Supplemental Digital Content is available in the text. Abnormal QT interval responses to heart rate (QT dynamics) is an independent risk predictor for cardiovascular disease in patients, but its genetic basis and prognostic value in a population-based cohort have not been investigated.QT dynamics during exercise and recovery were derived in 56 643 individuals from UK Biobank without a history of cardiovascular events. Genome-wide association studies were conducted to identify genetic variants and bioinformatics analyses were performed to prioritize candidate genes. The prognostic value of QT dynamics was evaluated for cardiovascular events  and all-cause mortality.KCNQ4 and KIAA1755. Genetic risk scores were not associated with cardiovascular events in 357 882 unrelated individuals from UK Biobank. We also did not observe associations of QT dynamics during exercise and recovery with cardiovascular events. Increased QT dynamics during recovery was significantly associated with all-cause mortality in the univariate Cox regression analysis , but the association was not significant after adjusting for clinical risk factors.Heritability of QT dynamics during exercise and recovery were 10.7% and 5.4%, respectively. Genome-wide association studies identified 20 loci, of which 4 loci included genes implicated in mendelian long-QT syndrome. Five loci did not overlap with previously reported resting QT interval loci; candidate genes included QT interval dynamics during exercise and recovery are heritable markers but do not carry independent prognostic information for clinical outcomes in the UK Biobank, a population-based cohort. Their prognostic importance may relate to cardiovascular disease cohorts where structural heart disease or ischemia may influence repolarization dynamics. The strong overlap between QT dynamics and resting QT interval loci suggests common biological pathways; however, nonoverlapping loci suggests alternative mechanisms may exist that underlie QT interval dynamics. Multiple lines of evidence indicate that abnormal dynamics of the QT interval, measured by the slope of the QT/RR profile, carries prognostic information in cardiac patients for cardiovascular mortality.9 The genetic architecture of QT dynamics has not been investigated and might inform biological mechanisms that underlie QT dynamics. Furthermore, the prognostic value of QT dynamics in a population-based cohort has not been evaluated.The ECG QT interval reflects the total duration of ventricular depolarization and repolarization and is a biomarker for cardiovascular risk and death, with an estimated heritability of ≈50% in twin-studies.An exercise stress test is a useful method to examine the dynamics of the QT interval in response to changes in heart rate. We first measured QT dynamics (approximated using RT dynamics) during exercise and recovery in 56 643 unselected individuals without a history of cardiovascular disease from the UKB study (UK Biobank). We next conducted genome-wide association studies (GWASs) in a subgroup of ≈52 000 individuals of European ancestry. We report for the first time the genetic architecture of QT dynamics, genetic risk score (GRS) results with cardiovascular outcomes, and evaluation of QT dynamics with cardiovascular events and all-cause mortality (ACM).Data Supplement. The UKB study has approval from the North West Multi-Centre Research Ethics Committee, and all participants provided informed consent.10 Data used in this study were part of UKB application number 8256, and anonymized data and materials generated in this work have been returned to UKB and can be accessed per request.The experimental design of the study is shown in Figure Data Supplement). In the discovery phase, genome-wide association results of ≈9.8 million single-nucleotide variations (SNVs) from ≈30 000 individuals of European ancestry from UKB were analyzed for each trait, QT dynamics during exercise, and recovery. All SNVs with P<1×10−6 were compiled and these were organized into regions of 1 Mb. The SNV with the lowest P value in each 1 Mb region was selected as the lead SNV. In total, 20 lead SNVs for QT dynamics during exercise and 7 for QT dynamics during recovery were taken forward into replication in ≈22 000 unrelated individuals. Twelve SNVs for QT dynamics during exercise formally replicated (P≤0.05/20=0.0025) and all had concordant directions of effect (Table P≤0.05/7=0.0071), all with concordant directions of effect  did not show evidence of population stratification or inflation. Regional plots are provided in Figure VI in the Data Supplement. Heritability estimations in the full dataset for QT dynamics during exercise and recovery were 10.7% and 5.4%, respectively, and their respective genetic correlation was 60%.An overview of the study results is provided in Figure SCN5A-SCN10A locus . This signal was ≈100 Kb downstream of the lead signal at this locus (rs7638275). For QT dynamics during recovery, one secondary signal was identified at the NOS1AP locus (rs16847548), at ≈163 kb upstream of the lead signal . Collectively, the lead and secondary variants explained 2.1% of the variance of QT dynamics during exercise (≈20% of heritability) and 0.6% (≈11% of heritability) for QT dynamics during recovery.Conditional analysis revealed one secondary independent signal for QT dynamics during exercise at the FOXN3) for QT dynamics during exercise : this locus at chromosome 14 was only genome-wide significant (P≤5×10-8) in males . No sex-specific loci for QT dynamics during recovery were identified. Altogether, 19 novel loci were associated with QT dynamics during exercise and 3 with QT dynamics during recovery. Two loci (PRKC1 and KCNE1) were common to both; thus, we identified 20 novel unique loci for QT dynamics . Two loci had reported genome-wide phenotype-genotype associations with another ECG marker: the CDKN1A locus with QRS and JT duration and the KIAA1755 locus with resting heart rate (Table V in the Data Supplement).Fifteen of the 20 loci discovered for QT dynamics overlapped with loci previously reported for resting QT interval from published GWAS  were all annotated as either intronic or intergenic (Table VI in the Data Supplement), and no variants were associated with changes in expression levels of nearby genes in the Genotype-Tissue Expression database.11 We explored the regulatory potential of the associated variants at these 5 loci using Hi-C datasets and observed significant chromatin interaction (RegulomeDB score=2b) at KIAA1755 with several genes in heart ventricles, brain, and adrenal gland (Table VII in the Data Supplement).The lead variants or close proxies at the 5 unique loci for QT dynamics during exercise . The variant rs751489327, a close proxy of the lead variant at CNOT1, was annotated as a frameshift variant (Table VI in the Data Supplement). The associated variants at KCNH2 and KCNE1 were predicted to be possibly damaging or deleterious by either sorting intolerant from tolerant or polymorphism phenotyping.12 Using COLOC,13 we observed evidence for colocalization (posterior probability >75%) between significant gene expression (expression quantitative trait locus) and QT dynamics GWAS signals at 2 loci: LITAF and PRKCA in left ventricular and atrial appendage tissue, respectively (Table VIII in the Data Supplement). Interestingly, variants at the PRKCA locus were discovered for both QT dynamics traits, and both demonstrated high degree of colocalization with the expression quantitative trait locus signal . The top expression quantitative trait locus variant for this locus in the heart left ventricle was rs11658550; this variant was in high linkage disequilibrium (r2>0.9) with the lead variants for QT dynamics during exercise (rs569258685) and recovery (rs12960410).At the remaining 15 loci for QT dynamics 5 lead variants or their close proxies mapping to Data Supplement). Several genes including SCN5A-SCN10A, SLC35F1, and KCNQ1 were indicated as prioritized genes (Table IXB in the Data Supplement). Table X in the Data Supplement presents an overview of potential candidate genes at each locus from all bioinformatic analyses and literature review. For these candidate genes, we found significant enrichments across several gene ontology terms including voltage-gated channel activity and cardiac muscle cell action potential. Ventricular fibrillation and torsade de pointes were indicated as the human phenotypes. With fewer genes for QT dynamics during recovery, the enrichments included several ventricular membrane repolarization processes (Table XI in the Data Supplement).Data-driven expression prioritized integration for complex traits analysis indicated significant enrichment in heart and ventricular tissue . Only QT dynamics during recovery was a significant predictor of ACM in the univariate Cox regression analysis: hazard ratio, 1.09 , P=2.28×10-5, but this did not remain significant after adjusting for covariates  QT dynamics during exercise has a significant heritable component (10%), with the recovery marker being less heritable (≈6%); (2) there is substantial genetic overlap between both QT dynamics markers and resting QT interval, and support for 5 loci for QT dynamics during exercise not previously reported for resting QT interval, and (3) QT dynamics during exercise and recovery were not associated with the occurrence of cardiovascular events or ACM in a population-based cohort.KCNQ1, KCNH2, SCN5A-SCN10A, and KCNE1). This may suggest that common variants in these genes do not only play a role in modulating resting QT interval but also QT dynamics, possibly by affecting rate-dependent channel kinetics. For example, KCNQ1 and KCNE1 are well known to confer kinetic properties on IKs currents required for rate adaptation of the cardiac action potential during exercise when β-adrenergic stimulation enhances IKs currents.14We demonstrate for the first time that QT dynamics during exercise and recovery are heritable; however, compared with the resting QT interval, the heritability is less and environmental factors likely have a more significant. Substantial overlap between loci for QT dynamics and resting QT interval suggests important genetic overlap. We observed variants at loci containing genes previously established to cause monogenic long-QT syndrome and encoding ion channels or channel-interacting proteins -alpha activity affects dephosphorylation of the SERCA-2 (sarcoplasmic reticulum Ca2+ ATPase-2) pump inhibitory protein PLB (phospholamban), and alters sarcoplasmic reticulum Ca2+ loading and the Ca2+ transient. The regulation of cardiac contraction is critically important during exercise and recovery and is tightly coupled with the electrical processes of the heart, which may be reflected in the QT dynamics possibly through recognized Ca2+ dependent mechanisms and stretch influencing QT interval.19From the loci that overlapped with resting QT interval, we found evidence for colocalization at the 20 and QT dynamics is thought to offer a more integrative measure of autonomic balance under stress than resting QT interval alone.21 This is supported by multiple lines of evidence showing that abnormal QT dynamics is an independent predictor of cardiovascular mortality in cardiac patients.9Previous work has shown that ventricular repolarization is strongly modulated by heart rate but also independently by autonomic nervous activity22 The variant rs796647867 at the FOXN3 locus was only associated with QT dynamics during exercise in men, suggesting that the genetic basis for QT dynamics during exercise may differ between sexes. FOXN3 is a candidate gene at the locus and is a member of the forkhead/winged-helix transcription factor family. The mechanism by which it could modulate QT dynamics is unclear.The QT dynamics and the response of autonomic nervous activity have shown to be different between females and males.8 cardiomyopathy,9 and postmyocardial infarction2 but not in a population-based sample as interrogated in our study. We found that QT dynamics was not an independent predictor of cardiovascular events and ACM. A review of the previous studies indicates only one used exercise stress data to measure QT dynamics during recovery.4 According to this study, QT dynamics was a predictor of mortality in n=2994 cardiovascular patients. Reported values for QT dynamics were higher compared with our findings . Increased QT dynamics are observed in patients at risk for cardiac death and arrhythmic events,23 and the fact that our values were lower may explain why QT dynamics were not associated with cardiovascular outcome or mortality in our cohort. It is possible that we measured lower values for QT dynamics because our population was healthier. However, it should be mentioned that they exercised at submaximal level only. It is well known that maximal workloads are superior in predicting cardiovascular risk and ACM.24 In addition, in our data the duration of the recovery period was shorter. The time-lag in the response of the QT interval to a sudden change in heart rate can take up to 2 minutes.25 The recovery period in our work lasted 1 minute, and it is, therefore, possible that the QT interval was not fully adapted resulting in a reduced QT/RR slope during recovery. QT interval dynamics reported in this work are specific for exercise and recovery. Importantly, QT dynamics has also shown to carry prognostic value when derived from the QT/RR relationship of 24-hour ECG recordings using linear regression between all QT and RR values. We did not have access to this data, but reported values in patients (0.17–0.22 for heart failure patients8 and 0.20 for cardiomyopathy9) were similar to QT dynamics during exercise observed in our cohort (0.17).The independent association between QT dynamics and cardiovascular events has been established in multiple studies including patients with heart failure,Several limitations in our study should be noted. First, from the 22 SNVs discovered, 14 formally replicated. The remaining identified SNVs we are reporting are from a GWAS of all samples and require formal replication in an independent data set. We also note our sample is relatively small compared to many other GWAS and this will limit power for discovery of loci. Second, the power to evaluate the predictive value of QT dynamics was limited as the number of events was low. This coupled with the small number of genetic variants (N=19) which only explained 2.1% of the variance of QT dynamics during exercise (≈20% of heritability), also affects the power of the GRS.In summary, we demonstrate for the first time that QT dynamics during exercise and recovery are heritable markers. Its genetic basis largely overlaps with resting QT interval; however, there may be additional biological mechanisms that specifically underlie QT dynamics having identified 5 novel loci specific to these traits. QT dynamics during exercise and recovery were not independent predictors of cardiovascular events or ACM as opposed to cardiovascular disease cohorts where structural heart disease or myocardial ischemia affect repolarization dynamics. Future studies will be required to evaluate the prognostic value of the GRS in cardiovascular cohorts.http://doi.org/10.5281/zenodo.438045.This research has been conducted using the UKB (UK Biobank) Resource (application 8256) and is supported by grant MR/N025083/1, by the National Institutes of Health Research (NIHR) Cardiovascular Biomedical Centre at Barts and The London, Queen Mary University of London (QMUL), by the People Programme of the European Union’s Seventh Framework Programme grant n° 608765 and Marie Sklodowska-Curie grant n° 786833, by the University College London Hospital Biomedicine NIHR, Barts Heart Centre Biomedical Research Centre. Dr Young is funded by the Medical Research Council (Grant code MR/R017468/1). This research utilized Queen Mary’s Apocrita High-performance cluster facility, supported by QMUL Research-IT. None.


However, clinical observations have revealed that ‘permissive’ hypercapnia, the deliberate allowance of respiratory produced CO2 to remain in the patient, can have anti-inflammatory effects that may be beneficial in certain circumstances. In parallel, studies at the cell level have demonstrated the profound effect of CO2 on multiple diverse signalling pathways, be it the effect from CO2 itself specifically or from the associated acidosis it generates. At the whole organism level, it now appears likely that there are many biological sensing systems designed to respond to CO2 concentration and tailor respiratory and other responses to atmospheric or local levels. Animal models have been widely employed to study the changes in CO2 levels in various disease states and also to what extent permissive or even directly delivered CO2 can affect patient outcome. These findings have been advanced to the bedside at the same time that further clinical observations have been elucidated at the cell and animal level. Here we present a synopsis of the current understanding of how CO2 affects mammalian biological systems, with a particular emphasis on inflammatory pathways and diseases such as lung specific or systemic sepsis. We also explore some future directions and possibilities, such as direct control of blood CO2 levels, that could lead to improved clinical care in the future.Carbon dioxide (CO More recently, however, it has come to be recognized that CO2 has many profound effects on biological systems which are not exclusively deleterious. Investigations have been two pronged, with molecular approaches studying protein and other compound interactions with CO2, with specific sensing mechanisms previously reviewed [2 in whole animals or patients has given us both mechanistic insights and a window on therapeutic possibilities. In this review, we focus on acute inflammatory processes, particularly in the context of the critically ill patient, and how exploiting the rapidly expanding knowledge base regarding CO2 interactions with biological processes may ultimately lead to preservation of life rather than the originally described toxicity.While suspected for several years beforehand, Scottish chemist Joseph Black 1728–1799) first proved that carbon dioxide  model [in vivo and in vitro through activation of protein kinase B [in vitro and in vivo models of pulmonary inflammation as well as to increase IL-8 expression [α was mediated by a pH-dependent mechanism rather than via CO2per se [Cell culture-based studies are critical to understanding how hypercapnia (HC) and its associated acidosis (HCA) may affect clinical outcomes in patients receiving mechanical ventilation for acute respiratory distress syndrome (ARDS). Several cell culture-based findings have described the effects of HC on pulmonary cells; however, few have unravelled the mechanism by which these effects occur. A 2017 review by Shigemura al cells . We haveal cells . In accoal cells –7. On thgnalling . A furthV) model . HC incrkinase B . ‘Permis2per se . Similar2per se .α pathway [Other signalling pathways are implicated in the mechanism by which HC controls cell functions, with studies noting effects on nucleosome and lipid metabolism in human airway epithelial cells , caspase pathway . What is2 concentration on gene expression in primary normal human bronchial epithelial cells. Genes linked to the immune response and nucleosome assembly were largely downregulated, while lipid metabolism genes were mainly upregulated in the hypercapnic state [One recent study reported the effects of COic state . Anotheric state ,16, althet al. [2 levels on endoplasmic reticulum folding of NaK-ATPase (NKA) [2-induced mitochondrial dysfunction possibly restoring alveolar epithelial barrier function in patients with ARDS and HC. Similarly, elevated CO2 levels were shown to promote activation of the ERK/AMPK/JNK axis in a human alveolar epithelial cell line resulting in epithelial sodium channel dysfunction [in vitro study of pulmonary cancer cells reported that the hypercapnic microenvironment of the tumour may result in chemoresistance to some therapies [Recently, there have been several attempts to further elucidate the mechanisms and pathways underlying how HC influences pulmonary cells. Bharat et al.  reportedet al. . Anotherse (NKA) . NKA is function . Disruptfunction . A recenherapies . These r3.2 0.12) has been found to display protective effects on the lung in a dose- and time-dependant manner [2. These protective effects consist of reducing microvascular permeability and preventing the build-up of excess protein content and haemoglobin in the bronchoalveolar lavage fluid (BAL) along with a decrease in alveolar fluid reabsorption [2 80–100 mmHg) in a rabbit model of VILI was also reported to prevent the increased levels of nitrates in the perfusate and BAL [2 0.05 and PaCO2 80–100 mmHg, respectively) subjected to VILI has shown improvements in lung compliance and oxygenation levels along with a decrease in histological lung damage [The effects of HC and HCA have been studied in a number of preclinical models relating to acute lung injury (ALI) . One modt manner  when comsorption ,23,25. H and BAL  which ha and BAL . Treatmeg damage .Table 12 can prevent p65 translocation [et al. also noted a decrease in the degradation of IκBα, another limiting step in the activation of NFκB [2 on gene expression, with modulation seen on various signalling pathways such as increased Wnt signalling in Caenorhabditis elegans, Drosophila melanogaster and various mouse tissues [Numerous studies have also noted a reduction in the presence of inflammatory cytokines in the BAL, including TNF-α, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein 2 (MIP-2) and IL-6 ,28. One location –28  enhanced vagus nerve activity to produce therapeutic effects in the lung [et al. showed that HC (FICO2 0.12) inhibits stretch-mediated activation of p44/42 MAPK signalling in alveolar epithelial cells which occurs through the inhibition of the metalloprotease ADAM17 [2 0.12) upregulates the α-tocopherol transporter (a potent anti-oxidant) and linked its benefits to the inhibition of pathogenic chemoattractant synthesis [A study by Xia the lung . They fothe lung . Otulakoe ADAM17 . Pharmace ADAM17 . The samynthesis 2, thereby ostensibly preserving normal physiological pH, in the same injury had a detrimental effect on lung injury severity [et al., in their studies, found that haem oxygenase-1, which is an anti-oxidant enzyme that protects tissue from inflammation injury, was upregulated by HC and blockade of this enzyme reversed the beneficial effects of HC [Another frequently used animal model of HC in the lung is the ischaemia-reperfusion model relevant to lung transplantations and to ARDS . Reductiseverity . It has severity . Furtherseverity . Using Hseverity . Wu et ats of HC . Attenuats of HC –35.et al. showed that pre-treatment with HC (FICO2 0.05) for 10 but not 60 min attenuated lung injury and inflammation in a murine LPS model [Pneumonia and systemic infections are common causes of ARDS. HC has been studied in a number of pathogenic ALI models with both beneficial and possibly detrimental effects being reported . In an LPS model .E. coli induced lung injury rat model, HCA attenuated airway pressure and improved lung compliance; however this time lung histologic injury was only improved when HCA was administered in combination with antibiotic therapy potentially highlighting that HCA effects can be affected by the type of infectious insult [E. coli induced ALI rat model improved lung physiology and attenuated lung histologic injury following neutrophil depletion in the model, suggesting that the beneficial effects of HCA in this acute setting are independent of any effect on neutrophils. [E. coli induced lung injury was through the inhibition of NFκB [In an s insult . In a sirophils. . Another of NFκB . HCA has of NFκB .2 0.05 over 6 h of ventilation) did not protect against E. coli induced lung injury in rats even with antibiotic therapy [E. coli instillation with or without antibiotic therapy in spontaneously breathing animals [E. coli administration in spontaneously breathing rats showed that HC at 5% for 24 h enhanced inflammation in the lung but reduced inflammation in the spleen [By contrast, other studies have reported that HCA  as it causes renal buffering of the HCA, that ultimately increases injury susceptibility [et al., whereby buffered HC was shown to be therapeutically ineffective against lung injury caused by systemic sepsis [In a murine ocytosis . HC expoocytosis . Finallyocytosis . Interestibility . This fic sepsis .2 has been shown to be beneficial in other models of ALI of aseptic aetiology. Both HC and buffered HC attenuated lung inflammation and cell death following paraquat-toxin induced ALI [et al. observed that HC exposure enhanced the ALI associated with gastric acid instillation in rats, though the levels may have been too high at 12% CO2 exposure given a cumulative effect of CO2 and gastric acid would lead to a very low and possibly directly injurious pH [COuced ALI . Overalluced ALI . Surfactuced ALI . Therapeuced ALI . By contrious pH .4.2 levels indicating that they were hypercapnic  which correlated with increasing ARDS severity [2 will ensure that even overventilated areas of the lung will have alveolar HC, whereas with permissive HC, i.e. HC due to reduced minute ventilation, there may still be areas of the lung that have alveolar normocapnia or even hypocapnia [2 and equal systemic HC by intentional hypoventilation would be useful in this regard.To avoid the incidence of VILI in the critically ill, the use of lung protective strategies  have been employed to mitigate the over-distension and resulting injury of the lung ,64 whichseverity . A retroseverity . In a seseverity ,73. A mopocapnia . Therefo5.in vitro, in vivo, and large cohort retrospective analyses of clinical trials the direct investigation of the effects of HC in various ailments has been brought forward to clinical trial. The use of protective ventilation (low VT 6 ml kg−1 predicted body weight) in pancreaticoduodenal surgery patients demonstrated a positive effect in terms of reducing lactate levels, atelectasis and hospital stay, while increasing oxygenation levels [2 concentration 50–60 mmHg) were permitted through a decrease in minute ventilation. The time to recovery of full cognitive function was reduced significantly in hypercapnic patients  and HC was deemed a feasible and effective method to speed up recovery from general anaesthesia induced by inhaled agents [2 60–70 mmHg) had lower inflammatory and tissue injury markers in serum and BAL, increased oxygenation index and decreased airway pressure [Given the positive effects demonstrated in various n levels . Permissd agents . In a rad agents . In patipressure . These r6.−1; pH > 7.15) compared with a control mechanical ventilation strategy  for the treatment of ARDS. The primary outcome was the number of ventilator free days (VFD) at day 28 post randomization. A total number of 340 patients had been projected for the PHARLAP study, but at the moment of termination 115 patients had been enrolled. Hodgson et al. [n = 3. Several studies have demonstrated a beneficial effect of hypercapnia in preserving lung compliance [The PHARLAP (Permissive Hypercapnia Alveolar Recruitment and Low Airway Pressure) trial implemented a low tidal volume mechanical ventilation strategy  increased cardiac output and cardiac index significantly in all participants and was tolerated (NCT02519517).2 in patients undergoing surgery in a reclined seated position which commonly results in an impaired haemodynamic stability at baseline. The results of this study demonstrated that mild HC prevented the reduction of cerebral oxygen saturation without changes in oxygenation index and haemodynamic parameters [In surgical procedures where positioning directly influences oxygenation, HC has also been investigated. Permissive HC was induced to 45 mmHg PaCOrameters .HCA increases pulmonary vascular resistance in a porcine model , which m2 levels on the mortality rate of cardiac arrest patients [A recently published retrospective report has determined the effects of COpatients . Patientpatients .In summary, these studies have indicated that the cardiovascular effects of HC are generally well tolerated with effects that may be beneficial in specific clinical situations. This has led to the initiation of several clinical trials to further investigate these therapeutic effects.8.et al. [et al. [et al. [Similar to the effects on the CVS, HC also demonstrates a potent augmentation of the cerebral blood flow (CBF) ; howeveret al. , who det [et al.  showed t [et al. , showingtiO2) after aneurysmal subarachnoid haemorrhage (SAH) was studied in six mechanically ventilated patients (NCT01799525). Respiratory rate and minute ventilation were reduced gradually in 15-minute intervals to reach a PaCO2 level of 40, 50 and 60 mmHg and then returned to baseline. It was observed that both CBF and StiO2 were gradually increased until PaCO2 level of 60 mmHg and after returning to baseline ventilator setting, this increment was maintained before gradually returning to baseline parameters without rebound effect. None of the patients suffered a secondary cerebral infarction. It was concluded that HC causes a global increase of brain perfusion and oxygen supply and it is safe and feasible for use in mechanically ventilated SAH patients [The effect of HC on CBF and brain tissue oxygen saturation (Spatients .2 50–55 mmHg) during the first 24 h of mechanical ventilation in the ICU improves neurological outcome at 6 months compared to standard care . The phase II arm of this study reported safety and feasibility of using mild HC in cardiac arrest patients [The TAME trial is a large multicentre phase III trial that is underway to determine whether a therapeutic mild HC improves neurological outcome at six months compared to standard care (normocapnia), in resuscitated cardiac arrest patients who are admitted to the ICU (NCT03114033). The main objective of this trial will be to determine whether targeted therapeutic mild HC (PaCOpatients .2) on cerebral perfusion in infants was examined in a study titled ‘Assessment of Cerebral Vasoreactivity Using Near-infrared Spectroscopy (NIRS) in Infants (VARO)’ (NCT02429154) [The effect of mild permissive HC (approx. 48 mmHg PaCO2429154) . This st9.2 60–75 mmHg, pH ≥ 7.20) and lower permissive HCA  levels are being investigated in preterm infants. Outcomes measured are alive VFD, all causes of death, BPD, post-natal steroid treatment for BPD.There have been some small clinical trials in neonates and the young paediatric setting demonstrating at least a lack of harm and suggestions of benefit ,112. Lat10.2 removal (ECCO2R) devices pump blood through an artificial circuit incorporating a gas exchanger with a large diffusing capacity that removes CO2 directly from the bloodstream independent of the lungs. ECCO2R is used to facilitate ultra-low tidal volume protective lung ventilation, as it allows the tidal and minute volumes to be further reduced while preventing excessive accumulation of CO2. Small studies have shown that ECCO2R does enable very low tidal volume ventilation strategies to be used in ARDS patients without extreme levels of acidosis, which may be effective in reducing lung injury [Extracorporeal COg injury  and to bg injury ,115.2 removal in ARDS [et al. [2R did not confer any improved survival but allowed for lower tidal volumes reducing the risk of VILI.As of 2018, there have been two randomized control trials evaluating extracorporeal CO in ARDS , with on in ARDS  finding  [et al.  who showet al. [−1 without ECCO2R is a potential option for severe ARDS patients, maintaining a similar hospital mortality rate when compared to a previous observational study using ECCO2R [2 is well tolerated when considering the potential morbidity/mortality associated with an increased tidal volume; an observational study performed by Needham et al. [−1 leads to a 23% higher mortality rate.Richard et al.  show thag ECCO2R . Howeverg ECCO2R . These sm et al.  showed a11.2, perhaps through utilization of small molecules that reproduce similar binding and influence. Candidate targets include aquaporins, soluble adenyl cyclases and various constituents of the NFκB and other pathways. This ability to have the best of both worlds, an enhancement of the beneficial effects of HC without its often simultaneous negative consequences, would be a significant boon in therapeutics for the critically ill and inflammation in general. Revolutionary approaches such a direct control of blood CO2 concentration will allow fine tuning of this new therapeutic paradigm. The future is indeed bright for this once maligned little ‘waste’ molecule.As it stands, there is much work to be done about the potential use of HC or HCA, but many reasons to be optimistic about both avenues of basic scientific investigation and emerging therapeutic options. The complex mechanisms of HC need to be tightly regulated to provide true benefit. There is also a gradual realization that, while pH effects may be non-specific, they are not exclusively deleterious, and may have a role to play in a strictly controlled medical environment. Of greater excitement is the emerging possibility of specifically targeting molecules that are under the control of CO


We also observed elevated levels of neurofilament light (NfL) chain protein in the plasma of veterans displaying excess [18F]AV1451 retention. These findings suggest an association linking blast injury, tauopathy, and neuronal injury. Further study is required to determine whether clinical, neuroimaging, and/or fluid biomarker signatures can improve the diagnosis of long-term neuropsychiatric sequelae of mTBI.Traumatic brain injury (TBI) is a risk factor for the later development of neurodegenerative diseases that may have various underlying pathologies. Chronic traumatic encephalopathy (CTE) in particular is associated with repetitive mild TBI (mTBI) and is characterized pathologically by aggregation of hyperphosphorylated tau into neurofibrillary tangles (NFTs). CTE may be suspected when behavior, cognition, and/or memory deteriorate following repetitive mTBI. Exposure to blast overpressure from improvised explosive devices (IEDs) has been implicated as a potential antecedent for CTE amongst Iraq and Afghanistan Warfighters. In this study, we identified biomarker signatures in rats exposed to repetitive low-level blast that develop chronic anxiety-related traits and in human veterans exposed to IED blasts in theater with behavioral, cognitive, and/or memory complaints. Rats exposed to repetitive low-level blasts accumulated abnormal hyperphosphorylated tau in neuronal perikarya and perivascular astroglial processes. Using positron emission tomography (PET) and the [ Concern exists over the role of traumatic brain injury (TBI) in the chronic cognitive and behavioral symptoms that may develop during or after military service . It is eSymptoms of post-traumatic neurodegeneration are variable but often include memory loss along with alterations in mood or personality, as well as poor impulse control and aggression. The pathology underlying these symptoms may represent Alzheimer’s disease (AD)-type dementia or another clinical entity known as chronic traumatic encephalopathy (CTE). CTE was first recognized in boxers and initially designated as “punch drunk syndrome” and later designated “dementia pugilistica” . ClinicaThere have been a few reports of pathologically confirmed cases of CTE in the military veteran population –22. In a18F]AV45 and the tau ligand [18F] AV1451. As an additional biomarker, we measured blood levels of neurofilament protein light chain (NfL), since elevated levels of NfL have been reported in human patients suffering from a variety of brain injuries including mTBI and neurodegenerative diseases AV45. Image acquisition began ~60 min post-injection and lasted for 20 min. MIM Software Version 6.1 was used to measure standard uptake value ratio (SUVr) for [18F]AV45 PET analysis in six predefined anatomically relevant cortical regions of interest ; the whole cerebellum used as a reference region AV1451 imaging was performed on day 2, after a 24-h washout period. Participants were injected with 370 MBq (10 mCi) of [18F]AV1451. Image acquisition began ~80 min post injection and lasted for 20 min [18F]AV1451 images were read by a neuroradiologist and deemed “positive” if the subject exhibited ligand retention in areas not known for non-specific or off target binding. SUVr images were calculated as the uptake at each voxel divided by the mean uptake within the inferior cerebellar gray matter ROI, which was identified from the T1-MPRAGE using the spatially unbiased atlas template of the cerebellum and brainstem (SUIT) v3.2 AV45, all veterans and members of the comparison group were identified as amyloid-negative as all ROIs SUVrs were below the cutoff level of 1.1. With regard to interpretation of [18F]AV1451 data, qualitative reads by a neuroradiologist (LK) led to identification of excess ligand retention in the brains of 5 of 10 veterans  vs. tau-negative veterans), we observed that the highest NfL levels were among veterans with excess [18F]AV1451 ligand retention . Future studies will be needed to determine whether there is any correlation in individual animals between serum NfL levels and the degree of CNS tauopathy.We also measured NfL levels in sera collected from rats at 4 or 10 months following blast exposure. We did not find differences between blast-exposed and controls at 10 months after blast exposure . At 4 moThe age-matched comparison group and veteran groups were significantly different in IQ, the comparison group having a higher average IQ . The groIn this study, we identified biomarker signatures in human veterans exposed to IED blasts in theater and rats exposed to repetitive low-level blasts in a shock tube. The veterans all reported histories of blast exposure and had chronic behavioral and cognitive complaints. The number of human blast exposures varied from as few as 1 to as many as 50 or more but were all associated with at least 1 mTBI by clinical history. The rat model was developed to mimic a level of blast exposure that would be associated with human mTBI or a subclinical blast exposure . Rats ex18F]AV1451 at the white/gray matter junctions, matching the anatomical distribution of tauopathy previously observed in postmortem studies of subjects with CTE [18F] AV1451 retention.The primary findings of this study reveal that: (1) repeated mTBI induced by blast-overpressure increased levels of p-tau-like immunoreactivity in the brains of rats from 6 weeks to 12 months following injury, with elevations observed in anterior cortex and hippocampus. Immunohistochemical staining revealed increased p-tau and somatodendritic redistribution during the same time frame. (2) Repeated mTBI induced by blast overpressure produced abnormal perivascular p-tau accumulation in astroglial processes surrounding blood vessels in rats. (3) 50% of veterans with a history of multiple blast exposure showed excess cortical retention of FAV1451 aAPOE genotype AV1451 retention in retired NFL players [18F] AV1451, and excess ligand retention was observed in the cerebellar, occipital, inferior temporal, and frontal regions [18F]AV1451 and FDDNP show different binding patterns and what is considered significant signal with one ligand  originates from regions that cannot be reliably assessed with [18F]AV1451 because these regions  are among those confounded by known patterns of non-specific or off-target retention of [18F]AV1451 AV1451 . MoreoveF]AV1451 ; others F]AV1451 , 81; andF]AV1451 . HoweverF]AV1451 . FurtherWith regard to cortical diffusivity analysis values and changes in the microstructure of white and gray matter, it is known that the accumulation of tau pathology  is correlated with disrupted cellular organization, including disruption of the columnar arrangement of cells, dendrites, and axon bundles in the cerebral cortex . One pos18F] AV1451. Moreover, the use of [18F]AV1451 for the in vivo detection of tauopathy in living veterans must be validated with postmortem neuropathological assessments. The pilot data in rats is also suggestive of an NfL elevation in serum at 4 months after blast exposure. While the present studies by themselves may not be conclusive they support an emerging consensus that NfL and other biomarkers may play some role in monitoring post concussive recovery and warrant further study in humans and in animal models.While these results are highly promising for developing a clinical biomarker signature that may one day support the diagnosis of CTE during life, there are limitations. The development of in vivo diagnostics of CTE is in its infancy and replicating these findings in larger cohorts with a range of TBI severity and frequency is warranted. Sample size in the current study precluded more detailed statistical analyses; however, the trends pointed toward higher plasma NfL levels in veterans who retained excess [Finally, we recognize that the healthy subject comparison group was made up of civilians who had no military exposure. The ideal control group would have been a group of non-blast exposed deployed veterans as well as veterans with no history of any TBI. However, we were not able to recruit such individuals at the time of this study. There are other studies that have performed imaging in veterans that have used as “healthy controls” civilian populations rather than non-blast or non-TBI military controls , 82 or hDefinitive studies will be required to establish both qualitative and quantitative associations between biomarker changes and chronic behavioral effects of blast. Anecdotal reports using other tauopathy ligands are also promising, and various ligands must be compared in the same patient at the same time in order to compare and contrast how these ligands perform , 84. TheSupplementary InformationSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Figure 8Supplementary Figure 9Supplementary Figure 10Supplementary Figure 11Supplementary Figure 12Supplementary Figure 13Supplementary Figure 14Supplementary Figure 15


Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne febrile illness with wide geographic distribution. CCHF is caused by infection with the Crimean-Congo hemorrhagic fever virus (CCHFV) and case fatality rates can be as high as 30%. Despite causing severe disease in humans, our understanding of the host and viral determinants of CCHFV pathogenesis are limited. A major limitation in the investigation of CCHF has been the lack of suitable small animal models. Wild-type mice are resistant to clinical isolates of CCHFV and consequently, mice must be deficient in type I interferon responses to study the more severe aspects of CCHFV. We report here a mouse-adapted variant of CCHFV that recapitulates in adult, immunocompetent mice the severe CCHF observed in humans. This mouse-adapted variant of CCHFV significantly improves our ability to study host and viral determinants of CCHFV-induced disease in a highly tractable mouse model. Hyalomma genus are the principal vector and reservoir for CCHFV and cases of CCHF closely follow the geographic range of Hyalomma ticks  is the cause of Crimean-Congo hemorrhagic fever (CCHF). CCHFV is among the most widely distributed hemorrhagic fever viruses with cases reported through Africa, the Middle East, Asia, and Southern and Eastern Europe . Ticks oma ticks . Climates Sweden . CCHF bes Sweden , and thes Sweden .-/-Ifnar1)   or throunar1-/-) . Infectinar1-/-) , althougnar1-/-) . Neverthnar1-/-) .-/-Rag2) and wild-type (WT) C57BL/6J mice to generate a mouse-adapted variant of CCHFV (MA-CCHFV). In contrast to the parental CCHFV strain, MA-CCHFV was able to cause severe disease in WT mice that was associated with replication to high titers in multiple tissues, severe pathology in the liver and a severe inflammatory immune response. Unexpectedly, we identified a significant sex-linked bias in disease severity with female mice largely resistant to severe disease. In addition, we found that both host innate and adaptive immune responses are necessary to survive MA-CCHFV infection. Cumulatively, we report here a mouse-adapted variant of CCHFV that recapitulates in WT mice many aspects of severe human cases of CCHF.We therefore sought to select a variant of CCHFV that was able to cause disease in fully immunocompetent mice. We serially passaged the clinical isolate, CCHFV strain Hoti, in mice deficient in adaptive immunity  route and for the first passage collected blood at 4 weeks post-infection (WPI). For passage 2, we inoculated a naive -/-Rag2 mouse with this blood and for remaining passages collected liver tissue when mice were exhibiting severe clinical signs of disease . Liver tissue was homogenized, clarified of large debris by centrifugation and inoculated IP into naive -/-Rag2 mice. At each passage, liver tissue from an individual mouse was passed into an individual naive mouse without purification or isolation . This serial passaging in -/-Rag2 mice was performed nine times during which we observed a decrease in time of onset of severe disease (> day 28 post-infection (PI) for passage 1 to <day 7 PI passage 9) (-/-Rag2 passage 4 (50). Upon infection of WT mice, this variant caused substantial weight loss and clinical disease in male WT mice  . We perfassage 4  and 9   . In contPI (<5%) . In addiPI (<5%) . During PI (<5%)  and signPI (<5%) . This wa50 to 10,000 TCID50 to determine the median infectious dose (ID50) and to evaluate whether there was a correlation between virus dose and disease severity, as has been seen with mouse-adapted Ebola virus . Similar to C57BL/6J mice  WT C57BL/6J and several other commonly used laboratory strains of mice demonstrated that MA-CCHFV infection results in a severe but rarely fatal infection. A lethal model of MA-CCHFV infection would have utility for studies evaluating antiviral therapeutics or therapeutic interventions that seek to prevent CCHFV-induced mortality. For several viral infections, younger mice exhibit more severe disease than older mice  and neonTo determine if MA-CCHFV had an increased ability to replicate and disseminate in wild-type mice, we evaluated viral loads in several tissues of adult male and female WT mice infected with parental CCHFV strain Hoti or MA-CCHFV. We necropsied mice shortly after infection (1 DPI), early acute disease (3 DPI), peak clinical disease (6 DPI), early convalescence (8 DPI) and when mice had resolved all overt clinical signs of disease (14 DPI). In the plasma, at day 1 PI, mice infected with either Hoti or MA-CCHFV had similar RNA titers (p>0.05) . In miceWe next evaluated viral RNA loads in the liver. At day 1 PI, male or female mice infected with parental strain Hoti or MA-CCHFV had similar viral RNA loads in the liver suggesting efficient dissemination to the liver independent of sex or virus strain . However-/-Ifnar1 mice  of day 6 and day 3 PI, respectively  that is proteolytically processed to produce a heavily glycosylated mucin-like domain (MLD), GP38 accessory protein, the envelope glycoproteins Gn and Gc and the medium non-structural protein NSm . Three mBunyavirales order, encoding a protein of over 3900 amino acids. It encodes the viral RNA-dependent RNA-polymerase (RdRp), along with domains for a zinc finger and leucine zipper  were also present in the passage 4 stock at a frequency of >80% of reads . These dof CCHFV . We founof CCHFV . The mutof CCHFV . Interesof CCHFV .-/-Ifnar1)   or transnar1-/-) . Thus, unar1-/-)  and ethinar1-/-)  suggesti-/-Ifnar1 and -/-Rag1 mice demonstrating that resistance to MA-CCHFV by female mice is age-dependent and requires both innate and adaptive host responses. Nevertheless, the relevance of this sex-linked bias in mice toward human disease is unclear. Although some studies have identified human males as more likely to become infected with CCHFV , wild-type C57BL/6J (stock #000664), BALBc/J (stock #000651), and 129S1 (stock #002448) were purchased from Jackson Laboratories. C57BL6/NCr (strain code 027) and outbred CD1 mice (strain code 022) were purchased from Charles River Laboratories. -/-Ifnar1 mice on the C57BL/6J background were from an in-house breeding colony. Mice were randomly assigned to study groups. Unless otherwise indicated, mice were all 6–8 weeks of age at time of infection except for the first passage in wild-type mice (passage 10) which utilized mice 3 weeks of age. Mice were humanely euthanized according to the following criteria: ataxia, extreme lethargy , bloody discharge from nose, mouth, rectum or urogenital area, tachypnea, dyspnea or paralysis of the limbs. Although animals were comprehensively evaluated for the above signs, animals that succumbed following MA-CCHFV infection were typically euthanized for extreme lethargy and dyspnea and in one mouse, ataxia was also present. For survival analysis, mice euthanized for severe disease were recorded as having succumbed +1 day to day of euthanasia. For ID50 calculations, mice with detectable anti-CCHFV Ig (A405nm >0.2) at a 1:400 dilution of serum were considered productively infected and ID50 calculated using the Mice were humanely euthanized, and piece of liver collected into a tube. A steel bead and 1 mL of L-15 media (ATCC) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin added. Tissue was homogenized at 30 hz for 1 min in a TissueLyser (Qiagen) then briefly spun at maximum RPM in a benchtop centrifuge to pellet large debris. Clarified tissue homogenate was then innoculated into naive mice via the IP route.50) assay. Virus stocks were sequenced with Illumina MiSeq-based deep sequencing to exclude contamination, including mycoplasma or other viral pathogens, and to identify mutations present. The sequence of MA-CCHFV has been deposited to Genbank (Accession #s MW058028 – MW058030). Mutations in mouse passaged CCHFV were identified by comparison to parental strain CCHFV Hoti sequenced in parallel. Minimum read depth at any mutation identified in the MA-CCHFV stock was 152 reads. For sequence comparison, we compared MA-CCHFV sequence to Afghan09 , ArD15786 , IbAr10200 , Turkey 2004 , Oman , UG3010 , and Hoti .Parental strain CCHFV Hoti was grown, titered, and sequenced as previously described . MA-CCHFAt time of euthanasia, whole blood was collected into lithium heparin treated tubes and blood chemistry analyzed with Preventive Care Profile Plus disks on Vetscan two analyzers (Abaxis). The complete blood chemistry data is available in the supplemental materials.At time of euthanasia, whole blood was collected into lithium heparin treated tubes (BD) via cardiac puncture. Plasma was separated by centrifugation and irradiated according to approved procedures to inactivate CCHFV. Plasma cytokine levels were analyzed by 23-plex mouse cytokine assay according to manufacturer’s instructions (Biorad). Plasma IFNα  and IFNβ levels were quantified in 1:10 dilutions of plasma by ELISA according to manufacturer’s instructions (PBL Assay).TCTACATGCACCCTGCTGTG, Reverse: 5’- AGCGTCATCAGGATTGGCAA and probe 5’- TGGGTGTCTGCTTTGGAACA were used in a one-step qRT-PCR reaction with either Quantifast reagents (Qiagen) for tissue RNA or LightCycler 480 RNA Master Hydrolysis Probes (Roche) for plasma RNA samples. Probe was labeled with a 5’ 6-FAM, ZEN internal quencher and 3’ Iowa Black quencher. Primers and probes were purchased from Integrated DNA Technologies. Reactions were run on a Quantstudio 3 or 5 instrument (ThermoFisher). Cycling conditions for Quantifast reagents were: 50°C for 10 min, 95°C for 5 min and 40 cycles of 95°C for 10 s, 60°C for 30 s. Cycling conditions for LightCycler 480 reagents were 61°C for 3 min, 95°C for 30 s and 45 cycles of 95°C for 10 s, 60°C for 30 s and 72°C for 1 s. An in vitro transcribed RNA standard curve was generated by T7 runoff transcripts of the CCHFV S segment and included in every run.RNA from mouse plasma was isolated using Qiamp RNA-mini isolation kit (Qiagen) and RNA from tissues isolated using the RNeasy mini isolation kit (Qiagen). Viral loads were quantified by qRT-PCR as follows: primers and probe specific for the CCHFV S segment: Forward: 5’- An ELISA to detect anti-CCHFV Ig was performed as previously described  with an An IFNγ ELISpot on splenocytes stimulated with peptides derived from the CCHFV NP was performed as before .Tissues were fixed in 10% neutral buffered formalin with two changes for a minimum of 7 days. Tissues were placed in cassettes and processed with a Sakura VIP-6. Tissue Tek on a 12 hr automated schedule, using a graded series of ethanol, xylene, and PureAffin (Cancer Diagnostics). Embedded tissues were sectioned at 5 μm and dried overnight at 42°C prior to staining. Specific anti-CCHF immunoreactivity was detected using a rabbit anti-CCHF NP (IBT Bioservices) at a 1:2000 dilution as the primary antibody and Vector Laboratories ImPRESS-VR anti-rabbit IgG polymer kit  neat as the secondary antibody. The tissues were processed for immunohistochemistry using the Ventana Ultra automated stainer using the Roche Tissue Diagnostics Discovery ChromoMap DAB detection kit . Tissue sections were scored by certified pathologists who were blinded to study groups. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study presents the first description of a lethal mouse model of CCHFV in immunocompetent mice. Development of a mouse-adapted strain of CCHFV, along with identification of differences in disease susceptibility based on sex of the animal, represent an important finding in the field of emerging viruses. The work is extremely well done, with defined study aims, clearly presented results and robust data covering the major aspects of disease pathology. Furthermore, the findings presented here are important given the interest and need for an immunocompetent small animal model of CCHFV disease. This work will greatly benefit the field in both basic understanding of CCHFV pathology as well as in ongoing antiviral and vaccine development.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Miles Davenport as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Dennis Bente (Reviewer #3).Thank you for submitting your article "Immunocompetent Mouse Model for Crimean-Congo Hemorrhagic Fever Virus" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:The development of a suitable animal model that mimic the human-specific disease Crimean Congo hemorrhagic fever has been difficult since the discovery of the disease in 1944. Immunocompetent small mammalian models are permissive to infection but refractory to disease. As a result, mice with knock outs in the interferon response have been used, which are not ideally suited of the immune system and vaccines. Hawman et al. present and characterize an immunocompetent mouse model for CCHF by adapting the CCHFV to the mouse. This is a valuable tool in pathogenesis, countermeasure, and virus evolution studies. This straightforward, well-written manuscript that also describes important sex differences in susceptibility to disease in mice infected with the mouse-adapted CCHFV. Overall, this study is a significant step forward for the field through its description of an immunocompetent mouse model. Comments to improve the text are listed below.Revisions:1) One critique of the study is the lack of data detailing the clinical disease of MA-CCHFV itself. While weight loss is a useful metric in this model, I would have liked to have seen an evaluation of clinical signs throughout the challenge period, i.e., clinical scores at each day, which would help the reader better understand any inherent variability in the model. For example, and it is touched upon in the results, the large error bars in Figure 1B indicate that the female mice infected with MA-EBOV have a much more variable disease course than male mice – a representation of clinical scores would help with the reporting of this data. It may also be useful to represent individual animals in these weight loss graphs rather than use the mean, again for clarity. A clearer understanding of the expected disease course and clinical signs in a MA-CCHFV infection from challenge to recovery is would benefit any future research understanding the efficacy of potential antiviral compounds or vaccines.2) Why were the passaged viruses not plaque -picked, and can the authors speculate on how the viral swarm/quasi species is influenced by the passaging? The authors comment on the non-synonymous mutations but not really how the SNP change from passage to passage.3) Results first paragraph: can the authors include the onset of disease for each passage in the supplementary information?4) Was there a reason than an intermediate dose  of MA-CCHFV was used when infecting multiple mouse strains rather than the 10,000 TCID50s used elsewhere?5) While MA-CCHFV was shown to be lethal in the 3wk old mice, do we have comparative data for for wt Hoti?6) The authors state that the clinical presentation was milder in females. I might have overlooked this in the manuscript, but is there a statistical side-by-side comparison of males vs. females in their different clinical parameters ? Figure 1?7) The authors state "NSm is dispensable for virus replication ". This is true for mammalian systems, however, not in mosquitoes, where it determines vector competence. Han et al., 2016 demonstrated a non-synonymous amino acid change in NSm after the mouse-passaged CCHFV strain IbAr 10200 was passaged back in ticks. This could be thought of a “reversion” back to a virus that can replicate in invertebrate and vertebrate system.8) The following paper is often overlooked in the CCHFV literature.:Host-passage-induced phenotypic changes in crimean-congo haemorrhagic fever virus Gonzalez et al., 1995. If possible, it would be good to cite the paper in the beginning of the Discussion.9) The authors refer to no "widely" approved vaccines for CCHF. Are there any approved vaccines? The word widely implies that there are vaccines that have limited distribution. Revisions:1) One critique of the study is the lack of data detailing the clinical disease of MA-CCHFV itself. While weight loss is a useful metric in this model, I would have liked to have seen an evaluation of clinical signs throughout the challenge period, i.e., clinical scores at each day, which would help the reader better understand any inherent variability in the model. For example, and it is touched upon in the results, the large error bars in Figure 1B indicate that the female mice infected with MA-EBOV have a much more variable disease course than male mice – a representation of clinical scores would help with the reporting of this data. It may also be useful to represent individual animals in these weight loss graphs rather than use the mean, again for clarity. A clearer understanding of the expected disease course and clinical signs in a MA-CCHFV infection from challenge to recovery is would benefit any future research understanding the efficacy of potential antiviral compounds or vaccines.Unfortunately, we did not collect clinical scores during these studies and thus the requested data does not exist. However, our subjective opinion is that weight loss correlated well with overt clinical signs of disease such as ruffled fur, hunched posture and lethargy. In addition to weight loss, we measured viral loads, inflammatory cytokines and liver enzymes that all correlated with disease severity. These objective parameters could be used as read outs for the efficacy of vaccines and antivirals.Although the error bars in Figure 1B are larger, cumulatively over the course of the studies we have observed consistently less disease in female mice although on occasion we do have female mice that have disease similar to male mice. We have provided the data as individual mice in 2) Why were the passaged viruses not plaque -picked, and can the authors speculate on how the viral swarm/quasi species is influenced by the passaging? The authors comment on the non-synonymous mutations but not really how the SNP change from passage to passage.We avoided plaque-picking virus as we wished to avoid any growth of virus in tissue culture until final generation of stocks. We had concerns that passaging in tissue culture may select against mouse-adapted variants. For example, a mouse-adapted variant may plaque poorly or not grow at all in human-cell based tissue culture and thus would be “lost” in this approach. We have further specified in the text that liver tissue was passed mouse to mouse without isolation or purification. However, we do agree that the viral swarm may be important and cannot claim that our passaging scheme is the only one that would have resulted in similar mouse adaptation. We have not done formal evaluations of the quasi-species when virus is grown in mouse liver tissue versus tissue culture. In addition, Table 1 does list the prevalence of identified mutations at passage 4, 9 and 11. By passage 4 all mutations except synonymous M A1502G are present at >80% of reads. M A1502G increased in frequency during passages.3) Results first paragraph: can the authors include the onset of disease for each passage in the supplementary information?We have included when we collected the sample in updated Figure 1—figure supplement 1 and the weight loss of each passage. Weight loss in these mice correlates with onset of other signs of disease. For this data it is important to consider that we did not weigh the piece of liver collected nor titer the amount of CCHFV in the homogenized sample and therefore inoculating virus dose at each passage is not uniform. It is also limited in that only one mouse was inoculated with each tissue sample at each passage. Thus, we caution drawing conclusions on the virulence of any particular passage based on this data alone. Importantly, we omitted to specify that following the first passage of CCHFV, CCHFV in the blood of the passage 1 mouse was transferred to the naïve passage 2 mouse. Thereafter liver tissue was used for all passages. We have updated the text accordingly.4) Was there a reason than an intermediate dose  of MA-CCHFV was used when infecting multiple mouse strains rather than the 10,000 TCID50s used elsewhere?While no inverse correlation between disease severity and virus dose was observed in B6 mice, we still had some concern that this may not hold true for other mouse strains. For example, with MA-EBOV an inverse correlation was seen in B6 but not BALBc mice . To minimize the number of mice used, rather than perform dose finding in multiple mouse trains we picked 1000 TCID50 as it was a middle ground between doses we knew to reliably cause disease in B6 mice. For further model development in any of these strains it may be important to perform similar dose finding studies.5) While MA-CCHFV was shown to be lethal in the 3wk old mice, do we have comparative data for for wt Hoti?We have not performed similar studies with CCHFV strain Hoti. We expect that infection of young WT mice with parental Hoti would not result in lethal disease as these younger mice still possess an intact, if not fully developed, type I IFN response. However, it is possible that young WT mice may show greater disease or viral replication with parental Hoti compared to adult mice.6) The authors state that the clinical presentation was milder in females. I might have overlooked this in the manuscript, but is there a statistical side-by-side comparison of males vs. females in their different clinical parameters ? Figure 1?We agree that this statistical comparison is important and have included it in Figure 1—figure supplement 2. Male mice had significantly greater weight loss than female mice on days 5, 6, 7 and 8 PI in this study. Similar findings were found in Figure 6 and 7 although since the major conclusions of those figures are comparison between WT and KO strains, we have not included the male and female WT statistical comparison in those figures.7) The authors state "NSm is dispensable for virus replication ". This is true for mammalian systems, howver, not in mosquitoes, where it determines vector competence. Han et al., 2016 demonstrated a non-synonymous amino acid change in NSm after the mouse-passaged CCHFV strain IbAr 10200 was passaged back in ticks. This could be thought of a “reversion” back to a virus that can replicate in invertebrate and vertebrate system.We agree and have added text to reflect this finding.8) The following paper is often overlooked in the CCHFV literature.:Host-passage-induced phenotypic changes in crimean-congo haemorrhagic fever virus Gonzalez et al., 1995. If possible, it would be good to cite the paper in the beginning of the Discussion.We have added this reference and expanded the Discussion on host-selective pressures for CCHFV.9) The authors refer to no "widely" approved vaccines for CCHF. Are there any approved vaccines? The word widely implies that there are vaccines that have limited distribution.There is a Bulgarian vaccine using inactivated CCHFV grown in mouse brains that is approved for use in that country. Although this vaccine appears protective it is unlikely this vaccine would ever achieve wider distribution due to safety concerns inherent to how it is manufactured.


Whether age has any impact on the risk of lymph node (LN) metastasis in patients with early-stage non-small cell lung cancer (NSCLC) remains controversial. Therefore, we aimed to objectively compare the risk of LN metastasis between elderly and young patients so as to justify for age-different extent of surgical resection for treating these patients. We retrospectively collected clinical data of patients undergoing lobectomy or segmentectomy with systematic hilar and mediastinal LN dissection for clinical stage IA peripheral NSCLC from January 2015 to December 2018. Both multivariate logistic regression analysis and propensity score-matched (PSM) analysis were applied to compare the risk of LN metastasis between elderly (>65 years old) and young (≤65 years old) patients. We finally included a total of 590 patients for analysis (142 elderly patients and 448 young patients). In the analysis of unmatched cohorts, young patients tended to have higher rates of hilar/intrapulmonary LN (13.4% VS 9.2%) and mediastinal LN metastasis (10.5% VS 6.3%) than elderly patients. In the multivariate analysis, age was found to be an independent predictor of both hilar/intrapulmonary (Odds ratio(OR) = 2.065, 95%confidence interval(CI): 1.049–4.064, P = 0.036) and mediastinal  LN metastasis. Moreover, in the analysis of well-matched cohorts generated by PSM analysis, young patients had significantly higher rates of hilar/intrapulmonary  and mediastinal LN metastasis  than elderly patients. Therefore, age remains to be an independent predictor of LN metastasis in early-stage NSCLC and age-different extent of surgical resection may be justified for these patients. With the advancement of computed tomography (CT), more and more early-stage NSCLCs are being found4. For the management of early-stage NSCLC, anatomic lung resection with systematic lymph node (LN) dissection (SLND) or sampling is recommended as the preferred option in the National Comprehensive Cancer Network guideline5. However, the extent of lung resection and lymphadenectomy for treating early-stage NSCLC depends largely on the characteristics of tumors such as tumor size, status of LN metastasis, histology as well as ground glass consistency6 and the patient’s physiological conditions.Lung cancer has become the leading cause of cancer and cancer-related death worldwide7 and the number of elderly patients continues to increase with the aging population. Moreover, significant difference of baseline characteristics between elderly and young lung cancer patients was observed8. However, current guideline still recommends the same surgical strategies for elderly and young patients except for elder patients with age-related or other co-morbidities which may lead to high risk of perioperative morbidity and mortality, of whom less extensive resection is suggested5. Hence, we are wondering whether there is evidence justifying age-different extent of surgical resection between elderly and young patients with early-stage NSCLC. As we know, from surgical perspectives, the optimal extent of surgical resection for early-stage NSCLC depends largely on the risk of intrapulmonary and mediastinal LN metastasis9. Therefore, it is of great value to compare the risk of LN metastasis between elderly and young patients with early-stage NSCLC so as to provide evidence justifying for age-different extent of surgical resection for elderly and young NSCLC patients. Previous studies comparing the risk of LN metastasis between elderly and young patients have reported different results due to significant patient selection bias11. Therefore, in this study, we aimed to draw an objective conclusion regarding the different risk of LN metastasis  between elderly and young patients by conducting both multivariate logistic regression analysis and propensity score-matched (PSM) analysis, so as to provide evidence for justifying age-different extent of surgical resection for elderly and young patients with early-stage NSCLC.It is reported that the age at diagnosis for majority of early-stage NSCLC patients falls into the range from 65 to 79 years old12. In our center, contrast-enhanced chest and upper abdominal CT, brain magnetic resonance imaging, bone scanning, and cardiopulmonary tests were routinely carried out for preoperative assessment of lung cancer patients13. Positive LN metastasis was defined as LN with a short-axis diameter >1 cm on transverse chest CT scan. Routinely, in our center, we would not perform invasive preoperative staging for resectable lung cancer patients. We set the following inclusion criteria for patient inclusion: 1) patients should undergo upfront lobectomy or segmentectomy with SLND without any preoperative therapy; 2) patients should be preoperatively evaluated with clinical stage IA disease without positive LNs on preoperative CT scan; 3) patients should have peripherally located lung cancer(defined as tumors in the outer one third of the hemithorax)14; 4) patients should be pathologically diagnosed as primary NSCLC. The exclusion criteria were also as follow: 1) patients who received preoperative chemoradiotherapy; 2) patients who were confirmed with synchronous multiple primary NSCLC or SCLC or secondary lung cancer.; 3) patients who had centrally located NSCLC; 4) patients who were intraoperatively found to have pleural metastasis. Our work was approved by the Ethics Committee of West China Hospital, Sichuan University. Because this study was a retrospective study and patients were analyzed anonymously in the study, the Ethic Committee of our hospital  waived the need for consent. Moreover, this study was carried out in accordance with the Declaration of Helsinki.This was a retrospective study analyzing clinical and pathological data of patients receiving lobectomy or segmentectomy with SLND in our center between January 2015 and December 2018. In our center, the standard procedures of SLND were performed in accordance with The European Society of Thoracic Surgeons guideline15.In this study, we analyzed the data of age, gender, radiographic features, pathologic findings, and LN metastasis status. The risk of LN metastasis was analyzed by age and patients were subgrouped into elderly group (>65 years old) and young group (≤65 years old) according to their age at diagnosis. Radiographic features included tumor location, tumor consistency (solid/part-solid/ground glass), and tumor size. Pathologic findings consisted of tumor histology and differentiation as well as the status of visceral pleural invasion(VPI). Patients in the study were staged based on the eighth edition of TNM staging system for NSCLC and LNs were further subgouped into N1(hilar/intrapulmonary LNs) and N2 LN stations16 to further validate our results based on well-matched cohorts. The following covariates were used for matching: gender, tumor size, histology, tumor differentiation and consistency. Moreover, we used the nearest neighbor method to match elderly and young patients at a ratio of 1:1 with a caliper width of 0.01. Here, the statistical significance was set as a two-sided P-value of <0.05.All statistical analyses were conducted by using the IBM SPSS software . We presented continuous data as mean ± standard deviation and categorical data as number with percentage. Chi-squared test or Fisher’s exact test was used to compare categorical data between groups while independent-sample Student’s t-test or the Mann–Whitney non-parametric U-test was applied for comparing continuous data. We applied multivariate logistic regression analysis to explore potential predictors of LN metastasis  for these patients, which was based on our discretion by adding all clinically relevant variables. Moreover, we further performed PSM analysis using the PSMATCHING 3.04 and R-2.15.1-win software as previously describedin situ, 42 patients as minimally invasive adenocarcinoma, 56 patients as T1a disease, 163 patients as T1b disease, 152 patients as T1c disease, and 162 patients were upstaged as T2a disease due to VPI (27.5%). The mean number of total dissected LN was 12.3 ± 5.8 and it was 4.1 ± 3.2 for N1 LNs and 8.2 ± 4.1 for N2 LNs. As for LN metastasis, 14.1% of the patients were confirmed to have positive LNs with a N1 LN metastasis rate of 12.4% and N2 LN metastasis rate of 9.5%.In total, 590 patients met our inclusion criteria and were enrolled in the study. The baseline characteristics of these patients are listed in Table The lymph node metastasis status of different age groups was presented in Fig. A multivariate regression analysis was conducted to identify potential risk factor of LN metastasis (both for N1 and N2 LNs) in clinical IA NSCLC patients by entering relevant factors into multivariate analyzing model  and young (≤65 years old) patients by including a total of 590 patients with clinical IA peripheral NSCLC. In the analysis of unmatched cohorts, we found that young patients tended to have a higher rate of N1 (P = 0.181) and N2 (P = 0.141) LN metastasis than elderly patients. However, significant patient bias such as gender, tumor size, histology, tumor differentiation, and tumor consistency was observed. Therefore, during multivariate regression analysis adjusting for above potential factors, age was found to be an independent predictor of N1  and N2  LN metastasis in patients with clinical IA peripheral NSCLC. Moreover, in the analysis of well-matched cohorts by PSM analysis, we further confirmed that young patients had a significantly higher risk of LN metastasis (both N1 and N2 LNs) than elderly patients.The status of LN metastasis remains to be an important factor determining the extent of lung resection and lymphadenectomy in the surgical management of early-stage NSCLC. However, the impact of age on the risk of LN metastasis remains controversial among previous literatures21. However, some of the previous studies also showed that age did not significantly influence the risk of LN metastasis in NSCLC23. Possible reason for the discrepancy were the patient inclusion criteria, analytical methods, and sample size. For example, some studies only focused on clinical stage I NSCLC23 while others focused on resectable NSCLC 20. Moreover, some studies calculated age as categorical data21 while others analyzed age as continuous data10. Nearly all these previous studies finding no correlation between age and LN metastasis had a relatively limited sample size (<300 cases)23. Therefore, it is reasonable that previous studies have drawn different conclusions about the comparison of LN metastasis between elderly and young patients.Our findings agreed with the majority of previous studies concluding that young age was an independent predictor of LN metastasis in NSCLC24 and young patients were found to have a significantly higher rate of hematogenous dissemination of lung cancer cells during surgery25 and worse outcome of distant metastasis dynamics26 than elderly patients, indicating that lung cancer in young patients may exhibited more malignant and higher aggressive features than that in elderly patients. Moreover, previous experimental studies also found that age-related alterations of the extracellular matrix may lead to the less invasive behavior of lung cancer in aged animal models27. As a result, considering the significant different risk of LN metastasis and metastatic behavior between elderly and young patient, age-different extent of surgical resection may be indicated for early-stage NSCLC patients. Previous evidence from retrospective studies showed that in elderly patients with early-stage NSCLC, sublobar resection may yield equivalent long-term outcomes to lobectomy28. Even though more elderly patients received sublobar resection, they may yield similar cancer-specific survival to young counterparts29. Moreover, in young patients with early-stage NSCLC, lobectomy was found to confer better survival than sublobar resection30. Taken together, we think that it may seem justifiable to provide compromised limited lung resection and lymphadenectomy to elderly patients with early-stage NSCLC especially for those with chronologic comorbidities whereas for young counterparts standard lung resection and lymphadenectomy should always be anticipated. However, one should be cautious when deciding limited lung resection and lymphadenectomy for elderly patients since they still have the chance of nodal upstaging despite of a very low rate.Since there is no study directly comparing the risk of LN metastasis between elderly and young NSCLC patients available yet, we conducted such study for the first time by applying both multivariate regression analysis and PSM analysis. Both multivariate regression analysis and PSM analysis confirmed that young age was significantly correlated with LN metastasis (both N1 and N2 LNs), which provided objective evidence that young age was an independent predictor of LN metastasis in clinical IA NSCLC. Previously, young age was also confirmed to be an independent risk factor of brain metastasis in NSCLC patientsOur study had several limitations. First, our study was a retrospective analysis and the retrospective study design could limit our analytical power. Second, the conclusions were drawn only based on LN metastasis rate not on survival analysis. Moreover, positron emission tomography (PET)/CT was not commonly applied in our study and it is unclear how our data and results would apply to cohorts staged with PET/CT. Finally, the exact extent of limited lung resection and lymphadenectomy for elderly patients needs to be further established. Therefore, further prospective well-designed studies with survival analysis are warranted to confirm our conclusions.In this study, we objectively compared the risk of LN metastasis (both N1 and N2 LNs) between elderly and young patients with clinical stage IA peripheral NSCLC. Young patients were found to have a significantly higher risk of LN metastasis than elderly counterparts in both multivariate regression analysis and PSM analysis after adjusting for confounding factors. Therefore, young age was an independent predictor of LN metastasis in early-stage NSCLC and age-different extent of surgical resection may be indicated for treating these patients.


PARP1 in lung cancer is still not clear. Analysis at allelic levels cannot fully explain the function of PARP1 on lung cancer. Our study aims to further explore the relation between PARP1 haplotypes and lung cancer.The carcinogenesis role of PARP1-SNPs were genotyped and PARP1-specific SNPs were imputed using IMPUTE and SHAPEIT software. The SNPs were subjected to allelic, haplotype and SNP-SNP interaction analyses. Correlation between SNPs and mRNA/protein expressions were performed.DNA and RNA were extracted from non-small cell lung cancer (NSCLC) tumor and adjacent normal fresh frozen tissue. Five PARP1  is able to differentiate the NSCLC tumor from normal tissues. Interaction between rs3219073, rs1805415, and rs1805414 were significantly associated with the NSCLC tumor with OR ranging from 3.61–6.75; 95%CI from 1.82 to 19.9; P-value<0.001.SNP imputation inferred the ungenotyped SNPs and increased the power for association analysis. Tumor tissue samples are more likely to carry rs1805414  and rs1805404  compared to normal tissues. Our study is the first study to show that haplotypes comprising of 5 SNPs on PARP1 haplotypes may serve as a better predictor in lung cancer development and prognosis compared to single alleles. TP53, EGFR, and KRAS have been identified and are currently used as biomarkers in clinical practice, but are only effective for selected lung cancer patients  were imported into GraphPad Prism 8. The normalized values for paired normal lung tissues were compared to normalized values for AC tissues and for SCC tissues using a paired and unpaired t-test. Data are presented as mean ±SEM. The purpose of this data was to functionally validate significant genotypes.Total of 83 subjects who were diagnosed as NSCLC were included in this study, among which 62 of them had paired tumor and normal tissues collected. The total number of samples is 145  were highly correlated with each other and together defined a block [32 Kilobase-pair] shown in PARP1 gene for the lung tumor tissue samples and lung normal tissue samples to examine the differences in the LD pattern of PARP1 SNPs between lung tumor (block size: 12 kilobase-pair) and normal tissues (block size: 32 kilobase-pair)  was more likely to present in normal tissue samples. Haplotypes “AATCCTCCGCGA”, “AGCGCTAGGCGA”, and “GGCGAGACGTTG” in the second cluster (in pink) were more likely to present in lung tumor tissue samples (AGCGCTAGGCGAG” and “GGCGAGACGTTGA” in the second cluster (in blue) compared to SCC lung tissue samples  was raised against the amino acids 764–1014 of PARP of human origin. The binding site of antibody covers part of PARP catalytic domain and whole PARP alpha-helical domain. The results showed that the expression of PARP1 was similar between lung tumor tissue samples compared to paired normal lung tissue samples. PARP1 was significantly overexpressed in AC tissue samples compared to paired normal lung tissue samples, but there was no significant difference when comparing SCC lung tissue samples to paired normal lung tissue samples  were significantly associated with both lung tumor and SCC tissue compared to normal lung tissues. SNP (rs1136410-G) was associated with lung tumor tissues compared to normal with a P-value of 0.062 . However, those SNPs could not differentiate between AC and normal tissues or between AC and SCC tissues at allelic/genotypic level.To comprehensively investigate the role of PARP1 and showed a significant interaction between rs3219073, rs1805414, and rs1805415 that contributed to an increased risk of AC and SCC. The strategic selection of these genotyped SNPs based on functional importance alongside imputed SNPs that share an LD block has the potential to identify complex biological links among SNPs derived from PARP1 functional domains and cancer processes. For example, rs3219073 is located in the DNA-damage checkpoint and implicated in DNA-repair pathways while rs1805414, and rs1805415 are in the DNA binding domain , and tested for association with lung cancer [AATCCTCCGCGA”, “AGCGCTAGGCGA”, “GGCGAGACGTTG”) that were more likely to present in lung tumor tissues compared to normal tissues Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(ZIP)Click here for additional data file.


KRAS was the most significantly enriched driver gene, with 4/50 (8%) of brain metastases harbouring non-synonymous mutations. Hotspot KRAS mutations were mutually exclusive from BRAFV600, NRAS and HRAS mutations and were associated with a reduced overall survival from the resection of brain metastases . Mutations in KRAS were clonal and concordant with extracranial disease, suggesting that these mutations are likely present within the primary. Our analyses suggest that KRAS mutations could help identify patients with primary melanoma at higher risk of brain metastases who may benefit from more intensive, protracted surveillance.Brain metastases are a major cause of melanoma-related mortality and morbidity. We undertook whole-exome sequencing of 50 tumours from patients undergoing surgical resection of brain metastases presenting as the first site of visceral disease spread and validated our findings in an independent dataset of 18 patients. Brain metastases had a similar driver mutational landscape to cutaneous melanomas in TCGA. However,  Importantly, epidemiological data suggest that patterns of metastatic dissemination may be partially determined by the clinical characteristics of the primary tumour.3Metastases to the central nervous system (CNS) are observed in ~60% of cutaneous melanoma patients developing disseminated disease and up to 90% at autopsy.4 Primary tumours in these ‘early brain metastasis’ cases were reported as thinner and of lower American Joint Committee of Cancer Stage when compared to other visceral metastases, challenging the current understanding of brain metastases as the final stage of tumour progression, and suggesting that these tumours could harbour distinct biological properties favouring early haematogenous dissemination to the brain.4 Our analyses of the mutational landscape of early brain metastasis highlights key molecular features that could inform future prognostic, surveillance and intervention strategies.Interestingly, 15–20% of brain metastases present as the isolated first visceral site of disease spread.n = 34), The Wellington School of Medicine (n = 8), New York University School of Medicine (n = 4) and Cambridge University Hospitals (n = 4) (discovery cohort). Samples from patients selected from The University of Queensland Australia and the Auckland region New Zealand  made up the external validation cohort. All neuro-resections were undertaken between 2008 and 2018 at the respective academic neurosurgical centres as part of routine clinical care. All cases were ethically approved by the local Institutional Review Boards, as well as by the Sanger Institute’s human materials and data management committee. All samples and clinical details are listed in Supplementary Table Patients with available archival paraffin-embedded melanoma brain metastases  were selected from prospectively maintained databases at The Melanoma Institute of Australia  were extracted from the publication by Zehir et al.6  et al.6 .n = 287) cancer-driver genes identified from analysis of the TCGA and ICGC cohorts  platform at the Wellcome Sanger Institute. MuTect (v1.1.7) and Sequenza (v2.1.2) were used to call somatic single nucleotide variants (SNVs) and copy number aberrations, respectively. Melanoma-driver SNVs called in the whole-exome-sequenced discovery cohort were orthogonally validated  using a custom gene panel designed to capture . Between-group differences in instantaneous risk were assessed by fitting univariate and multivariate Cox proportional hazards regression models and defining 95% confidence intervals (CIs) for relevant hazard ratios (HRs). Multivariate models were controlled for further predictors including sex, age, centre, BRAF and NRAS mutation status (as well as primary tumour characteristics where relevant), see Kaplan–Meier plots were used to compare survival of n = 25, 50%) and non-ulcerated  primary melanomas  (–T2 (n = BRAF were detected in 21 (42%) tumours, of which 15/21 (71%) were in the V600 hotspot  tumours and were all in hotspot positions on exons 2 (codons 12 and 13) and 3 (codon 61) and mutually exclusive from BRAFV600 mutations. Comparing the mutational landscape of brain metastases to that of cutaneous melanomas in the SKCM-TCGA dataset  vs 2% (7/358) in the entire SCKM-TCGA collection . Note that, although we identified 5 KRAS mutations within the discovery cohort, the KRASG115R mutation (occurring in association with a BRAFV600E-driver mutation in sample PD42113a) is exceptionally rare7 and was not considered pathogenic  in our dataset vs 2.1% (6/274) in extracranial melanoma metastases in SKCM-TCGA . The odds of observing a KRAS mutation in a given sample within the early brain metastases discovery cohort was approximately fourfold higher than in these three reference datasets , indicating that they likely represent clonal (early) driver mutations  signalling genes including BRAFV600, NRAS and HRAS, and this pattern of mutually exclusivity was also observed in KRAS-mutant melanomas within the SKCM-TCGA and SKCM-MSK datasets   to be somatic mutations see . We also% to be sKRAS-mutant brain metastases succumbed to disease, with a median overall survival from resection of brain metastasis of only 3 months, compared to 12 months in patients with resected KRAS wild-type brain metastases , although this did not meet the threshold for statistical significance after correction of clinical covariates likely due to the limited sample size  and a similar frequency of the key driver mutations, including BRAF (42%), NRAS (28%), NF1 (22%) and TP53 (18%). This is the first study to show significant enrichment of KRAS mutations in melanoma brain metastases as well as an association of KRAS mutations with adverse outcomes. The predominance of KRAS mutations in codons 12, 13 and 61 as well as the mutual exclusivity with other key drivers of MAPK signalling suggests that these likely represent important drivers in this context.This analysis represents the largest survey of mutation profiles of melanoma brain metastases. Consistent with the landmark melanoma sequencing studies ,NRAS, KRAS and HRAS mutated in 25, 2 and 1% of melanomas, respectively.5NRAS-mutant melanomas are recognised to be more aggressive and associated with poorer outcomes; however, very little is known about KRAS-mutant melanoma.8KRAS-mutant early brain metastases in our study generally emanated from thin and non-ulcerated primary melanomas  pathways are the two key downstream signalling pathways through which constitutively activated RAS exerts its pro-tumorigenic effects. MAPK pathway activation and brain metastases are inextricably connected and Kras can induce naevi and be a founder event in melanomagenesis.12 In one study, a KrasG12D allele was combined with alleles of p53 or Lkb1 and with a melanocyte-specific Cre driver to generate a model that developed melanoma with a penetrance of 100%.13 In this study, metastases were identified in lymph node, lung, liver and spleen but not in kidney or brain. It is therefore important to consider that, while we observe an increased frequency of KRAS mutations associated with early brain metastases, it is also plausible that KRAS mutations may play a more general role in metastases. Well-conducted in vivo studies will be needed to further uncover the potential for site-specific metastatic tropism of specific KRAS variants.Transgenic mouse models have established that oncogenic KRAS mutations as a predictive biomarker for the development of early brain metastases will ultimately require prospective validation in larger cohorts employing multivariate models, particularly assessing the predictive value of these mutations in relation to other clinical covariates.3The retrospective nature of this analysis could feasibly introduce a degree of selection bias, in particular by only identifying those patients with operable early brain metastasis we might have excluded a larger patient demographic with more widespread disease. Emerging evidence indicates that metastatic outgrowth may also depend on the interplay between cancer cells and the host stroma; however, such tumour-cell extrinsic factors would not be fully captured by this analysis. The identification of KRAS mutations. This observation has implications for deciphering the biology of site-specific metastatic pathogenesis and, if validated in larger prospectively curated cohorts, might influence prognosis, surveillance and interventions in patients carrying these somatic alterations.In summary, our analyses indicate that the patterns of melanoma recurrence may be at least partially determined by the tumour mutational profile and that up to 8% of patients developing early brain metastases may have tumours driven by oncogenic Supplemental Methods and FiguresSupplementary Tables 1–7