Although the use of ECCO2R for acute exacerbations of COPD is steadily increasing, solid evidence on its efficacy and safety is scarce, thus the need for a randomized controlled trial.Hypercapnic exacerbations are severe complications of chronic obstructive pulmonary disease (COPD), characterized by negative impact on prognosis, quality of life and healthcare costs. The present standard of care for acute exacerbations of COPD is non-invasive ventilation; when it fails, the use of invasive mechanical ventilation is inevitable, but is associated with extremely poor prognosis. Extracorporeal circuits designed to remove COmulticenter randomized controlled unblinded clinical trial including 284 (142 per arm) patients with acute hypercapnic respiratory failure caused by exacerbation of COPD, requiring respiratory support with NIV. The primary outcome is event free survival at 28 days, a composite outcome defined by survival in absence of prolonged mechanical ventilation, severe hypoxemia, septic shock and second episode of COPD exacerbation. Secondary outcomes are incidence of endotracheal intubation and tracheostomy, intensive care and hospital length-of-stay and 90-day mortality.2R justifies a randomized trial to evaluate whether patients with acute hypercapnic acidosis not responsive to NIV should undergo invasive mechanical ventilation  or be treated with ECCO2R to avoid invasive ventilation but be exposed to possible adverse events of ECCO2R. Owing to its pragmatic nature, sample size and composite primary outcome, this trial aims at providing valuable answers to relevant questions for clinical treatment of acute exacerbations of COPD.Acute exacerbations of COPD represent a significant burden in terms of prognosis, quality of life and healthcare costs. Lack definite evidence despite increasing use of ECCOClinicalTrials.gov, NCT04582799. Registered 12 October 2020, .The online version contains supplementary material available at 10.1186/s13063-021-05692-w. Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, resulting in a social and economic burden that is substantial and increasing , 2. Hype2 retention and increased work of breathing . However, ECCO2R-related complications were observed in almost half of the patients. A recent systematic review [2R in patients with hypercapnic respiratory failure across 12 studies and showed that the majority of patients were either successfully weaned from mechanical ventilation or sustained on NIV, avoiding intubation. However, this high success rate was associated with a high frequency of potentially severe complications [A recent matched cohort study  with hisc review  evaluate2R could increase event-free survival as compared to standard of care. Event free survival is defined as survival at day 28 free of septic shock, second episode of COPD exacerbation, occurrence of severe hypoxemia, and prolonged mechanical ventilation.Accordingly, the main objective of this randomized multicenter clinical trial is to test the hypothesis that in patients with acute life-threatening exacerbation of COPD, use of ECCOThis trial is a multicenter, prospective, randomized, controlled, unblinded clinical trial. The framework of the trial is superiority. The trial will include 284 patients with acute hypercapnic respiratory failure caused by exacerbation of COPD.Patients older than 18 and younger than 90 yearsDocumented clinical history of COPDICU admission for exacerbation of COPD requiring NIV supportarterial pH ≤ 7.25respiratory rate ≥30 breaths/minuse of accessory muscles or paradoxical abdominal movementsAfter two hours of NIV at least two of the following criteria for high risk of NIV-failure , 22 mustmean arterial pressure <60 mmHg despite infusion of fluids and vasoactive drugs2 fraction (PaO2/FiO2) ≤ 150 with FiO2 of less than 0.6 and PEEP of at least 5 cm H2Oratio of arterial-to-inspired oxygen O3; international normalized ratio (INR) >1.5; stroke or severe head trauma or intracranial arterio-venous malformation, or cerebral aneurysm, or central nervous system mass lesion within the previous 3 months; epidural catheter in place or expected to be positioned during the study; history of congenital bleeding diathesis; gastrointestinal bleeding within the 6 weeks prior to study entry; esophageal varices, chronic jaundice, cirrhosis, or chronic ascites; trauma);contraindications to anticoagulation  or fulminant hepatic failurediagnosis of acute or chronic neuromuscular diseasechronic mechanical ventilation prior to hospital admissionmoribund patientinclusion in other interventional trialsProlonged mechanical ventilation is defined as mechanical ventilation (invasive and non-invasive) applied for ≥ 14 days uninterrupted for more than 48 hours.Septic shock is defined by the presence of (a) sepsis  score of 2 points or more); (b) need for vasopressors to maintain a mean arterial pressure of 65 mmHg or greater; (c) serum lactate levels greater than 2 mmol/l (>18mg/dl) in the absence of hypovolemia.Severe hypoxemia is defined as occurrence of a ratio of arterial-to-inspired oxygen O2 fraction (PaO2/FiO2) ≤ 150 with FiO2 of less than 0.6 and PEEP of at least 5 cm H2O for at least 12 hours.Second episode of COPD exacerbation is defined as occurrence of sustained (≥24 h) increase in cough, sputum production, dyspnea and arterial pH≤7.30 .28-day all-cause mortality is defined at 672 hours from randomization. All patients will be classified as either “alive at Study Day 28” or, if dead, “dead at Study Day 28.” For example, day zero is the day of randomization and day 1 is the next day and encompasses all events that occur midnight-to-midnight, etc.The primary outcome is event free survival, defined as survival at day 28 free of any of the following: (a) prolonged mechanical ventilation; (b) development of septic shock; (c) occurrence of severe hypoxemia; (d) occurrence of a second episode of COPD exacerbation ; (e) 28-day all cause death. By analyzing the relevant epidemiological literature on COPD exacerbations –33 and oCumulative incidence of endotracheal intubationCumulative incidence of tracheostomyHospital length-of-stayICU length-of-stay90-day mortalityIncidence of adverse events as defined in the ‘Safety’ paragraph (see below)Secondary outcomes are the following:Informed consent will be taken by trained investigators. Before enrollment every patient must sign a written consent form for trial participation and for personal data processing. Anytime the patient is incapacitated and/or unable to fully understand the information provided, consent may be delayed .Central randomization by blocks stratified only by center. The randomization is centralized: a random sequence stratified by center will be generated in order to guarantee a balancing of the arms within the center; the sequence will be implemented following a block algorithm. The random sequence will be generated by a server run by the coordinating center. Participant sites will obtain the allocation of each enrolled patient upon enrolment, after checking for inclusion/exclusion criteria.For eligible patients, study data will be collected after enrollment Fig. . Study d2O and positive end-expiratory pressure (PEEP) of 5 cmH2O is recommended. IPAP may be then gradually increased by 2-5 cmH2O increments at a rate of approximately 5 cmH2O each 10 minutes until arterial pH>7.30 and patient tolerability has been reached. Inspiratory O2 fraction should be adjusted to maintain an O2 saturation of 88-92%.NIV should be applied after a maximum of 2 hours from ICU admission using ICU ventilators with NIV-dedicated software. Use of full-face mask is recommended. Initial inspiratory positive airway pressure (IPAP) of 10 cmHrespiratory rate >35 breaths/ min; arterial pH<7.25;2 greater than 60 mmHg;PaCO2 less than 60 mm Hg with an FiO2 greater than 60%;PaOrespiratory arrest or signs of patient distress with accessory muscle recruitment and paradoxical abdominal or thoracic motion.Escalation to invasive mechanical ventilation should be considered by the attending physician (not involved in the study) when two of the following occur for at least 2 hours (‘urgency criteria’):hemodynamic instability ;need for sedation for major agitation  ;acute neurological dysfunction (Kelly-Matthay score > 3) ;cardiac arrest.In addition, intubation will be immediately performed when any of the following is observed (‘emergency criteria’):and at least two of the following parameters are met:minute ventilation <15 l/min;2/FiO2 ratio > 150 to 200; with PEEP ≤ 5 cmH2OPaO2 ≤ 0.4;FiOpH > 7.30rapid shallow breathing index < 105 Patients should undergo a formal assessment of discontinuation from mechanical ventilation (invasive or non-invasive) and daily spontaneous breathing trials (SBT) will be performed to identify patients able to wean from mechanical ventilation once the cause of the exacerbation is adequately treated, the patient is hemodynamically stable (see above criteria for hemodynamic instability) and there are no signs of change in mental status , onset or worsening of discomfort, diaphoresis, signs of increased work of breathing  2≥90% and/or PaO2≥60 mmHg (PaO2 to take precedence if both available)SpORespiratory rate ≤30 bpmpH ≥ 7.35Heart rate ≥ 120% of the 6:00 a.m. rate for ≥5 minutesSignificant use of accessory musclesAbdominal paradoxDiaphoresisMarked dyspnea as subjectively assessed by the investigatorNo respiratory distress (defined as 2 or more of the following):Pressure support ventilation (PSV) will be used to gradually decrease mechanical ventilatory support. Patient will be defined as “weaned from mechanical ventilation” (invasive or non-invasive) if the following 4 criteria are met for the last 30 minutes during a spontaneous breathing trial (SBT):NIV is applied and managed with the same modalities and at the same conditions as in Group A (standard of care).2R device will be used . Blood flow is driven by a roller nonocclusive pump (0–450 mL/min) through a polymethylpentene artificial lung  that is connected to a fresh gas flow source delivering 100% oxygen at a constant rate of 8 L/min. A starting dose of heparin (80 IU/kg bolus followed by ∼18 IU/kg/ hour infusion) will be delivered by using a syringe pump included in the system. Continuous infusion of heparin is hence titrated to maintain values of activated partial thromboplastin time (aPTT) in the range 35-80 sec or aPTT ratio of 1.5-2.0; aPTT is checked approximately every 2-3 hours during the first 6 hours from therapy initiation; afterwards, aPTT is checked every 6 hours. ECCO2R will be continued for at least 24 hours.After setting NIV, percutaneous vascular access is provided using a double lumen catheter inserted in the internal jugular vein or in the femoral vein . An ECCOIntubation criteria are the same as in Group A (standard of care).2R must always precede weaning from mechanical ventilation. ECCO2R will be interrupted while keeping mechanical ventilation (invasive or non-invasive) when all of the following are achieved for at least 12 hours:respiratory rate<25 breaths/min;pH>7.35;2<20% of the baseline value;arterial PCOabsence of use of the accessory muscles or paradoxical abdominal movements.Weaning from ECCOrun formal assessment of discontinuation from mechanical ventilation (invasive or non-invasive) through a daily spontaneous breathing trial (SBT); (b) to be defined as “weaned from mechanical ventilation” (invasive or non-invasive) are the same as in Group A (standard of care).Criteria to (a) 2R is stopped and device (and catheter) removed in the following situations:Air embolus in the extracorporeal circuitCircuit clotting leading to an abrupt stop of extracorporeal blood flowDevice malfunction leading to abrupt stop of extracorporeal blood flow3)Severe thrombocytopenia  lasting longer than 6 hours are to be considered ‘treatment failure’ and lead to termination of the experimental protocol.Interruptions of ECCOAll concomitant therapies foreseen by the standard of care of enrolled patients are allowed.Post-trial care will be delivered according to the standard of care for AECOPD patients.ORany event that is fatal or immediately life threatening, permanently disabling, severely incapacitating or requires prolonged hospitalization ANDany event that may jeopardize the patient and requires medical or surgical intervention to prevent one of the outcomes listed above which the attending physician perceives may be directly related to enrollment in the clinical trial.Safety will be assessed by measuring the frequency of serious adverse events (SAE). A serious adverse event (SAE) in the trial is defined as:2R. SAE will be considered to be study-related if the event follows a reasonable sequence from a study procedure and could be produced by the study procedure. SAE will be considered not study related if they are thought to be primarily related to the underlying disease and its sequelae.Investigators will report severe adverse events to the clinical coordinator. An independent Data and Safety Monitoring Board (DSMB) will receive a detailed written report and evaluate whether the SAE is attributable to ECCO2R related adverse events (ECCO2R-AE) and classified as:mechanicaldifficulties upon catheter insertion (i.e. need for >2 attempts at catheter insertion during primary placement procedure);pump malfunction ;2R and/or circuit replacement);membrane lung clotting ;bleeding ;hemodynamic instability (see criteria in the ‘Treatment of participants’ paragraph);ischemic/gangrenous bowel;pneumothorax;2R);renal complications ;infectious complications ;thromboembolic complications ;thrombocytopenia ;2R).neurologic complications  achieves 90% power at a 0.05 significance level to detect a hazard ratio of 0.67 when the proportion surviving without events in the control group is 40% . TheThis study has been designed to demonstrate a relative increase of 35% in the event free survival (from 40% to 54%), assuming the above-mentioned parameters.2R . Frequent investigators’ meeting will be planned in order to motivate enrollment. The coordinating center will provide logistic support to all participating sites in order to ensure efficient enrolment.The study will be conducted at up to twenty sites within Italy. Three site visits for each center will be performed . Additional site visits will be performed as required. The coordinating center  will supervise all aspects of the study and will be available to participant sites 24 hours, 7 days. All sites must have significant experience in treating AECOPD and with ECCOMore specific data about sites can be obtained from the corresponding author.The study will be conducted in accordance with the current approved protocol. Independent study monitors will verify that the clinical trial is conducted, and data are generated, documented and reported according to the protocol and the current national and international regulations. Data quality will be assessed by the independent study monitors and frequent site visits (at least 3 visit/site/year) will be performed at each site, in order to ensure consistency throughout the duration of the trial.All data will remain property of the researchers and direct access to data will be granted only to authorized representatives of the regulatory authorities for inspective purposes.Trial results will be communicated to relevant groups  via publication into peer-reviewed journal(s).Statistical analysis will be conducted according to the intention to treat principles (ITT), analyzing the whole sample size of all randomized patients.Secondary analyses will be performed according to protocol adherence on “as treated” populations, with explorative and/or explicative purposes only.No interim analyses will be performed. No predefined subgroup analysis will be performed.The baseline characteristics of the patients will be described by group, using the most appropriate statistics: quantitative variables will typically be summarized using frequencies and percentages for appropriate categorizations and may also be summarized using descriptive statistics. For variables summarized with descriptive statistics, the following will be presented: N, mean, standard deviation, median, q1, and q3. Categorical variables will be presented using frequencies and percentages.No statistical comparisons between groups will be done on baseline variables.EFS will be estimated using the Kaplan-Meier estimator applied to the ITT population, and compared between those who received NIV plus ECCO2R and those who did receive only NIV using the stratified log-rank test.In addition, the Hazard Ratio (HR), along with 95% confidence intervals, will be estimated from a stratified Cox model with treatment group as a covariate.All components of the primary composite endpoint (singleton) will be examinated separately as independent proportion. The cumulative incidence of sepsis, hypercapnic respiratory failure, hypoxemic respiratory failure, prolonged mechanical ventilation >28days, tracheostomy and endotracheal intubation will be estimated with the Gooley method, to take into account the competitive risk of death, and compared with the Grey test. The Fine and Grey model will be used to estimate adjusted HR and their 95% CI. In addition, overall survival (OS) will be analyzed with Kaplan-Meier method and compared with the log rank test. A Cox proportional hazard model (if appropriate) will be used to estimate the Hazard Ratios (HR) and the 95% CI.Hospital length of stay and ICU length of stay will be described in each arm as median and interquartile range; they will be compared with the Wilcoxon rank sum test. The 95% confidence interval (CI) of the mean difference of measure will be estimated with a bootstrap method.Hospital mortality will be expressed as rate and a Chi square test will be used to evaluate the difference between the two groups.Incidence of adverse events and severe adverse events will be expressed as rate and a Chi square test will be used to evaluate the difference between the two groups.2R despite the lack of solid evidence [2R justifies our randomized clinical trial to evaluate whether patients with respiratory acidosis refractory to NIV should be intubated and take the risks associated with invasive mechanical ventilation, or should be connected to ECCO2R to avoid intubation, but run the risk of the potentially serious ECCO2R-related complications. Moreover, this trial may allow clinicians and hospital managers to know whether this treatment should be made available or should be avoided since inappropriate and associated to serious side effects.The consistency of the data from systematic reviews and matched cohort studies –14, and evidence  confirm One possible limitation of our study design is that it doesn’t include long-term patient-centered outcome measures; however, we feel that long-term outcome measures would add complexity to our pragmatic trial design and should be investigated in future trials, especially after clear evidence on shorter-term efficacy and safety are gathered.2R (combined with NIV) may significantly impact the natural history of COPD itself.The ORION trial, owing to its pragmatic nature, planned sample size and composite primary outcome is expected to provide valuable and definite answers to these important research questions. Hypercapnic exacerbation of COPD represent a significant clinical (prognosis), social  and economic  burden worldwide and the avoidance of invasive mechanical ventilation through the application of ECCOPresently, the interest of the scientific community for the subject is very high, and this work is thus potentially highly significant in the field of respiratory intensive care medicine.th, 2019.Latest protocol version: 2.0, November 25At the time of writing, this trial is not yet recruiting participants.st, 2023.Estimate date for recruitment completion: December 31Additional file 1.Additional file 2.Additional file 3.


Bacillus, a commonly recognized producer of multifarious antibiotics, to explore the role of phylogenetic patterns of biosynthetic gene clusters (BGCs) in mediating the relationship between antagonism and phylogeny. Comparative genomic analysis revealed a positive association between BGC distance and phylogenetic distance. Antagonistic tests demonstrated that the inhibition phenotype positively correlated with both phylogenetic and predicted BGC distance, especially for antagonistic strains possessing abundant BGCs. Mutant-based verification showed that the antagonism was dependent on the BGCs that specifically harbored by the antagonistic strain. These findings highlight that BGC-phylogeny coherence regulates the positive correlation between congeneric antagonism and phylogenetic distance, which deepens our understanding of the driving force and intrinsic mechanism of microbial interactions.Understanding the driving forces and intrinsic mechanisms of microbial competition is a fundamental question in microbial ecology. Despite the well-established negative correlation between exploitation competition and phylogenetic distance, the process of interference competition that is exemplified by antagonism remains controversial. Here, we studied the genus  Bacillus genomes is consistent with their phylogenetic relationship and that congeneric antagonism among Bacillus strains is positively correlated with phylogenetic distance.Interference competition exemplified by antagonism remains controversial. Using comparative genomic analysis and antagonistic assessments, this study shows that the distribution profile of biosynthetic gene clusters within  Competition between different species is generally categorized as exploitation competition, which involves the rapid consumption of a limited resource3, and interference competition, which refers to direct antagonistic interactions4. Microbial antagonism is driven by diverse toxins, such as broad-spectrum antibiotics and strain-specific bacteriocins6, and is recognized as a key element regulating populations and determining their success within diverse communities7. The stunning diversity of both the categories and functions of antimicrobial metabolites among different species, results in the extraordinary complexity of interference competition between microbes with different phylogenetic relationships8. Accordingly, illustrating the driving forces and mechanisms of antagonistic competition is crucial for understanding and predicting microbial behaviors during community assemblage and succession2.Microbes are naturally surrounded by taxonomically different ones with which they compete for scarce resources and space4. Despite the well-established negative correlation between exploitation competition and phylogenetic distance2, the process of interference competition is much more complicated and remains controversial. Russel et al. demonstrated that inhibition was more prevalent between closely related bacteria, and this negative correlation between antagonism and phylogeny was mediated by the overlap of the metabolic niche among different strains9. Conversely, other studies examined congeneric competition in Vibrio and Streptomyces and revealed that closely related strains competed less than phylogenetically distant strains, which was probably caused by the effect of the prior coexistence and distribution of secondary metabolites in different genomes11. Additionally, the positive relationship between kin discrimination and phylogeny was indicated within Bacillus subtilis, which was modulated by genes involved in antimicrobials and cell-surface modifiers13; however, this correlation was lost or even became a certain extent negative when more distantly Bacillus strains were tested for antagonism, probably being dependent on the demand of protecting public goods14. Taken together, the relationship between antagonism and phylogenetic distance with regard to microbes from different taxonomical scales or groups, as well as the involved biological mechanism, is still under debate, which limits both our understanding and application of these microbial interactions.Phylogenetic relatedness is considered to be closely associated with microbial competition, based on its determination of both primary and secondary metabolic profiles16, and also usually provide resistance against the self-produced antibiotic to protect the host cell18. Although the relevance (or lack of relevance) of BGCs to antagonism or phylogeny has been evaluated in diverse microbes21, the involvement of BGCs profile in mediating the relationship between interference competition and phylogeny, has not been well addressed. Here, we hypothesize that the correlation between BGC and phylogenetic distance can predict the pattern of congeneric antagonism among different taxonomic groups, as strains possessing higher BGC similarity should have a lower probability of inhibiting each other. To test this hypothesis, we referred to the Gram-positive Bacillus as the target genus, which is a commonly recognized producer of multifarious secondary metabolites25, including nonribosomal lipopeptides 23, nonribosomal polyketides 19, peptide-polyketide hybrid compound 24, and ribosomally synthesized and post-translationally modified peptides 26. Based on comparative genomic analysis, antagonistic assessments, and mutant-based verification, we show that the distribution profile of BGCs within Bacillus genomes is consistent with their phylogenetic relationship; accordingly, the congeneric antagonism among Bacillus strains positively correlates with phylogenetic distance, and this inhibition is dependent on the BGCs that specifically harbored by the antagonistic strain. Our results demonstrate that BGC-phylogeny coherence regulates the positive correlation between congeneric antagonism and phylogenetic distance.Biosynthetic gene clusters (BGCs) are responsible for the production of various secondary metabolites that contribute to interference competition between different microbesBacillus, we collected 4268 available Bacillus genomes covering 139 species from the NCBI database , a megaterium clade, and a circulans clade.BGCs are responsible for the synthesis of secondary metabolites involved in microbial interference competition. To investigate the relationship between BGC and phylogenetic distance within the genus 15 detected 49,671 putative BGCs in the 4268 genomes, corresponding to an average of 11.6 BGCs per genome  and PKS-NRPS Hybrids (0.7 vs. 0.0–0.2) classes, as compared with the three other clades  high genome sequencing quality that available for further BGC distance calculation; (ii) covering all Bacillus species; (iii) for each species, the representative genome(s) was/were chosen from each of the main branches in the phylogram of genomes within this species. We analyzed the interactive sequence similarity network of BGCs in these genomes by using the “biosynthetic gene similarity clustering and prospecting engine” (BiG-SCAPE)28. The (dis)similarity of paired BGCs was calculated based on a combination of three metrics, including the Jaccard index (JI), adjacency index (AI), and domain sequence similarity (DSS), which resulted in 1110 gene cluster families (GCFs) and 76 gene cluster clans (GCCs) of the 4877 putative BGCs . Genomes of phylogenetically close relatives (phylogenetic distance < 0.1) carried highly similar arsenal of BGCs , which are the two dominant groups within the genus Bacillus and have been explored to provide abundant secondary metabolites  showed weak antagonism against strains in the same subclade but exhibited an increased antagonistic ability to the out-clade species . Correspondingly, the antagonistic abilities of B. thuringiensis YX7 and B. mobilis XL40 (cereus clade) towards other Bacillus strains were also enhanced with increasing phylogenetic distance . Based on the results of the colony confrontation assays, a significant positive correlation between antagonism and phylogenetic distance was revealed . To further clarify whether this association was mediated by the BGC distribution pattern, we calculated the predicted BGC distance among all the tested paired strains . Interestingly, there was also a significant positive correlation between antagonism and the predicted BGC distance , suggesting that BGC profiling is likely to play a role in regulating interspecies antagonism.BGCs not only contribute to the synthesis of secondary metabolites but also usually afford self-tolerance to the antibiotic they encodelus Fig.  determinlus Fig. . The anttes Fig. ; the tarsubtilis clade, which harbored the most BGCs  and predicted BGC distance ; while within clades, this significant concordance between inhibition and phylogram was recovered in the subtilis clade but not in other three clades . Intriguingly, we found the correlation between antagonism and phylogenetic distance for each individual antagonistic strain, was positively associated with the predicted quantity of BGCs in this bacteria . This finding suggests that the antagonistic bacteria carrying abundant BGCs  tend to have a clear positive correlation between inhibition phenotype and phylogenetic distance, while those with fewer BGCs  show a weak or even irregular antagonistic pattern against diverse targets. Furthermore, the BGC-phylogeny coherence was similar among all the antagonistic strains , while the antagonism-BGC distance correlation revealed a positive association with the quantity of BGCs in bacteria , which can partially explain the weak correlation between antagonism and phylogenetic distance in strains possessing fewer BGCs.Furthermore, to check the positive antagonism-phylogeny correlation in a more defined system, we performed a fermentation supernatant assessment to test the inhibition between paired strains. This strategy can avoid potential bacterial nutrient competition and is feasible for a wider range of antagonistic strains since antagonism is not influenced by the growth speed or morphology of the colony , three polyketides 32, and one antimicrobial fatty acid 18. The antagonistic characteristics of SQR9 mutants deficient in each of the above BGCs and SQR9Δsfp with the 4’-phosphopantetheinyl transferase gene deleted and only bacilysin can be synthesized31   BGCs tended to have a lower probability and intensity of antagonism. It should be noted that not all the unique BGCs in antagonistic strains contributed to the inhibition of the target  Fig. . Furtherget Fig.  and the Origin of Species, that is, congeneric species are likely to compete more fiercely by means of their functional similarity33, has been examined in various organisms and has received both positive and negative support37. With regard to the microbial fierce competition exemplified by antagonism, Russel et al. found a negative correlation between inhibition probability and phylogenetic distance9; some other scientists discovered a positive relationship between antagonistic interaction (including kin discrimination) and phylogenetic dissimilarity in genus Vibrio7 and Streptomyces11, and species B. subtilis13. The present study demonstrated that antagonism tended to be positively correlated with phylogenetic distance within the genus Bacillus , the low number of the unique antibiotics , was not appropriate for statistical analysis and can impair the biological regularity of inhibition phenotype against diverse targets , but was weak or even not significant in those harboring fewer BGCs e.g., <8) Fig. . This di Fig. . T14. This discrepancy may be attributed to the following reasons: (i) The antagonism in this research were evaluated by colony confrontation and fermentation supernatant inhibition, which was dominated by diffusible secondary antibiotics within a comparatively longer distance; while the kin discrimination in Lyons & Kolter’s study was assessed based on swarm interaction, biofilm meeting, and halo formation14, which was likely to be dependent on closer cell association 13. (ii) The antagonism-phylogeny correlation in our study was calculated based on interactions between diverse antagonistic and target strains. Comparatively, the halo assay performed previously examined the inhibition of one indicated species (B. subtilis NCIB3610) by different testing strains14; as discussed above, the different BGCs distribution patterns  among distinct antagonistic strains can influence the inhibition phenotype and its relationship with phylogenetic distance. In general, we consider that Lyons & Kolter’s study has provided important knowledge with regard to kin discrimination and close contact, especially in a mixed bacterial population14; and our study focused more on the antagonistic interaction sensu lato, which can occur within a long distance.Noticeably, there are some differences between our finding and that reported by Lyons & Kolter, who demonstrated a negative correlation between kin discrimination and phylogenyBacilli possess an amazing capacity to synthesize a diverse range of secondary metabolites; previous studies have indicated the phylogenetic conservation of BGCs in the genus Bacillus and identified multiple species/clade-specific molecules44. Based on bioinformatics analysis of genomes from a larger scale, we also demonstrated the phylogenetic dependence of BGCs distribution pattern in the genus Bacillus , only closely associated species can share secreted cooperative goods, such as surfactin for swarming and biofilm formation14 or siderophores for iron acquisition47, while metabolic similarity decreases moderately or even changes irregularly with phylogenetic distance11. Consequently, closely related species can enjoy a higher benefit of public goods than the cost of resource competition and therefore exhibit a relatively weak antagonistic tendency, while distantly related species can hardly share public goods but still confront nutrient competition; thus, the relatively lower benefit than cost contributes to strong demand for congeneric inhibition. Over a broad phylogenetic range , all strains can barely exploit mutually cooperative goods; hence, their benefit-cost balance is predominantly driven by the cost of nutrient competition9. As a result, metabolic similarity facilitates the negative correlation between antagonism and phylogenetic distance. This dualistic relationship between interference competition and phylogenetic distance could be a consequence of natural selection, which impels microbes to balance cooperation and competition in an economical manner and plays an important role in regulating community assemblage and succession48.The cooperation-competition tradeoff among microbial interactions is usually the consequence of the balance between benefits, such as public goods sharing and cross-feeding, and costs, such as resource competition and stress resistanceBacillus, and this coherence acts as the main factor driving the positive correlation between congeneric antagonism and phylogenetic distance, especially in strains possessing abundant BGCs. We expect this positive association between congeneric antagonism and phylogeny is either pronounced11 or can be predicted in other genera with abundant BGCs. This study deepens our understanding of the driving forces and intrinsic mechanism of microbial interactions and provides implications for designing synthetic microbial communities and manipulating population assemblages for practical purpose.In conclusion, the present study demonstrates the consistency between the BGCs distribution and phylogenetic tendency within the genus Bacillus strains used in this study are listed in Supplementary Data Bacillus Genetic Stock Center (BGSC), 17 strains obtained from the Agricultural Culture Collection of China (ACCC) that originated from different environmental samples, 59 strains isolated from the plant rhizosphere by this laboratory, and 10 mutants of B. velezensis SQR9. All strains were grown at 30 °C in low-salt Luria-Bertani (LLB) medium ; when necessary, final concentrations of antibiotics were added as follows: 100 mg L−1 spectinomycin (Spc) and 20 mg L−1 zeocin (Zeo). To collect the fermentation supernatant for antagonism assessment, the bacterial strains were cultured in Landy medium49. The 16 S rRNA genes were amplified with the 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492 R (5′-GGTTACCTTGTTACGACTT-3′) primers and were subsequently Sanger sequenced. The taxonomic affiliations of these strains were determined through the EzBioCloud database and NCBI BLAST.All 89 Bacillus species were downloaded from the NCBI database using the ncbi-genome-download script (https://github.com/kblin/ncbigenome-download/) . Maximum likelihood (ML) phylogenetic trees of the 16 S rRNA sequence of the strains used in antagonism assessments were constructed by MEGA 5.0.In total, 4268 available genomes from 139 different Bacillus genomes were predicted using antiSMASH 5.0 software15. Considering that numerous congeneric strains shared highly similar BGC profiling, in order to compare the BGC distribution among different Bacillus groups more adequately, 545 representative Bacillus genomes covering all species with high sequencing quality, were further selected for BGC distance analysis  and hierarchical clustering (HCL) with the average linkage clustering method to view the whole dataset by the TIGR multiexperiment viewer . The BGC distances between different Bacillus genomes were then defined as the similarity at the level of GCFs and were directly extracted from the above clustering data. Connection of Bacillus genomes with different correlation between BGC distance and phylogenetic distance, was visualized using CIRCOS software .Biosynthetic gene clusters (BGCs) of all 4268 5 CFU mL−1) was spread onto LLB plates (10 × 10 cm) to be grown as a bacterial lawn. For the colony antagonism assay, 4 μL of the antagonistic strain culture (~108 CFU mL−1) was spotted onto plates covered by the target strain layer; for the fermentation supernatant inhibition assay, 150 μL of the filtration-sterilized extracellular supernatant of the antagonistic strain cultured in Landy medium for 48 h was injected into an Oxford cup and then placed on lawns of the target strain. The plates were placed at 22 °C until a clear zone formed around the spot, and the inhibition was scored. A heatmap showing the profile of the fermentation supernatant inhibition assay was built by the GraphPad Prism 8.3.0 (https://www.graphpad.com/). Each antagonism assay using wild-type strains includes three biological replicates, while each assay using mutants of strain SQR9 includes six biological replicates.The inhibition of antagonistic strains on target strains  region (~1000 bp), direct repeat (DR) sequence (~500 bp), and right flanking (RF) region (~1000 bp) were amplified from genomic DNA of strain SQR9 using the primer pairs LF-F/LF-R, DR-F/DR-R, and RF-F/RF-R, respectively. The PS cassette  was amplified with the primers PS-F/PS-R using p7S6 as a template52. The four fragments were fused using overlap PCR in the order of LF, DR, PS cassette, and RF. Subsequently, the fused fragments were transformed into competent cells of B. velezensis SQR9, and the transformants were selected via first-step screening on LLB plates containing Spc. The final mutants were obtained by combining LLB medium containing 10 mM p-Cl-Phe and the verified primer pair VF/VR. The primers used in this study are listed in Supplementary Data Marker-free deletion strains of the target BGC genes were constructed using the strategy previously described by Zhou et al.P-value < 0.01. Graphs of the correlation data were created using the “ggplot2” package in R; the gray shaded areas denote the 95% confidence intervals. The Duncan’s multiple rang tests (P < 0.05) of the SPSS version 22.0  was used for statistical analysis of differences among treatments.LM was performed in the R package (version 3.6.1) to assess the correlation between BGC distance and phylogenetic distance, inhibition phenotype and phylogenetic distance/predicted BGC distance, as well as the association of antagonism-phylogeny/BGC-phylogenetic distance/antagonism-BGC distance and the quantity of BGCs in antagonistic strains. Statistical significance was based on a Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesDataset 1Dataset 2Dataset 3Dataset 4Dataset 5Dataset 6Dataset 7Dataset 8Dataset 9Reporting Summary


New transient deficits with Protocol 1, 2 and 3 were noted in 38.96%, 34.31% and 31,08% of cases, and permanent deficits in 6.49%, 3.65% and 2.7% respectively. The average follow-up period was 6.8 years. OS was influenced by molecular class (p = 0.028), EOR (p = 0.018) and preoperative tumor growing pattern (p = 0.004). Multimodal surgical approach can provide a safer and wider removal of DLGG with potential subsequent benefits on OS. Further studies are necessary to corroborate our findings.Surgical management of Diffuse Low-Grade Gliomas (DLGGs) has radically changed in the last 20 years. Awake surgery (AS) in combination with Direct Electrical Stimulation (DES) and real-time neuropsychological testing (RTNT) permits continuous intraoperative feedback, thus allowing to increase the extent of resection (EOR). The aim of this study was to evaluate the impact of the technological advancements and integration of multidisciplinary techniques on EOR. Two hundred and eighty-eight patients affected by DLGG were enrolled. Cases were stratified according to the surgical protocol that changed over time: 1. DES; 2. DES plus functional MRI/DTI images fused on a NeuroNavigation system; 3. Protocol 2 plus RTNT. Patients belonging to Protocol 1 had a median EOR of 83% (28–100), while those belonging to Protocol 2 and 3 had a median EOR of 88% (34–100) and 98% (50–100) respectively ( Adult Diffuse Low-Grade Gliomas (DLGG) are rare genetically heterogeneous tumors accounting for up to 7% of primary brain tumors. DLGG generally affect young adults with no or only slight functional disorders.Patients with DLGG show an initial subclinical phase, followed by continuous tumor growth, infiltration of eloquent areas, progression to a higher grade of malignancy, which leads to neurological disability and death.12. To achieve this purpose, a personalized anatomo-functional planning and intraoperative strategy are crucial to have an optimal balance between a maximal and safe resection.The management of LGGs has radically changed over the time: from a “wait and see” attitude to maximal safe resection as the first step in DLGGs workflow14.Because functional neuroimaging tends to show limited reliability, intraoperative direct electrical stimulation (DES), especially in awake patients benefiting from real-time cognitive monitoring, is the best way to increase the extent of resection while sparing eloquent neural networks16. Recent studies have shown that awake mapping in association with real-time neuropsychological testing (RTNT), which reduces the uncertainty of negative mapping, resulted in higher extent of resection (EOR) and preservation of quality of life both for DLGGs involving language and extra-language functional networks17. Moreover, modern preoperative and intraoperative imaging techniques, along with surgical tools and developments in monitoring techniques, have advanced the potential magnitude of resection in eloquent areas18.Awake surgery (AS) represents the gold standard for cerebral brain mapping considering that, to date, this is the only technique that permits a direct identification of neural networks17, but to the best of our knowledge, there are no studies based on the combinatory effect of different techniques.These previous studies addressed the impact of each single approach on clinical and EOR parametersThe principal aim of this investigation was to analyze the effect on the EOR, and consequently on OS, of the evolution of intraoperative techniques, also considering the recent advances of the knowledge of molecular features of DLGG.Data from adult patients who underwent surgery for DLGG in a single institution between 2000 and 2018 were retrospectively analyzed.Age ≥ 18 yearsPreoperative MRI suggestive of supratentorial LGG near or involving the cortical and subcortical eloquent areasNo previous surgeryNo preoperative chemo- or radiotherapyAt least 18 months of follow-upObjective evaluation of EOR on MRI images in DICOM format based on T2-weighted MRI sequencesRevision of histopathological specimens according to the 2016 WHO Classification of Tumors of the Central Nervous SystemIntraoperative brain mapping and neurophysiological monitoring.Patients were enrolled according to the following criteria:Needle biopsies were excluded from the study.The tumor involvement of a cortical or subcortical eloquent was detected according to the relationships of DLGG to eloquent regions of the brain as predicted by the preoperative magnetic resonance imaging scan, functional MRI and Diffusion Tensor Imaging (DTI) data.The local Ethics Committee, Comitato Etico Unico Regionale del Friuli Venezia Giulia, approved this investigation . Considering that the study was retrospective, written consent to participate in the study was not applicable. Written informed consent was obtained for surgery. Consent in this form was decided and approved by the local ethics committee Regione Friuli Venezia Giulia, Italy. All research was performed in accordance with the Declaration of Helsinki.19.The surgical procedures were conducted under cortical and subcortical white matter brain mapping, according to the intraoperative technique previously described17. In addition to DES, RTNT was applied in the last series during AS17.The AS protocol was selected following the standard protocol in use during that specific time period at our institutionIn this investigation, the data were collected over about 20 years (from 2000 to 2018), during which the intraoperative technical protocol changed.Protocol 1 (January 2000–December 2004): patients underwent surgery with the aid of cortico-subcortical DES, neurophysiological monitoring , and intraoperative use of the NeuroNavigation (NN) system.Protocol 2 (January 2005–to present): Protocol 1 plus fMRI/DTI data on the T1-weighted/T2-weighted 3D MRI studies for intraoperative NN.Protocol 3 (January 2011–to present): Protocol 2 plus RTNT in AS.The following 3 consecutive intraoperative protocols were used during this time period:Protocol 1 and 2 were operated on AS or General Anesthesia (GA) based on the specific clinical and neuropsychological status of each subject.Patients belonging to 20.Magnetic resonance images in DICOM format were used to assess both pre- and postoperative tumor volume by using axial preoperative post-contrast T1-weighted MRI slices, pre and postoperative axial T2-weighted MRI studies. All pre- and postoperative tumor segmentations were performed manually across all MRI slices using the OsiriX software tool20. As previously described, higher levels of preoperative ∆T2T1 MRI index represent the prevalence of the diffusive growing mechanism with the tendency to infiltrate the functional cortical areas and subcortical pathways, thus limiting the achievable resection.The preoperative tumor growing pattern (infiltrative vs. expansive) was expressed as ΔT2T1 index that is preoperative volumetric tumor volume on T2-weighted MRI images—preoperative volumetric tumor volume on T1-weighted images20.The postoperative EOR was evaluated by using 3D T2-weighted MRI axial images as follows: (pre-operative tumor volume – post-operative tumor volume)/(pre-operative tumor volume)21. Molecular markers were evaluated as previously described22.All histological samples were reviewed according to the 2016 World Health Organization (WHO) classificationStatistically significant differences on distribution were evaluated performing chi-squared test for categorical variables, and t-test, Wilcoxon rank-sum tests or Kruskal–Wallis for continuous variables, as appropriate.Overall Survival (OS), progression free survival (PFS) and malignant progression free survival (MPFS) were estimated using the Kaplan–Meier approach. Univariate and multivariate Cox regression models were performed to identify the association between any variable and OS, PFS and MPFS as outcome variables, after the proportional hazards assumption had been verified.The primary endpoints were EOR and OS differences among the three groups.p = 0.05 in a multivariate analysis23.Retention in the stepwise model required the variable to be significant to a level of The Spearman’s rank correlation coefficient was computed to define the relationship between pre-operative ΔT2T1 MRI index and postoperative residual tumor computed on T2-weighted MR images.All analyses were conducted using Stata/SE software (version 14.0 Stata Corp.), and data were presented as HRs and 95% CIs.The local Ethics Committee, Comitato Etico Unico Regionale del Friuli Venezia Giulia, approved this investigation .Considering that the study was retrospective, written consent to participate in the study was not applicable. Written informed consent was obtained for surgery.Three hundred and fifty-two adult patients underwent surgery at our institution for primary supra-tentorial LGGs. A total of 288 patients met the inclusion criteria. Patient demographic, clinical and radiological data are summarized in Table Protocol 1: 77 patients (26.74%).Protocol 2: 137 patients (47.57%).Protocol 3: 74 patients (25.69%).Patients were surgically treated as follows:Overall, new postoperative deficits were noted in a total of 100 patients 34.72%). The incidence of permanent postoperative deficits was relatively low in all protocol groups. Post-surgical deficits were stratified according to each surgical protocol . Among patients operated on AS, mean EOR was higher in those in which the Protocol 3 was applied (p = 0.001)  in the study population. The mean EOR resulted to be statistically different between patient operated with AS when compared to those operated under GA (00 in thep < 0.001).A significant association was found between postoperative tumor volume on T2-weighted MRI images and ΔT2T1 value , molecular class (p = 0.028), EOR (p = 0.018) .The highest percentage of total resection (EOR 100%) was obtained amongst patients treated with Preoperative ΔT2T1 MRI index, EOR and the molecular class are confirmed as independent predictors for OS.Tumor recurrence was also seen in patients that underwent radical resection and amongst all molecular classes.The most relevant results we focused on were:38.In the last decades, DLGG management paradigms have evolved from “wait and see” strategy to a more active interventional approach that aims at reducing the risk of malignant transformation. Numerous studies strongly support that a more extensive resection of DLGGs is associated with improved overall survival time and tumor progression free survival time40.The milestone of LGGs surgery is the concept of maximal safe resectionOur results confirm that EOR is the strongest independent predictor for OS in DLGG: patients who underwent radical resection (EOR = 100%), when functionally possible, showed significant survival benefit.38.Association between postoperative tumor volume on T2-weighted MRI images and preoperative ΔT2T1 MRI index showed that the greater the preoperative ΔT2T1 MRI index, the less extensive was the possible resection; it highlights a possible predictive correlation between ΔT2T1 MRI index and EOR achievable as previously demonstratedA major volumetric difference between T2-weighted and contrast-enhanced T1-weighted MRI sequences suggests a greater propensity of the tumor to have a diffuse growing pattern and consequently to be less resectable.40. A personalized anatomo-functional planning is consequently mandatory17.The principal limiting factor of surgical radicality lies in the infiltrating nature of these tumors, which gradually overgrow into eloquent areas both at cortical and subcortical levelIn the modern era of glioma surgery, increasing technological advances in imaging methods , stimulation mapping, and/or somatosensory-evoked potential techniques, are enabling surgeons to maximize cytoreduction and minimize morbidity.41, we found no studies in literature that report the analysis of multimodal integrated protocols in the same cohort of DLGGs.Although there is growing literature supporting the accuracy and efficiency of individual techniques and methods18.Volumetric studies have shown the prognostic significance of EOR for survival outcomes but have not compared the impact of the various intraoperative imaging modalities and surgical strategies in assisting/optimizing the maximal safe resectionThis investigation assessed the efficacy of the combination of different techniques in surgery of DLGGs that are positioned in or close to eloquent areas both in terms of morbidity and EOR.44. Intraoperative use of a guided navigation system enriched with functional and DTI data used in Series 2 significantly increase the possible EOR. Patients in Series 1 had a mean EOR of 83%, while those in Series 2 had a mean EOR of 88% (p < 0.001).Specifically, neuroradiological techniques, such as fMRI and 3D reconstruction of white matter tracts, represent useful tools to preoperatively analyze the three-dimensional relationships between the tumor and the neighboring cortical functional areas and subcortical pathways, respectivelyProtocol 3) resulted in a higher EOR and lower permanent deficits. The data show that Protocol 3 was superior to Protocol 1 and 2 in improving the EOR. The median EOR and the number of radical resections is hence superior in patients belonging to Protocol 3 in comparison with those belonging to Protocol 1 and 2 (p < 0.001)  related to the brain area where the surgeon is working at that moment, thus guiding the surgical team. When a significant decrement of performance is noted, the surgeon can stop resection or move to another area, otherwise the surgical resection can be carried out without the uncertainty of “classical” negative mapping. As a matter of fact, RTNT can be defined as a continuous neuropsychological monitoring.Protocol 3 could represent a feasible support to the surgeon to safely maximize EOR in DLGGs surgery.Thus, a multimodal integrated protocol with those techniques  and strategies (AS and RTNT) can improve the chances of an extensive resection, which has repercussions on survival and recurrence. In light of that, 13.DES, both at cortical and subcortical levels, according to some experts of this field of surgery, represents the standard of care for DLGGs resection. It is used both for brain mapping and for monitoring neurologic performance, often in awake settingp = 0.006) supporting the importance of AS for DLGGs in eloquent areas. This is the sole technique enabling a direct identification of neural networks and thus maximizing the resection while minimizing the risk of permanent deficits39. RTNT is based on a continuous neuropsychological testing administered during the surgical resection in order to fill the functional information gap between two consecutive DES, thus aiming at exploiting at its maximum the potentiality of AS17.Our study showed that patients who underwent AS had a better EOR in comparison to those operated under GA  have become of crucial importance47.The 2016 WHO classification incorporated molecular findings into brain tumor diagnosis. Phenotypic and genotypic parameters in the diagnosis of DLGGs  and tended to also be larger in Diffuse Astrocytoma IDH1/2 mutated than in Diffuse Astrocytoma IDH1/2 wild-type . This difference could be related to a different tumor growing pattern.The median EOR was larger in Oligodendroglioma than in Diffuse Astrocytoma IDH1/2 wild-type  analysis, may better clarify tumor recurrence even for those cases with a radical resection51.T2w-FLAIR sequences were not available for both pre- and postoperative measures for some patients. We used the late postoperative scan to minimize overestimation of postoperative tumor volume due to edema or ischemia and to minimize potential differences between sequences40. Although intraoperative mapping of brain areas was shown to promote greater extent of resection and reduce functional deficits, this was shown only recently for some non-invasive techniques. We recognize that in this clinical setting the preoperative functional mapping by navigated transcraninical magnetic stimulation (nTMS) is an increasing valuable tool to stratify the individual risk of surgery-related deficits53. Our presurgical work-up, at the moment, does not include this technique that has become an important tool in the modern surgical glioma management.In addition, the cornerstone of LGGs surgery is based on the concept of maximal safe resectionIn closing, considering the higher interindividual variability of eloquent area arrangement and the wide potential displacement of functional sites, both at cortical level and subcortical pathways induced by the DLGG growing, a personal tailored multimodal integrated assessment of functional area should be developed to adopt in DLGG surgical field.Technological tools are clinically useful to improve the extent of resection. Multimodal integrated approaches combining awake craniotomy, RTNT, intraoperative brain mapping and functional imaging analysis can enhance maximal safe and efficient tumor resection with possible subsequent OS benefits.Combining volumetric studies and Next Generation Sequence investigations will provide a better understanding of DLGG evolution, paving the way for molecular neurosurgery.


The formation of stable, evolved (silica-rich) crust was essential in constructing Earth’s first cratons, the ancient nuclei of continents. Eoarchaean  evolved crust occurs on most continents, yet evidence for older, Hadean evolved crust is mostly limited to rare Hadean zircons recycled into younger rocks. Resolving why the preserved volume of evolved crust increased in the Eoarchaean is key to understanding how the first cratons stabilised. Here we report new zircon uranium-lead and hafnium isotope data from the Yilgarn Craton, Australia, which provides an extensive record of Hadean–Eoarchaean evolved magmatism. These data reveal that the first stable, evolved rocks in the Yilgarn Craton formed during an influx of juvenile (recently extracted from the mantle) magmatic source material into the craton. The concurrent shift to juvenile sources and onset of crustal preservation links craton stabilisation to the accumulation of enduring rafts of buoyant, melt-depleted mantle. Why Earth’s crust only started becoming widely preserved in the Eoarchaean, 500 Ma after planetary accretion, is poorly understood. Here, the authors document a shift to juvenile magmatic sources in the early Eoarchaean, linking crustal preservation to the formation of stabilising melt-depleted mantle. However, these models continue to be challenged by discoveries of intact remnants of Hadean crust5, recycled Hadean zircons in younger rocks8, and Hadean radiogenic Pb and 142Nd isotopic signatures in several cratons worldwide11, which indicate that potentially large tracts of Hadean crust survived the Hadean–Eoarchaean transition. An alternative view suggests that the Hadean Earth was surfaced by a predominantly mafic-ultramafic crust, and the Eoarchaean witnessed a fundamental change in crust-forming processes that produced the first widespread stable, evolved crust14. Testing this hypothesis has, however, proved challenging due to the scarcity and geological complexity of the ancient terranes that preserve a record of crust formation spanning the Hadean and Eoarchaean.The transition between the Hadean and Eoarchaean (approximately 4000 Ma) marks the time when evolved crustal rocks—the essential building material of continents—started to become widely preserved on Earth. The delayed arrival of this stable crust, over 500 Ma after planetary accretion, has traditionally been attributed to the wholesale destruction of pre-existing Hadean crust either by intense meteorite bombardment15. The mafic precursors of TTGs may be either: (i) juvenile reservoirs, which are remelted shortly after being extracted from the mantle and emplaced in the crust; or (ii) ancient reservoirs that have endured in the crust for hundreds of million years. The 176Lu-176Hf decay system is well-suited to characterising the source of TTGs, as the fractionation of Lu from Hf during partial melting results in the mantle (high 176Lu/177Hf) and crust (low 176Lu/177Hf) developing distinct time-integrated radiogenic Hf isotopic compositions. Consequently, Eoarchaean TTGs sourced from juvenile reservoirs inherited the near-chondritic Hf isotopic composition of the Eoarchaean mantle17, whereas Eoarchaean TTGs sourced from ancient reservoirs acquired less radiogenic (sub-chondritic) Hf isotopic signatures. Zircon is an ideal tool to study the Hf isotopic character of ancient TTGs as it is a ubiquitous phase in evolved magmas and preserves the Hf isotope signature of its parental melt owing to its low Lu/Hf (<0.001) and resilience to isotopic resetting. Coupling in situ U–Pb dating and Hf isotope analysis of zircon provides a time-integrated record of the source characteristics of TTGs, thus offering key insights into evolved crust-forming processes on the early Earth.Preserved evolved Eoarchaean crust is dominated by rocks of the tonalite–trondjhemite–granodiorite (TTG) suite. TTGs mostly form by a two-stage process: initial extraction of primary mafic crust through partial-melting of the mantle, followed by later partial melting of that mafic crust to form evolved magma18 and preserves Earth’s most complete temporal archive of Hadean evolved magmatism through 4370–4000 Ma detrital zircons14. The Yilgarn Craton therefore provides an important opportunity to examine the nature of evolved magmatism across the critical Hadean–Eoarchaean interval over which the majority of Earth’s first stable, evolved crust formed. New zircon U–Pb and Hf isotopic data reveal a link between the onset of crustal preservation in the Yilgran Craton and a fundamental shift in the source characteristics of evolved magmatism across the Hadean–Eoarchaean transition.Here, we focus on the Yilgarn Craton, Western Australia Fig. , which c19  from the Illaara Formation. A subset of 638 detrital zircons showing the least evidence for disturbance of their U–Pb and Hf isotope systematics are interpreted to best represent the age and Hf isotopic composition of their evolved magmatic source rocks  detrital zircons, the majority of which are found in approximately 3000 Ma sedimentary successions in four locations Fig. . The mos(t) values  and support previous studies of the least disturbed Jack Hills detrital zircons14 that suggest most of the earliest evolved magmas in the Yilgarn Craton were not derived from juvenile precursors. Instead, the least disturbed Hadean Jack Hills zircons and the majority of pre-3800 Ma zircons from the Illaara Formation collectively define an approximately linear εHf(t)–time array initiating at εHf(t) = −1.5 at 4400 Ma and evolving to εHf(t) = −3 to −7 by 3800 Ma  that was extracted from the mantle in the early Hadean19.Detrital zircons from the Illaara Formation with ages older than 3800 Ma are uncommon  and yield subchondritic Hf isotopic compositions. These unradiogenic Hf isotopic compositions equate to negative εHf Ma Fig. . This Hf(t)–time space that are anchored between an approximately chondritic endmember (εHf(t) = 0 to +1) and the extrapolation of the Hadean mafic crustal reworking trend defined by the pre-3800 Ma zircons. We interpret each of these vertical εHf(t)–time groups to result from the initial extraction of juvenile mafic magma from the mantle, its emplacement into the Hadean mafic crustal substrate sampled by the pre-3800 Ma zircons, followed by rapid reworking of both the juvenile and Hadean mafic crustal reservoirs to produce evolved magmas . However, the spread from suprachondritic (positive εHf(t)) to less radiogenic (negative εHf(t)) endmembers implies that these TTGs were not purely juvenile additions to the crust, but were emplaced within, and extensively reworked their Hadean crustal substrates24. In summary, the Hf isotope data from other ancient terranes are consistent with the more temporally complete archive of Hadean–Eoarchaean evolved magmatism preserved in the Yilgarn Craton and support a temporal link between crustal rejuvenation and the stabilisation of the Earth’s oldest cratons in the Eoarchaean.Although lacking extensive Hadean zircon archives, the preservation of Hadean Pb and t11 Fig. . Strikint11 Fig. , ref. 1726. The enhanced mechanical strength and buoyancy of this melt-depleted residue stabilises it against erosion by the convecting mantle. Roots of strongly depleted lithospheric mantle, locally >200 km thick, are found beneath most Archaean cratons and are widely interpreted to have facilitated the long-term preservation of the overlying crust28. This empirical evidence for a link between crustal stabilisation and the development of lithospheric mantle is supported by numerical models that demonstrate that even with a more vigorously convecting mantle on a hotter early Earth, the presence of lithospheric mantle substantially increases the preservation potential of crust30. Dating of the lithospheric mantle beneath Archaean cratons indicates that it mostly formed synchronously with the overlying crust, reinforcing the coupled development of stable crust and lithospheric mantle28. Importantly, lithospheric mantle xenoliths from several cratons with Re–Os mantle model ages in excess of 3600 Ma27 and rare exposures of ~3800 Ma melt-depleted mantle rocks31 provide independent evidence for the development of lithospheric mantle contemporaneously with the appearance of the first stable, evolved crust in the Eoarchaean.The temporal link between crustal rejuvenation and stabilisation is readily explained by the formation of lithospheric mantle beneath nascent Eoarchaean cratons. The removal of iron and volatile components during partial melting of the mantle to form juvenile magma produces a buoyant and rigid residue of melt-depleted lithospheric mantle14 or by meteorite impacts32  stretching, a lithospheric lid will begin to thin and segment, resulting in large-scale decompression melting of the underlying convecting mantle. Due to the negative feedback between strain migration and lithospheric stiffening that accompanies melt-extraction, rift migration incorporates large volumes of rigid, melt-depleted mantle into the lithosphere, triggering the stabilisation of cratons. This process develops distinct geodynamic settings for crust formation depending on the strength of the modelled lithosphere. For a high strength lithosphere, new crust is produced over broad mantle upwellings below thinned regions of a poorly mobile lid, in a setting analogous to early Archaean plateau-type terranes. In contrast, mobile-lid behaviour can develop under lower lithospheric strengths as rifting of the lid into discrete tectonic plates is compensated by the development of subduction-like lithospheric downwellings40. In the context of this modelling, our new findings provide important geochemical and geological evidence for a fundamental change in the geodynamic setting of crust formation during the Hadean–Archaean transition, which facilitated the extraction of juvenile melts, crustal reworking, evolved magmatism, and the production of stabilising melt-depleted lithospheric mantle, which were critical to forming Earth’s first cratons.A link between craton stabilisation and a change in global geodynamics is supported by recent numerical models describing the thermochemical differentiation of the lithosphere across the Hadean–Archaean transitionZircons were isolated from approximately 5 kg samples using standard magnetic and heavy liquid separation techniques. Zircons were cast into 25 mm round epoxy mounts, polished, and imaged using a Gatan PanaCL panchromatic CL detector housed at the University of Melbourne, Australia to characterise internal textures and aid in analytical spot placement.2 gas added to the aerosol stream prior to arriving at the torch to enhance sensitivity. Laser ablation was preformed using a 35 µm spot, a frequency of 8 Hz, and a fluence of approximately 4.5 J/cm2 . Hafnium isotopes were collected following the method outlined in ref. 41, which involved 30 s of background measurement and 60 s of ablation with a 1 s on-peak integration time. Dwell times for U, Th, and Pb isotopes on the iCAP TQ were 10 ms for 238U and 232Th, 20 ms for 208Pb, 70 ms for 207Pb, 40 ms for 206Pb and 30 ms for 204Pb and 202Hg. Isotopic data were reduced with the Iolite 3 software package42. The U–Pb data was reduced with the U_Pb_Geochron4 data reduction scheme of Iolite with a smooth cubic spline used to model down-hole fractionation. The Hf isotopes were reduced using the Hf_isotopes data reduction scheme of Iolite with Yb mass bias corrected assuming 173Yb/171Yb = 1.132685 (ref. 43) and Hf mass bias corrected assuming 179Hf/177Hf = 0.7325 (ref. 44).U–Pb and Hf isotopic data were collected via laser ablation split-stream inductively coupled plasma mass spectrometry at Monash University, Australia. The analytical set-up includes an ASI RESOLution 193 nm laser ablation system coupled to a Thermo Fisher iCAP TQ Triple Quadrupole inductively coupled plasma mass spectrometer for measurement of U and Pb isotopes and a Thermo Scientific Neptune Plus multicollector inductively coupled plasma mass spectrometer for measurement of Lu, Yb, and Hf isotopes. The ablated sample aerosol was split evenly using a Y-connector and transported to the mass spectrometers using polyurethane tubing with N45 was used to calibrate the U–Pb data and the Mud Tank zircon46 was used to calibrate the Hf isotope data. Secondary standards were used to validate U–Pb and Hf results and included the reference zircons 9150047, GJ148, Plešovice49, and QGNG50. The differences in the down-hole U–Pb fractionation behaviour of reference zircons with significantly different ages was accounted for by calibrating the U–Pb data for QGNG (1851 Ma) against OG1 (3465 Ma) and calibrating the U–Pb data for the younger reference zircons Plesovice (337 Ma), GJ1 (600 Ma), and Mud Tank (732 Ma) against 91500 (1063 Ma). The weighted mean U–Pb ages and 176Hf/177Hf values measured for the reference zircons agree with the published recommended values54, as do the stable Hf isotope ratios measured over all analytical runs56 ) at the time of crystallisation of the unknown zircons are calculated using their 207Pb/206Pb ages, the 176Lu decay constant of ref. 57, and the CHUR parameters of ref. 58. The kernel density estimation and histogram of zircon U–Pb ages and all weighted mean calculations were made using isoplotR59.Natural zircon reference materials were measured every 15 unknowns during each analytical run and the drift between reference material brackets was corrected using a spline function. OG1 zirconinst OG1 465 Ma anSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Enzyme-linked immunosorbent assay was used to measure KIF5A in CSF obtained from two independent biobanks comprising non-inflammatory neurological disease controls (NINDC), clinically isolated syndrome (CIS) and MS cases. CSF KIF5A expression was significantly elevated in progressive MS cases compared with NINDCs, CIS and relapsing–remitting MS (RRMS). In addition, levels of KIF5A positively correlated with change in MS disease severity scores , in RRMS patients who had documented disease progression at 2-year clinical follow-up. Copies of adenine risk alleles  corresponded with a higher proportion of individuals in relapse at the time of lumbar puncture (LP), higher use of disease-modifying therapies post LP and shorter MS duration. Our study suggests that CSF KIF5A has potential as a predictive biomarker in MS and further studies into the potential prognostic value of analysing MS susceptibility SNPs should be considered.There is increasing interest in the development of multiple sclerosis (MS) biomarkers that reflect central nervous system tissue injury to determine prognosis. We aimed to assess the prognostic value of kinesin superfamily motor protein KIF5A in MS by measuring levels of KIF5A in cerebrospinal fluid (CSF) combined with analysis of single nucleotide polymorphisms  contains supplementary material, which is available to authorized users. Multiple sclerosis (MS) is an immune-mediated inflammatory and neurodegenerative disease, presenting with clinical relapses [associated with inflammatory lesions within the central nervous system (CNS)], and/or progressive neurological decline  . Due to Neurofilaments (NF) are major structural proteins of the axonal cytoskeleton, whose phosphorylation improves structural stability . NFs in Both hypo- and hyper- phosphorylation of NFs within axons are recognised as pathological hallmarks of MS , 11 and rs12368653, rs703842, rs10431552, rs10877013 and rs6581155) at chromosome 12q13–14. This region spans 17 candidate genes, including KIF5A that confer susceptibility to MS . There was no significant difference in MS duration (measured from symptom onset) in patients homozygous for rs703842 risk SNP AA vs GG  and rs12368653 ; Supplementary Fig. 4]. However, when each allele subset  was normalised to correct for allele population frequency, only patients with copies of risk alleles (AG and AA) were documented as being in active relapse at the time of LP  received a DMT post LP, compared to those without copies (GG) . One limitation of the Bristol cohort is the absence of clinical data for IIH, which restricts the multivariate analysis. However, it has the advantage of being a single NINDC diagnosis group, whereas other MS biomarker studies have heterogenous control populations , 28.KIF5A is a neuronally enriched motor protein linked to anterograde axonal transport of various intracellular cargoes including phosphorylated NFs , 15. MouIn the Cardiff cohort, KIF5A was significantly elevated in CSF from progressive MS compared with RRMS and CIS in multivariate analysis. CSF KIF5A levels were significantly higher in patients who were in relapse at time of LP compared to patients who were not. This was not replicated in multivariate analysis, which may be due to the number of cases in relapse being underpowered. It is likely that acute inflammation during relapse could cause a higher influx of KIF5A into the CSF due to disruption at the blood brain barrier. This has also been found in studies examining NF-L as a marker of neuronal damage in MS .Our finding of higher KIF5A levels in progressive MS patients compared with RRMS suggest CSF KIF5A levels could be predictive for underlying axonal loss and progressive disability. This is further supported by our data showing levels of KIF5A positively correlate with change in MS disease severity scores , in RRMS patients who have documented disease progression at 2-year clinical follow-up. In progressive MS, patients become increasingly disabled over time as a result of axonal loss . It is brs12368653 and rs703842) that correlated with higher levels of dephosphorylated NFs, which are a hallmark of MS pathology [Other studies have highlighted that mutations in the KIF5A gene are directly linked to diseases with disturbed axonal transport and axonal loss –18. GWASathology , 23.rs703842 MS susceptibility allele. Data available from the 1000Genomes project has estimated the minor allele (G) frequency within a European population (sample size 1006) at 0.320. This may explain why significant differences were not detected in patients without the SNP (GG) compared with homozygotes (AA). The estimated minor allele (A) frequency for rs12368653 is 0.492. Patients homozygous for the rs12368653 adenine MS susceptibility allele had significantly shorter MS duration (time from documented symptom onset to LP), compared with patients with no copies. This may indicate that patients have a more aggressive disease course, reaching diagnostic LP sooner. This theory is supported by additional findings from the Cardiff cohort which demonstrate that when genotype subsets  are normalised to correct for allele population frequency, a higher percentage of patients with risk alleles (AG/AA) were in relapse at LP and went on to receive a DMT. Although of interest, it is important to note that the number of cases for rs703842 GG (n = 13) and rs12368653 AA (n = 18) were potentially underpowered (n < 22); genotype findings would therefore need validation in larger cohort studies to compensate for the minor allele frequencies of both rs703842 and rs12368653 [Results from our current study demonstrate CSF KIF5A levels are significantly different between patients heterozygous (AG) or homozygous (AA) for the 12368653 .rs12368653 and rs703842 SNPs are upstream of the KIF5A gene and form part of a locus control region, comprising 17 candidate genes [rs12368653 and rs703842 are not solely attributed to these individual SNPs and due to linkage disequilibrium, are linked with other SNPs around this gene locus at chromosome 12. Recent gene studies have indicated rs701006 as a lead MS susceptibility SNP within region 29 of chromosome 12 [Both te genes , 21. As te genes . It is losome 12 , 36. In rs703842, which is located at chromosome 12q13–14; a gene locus linked to MS susceptibility. SNP genotypes (rs703842 and rs12368653) alone showed differences with measures of MS disease activity, such as MS duration, proportion in relapse at the time of LP and DMT post LP. However, the findings require verification in a larger cohort and a full exploration of the prognostic value of all GWAS-identified MS susceptibility SNPs within this gene locus.Overall, this study has shown quantifiable differences in CSF KIF5A levels between NINDC, CIS, RRMS and progressive MS, suggesting levels are predictive of MS. In addition, CSF KIF5A levels correlated with measures of MS disease severity in RRMS patients who were documented as having increased disability scores over 2-year follow-up, suggesting KIF5A may have future prognostic potential in predicting disease progression. Of interest, differences in CSF KIF5A levels were detected based on SNP Supplementary material 1 (DOCX 53 kb)Below is the link to the electronic supplementary material.


Conventional transthoracic echocardiography (TTE) does often not accurately reveal pathologies in patients with acute myocarditis and preserved left ventricular ejection fraction (LVEEF). Therefore, we investigated the diagnostic value of two-dimensional (2D) speckle tracking echocardiography compared to late gadolinium enhancement (LGE) by cardiac magnetic resonance (CMR) imaging in patients with acute myocarditis and normal global LVEF.p < 0.001). Compared to controls, lower RLS values were detected predominantly in the lateral, inferolateral, and inferior segments in patients with acute myocarditis. Additionally RLS values were significantly lower in segments without LGE.31 patients (group 1) with the diagnosis of acute myocarditis confirmed by CMR according to the Lake Louise criteria and 20 healthy controls (group 2) were analyzed including global longitudinal strain (GLS) and regional longitudinal strain (RLS) derived by the bull’s eye plot. Although preserved LVEF was present in both groups, GLS was significantly lower in patients with acute myocarditis (group 1: GLS − 19.1 ± 1.8% vs. group 2: GLS − 22.1 ± 1.7%, In patients with acute myocarditis and preserved LVEF, a significant reduction of GLS compared to healthy subjects was detected. Further RLS adds important information to the localization and extent of myocardial injury. The clinical presentation of acute myocarditis is heterogeneous making the diagnosis challenging—especially in patients with preserved ejection fraction (LVEF) –5. The cThe prognosis of acute myocarditis is also variable . Acute mWhile historically endomyocardial biopsy has represented the diagnostic gold standard of acute myocarditis , the curLGE detected by CMR reflects myocardial injury, i.e., necrosis and fibrosis , 12. GräHowever, the quantification of LGE in patients with suspected myocarditis remains challenging because of the heterogeneity in presence, localization and intensity of LGE extent , 18. In Conventional TTE does often not accurately reveal pathologies in patients with acute myocarditis and preserved left ventricular ejection fraction (LVEF) . Novel iWe investigated the diagnostic value of global longitudinal strain imaging by echocardiography compared to myocardial injury detected by CMR imaging in patients with acute myocarditis and normal global LVEF. Myocardial injury was diagnosed by the presence of LGE and myocardial edema in CMR. To quantify myocardial injury established visual presence scores were used.We retrospectively identified 31 patients with acute myocarditis and preserved LVEF admitted to the University Hospital Regensburg between November 2015 and November 2019. The diagnosis of myocarditis was confirmed by typical clinical presentation and by typical findings in CMR. The so-called typical clinical presentation comprises “infarct-like” myocarditis including chest pain, elevation of troponin and creatinkinase levels, ECG abnormalities, and preceding signs of infection.In all the patients included typical symptoms as chest pain and elevation of troponin and creatinkinase (CK) levels were present. In 74.2% of patients ECG abnormalities were found and 83.9% had preceding signs of infection. CMR and TTE were conducted in the acute phase of myocarditis. Patients with a history of severe symptoms, e.g., sudden cardiac death, heart failure or reduced LVEF were excluded. In addition patients aged < 18 years and patients with poor TTE image quality were not included. Clinical data and blood samples of all participants were analyzed. The study was approved by the local ethics committee.20 healthy age- and gender-matched subjects were recruited to serve as a control group. The control group did not have a history of myocarditis and other cardiac diseases. All healthy volunteers gave written informed consent and were investigated by complete TTE. No external funding was obtained to support the study.TTE was performed in the first few days of acute myocarditis using IE33and Epiq CVx  ultrasound systems as well as S5-1 or X5-1  transducers. Routine two-dimensional (2D) cine loops were obtained and three apical views  were stored digitally. LVEF was calculated by a biplane Simpson’s method from apical four- and two-chamber views or if not applicable from an apical four-chamber view. TTEs were analyzed offline using the IntelliSpace QLAB software  by two experienced observers . The TTEs were studied independently by each observer.Speckle tracking analysis was performed on a frame-by-frame basis by automatic tracking of acoustic markers (speckles) throughout the cardiac cycle. The duration of systole was defined in the apical three-chamber view by marking the aortic valve opening and closure. The myocardial borders were traced from the apical four-, two-, and three-chamber views in the end-systolic frame of the 2D images to analyze global longitudinal strain (GLS).In accordance with the consensus document of the EACVI/ASE/Industry the region of interest (ROI) was defined by the endocardial border which symbolizes the inner contour of the myocardium . If necePeak systolic strain was determined as the maximum value of the peak negative strain during systole. By assessing the peak systolic longitudinal strain in all 17 longitudinal segments global longitudinal strain was provided by the software as the average value of the different segments  using a 32-channel phased-array receiver coil. CMR images were acquired during breath hold and with ECG-gating. The CMR imaging protocol included short axis fat-saturated T2-weighted sequences searching for myocardial edema and postcontrast late gadolinium-enhanced T1-weighted phase-sensitive inversion recovery (PSIR) sequences in short axis, vertical long axis, and horizontal long axis view for the detection of myocardial injury (LGE). T2STIR imaging was performed in all patients and analyzed visually, but not further quantified. According to the updated Lake Louise criteria we used T2-weighted images to search for edema and quantified LGE to detect non-ischemic myocardial injury . GadovisAs already described by Gräni et al. LGE extent was determined visually by the 17-segment model using two different scores : The vist test, continuous variables in unpaired data were compared with an unpaired t test. Further the chi-squared test was used for the gender comparison between the groups. A p value of < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS Version 25 . Categorical data are presented as absolute and relative frequencies whereas continuous variables are presented as mean ± standard deviation or as median (interquartile range) depending on the underlying distribution. Differences between continuous variables in paired data were tested with a paired p = 0.06). No significant differences were revealed concerning gender or comorbidities like arterial hypertension or valvular heart disease. BMI levels were significantly lower in the control group than in patients with myocarditis . 83.9% of patients with acute myocarditis had preceding signs of infection and in 74.2% ECG abnormalities were present. Coronary angiography was performed in 51.6% of patients with myocarditis and obstructive coronary artery disease was excluded in all patients. CMR has shown to provide a high sensitivity and specificity in cases of “infarct-like” myocarditis diagnosed according to the Lake Louise criteria. In our tertiary care center we are able to offer immediate access to CMR. Thus we could avoid coronary angiography in young patients without atherosclerotic risk factors [Baseline characteristics of the study population are depicted in Table  factors .Table 1BUsually several measurements of troponin and CRP have been performed in our institution. Peak troponin and creatinkinase levels were selected to present the maximum extent of myocardial injury. Median peak troponin level was 8310 pg/ml  and median maximum C-reactive protein (CRP) was 36.4 mg/l (10.2–83.4) in patients with acute myocarditis.p = 0.135, Table p = 0.021). Further diastolic dysfunction measured by E/E’ mean was significantly deteriorated in the myocarditis group compared to the control group (Table Ejection fraction was preserved in the myocarditis group (LVEF 58 ± 4%) as well as in the control group  compared to the control group  were revealed.In the present study, we focused on patients with acute myocarditis and preserved ejection fraction. The major findings of this study are:Gräni et al. demonstrated an incremental prognostic benefit of LGE in risk stratifying patients with suspected myocarditis . DespiteTherefore, Gräni et al. intended to compare LGE quantification methods including thresholding by 2, 3, 4, 5, 6 or 7 standard deviations above remote myocardium, the full width at half maximum (FWHM) technique, and visual quantification in patients with suspected myocarditis . Gräni eTherefore, we chose the LGE-VTS and LGE-VPS score to quantify myocardial injury in patients with acute myocarditis. In the present study, we could demonstrate significant associations between regional longitudinal strain and LGE-VTS score. The reason why LGE extent of 76–100% did not show significant results is probably the low number of segments with LGE extent of 76–100% (17 of 527 segments).In another study by Mewton et al. including 41 patients LGE extent—quantified by visual transmurality score—represented an independent predictor of MACE with Hazard Ratio (HR) 1.42 . AdditioBased on this evidence, it is important not only to describe the presence but also the extent of LGE in patients with acute myocarditis for instance using visual LGE quantification scores.In addition to myocardial fibrosis edema represents another important diagnostic criterion in acute myocarditis. Løgstrup et al. showed that in patients with acute myocarditis edema was mainly localized in the infero-postero-lateral segments assessed by CMR. In accordance with the CMR results segmental strain was also predominantly reduced in the infero-postero-lateral segments in 2D echocardiography . The preOur speckle tracking results also showed significant differences in the apical segments between the myocarditis and the control group. However, the strain values in the apical segments were in the normal range in both groups.n = 25) diagnosed by endomyocardial biopsy had a reduction in global longitudinal strain during the acute course of myocarditis. At follow-up during a period of 6.2 months in eight patients inflammation persisted and global longitudinal strain and strain rate were significantly lower in these patients compared to patients without inflammation [As described by Løgstrup et al. our results suggest global and segmental strain imaging adding valuable information compared to conventional 2D echocardiography in patients with acute myocarditis . Escher ammation .Therefore, deformation analysis by speckle tracking echocardiography should be added to routine diagnostics in patients with suspected acute myocarditis. A reduction of global longitudinal strain in addition with abnormalities in the lateral, inferolateral and inferior segments in the bull’s eye plot should be added to an algorithm for the diagnosis of myocarditis. The importance of speckle tracking echocardiography is even more pronounced in institutions with limited access to CMR.Caspar et al. detected myocardial dysfunction by 2D and 3D speckle tracking analysis in patients late after an acute episode of myocarditis and preserved LV function. Mean delay between the diagnosis of acute myocarditis and follow-up echocardiography was 21.7 ± 23.4 months [The present study included patients with acute myocarditis and speckle tracking analysis as well as CMR were performed in the first few days of myocarditis. Whether these pathologies in strain imaging and CMR remain during follow-up visits should be further investigated.Concerning strain imaging some methodological aspects warrant consideration. As a semiautomated method longitudinal strain analysis is accompanied by a learning curve and represents a potential source of measurement variability . FurtherFurther new evolving techniques like feature tracking by CMR could add valuable information in patients with suspected myocarditis. Recently Gräni et al. demonstrated an independent and incremental prognostic value over clinical features, LVEF and LGE in patients with myocarditis by the use of CMR feature tracking .Some limitations of the present study warrant consideration:First, our study represents a single-center experience with a retrospective study design. Second, we did not analyze circumferential and radial strain parameters. We chose to investigate the longitudinal strain parameters as it is known that in patients with acute myocarditis the most affected myocardial layer is the subepicardium and there the fibers are mainly directed in a longitudinal way , 29. FurThird, the 1.5 and 3.0 Tesla CMR systems were used to evaluate patients with suspected myocarditis which may possibly influence LGE quantification. Fourth, because of the retrospective study design we were not able to perform T2-mapping as a novel technique for quantifying myocardial edema.Fifth, the members of the control group had significantly lower BMI values compared to the patients with myocarditis albeit none of the groups had the diagnosis of obesity.In patients with acute myocarditis and preserved LVEF a significant reduction of GLS compared to healthy subjects was detected. Furthermore RLS adds important information to conventional TTE which could otherwise only be derived by CMR.