperative researches for malignant brain tumor . The resIDH-wild-type GBM based on the 2016 CNS WHO, molecular data available for analysis, and absence of IDH1/2 and H3F3A mutation, and 1p/19q codeletion. Out of 234 primary GBM initially collected from seven institutions participating in this study, 212 cases were enrolled as Cohort K1.The initial screening criteria from enrolled gliomas in KNBTG was as follows: local diagnosis of primary GBM and availability of genomic DNA for molecular analysis of the naïve specimens. Primary GBM was clinically regarded as the GBM arising de novo, with no known lower-grade precursor lesion. The inclusion criteria for Cohort K1 was as follows: central review of a histopathological diagnosis of (n = 8) were excluded. Finally, 140 cases were available for Step2 analysis as Cohort K2.To analyze the prognostic impact of CNAs, cases in which patients underwent radiotherapy (RT) of 50–65 Gy and concurrent TMZ were further selected from Cohort K1. Those treated with either RT alone, TMZ alone, or short-course RT plus TMZ were excluded from any survival analysis. Cases ineligible for treatment criteria and lacking in KPS score were excluded. In addition, those participating in clinical studies for testing experimental treatment add-on to RT plus TMZ IDH-wild-type GBM eligible for this investigation had reached an agreement with an experienced neuropathologist (Y.K.) and was according to the 2016 WHO classification for central nervous system tumors.All molecular-pathological diagnosis of All tissue specimens were obtained at the time of the initial treatment before chemoradiation between December 2006 and November 2017. Tumor genomic DNA was extracted with NucleoSpin Tissue kit  or DNeasy Blood & Tissue Kit , according to the manufacturer’s protocol. The same batch of DNA samples was used for each technique as follows.IDH1/2, TERT promoter, H3F3A, HIST1H3B, and TP53 were determined using the Sanger technique. The details of Sanger sequencing have been previously reported [MGMT promoter was assessed using quantitative methylation specific PCR (qMSP) following the bisulfite modification of tumor genomic DNA. The details of the qMSP protocol for testing MGMT methylation are described in SI.Mutational status of PDGFRA, EGFR, CDKN2A, PTEN, TP53, CDK4, MDM2, and NFKBIA genes. P088 kit was designed to assess mainly 1p/19q codeletion, and we used this kit only for the IDH-mutated-GBM to exclude oligodendroglial tumors. Based on previous publications, the CNA category was classified by the following thresholds: homozygous deletion (x ≤ 0.4), hemizygous deletion (0.4 < x ≤ 0.7), gain (1.3 ≤ x < 2.0), amplification (x ≥ 2.0) [To assess CNAs in GBM, we performed Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA KIT P105 (version D2) and P088 (version C2), in accordance with the manufacturer’s protocol  . The P10x ≥ 2.0) . Any othn = 577) were collected from cBioPortal for Cancer Genomics  [n = 550), whites accounted for a much higher percentage  than blacks  and Asians .An independent and extensive molecular data and clinical information including survival data and treatment of GBM in TCGA  , 11 and tal.org) , 5. BaseIDH-wild-type GBM. Recurrent, secondary, or IDH-mutant-GBMs along with those that lacked any such information were differentially excluded from this step. For the purpose of survival analysis in Step1, only one case was additionally excluded due to a lack of censoring data.Out of 577 cases initially collected, 359 cases were enrolled as Cohort T1, who were conclusively diagnosed with primary From Cohort T1, 152 cases receiving TMZ chemoradiation as initial treatment were selected for external validation (Cohort T2) . Those iTP53 was also downloaded from the portal. Further molecular data about MGMT methylation and various clinical information were available from the supplemental data of the previous publication by TCGA [IDH mutational status could be obtained from both data sources and obtainable results were fully cross-validated with each other. There were few data regarding TERT promoter mutation in any of the data sources.All data regarding CNA in each patient from TCGA were downloaded from the online resource provided in cBioPortal for Cancer Genomics on July 15th, 2018. To identify CNA profiles, we employed putative copy number calls generated by the GISTIC or RAE algorithms in the portal, such as “-2”, “-1”, “0”, “1”, and “2” , 11. Thep-value was < 0.05.All statistical analyses were performed using a JMP Pro version 13 software . The difference was considered significant if the Patients were subdivided into two groups on the basis of age (≥ 65 or < 65 years), preoperative KPS (≤ 70 or 80–100%), and extent of resection (< 90% or 90–100%) for the purposes of statistical analysis.p <  0.05, Fisher’s exact), LOR below − 2.0 and above 2.0 were respectively defined as association toward mutual exclusivity and co-occurrence. LOR from − 2.0 to − 1.5 and 1.5 to 2.0 were defined to have a tendency for these associations.Pair associations of molecular variables were evaluated using a two-tailed Fisher’s exact test and computing the log odds ratio (LOR). Concurrent with significant correlation  was defined as the interval between the initial operative day and the date of either death or the last follow-up date on which the patient was known to be alive, with a cut-off date of 28 February 2018 for the KNBTG cohort. Patients who were still alive at the last follow-up were considered as a censored event. The survival data were analyzed using the log-rank test and Cox regression analyses. In Cox analysis, the hazard ratio (HR) and p-value were computed. Variables with a significant p-value in univariate analysis were subsequently used to build a multivariate Cox model. Stepwise procedure was used for constructing multivariate Cox regression model for survival. After excluding non-significant factor one by one in the multivariate analysis, the remaining variables (p <  0.05) were considered to be independent predictors of survival. Extent of resection and TERT promoter mutation, MGMT promoter methylation, and TP53 mutation were observed in 125 cases (59.0%), 98 cases (46.2%), and 80 cases (37.7%), respectively. The major CNA, frequently observed in Cohort K1, were CDKN2A deletion (62.3%), EGFR amp/gain (56.1%), and PTEN deletion (44.3%), as shown in Table TERT promoter mutation, EGFR gain/amp, and PTEN deletion with each other. TERT promoter mutation also had a tendency of mutual exclusivity with PDGFRA amplification . Association toward mutual exclusivity was observed in the pairs of PDGFRA amplification/ EGFR amplification, CDKN2A deletion/CDK4 amplification, and TP53 mutation/MDM2 amp/gain. MGMT methylation status was not significantly associated with any genetic alterations. These mutually exclusive or co-occurring sets are shown in Additional file p-values from the Fisher’s exact test are provided in Additional file The molecular characteristics of each cohort are shown in Table EGFR, CDKN2A, and PTEN, in Cohort K1 were lower than that in Cohort T1, with the difference in excess of 20%. The triple overlapping presence of CNAs in EGFR, CDKN2A, and PTEN (termed triple CNA) especially showed the apparent distinction between Cohort K1 (24.1%) and T1 (70.5%). The frequencies of TP53 mutation and deletion were higher in Cohort K1 (57.5%) than in T1 (38.6%). The distribution of MGMT methylation status between the two cohorts was roughly equivalent (46.2% versus 41.5%). The patient molecular status of each cohort is shown in Fig. The frequencies of genetic abnormalities, especially in p <  0.001, log-rank test)  than Cohort T1 (p = 0.014) . Similar to that in Step1, OS of Cohort T2  was significantly shorter than that of Cohort K2  . Median OS was significantly different between cases in Cohort K2 with triple CNA (15.0 months) and without triple CNA (21.8 months) . Kaplan–Meier estimates according to triple CNA is shown in Fig. MGMT, and probable CNA profiles). After excluding non-significant factors by a stepwise procedure, triple CNA was found to be an independent prognostic factor along with age, extent of resection, NFKBIA, and MGMT in Cohort K2 . Kaplan–Meier estimates according to triple CNA is shown in Fig. MGMT, NFKBIA, and triple CNA), MGMT methylation and triple CNA remained as independent prognostic factors   Fig. d.n = 292) were subdivided into subgroups with or without the common prognostic markers in univariate analysis  to apply their adjustment, and Kaplan-Meier curve comparison for cohorts with log-rank test was shown in Fig. MGMT methylation status, generation, and NFKBIA status, survival differences remained significant in at least one of the subgroups representing presence or absence of these factors.  proved to be approximately three-fold higher in frequency in TCGA (70.5%) than KNBTG (24.1%). The prognostic impact of this finding was validated in both cohorts, which consisted entirely of GBM patients homogenously treated by standard chemoradiation. Although KNBTG cohort significantly showed the better prognosis than TCGA, its survival difference completely resolved after subclassifying all cases by triple CNA status, in contrast to other common prognostic molecular markers. Consequently, the discrepancies in the distribution of triple CNA in two cohorts could provide a full account of the cohort disparities of survival duration.In this study on NFKBIA deletion on survival was observed in both KNBTG and TCGA cohorts in the present study, and this result was in line with a previous report [EGFR, CDKN2A, and PTEN affected patients’ clinical outcome insufficiently by itself, these markers proved to commonly show a prognostic impact when combined. In another attempt to provide prognostic assessment based on CNA molecular subtype, the classification defined by chromosome 1 gain, 19 gain, and CDK4/MDM2 co-amplification was recently proposed through an analysis on the dataset from TCGA and German Glioma Network [IDH-wild-type GBM; however, their findings supported the impact of CNAs on survival. These clinical impacts of CNA profiles immediately raise a question of whether any molecular marker could make “authentic” GBMs further distinguishable from preexisting entities diagnosed by histopathological findings and IDH1/2 status. Our findings support the need to introduce molecular markers in the diagnostic criteria of GBM and re-evaluate the definition of GBMs.The prognostic impact of CNAs is still controversial; however, some of them have the potential to be related with clinical outcomes in GBM. The impact of s report . Even thEGFR mutations in pulmonary adenocarcinoma in Asia. Despite few data concerning GBM, the low prevalence of TP53 mutations in whites and homozygous deletion of CDKN2 gene in Japanese were reported [EGFR amplification in GBM. These findings are roughly compatible with our results in that the most striking disproportion of genetic aberrations is mainly in the loci of EGFR, CDKN2A, PTEN, and TP53. Our study may provide an additional evidence for the interregional molecular disproportion of GBM. Further study is needed to elucidate regional variances and its clinical value.The underlying reason for the unequal distribution of CNAs between Japan and TCGA cohorts is poorly understood; however, there are two presumptive causes. The first hypothesis is the interobserver variances in the histopathological diagnosis of GBMs. In practice, the diagnosis of GBM in our cohort was solely based on the universal guidelines in WHO classification. However, even the universal criteria may pose some limitations, and, hence, molecular marker seems to be of practical value in GBM diagnostics. The second hypothesis is the geographical variances in the frequencies of CNAs. The regional difference has been widely recognized in other malignant tumors such as lung adenocarcinoma , 30, brereported , 21, aloThis is an illustrative cohort study showing that different clinical outcomes across cohorts could be attributed to the uneven distributions of prognostic genetic backgrounds. We formed two independent cohorts consisting of population subjected to the same treatment, and successfully validated the common prognostic value of triple CNA. Notably, the survival discrepancy between the cohorts resolved after subclassifying these two cohorts by triple CNA. In population-based research, treatment options are submitted to physicians in local institutions and depend on the socioeconomical status of patients. Therefore, population-based cohort is reflective of the general population, but is likely to be a heterogeneously treated population, which makes it difficult to elucidate the clinical value of molecular markers. Our results indicate that some underlying selection biases remain even after adjusting the treatment backgrounds, and that one of those biases is the differences in prognostic CNA profiles. Along with our results, Cimino et al. showed an inherent bias that enrolled patients for the prospective cohort in clinical trial are liable to bear favorable molecular markers for survival, using molecular profiles by their original CNA subclassification : the proEGFR amplification and 7p gain is hard to be distinguished. Nonetheless, the putative copy number calls downloaded from cBioPortal was virtually equivalent to that generated by MLPA because focal gene copy number was equally estimated rather than the entire chromosomal aberrations. Our analyses incorporated hemizygous and homozygous deletion of CDKN2A as one of the molecular markers. The biological significance of hemizygous deletion is not relevant in the molecular pathway while CDKN2A homozygous deletion is regarded as one of the representative causes for disruptions of Rb1 pathway. When incorporating only homozygous deletion of CDKN2A for the definition of triple CNA, the prognostic impact of this combination is retained in the Japan cohort but not in the TCGA cohort (data not shown). Further investigation using homogeneous molecular techniques in independent larger cohorts is needed to validate our findings and select the optimal combination of CNAs. However, we believe that this study adequately addressed the clinical importance and geographical distribution of CNAs in GBMs.This study harbors the limitations as a retrospective analysis susceptible to selection biases; the initial inclusion criteria for molecular data availability probably reflected one of the selection biases. Instead, the cohorts in this study could be regarded as reflective of the general population with homogeneous treatment background. The other limitation is the different molecular biological techniques utilized in the two cohorts. We evaluated CNAs by MLPA, which is a reliable and cost-effective method, but lower throughput than the comprehensive high-throughput array of the TCGA cohort. The resultant CNA profiling in our cohort seems dependable, because interactive CNA relationships such as co-occurrence and mutual exclusivity showed mostly equivalent outcomes to previous reports  of genetic alterations showing co-occurrence or mutual exclusivity in KNBTG. Figure S2. Kaplan-Meier analysis of overall survival in Step1 between KNBTG and TCGA. (PPTX 281 kb)Additional file 2:Table S1. Dataset of patients with glioblastoma from Kansai Molecular Diagnosis Network for CNS tumors (n = 234). Table S2. Dataset of patients with glioblastoma from The Cancer Genome Atlas (n = 577). Table S3. Log odds ratio in pair association analysis for genetic aberrations. Table S4. P-value from two-tailed Fisher’s exact test in pair association analysis for genetic aberrations. Table S5. Univariate analysis for each molecular variable using Cox proportional hazards models in Step2. Table S6. Sequences of primers used for amplification and Sanger sequencing. Table S7. Sequences of primers used for quantitative methylation specific PCR. (ZIP 256 kb)


Ceratopteris thalictroides, for the detection of WGDs. High-quality RNA-seq data was used to infer the number of synonymous substitutions per synonymous site (Ks) between paralogs; Ks age distribution and absolute dating approach were used to determine the age of WGD events. Evidence of an ancient WGD event with a Ks peak value of approximately 1.2 was obtained for both samples; however, the Ks frequency distributions varied significantly. Importantly, we dated the WGD event at 51–53 mya, which coincides with the Paleocene-Eocene Thermal Maximum (PETM), when the Earth became warmer and wetter than any other period during the Cenozoic. Duplicate genes were preferentially retained for specific functions, such as environment response, further support that the duplicates may have promoted quick adaption to environmental changes and potentially resulted in evolutionary success, especially for pantropical species, such as C. thalictroides, which exhibits higher temperature tolerance.Whole-genome duplications (WGDs) are widespread in plants and frequently coincide with global climatic change events, such as the Cretaceous–Tertiary (KT) extinction event approximately 65 million years ago (mya). Ferns have larger genomes and higher chromosome numbers than seed plants, which likely resulted from multiple rounds of polyploidy. Here, we use diploid and triploid material from a model fern species,  Arabidopsis thaliana has undergone two recent WGDs and one whole genome triplication during the course of its evolution [Whole-genome duplication (WGD), or polyploidy, has long been considered as an important evolutionary force and often drives plant speciation ,2,3,4,5.volution ,7. Two Wvolution ,9.MADS-box family genes were retained after WGDs, especially MIKC-type genes [Massive WGDs in flowering plants have occurred at specific times during extreme environmental changes or extinction periods, including the Cretaceous–Tertiary (KT) boundary, and Paleocene-Eocene Thermal Maximum (PETM) ,10,11. Wpe genes ,16. Biaspe genes . Howevern = 57.0) greatly exceeds that in angiosperms (n = 15.99) and heterosporous ferns (n = 13.62) [Ks-based age distribution: one Azolla-specific and one at the base of core leptosporangiates in Azolla filiculoides and Salvinia cucullata [Equisetum giganteum and Ceratopteris richardii [Ferns (Monilophyta) constitute the second largest group of vascular plants and an early diverged lineage of land plants; they possess a higher frequency of polyploidy than seed plants. Previous studies have reported that the frequency of polyploidy in ferns varies from 31%–95%, while that in flowering plants varies by 15%–70% ,18. With= 13.62) . Moreove= 13.62) ,22. Li eucullata . WGDs weichardii . HoweverKs) between paralogs, which has been widely performed in lots of species [Ks age distribution; this data can be used to detect whether paleopolyploidizations have occurred in plants [Ks peaks), determine the time at which WGDs occurred, and identify whether gene retention is biased towards specific functional classes.Recent improvement in technology, such as transcriptome sequencing, has allowed for a relatively convenient and efficient alternative to evaluate paleopolyploid events because a large number of sequences can be obtained at a low cost . To dete species ,9,12. Win plants ,9. Most n plants . Howevern plants ,24. A WGCeratopteris is a pantropical genus of annual ferns. It is commonly used as a model for ferns because it is easy to culture and produces abundant spores. It also has an independent sporophyte, gametophyte generation, and short sexual cycle [C. thalictroides in previous published studies [Ks analysis. Additionally, we dated the WGD event using both Ks distribution peaks and the absolute dating approach. Furthermore, annotation of the biased gene retention following the WGD events was also analyzed. Our results will help improve the understanding of the genome history and evolutionary impact of WGD events in ferns.al cycle ,27. Here studies ,29, perfC. thalictroides sample reported by Shen et al. (2018) was identified as a diploid with a chromosome number of 2n = 78  is 2n = 117 . A total of 8364 paralogous pairs were identified in the diploid C. thalictroides, whereas 3088 paralogous pairs were identified in the triploid sample. We used Gaussian mixture models to confirm the WGD signature. We observed seven and five peaks in the diploid and the triploid, respectively. A significant Ks peak near 1.2 was detected by the fitted Gaussian mixture models for both samples, and several other smaller peaks were also fitted. We only focused on Ks values less than 2 because higher Ks values are uncertain due to Ks saturation and stochastic effects, which might provide misleading data for the mixture models. Thus, one ancient WGD event with a median Ks peak at approximately 1.2 occurred in the diploid and triploid C. thalictroides samples, indicating that the large peak present in C. thalictroides was a true WGD rather than stochastic variation.In this study, the distributions of D events . The difKs peak at approximately 1.2. The results showed that several GO terms were preferentially retained. A total of 111 GO terms were found to be significantly enriched (A gene ontology (GO) enrichment analysis was performed to explore the potential functions of retained duplicates following WGDs in the diploid with a higher quality for this study. The focus of the analysis was on the WGD event shared by the diploid and the triploid, with a enriched . The folenriched ; GO termKs distribution peaks and the absolute dating approach. Without changes of protein sequences, synonymous substitutions are considered to be putatively neutral and accumulate changes at a constant rate, which can be used to infer the age of WGDs. When a plant average Ks/year rate of 6.1 × 10−9 was used, the age of the WGD event with a peak value of approximately 1.2 was dated at 93–95 mya (Ks = 1.13–1.16) in the diploid and 96–101 mya (Ks = 1.17–1.23) in the triploid, with a confidence interval of 95%.We determined the age of the WGD events using both Ks distribution peaks method can introduce inaccuracies because it relies on the assumption of a strict molecular clock and synonymous substitution rates, and synonymous substitution rates are usually variable across different species. To improve the accuracy of our results, we further performed absolute dating of the diploid via phylogenomic analysis to infer the age of the WGDs. Using this method, we obtained an absolute age distribution with a clear peak at 52 ± 1 mya with a confidence interval of 95%  via genetic linkage mapping indicate that no ancient WGD events were detected in ichardii , or signte pairs ,33. In oectively , thus hiectively .Ks-based age distribution had an obvious peak near 1.2 in the diploid and the triploid samples of C. thalictroides  and 2.268 (triploid) in Ks frequency distribution of the diploid significantly differed from that of the triploid, the ancient WGD event detected is strongly supported, suggesting that ancient occurrences of polyploidy had a larger impact on the Ks age distribution than on recently formed polyploids, which can be disregarded.The ctroides , indicatr than 2 , althougr than 2 . The difC. thalictroides has indeed experienced an ancient WGD, as indicated by the obtained Ks-based age distributions. This event, which is prevalent but not limited to angiosperms, occurred more than once, and the recent polyploidy event has influenced Ks frequency distribution, but its weak interference on the detection of WGDs can be ignored.Our results reveal that Ks distribution peaks and the absolute dating approach to infer the dates of WGD events of C. thalictroides. Our Ks distribution peak analyses dated the ancient WGD event at 93–101 mya based on the assumption that C. thalictroides experienced a similar evolutionary rate with angiosperms. However, our results may not be completely accurate because absolute ages converted by Ks value are more dependent on the assumption of a strict molecular clock and the synonymous substitution rates used. Synonymous substitution rates are variable among different plants, and the rates are unknown for fern species [Ks values and absolute ages of E. giganteum can be converted to synonymous substitution rates, the date estimation in E. giganteum is largely uncertain [There are several different methods that can be used to estimate the age of a WGD event. In this study, we used both  species . AlthougKs analysis, we further dated the WGD event using an absolute age distribution, which is a combination of the Ks-based relative age distribution and phylogenetic analysis. This method uses a relaxed clock model that assumes a lognormal distribution of evolutionary rates, is based on several fossil calibrations from a broad taxonomic sampling, and the age estimates for all actual duplicates present an approximate lognormal distribution, which is more likely to obtain accurate estimates than Ks distribution peaks. Absolute age distribution analyses have been widely used in many studies [C. thalictroides shared similar evolutionary rates; the small confidence interval represents a small chance of error. Therefore, we trust the results from the absolute age distribution analysis and placed the WGD event at approximately 52 mya, which is much closer to the PETM than the KT boundary [E. giganteum, C. richardii (180 mya), Azolla, and an earlier WGD shared by core leptosporangiates [E. giganteum was estimated to be 75.16–112.53 mya , and the WGD event placed at 50–70 mya using a Ks distribution based on the assumption that E. giganteum shares similar evolutionary rates with angiosperms [Azolla and core leptosporangiates, no information was provided for the age of the WGD [−9 for the combination of Ks and absolute ages of C. thalictroides, which is higher than the average rate for plants (6.1 × 10−9) and cereals (6.5 × 10−9) and slightly lower than the range for Drosophila (15.6 × 10−9) and dicots (15 × 10−9) [C. thalictroides, which is an annual and has a short generation time [Therefore, to either confirm or refute the dating we obtained for the WGD event of the diploid based upon our  studies ,10,36. Fboundary . In prevangiates ,23,24. Aiosperms . In the  × 10−9) . The rapion time ,37.E. giganteum, and p. patens, are indicated to cluster around the KT boundary (60–70 mya), which led to the extinction of approximately 60% of the plant species on Earth [C. thalictroides was inferred at 51–53 mya, which is closer to the PETM than the KT boundary. As is common in other tropical species, C. thalictroides may be more susceptible to increases in temperature, which facilitates the occurrence of WGD during the PETM [2 concentration. Viewed as a representative of extreme cases, PETM, also known as the Late Paleocene Thermal Maximum (55 mya), reached exceptionally warmer and wetter climates than any other period on Earth [Polyploidy is usually considered to be an evolutionary dead end. However, WGDs might occur at specific times, such as during dramatic climate changes, providing novel opportunities for evolutionary success . The occon Earth ,10,36. Hthe PETM ,30,38,39on Earth . During Ceratopteris displays greater heat tolerance and intolerance to cooler temperatures than the temperate taxon [C. thalictroides, we discovered that genes were retained after WGDs and these were not random but preferentially retained. Genes preferentially retained after the WGD related to its tolerance to contribute to its adaption to extreme environments [C. thalictroides [C. thalictroides  (access on 1 August 2018) [In total, transcriptome data from nine species was obtained. For the diploid sample of , GigaDB . In the 00000000 , and thest 2018) .C. thalictroides unigenes were blasted against a core set of conservative orthologs in plant species from the OrthoDB database (www.orthodb.org) (access on 10 August 2018), and the number of complete and partially matched genes were recorded.BUSCO analysis was employed for the evaluation of the completeness level of the transcriptome assembly . Both seC. thalictroides, the Ks-based age distribution was performed as described previously [−5. A best match was considered significant if the alignment length was >100 amino acids and the expected value (E) was <1 × 10−15. Then, all pairs of sequences within each gene family were aligned using MUSCLE  with default parameters [Ks estimates were generated through maximum likelihood estimation with the CODEML program of PAML version 4.8  [Ks values, all Ks estimates for a particular duplication event were added to the Ks distribution, while the total weight of a single duplication event sums up to one [http://github.com/EndymionCooper/KSPlotting). Ks values from 0.1 to 5 were retained for subsequent analyses, and Gaussian mixture models were performed in the R package mclust  for the fitting of log-transformed Ks distributions [Ks/(2 × r) if the number of silent substitutions increased approximately linearly with time. The plant average Ks/year rate of 6.1 × 10−9 was applied to the WGDs of C. thalictroides [Ks frequency distributions of the diploids were compared with the triploid through the Wilcoxon matched pairs test [To examine candidate WGD events in eviously . An all-rameters . Ks estidon, UK) . A gene p to one . The appibutions ,57. The ctroides . Additioirs test .Ks values between 0.9 and 1.5 in the Ks distribution were collected. This resulted in 737 gene families. For each gene family, we retained the duplicates pair nearest the WGD peak boundaries as the representative homeologous pair, as multiple paralogous pairs might descend from the same gene duplication [C. thalictroides and seven other ferns, with an E-value of 1 × 10−10 [G. niponicum, W. prolifera, D. pilosella, C. chusana, A._aureum, O. odorata, and A. podophylla. A total of 706 orthogroups were collected. Maximum-likelihood trees were constructed using RAXML  under the GTRGAMMA model with 100 bootstrap replicates, and the paralogous pair of C. thalictroides that grouped together was used for further analyses. After removal of the orthogroups with gene trees in conflict with the species tree according to the Pteridophyte Phylogeny Group I system [C. chusana, as the Pteroids, was dated to 93.5 mya, and W. prolifera, as Woodwardia (family Blechnaceae), was dated to 55.8 mya [Absolute dating for the WGD was performed to determine the age of the event, similar to the analysis described by Vanneste et al. (2014) . A diagrlication . The ort × 10−10 . Based oI system , a totalI system . Fossil 55.8 mya ,63,64. T55.8 mya . A total55.8 mya .C. thalictroides occurs after a WGD and to understand whether the WGDs might be linked with extreme environmental changes. We obtained duplicate pairs with Ks values between 0.9 and 1.5, which lie under the WGD signature peak in the age distributions. GO enrichment analyses were performed using agriGO v2.0  with the singular enrichment analysis tool at default settings for the Fisher test  [As reported previously, genes are preferentially retained after WGDs ,66. We pficance) .C. thalictroides, BLASTP was performed to search the C. thalictroides protein transcript database with the Arabidopsis MADS proteins as queries. An E-value cutoff of 1 × 10−5 was used. Additionally, proteins with SRF-TF domains (PF00319) were obtained from the Pfam database, and the hidden Markov model (HMM) was also performed to identify new members of the MADS-box gene family. All candidate MADS-box genes were examined by using the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de/) and conserved domain databases to verify and remove incomplete MADS-box domains. We also removed redundant sequences with an identity higher than 99%.To identify putative MADS-box family genes in C. thalictroides and others, all putative MADS-box family genes in C. thalictroides and Arabidopsis were aligned using ClustalX , and manually adjusted. A Bayesian phylogenetic tree was constructed using the program MrBayes (v3.2.6) with the following settings: mixed model, 10,000 generations, and a sampling frequency of ten. The run was stopped when the standard deviation of the split frequencies was below 0.01 through adding generations and sampling frequency continually. A total of ten million generations were run and tree sampling density was 10,000 generations [Arabidopsis, Oryza sativa, Physcomitrella patens, Selaginellamoellendorffii, and C. thalictroides (Ceratopteris. CgMADS1 from the streptophyte green alga, Chara globularis, was used as the outgroup.To estimate the evolutionary relationship of MADS-box family genes retained in erations . The firerations . In addictroides , includi