P-values < 0.05) with reductions of 32% for ACQ5, 31% for ACQ6, 22% for ACQ7-FEV1, and 21% for ACQ7-PEF with high Pearson’s correlation coefficients of all scores (r between 0.96 and 0.99). ACQ7-FEV1 scores were significantly higher than others. Separated courses of lung function parameters showed nearly no change, but ACQ5 and ACQ6 as scores for symptoms and reliever medication improved constantly. ACQ5 and ACQ6 revealed higher percentages of patients classified as “controlled” than ACQ7-scores. In conclusion, with only a few missing data points, our results suggest feasibility of FEV1-self-measurements. Courses of symptom-related and lung function-related ACQ items differ clearly. Our results support the GINA recommendations to consider symptoms and lung function separately. FEV1-self-measurements for research purposes may be included with the ACQ, but in clinical practice seem to measure a different domain to symptomatic asthma control.The “Asthma Control Questionnaire” (ACQ) is a very common questionnaire for assessing asthma control. This study compares different ACQ versions in a self-monitoring program over a 12-week period combining them with patients' self-measurements of peak expiratory flow (PEF) and forced expiratory volume in one second (FEV1). The objective was to test the feasibility of FEV1-self-measurements and to compare ACQ versions regarding possible additional information given by lung function. In this prospective multicenter observational study 100 adult asthma patients, recruited at six family practices and two pulmologists' private practices in Germany, completed the ACQ weekly, performing self-measurements of PEF and FEV1. Seventy-six patients were included into final analysis with only 3% missing values. Scores for all ACQ versions improved significantly , which describes symptom severity and medication use. Looking at data from 76 participants, the researchers found that people did well measuring forced expiratory volume in one second, but they could not say whether this test was superior to another metric of lung function that considers peak expiratory flow. The results also showed that symptom scores and lung function assays seem to capture different aspects of the disease, meaning they’re worth considering separately rather than lumping together in a single test. Since then it is in use in a large number of countries being translated in 115 languages so far.11 This questionnaire is available in several versions. The original version includes not only questions about symptoms and reliever medication, but also an item on lung function. The authors of the ACQ recommend preferring measurements of FEV1 to measurements of peak expiratory flow (PEF) in usage with the ACQ, as the questionnaire has been validated with FEV1, but for practical reasons in daily disease management, they also accept PEF.11 Due to technical reasons, in the past it has not been possible for patients to perform self-measurements of FEV1 themselves at home. Still only a few but expensive portable devices are available. For all ACQ versions – whether including an item on lung function or not – an overall summary score is calculated. Previous studies, one of them done by Juniper et al. themselves, compared different ACQ versions and found extremely high correlation coefficients suggesting that adding or leaving out lung function makes little difference.16Therefore, in clinical routine assessing asthma control means to measure the patients' clinical symptoms and lung function parameters. To assess symptoms, there are several questionnaires in use, which ask about the frequency of typical asthma symptoms and use of reliever medications.7 In its 2017 update, GINA recommends long-term lung function-monitoring (PEF) for severe asthma cases and patients with poor perception of air flow limitation only.17In the 2015 update of its strategy for asthma management and prevention the “Global Initiative for Asthma” (GINA) advises to consider symptoms and lung function parameters separately, with lung function being no longer included among symptom measures numerically, but being mainly used for further risk assessment.In the study described below, we aimed to investigate, if weekly FEV1-measurements performed by patients at home over a 12 weeks period are feasible. Furthermore, we compared different versions of the ACQ to investigate whether inclusion of lung function provides additional relevant information, and whether it makes a difference if FEV1 is used instead of PEF.Of the 100 patients, who gave consent and were provided with asthma questionnaires and measurement devices at the beginning, 80 sets of records were returned. Three sets of records had been handed out to under-aged persons by mistake. Two patients quit the study prematurely because they spontaneously were free of symptoms. Six participants did not return their questionnaires and could not be contacted. Nine sets of records got lost by practices or patients. Furthermore, four more patients were excluded from the study: one patient had been given questionnaires because of his medical history only, but indeed had not been diagnosed securely, by neither a pulmologist nor a GP. For three patients no baseline values for ACQ7-FEV1 could be calculated. Finally, 76 patients were included into analysis. Overall, questionnaires were filled in very carefully by patients with only 3% missings of a total of 9880 item values. However, in 27 patients one or more ACQ items were missing for at least 1 week. 49 patients (64%) had filled in their sets of records without any missing data. The online 2. Fourteen persons (18%) were smokers. Eighteen patients (24%) had been diagnosed with asthma by a GP and 58 patients (76%) by a pulmologist. Fifty-two (68%) had undergone an asthma training program prior to the study with 92% of those participants being current non-smokers. 36 patients (47%) were recruited at general practitioners' practices, 40 (53%) at pulmologists' practices. The mean ACQ6 value at baseline was 1.3 with 34 (45%) of participants presenting with controlled asthma, mean FEV1 %-predicted in week 1 was 80.5%. In all, 92% were using a controller medication with 83% taking an inhalative corticosteroid, some in combination with other controllers. 59% took a reliever medication, one patient did not take any medication. Qualitative review of medication at baseline revealed an exceptional variety of treatment strategies. Some strategies seemed inconsistent with seven participants' treatment being incompatible with any guidelines.Characteristics of study participants are shown in Table p-values < 0.05; Fig. Over the 12-week period summary scores for all four versions of the ACQ improved significantly, both in complete cases and when replacing missing values  higher than for all other ACQ scores. ACQ–PEF summary scores tended to be higher than ACQ5 and ACQ6 scores but differences were not always significant. Yet, Pearson’s correlation coefficients of the four ACQ summary scores were very high . Findings for the other ACQ versions were similar. Correlations between puffs and lung function were low to moderate (r between 0.22 and 0.35 for week 2). Results were similar if evening measurements were analyzed instead of morning measurements.This led to an increasing difference in the courses of ACQ scores and isolated courses of FEV1 and PEF (biggest difference to ACQ seen in FEV1)  are likely to have increased adherence. Finally, we assessed feasibility only by counting missing. It could also be discussed, if improvement in asthma of some of the participants was derived from changes in their medication made at baseline visit. But as every patient diagnosed with asthma earlier on was offered to participate in the study, not only if consulting the doctor for problems with their asthma but for whatever reason, we think our study population represents a realistic sample of typical asthma patients with typical courses of their disease. Also, because of the broad selection criteria it is likely that patients represent typical asthma outpatients in the German health care system, although our sample of patients is not very large. Another argument against a medication derived change during the study is the persisting difference of lung function and symptom scores with lung function getting even slightly worse over a period of 12 weeks, which clearly stands against the improving symptom score only being caused by an effective change in medication. Within our study, it was not possible to investigate the validity of lung function self-measurements, e.g. by comparing these to regular spirometry in physicians' practices, but another study showed no significant difference between FEV1 at home and FEV1 using official spirometry at a physicians' offices .16 At the same time scores for symptoms and need for reliever medication improved significantly in contrast to both absolute values and the course over time of ACQ items for lung function, in particular for FEV1, which showed constant courses with even non-significant worsening (FEV1 showed 10% worse results than PEF). A recent study reporting a factorial analysis of ACQ6 and ACQ7 (with FEV1) found that the FEV1 item showed no relation to the latent factor derived from all other items and concluded that the factor structure of the ACQ7 remains unclear.19 It is likely that the very high correlations between different ACQ versions are mainly owing to the fact, that the majority of items (symptoms) is the same in all versions, with FEV1/PEF being only one of seven items. Lung function items do correlate with the other items but only moderately or weakly. They only have a little impact on ACQ7-scores, which rather mainly are influenced by patients' subjective concerns about their symptoms and by their reactions using their emergency devices according to emergency/self-management plans. The question is therefore, why put in this effort doing lung function measurements at all. So to us, it seems likely that symptomatic and lung function items could measure different dimensions of asthma control or disease status: on the one hand the subjective perceived present clinical control presented by symptoms and need of reliever medication, on the other hand the more objective unadulterated aspect of control, measured by lung function which may be an indicator of future risk regarding patients' prognosis for the disease.The very high correlation in scores of all ACQ versions meets the results of previous studies, which also presented usability of ACQ versions with and without lung function without loss of validity or change in interpretation.21 It could be considered to weight lung function items as such of overriding importance, so they would have more impact on ACQ7.Previous reviews showed equal benefits for patients using self-management programs with PEF-measurements or with symptom scores.20 But even as there is a lack of studies for FEV1, this is very questionable for both parameters if following the results of another review, which showed improving health outcomes for patients using action plans based on personal best PEF, in contrast to patients using plans based on PEF %-predicted.22Currently, the same coding rules are used for transforming raw values of FEV1 %-predicted and PEF %-predicted into an ACQ item. Maybe this should be changed, because in repeated observations FEV1 %-predicted on average was about 10% worse than PEF %-predicted if coded in the same way.A third alternative would be to present the ACQ6 summary score and lung function separately: the difference in courses of symptom scores and lung function items could be caused by on the one hand patients' individual and subjective perception of symptoms and deterioration. It is likely that improving scores of ACQ5 and ACQ6 express the effect of patients getting used to the procedure, e.g., as a learning effect in responding questions in a diary repeatedly. This can result in understanding the questions better and subsequently assigning better scores. Improving scores for symptoms and reliever medication can also be caused by patients getting used to stable asthma control and so being confirmed in their efforts positively. For lung function, this effect is not possible because you have to write down a value, which is not subjective.23 Implying this into interpretation of our study's results gives additional support to consider symptom scores and lung function separately.On the other hand, the stable courses of lung function parameters could be interpreted as a sign of these items being a helpful because objective and so unadulterated parameters in assessing asthma control. A previous study showed a discordance in patients' perception of asthma control and their actual asthma control comparing their personal impressions and perceptions with the results of another questionnaire to measure asthma control, the “Asthma Control Test”, with a high percentage of patients feeling controlled despite their test results showing an uncontrolled asthma.If this would be true, it would have potential consequences for the use of the ACQ. The very strong correlation between all four ACQ versions is raising the question, why lung function measurements should be done at all, if being integrated in summary scores according to current rules:Following this argumentation, we think symptoms and lung function both should be measured in clinical studies using the ACQ, for example, when investigating whether an intervention or treatment modifies only one or both of these aspects.24 it seems to be appropriate measuring both in clinical studies using the ACQ and rectifies additional costs by an expectable gain of knowledge. In self-management plans for patients ACQ so far has been used with PEF, although it was developed with FEV1, just because of regular FEV1-measurements being too expensive. More than this, there seems to be no additional benefit for patients from self-management plans with PEF-self-measurements than those involving symptoms only.21 Unless randomized trials could prove that interventions involving self-measurement of FEV1 lead to better patient-relevant outcomes, there is little argument for advocating their use.It could be questioned if in these cases there should be PEF- or FEV1-measurements or both. Because FEV1%-predicted and PEF %-predicted have been shown to differ systematically,In conclusion, we cannot give any recommendation to prefer FEV1 to PEF in use with the ACQ in clinical studies, but we think in connection with the ACQ lung function is more suitable for use in studies, where you can use a device measuring both, than for daily routine.The results of this study show the feasibility of regular FEV1-self-measurements performed by patients themselves at home monitoring their asthma control. However, following our data no recommendation can be given for preferring FEV1- to PEF-measurements. More than this, our results lend further support to the GINA recommendation to look at symptom scores and lung function separately. Further studies should investigate whether the ACQ7 including a lung function item should be considered a two-dimensional instrument. Self-monitoring of FEV1 is feasible, and for research purposes may be included with the ACQ questions, but in clinical practice measures a different domain to symptomatic asthma control.The study was approved by the Ethics Committee of the Medical Faculty of the Technical University Munich. It was run according to all relevant guidelines and procedures, especially in accordance to the professional code of medical doctors, the Declaration of Helsinki in its 2008 version and to the German Federal Data Protection Act. All patients gave written consent to their participation.This was a 12-week prospective multicenter observational study conducted by the Institute of General Practice of the Technical University Munich, Germany in 2011. Patients were recruited at six family practices and two pulmologists' private practices. All consecutive patients with a diagnosis of asthma consulting study practices for whatever reason who met inclusion criteria were informed about the study and offered to participate. Inclusion criteria were age ≥ 18 years, asthma diagnosis confirmed by spirometry in family practice or by a pulmologists via spirometry or bronchial provocation, signed declaration of informed consent and sufficient knowledge of German language. Patients were offered an incentive of €20 each for completing their asthma questionnaires. In addition, they could keep the used digital FEV1- and Peak-Flow-Meter (worth €70) after the study was finished.25 This questionnaire consists of five questions regarding the most frequent asthma symptoms in the previous week , one question regarding puffs of reliever medication and one question regarding lung function parameters before use of a bronchodilator.8 For every question, a score from zero to six is set up. For lung function parameters, measurements of FEV1 or PEF are transferred into percentage of nominal value and then into the same score from zero  to six (<50%). By adding all scores for single items and dividing the result by the number of items, summary scores are calculated to define the degree of asthma control. In our study, scores were calculated for four different versions of the ACQ: the ACQ5, consisting of five questions about symptoms only, the ACQ6, consisting of ACQ5 and one question about use of reliever medication, ACQ7-PEF, consisting of ACQ6 and PEF-self-measurements and ACQ7-FEV1, same as ACQ7-PEF, but conducted with FEV1-self-measurements instead. Cutoff points for asthma control are ≤ 0.75 for controlled asthma, 0.76–1.49 for partly controlled asthma and ≥ 1.5 for uncontrolled asthma and can be used the same for all four versions.26For assessment of asthma control the German version of the ACQ was used.Both, FEV1- and PEF-measurements were done by the patients themselves with the digital peak-flow-meter Vitalograph asma-1-monitor, which is a high quality and very reliable device saving up to 600 measurements. In the first week, patients measured FEV1 and PEF twice a day, mornings and evenings, by taking the best results out of three measurements at each time. This procedure was done to get reliable baseline values and to get patients used to the electronic device and possible differences to the readings by their usual mechanical devices. At the end of week one they answered the ACQ and made optional comments.28From week 2–12, patients did their measurements of FEV1 and PEF in the mornings and evenings once a week only, again taking the best of three results, completing the ACQ again once a week. The ACQ7-scores for week one were set up using the means of FEV1- und PEF-measurements in the morning. In weeks 2–12, the weekly gathered measurements in the morning were used. Besides, patients reported their medication use per week and important events like hospitalization. In addition, participants received an individual self-management-plan for adjusting their medication etc. in case of a worsening of their asthma including an individual emergency action plan. There was no further planned intervention such as control appointments or phone calls, except in case of exacerbations, when patients consulted the doctors. Severe exacerbations were defined by the need for oral glucocorticoids or even hospitalization.t-test was used to explore whether the different ACQ summary scores differed between each other for single weeks. A formal sample size calculation was not performed. Methods were performed in accordance with relevant regulations and guidelines.Data were entered into and processed with SPSS using all available data. Descriptive analysis was done on data using means (standard deviations) or absolute frequencies (percentages). Changes in ACQ scores over the 12-week period were analyzed using repeated measures analysis of variance. As about a quarter of the patients at some point had a missing value, we used multiple imputation (five runs) to investigate the influence of missing data. Pearson coefficients were used to quantify correlations. Consistency of the four ACQ scores was tested with Crohnbach’s α. The paired Prior to the performing of the study, we guaranteed to our patients that their data would not be transmitted outside, even not anonymously. Therefore, our medical ethics committee considers data sharing in this case as problematic.Supplementary tables


Background. Ulinastatin, identified as a urinary trypsin inhibitor, has been widely used in patients with inflammatory disorders. However, little is known about its effect on postoperative cognitive dysfunction (POCD). The aim of our current work is to review the current body of literature. Methods. A systematic literature search in PubMed and EMBASE was performed to identify randomized controlled trials. Incidence of POCD, MMSE score, and laboratory indicators  were selected as outcomes. Results. Five RCTs involving 461 elderly patients that underwent surgical operations were identified. The meta-analysis suggested no statistical difference of incidence of POCD between ulinastatin and control groups on postoperative day 1; but ulinastatin could significantly decrease the incidence of POCD on postoperative day 3 and day 7 when compared with control treatment. Ulinastatin was effective in improving the MMSE score on day 1, day 3, and day 7 after operation. IL-6 and S100β concentrations were lower up to postoperative day 2. The incidences of postoperative complications in ulinastatin groups were lower than control. Conclusion. Ulinastatin administration was effective in treating early POCD (postoperative day 3 and day 7) and reducing IL-6 and S100β concentrations within two days after operations. Studies with larger-scale and rigorous design are urgently needed. Postoperative cognitive dysfunction (POCD) is a common situation that may occur after any sort of surgery and defined by a drop in cognitive domain on a set of neuropsychological tests from before to after surgery; it broadly refers to a “deterioration in cognition temporally associated with surgical operation” . POCD imα), interleukin- (IL-) 1, interleukin- (IL-) 6, and C-reactive protein (CRP), has been shown to activate bone-marrow derived macrophages and contribute to the overall brain pathology after aseptic trauma  or ulinastatin or UTI68 or urinary trypsin inhibitor or MR20 or miraclid) and  screened each article independently and were blinded to the findings of the other reviewer. Following the prespecified inclusion criteria, two reviewers performed a rigorous screening to identify eligible articles. Data were collected from these selected articles using a standardized data collection sheet, which included first author, country, year of the publication, study design, cohort sizes, demographic characteristics of participants in different groups, details of intervention and control, and main outcomes.Discrepancies between two reviewers was resolved through discussion until a general consensus could be reached. The third review author (Jin-ming Zhang) was sought for opinions if a consensus could not be reached.To assess the risk of bias among our included studies, the Cochrane Collaboration's tool was utilized, which was based on seven items: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other sources of bias . Two revI2 test were used to assess the heterogeneity among studies (P > 0.1 and I2 indicate acceptable heterogeneity). The fixed-effect model was used for statistical analysis if no obvious heterogeneity existed; random-effect model was employed if apparent heterogeneity existed.For the incidence of POCD, risk ratio (RR) and the associated 95% confidence interval (95% CI) were calculated using the Rev Man 5.3 . Mean difference (MD) and the associated 95% CI were calculated for continuous variables using the same methodology. Before the combination of data from individual study, the chi-squared test and the Higgins Metaregression was performed to find the possible source of heterogeneity. Begg's rank correlation test and Egger's linear regression test were used to evaluate the publication bias if the number of included studies was greater than ten.An initial literature search yielded a total of 22 potential relevant citations including 7 from PubMed and 15 from EMBASE; 5 duplicates were deleted. At the stage of titles and abstracts screen, 12 articles were excluded because they were not related with ulinastatin in the treatment of POCD, and the remaining 5 articles were retrieved for a full-text review. Finally, 5 studies met our predetermined inclusion criteria .All the included studies were conducted in China and published from 2010 to 2015; each study was undertaken in a single center. These five RCTs –28 inclu Xu and colleagues assessed the neuropsychological state of enrolled patients using a brief battery of neuropsychological test. A postoperative deficit in any test was defined by a decline of 20% or more from the preoperative value of that test; any patient demonstrating a deficit in 2 or more tests was considered as having POCD . Ge and All the studies included the suggested randomization; four studies reported the method of random sequence generation. Only one study reported procedure of allocation concealment and double-blinding: study drugs were prepared by the hospital pharmacy in identical containers marked with the name of the project, the investigator's name, and consecutive numbers. In this study, patients and investigators were blinded to the infusion. Two studies reported number of drop-outs; the reasons for withdraw in different groups were similar. When it comes to selective reporting bias, all studies were judged low risk of bias because we only included studies which reported incidence of POCD, MMSE score. The judgement of risk of bias was presented in Figures P = 0.48); but ulinastatin could significantly decrease the incidence of POCD on postoperative day 3  and day 7  when compared with control treatment , day 3 , and day 7  after operation , CRP, and S100β remained to be further elucidated.To the best of our knowledge, this is the first systematic review and meta-analysis to assess the clinical effect of ulinastatin in the treatment of patients with early POCD. Five RCTs involving a total of 461 patients were identified by our current work. Based on the findings of this study, ulinastatin administration was effective in reducing the incidence of POCD and improving MMSE score. Compared with control treatment, incidences of all types of postoperative complications were lower in ulinastatin groups. However, the effect of ulinastatin in reducing proinflammatory cytokines . Future studies with longer follow-ups to validate the beneficial effect of ulinastatin are needed. Besides, only two included studies reported duration of operations; considering that some inflammatory mediators were directly associated with duration of operations, the results and conclusions may be sequentially confounded. Lastly, the funnel plots indicated obvious publication bias; this could be explained by the fact that all these trials were conducted in China and all these articles were published in Chinese academic journals; these works have only been performed in a Chinese population, clinical studies within western culture to evaluate the effect of ulinastatin are encouraged.β from the end of operation to postoperative day 2; results regarding the changes in CRP and TNF-α remained debatable. Further studies are need not only to determine the potential benefit but to understand the mechanisms involved in the mitigation of POCD by ulinastatin.In summary, ulinastatin administration showed remarkable effect in reducing the incidence of POCD and improving MMSE score. Ulinastatin could significantly reduce the concentrations of IL-6 and S100


Chronic mitral regurgitation (MR) is the most common cardiac valvular disease with more than 4 million people in the United States alone suffering from moderate or severe MR. Left untreated, chronic MR results in serious consequences. Surgical correction with mitral valve repair or replacement remains the mainstay of therapy for MR. Nevertheless, a large proportion of patients may not be offered treatment due to concerns over surgical risk. This unmet therapeutic need for a less invasive approach led to a marked explosion in the development of a variety of transcatheter approaches to treat mitral regurgitation in the past decade. The majority of these devices are based on principles learned from surgical mitral valve repair techniques. Inspired by the Alfieri surgical technique, the MitraClip edge-to-edge repair system is the most advanced technique with the highest safety and efficacy to date. In this review, we discuss the current status of the MitraClip repair system in the treatment of mitral regurgitation. Left untreated, chronic severe MR leads to left ventricular remodeling, myocardial dysfunction, heart failure, left atrial dilatation, atrial fibrillation and pulmonary hypertension. Medical therapy may alleviate symptoms; however, there is no proven benefit to medical therapy in terms of treating the underlying pathophysiology of MR or retarding its progression3. Therefore, surgical valve repair or replacement remains the standard of care for treating patients with chronic MR. Nevertheless, it is estimated that only about 20% of patients with significant MR are offered surgery with a large proportion of patients left untreated due to unavailability of surgical treatment or concerns about surgical risk, specifically those with advanced age and impaired left ventricular function. Therefore, there is a clear unmet need for patients with MR for a less invasive therapeutic alternative.Chronic mitral regurgitation (MR) poses a significant public health burden with more than 4 million people in the United States suffering from moderate or severe MR with nearly 250,000 new diagnoses of MR made each yearThe mitral valve is a complex structure that consists of several integrated components. This functional unit includes the mitral annulus, mitral leaflets, the subvalvular apparatus (papillary muscles and chordae tendineae), the left atrium and left ventricular myocardium. The mitral leaflets consist of a larger anterior leaflet and a smaller posterior leaflet, each subdivided into three scallops . Each leaflet is attached to the annulus and is connected to papillary muscles by a web of chordae tendineae. This subvalvular apparatus prevents the leaflets from prolapsing into the left atrium and is essential in maintaining ventricular shape and contractility. These components of the mitral apparatus are integral to the normal function of the valve and each is a potential venue for repair.In addressing the current therapy of MR, it is useful to distinguish degenerative (primary) from functional (secondary) MR  and 2. I4  . The guiThe MitraClip repair is performed under general anesthesia in the cardiac catheterization laboratory with fluoroscopy and transesophageal imaging guidance . The procedure consists of four main steps  and 6:  (1)Vascular access, transseptal puncture and steerable guide catheter insertion. (2)Steering of the guide catheter and clip delivery system (CDS) into the left ventricle. (3)Leaflet grasping and clip closure. (4)Clip deployment and system removal.After right heart catheterization is performed, transseptal puncture is performed under TEE guidance. This is a critical step of the procedure as the puncture site should be located in the posteroior-superior part of the interatrial septum, 3 to 4 cm above the plane of coaptation of the mitral valve leaflets. This is of course done under TEE guidance in two orthogonal views: the short axis that will determine anterior-posterior positions and in the long axis view that will determine superior-inferior relationship of the puncture. The puncture site should be posterior-superior location that allows adequate room in the left atrium for optimal orientation of the steerable CDS. Once the left atrium is entered with the Mullins sheath, unfractionated heparin is administered maintaining an ACT >250 seconds throughout the procedure. The left pulmonary vein is then cannulated using a multipurpose catheter and a 260 cm Amplatz Super stiff guidewire is left in place. The Mullins sheath is then exchanged for the 24 F guiding catheter and introduced into the left atrium over the guidewire. The dilator is then slowly and carefully retrieved to avoid air embolism. The clip delivery system, with a clip attached to its distal end, is then advanced into the left atrium, and the distal steerable part is manipulated in the atrium for obtaining a perpendicular and central position with respect to the mitral valve leaflets’ coaptation line. Under transesophageal echocardiographic guidance, the clip is steered until axially aligned and centered over the origin of the regurgitant jet. The clip is opened and the grippers are retracted. The correct trajectory of the clip and the perpendicularity of the two arms with respect to the mitral leaflet coaptation line are checked using echocardiography.Once the system has been aligned, the clip with opened arms is advanced into the left ventricle and under transesophageal echocardiographic (TEE) guidance the arms grasp the leaflets and the grippers are lowered such that the clip is partially closed to secure the leaflets. Stable grasp of both leaflets and MR reduction are carefully assessed using TEE. If the position is judged suboptimal by TEE evaluation, the clip can be reopened and repositioned. In the event that the clip must be withdrawn back into the left atrium, the arms can be inverted in the left ventricle and pulled back into the left atrium, avoiding entanglement of the clip with the chordae tendineae. If reduction of MR is deemed adequate and there is no significant mitral stenosis , the clip is deployed and the CDS is withdrawn. If significant residual MR is noted, a second clip can be implanted adjacent to the first clip at the location of the residual MR. Following final echocardiographic and hemodynamic assessment, the guide catheter is then removed and femoral venous access is closed as per operator preference. We favor using a “figure-of-eight” stitch.Post-procedural pharmacologic management includes aspirin 81 mg lifelong and clopidogrel 75 mg for three months in patients without atrial fibrillation. Patients with atrial fibrillation are generally prescribed aspirin and coumadin.7. Early complication events are primarily related due to need for periprocedural blood transfusion, while late events are primarily related to underlying heart failure or patient comorbidities.The MitraClip repair is associated with an overall complication rate of 15–19% at 30 days9. Other potential procedural complications include those secondary to transseptal puncture and air embolism. Therefore, careful echocardiographic guidance as well as careful aspiration and flushing of the steerable guide are essential to avoid such complications.Access site bleeding is a potential complication due to the large venous sheath size. In the EVEREST II trial, 13 percent of patients randomized to MitraClip required transfusion of ≥2 units of blood, compared to 45 percent of patients who underwent surgical mitral valve repair. In registries of MitraClip, the percentage of patients requiring ≥2 units of blood transfusion has been significantly lower, ranging from 0.9 to 3.9 percent6.Device embolization is an exceedingly rare complication. More commonly, partial clip detachment, defined as detachment of a single leaflet from the clip, may occur and requires surgical treatment. In EVEREST II study, 9 patients had partial clip detachment in the first 12 months, one additional patient developed partial clip detachment from years one to four. All patients subsequently underwent mitral valve surgeryThe development of clinically significant mitral stenosis is another potential complication of MitraClip implantation that tends to be poorly tolerated by patients. Careful hemodynamic assessment prior to clip release is important to avoid such complication.Multidisciplinary team approach comprising an interventional cardiologist, cardiac surgeon, and echocardiographer working together is essential for the proper selection of candidates for the transcatheter mitral valve repair.10. Currently, the MitraClip device is FDA approved in the US for patients with significant symptomatic primary (degenerative) MR who have been determined to be at prohibitive risk for mitral valve surgery by a heart team. Although MitraClip has been used to treat functional MR, no randomized controlled studies focusing on functional MR have been published to date. The COAPT trial , which is now underway in multiple sites, will be the first randomized controlled trial to evaluate the safety and efficacy of the use of the MitraClip device in patients with functional MR (see below).To establish clinical indication criteria, all patients undergo a standardized echocardiographic assessment protocol. Patients are required to meet basic criteria for intervention from the 2014 American College of Cardiology/American Heart association guidelines for valvular heart diseaseIn addition to meeting clinical indications, patients have to fulfill several anatomical criteria to qualify for MitraClip therapy . As disc2 to avoid the creation of mitral stenosis after clip deployment. For functional MR, it is important to determine whether the patient with ischemic cardiomyopathy has a need for concomitant revascularization. Lastly, patients with hemodynamic instability are unlikely to benefit solely from correction of their MR nor would patients who are truly in end-stage heart failure.To achieve optimal results in patients with a flail leaflet, the flail gap should not be greater than 10 mm with a flail width less than 15 mm on short-axis estimation. For patients with functional MR, leaflets coaptation length of at least 2 mm and coaptation length less than 11 mm are required in order to have adequate tissue to grasp with the clip. Leaflets with less coaptation length can possibly be grasped as well; however, data on long term outcome in this setting is limited. Furthermore, leaflets should be free of significant calcification in the grasping area. In addition, careful assessment of the subvalvular apparatus is needed and the presence of chordal tissue in excess may render orientation, recovery and clip stability cumbersome. It is also important to note that baseline mitral valve area should be greater than 4 cm5. After this first procedure, the MitraClip approach to percutaneous mitral valve repair was evaluated in the United States in the Endovascular Valve Edge-to-Edge Repair Study (EVEREST) trials.The first successful human MitraClip implant was performed in 2003 in a 56 year old woman in Venezuela with reduction of severe mitral regurgitation due to bileaflet prolapse to <2+ with concomitant clinical improvement11. This represented the initial experience with this novel technology that has a steep learning curve. The study enrolled a total of 107 patients with moderate-to-severe or severe (3 + ∕4 +) MR who met criteria for mitral valve surgery. Functional MR was present in 21% of patients; with the remainder having degenerative or combined degenerative and functional MR. Ninety percent of patients underwent successful clip placement with one third of the patients requiring placement of two clips. Acute procedural success, defined as placement of one or two clips with reduction of MR to <2+ occurred in 74% of patients with 66% were free from death, mitral valve surgery, or MR greater than 2+ at 12 months. The study demonstrated improvement in NYHA class III/IV symptoms from 55% to 8% at the end of 1 year. These outcomes were similar for both degenerative and functional MR. About 30% of patients required surgical intervention after clip placement at 3-year follow up with 84% success rate. The major adverse event rate was 9% with blood transfusions representing the majority of these events. No procedural mortality was encountered. Partial clip detachment occurred in 9% of patients, detected incidentally on 30-day echocardiography. All these patients were surgically treated successfully.EVEREST I trial was a prospective, multicenter, phase I study to evaluate the safety and feasibility of the MitraClip procedure6. The primary efficacy end point was freedom from death of any cause, surgery for MV dysfunction, and ≥3+ MR at 12 months. The primary safety end point was a composite of major adverse events within 30 days. The primary composite end point for efficacy was more frequent in the surgery group (73 versus 55%) due to higher rate of subsequent surgery for mitral valve dysfunction in the percutaneous group (20 versus 2%). Both approaches were associated with similar rates of overall mortality at one year (6% for both). Improvement in MR severity was seen in both groups with greater reduction in the surgical group. Presence of +3/+4 MR grade was similar at one year (21 versus 20%).Following the encouraging results of EVEREST I study, the MitraClip device was subsequently compared with surgical mitral valve repair or replacement in the EVEREST II randomized-controlled trial12. At five-year follow up, the primary efficacy end point in the as-treated population was reached in 44.2% in the percutaneous arm versus 64.3% in the surgical group (p = 0.01). The difference was driven by increased rates of 3+ to 4 + MR  and surgery  with percutaneous repair. The majority of surgical repairs after device therapy were encountered in the first 6 months (78% of surgeries); beyond 6 months, the rates of surgery and moderate-to-severe MR were comparable between groups. Similar rates of death in the percutaneous repair and surgical groups were reported at 5 years of follow up .The five-year follow up results of the EVEREST II trial was recently publishedP < 0.009). Similar improvements in functional and quality of life were demonstrated at 12 months in both groups which persisted at 5 years follow up.Left ventricular volumes and dimensions were significantly reduced from baseline in the two study groups with greater magnitude of reduction in the surgical group. At 5 year follow up, left ventricular dimensions were similar in both groups except for smaller left ventricular end-diastolic diameter in the surgical group . This was largely due to the higher rate of blood transfusion in the surgical group (8.8 versus 53.2 percent). The superiority of MitraClip with regard to safety end points persisted after adjusting for transfusion-related adverse events (0.7 vs 16.5%).13. All patients with LVEF ≤20%, unfavorable MV anatomy, LV end-systolic diameter >60 mm, and MV area <4 cm2 were excluded from the study. The observed mortality in patients with MitraClip was 7.7% at 30 days as compared to mean predicted mortality of 18.2% by STS score. Compared to matched high-risk controls who were treated medically, there was improved 1-year survival in addition to improvement in NYHA functional class, favorable ventricular remodeling and reduction in the hospitalizations for heart failure as compared to baseline. The favorable safety profile of the MitraClip eventually led to FDA approval in October 2013 for the reduction of “symptomatic MR >3+ due to primary abnormality of the mitral apparatus (degenerative MR) in patients who have been determined to be at prohibitive risk for MV surgery by a heart team”.It is important to note that both EVEREST I and II trials included patients considered at low and moderate surgical risk. High surgical risk patients were subsequently evaluated in the EVEREST II High-Risk registry which included 78 patients with elevated surgical risk with predicted mortality greater than 12% (measured by the Society of Thoracic Surgery (STS) calculator)Although it has been used successfully in functional mitral regurgitation, the role of MitraClip remains controversial in this population due to a lack of randomized data regarding the additive benefit of mitral valve repair in addition to optimal medical therapy. The COAPT trial, which is underway in multiple sites, will be the first randomized controlled trial to evaluate the potential benefit of MitraClip therapy in patients with heart failure and secondary MR who are at high surgical risk as compared to medical standard of care. Eligible candidates should be symptomatic with NYHA class 2 or greater and have grade 3 + ∕4 + MR due to cardiomyopathy of either ischemic or non-ischemic etiology. Patients should be considered high risk with predicted surgical mortality greater than 8% for inclusion. Primary endpoints include effectiveness at 1 year, defined as patients without recurrent heart failure hospitalizations, and a primary safety endpoint defined as freedom from all-cause mortality, stroke, worsening kidney function, and ventricular-assist device implantation or cardiac transplantation.Percutaneous mitral valve repair is an exciting new field with many devices at early developmental stages. The MitraClip repair system is the most advanced technique with the highest safety and efficacy to date, providing valuable alternative option to surgical intervention in patients with high or prohibitive surgical risk with degenerative mitral regurgitation. The role of the MitraClip in treating patients with functional mitral regurgitation remains unclear. The medical community is eagerly waiting for the results of the COAPT trial with hopes that it will lead to an expanded indication in the near future.


KCNJ2 gene. KCJN2 has particular characteristics that make it attractive for “directed” SSM.KCNJ2 has a sequence of 17,510 base pairs (bp), and a short coding region with two exons (exon 1=166 bp and exon 2=5220 bp), half of the mutations are located in the C-terminal cytosolic domain, a mutational hotspot has been described in residue Arg218, and this gene explains the phenotype in 60% of ATS cases that fulfill all the clinical criteria of the disease. In order to increase the diagnosis of ATS we urge cardiologists to search for facial and muscular abnormalities in subjects with frequent ventricular arrhythmias  and prominent U waves on the electrocardiogram.In 1977, Frederick Sanger developed a new method for DNA sequencing based on the chain termination method, now known as the Sanger sequencing method (SSM).  Recently, massive parallel sequencing, better known as next-generation sequencing (NGS),  is replacing the SSM for detecting mutations in cardiovascular diseases with a genetic background. The present opinion article wants to remark that “targeted” SSM is still effective as a first-line approach for the molecular diagnosis of some specific conditions, as is the case for Andersen-Tawil syndrome (ATS). ATS is described as a rare multisystemic autosomal dominant channelopathy syndrome caused mainly by a heterozygous mutation in the For this method, Sanger was awarded in 1980 with a second Nobel Prize in Chemistry, and nowadays this method is still known as the Sanger method of DNA sequencing, becoming a standard method in clinical genetics. The present opinion article wants to remark that, targeted SSM is still effective in specific clinical scenarios at a lower cost as a diagnostic method compared to new technologies for sequencing, one example is the detection of Andersen-Tawil syndrome (ATS).In 1977, Frederick Sanger developed a new method for DNA sequencing based on the chain termination method, where nucleotides in a single-stranded DNA molecules are determined by complementary synthesis of polynucleotide chains, based on the selective incorporation of chain-terminating dideoxynucleotides driven by the DNA polymerase enzyme2.Next-generation sequencing (NGS) technology, also known as massive parallel, high throughput or deep sequencing, is gradually replacing the traditional SSM as the first choice method for screening mutations in genetic cardiovascular diseases3 and Brugada syndromes4 has made this new genetic testing approach mandatory. The advantages of NGS versus the SSM in cases of genetic heterogeneity are undeniable, but NGS is still expensive and unaffordable for developing countries. The SSM remains the gold standard for sequencing short fragments of DNA (<1000 bases), previously amplified by PCR.The genetic heterogeneity in long QTOMIM) as a multisystem autosomal dominant channelopathy syndrome caused by a heterozygous mutation in theKCNJ2 gene (OMIM Entry - *600681) on chromosome 17q24.3. Periodic paralysis, ventricular arrhythmia, and distinctive dysmorphic features characterize it. Until 2015, the only gene thought to be affected was the potassium voltage-gated channel subfamily J member 25 (theKCNJ2 gene), which encodes the alpha subunit protein of the Kir2.1 channel composed of tetramers6. Mutations in this gene have been reported in 60% of clinically suspected cases (which are classified as ATS type 1)7. Less than 200 cases with theKCNJ2 gene affected have been described worldwide since the discovery of the first mutations in 20019. In 2014, a novel variant  in a second gene,KCNJ5, was associated with ATS in one Japanese patient taken from a cohort of 21 patients that had previously been screened negative for mutations inKCNJ2. TheKCNJ5 gene protein (potassium channel Kir 3.4 protein) has an interaction with theKCNJ2 protein that leads to a dominant negative effect in the channel formed, related to the ATS phenotype10. No additional cases ofKCNJ5 mutations in independent series of ATS patients have been reported; the frequency ofKCNJ5 mutations in ATS has to be determined in the future. With the widespread use of NGS, it is possible that in the next years we could discover new genes that explain part of the genetic heterogeneity observed in ATS, clarifying some of the 40% of clinically suspected negative cases that do not have a mutation inKCNJ2 (nowadays classified as ATS type 2)11.ATS, also named Long QT syndrome type 7, is described in the Online Mendelian Inheritance in Man database , and a coding region with near to 5,000 bp with two exons (exon 1=166 bp and exon 2=5220 bp). Also, half of the mutations are located in the C-terminal cytosolic domain, and have a mutational hotspot in the residue Arg218; as we have addressed before, this gene explains the phenotype in 60% of ATS cases, fulfilling the clinical criteria.Theet al.12 Recently, a second proband was diagnosed in a different Mexican family, finding the mutation p.Arg218Trp inKCNJ213.Fifteen years have passed since the first family with ATS in a Mexican population was reported by CanunKCNJ2 could be sequenced with the SSM14.A multidisciplinary approach is extremely useful to study suspicious cases of hereditary sudden death syndrome. For ATS, the team must include a cardiologist, a neurologist and a clinical geneticist. It is very important that each of these physicians had expertise in the evaluation of subjects with sudden cardiac death syndrome. After a common agreement on suspicion of ATS, the whole coding region and intron boundaries of the non-coding region inet al11 suggested that recognition of facial and limb dysmorphism  associated with ATS could help establish a correct ATS diagnosis. We believe that it is important that all cardiologists dealing with subjects with ventricular arrhythmias, specifically frequent ventricular premature beats in bigeminy, are aware of such distinctive phenotypic characteristics and also search for muscular disorders .Phenotypically, CanunThe SSM is nearly 40 years old, and it remains a useful molecular tool for genetic testing. It has its limitations because it is time-consuming, has limited use for long DNA fragments and is unable to detect sequences out of the region contemplated. We consider using “directed” SSM as first-line approach for diagnosis of suspected cases of ATS in places where NGS is not an option for genetic testing (due to low availability or high cost). KCNJ2 gene (that codes for the Kir2.1 channel protein) exhibiting autosomal dominant Mendelian inheritance, half the mutations are located in the C-terminal cytosolic domain with a hotspot at Arg218 (ATS type 1). Characteristic developmental abnormalities coupled to specific ventricular arrhythmias and/or muscular disorders can identify ATS patients who could then undergo SSM to identify the putative causative mutations in KCNJ2 with a potential 60% success. NGS could then be applied if the patient falls within the 40% of the cases that are not due to KCNJ2 (ATS type 2) with the hope to discover additional genes responsible for the disease and determine their frequency of occurrence. The opinion expressed by the authors is reasonable and advisable to physicians especially in cases where cost and access to NGS are problematic. Yet, it is likely a matter of time before cost and access to NGS improve and limit the choice of SSM for genetic testing. SSM has certainly served us well for the past 40 years and continues to serve us well in cases like ATS type 1 but it is inevitable that NGS will eventually make it obsolete. Until then, the authors make their case convincingly through this opinion article.The opinion article by Totomoch-Serra and colleagues suggests that a “targeted” Sanger Sequencing Method (SSM) is an appropriate first-line approach to diagnose Andersen-Tawil syndrome (ATS). Although Next-Generation Sequencing (NGS) technology, especially in cases of genetic heterogeneity, is gradually replacing the traditional SSM as the method of choice for genetic testing, its high cost and unavailability often preclude its routine use, particularly in developing countries. The authors maintain that for conditions that require sequencing of short fragments of DNA (<1000 bases), previously amplified by PCR, SSM remains the gold standard. In ATS, where 60% of the reported cases involve mutations of theWe have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al.refers to the usefulness of directed sequencing ofKCNJ2 by Sanger method for molecular diagnosis of Andersen-Tawil syndrome (ATS), a rare QT long syndrome associating ventricular arrhythmias, prominent U wave, dysmorphic facial or skeletal features and periodic paralysis.This opinion article by Totomoch-SerraRYR2 orTTN) where Sanger sequencing remains a long and expensive process. NGS can also be useful at second level when directed molecular diagnosis with Sanger method is negative. Sanger sequencing remains a useful method for directed sequencing of specific clinical syndromes with low genetic heterogeneity as ATS, especially in places where NGS is not accessible. In another hand, NGS is particularly useful and cost effective in inherited cardiomyopathies or channelopathies associated with high genetic heterogeneity (like dilated cardiomyopathy) or caused by mutations in larges genes  suggestive of ATS diagnosis that should direct toI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. It is a well written manuscript focused on use of Sanger technology in genetic diagnosis. Currently, despite to NGS technology allows a cost-effective analysis of hundreds genes in a reduced time, Sanger sequencing remains as gold standard for validation of variants identified using NGS, segregation of variants in family members, and analysis of small genes, such as KCNJ2.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Staphylococcus aureus (MRSA) from animals to humans is of great concern due to the implications for human health and the health care system. The objective was to investigate the frequency and duration of MRSA carriage in human volunteers after a short-term exposure in a swine farm. The experimental study included 34 human volunteers staying 1 h in a MRSA-positive swine farm in four trials. In two of the trials, the influence of farm work involving pig contact was studied using a crossover design. The quantities of MRSA in nasal swabs, throat swabs, and air samples were measured at different time points and analyzed in relation to relevant covariates. This investigation showed that, overall, 94% of the volunteers acquired MRSA during the farm visit. Two hours after the volunteers left the stable, the nasal MRSA count had declined to unquantifiable levels in 95% of the samples. After 48 h, 94% of the volunteers were MRSA-negative. Nasal MRSA carriage was positively correlated to personal exposure to airborne MRSA and farm work involving pig contact and negatively correlated to smoking. No association was observed between MRSA carriage and face touching behavior, nasal methicillin-susceptible Staphylococcus aureus (MSSA) carriage, age, or gender. The increase in human MRSA carriage among the volunteers with pig contact seems to be dependent on the increased concentration of airborne MRSA of the surrounding air and not directly on physical contact with pigs. MRSA was not detected in any of the throat samples.Transmission of methicillin-resistant IMPORTANCE The experimental approach made it possible to elucidate the contributions of airborne MRSA levels and farm work to nasal MRSA carriage in a swine farm. Short-term exposure to airborne MRSA poses a substantial risk for farm visitors to become nasal carriers, but the carriage is typically cleared within hours to a few days. The risk for short-term visitors to cause secondary transmissions of MRSA is most likely negligible due to the observed decline to unquantifiable levels in 95% of the nasal samples after only 2 h. The MRSA load in the nose was highly correlated to the amount of MRSA in the air and interventions to reduce the level of airborne MRSA or the use of face masks might consequently reduce nasal contamination. Staphylococcus aureus (MRSA) was described for the first time in 1961 and was for several decades almost exclusively found in humans. In 2005, a new variant of MRSA belonging to clonal complex 398 (CC398) was first described in pigs and pig farmers in France and the Netherlands  and Panton-Valentine leukocidin (PVL), which in contrast are found in the human CC398 MRSA lineage , due to negative implications for human health and the health care system. Several studies have reported an increased risk for being colonized or infected with LA-MRSA among persons working in the livestock industry –8, but i–There has been a steep increase in LA-MRSA cases in Denmark since 2004, primarily in persons with swine farm contact. Since 2012, patients with regular contact with livestock have been tested for MRSA carriage at hospital admission and isolated until negative MRSA results were confirmed. However, 33% of LA-MRSA infections in Denmark are not associated with livestock contact . In ordeS. aureus from surfaces to the nose , 30 to 39 years (n = 1), 40 to 49 years (n = 2), and ≥50 years (n = 2). In trials 1 to 3, 24 volunteers participated, whereas 22 participated in trial 4. The data set therefore contained 94 observations in total. Twenty-two volunteers participated in both trials 1 and 2, 12 volunteers participated in all 4 trials, 10 volunteers participated 3 times, four volunteers participated twice, and eight volunteers participated in one of the trials. Seven volunteers (22%) were male and seven volunteers (22%) were smokers .In total, 34 volunteers were enrolled in the study. The age distribution was as follows: 20 to 29 years  of the volunteers became positive after the 1-h stay in the stable. During the visits with high MRSA load in the air , the nasal samples of all 48 volunteers were MRSA-positive when leaving the stable (time [T] = 0). In trials 3 and 4, where all volunteers were passive observers and where the load of MRSA in the air was relatively low, 6/46 (13%) of the volunteers were MRSA-negative when leaving the stable. All strains were confirmed as LA-MRSA CC398 with Staphylococcus aureus (MSSA) population and were accordingly recorded as MSSA carriers. Nineteen of the volunteers (59%) were persistent MSSA throat carriers. Seven volunteers (21%) were MSSA carriers in both the nose and throat and identical spa types were found in both locations. Seven volunteers (21%) were MSSA-negative in both the nose and throat in all trials.Thirteen of the volunteers (41%) had a persistent nasal methicillin-susceptible 3 . The exposure level was significantly higher in trials 1 and 2  than in trials 3 and 4  which was as expected according to the initial investigations in the herd. There was a significant difference in exposure levels between trials 1 and 2, but not between trials 3 and 4. Being in the active group also had an effect on the personal exposure levels of the volunteers in trials 1 and 2, with the highest exposure in the groups taking pig samples. However, the difference was only statistically significant in trial 2 . On the other hand, five volunteers that had been exposed to similar concentrations were still positive after 24 h; one of these was also MRSA-positive after 48 h.The six volunteers who tested MRSA-negative immediately after leaving the stable in trials 3 and 4 had all been exposed to air levels in the range of 24 to 310 CFU MRSA/mThe effects of pig contact and face touching on nasal MRSA carriage were estimated using data from trials 1 and 2  positively correlated to personal exposure to airborne MRSA, whereas it was (ii) negatively correlated to smoking  than in the passive group . An increased respiratory rate can be expected among the volunteers in the active group, which might explain the higher nasal deposition of MRSA compared to the passive group. An increased nasal MRSA deposition can therefore be expected also in connection with other forms of farm work generating dust or increased physical activity.S. aureus carriage level associated with smoking has also been reported by others  samplers showed a significant correlation between the MRSA levels in the nose and air . There wRepeated sampling of the nasal cavity might influence the MRSA count by physically removing parts of the bacterial population, thus confounding an accurate assessment of bacterial loss. Other factors affecting the amount of nasal mucus, e.g., nasal secretion and sneezing induced by the farm visit, can also be expected to have impact on repeated nasal sampling. Only the anterior part of the nasal cavity was sampled in this study. Nasal lavage could have assessed a larger part of the MRSA population present in the nasal cavity but would probably have had an even higher confounding effect upon repeated sampling.GSP samplers are commonly used for dust sampling and have also been used for MRSA sampling . HoweverThe strength of this study is the experimental approach whereby it was possible to analyze the relative importance of work, direct contact with pigs, and air transmission for human MRSA contamination after a short-term visit to a swine farm. The study included data from a single swine farm and a single LA-MRSA strain and was based on MRSA-negative, healthy volunteers, primarily between 20 and 30 years old. In spite of these limitations, the conclusions can probably be extrapolated to several other groups, e.g., craftsmen, veterinarians, school classes, and other short-term visitors. However, the results may not be directly applicable to farm workers spending longer durations in swine farms, as longer colonization times have been observed in this group .This study shows that short-term visitors to MRSA-positive swine farms will experience transient contamination of the nasal cavity. The risk for short-term visitors to cause secondary transmissions of MRSA is most likely negligible, due to the observed decline to unquantifiable levels in 95% of the nasal samples after only 2 h.The MRSA load in the nose was highly correlated to the amount of MRSA in the air and interventions to reduce the level of airborne MRSA or the use of face masks might consequently reduce the nasal contamination.This study has shown that the nasal MRSA contamination level is positively correlated to the air level of MRSA and to farm work, and negatively correlated to the time passed since leaving the farm and smoking habit. No association was observed between MRSA contamination and face touching behavior, nasal MSSA carriage, age, or gender. An increased level of airborne MRSA had a higher impact on nasal MRSA contamination among volunteers performing farm work than on passive volunteers. This probably reflects the higher respiratory activity of active volunteers. MRSA contamination declined quickly after the farm visit and after 48 h MRSA could only be detected in 6% of the volunteers.Human volunteers were recruited through advertisements at the University of Copenhagen. Most of the volunteers were students within the fields of veterinary science and animal husbandry. In addition, a few staff members from the Statens Serum Institut participated. All volunteers participated in an information meeting, received written project information, and signed a declaration of informed consent. The participants filled in a questionnaire and were tested for MRSA carriage in the nose and throat. Subjects were eligible for participation if they (i) were healthy individuals above 18 years of age, (ii) tested negative for MRSA in the nose and throat, (iii) did not have professional exposure to swine, (iv) did not work in health care facilities, (v) did not have allergies to dust, (vi) had not used antibiotics during the last 3 months, and (vii) did not have skin diseases or wounds. The study was performed in accordance with the principles of the Declaration of Helsinki and was approved by the National Committee on Health Research Ethics (Protocol H-15013814).The study was conducted on an LA-MRSA-positive swine farm. We used the weaner section for our study, as initial investigations showed a low MRSA load the first week after weaning and a high MRSA load approximately 4 weeks after weaning, making it feasible to conduct experiments with different MRSA loads. The farm was visited in 2016 on two consecutive days in each of the weeks 14, 17, 21, and 39, here referred to as trials 1 to 4. In trials 1 and 2, we visited stables with pigs approximately 4 weeks after weaning (high MRSA exposure), whereas in trials 3 and 4, we visited stables with pigs 1 to 2 weeks after weaning (low MRSA exposure).Twelve and 11 volunteers visited the stable on each day in trials 1 to 3 and in trial 4, respectively. The influence of farm work with physical contact with pigs during the farm visit on subsequent MRSA carriage was studied in trials 1 and 2 using a crossover design. Six volunteers worked in groups of three persons inside the pens by taking nose and skin swabs from the pigs for 60 min, here referred to as the active group. Meanwhile, six volunteers in the passive group stayed in the corridor separating the pens and were instructed not to touch anything in the room. During the 60 min of the study, each member of the passive group registered the number of hand-to-face skin touches for one of the active volunteers. The volunteers allocated to the active group in trial 1 constituted the passive group in trial 2 and vice versa. In trials 3 and 4, all volunteers were passive observers and stayed in the corridor between the pens for 60 min without touching anything in the room.Before entering the farm, all volunteers washed their hands and changed clothes. The volunteers were dressed in a clean pair of boots and a disposable suit  covering the whole body, including the hair and leaving only the hands and the frontal part of the face exposed. When leaving the farm, the volunteers changed clothes and washed their hands. They were not allowed to smoke or wash their face within the first 2 h after leaving the stable.Swab samples were taken from the nose and throat of the volunteers using eSwab (Copan). Nasal samples were taken from the anterior part of the nose by rotating the same swab 5 times in each nostril. Throat samples were obtained by swabbing the palatopharyngeal arch and tonsils on both sides. Nasal and throat samples were taken 2 h before entering the stables and immediately after leaving the stable (T = 0). In addition, nasal samples were taken 1 and 2 h after leaving the stable in all trials, whereas corresponding throat samples were only collected in trial 1, as all throat samples turned out to be MRSA-negative. All samples were kept in a cooling box and cultivation was initiated approximately 3 h after leaving the stable. Additional nasal samples were taken the day after the visit (day 1), nasal and throat samples were obtained on day 2, and a final nasal swab was taken on day 7. If MRSA was detected in the final sample, an additional nasal sample was obtained 14 days after the farm visit. All samples were taken by the principal investigator except the nasal samples on day 1 and 7, which were taken by the volunteers themselves. The volunteers kept these samples at 4°C for up to 24 h before cultivation was initiated.From each sample, MRSA was quantified by making serial dilutions of the swab fluid (1 ml) with 0.9% NaCl added to 0.1% Triton X-100 (Sigma-Aldrich), followed by spread of 100 μl on one Brilliance MRSA 2 agar plate (Oxoid) and incubation at 35°C for 22 to 24 h. Furthermore, all samples were investigated for MRSA by enrichment in tryptic soy broth (Sigma-Aldrich) supplemented with 6.5% NaCl at 35°C for 16 to 24 h, followed by spread of 10 μl on Brilliance MRSA 2 agar plates and incubation at 35°C for 22 to 24 h. MRSA was identified as denim blue colonies. One colony from each volunteer at T = 0 was selected for molecular verification. In addition, one colony from each person showing growth of presumptive MRSA-colonies on day 1 or later, as well as all colonies showing an atypical phenotype, was verified by PCR.S. aureus (MSSA) was detected on SaSelect agar plates (Bio-Rad) as pink colonies after cultivation at 35°C for 22 to 24 h. Four presumptive S. aureus colonies from each volunteer were subcultivated on 5% blood agar plates for subsequent molecular analysis. The presence of MSSA was investigated from nasal and throat samples taken before and 2 days after the farm visit as well as from throat samples collected at T = 0, as initial investigations had shown that these contained predominantly MSSA.Methicillin-sensitive mecA, lukF-PV, scn, and spa, followed by spa typing  of the sampling events; the other participants were recorded as noncarriers (including transient carriers) following the recommendations by van Belkum et al. . The GSP samplers were unmounted outside the stable before leaving the farm, and all samplers were transported in closed boxes to the laboratory within 2 h after sampling for quantification of MRSA.Airborne MRSA was sampled using GSP (Gesamtstaubprobenahme) samplers . The GSP samplers with a Teflon filter  were mounted in the inhalation zone on the chest and were connected by flexible tubes to a pump adjusted to an airflow of 3.5 liters/min. In each trial, sampling was performed for approximately 60 min during the stay in the stable. The exact sampling time for each person was noted and used for calculation of exposure (CFU/m3).Extraction of dust particles was performed immediately after arrival at the laboratory as previously described . BrieflymecA, lukF-PV, scn, and spa, followed by spa typing  in the air was obtained from the personal GSP samplers.The following data regarding the volunteers were extracted from the questionnaire: age, gender, and regular smoking habit. During the farm visits in trial 1 and 2, allocation to group (active or passive role) and the number of face touches for the active group were recorded. The quantity of MRSA per ml of swab fluid was recorded at T = 0 as well as at 1 and 2 h after leaving the farm. In addition, the detection of MRSA after enrichment was recorded for all samples. The amount of MRSA . In addition, a model was evaluated using data from all 4 weeks, excluding the volunteers that had pig contact in trials 1 and 2 (model B). In this model, both trial number and person ID were included as random effects. The best model fit and hence the models to be analyzed and presented were found by a combined forward and backward selection process by selecting significant variables (P < 0.05). For model A, an interaction term could not be fitted due to quasiseparation of data. A scatter plot was made of the log-transformed values of airborne MRSA and MRSA carriage of the volunteers .All statistical analyses were performed with SAS Enterprise 6.1  and GraphPad Prism 7 . A multivariable analysis was performed using the MRSA counts per ml swab fluid when leaving the stable as the dependent variable. A multivariable model was evaluated using the GLIMMIX procedure (based on the Poisson distribution) and using person identifier (ID) as random effect. The estimated size of the random effect was presented using the median ratio parameter according to Larsen et al. . The air


Scientists have discovered various prognostic gene signatures (GSs) in different cancer types. Surprisingly, although different GSs from the same cancer type can be used to measure similar biological characteristics, often rarely is there a gene shared by different GSs. To explain such a paradox, we hypothesized that GSs from the same cancer type may be regulated by common regulatory motifs. To test this hypothesis, we carried out a comprehensive motif analysis on the prognostic GSs from five cancer types. We demonstrated that GSs from individual cancer type as well as across cancer types share regulatory motifs. We also observed that transcription factors that likely bind to these shared motifs have prognostic functions in cancers. Moreover, 75% of the predicted cofactors of these transcription factors may have cancer-related functions and some cofactors even have prognostic functions. In addition, there exist common microRNAs that regulate different GSs from individual cancer types and across cancer types, several of which are prognostic biomarkers for the corresponding cancer types. Our study suggested the existence of common regulatory mechanisms shared by GSs from individual cancer types and across cancer types, which shed light on the discovery of new prognostic GSs in cancers and the understanding of the regulatory mechanisms of cancers. In a given cancer type, a prognostic GS is a group of genes whose mRNA expression levels significantly associate with the tumor diagnosis, the tumor prognosis, or the therapeutic response of this cancer type2. Studying prognostic GSs thus greatly facilitates the understanding of tumor biology, the prediction of cancer progression, and the treatment of cancers3.Studying prognostic gene signatures (GSs) can revolutionize our understanding of cancers5. Many prognostic GSs from different cancer types were discovered thereafter. For instance, for breast cancer, scientists found the prognostic GSs such as the 70-gene GS6, the 76-gene GS7, the 21-gene GS8, etc. These GSs predict the breast cancer prognosis under different conditions. For instance, the 70-gene GS can predict the disease outcome in patients with 1–3 positive lymph nodes9 and can predict adjuvant chemotherapy response in early breast cancer10. The 21-gene GS has prognostic value in Asian populations with ER-positive and lymph node-negative breast cancer11, and can predict the response to neoadjuvant exemestane therapy in postmenopausal patients with the ER-positive breast cancer12.In 2002, the first studies of prognostic GSs were published14. We thus hypothesized that there may exist a shared regulatory mechanism by different GSs from the same cancer type, or even across different cancer types.An intriguing observation on the identified prognostic GSs in literature is that different GSs from the same cancer type seldom share genes. For instance, in breast cancer, the aforementioned 76-gene GS do not share any gene with the 21-gene GS, and only share one gene with the 70-gene GS. Although different GSs from the same cancer type rarely share genes, different GSs show similar prognostic performance in predicting the status of the cancer patientsTo test this hypothesis, we carried out a comprehensive motif analysis on the prognostic GSs from five cancer types . By de novo motifs discovery and comparison, we found that there exist shared motifs by GSs in individual cancer types as well as across cancer types. Nine of the twelve transcription factors (TFs) that likely bind to these shared motifs have reported prognostic functions in cancers. We further investigated the predicted cofactors of these TFs and found that 75% of the predicted cofactors may have prognostic or cancer-related functions. Moreover, we identified common microRNAs (miRNAs) that regulate genes in different GSs from an individual cancer type and even across cancer types. Several of these miRNAs are known prognostic biomarkers in the corresponding cancer types. Our study showed the existence of possible common regulatory mechanisms of GSs from individual cancer types and across cancer types, which shed light on the discovery of GSs in cancers and the understanding of the regulatory mechanisms of cancers.We collected prognostic GSs for five cancer types: breast cancer (7 GSs), colorectal cancer (5 GSs), leukemia (6 GSs), lymphoma (6 GSs), and lung cancer (5 GSs) (Supplementary Table 15 (hg38). The obtained sequences were repeat masked by RepeatMasker with the default setting (http://www.repeatmasker.org/). The de novo motifs were predicted by the SIOMICS 3.017 tool (http://hulab.ucf.edu/research/projects/SIOMICS/SIOMICS.html). SIOMICS can predict motifs without specifying the motif length and has been shown better performance than several popular methods16. In addition, it can predict cofactor motifs in the format of co-occurring motif groups, which we called motif combinations. We ran SIOMICS on the repeat-masked sequences in each GS with the default parameters except the parameter s = max , where T was set as the number of sequences in a GS. This was because the default parameter s = T*1% in SIOMICS intended to work for datasets with a large number of sequences (hundreds or thousands of sequences) instead of the small number of sequences in GSs we obtained here.For genes in each GS, we retrieved 1.5 kilobases (kb) of their upstream sequences relative to the gene translational start sites with the BioMart tool18 with the default parameters. For each predicted motif in one GS, all motifs from other GSs with the STAMP E-value smaller than 1e-08 were considered as its similar motifs. We used the more stringent cutoff 1e-8 instead of the widely used cutoff 1e-5 in previous studies19 to define similar motifs here, because we expected that the similarity of the predicted motifs by the same method should be high if they were the same motifs. With the defined similar motifs, we obtained all groups of similar motifs from different GSs per cancer type. We then defined shared motifs from the groups of similar motifs that contained predicted motifs from more than 50% of GSs from the same cancer type. For instance, with six GSs considered in leukemia, a shared motif in leukemia was required to be similar to at least one predicted motif in at least four GSs. For such a group of similar motifs, a shared motif was defined as the motif PWM of the motif in this group that was most similar to the known motifs in TRANSFAC20.We compared the predicted motifs by SIOMICS in each GS with those from all other GSs from the same cancer type. We compared motifs with their motif position weight matrices (PWMs), using the online version of STAMP21, with the default parameters. We randomly permuted the corresponding input sequences in each GS 20 times to obtain background sequences for HOMER. With the input sequences of genes in each GS and the corresponding background sequences, we ran HOMER with the motif length as 8, 9, 10, and 12, respectively. These motif lengths were determined by the length distribution of the motifs predicted by SIOMICS. We similarly compared these predicted motifs by HOMER with each shared motif predicted by SIOMICS.SIOMICS cannot predict motifs in small GSs well (e.g. GSs with no more than 10 genes). To corroborate the predictions by SIOMICS, and to study motifs in GSs where SIOMICS did not predict, we ran another motif discovery tool, HOMER V2To study the significance of the predicted motifs, we also ran SIOMICS with random sequences. For each GS, we generated random sequences by randomly permuting input sequences used by SIOMICS. That is, we randomized the order of nucleotides in input sequences to generate random sequences. We then predicted motifs in these random sequences with SIOMICS and HOMER. We repeated these steps 100 times for each GS.20. A shared motif was claimed to be similar to a known motif if the STAMP comparison E-value was smaller than 1e-05. We used 1e-5 because the two groups of motifs compared were from different sources and we may not be able to detect similar known motifs with the above stringent cutoff. We only considered the shared motifs with similar known motifs in TRANSFAC for further analyses, because we knew their functions better. In this way, we found the TFs and their functions for about 86% of the shared motifs.To obtain TFs that binds to shared motifs, we compared the shared motifs with known motifs in TRANSFAC22 (hg38). We obtained the “target genes” of each shared motif by scanning all 38478 sequences and their reverse complements with the motif PWMs. For a motif PWM, say M = (mij)k*4, where k is the motif length, we calculated the score of a DNA segment s1 s2 … sk as follows:si is the j-th type of nucleotides , and otherwise To study the function of the shared motifs, we obtained the putative target genes of a shared motif in the following way. We downloaded all 38478 human RefSeq genes (hg38) and retrieved the 1.5 kb upstream sequences of each gene from the University of California, Santa Cruz (UCSC) Genome Browseret al.23. In total, we had 1047 cancer-related gene sets, which contained 14196 genes with 13224 genes included in the above RefSeq gene set. We applied a binomial test to calculate the P-values of the significance of the overlap between a cancer-related gene set and the target genes of a motif combination. With a plethora of motifs combinations predicted, we could not study every motif combination. Instead, for each cancer type, we did the analyses with one randomly selected shared motif and all its corresponding motif combinations.To study the functions of target genes of motif combinations that contain the shared motifs, we obtained computationally inferred cancer-related genes sets (C4) and oncogenic cancer-related gene sets (C6) from Subramanian http://www.microrna.org, as in other studies24. For each predicted motif in a GS that was similar to a shared motif, we calculated the significance of the overlap between the target genes of this motif in this GS and each group of miRNA target genes by the hypergeometric test. The miRNAs with the hypergeometric testing P-value smaller than 0.05 were considered as candidate miRNAs that may regulate this GS. Since multiple shared motifs were identified in a GS, we claimed that a miRNA regulates this GS if the target genes of this miRNA significantly overlap with the target genes of more than 50% of the shared motifs in this GS. In other words, for each shared motif in this GS, the overlap of its target genes with the target genes of a miRNA that regulates this GS had a hypergeometric p-value smaller than 0.05. With the miRNAs that regulate each GS, we defined common regulating miRNAs of a cancer type as the miRNAs that regulated every GS in this cancer type. For each common regulating miRNA, we studied its prognostic functions and cancer-related functions in literature.To study miRNAs that bind to target genes of the shared motifs, we used miRNA target gene sets with the label of “Good mirSVR score, Conserved miRNA human” from We found that different GSs from the same cancer type seldom shared genes. We collected 7, 5, 6, 6, and 5 GSs from breast cancer, colorectal cancer, leukemia, lymphoma, and lung cancer, respectively. On average, there were 50, 57, 34, 74 and 47 genes in a GS from the corresponding cancer type, respectively .Since GSs from the same cancer type rarely shared genes while they characterized the same medical condition18 and defined similar motifs . We considered a group of similar motifs from each of the four GSs as a shared motif. We represented the shared motif for a group of similar motifs by one of the similar motifs in this group that had the smallest STAMP comparison E-value when compared with known TRANSFAC motifs. Considering the predicted SIOMICS motifs, we obtained the first shared motif in breast cancer (GGGSTGGG). Similarly, we identified other six shared motifs and their corresponding TFs . We compared these predicted motifs from different GSs by STAMPet al. studied 31 prognostic GSs from colorectal cancer and found that only five GSs had a significant association with prognosis25. They also found that all GSs had low reproducibility in independent datasets. Because of the above two reasons, we did not require a shared motif occur in all GSs per cancer type. Note that we also ran another tool HOMER21 to predict motifs. For the three GSs which have no prediction by SIOMICS, HOMER predicted motifs similar to the shared motifs , KLF6 (GBF), and EGR1 (KROX), respectively (the names in parentheses are the corresponding TRANSFAC TF names). Note that in order to show which TRANSFAC motifs were similar to the shared motifs, we used their corresponding TRANSFAC TF names in the following analyses. In leukemia, SIOMICS discovered two shared motifs that were similar to the motifs of MAZ and SP1. In lymphoma, SIOMICS found four motifs similar to the motifs of SP1, MAZ, GC and the CAC-binding protein, respectively. In the lung cancer, SIOMICS identified five shared motifs, which were similar to motifs of ZNF219, PATZ1 (MAZR), SP1, IKZF1 (Lyf-1), and the CAC-binding protein, respectively Table .From Table In addition to the significance of the shared motifs measured by their small P-values from SIOMICS Table , we furtTo corroborate the functionality of the predicted shared motifs and to investigate the potential functions of these shared motifs Table , we sear26. GC can inhibit human breast cancer cell proliferation and cancer cell-stimulated angiogenesis27. SP1 and MYC can modulate drug resistance of leukemia stem cells28. Moreover, five of the twelve TFs have been reported to be prognostic biomarkers in specific cancers, such as MAZ, KLF6 and BRF2 in breast cancer30, and ERG1 in colorectal cancer31. The aforementioned cancer-related functions of these TFs in their corresponding cancer types suggest that the predicted motifs are reliable and biologically significant.We found that the nine of the twelve TFs have cancer-related functions . We observed that 97.6% (328/336) of the target gene sets were significantly overlapped with the cancer-related gene sets . To assess the significance of the above overlaps, we also generated random gene groups. The number of genes in a random gene group was the same as that in the group of target genes of a corresponding motif combination. Genes in a random group were randomly selected from the 38478 RefSeq genes . We found that 80.0% (269/336) of actual target gene groups have smaller P-values than their corresponding random gene groups. The mean and median of the P-values from the random groups were 0.006 and 0.008, respectively, while that from actual target gene groups were 8e-5 and 0.001, respectively. Note that it was not surprising that certain random gene groups had low P-values, because about 34.4% (13224/38478) of the RefSeq genes considered were cancer-related genes.38. The cofactors PRDM1, EGR1, and TOPORS were shown to be tumor suppressors in colon cancer, leukemia, and lung cancer, respectively41. In addition, certain cofactors were verified to have prognostic functions in cancer. For instance, TP53 was shown to be a prognostic biomarker in breast cancer42. E2F was proved to be a prognostic biomarker43 in colorectal carcinoma. These facts corroborated the functions of the cofactor motifs and the shared motifs.Second, we searched literature for the functions of each cofactor in the motif combinations that contained shared motifs. With 469 cofactor motifs in the 336 motif combinations, it was time-consuming to study each cofactor in literature. Instead, in each type of cancers, we randomly picked one shared motif and all its motif combinations to do the analysis. There were in total 44 cofactors considered in five cancer types  identified in two of the five cancer types have general functions in cancers. For instance, TFAP2A can regulate tumor cell migration and apoptosis27. This protein complex is also able to inhibit the growth of human pancreatic cancer in immune compromised mice49. In another motif combination predicted in both breast cancer and colorectal cancer, KLF6 and SP1, these two TFs together initiate the transcription of CERS2 in human prostate carcinoma cells50. In colorectal cancer, the predicted combination of KLF6 and E2F1 co-exist in a protein complex51.Third, we studied whether the cofactors in the predicted motif combination indeed work together to regulate their target genes. For all twelve TFs in Table 52. Among these five TFs, we discovered that two of them, SP1 and KLF6 interact with their cofactors in the motif combinations: SP1 can interact with EGR1 and TFAP2A and KLF6 can interact with SP1 and GTF3C1. In colorectal cancer, we found that the cofactors of TFAP2A were PPARG and SP1. Both PPARG and SP1 were shown to interact with TFAP2A in BioGRID. All the above evidence suggested that cofactors in the motif combinations co-exist in cancer, which supported the functions of the shared motifs.Finally, we examined the cofactors of the TF that bound to the shared motifs using protein-protein interaction databases. We collected all cofactors of the seven TFs that were related to the seven shared motifs in breast cancer. Five of the seven TFs have interactive TFs in the experimentally verified TF interaction database BioGRIDIn summary, by studying the functions of the motif combinations and those of the cofactors, we proved that TFs binding to the shared motifs may function together with their cofactors in cancers, which supported the functionality of the predicted shared motifs and their cofactor motifs.53 and identified common miRNAs that may regulate GSs  of miRNAs were shown to be prognostic or potential prognostic biomarkers in breast cancer in literature (Table We predicted 61 common regulating miRNAs in breast cancer. We studied the functions of each of these miRNAs and found that about 90.2% (55/61) of them have breast cancer-related functions. For instance, hsa-mir-495 have been shown to be a novel therapeutic target for breast cancer55, hsa-mir-21 and hsa-mir-145 corporately regulate colon cancer growth and differentiation56. Compared with the well-studied breast cancer, we found a smaller number of prognostic biomarkers in other four cancer types (Table Similar to breast cancer, we found common regulating miRNAs in other four cancer types, and most of them have cancer-related functions Table . For ins57. This miRNA also promotes colorectal cancer cell migration and invasion by directly targeting DLC158. hsa-mir-106a promotes growth and metastasis of non-small cell lung cancer59, and its up-regulation plays an oncogenic role in pancreatic cancer60. In addition, hsa-mir-106a was significantly up-regulated in gastric cancer patients61. The above evidence showed that the identified common regulating miRNAs may have cancer-related function across different cancer types, which also suggested that the same miRNAs may regulate different GSs from the same or different cancer types.We noticed that 28 regulating miRNAs were shared by at least three types of cancers. For instance, two predicted common regulating miRNAs hsa-mir-106a and hsa-mir-106b, function in multiple cancer types. hsa-mir-106b expression determines the proliferation paradox of TGFB1 in breast cancer cellsWe discovered multiple shared motifs by GSs from five cancer types, which implied that GSs in individual cancer types and across cancer types may be regulated by common mechanisms. We studied the functions of the potential TFs that bind to these shared motifs and the functions of the corresponding cofactors of these potential TFs and found that 75% of the TFs and cofactors may have cancer-related functions and several even have prognostic functions. Similarly, we observed that GSs from the same or different cancer types may be regulated by the same miRNAs.25. This may be due to the limitation of the SIOMICS tool as well. As we pointed out above, SIOMICS cannot work well when there is no more than ten genes in a GS. This was the reason that we did not require a shared motif be shared by all GSs.SIOMICS did not find similar motifs to the shared motifs in all GSs from the same cancer type. This may be due to the quality of certain GSs used, as a previous study showed that certain prognostic GSs may be defined imperfectlyTo see whether our conclusions still hold with new GSs, we further studied additional GSs for each cancer type. We predicted motifs in one additional GS for each cancer type. As expected, the majority of the aforementioned predicted shared motifs were also identified in the additional GSs (Supplementary Table 25. We also notice that not all GSs used in this study contain the shared motifs, which may be because that not all GSs were of the same high-quality. Based on our observation that GSs from the same cancer type may be regulated similarly, scientists may in turn define GSs better. With more widely studied GSs collected in the future, we expect that we can discover the shared motifs in an even more robust and precise way.Scientists may define GSs more properly based on our studies. Previous studies showed that certain GSs may be defined improperlyThis study will also facilitate our understanding of the functions of TFs in cancer. About 75% of the TFs that potentially bind to the predicted shared motifs and their cofactor motifs may have cancer-related functions. Scientists can further refine prognostic TFs from the list of the predicted TFs in this study and further study the predicted common miRNAs to identify novel biomarkers in cancers.De novo motif discovery and motif comparison are still an open problem nowadays. With better defined GSs in cancers, improved motif discovery and comparison methods, we may find more precisely shared motifs across GSs in the same or across different cancer types. In addition, similar analyses can also be carried out for other types of cancers. We anticipate that there are also exist shared regulatory mechanisms underlying these GSs.supplementary information